Christian7e Experiments PDF

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EXPERIMENTS
Theory guides, experiment decides.
I. M. Kolthoff
I hear and I forget.
I see and I remember.
I do and I understand.
Confucius
The following experiments follow the order of coverage in the text, after introductory
experiments on the use of the analytical balance and volumetric glassware. Before
beginning experiments, you should review the material in Chapter 2 on the basic
tools of analytical chemistry and their use and the material in Appendix D on the
texts website on safety in the laboratory. It will also be helpful to review data
handling in Chapter 3, particularly signicant gures and propagation of errors, so
you will know how accurately to make each required measurement. You will also use
the spreadsheets described in that chapter and Chapter 16 for calibration plots and
unknown calculations, including precisions.
Video Resource. Professor Christopher Harrison from San Diego State University has a YouTube Channel of videos of different types of experiments, some illustrating laboratory and titration technique: http://www.youtube.com/user/crharrison.
You are encouraged to look at the ones dealing with buret rinsing, pipetting, and
aliquoting a sample, before you begin experiments. Also, you will nd useful the
examples of acidbase titrations illustrating methyl red or phenolphthalein indicator
change at end points. There are a few specic experiments that may be related to
ones you may perform from your textbook, for example, EDTA titration of calcium
or Fajans titration of chloride. If you perform an iodometric determination in which
iodine is titrated with thiosulfate using starch indicator, the glucose analysis using
titration video gives a good illustration of the starch end point.
Use of Apparatus
EXPERIMENT 1
USE OF THE ANALYTICAL BALANCE
Before beginning this experiment, familiarize yourself with the principles of the
balance and the general rules for weighing discussed in Chapter 2. The principles of
mechanical balances are presented on the text website.
Principle
You will use either an electronic balance or a mechanical single-pan balance (if you
have a double-pan balance, your instructor will provide instructions on its use). The
electronic balance is set to zero using the zero (tare) control bar or button before
weighing an unknown object. For mechanical balances, the zero point is determined
before weighing.
Solutions and Chemicals Required
None.
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EXPERIMENTS
Procedure
1. Zeroing or determination of the zero point. Dust the pan slightly with a small
brush. Color the doors of the balance.
(a) Electronic balance. Turn the balance on and touch the zero (tare) bar or
button. The reading should adjust to 0.0000 g.
(b) Mechanical balance. The balance has a knob that will rst partially release
the beam and the pan and then completely release them (two positions).
Release the beam and pan and adjust the zero reading. Arrest the pan and
the beam and then repeat the operation. Throughout the remainder of this
experiment, do all zero-point and rest-point determinations in duplicate;
thereafter, single determinations should sufce.
The zero or zero point of the balance will not remain constant. It may vary
by several tenths of a milligram from day to day, or even from hour to hour.
Hence, a new determination of the zero point must be made every time a series
of weighings is to be made or, if the zero has changed, rezero the balance.
2. Weighing of objects. Determine the weights of three test objects supplied by the
instructor. One of these may be the crucible cover to be weighed by difference in
part 3 below.
(a) Electronic balance. Make sure the balance is still zeroed. Place the test
object on the pan using paper or tongs. Close the doors and after the reading
stabilizes, record the weight to the nearest 0.1 mg.
(b) Mechanical balance. If more than an hour has elapsed, or if the balance has
been used by others since the zero point of the empty balance was adjusted,
it should be rechecked. Place the test object on the pan using paper or tongs
(never place or remove an object without the arrest engaged). Close the doors.
Move the beam arrest knob to the rst position. This partially releases the
beam but arrests it if it is so much off balance that it swings beyond a certain
position. This prevents damage to the knife edges when heavy objects are on
the pan and it is far off balance. Dial in weights from the weight knobs to the
nearest 0.1 g until the beam is in motion (near balance). Start with the larger
weights to nd the nearest 10 g, then the unit weights (nearest 1 g), and nally
the 0.1-unit weights (nearest 0.1 g). Then, completely release the beam and
read the weight to the nearest 0.1 mg from the vernier or digital counter after
it has come to rest. Record the total weight.
3. Weighing by difference. Obtain a crucible and cover (or any two objects of about
this weight) from your laboratory drawer. Weigh the cover of the crucible as in
part 2 and record this weight. Weigh the two objects combined and record this
weight. Remove the crucible cover and obtain the weight of the crucible. The
difference in the last two weighings represents the weight of the crucible cover.
This should agree within 0.5 mg with the weight obtained by direct weighing.
4. Electronic balance taring. Place the cucible cover on the pan, and push the tare
bar or button. The reading should come to zero. Then place the crucible on the
pan along with the cover, and record the weight. How close does this agree with
the weight for the crucible obtained in part 3?
EXPERIMENT 2 USE OF THE PIPET AND BURET
AND STATISTICAL ANALYSIS
Principle
Practice in the use of pipets is checked by weighing the amount of water delivered
in successive pipettings. Precision of delivery of different volumes is determined.
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USE OF APPARATUS
Similar experiments may be done with a buret. The experiments may also be used
for calculation (calibration) of the volumes of the glassware if the temperature of the
water is measured, as described in Table 2.4.
Cleaning solution; distilled water.
Procedure
1. Cleaning the glassware. Check your buret and pipets for cleanliness by rinsing
with distilled water and allowing to drain. Clean glassware will retain a continuous,
unbroken lm of water when emptied. If necessary, clean the buret with a detergent
and a buret brush. For pipets, try warm water, then detergent plus warm water. If
these do not work, use a commercial cleaning solution. Rinse the buret or pipet
several times with tap water and nally with distilled water. Leave the buret lled
with distilled water when not in use. Always check your volumetric glassware
for cleanliness before use, and clean whenever necessary. Burets if thoroughly
rinsed and lled with distilled water immediately after use should remain clean
for weeks; pipets, however, become contaminated easily and must be cleaned
frequently.
2. Pipetting. Practice lling a 25-mL pipet and adjusting the meniscus to the
calibration mark until you are procient. The forenger used to adjust the level
should be only slightly moistened. If it is too wet, it will be impossible to obtain
an even ow.
Weigh a clean, dry 50-mL Erlenmeyer ask and a rubber stopper to the
nearest milligram (0.001 g).
Transfer 25 mL water from the pipet to the ask; allow the water to run out
with the tip of the pipet touching the side of the ask, being careful that no water
splashes or is otherwise deposited on the neck of the ask. It is important that the
neck of the ask and the stopper remain dry throughout the experiment. The pipet
must be held vertically. Allow to drain for 10 s before removing the pipet. Do not
blow out the last drop. Replace the stopper and weigh.
Perform the procedure at least two times. Only the outside and the neck of
the ask need be dry for subsequent weighings. Calculate the standard deviation
and the range of delivery in parts per thousand as described in Chapter 3.
Repeat, using 1-, 5-, 10-, and 50-mL pipets. Compare the precision of
delivery for the different pipets.
3. Use of the buret. Check the stopcock (if ground glass) of your buret for proper
lubrication, and be sure the bore and tip are clear. Fill with distilled water and
place in the buret clamp; the water should be at room temperature. Displace any
air bubbles from the tip of the buret, and adjust to near the zero mark. Allow to
stand for a few minutes to check for leakage and read the initial volume to the
nearest 0.01 mL. Use a meniscus reader, and be careful to avoid parallax.
Using a procedure similar to that for the pipets, draw off about 5 mL into
the weighed ask. Touch the tip of the buret to the wall of the ask to remove the
adhering drop, and read the volume to the nearest 0.01 mL. Insert the stopper and
weigh to the nearest milligram. Repeat the operation, adding to the ask the next
5 mL water, and weigh again. Continue in this way until the entire 50 mL have
been weighed.1
1 To minimize evaporation errors, your instructor may direct you to rell the buret for each volume delivery (05,
010, . . . 050 mL) and deliver it into an empty (dry) ask.
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EXPERIMENTS
Subtract the weight of the empty ask from each of the succeeding weights
to get the weight of 5, 10, 15, and so forth, milliliters of water at the temperature
observed.
Repeat the entire procedure. At each approximate volume, the weight,
compared to the exact measured volume, should be within 0.03 g of that predicted
from the rst measurement. Recheck any points that are not.
4. Calibration of glassware. If your instructor wishes you to use the data from
these experiments to calibrate the volumes of the glassware, use Table 2.4 to
convert the weight in air to the volume. The temperature of the water delivered
must be determined; the Table 2.4 spreadsheet is on your text website, and you
can enter data in that by copying it to your computer desktop.
5. Statistics: The normal error curve. In this experiment, you will make 20
measurements of the weight of water delivered by a 1-mL pipet, as described
above, by adding successive pipet volumes to a small Erlenmeyer ask and
calculating the weight increase each time. Be sure to rapidly stopper the ask
each time. Plot the frequencies of the weights on graph paper to prepare a normal
error curve. Calculate the standard deviation and relate this to the plotted curve.
Do about 68% of the readings fall within one standard deviation?
EXPERIMENT 3 ANALYSIS OF VOLUMETRIC MEASUREMENTS
USING SPECTROPHOTOMETRIC MICROPLATE READERS
AND SPREADSHEET CALCULATIONS
Contributed by Professor Christopher Palmer, University of Montana
Objectives: This lab exercise introduces and reviews volumetric measurements, basic
statistical concepts, and use of spreadsheet software. It also serves to introduce the use
of the 96 well microplate format and the operation of spectrophotometric plate readers
for replicate measurements. Near infrared spectrophotometric measurements of water
are used to verify the performance of one or more pipettors and evaluate and improve
pipetting technique. The results are used to determine the volumetric accuracy and
precision of pipettors and to illustrate the use of signicant gures, condence interval
and propagation of error. Absorbance measurements of water are compared with that
of a phosphate buffer solution.
Overview: Pipettors often do not meet the manufacturers specications, and
pipetting technique can be a signicant source of error even when pipettors are
operating correctly. In order to reduce errors due to pipetting, the quality control system
in every laboratory should include verication of pipettor performance. The primary
method for pipettor calibration and verication is the gravimetric method, in which
the mass of a dispensed liquid of known density is determined. A secondary method
is to use spectrophotometry. Spectrophotometric methods utilize a solution containing
a known concentration of a highly-colored compound. Aliquots of that solution are
dispensed into a known volume of diluent, and the dispensed volume is calculated from
the measured absorbance. More recently, methods have been developed that utilize
spectrophotometric plate readers and near infrared absorbance of water, specically
the difference in absorbance at 970 nm and 900 nm (A970 A900 ).1 Commercial plate
readers that can determine the path length (or depth) of aqueous solutions in the plate
wells using the near infrared absorbance of the water are utilized. The linear relationship
between volume and path length is rst determined using a gravimetrically-calibrated
pipette that dispenses a known volume. Once the relationship has been determined,
the performance of additional pipettors can be rapidly veried.
1 D. G. Hafeman and C. T. Chow, Determination of Light Aborption Pathlength in Vertical-Beam Photometer,
U. S. Patent No. 5,959,738, September 28, 1999.
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USE OF APPARATUS
10
11
12
A
B
C
D
E
F
G
H
Spectrophotometric plate readers can read the absorbance (and in many cases
uorescence) of small volumes of sample in standard 96-well or 384-well plates. These
plates are simply an addressable array of wells or containers placed in a standard size
array. The wells in 96 well plates are arranged in 8 rows and 12 columns as shown in
Figure 1.1. The plate reader instrument is designed to read the absorbances of samples
placed in each of the wells. Because the plate reader is very fast, and because the
quantity of sample required for each well is small, the plate reader allows for rapid
standardization and replication of measurements for both standards and samples.
In this experiment, plates which have already been calibrated, using a gravimetrically calibrated pipet, to allow determination of volume from path length are utilized
to verify pipettor performance and skills in reproducibly delivering a xed volume
from the pipettor.
Procedure
1. Familiarize yourself with the operation and software on the plate reader instrument before pipetting any liquid into the plate reader. It is important to make
the readings within a few minutes of delivering the liquid to avoid errors caused
by evaporation. The plate will have been calibrated for volume vs. near infrared
absorbance of water, using a gravimetrically calibrated pipet to deliver the same
nominal volume as the pipet volumes being calibrated. If this has not been done,
your instructor will instruct you to do it.
2. Obtain a 20200 L pipettor, a 1001000 L pipettor, and a 96 well plate. Get
a small beaker of distilled water to dispense into the wells. Allow the water to sit
for a period to come to room temperature.
3. Select a volume between 150 and 200 L and dial this in to both pipettors. This
should be the same volume selected for the gravimetric measurements in Step 1.
Carefully pipette this volume of water into each well of column 5 on a plate using
the 20200 L pipettor and into each well of column 6 using the 1001000 L
pipettor.
4. Open the plate drawer on the spectrophotometer, place the plate in the correct
position in the open drawer, and close the drawer to introduce the plate into the
instrument.
5. Close all open data and method les and open the method le provided by the
instructor. The le should include a calibration data and graph for the plate.
Near infrared absorbance measurements will be made at 970 nm and 900 nm to
determine A970 A900 .
6. Following specic instructions for the instrument, use it to automatically read
and report the absorbance and path length for each of the wells in Columns 5
and 6.
Fig. 1.1.
Arrangement of wells on
a 96-well plate.
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EXPERIMENTS
7. Find and identify the data. The raw path length data and volumes calculated
from the gravimetric calibration data are reported, as are the mean, standard
deviation, and relative standard deviation (RSD) for each plate column. Save
the data to a USB ash drive, and manually copy the volume data into your
notebook.
8. Make a cursory check of the performance of the pipettor and your technique by
comparing the mean volume with the selected volume and by checking the RSD
for the 20200 L pipettor. The RSD should be < 1%. If it is greater than 1%,
practice to improve your technique. Do this using the wells in columns 1012,
and reading them as described in steps 67.
9. Repeat steps 38 using a different volume between 150 and 200 L for each
pipettor, and lling and reading plate columns 7 and 8.
10. Repeat steps 38 using the 20200 L pipettor and the same volume used in
step 9, but delivering the 50 mM pH 7.0 phosphate buffer provided instead of
water. Use plate column 9.
11. Clean the 96 well plate by rinsing with distilled water and methanol.
Lab Report
Open the data les and import the data into a spreadsheet program. Calculate the
mean, standard deviation, relative standard deviation and 95% condence interval
for each set of 8 measurements. Determine if there are any statistical outliers in
any of the data sets, and decide whether to exclude up to one measurement in each
data set.
Make a table that includes the selected volume, mean volume delivered, standard
deviation, and 95% condence interval for each set of replicates (5 sets in total). Make
a second table to include the calculated Student t and an indication as to whether there
is a signicant difference at 95% condence between the volumes delivered and the
volume selected on the pipettor for each set of data (5 sets in total). Discuss these
results. Report the calculated F-test values and an indication of whether there is a
signicant difference in the reproducibility when using the 20200 L pipettor vs.
the 1001000 L pipettor when delivering the same nominal volume. Repeat this
calculation for each of the volumes delivered (2 calculations/comparisons) and discuss
the implications of the results. Report the calculated Student t and an indication as to
whether there is a signicant difference at 95% condence between the volumes of
water and of buffer delivered when the pipettor is set to deliver the same volume (data
from sections 9 and 10). Discuss the implications of this result in terms of pipettor
performance and any potential systematic errors in the measurements.
Gravimetry
EXPERIMENT 4
GRAVIMETRIC DETERMINATION OF CHLORIDE
Principle
The chloride content of a soluble sample is determined by precipitating silver chloride
with added silver nitrate, ltering, drying, and weighing the precipitate. The Cl content
is calculated from the weight of AgCl.
Equation
Ag+ + Cl AgCl
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GRAVIMETRY
Solutions Required
1. Provided. Conc. HNO3 , conc. NH3 , dil. (3 M) HCl.
2. To prepare
(a) 0.1 M AgNO3 . Dissolve about 3 g solid AgNO3 (need not be dried) in about
180 mL distilled water. Store in a brown bottle. Do not get AgNO3 on your
hands. If you do, wash immediately with a sodium thiosulfate solution. If left
on the skin, brown-black spots (metallic silver) will appear within several
hours and will take 2 to 3 weeks to wear off.
(b) Wash solution. Add approximately 2 mL chloride-free concentrated nitric
acid to about 600 mL distilled water in a wash bottle.
Things to Do before the Experiment
1. Obtain your unknown from your instructor.
(a) Solution. Give your instructor a clean, labeled, 250-mL volumetric ask
with stopper. The ask should be cleaned and rinsed with three or four portions
of distilled water. It need not be dry. After receiving the unknown solution in
the ask, dilute to the mark with distilled water. Mix well (see Chapter 2 for
proper procedure).
(b) Solid. Obtain a soluble chloride sample from your instructor and place it in
a clean weighing bottle. The weighing bottle and cap are placed in a beaker
marked with your name and covered with a watch glass during the drying.
The weighing bottle should not be stoppered with the cap. Dry in the oven for
1 to 2 h at 120 to 160 C (about 1 h in a forced-air oven, or 2 h in a gravity
convection oven). Store in a desiccator after drying until ready for weighing
(wait until cooled before placing the cap on the bottle, to avoid creation of a
vacuum in the capped bottle).
2. Prepare three lter crucibles as described below.
3. Prepare the 0.1 M AgNO3 solution.1
Preparation of Crucibles
1. Sintered-glass lter crucible. Prepare three crucibles. Use crucibles of ne or
medium porosity and not coarse porosity. Clean each crucible from surface
contamination using soap and water, rinse, then place it in a crucible holder in
a suction ask. If chemical cleaning is required, follow the procedure at the end
of the experiment. With gentle suction, draw several small portions of distilled
water through the lter. Place three crucibles thus prepared in a beaker (marked
with your initials), cover with a watch glass, and put the beaker and crucibles in
the oven at 120 to 130 C for 1 to 2 h. With clean crucible tongs, transfer the hot
crucibles to the desiccator, cool for 30 to 40 min, and weigh accurately. Repeat the
heating for 0.5 h, cool, and weigh. Continue until weights constant within 0.3 to
0.4 mg are obtained.
2. Porcelain crucibles with glass ber lter paper. Place two glass ber lter
papers in the crucible to just cover the bottom of the porcelain crucible (Gooch
crucible). Wash with water as above and heat to constant weight (0.3 to 0.4 mg
is obtained).
1 Your laboratory will very likely have a collection bottle for leftover silver solutions and precipitates. Both
economy and pollution abatement make it important not to dump these materials down the drain.
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Procedure
1. Preparation of the sample
(a) Solution. Pipet three 50-ml, aliquots into three separate 500-mL beakers.
Dilute each sample with 100 to 150 mL distilled water. Add about 0.5 mL
concentrated nitric acid. Cover the beakers with clean watch glasses.
(b) Solid. After the dried sample has cooled in a desiccator for at least 30 to
40 min, weigh accurately triplicate samples of about 0.2 to 0.3 g into numbered
400-mL beakers. (Samples may contain carbonate, which is hygroscopic. If so,
your instructor will advise you to weigh the samples by difference.) Dissolve
the sample in distilled water and dilute to about 150 mL. Add about 0.5 mL
chloride-free concentrated nitric acid. (If carbonate-containing samples are
used, add slowly until the foaming action stops.)
2. Precipitation. Assume the solid sample to be pure sodium chloride, and calculate
the millimoles of silver nitrate required to precipitate the chloride. The slow
addition of silver nitrate to the solution is best accomplished by use of a buret,
with stirring. Wash the buret well with tap water, rinse with three or four portions of
distilled water, and nally ll it with approximately 0.1 M silver nitrate solution.
To each sample, add silver nitrate solution slowly with good stirring until an
excess of 10% over the calculated amount is added. Heat the suspensions nearly
to boiling, with frequent or constant stirring, to coagulate the silver chloride; the
stirring helps prevent bumping of the solution during heating and the danger of
loss of precipitate. Let the precipitate settle, and test for complete precipitation
by carefully adding a few drops of silver nitrate to the clear supernatant liquid; if
more precipitate or a cloudiness appears, add a few more milliliters silver nitrate
solution, stir well, heat, let the precipitate settle, and test again. Continue in this
way until precipitation is complete. Let the covered beakers with their contents
stand in the desk, protected from light,2 for at least 2 h before ltration; standing
overnight, or from one laboratory period to the next, may be required, and this is
all right as long as they are kept in the dark.
3. Filtration and washing of the precipitate. Decant the solution from the rst
sample through the rst weighed crucible, pouring the solution down a stirring
rod, and using gentle suction. The precipitate should be disturbed as little as
possible. To the precipitate in the beaker add about 25 mL of the wash solution,
stir well, let the precipitate settle, and decant the solution through the lter
crucible. The nitric acid wash solution replaces silver nitrate adsorbed on the
surface of the precipitate. An electrolyte is required to prevent peptization of the
sample. The nitric acid is volatilized during the drying step. Repeat the washing
by decantation four times, and nally bring the precipitate onto the lter; use
small portions of the wash solution for transfer. Remove with a rubber policeman
any solid particles adhering to the beaker. Continue washing the precipitate in the
crucible with the wash solution until the last portions of washings give a negative
test for silver ion. Silver ion is tested for by adding a drop of dilute hydrochloric
acid. Ten or more washings may be required. Filter and wash the second and the
third samples in the same way.
4. Drying and weighing of the precipitate. Place the crucibles containing the
precipitates in a covered beaker in the oven for 2 h at 120 to 130 C. Cool the
crucibles in the desiccator, and weigh accurately. Reheat for 1-h periods as
necessary to obtain weights constant within 0.3 to 0.4 mg.
2 When exposed to light, AgCl decomposes as follows: AgCl Ag + 1 Cl , resulting in a purple coloration
2
2
on the surface of the precipitate due to metallic silver, and a loss in weight. Some coloration cannot be avoided
and is not serious as long as strong light (especially sunlight) is avoided as much as possible.
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GRAVIMETRY
Cleaning the Crucibles

After completing the analysis, clean the crucibles as follows. Remove the cake of silver
chloride, then place the crucibles in a beaker, put about 5 mL concentrated ammonia
solution in each crucible, and cover the beaker with a watch glass. After 10 to 15 min,
transfer the crucible to the crucible holder in a suction ask and wash with several
portions of distilled water. If the lter plate is dark, empty and rinse the ammonia
from the suction ask and treat the crucible in the same way with a few milliliters
concentrated nitric acid. Finally, wash well with distilled water.
Calculations
1. Solution. Calculate and report the grams of Cl contained in your unknown.
Since one-fth of the sample was taken for each determination (50 mL out
of 250 mL), the weight determined in each aliquot must be multiplied by 5.
Report each individual result, the mean, and the relative standard deviation
in ppm.
2. Solid. Calculate and report the percent Cl in your unknown for each portion
analyzed. Report each individual result, the mean, and the relative standard
deviation.
EXPERIMENT 5 GRAVIMETRIC DETERMINATION OF SO3

IN A SOLUBLE SULFATE
Principle
Sulfate is precipitated as barium sulfate with barium chloride. After the ltering with
lter paper, the paper is charred off, and the precipitate is ignited to constant weight.
The SO3 content is calculated from the weight of BaSO4 .
Equation
SO4 2 + Ba2+ BaSO4
1. Provided. Dil. (0.1 M) AgNO3 , conc. HNO3 , conc. HCl.
2. To prepare. 0.25 M BaCl2 . Dissolve about 5.2 g BaCl2 (need not be dried) in
100 mL distilled water.
1. Obtain your unknown and dry. Check out a sample of a soluble sulfate from the
instructor and dry it in the oven at 110 to 120 C for at least 2 h. Allow it to cool
in a desiccator for at least 0.5 h.
2. Prepare crucibles. Clean three porcelain crucibles and covers. Place them over
Tirrill burners and heat at the maximum temperature of the burner for 10 to
15 min; then place in the desiccator to cool for at least 1 h. Weigh accurately each
crucible with its cover. Between heating and weighing, the crucibles and covers
should be handled only with a pair of tongs.
3. Prepare the 0.25 M BaCl2 solution.
Procedure
1. Preparation of the sample. Weigh accurately to four signicant gures, using
the direct method, three samples of about 0.5 to 0.7 g each. Transfer to 400-mL
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EXPERIMENTS
beakers, dissolve in 200 to 250 mL distilled water, and add 0.5 mL concentrated
hydrochloric acid to each.
2. Precipitation. Assume the sample to be pure sodium sulfate and calculate the
millimoles barium chloride required to precipitate the sulfate in the largest sample.
Heat the solutions nearly to boiling on a wire gauze over a Tirrill burner and
adjust the burner to keep the solution just below the boiling point. Add slowly
from a buret, drop by drop, 0.25 M barium chloride solution until 10% more than
the above calculated amount is added; stir vigorously throughout the addition. Let
the precipitate settle, then test for complete precipitation by adding a few drops of
barium chloride without stirring. If additional precipitate forms, add slowly, with
stirring, 5 mL more barium chloride; let settle, and test again. Repeat this operation
until precipitation is complete. Leave the stirring rods in the beakers, cover with
watch glasses, and digest on the steam bath until the supernatant liquid is clear.
(The initial precipitate is ne particles. During digestion, the particles grow to
lterable size.) This will require 30 to 60 min or longer. Add more distilled water
if the volume falls below 200 mL.
3. Filtration and washing of the precipitate. Prepare three 11-cm No. 42 Whatman
lter papers or equivalent for ltration; the paper should be well tted to the
funnel so that the stem of the funnel remains lled with liquid, or the ltration will
be very slow. See the discussion in Chapter 2 for the proper preparation of lters.
Filter the solutions while hot; be careful not to ll the paper too full, as the barium
sulfate has a tendency to creep above the edge of the paper. Wash the precipitate
into the lter with hot distilled water, clean the adhering precipitate from the
stirring rod and beaker with the rubber policeman, and again rinse the contents
of the beaker into the lter. Examine the beaker very carefully for particles of
precipitate that may have escaped transfer. Wash the precipitate and the lter
paper with hot distilled water until no turbidity appears when a few milliliters
of the washings acidied with a few drops of nitric acid are tested for chloride
with silver nitrate solution. During the washing, rinse the precipitate down into
the cone of the lter as much as possible. Examine the ltrate for any precipitate
that may have run through the lter.
4. Ignition and weighing of the precipitate. Loosen the lter paper in the funnels
and allow to drain for a few minutes. Fold each lter into a package enclosing
the precipitate, with the triple thickness of paper on top. Place in the weighed
porcelain crucibles and gently press down into the bottom. Inspect the funnels for
traces of precipitate; if any precipitate is found, wipe it off with a small piece of
moist ashless lter paper and add to the proper crucible. Place each crucible on
a triangle on a tripod or the ring of a ring stand, in an inclined position with the
cover displaced slightly. Heat gently with a small ame (Tirrill burner) until all
the moisture has been driven off and the paper begins to smoke and char. Adjust
the burner so that the paper continues to char without catching re. If the paper
inames, cover the crucible to smother the re, and lower the burner ame. When
the paper has completely carbonized and no smoke is given off, gradually raise
the temperature enough to burn off the carbon completely. A red glowing of the
carbon as it burns is normal, but there should be no ame. The precipitate should
nally be white with no black particles. Allow to cool. Place the crucible in a
vertical position in the triangle, and moisten the precipitate with three or four
drops of dilute (1:4) sulfuric acid. Heat very gently until the acid has fumed off.
(This treatment converts any precipitate that has been reduced to barium sulde
by the hot carbon black to barium sulfate.) Then, cover the crucibles and heat to
dull redness in the full ame of the Tirrill burner for 15 min.
Page 10
E11
GRAVIMETRY
Allow the covered crucibles to cool in a desiccator for at least 1 h and then
weigh them. Heat again to redness for 10 to 15 min, cool in the desiccator, and
weigh again. Repeat until two successive weighings agree within 0.3 to 0.4 mg.
Calculation
Calculate and report the percent SO3 in your unknown for each portion analyzed.
Report also the mean and the relative standard deviation.
EXPERIMENT 6 GRAVIMETRIC DETERMINATION OF NICKEL
IN A NICHROME ALLOY
Principle
Nickel forms a red chelate with dimethylglyoxime (DMG), which is quite suitable for
gravimetric analysis. Precipitation of the chelate is complete in an acetic acidacetate
buffer or in an ammoniacal solution. Acetate buffer is generally used when Zn, Fe,
or Mn is present in the alloy. The sample given to you is a nichrome alloy that has
Ni (approximately 60%), Cr, and Fe as the major constituents. Interference from Cr
and Fe is removed by complexation with tartrate or citrate ions. Precipitation is then
carried out in an ammoniacal solution. The Ni content is calculated from the weight of
the precipitate (see Table 10.2 for the formula).
Equation
Ni2+ + 2HDMG Ni(DMG)2 + 2H+
Solutions and Chemicals Provided

HCl (1:1), HNO3 (1:1), NH3 solution (conc. and 1:1), 1% DMG in ethanol, 20% citric
acid solution, 30% ethanol.
1. Obtain the alloy sample from your instructor.
2. Prepare three sintered-glass lter crucibles. Dry to a constant weight of 0.3
to 0.4 mg. (Repeated heatings, see Experiment 3.)
Procedure
1. Dissolving the sample. Weigh accurately triplicate 0.10- to 0.12-g samples of
the alloy. Place in separate numbered 400-mL beakers and add 25 mL each of
HCl (1:1) and HNO3 (1:1). Heat moderately (under the hood, please) until the
brown fumes are driven off40 to 50 min. Keep the beakers covered partially
with watch glasses. (You may notice some undissolved carbon particles in the
residue. If this occurs, then, using quantitative technique, lter the contents of
each beaker through Whatman lter paper No. 40 into separate 600-mL beakers.
Wash the 400-mL beaker several times with small amounts of water and transfer
the washings to the 600-mL beaker through the lter. Wash the lter several times
with small amounts of water. Remove the lter paper and wash the funnel and
stem with small amounts of water before removing from the receiving beaker.)
2. Precipitation. Add 4 to 6 mL citric acid solution and 100 to 150 mL water to
each beaker. Add conc. NH3 dropwise with stirring until the solution is slightly
basic (smell of ammonia in the solution). This should take 2 to 3 mL. Test to see
that the pH is about 8 by touching the wet stirring rod to pH paper. Be careful
that no drops adhere to the rod to keep solution loss negligible. If a precipitate is
Page 11
E12
EXPERIMENTS
formed, insufcient citric acid has been added. Dissolve the precipitate by adding
dropwise HCl (1:1, about 3 mL) until the pH is about 3. Add 1 to 2 mL more citric
acid solution. Check again by adding conc. NH3 . If no precipitate is formed add
HCl (1:1) until the solution is slightly acidic (no ammonia odor in solution). Heat
the beakers on the hot plate to about 70 to 80 C. Do not boil. Remove from the
hot plate and add about 50 mL 1% DMG solution dropwise with stirring. If a red
precipitate forms, add HCl (1:1) dropwise until it dissolves. The DMG should be
homogeneous throughout the solution before the precipitation is begun. Add NH3
(1:1) dropwise with stirring until the solution strongly smells of ammonia; this
may take 3 to 4 mL. A red precipitate is formed. It is important that the solution be
distinctly alkaline at this point. Since the nose may retain the odor of the ammonia
reagent, take care that the odor comes from the beaker. Test to be sure the pH is
greater than 8.5 by touching the wet stirring rod to pH paper to test the pH. This is
best done after the bulk of the precipitate is formed and allowed to settle. Cover
the beakers with watch glasses and set aside for at least 2 h (preferably overnight).
3. Filtering and washing the precipitate. Filter the precipitate through previously
weighed glass crucibles using gentle suction. After transferring the bulk of
the precipitate, wash the precipitate with lukewarm distilled water and transfer
quantitatively into the crucible. If traces of the precipitate (which are difcult to
transfer with a rubber policeman) stick to the sides of the beaker, dissolve in a few
drops of hot HCl (1:1) and reprecipitate by adding a few drops of 1% DMG and
NH3 (1:1). The precipitate now obtained will not stick and can be transferred to
the crucibles. Wash the precipitate in the crucible at least three to four times with
warm water. Finally, wash the precipitate once with 30% alcohol, which dissolves
any excess DMG from the precipitate.
4. Drying and weighing the precipitate. Place the crucibles in beakers (labeled
with your name and covered with watch glasses). Dry in the oven for 12 h at
110 to 130 C. Cool the crucibles in a desiccator (for 30 to 40 min) and weigh
accurately. Reheat for 1-h periods necessary to obtain weights constant to within
0.3 to 0.4 mg.
Calculation
Calculate and report the percent nickel in your unknown for each portion analyzed.
Report each individual result, the mean, and the relative standard deviation.
AcidBase Titrations
EXPERIMENT 7 DETERMINATION OF REPLACEABLE HYDROGEN
IN ACID BY TITRATION WITH SODIUM HYDROXIDE
Principle
One-tenth molar sodium hydroxide is prepared and standardized against primary
standard potassium acid phthalate (KHP). The unknown is titrated with the standardized
sodium hydroxide.
Equations
CO2H
KHP
CO2
+ OH
HA + OH A2 + H2O
CO2
CO2
+ H2O
Page 12
ACIDBASE TITRATIONS

1. Provided. KHP, 0.2% phenolphthalein solution in 90% ethanol, 50% NaOH
solution.
2. To prepare.1 0.1 M NaOH solution. This solution must be carbonate free. The
Na2 CO3 in NaOH pellets is insoluble in 50% NaOH solution. The precipitated
Na2 CO3 will settle to the bottom of the container after several days to a week,
and the clear, syrupy supernatant (approximately 19 M in NaOH) can be carefully
withdrawn or decanted from the container to obtain carbonate-free NaOH. The
solution is prepared in a borosilicate (Kimax or Pyrex) beaker (it gets very warm)
and after cooling is stored in a polyethylene bottle. Your instructor will provide
the carbonate-free 50% solution to you.
Distilled water free from carbon dioxide will be needed in this experiment. To
prepare this, ll a 1000-mL Florence ask nearly to the shoulder with distilled water,
insert a boiling rod with the cupped end down (the cup must be empty), heat to boiling
on a wire gauze over a Meker burner, and boil for 5 min. Cover the ask with an
inverted beaker and allow to cool overnight, or cool under the cold water tap. Prepare
an additional 800 mL the same way.
Fill a 500-mL bottle, with rubber stopper, half full of CO2 -free water, add 2.5 mL
clear 50% NaOH solution, stopper, and swirl to mix.2 Avoid exposing the solutions to
the atmosphere as much as possible. Finally, add more CO2 -free water to nearly ll
the bottle, stopper, and shake thoroughly.
The sodium hydroxide thus prepared is approximately 0.1 M. It will be standardized against primary standard potassium acid phthalate.
1. Obtain KHP and dry. Dry about 4 g primary standard potassium acid phthalate
in a weighing bottle at 110 to 120 C for 1 to 2 h. Cool in a desiccator for at least
30 to 40 min before weighing.
2. Obtain your unknown and dry. The unknown may be either a liquid or a solid.
If it is a solid, obtain it in a weighing bottle before the day of the experiment and
dry for 2 h at 110 to 120 C. Store in the desiccator. If the unknown is a liquid,
obtain it in a clean 250-mL volumetric ask and dilute to volume with CO2 -free
water. Mix well.
Procedure
1. Standardization of the 0.1 M NaOH solution against potassium acid phthalate.
Weigh accurately three portions of the dried potassium acid phthalate of about 0.7
to 0.9 g each, and transfer to clean 250-mL wide-mouth Erlenmeyer asks. (These
quantities are designed for titrations using 50-mL burets. If you are supplied
a 25-mL buret, your instructor will direct you to adjust the quantities of KHP
and the unknown accordingly.) The direct method of weighing, using a tared
weighing dish, may be used with this material. Dissolve each sample in about
50 mL CO2 -free distilled water. Rinse your buret with three small portions of
the 0.1 M NaOH solution, ll, and adjust to near zero. Record the initial volume
reading to the nearest 0.02 mL. Add 2 to 3 drops phenolphthalein indicator to each
KHP sample and titrate with the 0.1 M NaOH to a faint pink end point. The color
1 If you are to use this solution in Experiment 8 to standardize HCl, prepare 1 L instead of 500 mL.
2 If the NaOH were pure, only 2 mL (2 g NaOH) would be required for 500 mL of 0.1 M NaOH. Two and one-half
milliliters is required to compensate for the water and Na2 CO3 content of the pellets.
E13
Page 13
E14
EXPERIMENTS
should persist at least 30 s. Split drops at the end of the titration. Estimate the
buret reading to the nearest 0.02 mL.
Calculate the molarity of the NaOH to four signicant gures from the
weight of KHP used (three signicant gures if the molarity is slightly less than
0.1 M). Use the average of the results.
Keep the NaOH bottle stoppered with a rubber stopper, and protect the
solution from the air as much as possible to minimize absorption of CO2 . Proceed
as soon as possible to the determination of the unknown acid; standard sodium
hydroxide should be used within one week of standardization. After that time,
changes in molarity may have occurred, and restandardization will be necessary.3
2. Determination of replaceable hydrogen in an unknown acid. If the unknown
is a weak acid (e.g., KHP, acetic acid, or vinegar) you will be instructed to use
phenolphthalein indicator (color change same as above). If it is a strong acid, you
may use another indicator, such as chlorophenol red (color change from yellow to
violet).
If the unknown is a solid, dry it for 2 h at 110 C. Cool for 30 min. Your
instructor will inform you of the approximate weight of sample to take so that
about 30 to 40 mL NaOH will be used. If it is a KHP unknown, approximately 1 g
may be taken. Weigh three samples to the nearest 0.1 mg and transfer them into
numbered 250-mL wide-mouth Erlenmeyer asks. Dissolve in 50 mL CO2 -free
water, warming if necessary. Add two drops of indicator and titrate with 0.1 M
NaOH until the color change persists 30 s.
If the unknown is a liquid, transfer with a pipet three 50-mL aliquots from
the 250-mL volumetric ask and titrate as above.
Calculation
1. Solids. Calculate and report the percent of replaceable hydrogen or percent KHP
in your unknown for each portion analyzed.
2. Liquid. Calculate and report the milligrams replaceable hydrogen in your
unknown. Since one-fth of the sample was taken for each determination (50 mL
out of 250 mL), the weight determined in each aliquot must be multiplied by 5.
EXPERIMENT 8
OF SODA ASH
DETERMINATION OF TOTAL ALKALINITY
Principle
One-tenth molar hydrochloric acid is standardized against primary standard sodium
carbonate. Phenolphthalein is used to approximate the halfway point of the titration,
and then either modied methyl orange indicator or bromcresol green indicator is used
to detect the nal end point; the last indicator is used with boiling of the solution
near the end point to remove CO2 (see discussion of Figure 8.10). Alternatively,
the hydrochloric acid is standardized against standardized sodium hydroxide from
Experiment 6 using phenophthalein indicator. The soda ash sample is titrated with the
hydrochloric acid solution, with the addition of 2 mol hydrogen per mole Na2 CO3 .
Equations
CO3 2 + 2H+ H2 CO3

H2 CO3 H2 O + CO2
3 If you are to use this solution in Experiment 8 to standardize the HCl, your instructor will advise you to save the
remaining solution after completing this experiment. In that event, you should prepare and standardize the HCl
within one week of standardizing the NaOH.
Page 14
ACIDBASE TITRATIONS

1. Provided. 0.2% phenolphthalein in 90% ethanol, either modied methyl orange
(mixture of methyl orange and xylene cylanole FF available from Eastman Kodak
Co.) or 0.1% bromcresol green in 0.001 M NaOH, and either primary standard
Na2 CO3 or standardized 0.1 M NaOH.
2. To prepare. 0.1 M HCl. Concentrated hydrochloric acid has a density of 1.18
and contains 37% by weight HCl. Hence, about 4 mL concentrated acid should
be diluted to 500 mL to make 0.1 M acid. Measure about 0.5 mL more than
this amount in a 10-mL graduated cylinder and pour into water in a 500-mL
glass-stoppered bottle that is lled to the shoulder with distilled water.1 Shake
until the solution is homogeneous.
1. Obtain and dry the Na2 CO3 . If you are to standardize your HCl against
Na2 CO3 , obtain about 2 g primary standard Na2 CO3 in a weighing bottle. Place
the weighing bottle and cover (removed) in a 150-mL beaker marked with your
name and dry at 160 C for 2 h or more. Cool at least 30 min in a desiccator before
weighing.
2. Obtain and dry your unknown. Obtain the unknown in a weighing bottle and
dry as above for the Na2 CO3 .
Procedure
Standardize the HCl by either method 1 or method 2 below.
1. Standardization of HCl against Na2 CO3 . Weigh accurately (to 0.1 mg) the
weighing bottle containing the dried sodium carbonate. Keep the stopper in place
while weighing. Transfer quantitatively (for four-gure accuracy) about 0.2 g
to a clean 250-mL Erlenmeyer ask. The difference is the weight of sodium
carbonate taken. Anything between 0.16 and 0.24 g will be satisfactory. (This
procedure is called weighing by difference and is necessary because Na2 CO3 is
hygroscopic.) Weigh out a second and third portion in the same way. Add about
50 mL distilled water and one to two drops phenolphthalein indicator solution to
each ask.
The Na2 CO3 will be titrated rst to a phenolphthalein end point (pink to
colorless; 1 H+ added: CO3 2 + H+ HCO3 ) to approximate where the nal
end point should be (modied methyl orange or bromcresol green indicator; 1
more H+ added: HCO3 + H+ H2 CO3 ). Rinse the buret three times with small
portions (about 5 mL each) of the approximately 0.1 M acid prepared above, then
ll and adjust to near the zero mark. Record the volume to the nearest 0.02 mL.
Titrate the rst sample of the sodium carbonate, adding the acid no faster than
0.5 mL per second, swirling the ask constantly until the pink color disappears. At
this point, about half the total volume of acid necessary has been added (actually
a slight excess). Use this number to estimate where the nal end point will occur,
keeping in mind that slightly less acid should be required than has been added.
Continue the titration by either method (a) or method (b) below.
(a) Modied methyl orange indicator. Before beginning, check with your
instructor which indicator you are to use. Add two to three drops of modied
methyl orange indicator and titrate until the indicator color changes from
green to gray. The proper color can be more easily discerned by comparing
1 If you are to do Experiment 21, pH Titration of Unknown Soda Ash, prepare twice this amount and use this
standardization for that experiment.
E15
Page 15
E16
EXPERIMENTS
with the color of two drops of the indicator in a solution prepared by adding
0.20 g potassium acid phthalate in 100 mL distilled water. The pH of this
solution is 4.0, the same as the end-point pH in the presence of CO2 . Use the
same technique for the standardization and the unknown titrations.
(b) Bromcresol green indicator (alternative method). Add two to four drops
bromcresol green. The color change for this indicator is from blue through
pale green to yellow. Titrate to a blue-green color (just before the end point);
interrupt the titration at this point and boil the solution carefully for 2 to
3 min to drive off the carbon dioxide. The color should revert to blue. Cool
the solution to room temperature and continue the titration to the pale-green
color. This marks the nal end point.
Titrate the other two samples in the same manner as the rst and calculate
the molarity of the HCl from the weights of Na2 CO3 taken, remembering
that each carbonate has reacted with two protons. Use the mean of the three
determinations for calculations involving the unknown.
2. Standardization of HCl against standardized 0.1 M NaOH (alternate procedure).2
Rinse your 25-mL pipet with 0.1 M HCl, and add with the pipet 25 mL to a 250mL Erlenmeyer ask. Add two to three drops phenolphthalein indicator solution.
Rinse your buret with three small portions of the 0.1 M NaOH solution prepared
and standardized in Experiment 7. Fill and adjust to near zero. The NaOH is
placed in the buret rather than in the ask to protect it more from atmospheric
CO2 . Record the volume to 0.02 mL. Titrate the acid until a faint pink color is
obtained that lasts for at least 30 s. Repeat the titration two more times, using
your 50-mL pipet for the second and third titrations if less than 25 mL NaOH was
required in the rst. Calculate the molarity of the HCl.
3. Determination of sodium carbonate in soda ash. Accurately weigh out by
difference three samples of about 0.25 to 0.35 g each, dissolve each in about
60 mL water, and, titrate with 0.1 M HCl, following the same procedure with
respect to indicators and end point used in the standardization procedure against
Na2 CO3 or, if not used, the one specied by your instructor.
Calculation
Calculate the percentage of Na2 CO3 or Na2 O in your unknown for each portion
analyzed. Your instructor will specify which one is to be reported.
EXPERIMENT 9 DETERMINATION OF ASPIRIN
USING BACK TITRATION
Contributed by Professor Aleeta M. Powe, University of Louisville
This experiment is designed to illustrate techniques used in a typical indirect or back
titration. Standardized NaOH will be used to back titrate an aspirin solution and
determine the concentration of aspirin in a typical analgesic tablet.
Principle
Many reactions are slow or present unfavorable equilibria for direct titration. Aspirin
is a weak acid that also undergoes slow hydrolysis (i.e., each aspirin molecule reacts
2 The sodium hydroxide solution is a secondary standard, and any errors in standardization will be represented
in the standardization of the hydrochloric acid. The sodium hydroxide should be used within one week of
standardization. If this experiment is done before Experiment 7, this procedure can be used to standardize the
sodium hydroxide solution for that experiment.
Page 16
E17
ACIDBASE TITRATIONS
with two hydroxide ions) in a two-step reaction. The rst step of the reaction is
fast; the second step is very slow. To overcome the slow hydrolysis, a known excess
amount of 0.1M NaOH is added and allowed to react with the aspirin. The sample
is heated to speed the hydrolysis. Then, the excess NaOH is back-titrated with 0.1M
HCl. Standardized 0.1M NaOH solution is supplied. A diluted, standardized solution
of 0.1M HCl must be prepared.
Equations
COO
COOH
O
+ OH
O
(fast)
+ H2O
CH3
CH3
COO
COO
O
+ OH
+ CH3COO
(slow)
OH
CH3
Solutions and Chemical Required

1. Provided. Aspirin tablets, Ethanol, Hydrochloric acid, 37% wt%, Phenolphthalein indicator solution (1% in ethanol), Standardized 0.1M NaOH.
2. To Prepare. Standardized 0.1M HCl solution. The molarity of the standard HCl
and standard NaOH needs to be known only to three signicant gures.
Calculate the volume needed to prepare 0.1 M HCl from concentrated HCl. Prepare
and standardize the 0.1 M HCl (250mL) by titrating with the standard NaOH solution
using phenolphthalein indicator, as described in Experiment 7.
Procedure
1. Preparation of the sample. Use a mortar and pestle to crush an appropriate
amount of aspirin tables to produce a 1.5g sample. Transfer 0.5g samples into
three dry preweighed (to 1 mg) Erlenmeyer asks and weigh the asks to obtain
the sample weights. Add 20 mL ethanol and three drops of phenolphthalein to
each ask. Aspirin is only minutely soluble in water. So, the ethanol helps to
dissolve the aspiring tablet.
2. Titration of the sample. Titrate the rst aspirin sample with NaOH to a permanent
pink color. (The solution will be cloudy because of insoluble components in the
aspirin tablet.). This pink endpoint marks the completion of the rst fast step
of the hydrolysis, which quickly consumes one mole of NaOH. Add the known
excess amount of NaOH (10 mL + volume of NaOH used to rst endpoint).
Add boiling chips and heat the sample for about 10 min.15 min. to quicken
the slow hydrolysis step (do not boil the sample). Cool the sample approximately
5min.10 min. The solution should be dark pink. (If it is colorless, add more
phenolphthalein. If it remains colorless, add 10 mL NaOH and reheat.) The NaOH
that remains in the ask will be excess NaOH that has not reacted with aspirin.
Titrate the excess NaOH with 0.1 M HCl until the pink color disappears.
Page 17
E18
EXPERIMENTS
Calculation
Calculate the aspirin amount in mg. The total moles of NaOH minus moles of HCl
gives the moles of NaOH that reacted with the aspirin. Since there is a 2:1 ratio of
NaOH to aspirin used in the hydrolysis, half the moles of reacted NaOH will equal the
moles of Aspirin in the sample. Change moles of Aspirin to grams then to milligrams.
EXPERIMENT 10 DETERMINATION OF HYDROGEN CARBONATE
IN BLOOD USING BACK-TITRATION
Principle
About 95% of the total carbon dioxide in human blood exists as HCO3 , the remainder
existing as dissolved CO2 . The HCO3 concentration, for most clinical work, can
be used as a diagnostic aid. It is determined by adding an excess of 0.01 M HCl, to
volatilize the HCO3 as CO2 , swirling to allow the CO2 to escape, and then backtitrating the excess HCl with 0.01 M NaOH. The 0.01 M HCl and NaOH solutions are
prepared by diluting standardized 0.1 M solutions.
Equations
HCO3 + H+ H2 O + CO2
excess H+ + OH H2 O

1. Provided. 0.1% Phenol red (phenolsulfonphthalein) solution in 0.003 M NaOH,
1% saline (NaCl) solution in CO2 -free water (see Experiment 7), Antifoam A
(Dow Corning Corp.)
2. To prepare. Standard 0.1 M and 0.01 M HCl and 0.1 M and 0.01 M NaOH
solutions. The molarity of the standard HCl and standard NaOH needs to be
known only to three signicant gures. If you have prepared standard HCl or
NaOH in Experiment 7 or 8, use these for the present experiment. If you have
not, prepare 250 mL standard 0.1 M NaOH as in Experiment 8. Since only three
signicant gures are required, you may use one-tenth the amount of KHP for
titration, in which case the end point will occur at about 4.0 to 4.5 mL. A 10-mL
buret should be used in these titrations. Standardize a 0.1 M HCl solution by
titrating 5.00 mL of it with the standard 0.1 M NaOH as in Experiment 8. The
phenol red indicator may be used. If you have only a standard 0.1 M HCl solution,
use this to standardize 0.1 M NaOH solution.
Prepare 500 mL of 0.01 M HCl and 0.01 M NaOH solutions by diluting
50 mL of the 0.1 M solutions to 500 mL with the saline solution. These should be
prepared fresh on the day of use. The saline aids in the volatilization of the CO2
from the acidied solution by decreasing its solubility.
Prepare and standardize the 0.1 M HCl and 0.1 M NaOH. This will require drying primary standard KHP ahead of time if either standard HCl or NaOH is not
available.
Procedure1
1. Preparation of the sample. Either serum or plasma (oxalated or heparinized)
may be used for the determination. This may be freshly drawn blood from an
1
This determination may be performed on a macroscale using 5.00 mL acid and 1.00 mL sample, or using 2.00 mL
acid and 0.500 mL sample. In the former case, the back-titration will require about 2.4 mL of 0.01 M NaOH, and
in the latter case, about 0.7 mL.
Page 18
COMPLEXOMETRIC TITRATION
animal. (Do not do this yourself; your instructor will supply the sample.) See
Chapter 1 for a discussion of the differences between serum, plasma, and whole
blood. A 10- to 15-mL sample (20 to 30 mL whole blood) should be adequate
for triplicate determinations by a class of 30 students. Fluoride should be added
to prevent glycolysis, or breakdown of glucose, which can change the pH. The
uoride inhibits the enzyme catalysis causing glycolysis and stabilizes the pH for
about 2 h. The tube used for collecting the sample can be rinsed with a solution of
100 mg sodium heparin plus 4 g sodium uoride per 100 mL. The sample should
be kept anaerobically, that is, stoppered to keep out atmospheric CO2 . Since the
analysis should be done on the day the blood is drawn, the solutions should be
prepared ahead of time.
2. Preparation of comparison solution. Prepare a standard for color comparison at
the end point as follows. Place 6 mL of 1% saline solution in a 25-mL Erlenmeyer
ask and add 0.10 mL serum or plasma. Add two drops phenol red indicator,
insert a stopper, and rotate gently to mix the contents. The transition range of this
indicator is pH 8.4 to 6.7 (yellow to red). Because of the buffering capacity of the
blood, the end point occurs in this range.
3. Titration of the sample. The pooled serum or plasma sample will have been
prepared by touching the end of a stirring rod to some Antifoam A and rotating
it in the pooled sample. This will prevent excess foaming when the sample is
swirled. Place 0.100 mL of serum or plasma in a 25-mL Erlenmeyer ask and add
1.00 mL of 0.01 M HCl and 4 mL of 1% saline. Swirl the ask vigorously for at
least 1 min to allow the CO2 to escape. Add two drops of indicator and then titrate
with 0.01 M NaOH dropwise, but rapidly, until a pink color matching the standard
persists for at least 15 s. The NaOH may be added carefully with a graduated
1-mL measuring pipet and read to the nearest 0.01 mL.
The normal value of blood bicarbonate is about 26 meq/L (25 to 32 meq/L),
or 0.026 meq/mL. Meq HCO3 = mmol HCO3 . Since 0.1 mL blood was taken
for analysis, it should consume about 0.0026 mmol HCl, or 0.26 mL of 0.01
M HCl. Hence, since 1 mL of 0.01 M HCl was taken, about 0.74 mL should
remain unreacted, and the back-titration should take about 0.7 mL of 0.01 M
NaOH.
Calculation
Calculate the bicarbonate content of the sample in meq/L. (See Chapter 5 for denition
of milliequivalents of serum electrolytes.)
Complexometric Titration
EXPERIMENT 11
WITH EDTA
DETERMINATION OF WATER HARDNESS
Principle
Water hardness, due to Ca2+ and Mg2+ , is expressed as mg/L CaCO3 (ppm). The
total of Ca2+ and Mg2+ is titrated with standard EDTA using an Eriochrome Black
T indicator.1 A standard EDTA solution is prepared from dried (do not exceed 80 C)
Na2 H2 Y 2H2 O (purity 100.0 0.5%). If the sample does not contain magnesium,
MgEDTA is added to the titration ask to provide a sharp end point with the
1 If it is desired to titrate only Ca2+ , this can be done at pH 12 (use NaOH), where Mg(OH) is precipitated and
2
does not titrate. Hydroxynaphthol blue indicator is used.
E19
Page 19
E20
EXPERIMENTS
Eriochrome Black T, since calcium does not form a sufciently strong chelate with
the indicator to give a sharp end point. See the discussion in Chapter 9 for a more
complete description.
Equations
Titration:
Ca2+ + H2 Y2 CaY2 + 2H+

Ca2+ + MgY2 CaY2 + Mg2+
End point:
Mg2+ + HIn2 MgIn + H+

MgIn + H2 Y2
(red)
MgY2
(colorless)
(colorless)
+ HIn2 + H+
(blue)
The free acid parent of the indicator is H3 In, and that of the titrant EDTA is H4 Y.
1. Provided. 0.5% (wt/vol) Eriochrome Black T indicator solution in ethanol, 0.005
M MgEDTA (prepared by adding stoichiometric amounts of 0.01 M EDTA and
0.01 M MgCl2 ). The indicator solution should be prepared fresh every few days,
as it is unstable. A portion of the MgEDTA solution, when treated with pH 10
buffer and Eriochrome Black T, should turn a dull violet color; one drop 0.01 M
EDTA should change this to blue and one drop 0.01 M MgCl2 should change
it to red.
2. To prepare
(a) NH3 NH4 Cl buffer solution, pH 10. Dissolve 3.2 g NH4 Cl in water, add
29 mL conc. NH3 , and dilute to about 50 mL. The buffer solution is best stored
for long periods of time in a polyethylene bottle to prevent leaching of metal
ions from glass.
(b) Standard 0.01 M EDTA solution. Dry about 1.5 g reagent-grade
Na2 H2 Y 2H2 O in a weighing bottle at 80 C for 2 h. Cool in a desiccator for
30 min and accurately weigh (to the nearest milligram) approximately 1.0 g.
Transfer to a 250-mL volumetric ask. (This is the disodium salt of EDTA;
the free acid is insoluble). Add about 2.00 mL distilled, deionized water and
shake or swirl periodically until the EDTA has dissolved. EDTA dissolves
slowly and may take 0.5 h or longer. If possible, the solution should be
allowed to stand overnight before using. If any undissolved particles remain,
addition of three pellets of NaOH may aid dissolution, but there is danger of
adding metallic impurities. After the EDTA is dissolved, dilute to 250.0 mL
and shake thoroughly to prepare a homogeneous solution. Then, rinse a clean
polyethylene bottle with three small portions of the EDTA solution and
transfer the remainder of the solution to the bottle for storage. (Polyethylene
is preferable to glass for storage because EDTA solutions gradually leach
metal ions from glass containers, resulting in a change in the concentration of
free EDTA.) Calculate the molarity of the EDTA solution.
Dry the Na2 H2 Y 2H2 O and prepare the standard EDTA solution.
Procedure
Obtain a water sample from your instructor. Add with a pipet or a buret a 50-mL
aliquot of the sample to a 250-mL wide-mouth Erlenmeyer ask, add 2 mL of the buffer
Page 20
E21
PRECIPITATION TITRATIONS
solution, 0.5 mL of the MgEDTA solution, and ve drops of the indicator solution. (If
the unknown contains magnesium, addition of MgEDTA is not necessaryconsult
your instructor.) Avoid adding too much indicator with dilute solutions or the end-point
change may be too gradual. The indicator will not become wine red until magnesium
is added. The buffer should be added before the indicator, so that small amounts of
iron present in the water will not react with the indicator. [An end-point color change
from wine red to violet indicates a high level of iron in the water. After the titration,
add 1 g FeSO4 7H2 O to convert CN to harmless Fe(CN)6 4 .] If the water sample
is likely to contain copper, add a few crystals of hydroxylamine hydrochloride. This
reduces copper(II) to copper(I), which does not interfere.
Titrate with 0.01 M EDTA until the color changes from wine red through purple
to a pure blue. The reaction (color change) is slow at the end point, and titrant must
be added slowly and the solution stirred thoroughly in the vicinity of the end point.
A comparison solution for the proper color at the end point may be prepared by
adding 2 mL of pH 10 buffer to 50 mL distilled water, ve drops indicator, a few drops
MgEDTA, and a few drops EDTA.
If the end point for the rst titration is less than 10 mL, double the volume of
sample for the remaining two titrations.
Calculation
Calculate and report the hardness of the water as ppm CaCO3 for each portion analyzed.
Precipitation Titrations
EXPERIMENT 12 DETERMINATION OF SILVER IN AN ALLOY:
VOLHARDS METHOD
Principle
Primary standard AgNO3 is used to standardize a 0.1 M KSCN solution, using a ferric
alum indicator. The unknown silver alloy is analyzed by titrating with the standardized
KSCN solution.
Equations
SCN + Ag+ AgSCN
Fe3+ + SCN Fe(SCN)2+
(indicator reaction)
red

1. Provided. Primary standard AgNO3 , ferric alum indicator [KFe(SO4 )2 12 H2 O,
saturated solution], 6 M HNO3 (free of oxides of nitrogen). Nitric acid free from
lower oxides of nitrogen should be colorless and can be prepared, if necessary, by
boiling 1:1 HNO3 until NO2 is expelled.
2. To prepare. 0.1 M KSCN. Weigh approximately 5.0 g KSCN, dissolve in water
in a 500-mL or 1-L bottle and add 500 mL distilled water. Shake well to ensure
a homogeneous solution. This solution is approximately 0.1 M and will be
standardized against primary standard AgNO3 .
Prepare and standardize (below) the 0.1 M KCSN solution. This will require drying
and cooling AgNO3 .
Page 21
E22
EXPERIMENTS
Procedure
1. Standardization of KSCN solution. Obtain about 3 g primary standard AgNO3
in a weighing bottle from your instructor. Dry in the oven at 110 C for 1 to 2 h,
but no longer. Cool in a desiccator for 30 to 40 min. This will be weighed by the
direct method.
Place a clean, dry weighing dish on the balance and determine its weight to
the nearest 0.1 mg. Add 0.70 to 0.75 g AgNO3 to the weighing dish and determine
the increase in weight to the nearest 0.1 mg. Transfer quantitatively to a clean
250-mL wide-mouth Erlenmeyer ask. Keep away from strong light as much as
possible until ready to titrate. Weigh two other 0.70- to 0.75-g portions of AgNO3
and transfer to separate (numbered) 250-mL Erlenmeyer asks.
Add 50 mL distilled water to the ask ready for titration, 50 mL of 6 M nitric
acid free from lower oxides of nitrogen, and 2 mL ferric alum indicator. (Lower
oxides of nitrogen form nitroso complexes with Fe3+ that are red in color and
will interfere with the end point.) Fill your 50-mL buret with the KSCN solution,
record the initial volume to the nearest 0.01 mL, and titrate with constant vigorous
agitation until a faint reddish-brown color appears in the solution; this is more
easily seen if the precipitate is allowed to settle after each addition near the end
point. It will be helpful to compare the color with a solution made by adding
5 mL of 6 M nitric acid and 2 mL ferric alum to 75 mL water. The color must be
permanent after strong shaking. Titrate the other two AgNO3 samples in the same
manner and calculate the molarity of the KSCN solution from each titration. Use
the mean of the three determinations.
2. Determination of silver in an alloy.1 Obtain a sample of a silver alloy from
the instructor.2 Place in a 250-mL beaker, add 20 mL dilute (1:1) HNO3 , cover
with a watch glass, and warm on the steam bath in the hood until the alloy has
completely dissolved. When solution is complete, remove the watch glass, rinse
it off with a jet of water from your wash bottle, catching the rinsings in the
beaker, and continue heating with the beaker uncovered until all brown fumes
have disappeared and the solution is colorless. Cool to room temperature and
transfer the solution quantitatively to a clean 250-mL volumetric ask with the
aid of a funnel and stirring rod to pour the solution down. Rinse the beaker several
times with distilled water; adding the rinsings to the ask. Dilute to the mark and
shake thoroughly to ensure a homogeneous solution.
Transfer with a pipet two samples, 50 mL each, into 250-mL Erlenmeyer
asks, add 2 mL ferric alum indicator, and titrate with standard 0.1 M KSCN
solution to the appearance of a faint reddish-brown color, which is permanent
even after strong agitation. The two titrations should agree within 0.05 mL. If
they do not, pipet and titrate two more. When nished, put all silver-containing
solutions in a jar for this purpose.
Calculations
Calculate and report the number of grams silver in your alloy sample.
1 The unknown may instead be impure silver nitrate, in which case dissolve it in 50 mL water and proceed as in
the standardization of the KSCN above.

2 You may be instructed to weigh out 0.3 to 0.4 g of the alloy and then report the percentage of silver in
the sample.
Page 22
E23
PRECIPITATION TITRATIONS
EXPERIMENT 13 DETERMINATION OF CHLORIDE IN A SOLUBLE

CHLORIDE: FAJANS METHOD
Principle
The sample is titrated with a standard AgNO3 solution, using a dichlorouorescein
adsorption indicator end point.
Equations
Cl + Ag+ AgCl
1. Provided. 0.1% dichlorouorescein indicator solution, dextrin.
2. To prepare. Standard 0.1 M AgNO3 . Obtain from the instructor about 4.5 g
primary-standard-grade silver nitrate in a clean, dry weighing bottle; dry in the
oven at 110 C for 1 to 2 h, but no longer. This material will be used to prepare a
standard solution by the direct method. Cool for 30 to 40 min in a desiccator.
Using a weighing dish, weigh accurately (nearest 0.1 mg) about 4.3 g
AgNO3 . Transfer to a 250-mL beaker and dissolve in about 100 mL distilled
water. Carefully pour this solution into a 250-mL volumetric ask and rinse the
beaker several times with distilled water, adding the rinsings to the ask. Dilute
to the mark, shake well, and pour into a clean, dry 500-mL glass-stoppered amber
bottle. Shake again to ensure a homogeneous solution. Keep away from strong
light as much as possible. Store away from light. Calculate the molarity of the
solution.
1. Prepare the 0.1 M AgNO3 solution.
2. Obtain a sample of unknown chloride from your instructor and dry it in the oven
at 120 C for 1 h or longer. It is okay to dry it overnight. Cool in a desiccator at
least 30 to 40 min before weighing.
Procedure
Weigh out in the weighing dish three samples of about 0.25 to 0.30 g each (vary the
sample weights, but weigh each exactly), transfer to 250-mL Erlenmeyer asks, and
dissolve in about 50 mL distilled water. Add 10 mL 1% dextrin suspension (shake
well before using) and 10 drops dichlorouorescein indicator solution. The dextrin
prevents excessive coagulation of the precipitate at the end point. This keeps a larger
surface area for adsorption of the indicator, which enhances the sharpness of the end
point. Instead of a suspension of dextrin, 0.1 g of the solid may be added.
The pH of the solution should be between 4 and 10. If it is too acid (e.g., due to
hydrolysis of other chloride salts in the unknown), it may be neutralized by adding solid
CaCO3 until excess remains in suspension. The suspension does not interfere with the
end point. Results are low at pH values above 4 because the indicator tends to displace
chloride from the precipitate. (More accurate results are obtained by standardizing the
AgNO3 solution against dried reagent-grade NaCl, using the same conditions as for
the unknown titration.)
Titrate with 0.1 M AgNO3 . Thorough mixing by vigorous swirling of the ask
throughout the titration is essential to achieve a good end point. Do not let direct
sunlight strike the ask; if you are working next to a window on a very bright day, it
would be well to draw the shade. The end point is marked by a change from a pale
Page 23
E24
EXPERIMENTS
yellow to a distinct pink color. If the titration is overrun, add a few grains of NaCl or
KCl and practice the determination of the end point a few times before titrating the
second and third samples. When nished, put all silver-containing solutions in a jar
provided for this purpose.
Calculations
Calculate and report the percent Cl in your sample for each portion titrated.
Potentiometric Measurements
EXPERIMENT 14
DETERMINATION OF THE pH OF HAIR SHAMPOOS
Principle
A calibrated glass pH electrodeSCE pair is used to measure the pH of commercially
available shampoos, hair conditioners and rinses, and depilatories, both concentrated
and diluted 1:10. An estimate is made whether the pH is controlled primarily by
a buffered solution or a strong electrolyte. An evaluation is made of the relative
importance of what is probably the best pH for a shampoo and whydoes the pH
affect the cleaning power of the shampoo or does it affect the hair itself?
Equations
Electrode Response
E=k
2.303RT
pH
F

Provided. Standard pH 3, 5, 7, 9, and 11 buffers (approximateother values near
these may be used). Each member of the class should bring one or more samples of
commercially available hair shampoos, conditioners, rinses, and depilatories. These
may include dandruff shampoos and hair removers such as Veet or Nair. All the
samples will be shared by the class. There should be enough of each to provide about
15 to 20 mL to each student.
Procedure
1. Calibration of the electrodes. The experiment will be performed by rst calibrating the electrodes at pH 7 and making approximate pH measurements of
the various samples. The samples are then separated into ve groups: those with
pH < 4, pH 4 to 5.9, pH 6 to 8, pH 8.1 to 10, and pH > 10. The glass electrode
should have been soaked in 0.1 M KCl solution at least one day prior to its use;
store the electrode in dilute KCl solution when not in use. Calibrate the pH meter
for one group of samples at a time (pH 3 for the rst group, pH 11 for the last
group) using the procedure described by your instructor. (Your instructor may
decide to use only three buffers for calibration, e.g., pH 4, 7, and 9.) This will
consist essentially of adjusting the meter to read the pH of the standard buffer at
room temperature with the electrodes immersed in the buffer solution. Rinse the
electrodes with distilled water after calibration and blot with tissue paper; do not
wipe them as this may impart a static charge to the glass membrane. Be careful to
turn the pH meter to standby when removing electrodes from solution. If only a
small quantity of buffer is used, it would be better to discard it rather than chance
contamination of the entire supply.
Page 24
POTENTIOMETRIC MEASUREMENTS
2. pH Measurements of samples. Prepare a 1:10 dilution of each sample in a

100-mL beaker by pipetting 5.00 mL into 45.0 mL distilled water measured from a
50-mL buret. Measure the pH of rst the diluted sample and then the concentrated
sample. For the latter, if a small beaker is used (e.g., 25 mL) it may be possible
to make the measurement with 5 mL of sample; just enough to cover the bulb of
the glass electrode. Alternatively, a combination pHreference electrode may be
used, in which case the sample can be placed in a test tube and the single probe
dipped in this. (A combination electrode contains both a reference electrode and
a glass electrode in one probe. A salt bridge wick for the reference electrode is
above the pH glass bulb, and so the probe will have to be immersed a bit deeper to
make contact with both electrodes. There will be two wire leads from the probe,
one for each electrode.)
Immerse the electrode in the test solution and swish or agitate a few seconds.
Then, allow the pH reading to equilibrate and record to the nearest 0.01 pH. Rinse
the electrode well between measurements and blot off the water. It is best to make
the measurements from low pH to high, or vice versa. If the pH reading of any of
the samples falls outside the range of 4 to 10, the electrodes should be recalibrated
with a buffer closer to the sample pH (if depilatories are measured, their pH will
exceed 10 and a buffer at pH 11 should be used.)
Report
Arrange the samples into either shampoos or conditioners and rinses (depilatories
are separate). Arrange the samples in each group in order of increasing pH readings
of the concentrated samples. Your instructor may compare your pH readings with
the mean of the class. From the changes in readings on dilution, list whether you
think the pH of each solution was determined by a buffer system or by a strong
acid or base. Consult the reference J. J. Grifn, R. F. Corcoran, and K. K. Akana,
J. Chem. Ed., 54 (1977) 553 and include in your report a discussion of the relevance
of pH in hair cleansing or conditioning and in hair damage. Propose mechanisms for
the pH action. How do depilatories work? Within what pH range should childrens
shampoos be?
EXPERIMENT 15 POTENTIOMETRIC DETERMINATION
OF FLUORIDE IN DRINKING WATER USING A FLUORIDE
ION-SELECTIVE ELECTRODE1
Principle
Fluoride in a water sample is determined by measurement with a uoride ion-selective
combination electrode (contains reference electrode built in). First, you will determine
whether the electrode response is Nernstian over a wide range of concentrations. Then,
you will determine uoride in the unknown by comparing potential measurements
with standards over a narrower range, bracketing the unknown; a calibration curve
will be prepared.
Equations
2.303RT
log aF
F
2.303RT
log[F ] (if ionic strength is held constant)
= k
F
E=k
1
This experiment can also be used to determine % NaF or % SnF2 in toothpaste by preparing a suspension of the
paste in water and pipetting the supernatant, or to determine the uoride content of childrens uoride tablets or
drops. See T. Light and C. Cappucino, J. Chem. Ed., 52 (1975) 247.
E25
Page 25
E26
EXPERIMENTS

1. Provided. TISAB (total ionic-strength adjustment buffer) solution, which is
prepared with 57 mL glacial acetic acid, 58 g sodium chloride, and 4 g CDTA
(cyclohexylenedinitrilotetraacetic acid) in about 500 mL water, adjusted to pH 5.0
to 5.5 with 5 M NaOH and diluted to a total volume of 1 L. A 1:1 dilution of all
samples with this solution serves the following:
(a) It provides a high total ionic strength background, swamping out variations in
ionic strength between samples.
(b) It buffers the samples at pH 5 to 6. (In acid media, HF forms; while in alkaline
media, OH ion interferes in the electrode response.)
(c) The CDTA preferentially complexes with polyvalent cations present in water
(e.g., Si4+ , Al3+ , Fe3+ ), which otherwise would complex with F and change
its concentration.
2. To prepare
(a) Stock standard 0.1 M uoride solution. Dry about 1 g NaF at 110 C for
1 h, cool in a desiccator for 30 min. Weigh out 0.45 to 0.50 g of the dried NaF
(to the nearest milligram), transfer to a 100-mL volumetric ask, dissolve, and
dilute to volume with distilled deionized water. Shake thoroughly and transfer
to a polyethylene bottle (rinse with a few small portions rst). Fluoride tends to
adsorb on glass and should be stored in plastic containers. Caution: Fluoride
is poisonous. Handle with care. Commercially prepared uoride solutions
may be used.
(b) Linearity standards. By serial dilution of the stock solution with distilled
deionized water, prepare a series of solutions of about 102 , 103 , 104 , 105 ,
and 106 M uoride (calculate accurate concentrations). (Do not pipet by
mouth!) For example, dilute the stock solution 10:100, 1:100, and 1:1000 mL
to prepare the rst three solutions. Then, dilute the 104 M solution 10:100
and 1:100 mL to prepare the last two. Transfer to polyethylene bottles. These
solutions should be prepared on the day of use.
(c) Calibration standards. The unknown concentration will be within
1 103 and 1 102 M uoride, and a calibration curve will be prepared
using concentrations of uoride to bracket the unknown. Using the above
procedure, prepare additional standards of 2 103 and 4 103 M uoride
(calculate the accurate concentrations). Prepare on the day of use.
Prepare the stock NaF solution. This will require drying of NaF.
Procedure
1. Determination of range of response and range of linearity. Connect the electrode
leads to an expanded-scale pH meter. Add with a pipet 10 mL of the 106 M
standard solution and 10 mL TISAB solution to the small plastic beaker provided.
Place the electrode in the beaker. Stir the solution with a magnetic stirrer and
small stirring bar during measurement. You may make readings in pH units.
(1 pH = 59.2 mV at 25 C; arbitrarily take pH 0 as 0 mV). The meter should also
allow reading in millivolts. When a steady reading is obtained, record the value.
Rinse and blot the electrode and repeat, going from dilute to concentrated standard
solutions. Prepare a spreadsheet of the data, and chart mV vs. log C. Report the
slope in mV/decade, the intercept, and the r2 value. Report also the range of
linearity.
Page 26
E27
REDUCTIONOXIDATION TITRATIONS
2. Standardization for unknown. Record the readings of the standard solution from
1 103 M to 1 102 M, and plot a calibration curve as above over this range.
Enter the formula in one cell for calculating an unknown concentration from the
millivolt reading and the slope and intercept of the calibration curve.
3. Analysis of unknown. After preparing the calibration curve, obtain an unknown
uoride sample. This may be a synthetic solution, in which case obtain the
unknown in a 250-mL volumetric ask. Immediately dilute to volume with
distilled deionized water and transfer to a polyethylene bottle. Add 10 mL of the
unknown with a pipet to a small plastic beaker followed by 10 mL TISAB. Record
the mV reading as above. Make at least three separate runs (separate additions
and potential readings). Note: The unknown should be measured at the same time
the calibration curve is prepared. The mV scale should not be adjusted between
calibration and sample measurements. If it is, take a new reading of one of the
standards, and readjust to the original reading.
Calculations
From the spreadsheet calibration curve, determine the concentration of uoride in
the unknown solution. Report the results in parts per million uoride, along with the
standard deviation for the three measured samples.
ReductionOxidation Titrations
EXPERIMENT 16 ANALYSIS OF AN IRON ALLOY OR ORE BY
TITRATION WITH POTASSIUM DICHROMATE
Principle
An iron alloy or ore is dissolved in HCl and the iron is then reduced from Fe(III)
to Fe(II) with stannous chloride (SnCl2 ). The excess SnCl2 is oxidized by addition
of HgCl2 . The calomel formed (insoluble Hg2 Cl2 ) does not react at an appreciable
rate with the titrant. The Fe(II) is then titrated with a standard K2 Cr2 O7 solution to a
diphenylamine sulfonate end point.
Equations
Reduction:
2Fe3+ + Sn2+ (slight excess) 2Fe2+ + Sn4+ + Sn2+
Sn2+ + 2Hg2+ + 2Cl Sn4+ + Hg2 Cl2
(due to excess)
(white precipitate)
If too much Sn2+ is added, then

Sn2+ + Hg2 Cl2 Sn4+ + 2Hg0 + 2Cl
(Black Hg0 precipitate makes end-point determination impossible. The sample must
be discarded because Hg0 reacts with Cr2 O7 2 .)
Titration:
6Fe2+ + Cr2 O7 2 + 14H+ 6Fe3+ + 2Cr3+ + 7H2 O
1. Provided. 0.28% (wt/vol) of the sodium salt of p-diphenylamine sulfonate
indicator, 0.5 M SnCl2 in 3.5 M HCl (with mossy tin added to stabilize against
air oxidation: Sn4+ + Sn0 2Sn2+ ), saturated HgCl2 solution, conc. HCl, 6 M
HCl, conc. H3 PO4 -conc. H2 SO4 mixture (15 mL each added to 600 mL water and
cooled to room temperature), 0.1 M FeCl3 in 6 M HCl.
Page 27
E28
EXPERIMENTS
2. To prepare.1 Standard 1/60 M K2 Cr2 O7 solution (approx. 0.017 M). Dry about
3 g primary standard K2 Cr2 O7 in a weighing bottle at 120 C for at least 2 h.
Drying in the oven for longer periods of time (i.e., until the next lab period) will
do no harm. Place the K2 Cr2 O7 in the desiccator to cool for 30 to 40 min. Weigh
accurately to the nearest milligram, about 1.3 g in a weighing dish, and transfer
quantitatively to a 200-mL beaker. Dissolve in about 200 mL water and transfer
quantitatively to a 250-mL volumetric ask. Dilute to volume and mix thoroughly.
Rinse a clean 1-L bottle with three small portions of the solution and transfer the
remainder of the solution to the bottle for storage. Calculate the molarity of the
solution. K2 Cr2 O7 may also be prepared approximately and standardized against
electrolytic iron wire (primary standard) using the same procedure as given below
for an alloy sample.
1. Dry the necessary amount of K2 Cr2 O7 .
2. Obtain and dry or dissolve your unknown as required.
(a) Alloy sample. This may be in the form of a wire. Your instructor will
provide you with three separate (weighed) pieces of the unknown, each to be
placed in separate, labeled 500-mL Erlenmeyer asks containing 10 mL conc.
HCl. Cover with inverted 100-mL beakers and store these dissolving samples
in your desk overnight or longer. Alternatively, the samples can be dissolved
on the day of the experiment by heating on a hot plate or steam bath in 400-mL
beakers covered with ribbed watch glasses in the hood to hasten dissolution.
(After dissolution, rinse the cover glass and the sides of the beaker, catching
all the rinsings in the beaker. Use as little water as possible. The nal volume
should not be more than 50 mL.)
(b) Ore sample. Check out a sample of an iron ore from the instructor. Dry in
the oven at 110 to 120 C for at least 2 h; longer drying will do no harm.
Procedure
1. Reduction of the iron and trial titration. Before titrating your unknown, it is
advisable to perform one or two trial titrations. This can be done while the (ore)
samples are dissolving. Add approximately 10 mL 0.1 M FeCl3 solution in 6 M
HCl to a 600-mL beaker and add about 50 mL water. (This dilutes the sample
sufciently that when all the Fe3+ is reduced, the solution will be nearly colorless.
If the volume is less, then the pale green of the Fe2+ makes detection of complete
reduction more difcult). Place a ribbed watch glass on the beaker and heat nearly
to boiling on a hot plate in the hood; the solutions must be very close to the
boiling point, perhaps simmering gently, but not boiling violently since FeCl3 can
be lost due to volatilization. Add 0.5 M stannous chloride solution with a dropper
through the lip of the beaker until the color begins to fade; then, continue the
addition drop by drop, swirling the beaker and allowing each drop to react before
adding the next, until the solution is colorless. It will rst become pale yellow
and then will gradually turn more clear. It may never get completely colorless
but may instead go to a pale green due to the ferrous ion (this will depend on
1 If you are to use this solution also for Experiment 17 and/or 28, prepare 500 mL, taking accurately about 2.5 g
K2 Cr2 O7 , dissolving in 200 mL water, and transferring to a 500-mL volumetric ask. You need to save at least
150 mL for Experiment 17 and 100 mL for Experiment 28. The 250 mL you prepare for this experiment will be
enough if you are careful not to waste any.
Page 28
the amount of iron). Whichever you get (colorless or pale green) stop addition
and allow the solution to heat for two more minutes. If the yellow color returns,
add a few more drops of SnCl2 until it becomes colorless or pale green again.
Repeat dropwise addition of SnCl2 until the solution does not return to the yellow
color. At this point, add two drops excess, no more. (If more than two drops
are added, the stannous chloride can be oxidized with a few drops of potassium
permanganate solution and the above reduction process repeated.) Remove from
the hot plate, rinse down the cover glass and sides of the beaker, and cool quickly
to room temperature by immersing the bottom of the beaker in cold water. Two
to three samples may be taken this far together; the remainder of the procedure
must be carried out with each sample individually without interruption through
the titration. If any sample turns yellow again while awaiting its turn, it must be
reheated and sufcient stannous chloride added to discharge the color, with two
drops excess. Fill your 50-mL buret with the standard K2 Cr2 O7 and have it ready
for titration.
To one sample, which must be at room temperature, add 100 mL water
and then add rapidly 15 mL saturated mercuric chloride solution, previously
measured out, while stirring and immediately mix thoroughly. A slight, white
precipitate should form. If either a heavy gray or black precipitate (elemental
mercury) or no precipitate forms, too much or not enough (to reduce all the Fe3+ )
stannous chloride has been added; in either case, the sample must be discarded.
Mix for 2 min, then add 100 mL of the H3 PO4 H2 SO4 mixture and six to eight
drops diphenylamine sulfonate indicator. Titrate immediately with the K2 Cr2 O7
solution, stirring constantly, until the green color changes to a purple or violet blue
that remains for at least 1 min. (The acid mixture provides the protons consumed
in the titration reaction and forms a nearly colorless phosphate complex with the
Fe3+ titration product, which sharpens the end point.)
2. Alloy sample. The sample should by now be dissolved in the Erlenmeyer asks
(or in the heated 600-mL beakers). Adjust the volume to 40 to 60 mL with distilled
water. Heat nearly to boiling and follow the same procedure as used in the trial
titration, starting with addition of stannous chloride. All three samples can be
taken up to the point to just before the addition of mercuric chloride and then must
be treated one at a time up through the titration.
3. Ore sample. After cooling the dried sample in a desiccator for 30 to 40 min,
weigh out by difference (the iron ore may be hygroscopic) three samples; consult
the instructor as to the size of the samples. Transfer to 600-mL beakers. Add
10 mL water and swirl until the sample is completely moistened and in suspension;
then cover with ribbed watch glasses and add 10 mL concentrated HCl, pouring it
through the lip of the beaker. Heat on a hot-plate in the hood until the iron ore has
dissolved to give a clear, red-brown solution; with some samples there may be an
insoluble sandy residue, which may be disregarded. [Silica or insoluble suldes
(black) or silicates may remain.] The hot plate should be adjusted to keep the
solutions just barely at the boiling point; vigorous boiling should be avoided since
it may cause loss of material and excessive evaporation of acid. If necessary, add
6 M HCl to keep the volume about 20 mL. When all the iron has been dissolved,
the insoluble residue (if any) will be gray or white, with no black or reddish
particles, after adding stannous chloride to reduce the iron. When the solution
appears clear, add distilled water to bring the volume to about 50 mL and follow
the same procedure used in the trial titration starting with addition of stannous
chloride to the hot solution. All three samples can be carried up to the point just
before addition of mercuric chloride and then must be treated one at a time up
through the titration.
E29
Page 29
E30
EXPERIMENTS
Calculations
1. Alloy sample. Calculate and report the milligrams iron in each portion of the
unknown analyzed, along with the mean and the precision.
2. Ore sample. Calculate and report the percent iron in each portion of the unknown
analyzed, along with the mean and the precision.
EXPERIMENT 17 ANALYSIS OF COMMERCIAL HYPOCHLORITE

OR PEROXIDE SOLUTION BY IODOMETRIC TITRATION
Principle
The oxidizing power (percent NaOCl or H2 O2 ) of the solution is determined iodometrically by reacting it with an excess of iodide in acetic acid solution and titrating the
iodine produced (I3 in the presence of excess iodide) with standard sodium thiosulfate
solution. The sodium thiosulfate is standardized against primary standard potassium
iodate, and a starch indicator is used.
Equations
Standardization of Na2 S2 O3 :
IO3 + 8I + 6H+ 3I3 + 3H2 O
I3 + 2S2 O3 2 3I + S4 O6 2
Sample titration:
or
ClO + 3I + 2H+ Cl + I3 + H2 O
Mo(VI)
H2 O2 + 3I + 2H+ 2H2 O + I3
catalyst
I3 is titrated with S2 O3 2 as above.

Solution and Chemicals Required
1. Provided. 6 M HCl, KI, primary standard KIO3 , glacial acetic acid, 3% ammonium molybdate solution (for peroxide samples), Na2 CO3 , dil. (1:4) H2 SO4 .
2. To prepare
(a) Starch solution. Prepare a 1% solution by mixing 0.5 g soluble starch with
2 to 3 mL of distilled water; add a pinch of HgI2 . Pour this mixture into 50 mL
boiling distilled water with stirring and continue heating for 2 to 3 min until
the solution is clear or only faintly opalescent. Cool to room temperature. The
HgI2 stabilizes the starch indenitely; otherwise, it should be prepared fresh
on the day of use. Note: Approximately 0.4 g of the commercial indicator
Thiodene may be used in place of the prepared starch solution.
(b) Standard 0.01 M KIO3 solution. This will be used to standardize the
Na2 S2 O3 solution. (If you prepared a standard 1/60 M K2 Cr2 O7 solution
in Experiment 16, you may titrate 50-mL aliquots of this iodometrically
for the standardization of the Na2 S2 O3 . If so, consult your instructor for
directions.) A standard solution of KIO3 is prepared and aliquots of this are
titrated with the Na2 S2 O3 solution. This procedure is used instead of titrating
individually weighed portions of KIO3 . The reason is that KIO3 has a low
equivalent weight and only about 0.1-g portions can be titrated. Hence, it is
Page 30
more accurate to prepare a standard solution. This requires special care in the
accurate preparation of the solution since only one solution is prepared.
Dry about 1.5 g primary standard KIO3 at 120 C for 1 to 2 h and cool in a
desiccator for 30 to 40 min. Accurately weigh (to the nearest 0.1 mg) 1.0 to
1.4 g and dissolve in a small amount of distilled water in a 200-mL beaker.
Quantitatively transfer, with rinsing, to a 500-mL volumetric ask, using a
glass funnel and stirring rod to direct the solution into the ask. Dilute to the
calibration mark. Calculate the molarity of the solution.
(c) 0.1 M Na2 S2 O3 solution. Boil about 1200 mL distilled water for 5 to 10 min
to ensure sterility and to expel carbon dioxide. Cool to room temperature.
(Sodium thiosulfate solutions are subject to bacterial attack, which may change
the molarity after a time. Thus, all water and glassware used to prepare and
store the solution should be sterilized. If any turbidity or bacteria or mold
growth appears, the solution should be discarded. Removal of carbon dioxide is
also benecial, because thiosulfate is more stable in neutral solution.) Sterilize
a 0.5-L bottle with hot distilled water, near boiling, and then rinse with the
cool boiled distilled water. Weigh out on a watch glass, using a rough balance,
12.5 g sodium thiosulfate crystals, Na2 S2 O3 5H2 O. Transfer to the sterilized
bottle, add 500 mL of the freshly boiled and cooled distilled water, add 0.05 g
sodium carbonate, and shake thoroughly until the solution is homogeneous.
(A small amount of sodium carbonate is added to keep the solution neutral or
slightly alkaline and thereby stabilize it against decomposition to elemental
sulfur.) Store in a refrigerator if possible, but let warm up to room temperature
before using.
1. Dry the required amount of primary standard KIO3 .
2. Prepare the 0.1 M Na2 S2 O3 solution. Although this can be prepared on the day
of the experiment, it is preferable to prepare it at least a day before it is to be
standardized. The solution tends to lose some of its titer right after preparing.
Procedure
1. Standardization of the Na2 S2 O3 solution. The solution should be standardized
on the day of the experiment. Consult your instructor if you are to use the standard
K2 Cr2 O7 solution from Experiment 16 for standardization. Otherwise, proceed as
follows.
Rinse the 50-mL buret several times with small portions of the thiosulfate
solution and ll it with thiosulfate solution. Adjust to near the zero mark and record
the volume reading to the nearest 0.02 mL. Add with a pipet a 50.00-mL aliquot
of the potassium iodate solution to a clean 250-mL wide-mouth Erlenmeyer ask.
Add about 2 g solid potassium iodide and swirl to dissolve. Add, with rapid
mixing, 5 mL dilute H2 SO4 . Mix thoroughly.
Titrate immediately with thiosulfate solution. (In strongly acid solution, the
excess iodide is rapidly air-oxidized to I3 , and so the titration must be performed
quickly.) Thorough, continuous mixing throughout the titration is essential; the
thiosulfate must not be allowed to accumulate in local excess in the acid solution
or else some decomposition into H2 SO3 and S may occur. Titrate until the yellow
color (due to I3 ) almost disappears. It will become a pale yellow. Then, add 2
to 3 mL starch solution and titrate until the blue color just disappears (properly
done, this should occur within 0.5 mL after adding the starch solution).
E31
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E32
EXPERIMENTS
The standardization should be repeated until you are sure of the titration
volume to within one part per thousand (e.g., 0.03 mL at a titration volume of
30 mL). Calculate the molarity of the Na2 S2 O3 solution.
2. Determination of hypochlorite or H2 O2 in unknown1
(a) Weighing and diluting the unknown. Roughly calibrate a weighing bottle
by pouring into it about 12 mL water and noting the level to which it lls
the bottle. Empty and thoroughly dry the weighing bottle and weigh it to the
nearest milligram. The hypochlorite or peroxide solutions to be analyzed will
be supplied in the commercial bottles, tted with siphon delivery tubes. Clean
off any solid crust on the tip and discard a few drops to ush out the tip.
Withdraw about 12 mL into the calibrated and weighed weighing bottle; it is
essential that the upper portion of the bottle, particularly the ground-glass part,
remain dry. Replace the stopper and weigh to the nearest milligram. Empty
the weighing bottle into a 250-mL volumetric ask containing about 100 mL
water, using a funnel. Wash out the weighing bottle and the funnel with a
jet of water from your wash bottle, catching the rinsings in the volumetric
ask. Dilute to the mark and mix thoroughly. Transfer with a pipet three
50-mL aliquots of the solution into 250-mL Erlenmeyer asks containing
about 50 mL water; rinse down the walls of the asks in such a way as to form
a layer of water above the sample. From this point on, handle each sample
individually through the remainder of the procedure.
(b) Titration. Fill your buret with standard 0.1 M sodium thiosulfate solution.
Measure out and have ready 10 mL glacial acetic acid, 2 g potassium iodide,
and, if the sample is a peroxide, a dropping bottle containing 3% ammonium
molybdate catalyst solution. Hypochlorite requires no catalyst. When ready
to titrate, add glacial acetic acid, potassium iodide, and if the sample is
a peroxide, add three drops of the catalyst solution. Titrate immediately,
swirling the ask constantly. When the color has faded to a pale yellow,
add about 2 mL starch solution and continue the titration drop by drop
until the solution just becomes colorless. Complete the other samples in the
same way.
Calculation
Calculate the percentage by weight of NaClO or H2 O2 in the solution and the relative
standard deviation. (Note: Commercial bleach should contain at least 5.25% NaClO.
If less than this is present, it cannot be called bleach.)
EXPERIMENT 18
IODOMETRIC DETERMINATION OF COPPER
Principle
A copper metal sample is dissolved in nitric acid to produce Cu(II), and the oxides
of nitrogen are removed by adding H2 SO4 and boiling to SO3 fumes. The solution
is neutralized with NH3 and then slightly acidied with H3 PO4 . [The H3 PO4 also
complexes any iron(III) that might be present and prevents its reaction with I .]
Finally, the solution is treated with excess KI to produce CuI and an equivalent
amount of I3 , which is titrated with standard Na2 S2 O3 solution, using a starch
indicator. KSCN is added near the end point to displace absorbed I2 on the CuI
by forming a layer of CuSCN. For best accuracy, the Na2 S2 O3 is standardized
1 This experiment should be completed in a single laboratory period by all students, including calculation and
reporting of results. The hypochlorite and peroxide solutions are subject to decomposition, with a resultant change
in concentration. The instructor may take the average of the class results as the correct value, or he or she may
perform an analysis alone for comparison.
Page 32
against high-purity copper wire since some error occurs from reduction of copper(II)
by thiocyanate.
Equations
2Cu2+ + 5I 2CuI + I3
I3 + 2S2 O3 2 3I + S4 O6 2
1. Provided. 6 M HNO3 , conc. H2 SO4 , 3 M H2 SO4 , conc. H3 PO4 , 6 M NH3 ,
KSCN.
2. To prepare.
(a) Starch solution. Prepare the day of the experiment as described in Experiment 17, or use Thiodene indicator.
(b) 0.1 M Na2 S2 O3 solution. Prepare as described in Experiment 17.
Prepare the 0.1 M Na2 S2 O3 solution. Although this can be prepared on the day of the
experiment, it is preferable to prepare it at least a day before it is standardized. The
solution tends to lose some of its titer right after preparing.
Procedure
1. Standardization of the 0.1 M Na2 S2 O3 solution. The same procedure is used as
will be used for analyzing the sample. Weigh out three 0.20- to 0.25-g samples
of pure electrolytic copper foil and add to 250-mL Erlenmeyer asks. In a hood,
dissolve in 10 mL 6 M HNO3 , heating on a steam bath if necessary. Do in a fume
hood. Add 10 mL conc. H2 SO4 and evaporate to copious white SO3 fumes. Cool
and add carefully 20 mL water. Boil 1 to 2 min and cool. Add 6 M NH3 dropwise
with swirling of the sample solution until the rst dark blue of the Cu(NH3 )4 2+
complex appears. Then, add 3 M H2 SO4 until the blue color just disappears,
followed by 2.0 mL conc. H3 PO4 . Cool to room temperature.
From this point, each sample must be treated separately. Dissolve about
2 g KI in 10 mL water and add to one of the asks. Titrate immediately with the
Na2 S2 O3 solution until the yellow color of I3 almost disappears. Add 2 to 3 mL
of the starch solution or approximately 0.4 g Thiodene indicator, and titrate until
the blue color begins to fade (should be less than 0.5 mL). Finally, add about 2 g
KSCN and continue the titration until the blue color just disappears.
Repeat with the other two samples. Calculate the molarity of the Na2 S2 O3 .
2. Determination of copper in an unknown. Add 10 mL of 6 M HNO3 to each of
three clean 250-mL Erlenmeyer asks and take these to your instructor, who will
add an unknown sample to each. (This unknown may be copper foil as used in
the standardization.) Dissolve each and titrate as described for the standardization
of Na2 S2 O3 . Note: In place of H3 PO4 , approximately 2 g ammonium biuoride,
NH4 HF2 or NH4 F HF, may be added to complex any iron and at the same
time adjust the solution to the proper acidity. This experiment is also suitable for
determining copper in about 0.3 g brass.
Calculation
Calculate the grams copper in each unknown sample and report the values of each
and the relative standard deviation. (If a weighed brass sample is analyzed, report the
percent copper.)
E33
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E34
EXPERIMENTS
EXPERIMENT 19 DETERMINATION OF ANTIMONY

BY TITRATION WITH IODINE
Principle
Antimony(III) is titrated to antimony(V) in neutral or slightly alkaline solution with
iodine to a blue starch end point. The iodine is standardized against primary standard
arsenic(III) oxide. Tartaric acid is added to complex the antimony and prevent its
hydrolysis to form insoluble basic salts such as SbOCl and SbO2 Cl (which form in
slightly acid and neutral solution).
Equations
Standardization:
H2 AsO3 + I3 + H2 O HAsO4 2 + 3I + 3H+ (pH 8)
Sample titration:
SbOC4 H4 O6 + I3 + H2 O SbO2 H4 O6 + 3I + 2H+
1. Provided. Primary standard As2 O3 , 1 M NaOH, Na2 CO3 , NaHCO3 , KI, tartaric
acid, 1 M HCl. For stibnite ore: KCl, 0.1% (wt/vol) methyl red indicator in 60%
ethanol, conc. HCl, 6 M HCl, 6 M NaOH.
2. To prepare
(a) Starch solution. Prepare as described in Experiment 17, or use Thiodene
indicator.
(b) 0.05 M iodine solution. Weigh 6.5 g iodine crystals and 20 g potassium
iodide. Grind the iodine in a mortar with repeated small portions of the
weighed KI crystals and water, pouring off the solution frequently into a
glass-stoppered bottle until the solids are completely dissolved. [I2 is only
slightly soluble in water, but forms soluble KI3 (I3 complex) in the presence
of excess KI.]
Avoid pouring undissolved iodine into the bottle. Dilute the solution to about
500 mL and mix thoroughly. Check for any undissolved iodine. Preferably
let stand overnight before standardizing to ensure complete dissolution of the
iodine. Alternatively, before diluting the solution, add more KI until all iodine
is dissolved.
1. Obtain and dry your unknown. Obtain a sample in a weighing bottle from your
instructor and dry at 110 to 120 C for at least 1 to 2 h. Cool for at least 30 to
40 min before weighing.
2. Dry the As2 O3 . Obtain and dry about 1 g primary standard As2 O3 at 110 to
120 C for 1 to 2 h. Cool in a desiccator at least 30 to 40 min before weighing.
3. Prepare the 0.05 M I2 solution. If your unknown is a stibnite ore that will
require some time to dissolve (as opposed to a water-soluble synthetic sample), it
is advisable to also standardize the iodine solution before the day of the experiment
to allow sufcient time to complete the experiment (procedure below).
Procedure
1. Standardization of iodine solution. Weigh directly and accurately three 0.15- to
0.20-g portions of dried primary standard As2 O3 . Transfer to 250-mL Erlenmeyer
asks and dissolve in 10 to 20 mL 1 M NaOH, heating if necessary to aid
Page 34
dissolution. No undissolved particles should remain. Cool and add 1 M HCl until
the solution is just acidic to litmus paper. Add 3 to 4 g solid NaHCO3 . No further
CO2 evolution should occur on addition of the last portion of NaHCO3 . If it does,
add more NaHCO3 . The pH of the solution should be 7 to 8. Wash the walls of the
ask down and add 50 mL water and 3 mL starch solution or about 0.4 g Thiodene
indicator. Titrate with the iodine solution to the appearance of the rst tinge of
blue that persists for at least 30 s. From the three titrations, calculate the molarity
of the I2 solution. Use the average of the results.
2. Determination of antimony in unknown
(a) Water-soluble synthetic sample. Consult your instructor for the proper
size sample to weigh so that it will contain about 2 mmol antimony. Weigh
three portions of the dried unknown and dissolve in 50 mL water in 500-mL
Erlenmeyer asks. Dissolve 4 g NaHCO3 and 2 g tartaric acid in 100 mL water
and add this solution to the antimony solution. The solution should be clear
at this point with no hydrolyzed antimony chloride. Add 3 mL starch solution
and titrate to a blue color that persists at least 30 s.
(b) Insoluble stibnite ore. Consult your instructor for the proper size sample
to weigh so that it will contain about 2 mmol antimony. Into the dry 250-mL
beakers weigh accurately triplicate samples of the dried ore. Add about 0.3 g
nely powdered potassium chloride, nearly cover the beaker with a watch
glass, and carefully add 10 mL conc. hydrochloric acid by pouring it down
the side of the beaker. (A high concentration of chloride is necessary to
prevent hydrolysis during the dissolution before tartaric acid is added. SbCl3
is formed.) Warm (do not boil) in the hood until the ore is decomposed;
the mixture should no longer give an odor of hydrogen sulde, and any
residue (silica) should be white or only slightly gray. A stibnite ore consists of
antimony sulde, Sb2 S3 , silica, and small amounts of other substances. When
all the antimony is dissolved, no more hydrogen sulde should be evolved.
Do not allow the solution to evaporate to dryness, which might result in loss
of antimony trichloride; add more HCl as necessary. When decomposition is
complete, add 3 g nely powdered tartaric acid and continue the heating for
10 to 15 minutes. Add water in portions of about 5 mL with good stirring
until the solution is diluted to about 100 mL. (The solution must be diluted
slowly, since some of the antimony may be hydrolyzed by local excesses
of water.) If, during dilution, a red-orange precipitate (Sb2 S3 ) appears, heat
gently until the precipitate has dissolved before continuing the dilution. If a
white precipitate of basic salts forms, the determination should be discarded.
When the dilution is complete, boil the solution for 1 min.
Rinse off the watch glass into the solution, and carefully neutralize the
solution with 6 M sodium hydroxide by using a few drops of methyl red
indicator. Then, add 6 M HCl dropwise until the solution is just acidic,
carefully avoiding an excess.
In 600-mL beakers or 500-mL Erlenmeyer asks, prepare solutions containing 4 g sodium bicarbonate in 200 mL water. Pour the sample into the
sodium bicarbonate solution, avoid loss by effervescence, and rinse several
times with a stream of water from the wash bottle to obtain a complete
transfer of the solution. Add 3 mL starch indicator or about 0.4 g Thiodene
indicator and titrate with standard iodine solution to the appearance of the rst
permanent blue color. A fading or indistinct end point is due to insufcient
sodium bicarbonate in the solution to consume the acid produced in the titration. Add 1 g additional NaHCO3 and complete the titration to a permanent
blue color.
E35
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E36
EXPERIMENTS
Calculation
Calculate and report the percent Sb2 O3 for each portion of your sample analyzed.
Report also the mean of your values and the precision.
EXPERIMENT 20 MICROSCALE QUANTITATIVE ANALYSIS OF
HARD-WATER SAMPLES USING AN INDIRECT POTASSIUM
PERMANGANATE REDOX TITRATION1
Principle
The calcium in an unknown hard-water sample is precipitated as calcium oxalate in
ammonia solution, and the precipitate is quantitatively ltered and washed, and is then
dissolved in dilute sulfuric acid. The oxalic acid is titrated with standardized potassium
permanganate solution.
Note: This experiment may serve as a template for microscale versions of some
other experiments in the text. Statistical comparison of the microscale experiment with
a similar conventional macroscale experiment using a 50-mL buret shows comparable
precision.1 But there is a slight negative determinate error in the microscale experiment, averaging about 50 ppm for ca. 500-ppm samples, probably due to physical loss
of some of the precipitate, using a large funnel. Richardson suggests using a microfunnel to minimize this problem. For additional information on microscale titrations,
see M. M. Singh, C. B. McGown, Z. Szafran, and R. M. Pike, J. Chem. Educ., 77
(2000) 625.
Equations
Ca2+ + C2 O4 2 CaC2 O4
CaC2 O4 + 2H+ Ca2+ + H2 C2 O4
5H2 C2 O4 + 2MnO4 + 6H+ 10CO2 + 2Mn2+ + 8H2 O
1. Provided. Conc. H2 SO4 , conc. HCl, conc. HNO3 , primary standard Na2 C2 O4
(dried at 120 C for 1 h), 0.35 M (NH4 )2 C2 O4 , 0.10 M AgNO3 , dil. (1:10) NH3
solution, methyl red (0.02% in 60% ethanoldissolve rst in the ethanol portion).
Provide concentrated acids and AgNO3 in dropper bottles.
2. Approx. 0.2 M KMnO4 . One liter is enough for 30 students. Prepare as follows
(see Section 14.6). The preparation may be scaled up for more students. Calculate
the weight of potassium permanganate required to make 1 L of 0.02 M solution.
Weigh out in a weighing dish about 0.05 g more than this amount. Transfer to
two 600-mL beakers, placing about half of the permanganate in each, add 500 mL
of distilled water to each, cover with watch glasses, heat to boiling, and boil
gently for 1 to 2 min, not longer. (Longer boiling, 0.5 to 1 h, is desirable but will
evaporate the solution and change its concentration. The solution may be heated
for a longer period at a temperature just below its boiling point.) Allow to stand
for at least 24 h before proceeding with the next step. Keep covered with watch
glasses at all times to exclude dust and vapors, and to retard evaporation.
Mount a sintered-glass lter in a lter ask, and lter the permanganate
solution through the crucible. Do not stir or swirl the solution; a sediment will have
settled to the bottom that would clog the lter and make ltration very slow. For
this reason, discard the last few milliliters of the solution from the rst beaker. Use
1
Courtesy of Professor J. N. Richardson, M. T. Stauffer, and J. L. Henry Shippensburg University. Details of
statistical analysis of the experiment in J. Chem. Educ., 80(1) (2003) 65.
Page 36
a lter trap; if any tap water backs up from the suction pump, the permanganate
will be contaminated. Pour the ltered solution into a clean brown glass bottle
with glass stopper and shake until homogeneous. The solution must never come
into contact with organic material, including corks and rubber stoppers.
3. Unknown hard-water solution. Prepared by dissolving ca. 20 g of dried CaCO3
in a minimum volume of 1 M HCl. To this solution is added a few drops of methyl
red indicator, followed by dropwise addition of 1 M NaOH until the methyl red
end point is noted (red to yellow). The resulting solution is then quantitatively
transferred to a 2.000-L volumetric ask and diluted to the mark followed by
thorough mixing. Student unknowns may then be dispensed from a buret into
individual 100.00-mL volumetric asks. Typical aliquots range from 10.00 to
20.00 mL, resulting in diluted unknowns with concentrations ranging from ca.
400 to 800 ppm Ca2+ .
Microburet
Constructed using a 2.000-mL graduated pipet, 2-cm length of latex tubing, 10-mL
plastic syringe barrel, and an automatic delivery pipet tip. A detailed illustration of
the microburet, as well as documentation regarding its construction, are provided
in M. M. Singh, C. B. McGown, Z. Szafran, and R. M. Pike, J. Chem. Educ., 75
(1998) 371.
Procedure
1. Standardization of KMnO4 . Each bench prepares 200 mL of dilute (1:20 v:v)
sulfuric acid using concentrated H2 SO4 . (Caution: Acid should be added slowly to
water with stirring.) Remove dissolved gases from this solution by boiling for 5 to
10 min with a glass stirring rod in the beaker to keep solution from bumping. Cool
the solution to room temperature using an ice bath and store in a tightly capped,
appropriately labeled polypropylene bottle. All students at a bench will share this
solution (with typically three or four students occupying a lab bench). Do not
proceed past this point unless the standardization titration will be attempted the
same day.
To a 100.00-mL volumetric ask, each bench adds 0.5000 g of primary
standard Na2 C2 O4 , dissolves it in the dilute H2 SO4 prepared previously, and
dilutes to the mark, mixing thoroughly. Again, this solution is shared among all
members of a lab bench. From this point on, each student works individually.
Rinse a 2-mL microburet with the primary standard solution and then ll it with
the solution. Transfer 1.500 to 2.000 mL of the solution into a clean 30-mL beaker,
recording the volume delivered exactly. Dilute to a total volume of about 5 mL
using the dilute H2 SO4 . Prepare three more samples this way, using a different
volume of Na2 C2 O4 solution each time.
Clean a 2-mL microburet, rinse with and then ll with the 0.02 M KMnO4
solution. Calculate the approximate volume of this titrant needed to reach an end
point for the primary standard sample with the smallest volume. Add rapidly all
but about 0.2 mL of this amount from the buret, with constant but gentle stirring
with a stirring rod. Let this solution stand until the color disappears, which may
take a minute or two. Heat the solution to 55 to 60 C, and complete the titration
at this temperature (the temperature can be monitored by a thermometer, or one
can watch for initial formation of steam). The remaining titrant is added dropwise,
allowing each drop to react before adding the next. The end point is the rst
perceptible pink that persists for at least 30 s. Repeat this procedure for each of the
remaining samples of Na2 C2 O4 . Calculate the molarity of the KMnO4 for each
titration, along with the average molarity and standard deviation.
E37
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E38
EXPERIMENTS
2. Analysis of an Unknown Hard-Water Sample. Each student is assigned a labeled

100.00-mL volumetric ask containing an unknown hard-water sample. Add
distilled water to the mark and invert the ask to mix thoroughly. Use a microburet
to transfer an aliquot of hard water of about 1 mL into a clean 30 mL beaker. The
volume transferred should be recorded to the precision of the buret. To this sample
add 10 mL of distilled water, 7 drops of concentrated HCl, and a drop or two of
methyl red indicating solution. Heat the resulting solution to nearly boiling, and
add 1 mL of 0.35 M (NH4 )2 C2 O4 . (Note: students typically obtain this solution
from a community macroburet or from a small graduated cylinder because they
are only concerned with having an excess of oxalate ion in the solution.)
Prepare and ll a microburet with dilute (1:10 v:v) NH3 solution. Add this
solution dropwise to the unknown sample in the beaker, noting that the solution
should become cloudy as the precipitate (CaC2 O4 ) starts to form. Continue the
addition of NH3 solution until the solution becomes alkaline to methyl red as
indicated by a change from pink to pale yellow. Addition of excess NH3 solution
should be avoided. Cover the beaker with Paralm, and allow the precipitate to
digest overnight. If the pink returns, add more NH3 solution to obtain the methyl
red end point. Repeat this procedure for two more hard-water samples, using a
slightly different aliquot size each time.
Gravity lter the precipitated CaC2 O4 in each sample through 42.5-mm
diameter No. 42 or equivalent Whatman lter paper seated in a long-stem lter
funnel.2 Note that transfer of the precipitate must be quantitative. Wash the
ltered precipitate with cold distilled water until the ltrate is clear upon addition
of HNO3 /AgNO3 . This is done by collecting a few drops of ltrate in a test
tube and adding a drop or two of the acid, followed by a drop or two of AgNO3
solution.
Remove the lter paper from the funnel and place in a clean 30-mL beaker.
Add about 3 mL of 1:10 (v:v) sulfuric acid (previously boiled as in step 1). Then
stir the mixture until the solid CaC2 O4 is dissolved and the lter paper is torn
apart. Add about 10 mL of distilled water to the mixture, and heat to just below
the boiling point. Titrate the mixture with the standardized KMnO4 solution as
follows: (1) add 0.10 mL of titrant and allow the mixture to stand until the color
fades, and (2) continue the titration as normal until an end point is reached. The
temperature of the solution should be maintained above 55 C for the duration of
the titration. Repeat this procedure for each of the other unknown samples.
Calculation
Using the data obtained, along with a balanced chemical reaction equation, calculate
the concentration of calcium in parts per million for each trial. Report the mean and
standard deviation.
Potentiometric Titrations
EXPERIMENT 21
pH TITRATION OF UNKNOWN SODA ASH
Principle
The unknown soda ash is titrated with standard HCl using a potentiometric (pH)
end point measured with a pH meter using a pH glass electrodesaturated calomel
reference electrode combination. The end-point breaks are compared with indicator
color changes.
2 Improved recovery may be achieved by using a smaller funnel to t the paper.
Page 38
E39
POTENTIOMETRIC TITRATIONS
Equations
CO3 2 + H+ HCO3
(phenolphtalein end point)
HCO3 + H H2 O + CO2
(methyl purple end point)
Note that between the rst and second end points, a gradual decrease in pH due to the
HCO3 /CO2 buffer system will occur. This will give a poor visual end point, unless
the buffer couple is destroyed. In practice, the visual titration used for standardization
is continued until the methyl purple end point is reached, at which time the solution is
gently boiled to remove the CO2 , leaving only the remaining HCO3 , which is then
titrated to completion (see Chapter 8 for a more detailed discussion).
1. Provided. 0.2% phenolphthalein in 95% ethanol, 0.1% methyl purple in water,
primary standard Na2 CO3 , standard pH 7 buffer.
2. To prepare. Standard 0.1 M HCl solution. Use the solution prepared in Experiment 8. If this solution is not available, prepare and standardize 500 mL as
described in Experiment 7. Alternatively, the acid may be standardized against
the primary standard Na2 CO3 by pH titration as described below for the unknown
soda ash.
Prepare and standardize the HCl solution. This will require prior drying of primary
standard Na2 CO3 .
Obtain the unknown soda ash from your instructor and dry for at least 2 h at
160 C. Cool at least 30 min in a desiccator before weighing.
Procedure
The glass electrode to be used for pH measurements should have been soaked and
stored in 0.1 M KCl for at least one day prior to its use. Always store the electrode in
KCl solution when not in use. Calibrate the pH meter as described by your instructor,
using the pH 7 standard buffer. This will consist essentially of adjusting the meter to
read pH 7.00 with the electrodes immersed in the buffer solution.
1. Trial titration. The purpose of this titration is to locate quickly and approximately
the two end points. Weigh accurately by difference a dried sample of unknown
soda ash (0.2 to 0.3 g) and add it to a 400-mL beaker containing a magnetic
stirring bar. Add approximately 50 mL water and a few drops phenolphthalein
indicator. The indicators are for the purpose of making a comparison between the
potentiometric end points and the indicator color changes. Place the beaker on a
magnetic stirrer, immerse the electrodes, and start the stirrer, being careful not to
touch the electrodes to the stirring bar. Titrate with standard HCl, taking readings
about every 2 mL. After the phenolphthalein color disappears, add a few drops
methyl purple indicator and titrate at 2-mL increments until the second end point
is reached. Add a few increments beyond the end point. The correct color for the
second end point can be determined by comparison with the color of a few drops
of the indicator in a solution of 0.20 g potassium acid phthalate in 100 mL water.
Prepare a spreadsheet to plot your titration curve of pH (ordinate) versus volume
of HCl (abscissa), and also the rst derivative plot, and locate the approximate
end points. See Chapters 8 and 14 and your text website for those chapters for the
preparation of a spreadsheet for derivative titrations.
Page 39
E40
EXPERIMENTS
2. Final titration. Weigh accurately another sample of the unknown and titrate as
before, but make pH readings every 5 mL to within 3 mL of each end point (both
sides of end point). Then, make readings of 0.50- to 1-mL intervals within 1 mL of
the end point. Near the end point, take readings as quickly as possible because the
pH will tend to drift as CO2 escapes from the solution. Note and record the points
at which the indicators change color. Use the spreadsheet you prepared above to
plot your titration curve and its rst derivative. Print out the titration curve and
indicate on this curve the range in which the indicators change color. Determine
the end point from the second inection point of the curve. Repeat the titration
on two more portions of the unknown. Be sure to rinse the electrodes between
titrations.
Calculations
Calculate and report the percent Na2 CO3 and Na2 O in your unknown for each portion
analyzed. Hand in the plots of the titration curves with your report. Report also the
mean percent value and the precision.
EXPERIMENT 22 POTENTIOMETRIC TITRATION OF A MIXTURE
OF CHLORIDE AND IODIDE
Principle
The mixture is titrated with a standard solution of silver nitrate, and the potentiometric
end points are indicated with a silver wire electrodeglass electrode pair using a
pH meter for potential measurements. Because the pH during the titration remains
essentially constant, the glass electrodes potential remains constant, and this electrode
serves as the reference electrode; this eliminates the necessity of preparing a chloridefree salt bridge for the reference electrode. AgI (Ksp = 1 1016 ) precipitates rst
since it is less soluble than AgCl (Ksp = 1 1010 ). The AgCl starts precipitating near
+
10
+
the equivalence point of the iodide
titration (when [Ag ] [Cl ] = 1 10 ; [Ag ]
16
8
at the iodide equivalence point is 1 10
= 1 10 M). The potential (i.e., pX)
rise of the iodide titration curve will level off at the point when the chloride starts
precipitating, that is, near the iodide equivalence point inection. This will be followed
by the typical S-shaped chloride potentiometric end point. The error in determining
the iodide end point is small if it is taken at the point at which the potential levels off.
(It should be noted that while mixtures of chloride and iodide can be titrated, mixtures
of bromide with either chloride or iodide cannot generally be titrated because of mixed
crystal formationsee isomorphous replacement in Chapter 10.)
Equations
I + Ag+ AgI
Cl + Ag+ AgCl

0.1 M standard AgNO3 . Prepare as described in Experiment 13.
Dry the primary standard AgNO3 for 1 to 2 h at 110 to 120 C (no longer). Store in a
desiccator until ready for weighing.
Obtain and dry your unknown at 120 C for 1 to 2 h. Store in desiccator until
ready for weighing.
Page 40
E41
SPECTROCHEMICAL MEASUREMENTS
Procedure
Weigh directly three 0.5- to 0.6-g samples of the dried unknown into 400-mL beakers.
Dissolve in 150 mL distilled water, add a magnetic stirring bar, and place the beaker
on a magnetic stirrer. (Dissolve and titrate only one portion at a time to minimize
air oxidation of the iodide.) Immerse the electrodes in the solution, taking care that
they do not hit the magnetic stirrer. Connect the silver wire electrode to the reference
terminal of the pH meter and the glass electrode to its usual terminal.1 Stir the solution
and titrate the sample with the standard AgNO3 . Take pH readings (actually pX), or
millivolt readings, at 2-mL increments until the change is greater than 0.4 pH unit or
25 mV. Then add 0.2-mL increments. After the rst end point is reached, add 2-mL
increments until the second end point is approached and then 0.2-mL increments.
Using a spreadsheet, plot the potential versus volume of AgNO3 and determine the
end point for the iodide and the chloride (inection point of second potential break).
Use these values to estimate the end point for the other two samples and repeat the
above procedure for these samples. Titrant may be added rapidly up to within 2 or
3 mL of the end point. Be sure to rinse the electrodes between titrations.
Calculations
Calculate and report the percent iodide (from the volume required to reach the rst
end point) and chloride (from the volume required to go from the rst end point to the
second end point) in your unknown for each portion analyzed. Hand in the plots of the
titration curves with your report.
Report also the mean values and the precision.
Spectrochemical Measurements
EXPERIMENT 23 SPECTROPHOTOMETRIC
DETERMINATION OF IRON
Principle
A complex of iron(II) is formed with 1,10-phenanthroline, Fe(C12 H8 N2 )3 2+ , and
the absorbance of this colored solution is measured with a spectrophotometer. The
spectrum is plotted to determine the absorption maximum. Hydroxylamine (as the
hydrochloride salt to increase solubility) is added to reduce any Fe3+ to Fe2+ and to
maintain it in that state.
Equations
4Fe3+ + 2NH2OH 4Fe2+ + N2O + 4H+ + H2O
1
2+
3 Fe
Fe2+/3
1,10-phenanthroline
tris(1,10-phenanthroline) iron(II)
1 The silver electrode is actually the indicating electrode and the glass electrode is the reference electrode. But
most glass pH electrodes require a special plug and will not t the reference terminal. The above arrangement is
satisfactory and simply means the potential will be of the opposite sign from usual and change in the opposite
direction.
Page 41
E42
EXPERIMENTS

1. Standard iron(II) solution. Prepare a standard iron solution by weighing 0.0176 g
ferrous ammonium sulfate, Fe(NH4 )2 (SO4 )2 6H2 O. Quantitatively transfer the
weighed sample to a 250-mL volumetric ask and add sufcient water to
dissolve the salt. Add 0.7 mL conc. sulfuric acid, dilute exactly to the mark with
distilled water, and mix thoroughly. This solution contains 10.0 mg iron per liter
(10 ppm); if the amount weighed is other than specied above, calculate the
concentration.
2. 1,10-Phenanthroline solution. Dissolve 25 mg 1,10-phenanthroline monohydrate in 25 mL water. Store in a plastic bottle.
3. Hydroxylammonium chloride solution. Dissolve 10 g hydroxylammonium chloride in 100 mL water.
4. Sodium acetate solution. Dissolve 10 g sodium acetate in 100 mL water.
Procedure
Into a series of 100-mL volumetric asks, add with pipets 1.00, 2.00, 5.00, 10.00, and
25.00 mL of the standard iron solution. Into another 100-mL volumetric ask, place
50 mL distilled water for a blank. The unknown sample will be furnished in another
100-mL volumetric ask. To each of the asks (including the unknown) add 1.0 mL
of the hydroxylammonium chloride solution and 5.0 mL of the 1,10-phenanthroline
solution. Buffer each solution by the addition of 8.0 mL of the sodium acetate solution
to produce the red color of ferrous 1,10-phenanthroline. [The iron(II)phenanthroline
complex forms at pH 2 to 9. The sodium acetate neutralizes the acid present and adjusts
the pH to a value at which the complex forms.] Allow at least 15 min after adding
the reagents before making absorbance measurements so that the color of the complex
can fully develop. Once developed, the color is stable for hours. Dilute each solution
to exactly 100 mL. The standards will correspond to 0.1, 0.2, 0.5, 1, and 2.5 ppm iron,
respectively.
Obtain the absorption spectrum of the 2.5-ppm solution by measuring the
absorbance from about 400 to 700 nm (or the range of your instrument). Take readings
at 25-nm intervals except near the vicinity of the absorption maximum, where you
should take readings at 5- or 10-nm intervals. Follow your instructors directions
for the operation of your spectrophotometer. The blank solution should be used
as the reference solution. Plot the absorbance against the wavelength and select
the wavelength of the absorption maximum. This may be done with a spreadsheet.
From the molar concentration of the iron solution and the cell pathlength, calculate
the molar absorptivity of the iron(II)phenanthroline complex at the absorption
maximum.
Prepare a calibration curve by measuring the absorbance of each of the standard
solutions of the wavelength of maximum absorbance. Measure the unknown in the
same way. Using a spreadsheet, prepare a calibration curve by plotting the absorbance
of the standards against concentration in ppm. From this plot and the unknowns
absorbance, determine the nal concentration of iron in your unknown solution.
Perform the calculations of the measured concentration by entering the formula in
a cell of the spreadsheet using the slope and intercept values and the measured
absorbance. (See Chapters 3 and 16 for preparing the spreadsheet.) Report the number
of micrograms of iron in your unknown along with the molar absorptivity and the
spectrum of the iron(II)phenanthroline complex.
Page 42
E43
EXPERIMENT 24 SPECTROPHOTOMETRIC DETERMINATION OF IRON

IN VITAMIN TABLETS USING A 96 WELL PLATE READER
Objective: The objective of this lab exercise is to determine the iron content in a
commercial vitamin tablet using ultravioletvisible spectrophotometry.
Overview: UVVis spectrophotometry is the analytical method that takes
advantage of the fact that many substances absorb light in the ultraviolet (UV) to visible
(Vis) region of the electromagnetic spectrum. Because light absorbance is proportional
to the concentration of the absorbing substance (Beers Law), spectrophotometry is
well-suited for quantitative analysis and is widely used in industrial, environmental
and medical laboratories. One disadvantage is that not all substances absorb UV or
Vis light. To get around this problem, chemical reactions have been developed that
convert the analyte to an absorbing species. This approach will be used to analyze for
iron in a vitamin tablet. The tablet is dissolved in acid and the iron is reduced from
Fe3+ to Fe2+ with hydroquinone and then complexed with o-phenanthroline to form
an intensely colored complex. The absorbance maximum (max ) of the complex is at
508 nanometers (nm). The reactions are shown below:
2Fe3+ + HO
OH
Hydroquinone
O + 2H+
2Fe2+ + O
Quinone
N
Fe2+
Fe2+ + 3
N
o-Phenanthroline
N
3
Procedure
A. Overview: The concentration of the Fe-phenanthroline complex is determined
by measuring the absorbance at 508 nm. Absorbance is equal to log (I/I0 )
where I is the light transmitted through the sample and I0 is the light transmitted
through a blank solution, usually the pure solvent (DI water in this case). To
quantify the iron complex we need to generate a calibration curve using solutions
of known concentrations of the analyte. The colored complex is stable, so all
solutions may be prepared in advance and all the absorbance measurements done
together.
B. Required reagents
1. Hydroquinone: Freshly prepared solution containing 10 g/L in water. Store
in an amber bottle.
2. Trisodium citrate: 25 g/L in water.
3. o-Phenanthroline: Dissolve 2.5 g in 100 mL of ethanol and add 900 mL of
water. Store in an amber bottle.
Page 43
E44
EXPERIMENTS
4. Standard Fe (0.08 mg Fe/mL): This is the stock solution for preparing calibration standards. Dissolve 0.28 g of reagent-grade Fe(NH4 )2 (SO4 )2 6H2 O
in DI in a 500-mL volumetric ask containing 0.5 mL of 98 wt % H2 SO4 .,
and dilute to volume. Record the actual weight to the nearest 0.1 mg. Another
reminder, always add acid to water, not vice versa.
C. Vitamin preparation
1. Obtain vitamin tablets and record the sample number in your notebook. Place
one vitamin tablet in a 250 mL ask or beaker and boil gently in a hood or
close to an air intake with 25 mL of 6 M HCl for 15 min. Filter the solution
directly into a 100 ml volumetric ask with a lter funnel and properly folded
lter paper. Wash the beaker and lter several times with small portions
of dilute HCl (23 mL of 6 M HCl per 100 mL DI water) to complete a
quantitative transfer. Allow the solution to cool, ll to the mark with dilute
HCl, and mix well. Dilute 1.00 mL of this solution (using micropipettors) to
the mark with DI water in a 10 mL volumetric ask. This solution will be
treated and analyzed in the spectrophotometer.
2. Repeat this procedure for two additional vitamin tablets for a total of three
replicates (Do not attempt to do all three vitamin samples simultaneously). Be
sure to number or otherwise identify each solution. Also, remember to record
any observations in your lab notebook.
3. Determine how much citrate buffer is required to increase the pH to 3.5, the
pH at which phenanthroline effectively binds Fe, as follows. Pipet 1.00 mL
of one of the vitamin solutions into a beaker. With a Pasteur pipet, determine
how many drops of citrate solution are needed to bring the 1.00 mL of solution
to pH 3.5 (use pH paper sparingly). If the solution is too acidic (pH < 3.5),
the o-phenanthroline will not effectively complex the Fe2+ . If too basic, iron
hydroxide could precipitate out of solution.
4. It is now time to reduce and complex the Fe in the vitamin solutions. Pipet
1.00 mL of solution from the 10 mL ask above into a 10 mL volumetric
ask. Add the required amount of citrate solution, 0.200 mL of hydroquinone
solution, and 0.300 mL of o-phenanthroline solution; dilute to the mark with
DI and mix well. Pour into a separate container and set aside. Repeat for the
two additional vitamin solutions.
5. Make up a blank solution by diluting 1.00 mL of the vitamin solution to
10.00 mL with no reagents added (sample blank). Do this with only one
solution, no need to do all three. Also prepare another blank that contains all
the reagents but no Fe (reagent blank).
D. Standards preparation
1. Pipet 1.00 mL of the standard Fe stock solution into a beaker. As before, add
sodium citrate solution 1 drop at a time until a pH of 3.5 is reached and
record the volume of citrate required.
2. Pipet a fresh 1.00-mL aliquot of Fe standard into a 10 mL volumetric ask
and add the same number of drops of citrate solution as required in Step D-1.
Add 0.200 mL of hydroquinone solution and 0.300 mL of o-phenanthroline
solution, dilute to the mark with DI water and mix well.
3. Prepare three more solutions from 0.500, 0.200 and 0.100 mL of Fe standard.
Use the sodium citrate solution in proportion to the volume of Fe solution.
It might be wise to check the pH of these solutions with pH paper just to make
sure. Allow all Fe-phenanthroline solutions to stand for at least 10 min.
Page 44
E. Spectrophotometric measurements
1. Obtain a clean 96 well plate. Using a 20-200 L pipettor, pipette 200 L of
the reagent blank, standards, sample blank and vitamin samples into the wells
in the following pattern:
Column 1 (A-H) Reagent Blank
Column 2 (A-H) First standard
Column 3 (A-H) Second Standard
Column 4 (A-H) Third standard
Column 5 (A-H) Fourth Standard
Column 6 (A-H) Distilled Water
Column 7 (A-H) Vitamin Sample 1
Column 10 (A-H) Vitamin Sample 1 diluted 1/2 in distilled water.
Column 11 (A-H) Sample Blank
2. Place the 96 well plate in one of the plate readers. Open the le/method
provided by the instructor, and follow instructions to read the plate. The
absorbance data for each of the wells will be read and reported. The data will
appear in a table with the raw data, the mean for each column, and the relative
standard deviation for each column. Export this data to a le with a unique le
name and store on a USB ash drive. Copy the results to your notebook.
Lab Report
Include a table with the average absorbances and their standard deviations as provided
by the plate reader for the blanks, standards and samples. Also include in the table
the blank-corrected absorbances for the standards and the samples, with precision
propagated from the standard deviations for the raw absorbances. Note that the
standards should only be corrected for the reagent blank whereas the samples should
be corrected for the sample and reagent blanks.
Using a spreadsheet program, make a graph of absorbance versus the Fe
concentration (M) of the calibration standards. Find the slope, intercept, correlation
coefcient and standard error using the regression routines in the spreadsheet program.
Include the plots and regression results.
With the linear equation from the calibration curve above and the blank-corrected
absorbance of the vitamin solutions, nd the concentration of Fe in the three tablet
solutions. Estimate the uncertainty in each concentration using the linear regression
results.
Calculate the mg Fe/tablet for each replicate and propagate the error, using
the concentration uncertainty determined previously and the expected uncertainties
for the volumetric glassware and pipettes, to obtain the uncertainty for this result.
In a table, report the results and uncertainties for each replicate as well as the
overall mean, standard deviation, and condence interval calculated for the three
tablets.
Compare the individual values for the three tablets and discuss whether you can
conclude that there is a signicant difference in the Fe/tablet between tablets using
your measurements.
Briey discuss the uncertainty in the results for a single tablet preparation and for
the three tablets. Consider whether the standard deviations of the absorbance readings
are representative of the overall random error in the experiment.
E45
Page 45
E46
EXPERIMENTS
EXPERIMENT 25 DETERMINATION OF NITRATE

NITROGEN IN WATER1
Principle
Nitrate, NO3 , is reacted with phenoldisulfonic acid to give a yellow color with an
absorption maximum at 410 nm. Chloride interference is removed by precipitating the
chloride. Nitrite, NO2 , levels in excess of 0.2 mg/L cause positive interference, but
these concentrations rarely occur in surface waters.
1. Provided. 1 M NaOH solution; conc. NH3 , phenoldisulfonic acid reagent prepared by dissolving 25 g phenol in 150 mL conc. H2 SO4 , adding 75 mL fuming
H2 SO4 (15% free SO3 ), stirring well, and heating for 2 h on a hot-water bath.
2. To prepare
(a) Silver sulfate solution. (This will not be required if synthetic chloride-free
unknowns are provided.) Dissolve 0.44 g Ag2 SO4 , free from nitrate, in 100 mL
distilled water. One milliliter is equivalent to 1 mg chloride.
(b) EDTA solution. Prepare a paste of 50 g Na2 EDTA 2H2 O in 20 mL water,
add 60 mL conc. NH3 , and mix well to dissolve the paste.
(c) Stock nitrate solution, 100 mg/L N. Dissolve 0.722 g anhydrous KNO3
and dilute to 1 L.
(d) Standard nitrate solution, 10 g/mL N (44 g/mL NO3 ). Dilute 50 mL
of the stock solution to 500 mL with distilled water.
Procedure
1. Removal of chloride interference. (This step may be eliminated if synthetic
chloride-free nitrate unknowns are prepared in distilled water.) Small amounts of
chloride cause negative interferences. If the chloride content is above 10 mg/L,
the chloride should be removed. (Your instructor will provide an estimate of the
chloride concentration.) Treat a 100-mL sample with an equivalent amount of
silver sulfate solution and remove the precipitated silver chloride by centrifugation
or ltration. If necessary, coagulate the precipitate by heating the solution or by
allowing to stand overnight away from strong light (only if sample is free from
nitrifying organismssee below).
2. Determination of nitrate. The sample should not exhibit appreciable color. To
prevent any change in the nitrogen balance through biological activity, natural
waters should be analyzed promptly after sampling. They may, however, be
stored near freezing by adding 0.8 mL H2 SO4 /L as preservative. If the sample is
acidied, it should be neutralized just before the analysis is started.
Neutralize the chloride-free prepared sample from above or a 100-mL fresh
sample if already chloride free to about pH 7 with dilute NaOH. Transfer to a
casserole and evaporate to dryness. Mix the residue with 2.0 mL phenoldisulfonic
acid reagent, using a glass rod to help dissolve the solids; heat on a hot-water bath
if necessary to aid dissolution. Dilute with 20 mL distilled water and then add 6 to
7 mL ammonia until maximum color is developed. If a occulent hydroxide forms,
dissolve it by adding the EDTA reagent dropwise with stirring. (Alternatively, the
1
Nitrate may also be determined uorometrically by reacting with uorescein in conc. H2 SO4 and measuring the
uorescence quenching of the uorescein. See J. Chem. Ed., 51 (1974) 682.
Page 46
E47
sample may be ltered.) Transfer the clear solution to a 50-mL volumetric ask
and dilute to volume with distilled water.
Prepare standards in the same manner, using the same volumes of reagents, by
evaporating 10, 25, and 50 mL of the standard nitrate solution, respectively; these
represent 0.10, 0.25, and 0.50 mg N, respectively. Omit the chloride precipitation
step. Prepare a blank using the same volumes of reagents.
Read the absorbance of the solution at 410 nm, correct for the blank, prepare
a calibration curve, and calculate the concentration of nitrate nitrogen in your
sample in mg/L.
As little as 1 g nitrate nitrogen can be detected, representing 0.01 mg/L in
a 100-mL sample. The nitrate concentration in drinking water usually falls below
10 mg/L. If concentrations are high, measurements can be extended sixfold by
measuring at 480 nm, or twofold by diluting prepared samples to 100 mL instead
of 50 mL.
EXPERIMENT 26 SPECTROPHOTOMETRIC DETERMINATION

OF LEAD ON LEAVES USING SOLVENT EXTRACTION
Principle
Lead on the surfaces of leaves is dissolved by shaking with nitric acid solution. The
lead is extracted as the dithizone complex into methylene chloride at pH above 9. The
intensity of the color of the complex is measured spectrophotometrically and compared
to a calibration curve prepared similarly from lead standards to calculate the amount
of lead. Sulte is been added as a masking agent to eliminate most interference from
other metals1 .
Equation
1
2+
2 Pb
NHNH
C S
N
green
Pb2+/2
NHN
C S
+ H+
red

1. Provided. 1 M HNO3 , 0.1 M HNO3 , thymol blue indicator solution (0.1% in
water), 2 M NH3 solution, ammonia sulte solution (350 mL conc. NH3 solution,
and 1.5 g Na2 SO3 diluted to 1 L; the pH is about 11).
2. To prepare
(a) Stock 1000-ppm standard lead solution. Dissolve 0.160 g Pb(NO3 )2 and
dilute to 100 mL in a volumetric ask.
(b) Standard 10-ppm working solution. On the day of the experiment, dilute
1 mL of the stock solution to 100 mL in a volumetric ask.
(c) Dithizone solution. Dissolve 7.5 mg dithizone in 300 mL methylene chloride. This should be prepared fresh on the day of the experiment.
1 Normally, cyanide is also added to mask other metals and provide maximum selectivity. But for safety reasons,
this is omitted from the masking solution for this experiment. For illustrative purposes, you can assume the
measurements are due soley to lead.
Page 47
E48
EXPERIMENTS

Collect leaf samples. These can be from trees near a road or highway and from some
that are more isolated for comparison. Collect at least two large leaves from each tree
and place each in a clean plastic bag and seal. The leaves selected should be reasonably
free from dirt or other visible contamination. Record the location of the leaves. Your
instructor will advise you of the number of trees you should sample.
Procedure
1. Preparation of calibration curve. This should be done at the time the samples are
to be analyzed. The chelate formation and solvent extraction are to be performed
in clean 6-oz vials with caps. To each of six labeled vials add with pipets 0
(blank), 2, 4, 6, or 8 mL of the 10-ppm lead standard and sufcient water to bring
the volume to about 20 mL. Add about 60 mL of the ammoniasulte solution
using a graduated cylinder and 25 mL of the CH2 Cl2 dithizone solution using
a pipet (not by mouth). Stopper the vial and shake for about a minute. Using a
pipet, withdraw most of the heavier methylene chloride layer and lter through
dry lter paper (Whatman No. 40) into a dry Bausch and Lomb Spectronic 20
measuring tube or the equivalent, or centrifuge before transferring. (The samples
should be prepared for measurement now so they can be measured when the
standards are.)
Using one of the standards, measure the absorbance from 400 to 600 nm in
20-nm increments to determine the wavelength of maximum absorption. Using
this wavelength, measure the absorbance of each standard, using the blank to zero
the instrument. Plot absorbance against micrograms of lead taken to prepare a
calibration curve, using a spreadsheet (Chapters 3 and 16).
2. Determination of lead on leaves. For each plastic bag containing a leaf sample,
heat 20 mL of 0.1 M HNO3 to about 70 C. Add 20 mL to each bag, close, and
shake for about 2 min. Pour into clean 100-ml- beakers. Add one drop thymol
blue indicator solution to each, followed by dropwise addition of 2 M NH3 until
the indicator color change is complete (to blue) and add a couple of extra drops.
The solution should smell of ammonia. Then, add 60 mL of the ammoniasulte
solutionand then add with a pipet 25 mL of the CH2 Cl2 dithizone solution and
proceed with the extraction measurement as with the standards.
Calculation
Blot each leaf dry, place on a sheet of paper, and trace the outline of the leaf. Cut
out the leaf outline and weigh on an analytical balance to three gures. Cut out a
10-cm 10-cm (100 cm2 ) square from the same paper and weigh. Calculate the area
of the leaf in cm2 .
From the measured absorbance of each sample and the calibration curve, calculate
the micrograms of lead on the leaf and report the amount of lead in g Pb/100 cm2
leaf. Is there any correlation of lead content with proximity of the tree to a roadway?
EXPERIMENT 27 SPECTROPHOTOMETRIC DETERMINATION OF
INORGANIC PHOSPHORUS IN SERUM1
Principle
The inorganic phosphorus in a protein-free ltrate is reacted with ammonium molybdate [Mo(VI)] to form ammonium phosphomolybdate. This is reduced with a mild
1
A synthetic serum sample may be prepared as described in Experiment 34, footnote 1, and adding 60 g of
albumin as a source of protein (e.g., bovine serum albumin, BSA.) Serum contains about 6% (w/w) protein. The
solution will contain about 0.41 mg P/dL, which can be varied from unknown to unknown.
Page 48
reducing agent to produce molybdenum blue, a heteropoly molybdenum(V) species.

Molybdates are not reduced under these conditions. The blue color of the solution is
measured spectrophotometrically.
Equations
7H3 PO4 3 +12(NH4 )6 Mo7 O24 +51H+ 7(NH4 )3 PO4 12MoO3 +51NH4 + +36H2 O
(NH4 )3 PO4 12MoO4 + mild reducing agent Mo(V) species (blue)
[Although normal ammonium molybdate (NH4 )2 MoO4 can be crystallized, the common crystalline form is (NH4 )6 Mo7 O24 4H2 O or 3(NH4 )2 O 7MoO3 4H2 O.]
1. Provided. 5% (wt/vol) Trichloroacetic acid solution; 5 M H2 SO4 ; aminonaphtholsulfonic acid reducing solution prepared as follows: Add 0.50 g 1,2,4aminonaphtholsulfonic acid and 5.0 mL sodium sulte solution (20 g anhydrous
Na2 SO3 /100 mL) to 195 mL sodium bisulte solution (15 g NaHSO3 /100 mL) in
a brown glass-stoppered bottle. Stopper and shake until the powder is dissolved.
If solution is not complete, add 1-mL increments of sodium sulte solution with
continued shaking until solution is complete. Avoid excess sodium sulte. Store
in refrigerator. The solution is stable for about 1 month.
2. To prepare
(a) Stock phosphorus standard solution (100 mg/dL P). Dissolve 0.439 g
KH2 PO4 in water and dilute to 100 mL in a volumetric ask.
(b) Working phosphorus standards. Add with a pipet 1 mL of the stock solution to a 100-mL volumetric ask and dilute to volume with 5% trichloroacetic
acid (TCA). (CAUTION: TCA is very corrosive. Avoid contact with the skin.
It should never be pipetted by mouth.) This contains 1 mg/dL phosphorus and
will be used to prepare the serial standards. Transfer with a pipet 2 and 5 mL
of this solution into 10-mL volumetric asks and dilute to volume with 10%
TCA. You now have standards of 0.2, 0.5, and 1 mg/dL P. These correspond
to serum concentrations of 2, 5, and 10 mg/dL, respectively, in the procedure
below, since the sample is diluted 1:10.
(c) Ammonium molybdate solution. Dissolve 0.62 g ammonium molybdate,
(NH4 )6 Mo7 O24 4H2 O, in 2.0 mL water and add 8 mL of 5 M H2 SO4 . The
solution should be stable indenitely. Discard if blanks show a blue color.
Procedure
1. Serum. Perform the analysis in duplicate. Place 9.50 mL of 5% TCA in a 12mL centrifuge tube. Add 0.500 mL serum, mix well, and let stand for 5 min.
Centrifuge at 1500 rpm until the supernatant is clear (ca. 5 min). If a centrifuge
is not available, the sample should be ltered through dry Whatman No. 42 lter
paper into a dry beaker. Since the lter paper may contain reducing impurities,
the blank should be prepared using ltered 5% TCA.
Transfer 5.00 mL of the clear supernatant to a 15 150 mm test tube.
Prepare a blank and standards by pipetting 5.00 mL of 5% TCA and of the 0.2,
0.5, and 1.0 mg/dL P standard solutions into four separate test tubes. To all the test
tubes, add 1.00 mL of the molybdate reagent and mix well. Finally, add 0.40 mL
of the aminonaphtholsulfonic acid reagent and mix well. Allow to stand for 5
to 10 min or longer and measure the absorbance for each solution in a cuvet
at 690 nm, setting the zero absorbance with distilled water. Subtract any blank
reading from all standard and sample readings.
E49
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EXPERIMENTS
Plot the net absorbance of the standards against concentration using a

spreadsheet (Chapters 3 and 16). From this plot and the net absorbance of the
sample, determine the concentration of phosphorus in the protein-free ltrate.
Multiply by 20 to obtain the concentration in the original serum sample. The
normal range of phosphorus in serum is about 3.0 to 4.5 mg/dL for adults and 4.5
to 6.5 mg/dL for children.
EXPERIMENT 28 SPECTROPHOTOMETRIC DETERMINATION

OF MANGANESE AND CHROMIUM IN MIXTURE
Principle
Manganese and chromium concentrations may be determined simultaneously by
measurement of the absorbance of light at two wavelengths, after the metals have been
oxidized to Cr2 O7 2 and MnO4 . Beers law has been shown to apply closely if the
solutions are at least 0.5 M in H2 SO4 . Cr2 O7 2 has an absorption maximum at 440 nm
and MnO4 has one at 545 nm. (A somewhat more intense maximum is at 525 nm,
but there is less interference from Cr2 O7 2 at 545 nm.) Equations similar to Equations
16.16 and 16.17 are solved for the unknown concentrations from the measured
absorbances at the two wavelengths. The four constants (b = k) are determined by
measurements of absorbance at the two wavelengths using pure solutions of known
concentration; a calibration curve is prepared at each wavelength for both Cr2 O7 2
and MnO4 and the slopes of the curves (A versus C) are used to obtain an average k
value.
Equations
The unknown contains Cr3+ and Mn2+ . The former is oxidized to Cr2 O7 2 by heating
with peroxydisulfate (persulfate) in the presence of a silver catalyst:
Ag+
2Cr3+ + 3S2 O8 2 + 7H2 O Cr2 O7 2 + 6SO4 2 + 14H+

Mn
2+
is oxidized in part by peroxydisulfate, but also by periodate:

Ag+
2Mn2+ + 5S2 O8 2 + 8H2 O 2MnO4 + 10SO4 2 + 16H+

2Mn
For the mixture,
2+
+ 5IO4 + 3H2 O 2MnO4 + 5IO3 + 6H+
A440 = kCr,440 CCr + kMn,440 CMn

A545 = kCr,545 CCr + kMn,545 CMn
The k values are determined from the slopes of the calibration curves of the pure
solutions:
kCr,545 = A545 /CCr
kCr,440 = A440 /CCr
kMn,440 = A440 /CMn kMn,545 = A545 /CMn
1. Provided. 18 M H2 SO4 , K2 S2 O8 , KIO4 , AgNO3 .
2. To prepare
(a) Standard 0.002 M MnSO4 solution. Dry about 1 g MnSO4 at 110 C
for 1 h, cool for 30 min, and weigh out about 0.08 g (to the nearest tenth
milligram). Transfer to a 250-mL volumetric ask, dissolve, and dilute to
volume. Calculate the molarity of the solution and the concentration of Mn in
mg/L (ppm).
Page 50
(b) Standard 0.0178 M K2 Cr2 O7 solution. Use the solution prepared in

Experiment 16 or prepare 100 mL as directed there (weigh to the nearest
milligram). Calculate the molarity of the solution and the concentration of Cr
in mg/L (remember there are two Cr atoms per molecule of K2 Cr2 O7 ).
(c) 0.1 M AgNO3 . Dissolve about 0.2 g AgNO3 in about 12 mL water.
Prepare the standard MnSO4 and K2 Cr2 O7 solutions. This will require drying MnSO4
and K2 Cr2 O7 .
Procedure
1. Calibration (determination of k values). Note: The absorbance of the calibration
solutions and the unknown should be read at the same time. Therefore, get all
solutions prepared before making any readings. They are all sufciently stable
that they could be allowed to set until another laboratory period but it is best
not to.
(a) Manganese. Add with pipets aliquots of 10, 15, and 25 mL of the standard
MnSO4 solution into three different 250-mL Erlenmeyer asks. Add distilled
water to bring the volume in each ask to about 50 mL. To each ask add 10 mL
conc. H2 SO4 (CAREFULLY, using a graduated cylinder) and 0.5 g solid KIO4
(potassium periodate or metaperiodate, depending on the manufacturer). Heat
each to boiling for about 10 min, cool, transfer quantitatively to 250-mL
volumetric asks, and dilute to the mark with distilled water. Determine the
absorbance of each solution at 440 and 545 nm, using 0.5 M H2 SO4 as a
blank solution. Permanganate solutions containing periodate are stable. The
absorbance at 440 nm will be less than 0.1 and, hence, the spectrophotometric
error (precision) will be large (see Figure 16.29). But this is acceptable, in fact,
desirable, because the correction for manganese absorption at this wavelength
is small; that is, a relatively large error in determining a small correction
results in only a small error.
(b) Chromium. Add 10-, 15-, and 25-mL aliquots of the standard K2 Cr2 O7
solution to 250-mL volumetric asks, add about 100 mL distilled water and
10 mL conc. H2 SO4 , mix thoroughly, and dilute to 250 mL with distilled
water. Determine the absorbance of each solution at 440 and 545 nm, using
0.5 M H2 SO4 as the blank solution. The absorbance in this case will be small
(<0.1) at 545 nm.
(c) Determination of k values. Using a spreadsheet, plot absorbance versus
concentration in units of mg/L for each solution at each wavelength, and plot
the least-squares straight line through each set of data points. The lines should
intercept at zero absorbance and zero concentration; under Chart Options, you
can instruct Excel to plot the intercept at zero. The slopes of these lines are
the coefcients (k = A/C) to be used in determining the concentrations of
chromium and manganese in the unknown. These slopes relate absorbance
and concentration for the instrumental parameters used. Therefore, one should
use the same instrument, cuvets, cuvet position, and volumes of solutions for
all determinations in this experiment.
2. Analysis of unknown. Obtain a mixture of Mn2+ and Cr3+ or Cr2 O7 2 unknown
in a 250-mL volumetric ask and dilute to volume. Transfer with a pipet three
50-mL aliquots into 250-mL Erlenmeyer asks. The procedure may be stopped
up to this point. But once the peroxydisulfate is added, the oxidation should be
completed.
E51
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E52
EXPERIMENTS
To each ask add 5 mL conc. H2 SO4 (BE CAREFUL!), then mix well. Add
about 1 or 2 mL of 0.1 M AgNO3 solution and 1.0 g solid potassium peroxydisulfate
(K2 S2 O8 ). BE CAREFUL! PEROXYDISULFATE IS A STRONG OXIDIZING
AGENT THAT CAN REACT VIOLENTLY WITH REDUCING AGENTS!
USE ONLY AS DIRECTED. Do not spill. Dissolve peroxydisulfate and heat the
solution to boiling and boil gently for about 5 min. Cool the solution and then add
0.5 g KIO4 . Again heat to boiling for 5 min.
Cool each solution to room temperature, quantitatively transfer to 250-mL
volumetric asks, and dilute to volume. The solutions at this point (or before
dilution) are stable and can be saved until another laboratory period if necessary.
Also save the serial standards to calibrate the instrument at the same time.
From the unknown absorbances at the two wavelengths, calculate the parts
per million of Cr and Mn in your unknown using Beers law for the mixture and
the determined constants. Use a spreadsheet similar to the one given in Chapter 16
and in your text website to perform the mixture calculation. The calculated results
will have the same units as used in determining the constants. Keep in mind the
dilutions made. Report the results for each portion analyzed.
EXPERIMENT 29 SPECTROPHOTOMETRIC DETERMINATION OF

MANGANESE IN STEEL USING A 96 WELL PLATE READER
Objective: An accurate analysis of Mn in steel is be performed in this laboratory
exercise. Manganese is an important steel alloying element that regulates steel hardness
and other properties. Since steel is commonly used in large structures that require high
strength materials, accurate determination of Mn and other alloying elements is very
important to ensure that future bridges, skyscrapers, etc. do not collapse.
Background: Small amounts of manganese can be determined spectrophotometrically by the oxidation of Mn(II) to the intensely colored permanganate ion (MnO4 ),
using potassium periodate (KIO4 ) as the oxidizing agent. The oxidation reaction is:
2Mn2+ + 5IO4 + 3H2 O 2MnO4 + 5IO3 + 6H+
The Mn content is determined in a spectrophotometer from the absorbance of the
permanganate solution at 525 nm. We will use a 96-wellplate reader to compare
solutions. Permanganate solutions that contain an excess of periodate are quite stable.
The method is quite specic for manganese, with few interferences from colored ions.
In order to correct for colored species, a blank sample solution is prepared without the
oxidation step. Nitric acid is used to dissolve the steel sample. Some steels contain
carbon, and peroxydisulfate is used to oxidize this. Iron(III) is eliminated as a source of
interference (it absorbs appreciably where the permanganate absorption is measured)
by complexation with phosphoric acid, forming ferric phosphate complexes which do
not absorb light at 525 nm. The standard-addition method is used for calibration to
calculate the amount of manganese in the sample from its net absorbance.
Procedure
(The sample preparation procedure was adapted from Skoog et al., Fundamentals of
Analytical Chemistry 5th ed.) The dissolution, manganese oxidation and iron masking
procedure is based on the classic works of H. H. Willard and L. H. Greenhouse,
J. Amer. Chem. Soc., 39 (1917) 2366 and J. Soc. Chem. Ind., 37 (1918) 41A.
Page 52
E53
1. Preparation of Mn standard solution: Accurately weigh 50 mg (to 0.1 mg)

of Mn metal into a 50-ml beaker. Slowly add 10 mL of 6 M nitric acid. Boil
gently to eliminate oxides of nitrogen, KEEPING THE BEAKER IN A FUME
HOOD OR NEXT TO THE AIR INTAKE VENT. Cool; then transfer the solution
quantitatively to a 500-mL volumetric ask. Dilute to the mark with DI water
and mix thoroughly. Calculate and record the concentration of this Mn standard
in mg/mL.
2. Preparation of unknown steel solutions and standard addition solutions
1. Sample digestion: Obtain a steel sample and record the sample number.
The sample does not require drying. Accurately weigh 2.0 g of the steel
sample. Place in a 250 mL ask. Two additional replicate samples should also
be weighed at this time. Add 50 mL DI water and then slowly, in a hood
or with other safe ventilation, add 50 ml concentrated (16 M) HNO3 to each
ask containing the steel sample. After any violent bubbling subsides, boil
gently in a hood or with other safe ventilation for about 5 min. to extract Mn
completely from any slight steel residue that may remain. Cool the solution
below boiling and cautiously add 2 g ammonium persulfate to oxidize carbon
in the solution. Boil the ask contents gently for 10 minutes to destroy the
excess persulfate. If the solution is pink or has a black deposit of MnO2 , add
0.1 g of NaHSO3 and heat for 5 min. to reduce any Mn that is not in the
Mn2+ state. Do not attempt to dissolve all 3 replicate samples simultaneously.
2. Sample Aliquots: Transfer each sample solution quantitatively to a 500-mL
volumetric ask. Dilute to the mark with DI water and mix well. Use a
25.00-mL glass pipet to transfer four aliquots of one of the sample solutions to
4 beakers. These will be used to generate the blank, sample 1 and two standard
addition solutions as described in Table 1 below. Transfer a 25.00 mL aliquot
of each of the other two samples to two separate beakers. These will be used
to generate the replicate sample analysis solutions.
3. Standard Addition: Select two of the four 25.00 mL aliquots of the rst
sample. Add 5.00 mL of the Mn standard to one, and 10.00 mL of the standard
to the other. Be sure to keep track of which beakers contain which volume of
added standard.
4. Oxidation: Add 5 mL of 85% H3 PO4 to each of the six beakers containing
the aliquots and standards. Set aside one of the four 25.00 mL aliquots of
the rst sample without standard added. Add 0.4 g of KIO4 to each of the
other 5 beakers. Boil each solution gently for 5 min., cool, and transfer
quantitatively to 100-mL volumetric asks. Fill to the mark with DI water
and mix well. Record all observations in your lab notebook. Calculate and
Table 1
Treatment of aliquots from Sample 1
Aliquot
1
2
3
4
This
Volume of 85%
H3 PO4 (ml)
Volume of standard
Mn added (ml)
Weight of KIO4 , g
5
5
5
5
0.00
0.00
5.00
10.00
0.0
0.4
0.4
0.4
is the blank solution. No oxidant is added so no permanganate will be formed.
Page 53
E54
EXPERIMENTS
record the concentration of added Mn in each of the two standard addition

solutions.
3. Spectrophotometric measurements
A. Get a 96-well plate and pipette 200 L into wells as follows:
Column 1 (A-H) Sample 1 Aliquot 1 (Blank, without KIO4 )
Column 2 (A-H) Sample 1 Aliquot 2
Column 3 (A-H) Sample 1 Aliquot 3 (5.00 mL Standard Addition)
Column 4 (A-H) Sample 1 Aliquot 4 (10.00 mL Standard Addition)
Column 5 (A-H) Sample 2
Column 6 (A-H) Sample 3
B. Go to the plate reader and open the method provided by the instructor.
Following specic instructions for the instrument and software, enter the
concentration of Mn added to the two standard addition solutions. Read the
absorbances for each well in the plate.
C. The plate reader will report the blank-corrected absorbance readings (using
column 1 for the blank) for each cell in columns 26. The system may
also generate a calibration plot using the absorbance results for aliquots 24
of Sample 1 and the standard addition concentrations entered in step B.
Also reported in the tables are the concentrations of Mn in Sample 1 (from
the x intercept of the calibration plot) and the concentrations of samples 2
and 3 (from the absorbance readings and the slope of the calibration plot).
Copy the calibration plot and paste it in a word processing document. Copy
the data in the tables and paste them in a spreadsheet. Save both les to
a USB drive.
Lab Report
Include a table of the corrected standard addition and sample absorbances. Make up
a standard-addition plot of the blank-corrected data in a spreadsheet program. Use
a least squares linear regression to determine the equation of the best line through
the measured data points. With the slope from this equation and the actual sample
absorbances (blank corrected), determine the concentration of Mn in the steel solutions.
Compare the calibration plot and Mn results with those generated by the plate reader
software.
Calculate the wt% Mn in the unknown steel samples. Report the wt% Mn of each
replicate, the overall average, SD, RSD and 95% condence interval in a table. Discuss
any possible sources of uncertainty and error in the analysis. Include a discussion of
the uncertainty in the absorbance readings, and to what extent this adds to the overall
uncertainty in the experiment (i.e., compare the standard deviation in the absorbance
readings for the individual samples to the standard deviation in the results for the three
replicates).
EXPERIMENT 30 ULTRAVIOLET SPECTROPHOTOMETRIC
DETERMINATION OF ASPIRIN, PHENACETIN, AND CAFFEINE
IN APC TABLETS USING SOLVENT EXTRACTION
Principle
APC tablets are a mixture of aspirin, phenacetin, and caffeine. Each of these substances
has characteristic absorption in the ultraviolet region, with the principal maxima lying
at 277 nm for aspirin, 275 nm for caffeine, and 250 nm for phenacetin. In the procedure,
a powdered tablet is dissolved in methylene chloride, and the aspirin is separated from
the phenacetin and caffeine by extracting it into aqueous sodium bicarbonate solution.
Page 54
E55
The separated aspirin is back-extracted into methylene chloride by acidifying the

aqueous layer and is then measured spectrophotometrically at 277 nm. The phenacetin
and caffeine that remain in the original methylene chloride layer are determined in
mixture as described in Chapter 16 (Equations 16.16 and 16.17).
Equations
NHCOCH3
CO2H
OCOCH3
N
CH3 O
OC2H5
aspirin (A)
(acetylsalicylic
acid)
phenacetin (P)
(acetophenetidin)
A
P
C
O3
HC
CH2Cl2
H+
O
N
CH3
caffeine (C)
(Theine; 1,3,7trimethylxanthine)
A (277 nm)
CH
2C
l2
P (250 nm)
+
C (275 nm)

1. Provided. CH2 Cl2 , 4% (wt/vol) NaHCO3 solution (chilled), conc. HCl, 1 M
H2 SO4 .
2. To prepare1
Standard solutions. Prepare individual standard solutions of about 100 mg/L,
20 mg/L, and 10 mg/L each for aspirin, phenacetin, and caffeine in methylene
chloride as follows. Weigh about 25 mg (to the nearest 0.1 mg) of each, transfer to
100-mL volumetric asks, dissolve, and dilute to volume with methylene chloride.
Dilute 2 and 1 mL of this solution to 25 mL in 25-mL volumetric asks to prepare
the 20 and 10 mg/L solutions, respectively.
Procedure2
Weigh accurately and record the weight of one tablet. This should be equivalent to
about 220 mg aspirin, 160 mg phenacetin, and 30 mg caffeine. To minimize required
dilutions and save on solvents, cut the tablet into quarters and weigh out a one-quarter
portion to be analyzed. Crush to a ne powder in a beaker. Add, with stirring, 20 mL
methylene chloride; then transfer the mixture quantitatively to a 60-mL separatory
funnel, rinsing all particles in with a little more methylene chloride. Extract the aspirin
from the methylene chloride solution with two 10-mL portions of chilled 4% sodium
bicarbonate to which has been added two drops hydrochloric acid, and then with
one 5-mL portion of water. Wash the combined aqueous extracts with three 10-mL
portions of methylene chloride and add the methylene chloride wash solutions to
the original methylene chloride solution. Leave the aqueous extract in the separatory
funnel. Filter the methylene chloride solution through paper previously wetted with
1 Caffeine and phenacetin are available from Sigma-Aldrich.
2
Aspirin tends to decompose in solution, and analyses should be performed as soon as possible after preparing
solutions.
Page 55
E56
EXPERIMENTS
methylene chloride (to remove traces of water) into a 50-mL volumetric ask and
dilute to the mark with methylene chloride. Then dilute further a 1-mL aliquot of this
solution to 50 mL with methylene chloride in a volumetric ask.
Acidify the bicarbonate solution (aqueous extract), still in the separatory funnel,
with 6 mL of 1 M sulfuric acid. This step should be performed without delay, to avoid
hydrolysis of the aspirin. The acid must be added slowly in small portions. Mix well
only after most of the carbon dioxide evolution has ceased. The pH at this point should
be 1 to 2 (pH test paper). Extract the acidied solution with eight separate 10-mL
portions of methylene chloride and lter through a methylene chloride-wet paper into
a 100-mL volumetric ask. Dilute to volume. Then, dilute further a 5-mL portion of
this solution to 25 mL with methylene chloride in a volumetric ask.
Record absorbance versus wavelength curves for the standard solutions and
unknown solutions between 200 to 300 nm. (This step may be deleted if you do
not have a recording ultraviolet spectrophotometer.) Does the wavelength of 277 nm
appear to be the most suitable wavelength for the determination of aspirin? Do the
wavelengths of 250 and 275 nm appear to be the best wavelengths for the measurement
of the absorbance for the mixture of phenacetin and caffeine? Explain.
Using the absorbances of the standard and the unknown aspirin solution at
277 nm, calculate the percent aspirin in the APC tablets and the number of milligrams
of aspirin per tablet keeping in mind the dilutions.
To calculate the concentrations of phenacetin and caffeine, the absorbances of
the phenacetin and caffeine standards and of the methylene chloride extract of the
sample must all be read at both 250 and 275 nm. Using these absorbances, calculate
the percent phenacetin and caffeine in the APC tablets and the milligrams of each per
tablet. See Chapter 16 for the spectrophotometric determination of mixtures. Use a
spreadsheet similar to the one in Chapter 16 and your text website to do the mixture
calculations.
EXPERIMENT 31 INFRARED DETERMINATION OF A MIXTURE
OF XYLENE ISOMERS
Principle
Meta- and para-xylene are determined in mixtures using ortho-xylene as an internal
standard, to compensate for variation in cell length between runs. The infrared
spectrum of the unknown mixture is recorded and the relative height of peaks of the
two compounds are compared with those of standard mixtures, using the baseline
technique.
1. Provided. Ortho-, meta-, and para-xylene.
2. To prepare. Meta-xylene/para-xylene standards. Prepare a series of standards
(use available burets), all containing 30% (vol/vol) o-xylene as internal standard,
by mixing the appropriate volumes of the three isomers to give 25, 35, and
45 vol % of m-xylene. The corresponding concentrations of p-xylene will be 45,
35, and 25%, respectively.
Procedure
Consult your instructor on the proper operation of your instrument. Handle the infrared
cell carefully, avoiding contact with water and the ngers. Fill the cell with pure
m-xylene and obtain a spectrum on this from 2 to 15 m, being sure to record the
last peak just before 15 m (692 cm1 ). Each time you run a sample, be sure to
check 0% T by placing a card in the sample beam and adjust the pen to 0% T.
Page 56
ATOMIC SPECTROMETRY MEASUREMENTS
Empty the cell, rinse and ll with p-xylene, and run a spectrum on this. Repeat for
o-xylene. Run spectra on each of the standard mixtures. From the spectra of the pure
substances, choose a peak of each isomer to measure. Using the baseline method (see
Figure 16.11), measure P0 /P for the peak for each compound. Prepare a calibration
curve of the ratio of log(P0 /P)meta / log(P0 /P)ortho and of log(P0 /P)para / log(P0 /P)ortho
versus concentration for the meta and para isomers, respectively. See Chapter 20 and
your text website for spreadsheet preparation using an internal standard.
Obtain an unknown mixture of meta and para isomers from your instructor.
Prepare a mixture of this with o-xylene by adding 70 parts of the unknown to 30
parts o-xylene. Run the spectrum on this mixture and, using the baseline method
and the same peaks as before, measure P0 /P for the three compounds and calculate
log(P0 /P)/ log(P0 /P)ortho for the two unknown isomers. Compare with the calibration
curve to determine the percent concentrations of the meta and para isomers; use
the spreadsheet for calculations. Remember to divide by 0.7 to convert to initial
concentrations.
EXPERIMENT 32 FLUOROMETRIC DETERMINATION
OF RIBOFLAVIN (VITAMIN B2 )
Principle
Riboavin is strongly uorescent in 5% acetic acid solution. The excitation and
uorescence spectra are obtained to determine the wavelengths of excitation and
emission to use, and an unknown is determined by comparison to standards.
Chemicals and Solutions Required
Riboavin standards. Prepare a 100-ppm riboavin stock solution by accurately
weighing about 50 mg riboavin, transferring to a 500-mL volumetric ask, and
diluting to volume with 5% (vol/vol) acetic acid. This should be stored in a cool,
dark place. On the day of the experiment, dilute an aliquot of this 1:10 to obtain
a 10-ppm working standard solution. Dilute aliquots of this with 5% acetic acid to
prepare standards of 0 (blank), 0.2, 0.4, 0.6, 0.8, and 1.0 ppm riboavin.
Procedure
Record the excitation and emission spectra of the 0.6-ppm solution to determine the
best excitation wavelength and best detection wavelength. If a lter instrument rather
than a recording spectrouorometer is used, take readings with different arrangements
of the lters to give the maximum reading. Using these wavelengths, set the instrument
gain to give a reading of 100% with the 1-ppm solution. Read the uorescence of
the other standards and prepare a calibration curve. Obtain your unknown in a 50-mL
volumetric ask and dilute to volume with 5% acetic acid. Read the uorescence
of this, and, from the calibration curve, calculate the micrograms riboavin in your
unknown. Use a spreadsheet.
Atomic Spectrometry Measurements

EXPERIMENT 33 DETERMINATION OF CALCIUM BY ATOMIC
ABSORPTION SPECTROPHOTOMETRY
Principle
The effects of instrumental parameters and of phosphate and aluminum on calcium
absorption are studied [see, e.g., W. Hoskins et al., J. Chem. Ed., 54 (1977) 128].
Calcium in an unknown synthetic or serum sample is determined by comparing the
absorbance with that of standards.
E57
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E58
EXPERIMENTS

500 ppm Ca, 4% SrCl2 , 2000 ppm NaCl, 100 ppm phosphate, ethanol.
1. Provided. Ethanol, 4%, SrCl2 solution (wt/vol), stock solution of 140 meq/L Na
and 4.1 meq/L K (see footnote below).
2. To prepare
(a) 500 ppm Ca stock solution. Dissolve 1.834 g (accurately weighed)
CaCl2 2H2 O in water and dilute to 1 L in a volumetric ask. Dilute this 1:10
to prepare a 50-ppm stock solution. Use this to prepare the solutions required
below. (Commercial 1000-ppm Ca2+ solutions may be used.)
(b) 2000-ppm Na stock solution. Dissolve 0.51 g NaCl in 100 mL water.
(c) 100 ppm phosphate. Dissolve 0.15 g Na2 HPO4 in 1 L water.
(d) 100-ppm Al stock solution. Dissolve 0.18 g Al2 (SO4 )3 K2 SO4 24H2 O
in 100 mL water. (AlCl3 may be used, but take care when adding water.)
Study of Instrumental Parameters
Follow your instructors directions for the operation of the instrument. If you have a
single-beam instrument, the hollow-cathode lamp should be allowed to warm up for
30 min before the experiment. A few minutes should be adequate with a double-beam
instrument. An airacetylene ame should be used with a premix burner.
1. Burner height. Adjust the fuel and support gas pressures until the ame is near
stoichiometric (just a slight yellow color to the ame). Then, turn up the fuel
pressure to impart a strong yellow glow to the ame (fuel rich). The yellow glow
is due to unburned carbon particles in the rich ame. In a lean ame, an excess
of oxidant is present and the ame appears blue. Prepare and aspirate a 5-ppm
calcium solution and note its absorbance at 422.67 nm. Adjust the wavelength
setting to obtain maximum absorbance. The monochromator is now set exactly
at the calcium line. With the burner height adjusting knob, raise the burner so
that the light beam just passes over the tip of it (base of the ame). Use distilled
water to zero the instrument, and then measure the absorbance of the 5-ppm
calcium solution. Lower the burner in increments (six to eight steps) and record
the absorbance at each height.
Plot the absorbance against heights of observation in the ame and select
the optimum height.
2. Fuel/air ratio. Hold the air pressure constant and adjust the fuel pressure in
increments from a very fuel-rich to a lean ame. Record the absorbance of 5 ppm
Ca at each increment.
Select the optimum fuel pressure and vary the air pressure in a similar
manner. Plot absorbance against gas pressure for both the fuel and the air, noting
the pressure setting of the one held constant. Select the optimum fuel, and air
settings. Is this a rich, stoichiometric, or lean ame?
Interference Studies
1. Effect of phosphate. Prepare a solution containing 5 ppm Ca and 10 ppm phosphate. Record the absorbance of this solution, using the optimum conditions
determined above, and compare to that of 5 ppm Ca. Explain the results.
Prepare a solution containing 5 ppm Ca, 10 ppm phosphate, and 1% SrCl2 .
Prepare also a solution of 5 ppm Ca and 1% SrCl2 . Record the absorbance
of the solutions. Compare the absorption of the rst solution with that of the
phosphate-containing solution above and with that of the 1% SrCl2 -containing
solution. Explain.
2. Effect of sodium. Prepare a solution containing 5 ppm Ca and 1000 ppm Na.
Record the absorbance and compare with that of 5 ppm Ca. Explain any difference.
Page 58
ATOMIC SPECTROMETRY MEASUREMENTS
3. Effect of aluminum. Prepare a solution containing 5 ppm Ca and 10 ppm Al.

Record the absorbance and compare with that of 5 ppm Ca by itself. Suggest a
possible reaction for the results.
Determination of Calcium in an Unknown
(The method of standard additions below may be used instead of the following
procedures.)
1. Synthetic unknown. Obtain an unknown from your instructor and dilute to give
a concentration of 5 to 15 ppm Ca. Prepare a series of calcium standards of 0, 2.5,
5, 7.5, 10, and 15 ppm from the 50-ppm stock solution. If the unknown contains
phosphate, add SrCl2 to standards and the unknown to give a nal concentration of
1%. Record the absorbance (or % absorption and convert to absorbance) of these
and prepare a calibration curve of absorbance versus concentration. Determine
the concentration of the unknown in the usual manner.
2. Serum. Calcium in serum or an articial serum as described in the footnote to
Experiment 34 is determined by diluting 1:20 with 1% SrCl2 solution. The normal
calcium content of serum is about 100 ppm, and so that analyzed solution contains
about 5 ppm Ca. Sodium and potassium equal to that in the sample are added to
the standards.
Add 0.5 mL of the unknown serum or the articial serum to a 10-mL
volumetric ask and dilute to volume with 1% SrCl2 . (If the method of standard
additions is to be used also for comparison, dilute 2.5 mL of unknown to 50 mL
with 1% SrCl2 ). Prepare standards of 0, 3, 4, 5, 6, and 8 ppm Ca, each also
containing 1% SrCl2 , 6.9 meq/L Na, and 0.21 meq/L K.1
Prepare a calibration curve from the absorbance of the standards and from
this determine the concentration of calcium in the unknown. Use a spreadsheet.
Method of Standard Additions
This procedure may be used, instead of the one above, to analyze the unknowns, and
it illustrates the usefulness of the method of standard additions for compensating for
matrix effects. Dilute your unknown as described above, using distilled water to give
a concentration of about 5 ppm Ca (1:20 for serum, e.g., 2.5 mL diluted to 50 mL with
1% SrCl2 ). Transfer with a pipet separate 10.0-mL aliquots of the diluted unknown to
three separate clean and dry test tubes or asks. Add to these 50.0, 100, and 150 L,
respectively, of the 500-ppm standard calcium solution (or 25.0, 50.0, and 75.0 L of a
1000-ppm solution if available). This results in an increase in the calcium concentration
in the diluted unknown of about 2.5, 5.0, and 7.5 ppm, respectively, depending on the
exact concentration of the standard, and brackets the unknown. The volume changes
can be considered negligible. Use an appropriate syringe microliter pipet if available
(e.g., a 50-L Eppendorf pipet or Finn-pipette) or else a 0.1-mL graduated measuring
pipet. For best accuracy, the pipet should be calibrated (see Chapter 2).
Zero the instrument with distilled water and aspirate the diluted unknown and
the standard addition samples. The absorbance increases in the latter are due to the
added calcium. Using a spreadsheet, prepare a plot of absorbance against added
concentration of calcium (starting at zero added, i.e., the sample). From the x-axis
intercept of the plot, determine the concentration of calcium in the diluted unknown.
See the spreadsheet in Chapter 17 for preparation of a standard additions plot and
unknown calculation. Calculate the concentration in the original sample. How does
this method account for phosphate interference?
1 A stock solution of 140 meq/L Na and 4.1 meq/L K (20 times the concentration in the standard solution) can be
prepared by dissolving 8.1 g NaCl and 0.21 KCl in 1 L water. This contains the normal levels of Na and K in
serum and compensates for ionization interference due to these elements in the serum. Dilute this solution 1:20
in the standards.
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EXPERIMENTS
EXPERIMENT 34 FLAME EMISSION SPECTROMETRIC

DETERMINATION OF SODIUM
Principle
The intensity of sodium emission in a ame at 589.0 nm is compared with that of
standards. If an internal standard instrument is available, the ratio of sodium to lithium
emission is measured.
1. Stock standard NaCl solution (1000 ppm Na). Dry about 1 g NaCl at 120 C for
1 h and cool for 30 min. Weigh and dissolve 0.254 g NaCl in water and dilute to
1 L. Care must be taken to avoid sodium contamination, especially from the water
and glassware. A blank must be run to correct for sodium in the water.
2. LiNO3 internal standard solution (1000 ppm Li). (Lithium is not required if a
direct-intensity instrument, rather than an internal standard instrument, is used.)
Dissolve 0.99 g LiNO3 in water and dilute to 100 mL.
3. Working standard solutions1
(a) Direct-intensity instrument. Prepare standards of 0, 10, 20, 30, and 40 ppm
Na by diluting 0, 5, 10, 15, 20, and 25 mL of the stock NaCl solution to 50 mL.
(It may be better with some instruments to prepare solutions ve times more
dilute than this by adding 0, 1, 2, 3, 4, and 5 mL of solution to the asks.
This may result in a more linear calibration curve. Follow your instructors
directions.)
(b) Internal standard instrument. Prepare the same solutions as above for
the direct-reading instrument, but add 5 mL of the stock lithium solution to
each ask. (This results in 100 ppm Li in each solution. The recommended
concentration may vary from one manufacturer to another, and so your
instructor may direct you to add a different amount.)
Dry the NaCl at 120 C for 1 hour and cool in a desiccator.
Procedure
Have your instructor add your unknown to a 100-mL volumetric ask.
1. Direct-reading instrument. Dilute your unknown to volume with water. Follow
your instructors directions for the operation of the instrument. Several atomic
absorption instruments can be used for measuring emission. Set the zero reading
while aspirating distilled water (the blank). Aspirate each standard and the
unknown and record their emission intensity readings. With some instruments,
the 100% reading is set with the most concentrated standard.
1 The unknown may be simply a sodium chloride solution, or it may be serum. If serum is analyzed, then the
standards should be prepared over a narrower concentration range to better bracket the unknown. Sodium in
serum (approximately 140 meq/L, or 3200 ppm Na) may be determined by simple 1:100 dilution (e.g., 0.1 mL
diluted to 10 mL) or 1:500 if required by the instrument. An alternative unknown is an articial serum prepared
by dissolving the following salts in water and diluting to 1 L:
NaCl
KCl
KH2 PO4
CaCl2 2H2 O
MgSO4 7H2 O
8.072 g
0.21 g
0.18 g
0.37 g
0.25 g
This contains 138.1 meq/L Na, which can be varied from unknown to unknown. Bovine serum albumin (60 g)
may be added as a source of protein. Serum contains about 6% (wt/wt) protein.
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SOLID-PHASE EXTRACTION AND CHROMATOGRAPHY
Using a spreadsheet, plot the emission readings for the standards against
concentration and determine the concentration in the unknown solution from
the calibration curve. From this, calculate the micrograms of solution in your
unknown if it is water, and ppm or meq/L if it is serum (see footnote 1).
2. Internal standard instrument. Add the same amount of lithium solution to
your unknown as was added to your standard, and dilute to volume with water.
Follow your instructors directions for the operation of the instrument. The
lithium emission line is 670.8 nm. Prepare a calibration curve as above for the
direct-reading instrument, but record the ratio of the Na/Li emission intensities.
Determine the concentration of sodium in the unknown solution and report
micrograms of sodium in the unknown if it is water, and ppm or meq/L if it
is serum (see footnote 1). Use a spreadsheet as described in Chapter 20 for the
internal standard plot and calculation.
Solid-Phase Extraction and Chromatography

EXPERIMENT 35 SOLID PHASE EXTRACTION
WITH PRECONCENTRATION, ELUTION,
AND SPECTROPHOTOMETRIC ANALYSIS
Contributed by University of Washington: Thomas C. Leach and
Professor Robert E. Synovec
Introduction and Theory
The purpose of this laboratory experience is to introduce the student to the concepts
and methodology involving solid phase extraction (SPE) of an analyte of interest from
an initial solution, preconcentration and elution of that analyte, and then absorbance
spectrophotometric analysis of the preconcentrated analyte solution. The basic idea is
to develop the skill and knowledge in order to take a sample in which an analyte
of interest is initially at too dilute of a concentration to analyze directly, and
then to preconcentrate the analyte, to obtain a signicantly more concentrated
sample that is amenable to analysis. In this experiment, the test analyte is 2-((4(Dimethylamino)phenyl)azo)benzoic acid or Methyl Red (F.W. = 269.31), which is a
weak acid, so it is necessary to work in acidic solution conditions to keep Methyl Red
protonated (in the neutral form), thus it will behave like a typical organic compound.
Also, in the acidic form the Methyl Red solution is red, and is easily observed if at
sufcient concentration.
The structure of the Methyl Red indicator is shown below.
O
OH
CH3
N
N N
CH3
Methyl Red (C15 H15 N3 O2 )

This compound is minimally soluble in pure water. It is supplied to students
sufciently diluted for the purposes of preparing a standard curve and spiking the
sample.
The experiment proceeds is as follows. The initial sample solution containing
Methyl Red is at a sufciently low concentration so it is barely perceptible that
there is Methyl Red present, and absorbance measurements of this initial sample will
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EXPERIMENTS
conrm that this initial sample would benet from SPE-based preconcentration prior
to analysis via absorbance. Additionally, in a separate measurement, the initial sample
will be spiked with Methyl Red to perform quantitative analysis following the Standard
Addition Method (SAM) procedure. Preconcentration of both the initial sample and
spiked sample is performed using separation SPE cartridges (300 mg Hi-Cap C18).
These SPE column cartridges contain C18, a very non-polar stationary phase on a
solid substrate (like a liquid chromatography stationary phase). The basic principle
involved with SPE-based preconcentration is to rst extract the analyte from a large
volume of an initial sample solvent onto the stationary phase, and second, to extract
(i.e., remove) the analyte from the stationary phase using a much smaller volume of
an eluting solvent. Then, the preconcentration factor (P), i.e., the factor by which the
initial sample has had its concentration amplied, is the ratio of the initial sample
volume (Vi ), divided by the eluting solvent volume (Vf ), or P = Vi /Vf . This theory
is summarized as follows:
Goal of preconcentration:
[MR]f [MR]i
(1)
where [MR]f is the concentration of Methyl Red following preconcentration and [MR]i
is the concentration of Methyl Red in an initial sample. In terms of the preconcentration
factor,
[MR]f = P[MR]i
(2)
Substituting in for concentration,
molef /Vf = P (molei /Vi )
(3)
molef = molei
(4)
P = Vi /Vf
(5)
Since ideally,
then the preconcentration factor is,
Therefore, selecting a Vi Vf will ideally provide a large preconcentration factor.
Experimentally, it is very important to reproduce the same P from one sample to
another in order to obtain accurate quantication.
In this experiment, Methyl Red in the acidied neutral form is sufciently nonpolar so it is quantitatively extracted from the initial aqueous sample matrix (polar and
acidied solvent) since the SPE stationary is also non-polar (C18). Then, the Methyl
Red is quantitatively eluted using ethanol (a solvent that is sufciently non-polar
relative to the initial aqueous sample matrix). This eluted sample is then analyzed
(following pH adjustment) using absorbance spectrophotometry. The preconcentration
factor P ideally will be 50 since the initial volume, Vi , is 500 mL and the nal
volume, Vf , of the eluted sample is 10 mL. The spiked sample is analyzed similarly,
and the SAM is used for quantication. Additionally, a traditional calibration plot
of Methyl Red is prepared using standard solutions. The slope of the calibration
plot (for the standards) is compared to the slope for the SAM plot. If the sample
matrix does not interfere with detection of the analyte, agreement between the slopes
should be obtained. A discrepancy between the slopes of the two plots indicates that
matrix interferences may be present and the SAM would be the preferred method
of analysis.
Prelab Preparation
At least one day before requesting a SPE sample, clean and drain and label a 2 L
bottle. Attach an approximately two foot length of exible PVC tubing to the air line
nozzle in one of the hoods and blow air into the bottle with the air valve turned to a
Page 62
fully opened position until all of the water in the bottle has evaporated. Weigh the dry
bottle on a balance with at least a 10 kg capability and record this tare weight.
Clean and dry two vials capable of holding 30 mL to give to the lab assistant
at the time the SPE sample is requested. Label the vials with the name and account
number when requesting the SPE sample.
One lab period after submitting the vial, the SPE sample will be issued in a plastic
test tube. This sample represents two liters of original sample that has been issued in
concentrated form to facilitate transport and handling. (This is a similar to buying frozen
orange juice. It is shipped to the store in small containers to facilitate transportation
and storage then diluted to a gallon to reconstitute it to its original strength.)
Students will also receive a 5.57 106 M pink reference solution of Methyl Red
in one of the vials that was turned in to the lab assistant, and a clear water/ethanol/acetic
acid make-up solution in the other vial.
When getting the unknown sample also obtain two High Capacity SPE columns
from the lab assistant. 5.0 mL syringes should be available in the lab.
Sample Preparation
The density of the sample after dilution will be considered to be exactly 1 kg/L. After
receiving the unknown sample from the lab assistant, transfer it quantitatively to the
two liter bottle. Add deionized water to the bottle to bring the nal weight of the
bottle and sample to a weight of approximately 2,000 g plus the tare weight of the
bottle. [Note: the weight of the water with sample does not need to be exactly 2,000 g,
but the exact weight to the nearest gram must be measured and recorded so that
the concentration of methyl red can be calculated based on a sample volume of 2 L
(2,000 g). In the Calculating and Reporting Results section, the nal calculations will
adjust for the difference in actual weight of the sample from 2,000 g.]
Sample Aliquot Preparation
Rinse a clean 100 mL beaker three times with small portions of the sample.
Fill the beaker with the sample.
Use the sample in the beaker to rinse a clean 500 mL volumetric ask and long
stem funnel three times. Using the long stem funnel ll a 500 mL volumetric asks
almost to the mark with sample from the 2 L bottle. Use a plastic pipet to ll the
500 mL ask to the mark with the sample in the 100 mL beaker.
Pour the 500 mL aliquot of sample in the volumetric ask into a 500 mL
Erlenmeyer ask. You will now rinse the ask to obtain quantitative transfer.
Quantitatively pipet 10 mL of deionized water into a clean 100 mL beaker. While
turning the 500 mL volumetric ask pour the deionized water from the beaker down
the neck of the ask so water coats the entire surface of the ask. Transfer all of
this rinse water into the Erlenmeyer ask. Repeat this process twice more so that the
volumetric ask gets rinsed out quantitatively with a total of 30 mL of deionized water
that is transferred into the Erlenmeyer ask.
Quantitatively pipet 10 mL of the water/ethanol/acetic acid make-up solution
into the Erlenmeyer ask to result in a total volume of 540 mL in the Erlenmeyer ask.
Spiked Aliquot Preparation
Fill the 500 mL volumetric ask with sample as was done for the sample preparation
step above, being sure to rst rinse the ask three times with the sample solution in
the 100 mL beaker before lling it. Transfer the entire volume to a second 500 mL
Erlenmeyer ask. As with the sample aliquot, quantitatively pipet 10 mL of deionized
water into a clean 100 mL beaker. While turning the 500 mL volumetric ask pour the
deionized water from the beaker down the neck of the ask so water coats the entire
surface of the ask. Transfer all of this rinse water into the Erlenmeyer ask. Repeat
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EXPERIMENTS
this process twice more so that the volumetric ask is rinsed out quantitatively with a
total of 30 mL of deionized water that is transferred into the Erlenmeyer ask.
Quantitatively transfer 10 mL of the 5.57 106 M reference solution to the
Erlenmeyer ask to result in a total volume of 540 mL in the Erlenmeyer ask.
Solid Phase Extraction
In the hood place a few milliliters of concentrated acetic acid in a small (30 mL)
beaker or vial. Using an adjustable pipet add 1 mL of concentrated glacial acetic acid
to the Erlenmeyer asks containing the sample and spiked sample.
Set up one of the 500 mL Erlenmeyer asks containing the sample using clamps
and a ring stand so the ask is secure and tips at an angle so, as it empties, the last few
milliliters of liquid can be easily sucked out of a single location.
Plug in vacuum tubing to an aspirator and attach Tygon tubing to the free end.
Place a two inch piece of Tygon tubing on the end of a graduated 1 mL pipet.
Mix 1 mL of acetic acid with 500 mL of deionized water (DIW). (This makes a
0.2% acetic acid solution, which will equal the acetic acid concentration of the sample
when it is extracted.)
Prior to connecting them to the rest of the experimental apparatus, condition
two SPE cartridge columns by pushing 5 mL of ethanol through each of them with a
syringe, then rinse the syringe with the 0.2% acetic acid solution and push 5 mL of
0.2% acetic acid through each one. The syringe will only lock into one end of the SPE
column, which is referred to as the large port (top), with the small port (bottom) of the
SPE column serving as the drain to waste for the conditioning step.
To nish setting up the experimental apparatus, insert the large port (top) of one
of the conditioned SPE columns into the Tygon tubing leading to the vacuum and
the small end into the Tygon tubing leading to the 1 mL graduated pipet. The small
port (bottom) of the SPE column is connected to the larger Tygon tubing leading to
the vacuum. Note that the ow of liquid in the conditioning step is reversed from the
preconcentration step. See the picture below.
Tygon Tubing
(3/16 I.D.)
Tygon Tubing
(1/8 I.D.)
1 mL Serum
Pipet
SPE Cartridge
Vacuum Tubing
Methyl Red Solution in
500 mL Erlenmeyer Flask
Place the 1 mL plastic graduated pipet into the Erlenmeyer ask so the tip of the
pipet goes into the deepest portion of the ask. Stir the solution well with the 1 mL
graduated pipet to mix.
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Turn on the aspirator to draw the sample through the SPE column. After drawing
the sample through the SPE column the aspirated solution goes directly into the
aspirator and down the drain. The Methyl Red is trapped directly on the column and
no aspirator trap is necessary. While the sample is being extracted onto the column
prepare the standards as described in the section: Standard Calibration Plot for
Absorption Coefcient Determination
When only one or two milliliters of sample is left in the Erlenmeyer ask, rinse
the sides of the ask down with 10 mL of DIW quantitatively pipetted into a 100 mL
beaker as was done with the rinsing of the 500 mL volumetric asks. Aspirate this
rinse through the SPE column until no liquid remains in the ask. Repeat this process
two more times. At this point 571 mL have been passed through the SPE column.
[500 mL original sample + 30 mL rinse for the volumetric ask + a 10 mL aliquot of
water/ethanol/acetic acid make-up solution + 1 mL acetic acid + 30 mL rinse for the
Erlenmeyer ask].
After the sample aliquot is collected on one column, remove that column and
insert the second column in between the vacuum tubing and graduated pipet. Place
the 1mL graduated pipet into the Erlenmeyer ask containing the spiked aliquot so
the tip of the pipet goes into the deepest portion of the ask. Stir the solution with the
graduated pipet to mix.
As before with the sample, when only one or two milliliters of sample is left in the
Erlenmeyer ask, rinse the sides of the ask down with 10 mL of DIW quantitatively
pipetted into a 100 mL beaker. Aspirate through the SPE column until no liquid
remains in the ask. Repeat this process two more times. At this point 571 mL have
been passed through the SPE column. [500 mL original sample + 30 mL rinse for the
volumetric ask + a 10 mL aliquot of the 5.57 106 M reference solution + 1 mL
acetic acid + 30 mL rinse for the Erlenmeyer ask].
Removal of sample and spike solution from the SPE column
After removing both columns from the vacuum system, draw exactly 4 mL ethanol into
a 5 mL syringe. Connect the syringe to the column containing the sample and place
the other end of the column into the mouth of a 10 mL volumetric ask. Slowly push
the ethanol through the column into the 10 mL volumetric ask. Repeat this process
with an additional aliquot of exactly 3 mL of ethanol. Some methyl red analyte may
appear to remain on the column. Do not attempt to remove it using any additional
ethanol rinses because this will bias the analysis, and indeed, application of the SAM
provides correction for this effect.
In the hood place a few milliliters of concentrated HCl in a small beaker or vial
(30 mL). Use an adjustable pipet to add exactly 0.5 mL of concentrated HCl to the
10 mL volumetric ask. Using a plastic dropper add deionized water to the ask to
bring the contents to the mark. Stopper the ask and invert ten times to mix the sample.
Transfer the sample to a glass vial and cap it.
Rinse the 10 mL volumetric ask with DIW and repeat the removal process with
the spiked solution, placing it in a separate vial that will then be capped.
Standard Calibration Plot for Absorption Coefficient Determination
The presence of matrix effects may alter the absorption coefcient of the analyte in
a sample. One benet of extracting analyte from the sample and then rediluting it in
pure ethanol and water is to minimize matrix interferences. To measure the extent to
which matrix effects may still be present after the sample (and spiked sample) have
been extracted then rediluted to 10 mL, a plot using the SAM is used and compared
with a standard curve made with dilutions of the reference solution with deionized
water.
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EXPERIMENTS
Prepare ve reference standard dilutions of the 5.57 106 M reference solution

with:
1.
2.
3.
4.
5.
4 mL reference
3 mL reference with 1 mL DIW
4 mL DIW (use as a blank)
Spectrophotometric Analysis
Use your spectrometer to measure the absorbance of the standards, sample and spike
at 520 nm or select a wavelength near that value where the maximum absorbance is
observed. Use the prepared blank in the dilution 5) above to calibrate the spectrometer.
The original volume of the spike added and the nal volume of the extract are the
same (10 mL each), so the increase in concentration due to the addition of Methyl Red
in the spiked extract will equal the concentration of Methyl Red in the reference, that is
5.57 106 M. Plot a two point SAM line for the absorbance vs. molar concentration
for the sample and spiked sample extract solutions. Also, plot the absorbance vs. molar
concentration for the ve reference standards (including the blank) prepared. From
the slope of the ve point standard line and the slope of the SAM line, determine and
compare the molar absorptivity obtained by each method. Were any signicant matrix
effects seen between the extracted samples and the diluted reference solution in this
experiment?
Calculating and Reporting Results
Divide the y intercept of the two point line (SAM plot) by its slope to determine the
molar concentration of Methyl Red in the 10 mL of extract. Multiply this value by
the volume of the extract (10 mL) to determine the total number of moles of Methyl
Red in 500 mL of the sample. Assuming the density of the sample is exactly 1 g/mL,
divide the 500 g weight of sample analyzed by the weight of sample you measured in
the sample bottle in grams to determine the fraction of total sample used for analysis.
Divide the total moles of Methyl Red in 500 mL by this fraction to determine the total
moles of Methyl Red in the sample.
Using the total moles of Methyl Red in the sample calculate and report the
molarity, M, of Methyl Red in exactly 2 liters of sample.
Waste Disposal
All of the waste generated in this experiment can go down the drain after it is
neutralized to a pH above 5. Add some sodium bicarbonate to 600 mL beaker and ll
it half way with water. Add the waste slowly to the beaker and allow it to react. If
excess bicarbonate is left in the bottom of the beaker the waste can go down the drain.
If all of the bicarbonate has been dissolved add more until it falls to the bottom of the
beaker in excess.
Supplemental Information for Instructors for the SPE Experiment
0.3 g Methyl Red (F.W. 269.309) to the nearest 0.1 mg is dissolved in 100 mL ethanol
for a 0.01114 M solution (Solution A).
A 1/10 dilution of Solution A in ethanol is made for a 0.03% or 1.114 103 M
solution (Solution B).
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A 1/200 dilution of Solution B in 0.5% Acetic acid is made for a 5.57 106 M
(Working Solution).
The Working Solution is used for making unknowns and spiking solutions.
5 to 45 grams of the Working Solution is issued to students as an unknown
sample. See Note Below.
Students dilute their unknown to 2000 grams for the SPE analysis. Scoring of
results is less stringent than the error that is introduced due to the deviation of the
density of water from 1 g/mL at normal temperatures.
A 0.5% Ethanol0.5% Acetic acid solution is issued to students as a Make-Up
solution.
Note: On 11/3/2011 the temperature in the laboratory was 24.5 deg C. Water
density at this temperature is 0.997171 g/mL.
Four times a Class A 200 mL 0.1 mL volumetric ask was tarred, lled to the
mark with Methyl Red working solution and weighed with the Methyl Red.
The weights and densities were
199.411 g
199.382 g
199.351 g
199.372 g
Avg. density
0.997055 g/mL
0.996910 g/mL
0.996755 g/mL
0.996860 g/mL
0.996895 g/mL
The density of water is 0.997171 g/mL at 24.5 deg C.

The difference in the average density between the solution and water was:
0.996895 0.997171 = 0.000276
(0.000276/0.997171) 100 = 0.0277%
The possible percent error in the class A 200 mL volumetric ask is:
(0.1 mL/200 mL) 100 = 0.05%
Since the per cent different in density between the Methyl Red working solution
and water is less than the uncertainty of the volumetric glassware, used the density of
the Methyl Red working solution is assumed to be the same as that of water.
EXPERIMENT 36 THIN-LAYER CHROMATOGRAPHY
SEPARATION OF AMINO ACIDS
Principle
The amino acids are separated on a TLC sheet, e.g., silica gel, using a choice of two
developing solvents. The locating reagent is ninhydrin.
Provided
(a) Developing solvent: No. 1, butyl alcoholacetic acidwater (80:20:20
vol/vol); No. 2, propyl alcoholwater (7:3 vol/vol).
(b) Locating reagent: Ninhydrin solution0.3% ninhydrin (1,2,3triketohydrindene, Eastman No. 2495) in butyl alcohol containing 3%
glacial acetic acid.
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EXPERIMENTS
Chromatographic Equipment
Fisher Scientic TLC Kit A or equivalent TLC sheets, sprayer (for application of
reagent), developing apparatus (e.g., Fisher Scientic TLC Kit A: www.sheredu.com
or equivalent).
Procedure
Obtain an unknown mixture from your instructor. The mixtures to be separated
will contain approximately 1 mg of each amino acid per milliliter 0.5 M alcoholic
hydrochloric acid solution (see discussion below). Spot approximately 1 L of the
sample solution on the Chromagram sheet about 2 cm from the lower edge. (Activation
of the sheet is not necessary for this separation.) The unknown should contain acids
whose Rf values are sufciently different with the solvent system used so that they
will be readily separated. Your instructor will advise you which standard amino acid
solutions to run with your unknown so that you can distinguish between two amino
acids that might have an Rf value close to one in your mixture. Allow 15 to 20 min
drying to ensure complete evaporation of the hydrochloric acid.
Develop the Chromagram sheet in the solvent of choice for a distance of 10 cm
or for approximately 90 min. (See the list of Rf values in the table below and follow
your instructors directions for the solvent of choice for your unknown mixture.) Dry
the developed Chromagram sheet and spray with the ninhydrin solution. Heat gently
for several minutes until separated zones appear clearly visible.
Results
The table following lists the approximate Rf values obtained when the two solvent
systems are applied to the separation of 13 different amino acids. From this table and
the standard acids you run, determine what amino acids are present in your unknown.
Discussion
Due to the limited solubility of various amino acids, great care must be taken in the
preparation of the sample before applying chromatography. The use of alcoholic 0.5
M HCl is suggested in the procedure. However, it may become necessary to add
signicant amounts of water to achieve solubility. When this is the case, it becomes
extremely important to keep the spot area small and to allow enough time for complete
evaporation of the spotting solvents before starting development.
When a greater degree of resolution is required, it is possible to improve
results by using the two-dimensional technique of development. The spot containing the components to be separated is placed in the lower left-hand corner of a
20 20-cm sheet, 2 cm from each edge. The sheet is developed in the normal fashion,
removed, and dried. It is then turned counterclockwise 90 and developed again
in a different solvent system to separate components that were not resolved in the
rst migration.
The Rf values of separated amino acids depend on a number of factors, including
the concentrations of the amino acids as well as other components in the sample
mixture. It is for this reason that standards used should be as nearly like the actual
samples as possible.
The visualization with ninhydrin has limits of detection that may vary from 0.01
to 0.5 g, depending on the particular amino acids as well as the method of separation
employed. A degree of ninhydrin color stabilization can be achieved by spraying the
visualized TLC sheet with the following solution: 1 mL saturated aqueous copper
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E69
nitrate solution dissolved by 0.2 mL 10% nitric acid in 100 mL ethanol (95%). The
sheet is then exposed to ammonia vapors and a red copper complex is obtained. The
color is stable only in the absence of acid.
Amino Acid
Approx. Rf Value
Developing Solvent
No. 1
Approx. Rf Value
Developing Solvent
No. 2
0.29
0.15
0.20
0.12
0.33
0.22
0.57
0.10
0.47
0.25
0.55
0.52
0.40
0.50
0.15
0.43
0.22
0.40
0.39
0.69
0.20
0.63
0.45
0.71
0.69
0.60
Alanine
Arginine monohydrochloride
Asparagine
Cystine
Glutamic acid
Glycine
Leucine
Lysine
Methionine
Serine
Tryptophan
Tyrosine
Valine
EXPERIMENT 37 GAS CHROMATOGRAPHIC ANALYSIS

OF A TERTIARY MIXTURE
Principle
A mixture of pentane, hexane, and heptane is separated by gas chromatography.
A number of different types of columns can be used, and a simple thermal conductivity
or hot wire detector is satisfactory. The instrument response for each compound is
calibrated by running a standard mixture of the compounds. The order of separation is
determined by running the individual compounds.
An alternative experiment is to analyze a two-component mixture of n-hexane
and n-heptane, and use n-pentane as an internal standard.
1. Provided. Acetone.
2. To prepare. Standard mixture. Prepare the following standard mixture: n-pentane
(5.00 mL), n-hexane (10.00 mL), n-heptane (15.00 mL). All mixtures should be
fresh the day of use and kept in plastic-stoppered vials. Alternatively, the standards
may be weighed and results reported on a weight/weight basis (or use density to
calculate weights from volumes).
Caution. Exercise great care not to damage the Hamilton syringes. Syringes
require proper cleaning and handling to give consistent results. After each use,
remove the plunger, rinse thoroughly with acetone, and allow to dry. Insert the
syringe through a septum on one end of a glass tube and insert a clean syringe
containing acetone into the other end. Force acetone through the tubing and the
dirty syringe until clean. Dry by drawing air through the barrel of the syringe with
a water aspirator. The glass will appear frosted when completely dry.
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EXPERIMENTS
Procedure
Obtain an unknown mixture from the instructor. Check the instrument instructions
and your instructor regarding the operation of the chromatograph. Do not make any
temperature adjustments. Using the appropriate syringe for the instrument, obtain
chromatograms of each separate component of the mixture, three chromatograms of
your standard mixture, and three chromatograms of your unknown.
Having obtained the necessary data, leave the instrument in the manner prescribed
by the instructor. Especially important is the decrease in gas ow and adjustment of
lament conditions to a stand-by state. Also, clean, rinse, and dry with acetone all
syringes and vials.
Analysis of Data
The peak areas on the chromatographic curves are equal to height times width
at half-height.1 Make measurements with a millimeter ruler. Your instrument may
automatically print peak areas for each compound, in which case, use these.
Individual components are recognized by the positions of their peaks with respect
to the origin (the retention time).
The standard mixture is used for quantitative calibration. Since the detector
does not exhibit equal response to all compounds, then a calibration factor must be
determined for each, using the standard mixture. One simple way of calibration is
just to make a direct comparison of the absolute peak area for each compound with
its percent in the mixture. Thus, if 25% compound A gives a peak area of 40, then a
peak area of 80 for that compound would correspond to 50% A. Obviously, greatest
accuracy would be obtained by preparing a calibration curve of area versus percent
compound over a range of percentages for each compound. This would compensate
for the fact that the net volume of a given total volume of the individual compounds
may vary with the composition.
From the average of the measured chromatographic areas for each compound of
the unknown, calculate the average volume percentage of each compound.
Internal Standard Calibration
Your instructor may give you a two-component unknown of n-hexane and n-heptane,
and direct you to add n-pentane to all solutions as an internal standard. In this case,
prepare the standards as before, but for the unknown, take 25.00 mL and add 5.00 mL
of n-pentane. Calculate the ratios of the analyte to n-pentane areas for the calibration.
See Chapter 20 for a description of the use of internal standards.
EXPERIMENT 38 QUALITATIVE AND QUANTITATIVE
ANALYSIS OF FRUIT JUICES FOR VITAMIN C USING
HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY1*
Principle
Samples of several juices are chromatographed directly on a strong-base anion
exchange column using a UV detector at 254 nm. By comparison of retention times
with that of a vitamin C standard (l-ascorbic acid), the presence or absence of vitamin C
1 In place of determining the areas under the peaks by measurement and using these for the computations, a
somewhat more accurate method is to photocopy the chromatograms rst and then cut out from the original
chromatograms carefully with scissors each peak area. These pieces are then weighed on the analytical balance
and the weights are used in the calculations. If you use this method, hand in the cutouts with your report. The
method of choice is electronic integration.
1*
The oxidized form of ascorbic acid, dehydroascorbic acid, does not absorb in the UV region and is not detected
by this method. For more quantitative results, the samples should be treated with a reducing agent such as H2 S
[see Roe et al., J. Biol. Chem., 174 (1984) 201] to reduce any dehydroascorbic acid.
Page 70
is ascertained. Peak area measurements are used for quantitative determination of the
concentration of vitamin C in those juices in which it is found.
Solutions and Reagents Required
1. Provided. Mobile-phase solution (1.36 g KH2 PO4 /L distilled deionized water),
l-ascorbic acid.
2. To prepare. Vitamin C standard. Weigh 50 mg of l-ascorbic acid to the nearest
0.1 mg, dissolve in a 50-mL volumetric ask, and dilute to volume with distilled
deionized water. Prepare the day of use. This is a 0.1% solution. Prepare serial
dilution of this to obtain also 0.05% and 0.02% standards.
Procedure
1. Calibration. Consult your instructor for the operation of your instrument, including the proper attenuation. It will contain a column of a strong-base anion exchange
resin. At a typical pressure of 1000 psi, the ow rate will be about 0.5 mL/min.
Using a 10- or 25-L syringe, inject a 10-L aliquot of the 0.02% vitamin C
standard and record the chromatogram. Repeat for the 0.05% and 0.1% standards.
Measure the retention times and the areas. The peak areas may be obtained from
the height times the width at half-height for each. Alternatively, the peaks may be
cut out and weighed. Or, the instrument may print out the integrated areas. Using
a spreadsheet, plot a calibration curve of the peak area against concentration.
2. Unknown. Your instructor will provide you three or more unknown juices. These
may be such products as Hi C drinks, grape drinks, orange juice, and the like.
Inject 10-L portions of each and record the chromatograms. Several peaks may
be obtained. If you have time, run at least two chromatograms on each. From a
comparison of retention times, identify the juices that contain vitamin C. Measure
the areas of the vitamin C peaks, and from the calibration curve determine and
report the concentrations in the unknowns. If two or more chromatograms of the
unknowns were run, report the average concentration and the standard deviation
if more than two. The vitamin C peak may be partially overlapped by another. In
this case, extrapolate to the baseline and measure the area from the baseline.
EXPERIMENT 39 ANALYSIS OF ANALGESICS USING

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY
Principle
The compositions of the analgesics Bufferin, Anacin, Empirin, and several brands
of aspirin tablets are qualitatively determined by comparing their chromatograms
with those of standard aspirin, phenacitin, and caffeine samples. The aspirin content
of each is quantitatively determined by comparison of peak areas with those of
standards. Separations are performed on a strong anion exchange column, using a
UV detector.
Solutions and Reagents Required
1. Provided.1 Aspirin (acetylsalicyclic acid), phenacetin (acetophenetidin), caffeine (theine; 1,3,7-trimethylxanthine), methanol, mobile-phase solution (0.01 M
sodium borate +0.005 M ammonium nitrate). See Experiment 30 for the structures
of aspirin, phenacetin, and caffeine.
1 Caffeine and phenacetin are available from Sigma-Aldrich.
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EXPERIMENTS
2. To prepare2
(a) Stock solutions of aspirin, phenacetin, and caffeine. Weigh 50 mg each of
phenacetin and caffeine to the nearest milligram and transfer to labeled 50-mL
volumetric asks. Dissolve in methanol and dilute to volume. Weigh about
150 mg aspirin to the nearest 0.1 mg, transfer to a labeled 50-mL volumetric
ask, dissolve in methanol, and dilute to volume with methanol. Prepare on
the day of use.
(b) Aspirin working standards. Prepare serial dilutions of the stock 300 mg/dL
solution to obtain standards of about 200, 100, and 50 mg/dL in methanol.
Procedure
1. Calibration. Consult your instructor for the operation of your instrument, including the proper attenuation for each sample. It will contain a column of a strong
anion exchange resin. At a typical operating pressure of 1000 psi, the ow rate
will be about 0.5 mL/min.
Using a 5- or 10-L syringe, inject a 5-L aliquot of each 100 mg/dL
standard solution to obtain chromatograms for aspirin, phenacetin, and caffeine. Similarly, obtain chromatograms for the serial aspirin standards (one
chromatogram for each, unless you have time for more).
Measure the retention times. For the aspirin chromatograms, also measure
the areas. The peak areas may be obtained from the height times the width at
half-height for each. Alternatively, the peaks may be cut out and weighed. Or
the instrument may print out the integrated areas. Using a spreadsheet, plot a
calibration curve of peak area against concentration.
2. Unknowns. Your instructor will provide you with ve tablets each of Bufferin,
Anacin, Empirin, Bayer aspirin, and one or two other brands of aspirin. Prepare
solutions of each in methanol by grinding the ve tablets with a mortar and
pestle, weighing to the nearest 0.1 mg about 175 mg, and transferring to a 50-mL
volumetric ask. Dissolve in methanol and dilute to the mark. Mix well and allow
any residue to settle for about 10 min, then obtain chromatograms as above, taking
5-L aliquots. If time permits, run two chromatograms on each. It will take about
10 to 15 min for each chromatogram.
From a comparison of retention times, identify the components of each
analgesic. Measure the areas of the aspirin peaks, and from the calibration curve
determine and report the concentrations in the various tablets. If there is any
overlap of the aspirin peak with another, extrapolate to the baseline and measure
the area from the baseline. If two chromatograms were run, report the average
concentration.
Mass Spectrometry
EXPERIMENT 40 CAPILLARY GAS
CHROMATOGRAPHY-MASS SPECTROMETRY
Adapted from experiment courtesy of Thomas Leach,
University of Washington
Introduction
The most powerful tool in the analytical laboratory for the characterization of
complex mixtures of organic molecules is the capillary gas chromatograph-mass
2 Aspirin tends to decompose in solution, and measurements should be made as soon as possible after preparing
solutions.
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MASS SPECTROMETRY
spectrometer or GC-MS. Its extraordinary capabilities are attained by separating

the components of a mixture using capillary gas chromatography, a high resolution
chromatographic technique, combined with detection by mass spectrometry to obtain
molecular information about the eluted compounds by comparing mass spectra with
MS databases.
A typical column is 30 meters long with an internal diameter of 0.25 mm. It has
a 0.25 micron lm on the inside wall containing the stationary phase made up of a
polysiloxane polymer shown below. The two silicon bonds are linked by oxygen to
form a chain, leaving two silicon bonds (the vertical bonds) free to attach to organic
functional groups. In one common variation encountered, 95% of these bonds are
methyl groups and 5% of them are phenyl groups. The name for the polymer is
5% Phenyl/95% Dimethyl Polysiloxane (manufacturer names for these include DB-5,
HP-5, and Rtx-5).
Si
Si
Si
Some material injected into the column may, over time, contaminate the inner
layer of the stationary phase near the head of the column. In that case, the rst few
inches of the column may sometimes be cut to improve its performance. Therefore,
the column may be shorter than 30 meters in practice.
The column can operate at temperatures between 60 deg C and 325 to 350
deg C.
As each component leaves the chromatography column it enters a rapid scan
mass spectrometer where it is fragmented by electron ionization. The mass-to-charge
(m/z) ratio of each fragment is measured. Differing compounds will break into different
patterns of fragments with different m/z ratios.
An essential element of this instrument is a computer with a graphics display
terminal. In addition to controlling the operating parameters of the gas chromatograph
and the mass spectrometer, the data system allows the progress of the analysis to be
monitored as it takes place, writes all of the data to permanent storage on disk, and
provides a variety of routines for post run analysis of the data.
Experiment
In this experiment, you will separate two or more compounds, along with an internal
standard to correct for experimental variations such as injection volume. You will
inject air or butane to determine the time of a non-retained substance (t0 ). Your
instructor will give directions on how to operate your particular GC-MS system,
including the software for controlling the system and for collecting and processing
data. You will plot a conventional chromatogram in the total ion current mode, i.e.,
record a total ion chromatogram. And you will take the mass spectrum at the peaks
of interest and identify the compounds. The NIST (National Institute of Standards
and Technology) database created in 2008 of ion fragmentation patterns is available
to identify compounds from their fragmentation patterns in the mass spectrometer
by comparing them to fragmentation patterns collected from numerous other mass
spectrometers by the National Institute of Standards and Technology.
You should be able to print the chromatogram and the mass spectra for your
report. You will be given standards marked, e.g., A and B, to prepare a calibration
curve for the two peaks identied as A and B. The program will quantitate the peaks
that appear at the retention times within specied windows, adjust responses to their
relative response to the internal standard, match the compounds selected with the
qualier ions and display a report on the screen.
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EXPERIMENTS
Report
1. Turn in a TIC for the air or butane injection. Report the retention time measured.
This represents the time of a non-retained substance in the column (t0 ).
2. Report the retention time and identity of each compound quantitated.
3. Provide the total ion chromatograms (TIC) for the standards and the sample runs.
4. Provide a calibration curve plot, along with the equation of the best-t line, that
is used to determine the concentration of each unknown.
5. Circle the parent ion peaks and qualitative ion peaks in the fragmentation patterns
that appear with each analyte in the sample.
6. Report the concentrations of the unkowns in the sample.
7. Print out the summary report.
Kinetic Analysis
EXPERIMENT 41 ENZYMATIC DETERMINATION
OF GLUCOSE IN BLOOD1
Principles
A protein-free ltrate of a 0.5-mL sample of whole blood, serum, or plasma is prepared
by precipitating proteins with zinc hydroxide. The glucose in an aliquot of this is
reacted with a mixture of the enzymes glucose oxidase and horseradish peroxidase,
and the hydrogen peroxide produced is coupled with the chromogenic hydrogen donor
o-dianisidine to form a color with absorption maximum at 540 nm.
Equations
glucose oxidase
glucose + O2 + H2 O gluconic acid + H2 O2

horseradish
H2 O2 + o-dianisidine chromogen (absorption max 540 nm)

peroxidase
Solutions and Chemicals Required2

1. Provided
(a) Zinc sulfate solution, 2.2% (from ZnSO4 7H2 O); 3 M H2 SO4 .
(b) Barium hydroxide solution, saturated. Add about 80 g Ba(OH)2 8H2 O
to 1 L boiling water. Remove heat, insert a stopper with a soda lime trap, mix
thoroughly, and set aside for several days to settle. At 25 C, the solution will
be about 0.22 M.
(c) Barium hydroxide solution 0.06 M. Dilute 270 mL of the saturated barium
hydroxide solution to 1 L with CO2 -free (boiled and cooled) water. A volume
of 9.00 0.10 mL of this should be required to neutralize 10.00 mL of
the zinc sulfate solution. Test by adding 10.00 mL zinc sulfate and 25 mL
water to a 100-mL Erlenmeyer ask, adding two drops 0.2% phenolphthalein
See footnote 3, concerning aqueous unknowns and commercial serum preparations.
2 Commercially
packaged reagents are available, which eliminates much of the solution preparation.
Page 74
KINETIC ANALYSIS
solution (in alcohol), and titrating with the barium hydroxide solution to a
faint but permanent pink color. Dilute one or the other solution to proper
equivalence.
2. To prepare
(a) Glycerolbuffer solution, pH 7.0. Dissolve 0.35 g Na2 HPO4 and 0.21 g
KH2 PO4 in 60 mL water and add 40 mL reagent-grade glycerol.
(b) Enzyme reagent. Grind 250 mg glucose oxidase, 5 mg horseradish peroxidase, and 1 mL of the glycerolbuffer solution in a clean dry mortar. Wash
into a 100-mL graduated cylinder using the glycerolbuffer solution, and
bring to a volume of 100 mL with the same solution. Filter into a clean and
dry ask. A Buchner funnel with light suction will facilitate the ltration.
Dissolve 10 mg o-dianisidine in 1.0 mL absolute methanol by intermittent
shaking. Pour into an amber bottle and add the ltered enzyme solution. Pour
the initial portion rapidly to prevent precipitation of the chromogen. This
reagent is stable for 3 weeks when stored in a refrigerator.
(c) Glucose standards. Prepare a 1% stock solution by dissolving 1 g (accurately weighed) of oven-dried (110 C, 1 h) reagent-grade glucose in water and
diluting with 0.25% benzoic acid solution in a volumetric ask. The benzoic
acid acts as a preservative.
Prepare working standards containing 100, 200, 300, and 400 mg glucose
per 100 mL by dilution of the stock solution with 0.25% benzoic acid (e.g.,
5.0, 10.0, 15.0, and 20.0 mL diluted to 50 mL).
Dry the glucose at 110 C for 1 h and cool in a desiccator.
Procedure
Add with a pipet a 0.5-mL blood, serum, or plasma sample3 into a 50-mL Erlenmeyer
ask and add 4.5 mL 0.06 M barium hydroxide solution. Swirl to mix and then slowly
add 5.00 mL 2.2% zinc sulfate solution. Mix thoroughly and allow to stand 5 min.
Filter into a dry ask, or centrifuge in a dry centrifuge tube.
Prepare a blank ltrate and standard ltrates in the same manner by using 0.5 mL
water for the blank and 0.5-mL aliquots of the working standards.
Add with a pipet a 0.2-mL aliquot of each of the ltrates into a clean, dry test
tube, and at 30-s intervals add 1.00 mL enzyme reagent. Immediately place in a 37 C
water bath. (Reactions may be run for 45 minutes at room temperature with some
loss in sensitivity.) Run duplicate aliquots of the unknown ltrate. After exactly 30
minutes, remove the tubes one at a time (30-s intervals) and add 50 mL 3 M H2 SO4 .4
The sulfuric acid effectively stops the enzyme reaction.
After 5 min, but before 1 h, determine the absorbance of the solutions at 540 nm,
using the blank as reference. Using a spreadsheet, construct a calibration curve and
report the mg/dL glucose in your sample.
3 Standard commercial control serum preparations (e.g., Versatol-www.innov-research.com) may be used.
Preparations with abnormal glucose concentrations are available, so the range of normal (ca. 90 mg/dL) to
abnormal (300 mg/dL or greater) levels can be covered.
Alternatively, aqueous glucose unknowns may be used, in which case the protein precipitation step is eliminated
and 9.5 mL water is added in place of the barium hydroxide and zinc sulfate solution.
4 The reaction is pseudo rst order at low concentrations of glucose, but a slower rate is observed at high
concentrations of glucose. Thus, the absorbance may not be a linear function of incubation time at high
concentrations.
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EXPERIMENTS
Flow Injection Analysis

EXPERIMENT 42 CHARACTERIZATION OF PHYSICAL PARAMETERS
OF A FLOW INJECTION ANALYSIS SYSTEM
S(0.002% BTB)
mL/min
Carrier
1.2
Orange-orange
1.2
Orange-orange
Injection
valve
0.01 M borax reagent
620
nm
Waste
0.01 M borax
Pump
Principles
The purpose of this experiment is to learn the operation of your ow injection apparatus
and to estimate (a) the ow rates of the carrier and reagent streams, (b) the volumes
of the sample injection loop and of the ow channels, (c) the dispersion, Dmax , of the
sample plug at the peak maximum (two-line and single line), and (d) the maximum
sampling frequency. The two-line system is used for all experiments, except for a
single-line dispersion experiment.
Equations
D = C0 /C = H 0 /H
Dmax = C0 /Cmax = H 0 /H max
H 0 is the recorded steady-state signal for pure sample, and H max is the recorded peak
maximum for the injected sample.
1. Provided. Stock 0.1 M borax solution (Na2 B4 O7 ), stock 0.4% (wt/vol) bromothymol blue (BTB) solution (0.4 g dissolved in 25 mL 96% ethanol and made
to 100 mL with 0.01 M borax). NOTE: The bromothymol blue should not be
injected in acid solution since the acid form of the indicator adsorbs on the
plastic tubing.
2. To prepare. Dilute 100 mL of 0.1 M borax solution to 1 L to prepare 0.01 M
working solution. Dilute 1 mL of the 0.4% bromothymol blue solution to 200 mL
with the 0.01 M borax solution. This 0.002% BMT solution will serve as
the working solution. The absorbance of this solution in a 1-cm-long cell is
about 1.2.
Assembly of Apparatus
Assemble the peristaltic pump tubes, injector, reactor, and detector as described in
the instrument operation manual or by your instructor. There will be three peristaltic
pump tubes, one for the carrier (0.89 mm i.d., orangeorange color-coded stops), one
for the reagent (0.89 mm i.d., orangeorange), and one for the sample (0.89 mm i.d.,
orangeorange).
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FLOW INJECTION ANALYSIS
Connect the sampling tube to the inlet of the injection loop and the sample pump
tube to the outlet of the loop (the sample will be drawn into the loop by means of
the pump).
Procedure
1. Checking the ow system. Turn on the detector and recorder and allow to warm
up. Fill the carrier bottle and the reagent bottle with the 0.01 M sodium borate
solution. Fill a small beaker approximately half full with the 0.002% bromothymol
blue solution. Place the appropriate tubes in the bottles. Place the injector in the
load position and turn on the peristaltic pump. The channels will ll and solution
will exit to the waste bottle. The blue sample solution will ll the injector sample
loop. Allow to ow until all air bubbles are gone. NOTE: Occasionally an air
bubble may get stuck in the detector, as indicated by a deection on the recorder.
This is most conveniently dislodged by introducing a large air bubble in the carrier
stream by momentarily pulling the carrier tube out of the carrier solution.
Set the detector at 620 nm. The injected sample should provide a peak
maximum of about 0.15 absorbance unit with a 1-cm path ow cell. Adjust your
recording device (strip chart recorder or computeruse a chart speed of 0.5 cm/s
if a recorder) to accommodate this range. With the carrier being pumped, turn
the injection valve to the inject position. Note the blue sample plug as it passes
through the channels. If sample continues to be aspirated (to waste) in the inject
position of your valve, in order to conserve sample, the sampling tube may be
removed from the sample solution, causing air to be aspirated. When the sample
reaches the detector, there will be a deection followed by return to baseline.
If necessary, adjust the recording sensitivity so that the deection will be about
two-thirds full scale. After the sample has passed the detector, turn the valve to
the load position to rell the sample loop. Continue the ow until the sample
liquid reaches the end of the injector waste tube (this assures proper ushing of
the sample loop with the new sample), then inject the sample and record the peak.
Continue several injections in this manner until you are satised the system is
operating properly, the detector is not drifting, and reproducible peaks are being
obtained. You are now ready to perform the experiment.
2. Determination of ow rates. Fill a 10-mL graduated cylinder with distilled
water, record the volume, and insert the carrier tube in the cylinder. Turn on the
pump and simultaneously start a stop watch (or begin timing with a clock). Pump
for 5 min, remove the tube, and turn off the pump. Record the volume of water
remaining in the cylinder. Calculate the ow rate in milliliters per minute.
Perform a similar experiment for the reagent ow and then for the sample
ow (injector in the load position).
Finally, measure the ow rate of the waste stream from the detector, by
collecting the waste for 5 min.
If pump tubing of equal internal diameter is used for all channels, the ow
rates should be similar. Also, the ow rate of the waste stream should total the
combined ow rates of the carrier and reagent streams.
Note that ow rate is directly proportional to the square of the internal radius
of a pump tube, so rates can be appropriately adjusted by changing the pump tubes
(i.e., ow is proportional to cross-section area = R2 ).
3. Estimation of sample loop volume. With the pump turned on, place the valve in
the load position and remove the sampling tube from the sample. This will allow
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EXPERIMENTS
4.
5.
6.
7.
8.
9.
the loop to ll with air. Then, insert the tube in the sample solution and, with a
stopwatch, measure the time from when the sample just enters the loop to when
it leaves the loop. Perform the measurement several times and take the average.
From a knowledge of the sample ow rate determined above and the measured
time to ll the loop, calculate the sample loop volume in microliters. NOTE: This
is an estimate and will not include the dead volume of the holes in the injector
rotor where the loop is connected.
Estimation of the ow system volume. Fill the sample loop with air and inject the
air into the carrier. Measure the time to reach the merging point of the carrier and
reagent streams. From a knowledge of the carrier ow rate determined above and
the measured time, calculate the volume from the injection valve to the merging
point, in microliters. Perform a similar experiment, but measure the time for the air
to go from the merging point to the detector. From a knowledge of the combined
ow of the carrier and reagent streams in the reactor module determined above
and the measured time, calculate the volume from the reactor module inlet to the
detector, in microliters.
Determination of total dead time. With the recording device on, simultaneously
inject a sample and start a stopwatch. Measure the time for the sample to reach
the detector, that is, the time for the recorder to begin deection. This represents
the dead time from injection to initial measurement.
Time to ush sampling tube and sample loop. When injecting different samples,
it will be necessary to ush the previous sample completely from the sampling
tube and the injection loop. Determine the time required to just ush the previous
sample by introducing air into the tube and then reinserting the tube in the sample
solution with the pump running. Measure the time for the sample solution to reach
and ll the sample loop. Use at least three times this time for introduction of each
sample in all future experiments, to allow adequate washing of the loop by the
new sample.
From a knowledge of the sample ow rate, also calculate the volume of
solution required to ush the line.
Determination of dispersion: Two-line system. Adjust the recording sensitivity
so the peak deection for an injected sample is about one-fth full scale. Make
several injections of the sample and determine the average peak height. This
represents H max . Turn the pump off and insert both the carrier tube and the reagent
tube in the sample solution. Turn the pump on and record the signal until a
steady-state signal is obtained. This represents H 0 . NOTE: Alternatively, H 0 may
be determined by inserting the exit waste tube in the sample solution and reversing
the pump ow to ll the cell with sample solution. Calculate the dispersion at the
peak height, Dmax . NOTE: If transmittance is recorded rather than absorbance,
convert the readings to absorbance for calculating Dmax .
Determination of dispersion: Single-line system. Also determine the dispersion
by clamping the reagent tube to convert to a single-line system; remove the tube
from the pump. Determine the dispersion as above and compare with that of the
two-line system.
Determination of maximum sampling frequency. Expand the recording time
axis 10-fold. Inject a sample and record the rise and fall of the peak. Measure
the distance from baseline to baseline on the peak and convert this to seconds.
This will represent the minimum time between injections. Report the maximum
sampling frequency in samples per hour.
At the end of the experiment, wash the system thoroughly by pumping
distilled water for a few minutes. Include ushing the sampling tube and valve
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E79
loops. Then release the pump cassettes and pump tubes. If the system is not to be
used for an extended time, your instructor will instruct you to empty it.
In your report, list all instrument parameters and solutions employed.
EXPERIMENT 43 SINGLE-LINE FIA: SPECTROPHOTOMETRIC

DETERMINATION OF CHLORIDE
mL/min
0.002 M Hg(SCN)2
1.2
Orangeorange
Injection
valve
0.125 M Fe3+
0.125 M HNO3
480
nm
Waste
Pump
Principle
The analytical procedure is based on the following reactions:
Hg(SCN)2 + 2Cl HgCl2 + 2SCN
2SCN + Fe3+ Fe(SCN)2 +
The carrier stream contains Hg(SCN)2 and Fe(III). The chloride of the injected sample
reacts with Hg(SCN)2 , liberating SCN , which in turn forms with Fe(III) the redcolored complex ion Fe(SCN)2 + , which is measured spectrophotometrically at 480 nm.
The height of the recorded absorbance peak is then proportional to the concentration
of chloride in the sample. Besides Fe(SCN)2 + , other (higher) complex ions between
Fe(III) and SCN might be formed, causing nonlinearity in the calibration curve at
high concentrations.
Perform Experiment 42 rst to characterize and learn how to use the system, or
else read through it.
1. Reagent. The carrier stream is prepared by dissolving 0.157 g of mercury(II)
thiocyanate, 7.6 g of iron(III) nitrate, 0.8 mL of concentrated nitric acid, and 40 mL
of methanol in water, making the nal volume up to 250 mL.
2. Standard solutions. Standard solutions in the 5- to 75-ppm Cl range are made by
suitable dilution of the stock solution containing 1000 ppm Cl (0.165 g of sodium
chloride in 100 mL).
Procedure
Assemble the ow injection apparatus in the single-line mode as described by the
manufacturer or your instructor. Use 0.89-mm i.d. pump tubing for the carrier and
the sampling tubes (orangeorange color-coded stops on the peristaltic pump tubing).
This should provide a ow rate of about 1.15 mL/min when using 25 rpm for the
peristaltic pump.
Turn on the detector and allow to warm up for several minutes to stabilize. If
a monochromator is used, set to 480 nm. (Since the color produced is specic for
the analyte, a simple visible light sourcedetector system may be used without a
monochromator.) Use injections of the highest concentration standard into the pumped
reagent carrier to adjust the recording sensitivity to give about 75% deection. Use a
recorder speed of about 0.5 cm/s, if using a strip chart recorder. Each time the injection
loop is lled with a new solution, it should be ushed with at least three loop volumes
of the solution before lling with the aliquot to be injected. That is, run 100 L through
a 25-L loop and then stop.
Page 79
E80
EXPERIMENTS
Reagent carrier solution is pumped through the system and the individual chloride
standards are injected successively in triplicate, yielding a series of peaks for each.
Determine the precision of the procedure by injecting a single standard (in the middle
concentration range) 10 times. Report the percent relative standard deviation.
Obtain an unknown from your instructor and inject at least three times to obtain
an average peak height.
Prepare a calibration curve from the peaks recorded for the standard solutions
and, from this, calculate the concentration of chloride in your unknown solution.
At the end of the experiment wash the system thoroughly by pumping distilled
water for a few minutes. Include ushing the sampling tube and valve loop. Then
release the pump cassettes and pump tubes. If the system is not to be used for an
extended time, your instructor will instruct you to empty it.
In your report, list all instrument parameters and solutions employed.
EXPERIMENT 44 THREE-LINE FIA: SPECTROPHOTOMETRIC
DETERMINATION OF PHOSPHATE
mL/min
Carrier
1.2 Orangeorange
Injection
valve
H2O
0.0025 M (NH4)2Mo7O24
0.6
Orangewhite
0.6
Orangewhite
660
nm
Waste
0.2 M HNO3
0.25% (w/v) ascorbic acid
Pump
Principle
The analytical procedure is based upon the following reactions:
7H3 PO4 + 12(NH4 )6 Mo7 O24 + 51H+ 7(NH4 )3 PO4 12MoO3 + 51NH4 + + 36H2 O
Mo(VI) YELLOW COMPLEX + ascorbic acid Mo(V) BLUE COMPLEX
It is based on the same procedure as Experiment 27. The carrier is distilled water.
Reagent 1 is a 0.0025 M solution of heptamolybdate. Reagent 2 is 5% ascorbic acid
(wt/vol) in a 10% aqueous solution of glycerine. The glycerine aids in preventing the
colored complex from adhering to the walls of the ow-through cell upon injection of
the sample into the carrier stream. The sample merges successively with the reagents
and passes through the reaction coils. The phosphate in the sample combines with
the heptamolybdate, forming a yellow-colored complex. This yellow complex then
reacts with the ascorbic acid, which reduces the molybdenum from the +6 state to the
+5 state, forming a blue-colored complex, which has an extremely high absorptivity
and is measured spectrophotometrically at 660 nm. The height of the recorded peak
is proportional to the concentration of phosphate. The calibration curve linearity and
slope depend upon the extent of reaction, that is, how much of the blue complex
has been formed. This is a function of the kinetic nature of the FIA procedure in
that the reaction may not reach its steady-state value, but rather some fraction of the
steady-state value, since it is a slow reaction. The extent of reaction is dependent upon
the particular reaction system. Addition of antimony(III) catalyzes the reduction by
ascorbic acid.
Perform Experiment 42 rst to characterize and learn how to use the system, or
else read through it.
Page 80

Reagent 1. In a 100-mL volumetric ask, place approximately 50 mL of
distilled water and carefully add 1.3 mL of concentrated nitric acid. To this add 0.309 g
of ammonium heptamolybdate, (NH4 )6 Mo7 O24 4H2 O, and dilute to the mark with
distilled water. This results in a solution that is 0.2 M in nitric acid and 0.0025 M in
heptamolybdate.
Reagent 2. In a 100-mL volumetric ask place 5 g of ascorbic acid, approximately 50 mL of distilled water, and 10 mL of glycerine. Mix thoroughly prior to
diluting to the mark with distilled water. This results in a solution that is 5% (wt/vol)
in ascorbic acid.
Standard solutions. Standard solutions of phosphate (as P) in the 10- to 100ppm P range are made by suitable dilution of a stock solution containing 100 ppm P
(0.440 g of anhydrous KH2 PO4 per liter).
Procedure
Assemble the injector, reactor, and detector as described by the manufacturer or your
instructor. Orangewhite color-coded peristaltic pump tubing (0.64 mm i.d.) should
be used for reagents 1 and 2, while orangeorange (0.89-mm i.d.) pump tubing
should be used for the H2 O carrier and sample. The sample injection size should
be approximately 25 L. This corresponds to a sample loop length of approximately
13 cm when using 0.5-mm inside diameter Micro-line tubing. Each time the injection
loop is lled with a new solution, it should be ushed with at least three loop volumes
of the solution before lling with the aliquot to be injected. That is, run 100 L through
a 25-L loop and then stop.
Turn on the detector and allow it to warm up for several minutes to stabilize. If
a monochromator is used, set to 660 nm. (Since the color produced is specic for the
analyte product, a simple visible white light source detector may be used without a
monochromator.) Use a chart speed of approximately 0.5 cm/s if a strip chart recorder
is used. With the water carrier and the two reagents being pumped, inject the highest
concentration standard solution. Adjust the recording sensitivity to give about 75%
deection with this standard.
Inject the individual phosphate standards successively in triplicate, thereby
yielding a series of peaks for each standard. The precision of the procedure is
determined by injecting the 50-ppm standard 10 times. Report the percent relative
standard deviation.
Obtain an unknown from your instructor and inject it at least three times to
obtain an average peak height. Using a spreadsheet, prepare a calibration curve from
the peaks recorded for the standard solutions. From this calibration curve, calculate
the concentration of phosphate in your unknown solution.
Your report should include all instrument operation parameters, as well as
reagents and solutions used in performing the experiment.
Catalyzed Reaction
The sensitivity of this reaction can be enhanced through the use of a catalyst or by
using stopped-ow techniques. Antimony(III) catalyzes the reaction, and phosphorus
concentrations in the 1- to 10-ppm range can be analyzed with this system without the
need for elevated temperatures for increasing reaction rate (the uncatalyzed reaction is
actually quite slow, requiring up to 10 min for complete reaction to occur). To perform
the catalyzed reaction, a stock antimony(III) solution is prepared by placing 1.10 g
of potassium antimony(III) tartrate, KSbOC4 H4 O6 12 H2 O, in a 100-mL volumetric
ask and diluting to the mark with distilled water. This makes a 0.033 M or 4000-ppm
solution in antimony(III). A 2.5-mL aliquot of this solution is then added during the
preparation of reagent 2, prior to diluting to the mark, yielding a 5% ascorbic acid
E81
Page 81
E82
EXPERIMENTS
solution, which is also 100 ppm in antimony(III). Appropriate dilutions of the 100-ppm
phosphate standard solution are then made to yield concentrations from 1 to 10 ppm.
The experiment is then run in the same manner as before, substituting the new ascorbic
acid solution containing antimony and the new phosphate standards. The recorder
sensitivity is adjusted using the 10-ppm standard.
Shutdown Procedure
At the end of the experiment, ush the system thoroughly by pumping distilled water
through all pump tubes for several minutes. After this has been done, release the
clamps that hold the pump tubes and release the pump tubes from around the pump
rollers. If the system is not to be used for an extended time, your instructor will advise
you as to the proper procedure for emptying it.
Compare with Manual Spectrophotometry
You made triplicate measurements of each standard and the unknown in this experiment. About how long did it take to make these measurements (once the reagents were
prepared)? You will have probably performed some spectrophotometric experiments.
How long do you think the same number of measurements would take using conventional spectrophotometry, considering the cuvet has to be rinsed for each measurement.
How much reagent would be consumed for each measurement compared with the
amount used in this experiment?
Team Experiments
EXPERIMENT 45 METHOD VALIDATION
AND QUALITY CONTROL STUDY
Goal
Your instructor will select one experiment for teams to perform validation studies.
An example is a gas chromatography experiment such as Experiment 37, but for one
analyte. A ow injection analysis (FIA) experiment, such as Experiment 43, would be
a good choice as well, since multiple measurements can be made rapidly. The team will
determine linearity, accuracy, precision, sensitivity, range, limit of detection, limit of
quantitation, and robustness (repeatability) of the method. In addition, a control chart
will be prepared over at least one laboratory period. The instructor will have available
a reference standard to use for accuracy studies. Plan for two laboratory periods for
the completed study. A report of the method will be prepared and documented. Before
beginning the experiment, you should review method validation in Chapter 4.
Equation
Response factor (RF) = (signal y intercept)/concentration
Prepare the solutions needed for the selected experiment that are not provided. You
will need to prepare a variety of concentrations of standards. Some of these may be
prepared as you perform the different parts of the experiment.
Procedure
Work in teams of four. Pairs in the team will work together on the different tasks
to complete the project. Team A will prepare the calibration curve and determine
linearity, accuracy, and repeatability. They will also prepare the control chart. Team B
will determine precision, sensitivity, range, limit of detection, and limit of quantitation.
Page 82
TEAM EXPERIMENTS
(These, assignments may be adjusted if the teams and the instructor agree on the
assignments, planning for timely completion.) The whole team will prepare the report
and sign and date it. The team will be graded together on the thoroughness and care of
the study, and of the report. This is a good exercise in teamwork.
Team A
Prepare the Calibration Curve. It is preferable to measure each standard three times
and plot the average; the standard deviation of each point can be calculated, and the
range of one standard deviation or the range of data for each point can be marked on
the calibration curve. Using Excel, plot the trendline, with the regression line equation
(slope and intercept).
Linearity. Calculate r2 and the y intercept as a percentage of the midrange response.
Calculate the response factor (RF) for each experimental point on the line, and using
Excel, plot the RF vs. concentration. From the slope (RF/unit concentration), calculate
the RF change over the range of experimental points and calculate this as a percentage
of the average RF value.
Accuracy. Obtain the reference standard from your instructor. You will not know its
concentration until after you make measurements and provide the results. Measure it
seven times and calculate the standard deviation. Report the results to your instructor
and obtain the true value and the standard deviation if known.
Repeatability. Make small variations in one or more parameters in the experiment that
should not proportionally or directly impact the measurement. This could be reagent
concentration, the pH, time of reaction, size of measured sample (e.g., increase the
injected volume of a standard in a chromatography measurement by 10%, adjusting
for the increased quantity injected in calculating the predicted response.)
Control Chart. You will probably prepare this on the second day, after the method
has been developed. You will be given an unknown (blind) sample. Make a measurement on it every 20 min throughout one laboratory period, intermittently with other
measurements the team is making; you should make at least eight measurements. Plot
the determined concentration vs. time of day. After you have done this, and shown it
to your instructor, obtain the known value from the instructor, and draw a horizontal
line on the chart at that concentration. From the precision that Team B has determined
for the method (at midrange), draw lines for inner and outer control limits at 2 and 2.5
standard deviations, respectively. Are your values within the control lines? Is there a
trend in one direction?
Team B
Precision. Take a standard at the low end, the midrange, and the high end of the
calibration curve. Measure each seven times and calculate the standard deviations.
Sensitivity. Take two standards that are about two standard deviations apart, in the
midrange of the calibration curve, measuring each three times. Report the mean and
standard deviations. Can you distinguish them? How many signicant gures should
you report?
Range. Select an acceptable precision within the measurement range. This will depend
on what the precision is at midrange. Lets take the acceptable precision to be twice that
precision. From the precision determined at the low and high ends of the calibration
curve, is the calibration curve within the acceptable range? If so, do more experiments
at the low end, taking smaller concentrations and determining the standard deviation
to establish what the lower range is.
E83
Page 83
E84
EXPERIMENTS
Limit of Detection. Make seven blank readings. Calculate the (absolute) standard
deviation. Measure a standard seven times that should give a response 5 to 10 times
the standard deviation above the blank signal. Calculate the detection limit as the
concentration that gives a net response of 3 standard deviations above the blank.
Limit of Quantitation (LOQ). Calculate the LOQ two ways. First, calculate the
concentration that gives a response 10 blank standard deviations above the blank
signal. Also, determine the concentration that gives a relative standard deviation of
15%. (You may have this information in the range study above.)
Report
Prepare a report on validation of the method and the quality control (control chart).
Document with original data, which may include copies of recordings, Excel printouts,
calculations, literature references, and so forth. Make some conclusions about the
validity of the method.
EXPERIMENT 46 PROFICIENCY TESTING: DETERMINATION
OF Z VALUES OF CLASS EXPERIMENTS
Goal
To calculate z values for all the class results of one or more experiments, and to
compare the z value of your results with those of the remainder of the class. Review
prociency testing in Chapter 4 before beginning the experiment.
Equation
z=
where
Xi
X
s
X i = mean of your results

X = true value, or the class mean
s = standard deviation of the true value
or of the class mean (the class standard deviation may be used for the true value if
needed. You will calculate the class standard deviation).
Procedure
Your instructor will provide you with coded results for the entire class. You will know
the identity for the code of only your experiment. The instructor will either provide
the true value (used for grading the experiment) or ask you to use the class average
(which you will calculate) to determine the z value.
Calculations
Calculate the z values for each coded experiment. Arrange them from high (+) to low
(). Plot them, using Excel, in bar form (like Figure 4.4). Identify your value, and
give the chart to your instructor. Would you accept your result as accurate?
Page 84

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Christian7e both.tex V2 - 07/29/2013 9:35 A.M.

USE OF THE ANALYTICAL BALANCE

Christian7e both.tex V2 - 07/29/2013 9:35 A.M.

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010, . . . 050 mL) and deliver it into an empty (dry) ask.

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U. S. Patent No. 5,959,738, September 28, 1999.

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Christian7e both.tex V2 - 07/29/2013 9:35 A.M.

GRAVIMETRIC DETERMINATION OF CHLORIDE

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Christian7e both.tex V2 - 07/29/2013 9:35 A.M.

Christian7e both.tex V2 - 07/29/2013 9:35 A.M.

Cleaning the Crucibles

EXPERIMENT 5 GRAVIMETRIC DETERMINATION OF SO3

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Ni2+ + 2HDMG Ni(DMG)2 + 2H+

Solutions and Chemicals Provided

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Solutions and Chemicals Required

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DETERMINATION OF TOTAL ALKALINITY

CO3 2 + 2H+ H2 CO3

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Solutions and Chemicals Required

standardization for that experiment.

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Solutions and Chemical Required

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Solutions and Chemicals Required

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DETERMINATION OF WATER HARDNESS

does not titrate. Hydroxynaphthol blue indicator is used.

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Ca2+ + H2 Y2 CaY2 + 2H+

Mg2+ + HIn2 MgIn + H+

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Solutions and Chemicals Required

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the standardization of the KSCN above.

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EXPERIMENT 13 DETERMINATION OF CHLORIDE IN A SOLUBLE

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DETERMINATION OF THE pH OF HAIR SHAMPOOS

Solutions and Chemicals Required

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2. pH Measurements of samples. Prepare a 1:10 dilution of each sample in a

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Solutions and Chemicals Required

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If too much Sn2+ is added, then

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EXPERIMENT 17 ANALYSIS OF COMMERCIAL HYPOCHLORITE

I3 is titrated with S2 O3 2 as above.