Chapter- 4 :

Reagents (solution) used in clinical

laboratories

1
Objective.

After completion of this chapter, the student will be able to:

 Define different terminologies related to reagent &
chemicals.
 know different methods of expression of strength of
solution
 Carry out different calculations.
 Describe safe handling of chemicals & reagents.
 Explain about standard & staining solutions .
2
Out line

4.1. Definitions
4.2. Types of solution
4.3 Expression of the strength of solution
4.4. Reagent solutions
4.5. Grades of chemicals
4.6. Standard solutions
4.7. Staining solutions
3
4 : Reagents (solution) used in
clinical laboratories
4.1. Definitions:
Solution
 A solution: - is a homogenous mixture of two or more
components that can be varied in composition within
certain limit.
 Every solution consists of two parts, the solvent

and solute
 Solvent: - is the substance that dissolves the solute.
The solvent in most cases are liquid.

4
Reagents (solution) ……………..

 Liquid solvent are divided in two:-

 Polar solvents, like water, alcohol
 Non-polar solvent like
chloroform,benzene,ether,CCl4

5
Reagents (solution) ……………..

 Solute:-is the substance that is dissolved in solvent, the

solute can be solids, liquid or gases.
 If a solid or gas is mixed with a liquid, the liquid is
considered as solvent.
 If the solution is a mixture of two liquid, the
component present in large amount is considered
as the solvent.

6
4.2. Types of solution
1.Solid solution: - are mixtures of two or more solids and
they are dispersed or mixed randomly throughout one
another resulting a heterogeneous mixture. Eg. Alloys
2.Gaseous solution :-is a homogeneous mixture of two or
more gases
E.g. Air is a solution of O2, N2, H2, CO2 & other gases.
3. Liquid solution: - is a solution where the solvent liquid
& solute are also liquid but it can be solid or gas.
7
4.3 Expression of the strength of solution

Expression of the strength of solutions is broadly divided

into two
1.Relative Expressions of the concentration
2. Quantitative expression of the concentration.
1. Relative expressions of the concentrations
a. Dilute solution
- Small amount of solute dissolved in large amount of
solvent
Example, 00001M NaOH, 1% H2SO4

8
Expression of the strength of …….

c. Concentrated solution
Large amount of solute dissolved in a small amount of
solvents Example, 98% H2SO4.
c. Saturated
 It is one in which a given volume of solvent has

dissolved all the solute that it can under a given

condition of temperature and pressure.
d. Supper saturated solutions.
 A super saturated solution is a solution which holds more
solute than it can hold normally at a given temperature
and pressure.
9
 2. Quantitative expressions of
concentration of solution
 The concentration of solutes in a solution can be
expressed quantitatively in physical units or chemical
units.
A. Physical unit:
 Percentages

 Parts per unit

 Density

 Specific gravity

10
Quantitative expressions…..

1. Percentage
a. Percentage weight by weight % w/w
 This is the number of parts of solute by weight
per 100 parts solution by weight.
Example: - 37gm%w/w HCL means each 100 gm of
HCL solution contain 37gm of HCL and the rest 63
gm solvent (water)

11
Quantitative expressions…..

b. Percentage weight by volume (%w/v)

 It is the number of parts of solvents by weight per 100
parts of solution by volume.
Example:- 98 gm% w/v glucose solution means,
98gm glucose was dissolved in 100 ml solution.
c. Percentage volume by volume % V/V
 It is the number of parts of solute by volume per 100
parts of solute.
Example :- 70% V/V ethanol means,70ml of ethanol was
mixed with 30ml of water.
12
Exercises -1
 How do you prepare an aqueous solution of 30gm%
W/V NaOH?
 Prepare 4 liter of 20% W/V trichloroacetic acid (TCA).

13
 Quantitative expressions…..
2. Parts per unit.
 This is to express the number of parts by volume or

by weight of solute per given volume or weight of the

solution.
 For example:- If 1m ethanol (pure) is mixed with

999 ml of water, to express the number of parts of

ethanol, It can be said that ethanol solution is
1:1000
Exercise-2
If you are asked to prepare 1000ml of a mixture of acetic
acid and methanol. If the procedure says mix 1 parts of
acetic acid with 3part of methanol. How can you prepare?14
Quantitative expressions…..
B. Chemical units.
 Commonly used chemical unit to express the quantity of
solutes in a given solution are:-
 Mole

 Equivalents

 Molarity

 Normality

 Molality

1. Mole.
 1 mole of a give substance is its atomic weight or
molecular weight expressed in gram.
15
Quantitative expressions…..

For example :-1mole = 6.02 x 1023 particle of

substance.
1 mole of Na= 23 grams = 6.02 x 1023
1mole of glucose C6H12O6= 180gm
Since, atomic weight of C=12, H=1, O=16
Example:- How can you prepare 2mole of glucose per liter
of aqueous solution?

16
Quantitative expressions…..

Solution
1 mole C6H12O6= its molecular weight
1mole C6H12O6 = 180gm
2mole = ?
180gm x 2moles = 360gm
1 mole
* 360 gm glucose is dissolved in one liter of solution to give
2 moles of glucose.

17
 Quantitative expressions…..
 Exercises
 How can you prepare 1.5 mole of HCL in litter?
 How can you prepare 70 mole/L NaOH solutions?

Molar solution (Molarity )

 A molar solution is the solutions which contains 1 mole of
the solute per litter of solution. Or a molar solution which
contains one gram molecular weight of a solute per liter of
solution.
Simple formula to calculate Morality
Morality (M) = Actual Mass in grams
Mole. Wt x Vol in liter
18
Quantitative expressions…..

Example-1: How do you prepare 1 molar solution of NaOH.

Mol wt of NaOH= 40 gm?

Solution: - by dissolving 40gm NaOH in 1000ml of aqueous
solution
Example-2:-What is the morality of NaOH, if 20gm NaOH is
dissolved in 200ml of solution?

19
Quantitative expressions…..

Solution
Actual mass = 20gm
Molecular wt = 40gm
Volume = 0.2 lit (200ml)
M =Actual Mass
Mol.wt x vol in liter
M= 20gm
40x 0.2 = 2.5M Na OH

20
Quantitative expressions…..

Normal solution (Normality)

 - Normality is defined as the number of equivalent of solutes
that are present in one liter of solution. If 1 gm equivalent
H2SO4 is dissolved in one liter of solution it is called Normal
solution of H2SO4 or 1N H2SO4.
 NB.One equivalent of substance is its equivalent
weight expressed in grams.
Normality = Actual Mass in grams
Equivalent weight x vol. in liter
Weight to be dissolved = equivalent weight x vol. in L x Normality.

21
Quantitative expressions…..
Example-1 what is the normality of KOH. If 5.6gm KOH is
dissolved in 1 liter
of solution?
Solution
Actual mass = 5.6gm
Volume = 1 lit
N= Actual Mass in gram
Equ. Wt x vol in lit
= 5.6gm = 0.1NKOH
56 x 1

22
Quantitative expressions…..

Example-2. Prepare 0.2N NaOH solution in 1 liter.

V= 1 liter
Actual mass=?
Normality = 0.2N
Equiv. wt= 40
N= Actual mass in gram
Equ. Wt x vol in liter
0.2 = x
40x1
Actual mass = 8gm
There for 8gm is dissolved in a liter of solution to give
0.2N NaOH. 23
Quantitative expressions…..

Molality(m) = moles of solute

mass of solvent (kg)

Example : What is the molality of a 5.86 M ethanol

(C2H5OH) solution whose density is 0.927 g/mL?

24
 Quantitative expressions…..

 Assume 1 L of solution:
 5.86 moles ethanol = 270 g ethanol
 927 g of solution (1000 mL x 0.927 g/mL)

mass of solvent = mass of solution – mass of solute

= 927 g – 270 g = 657 g = 0.657 kg

moles of solute 5.86 moles C2H5OH

m = = = 8.92 m
mass of solvent (kg) 0.657 kg solvent

25
Dilution of solution

 Dilution is the process by which the concentration of or

activity of a given solution is decreased by the addition of
solvent.
 A dilution represents the ratio of the concentrated or
stock material of the total final volume of a solution. i.e
Dilutions are usually expressed as 1 unit of the final
solution.
 Adding a diluents such as water or saline, which
contains none of the material being diluted, is used to do
this.
 Dilution has two parts: diluents and solute.
26
Dilution …

Dilution is used to prepare

 Working solution from stock solution

 Measurable concentration of a sample

 Whenever a solution is diluted, it is volume is

increased and its concentration decreased, but the
total amount of solute remains unchanged.
 The following equation is used to determine the
volume (V2) needed to dilute a given volume of (V1)
of solution of known concentration (C1) to the desired
lesser concentration (C2)
C1 x V1 = C2 x V2
27
Dilution …
Example: to make 45 ml of 30% solution from 70%
solution
C2= 30%
V2= 45ml
C1= 70%
V1= C2 x V2
C1
V1= 30 x 45 = 19.3ml
70
There fore 19.3ml of 70% solution must be diluted with
25.7ml of distilled to obtain 45ml of 30%solution.

28
Methods of dilution

A. Simple dilution
 A general process of preparing less concentration
solutions from a solution of greater concentration.
E.g.,
if you want to prepare 1:10 dilution
 Take 1 ml solute
 Take 9 ml of solvent
 Then mix
29
Methods of dilution …

B. Serial dilution
 It is defined as multiple progressive dilutions ranging
from more concentrated solutions to less concentrated
solutions.
 Example ???

30
Dilution…..

Diluting body fluids and calculating dilution.

In the laboratory it is frequently necessary to dilute body fluids.
Diluting body fluids
Examples: To make 8ml of a 1 in 20 dilution of blood:
Volume of blood required 8 = 0.4ml
20
There fore to prepare 8 ml of a 1 in 20 dilution add 0.4ml of blood to
7.6ml of diluting fluid.
Exercise
If it desired to make 500ml of 0.2M solution of HCl from a 1.00 molar
stock solution. How can we make it?

31
4.4. Reagent solutions

 A reagent or reactant is a substance or compound

consumed during a chemical reaction.
 A reagent is more specifically "a test substance that
is added to a system in order to bring about a reaction
or to see whether a reaction occurs".
 Such a reaction is used to confirm the presence of
another substance.

32
Reagent …

 Reagent

 Any substance employed to produce a chemical

reaction. In medical laboratory, this is used in
conjunction with a given specimen to produce a
measurable or noticeable change.
 The accuracy of tests depends to a great extent on
the accuracy of the reagents used.

33
4.4.1. Types of reagent solutions
1. Stock reagent solution
 Is a concentrated reagent solution which is diluted to
prepare a working solution.
 Has a longer shelf life and occupies less space in
storage than the working solution.
2. Working –reagent solution
 Can be diluted from the stock solution or prepared
directly from the reagent chemical following the
recommended procedures.

34
Types of reagent …

3. Saturated solution
 Is a solution in which further solute can not be
dissolved
 A saturated solution must have the deposits of the
solute at the bottom of the reagent bottle.

35
4.4.2. REAGENT HANDLING
1. Personal safety precautions
 Assume all chemicals are potentially harmful to a

certain degree.
 Be aware of all routes of entry to the body.

 Avoid direct contact with any chemical.

2. General reagent handling precautions

 All containers of chemicals must be clearly labeled

 Read the label carefully

 Before picking
 After picking
 Before removing a reagent
 Before turning the container 36
Reagent …

Only remove approximately what is needed.

 Excess reagent should be placed in proper

container
 Always add a reagent slowly and observe the reaction
 Always pour more concentrated solutions slowly into
water or less concentrated solutions
3. Labeling of reagents and solutions
 Every reagents and solution in the laboratory should be
labled appropriately .

37
4.5. Grades of chemicals
 Chemicals are produced in various purities or grades.
Some of these includes:
1. Reagent grade or Analytical grade(AR) grade
 Are of a high degree of purity.
 Often used in the preparation of reagents in

clinical laboratory.
2. American chemical society(ACS)
 Are reagent grade chemicals that meet the

standards of ACS.
 Labeled as ACS

38
Grade of …

3. Chemically pure grade(CP) grade:

Sufficient for different analysis in the clinical

laboratory
Since their designation does not reveal the limits of

impurities tolerated, not acceptable for research

purposes
Usually used when higher purity biochemical are not

available.
4. USP and NF grade
 Are reagents that meet the specifications of united
states pharmacopeia(USP) or the National
formulary(NF)
 Are less pure than the CP grade.
39
 Grade of …
5.Purified, practical or pure grade
 Are not generally used in the clinical laboratory but
may be used as starting materials for synthesis of
other chemicals of better purity.
6.Technical or Commercial grade
 Are used only for industrial purposes and are not
generally used in the preparation of reagents for the
clinical laboratory.

40
4.6. Standard solutions
 These are solutions in which the concentration of a
given chemical is precisely known and are used to
determine the value of an identical chemical with
unknown concentration of a given solution.
 Chemicals that are used to prepare these solutions
should be of analytical grade.
 Since poor standard solutions cause errors in the
estimation of the intended substances.
 their accurate preparation is of paramount
importance in order to obtain accurate and precise
laboratory findings in medical laboratories.
41
Standard……

1. Primary standard solution

 Primary standard solution is a chemical solution

that has the highest purity and can be used directly

for the exact measurement of substances of
unknown concentration in a given solution.
 Primary standard solution should be made of

substances that are:

 Free of impurities,
 Stable on keeping in solid state and in solution,
 Able to be accurately weighed or measured to

give a solution of exactly known concentration,

 Not hygroscopic (does not absorb moisture)

and vaporize at 20Oc. 42

Standard……

2. Secondary standard solutions

 Secondary standard solutions are solutions of

lower purity and their concentrations are

determined by comparison to primary standard
solutions.
 Secondary standard solutions are used for

analytical procedures after their concentration is

already determined. Some examples of these
solutions are nitric acid, hydrochloric acid, sulfuric
acid, etc.

43
Standard….
 In the preparation of secondary standard solutions the
following points should be taken into consideration:
 Using analytical balance for weighing.

 Dissolving the weighted substance in the

prescribed volume of solvent.

 Determining the exact concentration by

comparison against a primary standard solution.

 Diluting stock secondary standard solutions using

exact measurements.

44
4.7. Staining solutions
 Staining solutions are solutions that contain colored
dyes.
 These solutions can contain basic, acidic or neutral
dyes.
 Different stains are used in medical laboratories to
give an artificial color for the substances to be
identified from a given biological specimen (whole
blood, body fluids, urine, etc.).
 The substances may be identified by their
characteristic reaction with the staining solutions.

45
Staining …

 Based on their reaction, there are three kinds of

stains:
1. Basic stains
2. Acidic stains
3. Neutral stains

46
Staining …

1. Basic Stains
 Are stains in which the colouring substance is contained
in the base part of the stain and the acidic part is
colourless.
Example : Methylene blue stain, Safranin, Genetian
violet, Carbolfuchsin etc.

47
 Staining …
2. Acidic Stains
 Are stains in which the colouring substance is contained
in the acidic part of the stain and the base part is colourless.
E.g. eosin.
3. Neutral Stains
 Are stains in which the acidic and basic components of
stains are coloured.
 Neutral dyes stain both nucleic acid and cytoplasm. e.g
Giemsa’s stain, Wright’s stain.
48
Staining………
 Different types of blood cells, bacteria, parasites, and
tissues together with their cellular elements can be
stained by using appropriate types of stains
(differential stains) such as Giemsa stain, Wright
stain, Gram stain, Leishman stain, Acid Fast Stain,
etc.
 Simple stains are used to reveal the morphology
(shape, size and content) of an organism(s) and
single dye is utilized for the procedure.

49
Summary questions

1. Describe safe handling of strong acid.

2. Describe safe handling of Giemsa stain.
3. Explain about standard & staining solutions .
4. Explain the use of solutions in medical laboratory

50
References

1. Linne Jean Jergenson, Basic techniques of medical

laboratory 4th ed. 2000.
2. WHO, Manual of basic techniques for a health
laboratory 2000.
3. Chees brough M.District Laboratory manual for
tropical courtiers, Cambridge Univerity press, 2000 (Vol
).
4. Chees brough M.District Laboratory manual for
tropical courtiers, Cambridge Univerity press, 2000 (Vol
II).
5. Seyoum B. Introduction to medical laboratory
technology students lecture note series 2002. 51
Next chapter will be on : STERILIZATION AND
DISINFECTION

52