NFS-209: Questions

1. State the general expression of Beer-Lambert’s law with its limitations.

[2019, 2018]

The Beer-Lambert law (or Beer's law) is the linear relationship between absorbance
and concentration of an absorbing species. The general Beer-Lambert law is usually
written as:
A = λ (lambda) * b * c
Where,
A is the measured absorbance,
λ (lambda) is a wavelength-dependent absorptivity coefficient
b is the path length of light in the absorbing species, and
c is the analyte concentration.
When working in concentration units of molarity, the Beer-Lambert law is written
as:
A = ε (epsilon) * b * c
Where,
ε (epsilon) is the wavelength-dependent molar absorptivity coefficient with units
of M−1 cm−1.

The linearity of the Beer-Lambert law is limited by chemical and instrumental

factors. Causes of nonlinearity include:
• Deviations in absorptivity coefficients at high concentrations (>0.01M) due to
electrostatic interactions between molecules in close proximity
• Scattering of light due to particulates in the sample
• Fluorescence or phosphorescence of the sample
• Changes in refractive index at high analyte concentration
• Shift in chemical equilibria as a function of concentration
• Non-monochromatic radiation, deviations can be minimized by using a relatively
flat part of the absorption spectrum such as the maximum of an absorption band
stray light

2. Depict the relationship between transmittance and absorbance. [2019,

2018, 2017]

The amount of radiation absorbed may be measured in a number of ways:

Transmittance, T = P/P0
% Transmittance=%T - 100 T
Absorbance, A = log10P0 /P
A = log10 I / T A = log10 100 / % T
A = 2− log10 % T
The last equation, A = 2 – log10 %T, is worth remembering because it allows you
to easily calculate absorbance from percentage transmittance data. The
relationship between absorbance and transmittance is illustrated in the following
diagram:
 So, if all the light passes through a solution without any absorption, then
absorbance is zero, and percent transmittance is 100%. If all the light is absorbed,
then percent transmittance is zero, and absorption is infinite.

3. Describe photo-multiplier tube as detector. [2019]

Or, describe photo-multiplier tube as detector with its merits and demerits.
[2018]

Photomultiplier tube is a vacuum tube utilizes the multiplication of electrons by

secondary emission to measures low light intensities. It is useful in optical
instrument to measure
- Intensity of faint light and
- Minute flashes of light.

A photon of radiation entering the tube, strikes the cathode, causing the
emission of several electrons. These electrons are accelerated towards the first
dynode (which is 90V more positive than the cathode). The electrons strike the
first dynode, causing the emission of several electrons for each incident
electron. These electrons are then accelerated towards the second dynode, to
produce more electrons, which are accelerated towards dynode three and so
on. Eventually, the electrons are collected at the anode. By this time each
original photon has produced 106 − 107 electrons. The resulting current is
amplified and measured.

Merits of Photomultiplier:
1) Higher sensitivity for UV and visible radiation
2) Lower dark current
3) Wide spectral response
4) High stability

Demerits of Photomultiplier:
 1) Mechanically fragile
2) Shapes and sizes are limited and are physically large
3) Need stable high voltage power supply
4) Responsivity affected by magnetic fields, hence it requires magnetic
shielding in critical application
5) Expensive and sometimes requires cooling for noise reduction

4. Delineate the components of Atomic absorption spectroscopy. [2019,

2018]

Atomic-absorption (AA) spectroscopy is an analytical method for the

determination of various elements, bases upon the absorption of electromagnetic
radiation by free atoms of the element. It uses the absorption of light to measure
the concentration of gas-phase atoms.
Components of Atomic absorption spectroscopy:
 Light source: The light source is usually a hollow-cathode lamp. It emits
radiation at the resonance frequency of the analyte element.
 Atomizer: AA spectroscopy requires that the analyte atoms be in the gas
phase. Ions or atoms in a sample must undergo desolation and
vaporization in a high temperature source such as a flame or graphite
furnace. Flame AA can only analyse solutions, while graphite furnace AA
can accept solution, slurries, or solid samples.
 Monochromator: AA spectrometer use monochromators and detectors for
UV and visible light. The main purpose of the monochromator is to select
the absorption line and isolate from background light due to interferences.
Simple dedicated AA instruments often replace the monochromator with a
band pass interference filter.
 Detector: Photomultiplier tubes are the most common detectors for AA
spectroscopy.

5. State the working principle of AAS. [2019]

Or, what is Atomic Absorption Spectroscopy (AAS)? Describe the mode of action
of AAS with diagram. [2016]

Atomic-absorption (AA) spectroscopy is an analytical method for the

determination of various elements, bases upon the absorption of electromagnetic
radiation by free atoms of the element. It uses the absorption of light to measure
the concentration of gas-phase atoms.
Working Principle of AAS:
- A flame of air-acetylene or N2O-acetylene is produced on a slot-burner.
- The radiation from the HCL is focused on to the centre of the flame and about
5-10 mm above the burner slot.
- Sample solution is drawn through a nebulizer into mixing chamber where it
mixes with acetylene gas and a support gas, either air or nitrous oxide, and
passes to the flame for atomization.
- The free atoms, formed in the flame, of the analyte element absorb radiation
of the HCL. It is to be noted that the absorption of radiation is proportional to
the concentration of the analyte element in solution.
- The attenuated radiation then passes through a monochromator, adjusted to
the wavelength of the analyte element, and falls on a detector, usually
photomultiplier tube.
- The output of the detector is amplified and processed by electronic circuitry
before the result is finally displayed on a suitable readout.
6. Describe different types of filters that are used as wavelength selector.
[2019]

Filters allow transmission of a narrow band of wavelengths through it and absorb

most of the radiation of other wavelengths. Filters are normally used in
colorimetry, fluorometry, flame photometry, microplate reader and in other
optical instruments. There are three types of filters, i) glass, 2) gelatin and 3)
interference filters.
 Glass filters: These are pieces of coloured glass. The colour is produced by
incorporating oxides of vanadium, chromium, iron, nickel and copper in
glass. These niters are not affected by heat, but its disadvantage lies in the
broad bandwidth of the transmitted light.
 Gelatine filters: A suitable dye mixture is incorporated into gelatin then
converted into a thin sheet, and sandwiched between a pair of glass plates.
It is affected by heat, so a heat shield is to be used between the filter and
the source.
 Interference filters: It consists of two parallel glass pales, soldered
internally and separated by a thin film, of cryolite. It eliminates undesired
radiation by the interference of light waves, rather than absorption.

7. State the working principle of inductively couple plasma mass spectrophotometer

(ICPMS). [2019]

8. Describe bomb calorimeter. [2019, 2018]

An instrument which measures the heat released or absorbed during a chemical

reaction. It is used for determining the potential energy of organic substances,
including those in foods. It consists of a hollow steel container, lined with
platinum and filled with pure oxygen, into which a weighed quantity of substance
is placed and ignited with an electric fuse; the heat producers absorbed by water
surrounding the bomb and, from the rise in temperature, the calories liberated are
calculated.

Principle:
Bomb calorimeters-operate on the principle of direct calorimetry, measuring the
heat liberated as the food bums completely.
- Food is sealed within a chamber and charged with oxygen at high pressure
electrical current moving through the fuse at the tip ignites the food-oxygen
mixture.
- Because the calorimeter is fully insulated from the ambient environment, the
increase in water temperature directly reflects the heat released.
- Each substrate, carbohydrates, lipid, and protein, releases a different amount
of energy called its heat of combustion.
 - The reaction takes place in a sealed metal container called bomb is placed in
the water in an insulated container.
- A thermometer is inserted through the lid of the container so that its bulb is
below the water surface.
- When a reaction occurs in the bomb of the calorimeter, the heat of the reaction
flows across the walls of the bomb to the water. The temperature difference of
the water is measured. Here the heat flow into the metal parts of the
calorimeter must be taken into account.

The bomb has a fixed mass and specific heat. The mass of the bomb multiplied
by its specific hearts sometimes termed the calorimeter constant, denoted by the
symbol C with units of joules per degree Celsius. The calorimeter constant is
determined experimentally and will vary from one calorimeter to the next. The
heat flow of the bomb is:
Qbomb = C x ∆t
Once the calorimeter constant is known, calculating heat flow is a simple matter.
The pressure within a bomb calorimeter often changes during a reaction, so the
heat flow may not be equal in magnitude to the enthalpy change.

9. What is brix refractometer? [2018]

10. What are the uses of brix refractometer and desiccator in laboratory analysis?
[2019]

11. Delineate the working principle of a pH meter. [2019, 2018]

12. Discuss the factors that affect the resolving power of a microscope. [2018, 2017]

Resolving power of your microscope determines how well specimen details is

preserved during the magnification process. It is important in the quality of
observation and depends on the ability to distinguish 2 close points of a sample
as separate. The shortest distance which 2 points of the sample can be
distinguished individually is called the limit of resolution. This limit is determined
by-
- The quality of the objective lens,
- The refractive index of the medium (air or oil) separating the sample from the
objective lens and
- The quality of the illumination (its wavelength and appropriate focusing on the
specimen).

For instance, the limit of resolution is 1 mm for a 10X objective, and 0.2 mm for a
100X objective.

13. Discuss the three basic concepts involves in microscopy. [2019]

14. Why immersion oil is used during microscopic examination. [2018, 2017]

15. State the applications of centrifuge machine in laboratory techniques? [2019,

2018]
Or, describe applications centrifuge. [2015]
 A centrifuge is a piece of equipment that uses the centrifugal force and puts a
substance in rotation around a fixed axis in order to separate a fluid from a fluid
or from a solid substance. Generally, a motor drives the rotary motion of the
sample. There are many different kinds of centrifuges, often used for various
specialised purposes.
Centrifuges are generally used to-
 Clarify a solution containing fine solid particles,
 Break down emulsions of two immiscible liquids from each other,
 Collect solids from slurry,
 Obtain "solid" particles from a liquid-solid mixture,
 Separate two liquids of different densities,
 Perform research on atomic and cellular level.

16. Describe thin layer chromatography. [2019]

17. Why might you choose to use HPLC rather than traditional low-pressure column
chromatography? [2019]

18. Delineate the procedure of indirect enzyme immunoassay.

19. Elucidate hollow cathode lamp as a source of radiant energy. [2018]

The most common radiant energy source for atomic absorption

spectrophotometry is the hollow cathode lamp.
It consists of
- A tungsten-and
- A cylindrical cathode sealed in a glass tube filled with neon or argon gas at low
pressure.

The cathode of individual lamp is constructed of the metal whose line spectrum
is required. The cylindrical configuration of the cathode is to keep the radiation
in a limited region of the tube. A quartz window is provided to pass UV radiation.
The cathodes are normally made of single metal, but cathodes of some lamps
consists of a mixture of more than one metal. Such lamps permit the analysis of
more than a single element. Two-element lamps are Ca/Mg, Cu/Zn and Na/K; and
three-element lamp is Ca/Mg/Zn.

20. Describe monochromator. [2018]

All monochromators contain the following component parts,

1. An entrance slit
2. A collimating device
3. A dispersing device
4. A focusing device
5. An exit slit

Working Principle:

1. An entrance slit admits wide band radiation from the source of radiant energy.
2. A collimating device, either a lens or a mirror, produces parallel beam of
radiation.
3. A dispersion device, cither a prism or a grating, resolves' light into its
component wavelengths,
4. A focusing lens or mirror, focuses the dispersed beam towards exit slit,
 5. An exit slit allows desired monochromatic beam through it

21. State the working principle of spectrophotometry. [2018]

Or, State the working principle of UV-visible spectrophotometer. [2017]

The following schematic diagram is of a double-beam UV-Visible

Spectrophotometer.

In a UV-Visible Spectrophotometer there are two light sources; one for UV region,
deuterium/hydrogen lamp, and other for visible region, tungsten halogen lamp.
Usually, in routine investigation, either of the two is needed at a time. However,
at the time of complete wavelength scan the sources are changed automatically.
A double beam instrument employs some type of beam splitter/ chopper prior to
the cell compartment. One beam is directed through the ‘blank’ (or preference
cell) and the other through the sample. These two beams are then compared
continuously or alternately many times in a second by the instrument, and then
display the results.

22. Describe sample introduction methods used in chromatography. [2018]

23. What is a chromatogram? How is it interpreted? [2018]

24. What are the steps in calibration of a GC system? [2018]

25. What do you understand by ‘sensitivity’ and ‘accuracy’ of an instrument? Explain

with example how the sensitivity of an instrument can be measure. [2017]
Or, define accuracy and sensitivity of measurement. [2014]

The sensitivity of an analytical method can be defined as the slope of the

calibration curve, that is, as the ratio of change in the instrument response with
a change in the analyte concentration. Other definitions are also used. In AAS,
sensitivity is defined as the concentration of analyte that produces an absorbance
of 0.0044 (an absorption of 1%).
For example, once the relationship between the signal and analyte concentration
(i.e., the calibration curve) has been established, the linear working range of the
method can be determined. The range is that interval between (and including) the
lowest and highest analyte concentrations that have been demonstrated to be
determined with the accuracy, precision, and linearity of the method. Linear
working ranges vary greatly among instrumental methods and may depend on the
instrument design and the detector used, among other factors.

Accuracy is a measure of how close a measured analytical result is to the true

answer. For most analytical work, the “true answer” is not usually known. We often
work with an “accepted” true value or “accepted reference value.” Accuracy is
evaluated by analysing known, standard samples.

26. Describe the advantages of different instrumental methods of analysis. [2017]

27. Mention importance of ‘amplifier’ in instrumental analysis. [2017]

Electronic amplifiers are used mainly to increase the magnitude (of voltage,
current, or power) of an electrical signal. A linear amplifier provides signal
amplification with little or do distortion, so that the output is proportional to the
input. In photometry most detector signals must be amplified by several orders
of magnitude in order to properly display the result. An amplifier takes an "input".
The amplification factor (ratio of output to input) is called “the gain” of the
amplifier. The input and output signals are normally electrical voltage.

28. Name the sources of Renewable Energy (RE). In your opinion which is the most
convenient for Bangladesh and why? [2017]

29. Identify various scopes of RE utilization in Bangladesh. [2017]

30. From your point of view, among the various initiative taken by Bangladesh
Government to popularize renewable energy within the country, which one will
be most effective from all aspects and why? [2017]
31. What is detector? Explain why detector is used in spectrophotometer. [2017]

A detector is a transducer that converts one type of energy to another. In optical

instrument, it converts radiant energy into an electrical signal. An ideal detector
generates an electrical signal quantitatively related to the radiant power or
number of photons striking it. It is used in the Spectrophotometer in order to
measure quantitatively the absorption of light by a sample solution Examples of
detectors are phototube, photomultiplier tube, thermocouple barrier layer cell
and the like

32. Discuss in short various detector commonly used in spectrophotometer. [2017]

A detector is a transducer that converts one type of energy to another. In optical

instrument, it converts radiant energy into an electrical signal.
Examples of detectors are phototube, photomultiplier tube, thermocouple barrier
layer cell.
Phototube
Phototube (Photocell) is an electron tube with a photosensitive cathode that emits
electrons when illuminated and an anode for collecting the emitted electrons.
Photomultiplier
Photomultiplier tube is a vacuum tube utilizes the multiplication of electrons by
secondary emission to measures low light intensities. It is useful in optical
instrument to measure intensity of faint light and minute flashes of light.
Thermocouple
Thermocouple is temperature-measuring device consisting on two wires of
different metals joined at each end. Thermocouples used in infrared region
typically consist of a blackened gold foil-tellurium metal pin junction that
develops a temperature dependent voltage.

33. What do you mean by ‘Tungsten Halogen Lamp’?

The tungsten filament (or tungsten halogen) lamp is commonly employed as a

source of radiant energy in the visible as well as in the near-infrared and near-
ultraviolet region. Under normal operating conditions, this lamp emits continuous
 radiation between 325 - 3000nm with a peak energy at about 950nm. It is an
incandescent lamp with tungsten filament and enclosed in an evacuated glass
envelope. Tungsten-halogen lamps contain a small amount of iodine in a quartz
"envelope" which also contains the tungsten filament. The iodine reacts with
gaseous tungsten, formed by sublimation, producing the volatile compound WI2.
When molecules of WI2 hit the filament they decompose, redepositing tungsten
back on the filament. The lifetime of a tungsten halogen lamp is approximately
double that of an ordinary tungsten filament lamp. With the quartz envelope the
lamp can be operated at a higher temperature.

34. Define gas chromatography. Elaborate on various instrumental components of a

gas chromatograph with a schematic diagram. [2017]
35. Define UPLC of HPLC. Delineate the difference that made UPLC better option for
analysis in molecular biology over HPLC. [2017]
36. Describe the principle of flame AAS and graphite furnace AAS. [2017]
37. Describe the criteria which should be considered at the time of choosing an
instrument for any biochemical measurement. [2016]
38. State the application of instrumental methods. [2016]
39. Name different types of error which may evolve during an instrumental analysis.
[2016]
40. Explain why a method blank is used in instrumental analysis. [2016]
41. Define Renewable Energy (RE). Briefly discuss different types of RE by mentioning
their source and technology used. [2016]
42. Differentiate between Renewable Energy and conventional energy. Justify which
one of the two source of energy you prefer for your daily use. [2016]
43. Elaborate on various initiatives adopted by Bangladesh Government to make
Renewable Energy popular among the country people. [2016]
44. Define chromatography. Briefly discuss different types of chromatography based
on separation technique. [2016]
45. What is HPLC? Illustrate the components of HPLC with diagram. [2016]
46. What is pH meter? Mention the application of pH meter. [2016]
47. Name with example different types of analytical techniques in analytical
chemistry. [2016]
48. Define resolving power of microscopy and briefly explain the factors affecting it.
[2016]
49. State the working principle of a compound microscope. [2016]
50. Short notes on the following:
a) Transducer [2016]
b) Wavelength vs. Wave number [2016]

Wavelength Wave Number

It is the linear distance between the It is the number of oscillations of a
two adjacent crests (or troughs) of a wave occurs m one centimetre (per
propagating transverse wave. second).
It is expressed as, It is expressed as.
𝝺 =c/f V = 1/𝝺
Wavelength is equal to the velocity Wave number is the reciprocal of the
by frequency. wavelength expressed in centimetre.
Various units of wavelength include: Units of wave number is reciprocal of
A (Angstrom), nm (nanometer), µ the unit of wavelength.
(micron)

c) Sensitivity vs Selectivity [2016]

51. Describe chromatography [2015]
52. List application of gas liquid chromatography. [2015]
53. What are the advantages in the instrument based research analysis? [2015]
54. Explain the role of monochromator in spectrophotometer. [2015]
55. What is column? Write in details about different types of column. [2015]
56. Write about an instrument which uses high speed rotator to separate substance
of different densities. [2015]
57. What is micro centrifuge?[2015]

58. What is primary energy? [2015]

59. Describe with diagram the operation of solar power system. [2015]
60. Explain the importance of Transducer in instrumental method of analysis. [2014]
61. Describe spectroscopy with diagram. [2014]
62. Explain with diagram UV and infrared light sources for spectroscopy. [2014]
63. What is pH and pH measurement? [2014]
64. Describe the role of electrodes in pH measurement. [2014]
65. What is renewable energy and sources of renewable energy? [2014]
66. Describe solar cooking and E-bag cooking. [2014]
67. Write short notes on:
a) PMT [2014]
b) Quenching [2014]
c) Different types of electrodes for pH measurement [2014]