SUBJECT: INSTRUMENTAL METHODS OF ANALYSIS

(BP705P)

PRACTICAL MANUAL BOOK

 LAB MANUAL
INSTRUMENTAL METHODS OF ANALYSIS
(BP705P)
 INDEX

Sr.
Title of experiments Page no.
no.

Determination of absorption maxima and effect of solvents on 1-8

1.
absorption maxima of organic compounds
2. Estimation of dextrose by colorimetry 9-14

3. Estimation of sulfanilamide by colorimetry 15-20

Simultaneous estimation of ibuprofen and paracetamol by UV 21-27

4.
spectroscopy
5. Assay of paracetamol by UV- Spectrophotometry 28-32

6. Estimation of quinine sulfate by fluorimetry 33-37

7. Study of quenching of fluorescence 38-42

8. Determination of sodium by flame photometry 43-47

9. Determination of potassium by flame photometry 48-52

10. Determination of chlorides and sulphates by Nephelo turbidimetry 53-58

11. Separation of amino acids by paper chromatography 59-64

12. Separation of sugars by thin layer chromatography 65-68

13. Separation of plant pigments by column chromatography 69-72

14. Demonstration experiment on HPLC 73-76

15. Demonstration experiment on Gas Chromatography 77-82

 BP705P. INSTRUMENTAL METHODS OF ANALYSIS

Syllabus

1. Determination of absorption maxima and effect of solvents on absorption maxima of

organic compounds

2. Estimation of dextrose by colorimetry

3. Estimation of sulfanilamide by colorimetry

4. Simultaneous estimation of ibuprofen and paracetamol by UV spectroscopy

5. Assay of paracetamol by UV- Spectrophotometry

6. Estimation of quinine sulfate by fluorimetry

7. Study of quenching of fluorescence

8. Determination of sodium by flame photometry

9. Determination of potassium by flame photometry

10. Determination of chlorides and sulphates by Nephelo turbidimetry

11. Separation of amino acids by paper chromatography

12. Separation of sugars by thin layer chromatography

13. Separation of plant pigments by column chromatography

14. Demonstration experiment on HPLC

15. Demonstration experiment on Gas Chromatography

 Experiment No. 1

AIM

Determination of absorption maxima and effect of solvents on absorption maxima of organic

compounds.

PRINCIPLE

The solvent dissolves the drug substance and exerts an intense influence on the quality and

shape of the UV-visible spectrum. Hence the absorption spectrum of drug substance changes

mostly as per the change of solvent that has been used to dissolve the drug substance. Here,

the change in either wavelength (absorption maxima) or absorption intensity is monitored by

changing the different solvents.

THEORY

Ultraviolet–visible spectroscopy or ultraviolet–visible spectrophotometry (UV–Vis or

UV/Vis) refers to absorption spectroscopy or reflectance spectroscopy in part of the

ultraviolet and the full, adjacent visible regions of the electromagnetic spectrum.

Absorption spectroscopy deals with the spectroscopic techniques that measure the

absorption of radiation, as a function of frequency or wavelength, due to its interaction with a

sample. The sample absorbs energy, i.e., photons, from the radiating field. The intensity of

the absorption varies as a function of frequency, and this variation is the absorption spectrum.

Absorption spectroscopy is employed as an analytical chemistry tool to determine the

presence of a particular substance in a sample and, in many cases, to quantify the amount of

the material present. Infrared and ultraviolet–visible spectroscopies are particularly common

in analytical applications. Absorption spectroscopy is also employed in studies of molecular

and atomic physics, astronomical spectroscopy and remote sensing.

There are a wide range of experimental approaches for measuring absorption spectra.

The most common arrangement is to direct a generated beam of radiation at a sample and
detect the intensity of the radiation that passes through it. The transmitted energy can be used

to calculate the absorption. The source, sample arrangement and detection technique vary

significantly depending on the frequency range and the purpose of the experiment.

Lambda max or absorption maxima (λmax)

Lambda max refers to the wavelength along the absorption spectrum where a substance has

its strongest photon absorption. Simply, the wavelength at which a substance displays

maximum absorption is called as lambda max (figure 1). Different compounds may have very

different absorption maxima and absorbances. Intensely absorbing compounds must be

examined in dilute solution (absorbance value less than 1), so that significant light energy is

received by the detector, and this requires the use of completely transparent (non-absorbing)

solvents. The most commonly used solvents are water, ethanol, hexane and cyclohexane.

Solvents having double or triple bonds, or heavy atoms (e.g. S, Br & I) are generally avoided.

Figure 1: Typical example of unknown sample depicting absorption maxima at 279.50 nm

The Beer–Lambert:

The Beer–Lambert law states that the absorbance of a solution is directly proportional to the

concentration of the absorbing species in the solution and the path length.[3] Thus, for a fixed
path length, UV/Vis spectroscopy can be used to determine the concentration of the absorber

in a solution.

Ultraviolet–visible spectrophotometer

The instrument used in ultraviolet–visible spectroscopy is called a UV/Vis

spectrophotometer. It measures the intensity of light after passing through a sample and

compares it to the intensity of light before it passes through the sample.

Choice of solvents

Every solvent is supposed to exhibit UV-vis absorbance cut-off wavelength. The solvent cut-

off is the wavelength below which the solvent itself absorbs all of the light. So when

choosing a solvent student has to be careful of its absorbance cut-off. If the solvent is

showing cut-off near the absorption maxima of the substance under examination, another

solvent is to be chosen.

Solvent UV Absorbance Cut-off (nm)

Water 180

Ethanol 205

Toluene 285

Dimethyl formamide 267

Acetone 329

Benzene 278

Applications:

1. UV/Vis spectroscopy is routinely used in analytical chemistry for the quantitative

determination of different analytes, such as transition metal ions, highly conjugated

organic compounds, and biological macromolecules.

 2. Solutions of transition metal ions can be colored (i.e., absorb visible light) because d

electrons within the metal atoms can be excited from one electronic state to another.

The colour of metal ion solutions is strongly affected by the presence of other species,

such as certain anions or ligands. For instance, the colour of a dilute solution of

copper sulfate is a very light blue; adding ammonia intensifies the colour and changes

the wavelength of maximum absorption (λmax).

3. Organic compounds, especially those with a high degree of conjugation, also absorb

light in the UV or visible regions of the electromagnetic spectrum.

4. UV/Vis can be applied to determine the kinetics or rate constant of a chemical

reaction.

5. Spectroscopic analysis is commonly carried out in solutions but solids and gases may

also be studied.

REQUIREMENTS

Apparatus: Glass beakers, Measuring flasks, Whatmann filter paper, Measuring cylinder,

etc.

Chemicals: Paracetamol, Distilled water, Ethanol, 0.1 N NaOH, 0.1N HCl etc.

PROCEDURE

1. Weigh accurately about 10 mg of Paracetamol and dissolve in sufficient quantity of

solvent (2/3 volume), in 100mL of measuring flask, shake well to dissolve completely

and makeup the volume upto mark to prepare 100 ppm of stock solution.

2. Pipette out 1 mL of solution from stock solution and add to 10 mL measuring flask

and make up the volume with fresh solvent to prepare 10 ppm of solution.

3. Scan the solution in UV visible spectrophotometer to obtain the absorption maxima.

Observation

Solvent λmax (nm)

Distilled water

Ethanol

0.1 N NaOH

0.1N HCl

RESULTS

The absorption maxima (λmax) of paracetamol in various solvents like distilled water,

ethanol, 0.1 N NaOH, and 0.1N HCl was observed and compiled.

MCQ’s
1. When absorption intensity of compound is decreased it is called
A. Red shift (Bathochromic shift)

B. Blue shift (Hypsochromic shift)

C. Hypochromic shift

D. Hyperchromic shift

2. When absorption maximum of a compound shift to longer wavelength, it is known as

A. Red shift (Bathochromic shift)

B. Blue shift (Hypsochromic shift)

C. Hypochromic shift

D. Hyperchromic shift

3. If solvent is polar and electron transition is nπ∗ which type of shift are seen in graph?

A. Hypsochromic shift

B. Red shift
 C. Blue shift

D. A and C

4. If solvent is non-polar and electron transition is nπ ∗which type of shift are seen in

graph?

A. Hypsochromic shift

B. Red shift

C. Blue shift

D. A and C

5. Which of the following compounds does not absorb light in the UV/visible spectrum?

A. Aspirin

B. Paracetamol

C. Phenobarbitone

D. Chloral hydrate

Questions

1. What is principle of UV visible spectroscopy?

Ans: UV Spectroscopy uses ultraviolet light to determine the absorbency of a substance.

In simple terms, the technique maps the interaction between light and matter and

measures. As matter absorbs light it undergoes either excitation or de-excitation, which

generates what is known as a spectrum. This allows scientists to measure the rate at which

a beam of light weakens after passing through a substance. Absorption spectroscopy is

complementary to fluorescence spectroscopy, in that fluorescence deals with transitions

of electrons from the excited state to the ground state, while absorption measures

transitions from the ground state to the excited state.

2. Enlist factors affecting UV visible spectroscopy.

 Ans:

i. Solvent

ii. sample PH

iii. sample concentration

iv. sample temperature

v. effect of conjugation

3. Define chromophore and auxochrome?

Ans:

(a) CHROMOPHORE: The term chromophore was previously used to denote a

functional group of some other structural feature of which gives a color to compound.

For example- Nitro group is a chromophore because its presence in a compound gives

yellow color to the compound. But these days the term chromophore is used in a

much broader sense which may be defined as “any group which exhibit absorption of

electromagnetic radiation in a visible or ultra-visible region “It may or may not impart

any color to the compound. Some of the important chromophores are: ethylene,

acetylene, carbonyls, acids, esters and nitrile groups etc.

Types of chromophores: Two types of chromophores are known.

1. Chromophores in which the groups have π electrons undergo π-π* transitions. For

examples:-ethylenes, acetylenes etc.

2. Chromophores having both π- electrons and n (non-bonding) electrons undergo

two types of transitions. i.e., π-π* and n-π*, for examples: - carbonyls, nitriles, azo

compounds and nitro compounds etc.

(b) AUXOCHROMES: It is a group which itself does not act as a chromophore but when

attached to a chromophore, it shifts the adsorption towards longer wavelength along

with an increase in the intensity of absorption. Some commonly known auxochromic

 groups are: -OH, -NH2, -OR, -NHR, and –NR2. For example: When the auxochrome

–NH2 group is attached to benzene ring. Its absorption change from λ max 225 (ɛmax

203) to λmax 280 (εmax1430)

All auxochromes have one or more non-bonding pairs of electrons. If an auxochromes

is attached to a chromophore, it helps is extending the conjugation by sharing of non-

bonding pair of electrons

4. What is Bathochromic shift?

Ans: It is a change of spectral band position in the absorption, reflectance, transmittance,

or emission spectrum of a molecule to a longer wavelength (lower frequency). Because

the red color in the visible spectrum has a longer wavelength than most other colors, the

effect is also commonly called a red shift.

5. What is Hypsochromic shift?

Ans: It is a change of spectral band position in the absorption, reflectance, transmittance,

or emission spectrum of a molecule to a shorter wavelength (higher frequency). Because

the blue color in the visible spectrum has a shorter wavelength than most other colors, this

effect is also commonly called a blue shift.

 Experiment No. 2

AIM: To perform estimation of dextrose by colorimetry

PRINCIPLE

P-hydroxybenzoic acid hydrazide (PHBAH) reacts with dextrose in alkaline medium, which

gives colour products, these colored products can be analysed by analytical technique of

colorimetry. Dextrose reacts with PHBAH reagent and produces colored product; the

absorbance of which is proportional to concentration of dextrose being analysed.

THEORY

Colorimetry or (colourimetry) is a technique used to determine the concentration of colored

compounds in solution. Colorimetry is the measurement of concentration of analyte in a

solution by determining absorption of a particular wavelength. A colorimeter is a device used

to test the concentration of a solution by measuring its absorbance of a specific wavelength of

light. To use this device, different solutions must be made, and a control (usually a mixture of

distilled water and another solution) is first filled into a cuvette and placed inside a

colorimeter to calibrate the machine. Only after the device has been calibrated you can use it

to find the densities and/or concentrations of the other solutions.

The technique is based upon Beer-Lambert’s law. Overall the process is accomplished

with the measurement of analyte in a colored solution or development of color that appears in

the solution following reaction with specific reagent. In alkaline medium p-hydroxybenzoic

acid hydrazide (PHBAH) reacts with dextrose that give products which can be assayed by

colorimetry. Dextrose reacts with p-hydroxybenzoic acid hydrazide (PHBAH) reagent to give

coloured product; the absorbance noted is proportional to concentration of sugar.

Applications of colorimeters

1. Besides being valuable for basic research in chemistry laboratories, colorimeters have

many practical applications. For instance, they are used to test for water quality, by
 screening for chemicals such as: chlorine, fluoride, cyanide, dissolved oxygen, iron,

molybdenum, zinc hydrazine etc.

2. They are also used to determine the concentrations of plant nutrients (such as

phosphorus, nitrate and ammonia) in the soil or haemoglobin in the blood and to

identify substandard and counterfeit drugs.

3. In addition, they are used by the food industry and by manufacturers of paints and

textiles. In these disciplines, a colorimeter checks the quality and consistency of

colors in paints and fabrics, to ensure that every batch comes out looking the same.

REQUIREMENTS

Apparatus: Test tubes, Burette, Colorimeter, and Water bath, etc.

Chemicals: Dextrose: 5mg, Sodium hydroxide: 3-5 pellets, p- hydroxybenzoic acid

hydrazide (PHBAH): 50 ml

PROCEDURE

Alkaline solution

Put 3-5 pellets of sodium hydroxide in about 50 ml of water, stir to mix well.

Diluent

Add 10.0 ml of p-hydroxybenzoic acid hydrazide (PHBAH) solution with 30.0ml of water

along with few drops of Sodium hydroxide.

Standard stock solution preparation

Weigh 5.0 mg of dextrose; add in a test tube containing 15.0 ml of water, 2-3 drops of

sodium hydroxide and 5.0 ml of p-hydroxybenzoic acid hydrazide (PHBAH) solution, and

mix well.

Working standard solution preparation

Dilute 5.0ml of stock solution with 15.0 ml of diluent.

Sample solution preparation

Take a test tube, add 1.0 ml of sample with burette, add 4.0 ml of water to it, and shake

gently to mix. Keep the above sample solution for 30 -35 minutes at normal temperature.

Again shake gently, then add 5.0 ml of p-hydroxybenzoic acid hydrazide (PHBAH) coloured

solution and few drops of sodium hydroxide, mix well and keep the test tube in water bath in

boiling mode. After about 5 minutes withdraw test tube from water bath, allow to cool, then

add 10 ml of water and mix well.

Instrumentation

Before measuring absorbance by colorimeter clean the glass tube with water, and ensure zero

absorbance with diluent.

To finalize the light source, take sample solution in glass tube and keep first in blue light

source and repeat with the available light source simultaneously recording absorbance.

Use the light source with highest absorbance and record the absorbance for the followings.

1. Standard stock

2. Working standard solution

3. Sample of unknown concentration.

Note the above three different absorbance. Now plot the graph of two known solution (1 is

standard stock, 2 is working standard) i.e. absorbance (Y-axis) against concentration (X-

axis), plot a straight line in between two points and find the concentration of unknown sample

with the sample absorbance.

CONCLUSION

The chelate formed from the reaction of sucrose and p-hydroxybenzoic acid hydrazide

(PHBAH) in alkaline medium is measured by colorimeter which is the basis of quantifying

the amount of sucrose present in sample.

MCQs
1. What is the name of an instrument used to measure the absorbance of a coloured

compound in solution?

A. Colorimeter.

B. Colourymeter

C. Coulometer.

D. Calorimeter.

2. The dichromate ion absorbs light of wavelength close to 500 nm. Based on this

information, what can you conclude?

A. The dichromate ion absorbs outside the visible region.

B. Solutions of the dichromate ion are colourless

C. The dichromate ion absorbs in the ultraviolet region.

D. The dichromate ion absorbs within the visible region.

3. The wavelength of absorption is 495 nm. In what part of the electromagnetic spectrum

does this lie?

A. Ultraviolet-visible.

B. Microwave.

C. Infrared.

D. Radiowave.

4. Which part of the spectrophotometer is adjusted to select the desired wavelength?

A. Light source.

B. Filter.

C. Sample.

D. Photo detector.

5. The plot of absorption verses wavelength for a particular compound is referred to as the

A. Path length.
 B. Emission spectrum.

C. Absorption spectrum.

D. None of these.

Questions

1. What is principle of colorimetry?

Ans: The working principle of the colorimeter is based on Beer-Lambert's law, which

states that the amount of light absorbed by a color solution is directly proportional to the

concentration of the solution and the length of a light path through the solution.

2. What do you mean by Colorimeter?

i. Ans: The colorimeter instrument is very simple, consisting merely of a light

source (lamp), filter, cuvettes, and photosensitive detector to collect the

transmitted light. Another detector is required to measure the incident light; or a

single detector may be used to measure incident and transmitted light, alternately.

The latter design is both cheaper and analytically better, because it eliminates

variations between detectors. The filter is used here to obtain an appropriate range

of wavelengths within the bands, which it is capable of selecting.

3. What are the components of colorimetry?

Ans: The components of colorimetry are;

i. a light source (often an ordinary low-voltage filament lamp);

ii. an adjustable aperture;

iii. a set of colored filters;

iv. a cuvette to hold the working solution;

v. a detector (usually a photoresistor) to measure the transmitted light;

vi. a meter to display the output from the detector.

4. What are the advantages of colorimetry?

 Ans: It is fast, economical, and has a simple operation of a spectrometer. To handle the

Colorimeter, experienced person is not the requirement. The method is fast and

convenient to that of the gravimetric or volumetric processes, and they are easily

optimized for automation.

5. What are applications of colorimetry?

Ans: Colorimeters are widely used to monitor the growth of a bacterial or yeast culture.

They provide reliable and highly accurate results when used for the assessment of color in

bird plumage. They are used to measure and monitor the color in various foods and

beverages, including vegetable products and sugar. Certain colorimeters can measure the

colors that are used in copy machines, fax machines, and printers. Besides being used for

basic research in chemistry laboratories, colorimeters have many practical applications

such as testing water quality by screening chemicals such as chlorine, fluoride, cyanide,

dissolved oxygen, iron, molybdenum, zinc, and hydrazine.

 Experiment No. 3

AIM

Estimation of sulfanilamide by colorimetry

PRINCIPLE

Principle involved in the estimation of sulphanilamide is based on formation of derivative of

para-amino benzoic acid analogue. This derivative can be analysed calorimetrically at

wavelength of 510 nm.

THEORY

Colorimetry or (colourimetry) is a technique used to determine the concentration of colored

compounds in solution. Colorimetry is the measurement of concentration of analyte in a

solution by determining absorption of a particular wavelength. A colorimeter is a device used

to test the concentration of a solution by measuring its absorbance of a specific wavelength of

light. To use this device, different solutions must be made, and a control (usually a mixture of

distilled water and another solution) is first filled into a cuvette and placed inside a

colorimeter to calibrate the machine. Only after the device has been calibrated you can use it

to find the densities and/or concentrations of the other solutions.

The technique is based upon Beer-Lambert’s law. Overall the process is accomplished

with the measurement of analyte in a colored solution or development of color that appears in

the solution following reaction with specific reagent.

Applications of colorimeters

1. Besides being valuable for basic research in chemistry laboratories, colorimeters have

many practical applications. For instance, they are used to test for water quality, by

screening for chemicals such as: chlorine, fluoride, cyanide, dissolved oxygen, iron,

molybdenum, zinc hydrazine etc.

 2. They are also used to determine the concentrations of plant nutrients (such as

phosphorus, nitrate and ammonia) in the soil or haemoglobin in the blood and to

identify substandard and counterfeit drugs.

3. In addition, they are used by the food industry and by manufacturers of paints and

textiles. In these disciplines, a colorimeter checks the quality and consistency of

colors in paints and fabrics, to ensure that every batch comes out looking the same.

Requirements:

Chemicals: Sulphanilamide, 0.1 N NaOH solution, Conc. H2SO4, Ethanol Solution (95%v/)

Distilled water.

Apparatus: Beaker, Volumetric flask, Pipette, Spectrophotometer.

Theory: Sulphanilamide is a para-amino benzoic acid analogue. It is estimated

calorimetrically after derivatization at 510 nm. Visible spectrophotometry is one of the most

employed methods of pharmaceutical analysis. It involves the amount of radiation absorbed

by substance in a solution. The instrument that measures the ratio or the function of the ratio

of the intensity of the two beams of light in the visible region is called as colorimeter.

Single Point Standardization:

The single point procedure involves the measurement of the absorbance of a sample solution

and of a standard solution of the reference substance.

The standard and sample solutions are prepared in a similar manner; ideally the concentration

of the standard solution should be close to that of the sample solution.

The concentration of the substance in the sample is calculated from the proportional

relationship that exists between absorbance and concentration.

Procedure:
1. Weigh accurately about 0.15 gm of Sulphanilamide and add 1 ml of conc. sulphuric acid

in 100 mL measuring flask. Now add 2 ml of 0.1 N NaOH solutions and make the volume

up to 100 ml with distilled water and shake for 15 to 20 minutes.

2. Pipette out 10 ml of this solution and make the volume up to 100 ml with distilled water.

3. Again, pipette out 10 ml of this solution and add 10 ml of ethanol solution 95% v/v and

make the volume up to 100 ml with distilled water.

4. Analyse calorimetrically by setting the filter to 510 nm.

Observation Table:

Sr. no. Name of sample Wavelength (λ) Absorbance

1.

2.

Calculation: 𝐴 = 𝑎 × 𝑏 × 𝑐

A = Absorbance

a = Absorptivity

b = Path length (1cm)

c = Concentration of absorbing substance

Result

Observed % purity of given sample was found to be = ____________ %.

Conclusion:

The quantitative spectrophotometric analysis and the estimation of impurity level in the

drug are determined by colorimetric technique.

Precautions: Wait to initialize the spectrophotometer. Handle the cuvettes carefully.

Handle apparatus with care. Instruments must be used carefully.

MCQs

1. Colorimeter is applied only in relation to_?

A. X rays.

B. Visible light

C. IR Rays.

D. UV light.

2. Which of the following is not an application of colorimeter?

A. Analysis of paint.

B. Analysis of ink.

C. Analysis of cosmetic.

D. Composition detection.

3. Colorimeters are used in applications where great accuracy is required.

A. True

B. False

4. Colorimeters are used to determine the concentration of solutions.

A. True

B. False

5. Which of the following is the purpose of balance indicator in double beam photometer or

colorimeter?

A. Selects a particular wavelength

B. Splits the wavelength selected into two equal beams

C. Detects and indicates the amount of light falling on it

D. Indicates the difference between the output of two photometers

Questions

1. Describe laws governing the absorption of radiation in colorimetry.

Ans: The absorption of radiation in colorimetry is based on Beer-Lambert's law, which

states that the amount of light absorbed by a color solution is directly proportional to the

concentration of the solution and the length of a light path through the solution.

Beer law: The intensity of beam of monochromatic light decreases exponentially with

increase in the concentration of absorbing species.

Lambert's law: The intensity of beam of monochromatic light decreases exponentially

with increase in the thickness of the medium.

2. Explain the properties of sources of light used in colorimetry.

Ans: SOURCE OF LIGHT: The visible spectrum ranges from 400nm to 800 nm.

Hence, any lamp source, which gives adequate intensity of radiation over the entire

wavelength region, can be used. The requirements of a source of light for colorimeter

are:

i. It should not observe fluctuations.

ii. It should offer radiation in between 400nm -800nm.

iii. It should provide adequate intensity.

3. What are sources of light used commonly in colorimetry?

Ans: The following are the sources of light used commonly.

i. Tungsten lamp: As it satisfies the above criteria, this lamp finds its place in most

of colorimeter spectrophotometer. The lamp consists of a tungsten filament in a

vacuum bulb similar to the ones used domestically. However, it offers sufficient

intensity.

ii. Carbon arc lamp: For a source of very high intensity, carbon arc lamp can be

used. It also provides a whole range of visible spectrum.

4. Describe the filters used in colorimetry?

Ans: Two types of filters are used in colorimetry.

i. Absorption filters: These filters are made up of glass, coated with pigments or

they are made up of dyed gelatin. They absorb the unwanted radiation and

transmit the rest of the radiation, necessary for colorimetry.

ii. Interference Filters: This filter is known as Fabry – Perot filter. The features

include (1): It has dielectric spacer film made up of CaF2, MgF2 or SiO, between

two parallel reflecting silver films.

5. What do you mean by monochromators?

Ans: Monochromators are better and more efficient than filters in converting a

polychromatic light or heterochromatic light into monochromatic light.

A monochromator has the following components

1. Entrance slit (to get narrow source).

2. Collimator (to render light parallel).

3. Grating or prism (to disperse radiation).

4. Collimator (to reform the images of entrance slit).

5. Exit slit (to fall on sample cell).

 Experiment No. 4

AIM: Simultaneous estimation of ibuprofen and paracetamol by UV spectroscopy.

Ibuprofen (Ibu) (400mg) + Paracetamol (Para) (325mg) tablet

PRINCPLE:

Estimation of combined dosage form such as ibuprofen and paracetamol tablet can be done

using the Vierodt’s method. Here two absorbing drugs, display absorption at the wavelength

of each other, and the concentrations of both the drugs can be analysed by technique of

simultaneous equation method called as Vierodt’s method.

THEORY

Simultaneous equation method

If a sample contains two absorbing drugs (X and Y) each of which absorb at the Lambda Max

of the other, it may be possible to determine both drugs by the technique of simultaneous

equation method ( Vierodt’s method) Provided that certain criteria apply.

The Information required is;

a) Absorptivities of X at lambda 1 and lambda 2, ax1 and ax2 respectively.

b) Absorptivities of Y at lambda 1 and lambda 2, ay1 and ay2 respectively.

c) The absorbances of diluted sample at lambda 1 and lambda 2, A1 and A2

respectively.

Let Cx and Cy be the concentrations of X and Y respectively in a diluted sample.

Two equations are constructed based upon the fact that at lambda 1 and lambda 2 the

absorbance of the mixture is the sum of the individual absorbances of X and Y.

1. At lambda 1

A1= ax1 b cx + ay1 b cy--------------------------------------------- (1)

2. At lambda 2

A2= ax2 b cx + ay2 b cy--------------------------------------------- (2)

For measurements in 1 cm cell, b =1

Rearrange equation (2)

Cy = A2- ax2 cx/ay2

Substituting for Cy in equation (1) and rearranging

Cx = A2 ay1-A1ay2 / ax2 ay1- ax1 ay2 -------------------------- (3)

Cy = A1 ax2-A2ax1 / ax2 ay1- ax1ay2---------------------------- (4)

Absorptivity: E 1%, 1 cm

A = abc------------------------------- (5).

A - Absorbance

a = absorptivity (Extinction coefficient)

b = path length

c = concentration

Rearranging equation (5)

a = A/bc------------------------------- (6).

b=1cm

c=1% (1gm/100mL)

a = A/c (1gm/100mL) ---------------------------------- (7).

Procedure:

Selection of common solvent

After studying solubility of both the drugs in various solvents, select a common solvent in

which both drugs (Ibuprofen and Paracetamol) are soluble.

Preparation of stock standard solutions of Ibu and Para

Ibu 40 and Para 32.5 mg were transferred into two separate 100 mL volumetric flasks to

prepare (400 and 325 µg/mL). Withdraw 0.1mL and dilute upto 10 mL with fresh solvent to

prepare (4 and 3.25 µg/mL).

Selection of analytical wavelengths

Above two solutions were scanned separately between 400-200 nm. The overlain spectra of

both drugs were recorded. λmax of both the drugs were recorded (λ1 and λ2).

Linearity study of Ibu and Para

Linearity of Ibu and Para was recorded as function of absorbance (absorbance should be less

than 1). Calibration curve was constructed by plotting absorbance versus concentration.

Concentration of Ibu Absorbance Concentration of Absorbance

(µg/mL) Para (µg/mL)

Determination of E (1%, 1cm) values of both drugs at selected wavelengths

E (1%, 1cm) values of these drugs were calculated using following formula;

𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒
E (1%, 1cm) =
𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 (𝑔/100𝑚𝐿)

Sr no. Absorptivity at λ1 Absorptivity λ2

1. Ibu Para Ibu Para

 2.

3.

4.

5.

6.

Mean ax1 =_______ ay1 =_______ ax2 =_______ ay2 =_______

Formula:

𝐀𝟐 𝐚𝐲𝟏 − 𝐀𝟏𝐚𝐲𝟐
𝐂𝐱 (𝐈𝐛𝐮) =
𝐚𝐱𝟐 𝐚𝐲𝟏 − 𝐚𝐱𝟏 𝐚𝐲𝟐

𝐀𝟏 𝐚𝐱𝟐 − 𝐀𝟐𝐚𝐱𝟏
𝐂𝐲 (𝐏𝐚𝐫𝐚) =
𝐚𝐱𝟐 𝐚𝐲𝟏 − 𝐚𝐱𝟏𝐚𝐲𝟐

Where,

A1 and A2 are absorbance of mixture at λ1 and λ2;

ax1 and ax2, are E(1%, 1cm) of Ibu at λ1and λ2, respectively;

ay1 and ay2, E (1%, 1cm) of Para at λ1and λ2, respectively;

Cx and Cy are concentrations of Ibu and Para in mixture.

Result:

The percentage purity of Ibuprofen and Paracetamol in combination form by

Spectrophotometric method was found to be _______%w/w and _________%w/w

respectively.

MCQs

1. Which of the following statement is incorrect regarding UV-visible spectrophotometry?

A. It gives a measure of the amount of light absorbed by a sample.

 B. The sample is exposed to UV-visible light of different wavelengths.

C. It is used to determine the concentration of a compound in solution.

D. The amount of UV-visible light absorbed by a sample during UV-visible

spectrophotometry is called its absorbance.

2. The photomultiplier tube is a widely used ---------------in UV-Vis spectroscopy.

A. Wavelength selector

B. Detector

C. Signal processors

D. Cuvette

3. Select the wavelength range corresponding to UV-visible region.

A. 400-800 nm

B. 200-800 nm

C. 25 μm-2.5 μm

D. 2.5 μm – 1mm

4. The spectrophotometer is made up of-

A. Spectrometer

B. Photometer

C. both of these

D. none of these

5. Spectrophotometers measure intensity as a function of the wavelength of the

A. Light source

B. Colour

C. Sound

D. none of these

Questions:
1. What is simultaneous equation method of analysis?

Ans: If a sample contains two absorbing drugs (X and Y) each of which absorb at the

Lambda Max of the other, it may be possible to determine both drugs by the technique

of simultaneous equation method ( Vierodt’s method). Vierodt’s method is typically

applied to estimate drug combinations that contain two drugs or more than two drugs in

combined dosage form.

2. What is multicomponent analysis?

Ans: In multicomponent systems, specific analytical methods are required to

determine the concentrations of individual components in the presence of interfering

substances. Ultraviolet and visible spectrometric methods have widely been

developed for the analysis of drugs in mixtures and pharmaceutical preparations.

3. What are the three main components of a spectrophotometer?

Ans: A spectrophotometer consists of three primary components: a light source,

optics to deliver and collect the light, and a detector.

4. Which detector is used in UV Visible Spectroscopy?

Ans: photomultiplier tube: The photomultiplier tube is the most popular detector used

in UV- Visible spectroscopy. It comprises of a photosensitive cathode, anode and

several dynodes. Photons entering the tube strike the cathode resulting in emission of

electrons.

5. What is the difference between colorimeter and spectrophotometer

Ans: A colorimeter is generally any tool that characterizes color samples to provide

an objective measure of color characteristics. In chemistry, the colorimeter is an

apparatus that allows the absorbance of a solution at a particular frequency (color) of

visual light to be determined. Colorimeters hence make it possible to ascertain the

concentration of a known solute, since it is proportional to the absorbance.

A spectrophotometer is a photometer (a device for measuring light intensity) that

can measure intensity as a function of the color, or more specifically, the wavelength

of light. There are many kinds of spectrophotometers. Among the most important

distinctions used to classify them are the wavelengths they work with, the

measurement techniques they use, how they acquire a spectrum, and the sources of

intensity variation they are designed to measure. Other important features of

spectrophotometers include the spectral bandwidth and linear range. The most

common application of spectrophotometers is the measurement of light absorption.

 Experiment No. 5

AIM: To perform assay of paracetamol by UV- Spectrophotometry.

PRINCIPLE

Assay of paracetamol tablet is based on determination of absorption intensity of the solution

prepared as per procedure of Indian Pharmacopeia. Absorption of radiation, as a function of

frequency or wavelength, is measured, this absorption value is proportion to the concentration

of the drug substance (paracetamol) being analysed.

THEORY

Ultraviolet–visible spectroscopy or ultraviolet–visible spectrophotometry (UV–Vis or

UV/Vis) refers to absorption spectroscopy or reflectance spectroscopy in part of the

ultraviolet and the full, adjacent visible regions of the electromagnetic spectrum.

Absorption spectroscopy deals with the spectroscopic techniques that measure the

absorption of radiation, as a function of frequency or wavelength, due to its interaction with a

sample. The sample absorbs energy, i.e., photons, from the radiating field. The intensity of

the absorption varies as a function of frequency, and this variation is the absorption spectrum.

Absorption spectroscopy is employed as an analytical chemistry tool to determine the

presence of a particular substance in a sample and, in many cases, to quantify the amount of

the material present. Infrared and ultraviolet–visible spectroscopies are particularly common

in analytical applications. Absorption spectroscopy is also employed in studies of molecular

and atomic physics, astronomical spectroscopy and remote sensing.

REQUIREMENTS

Apparatus: Beaker, Volumetric flask, Pipette etc.

Chemicals: Paracetamol tablets, 0.1N Sodium hydroxide, distilled water etc.

PROCEDURE:
Assay: Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing

about 0.15 g of Paracetamol, add 50 ml of 0.1 M sodium hydroxide, dilute with 100 ml of

water, shake for 15 minutes and add sufficient water to produce 200.0 ml. Mix, filter and

dilute 10.0 ml of the filtrate to 100.0 ml with water. To 10.0 ml of the resulting solution add

10 ml of 0.1 M sodium hydroxide, dilute to 100.0 ml with water and mix. Measure the

absorbance of the resulting solution at the maximum at about 257 nm (2.4.7). Calculate the

content of C8H9NO2 taking 715 as the specific absorbance at 257 nm.

Observations

S. no. Weight of tablet (gm) S. no. Weight of tablet

1. 11. (gm)

2. 12.

3. 13.

4. 14.

5. 15.

6. 16.

7. 17.

8. 18.

9. 19.

10. 20.

Average weight (gm)

Label claim of given tablet = _______________ gm

Weight of twenty tablets = _____________ gm

Average weight of each tablet = ____________ gm-------- (A)

Average weight of each tablet (A) contains = 0.500 gm of paracetamol

x gm of powder contains = 0.15gm of paracetamol

0.15 × (𝐴)
x= = _________ gm --------- (B)
0.500

Wavelength Absorbance

257 nm

(𝐵) 10 10
% Dilution factor= × × × 100 = _________%w/v----------------(C)
200 100 100

Formula

𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑤𝑒𝑖𝑔ℎ𝑡 100

% Purity = × × (𝐶)
𝐸 1% 1 𝑐𝑚 𝐿𝑎𝑏𝑒𝑙 𝑐𝑙𝑎𝑖𝑚

% Purity = _____% w/w

Result: The Percent purity of the paracetamol tablet was found to be_____% w/w.

MCQ’s
1. Beer Lambert’s law gives the relation between which of the following?

A. Reflected radiation and concentration

B. Scattered radiation and concentration

C. Energy absorption and concentration

D. Energy absorption and reflected radiation

2. In which of the following ways, absorption is related to transmittance?

A. Absorption is the logarithm of transmittance

B. Absorption is the reciprocal of transmittance

C. Absorption is the negative logarithm of transmittance

 D. Absorption is a multiple of transmittance

3. Beer’s law states that the intensity of light decreases with respect to __________

A. Distance

B. Concentration

C. Volume

D. Composition

4. Beer’s law states that the intensity of light decreases with respect to __________

A. Distance

B. Concentration

C. Volume

D. Composition

5. What is the unit of absorbance, which can be derived from Beer Lambert’s law?

A. L mol-1 cm-1

B. L gm-1 cm-1

C. cm

D. No unit

Questions

1. What is principle of Ultraviolet-visible spectroscopy?

Ans: Ultraviolet-visible spectroscopy (UV-Vis spectroscopy) makes use of absorption

spectroscopy in ultraviolet and visible wavelength ranges―180–380 nm and 380–750

nm, respectively―for characterizing molecules. This is one of the most basic

techniques that need to be conducted while characterizing an analyte. All the major

classes of biomolecules contain certain light absorbing functional groups known as

chromophores. Upon absorbing UV/Vis light, these chromophores get excited from
 ground state to a higher energy level, thus giving out characteristic spectra, aiding in

the identification of specific biomolecules.

2. Which lamp is used in UV spectroscopy?

Ans: Deuterium lamps are always used with a Tungsten halogen lamp to allow

measurements to be performed in both the UV and visible regions. Also known as

quartz Iodine lamps, this measure most effectively in the visible region from 320 -

1100 nm.

3. What are the components of UV Visible Spectrophotometer?

Ans: There are four basic components to a simple single beam UV/Vis

spectrophotometer; a light source, a monochromator, a sample, and a detector.

4. What are the 7 visible spectrum colors?

Ans: A commonly taught acronym that helps people remember the colors in the

visible spectrum is (ROY G BIV) which stands for Red, Orange, Yellow, Green,

Blue, Indigo, and Violet.

5. Does cuvette size affect absorbance?

Ans: Yes, The absorbance is directly proportional to the concentration (c) of the

solution of the sample used in the experiment. The absorbance is directly proportional

to the length of the light path (l), which is equal to the width of the cuvette.
 Experiment No. 6

Aim:

Estimation of Quinine sulfate by Fluorimetry

Principle:

Fluorescence is the phenomenon of emission of radiation when there is transition from singlet

excited state to singlet ground state. The wavelength of absorbed radiation is called as

excitation wavelength and that of emitted radiation is called as emission wavelength.

Fluorescent intensity is directly proportional to concentration of substance. Fluorometer is the

instrument used to measure fluorescence with a wavelength varying between 240 nm to 800

nm.

Requirement:

Apparatus: Volumetric flask, Beaker, Pipette

Chemicals: Quinine sulphate, 0.1N H2SO4, Distilled water

Instrument: Fluorometer

Preparation of Standard Solution:

1) Weigh accurately 100 mg of Quinine sulphate powdered drug

2) Dissolve in 100 mL of 0.1 N H2SO4 to get Stock solution (1000 μg/mL)

3) Take 10 mL of stock solution and dilute to 100 mL with 0.1 N H 2SO4 (100 μg/mL)

4) Again add 1 mL of above solution and dilute to 100 mL with 0.1 N H2SO4 (10 μg/mL)

5) Prepare concentration of 0.5, 1, 1.5, 2, and 2.5 μg/mL from above solution and dilute to

10 mL with 0.1 N H2SO4.

Preparation of Sample Solution:

1) Pipette out 1 mL of given sample solution and make up the volume to 10 mL with with

0.1 N H2SO4
2) Switch on the instrument, set the excitation and emission filters at the wavelength 360 to

460 nm respectively

3) Set the Fluorescent Intensity (FI) to 0 % by using 0.1 N H 2SO4 as blank and 100 % by

using highest concentration of the standard solution 2.5 μg/mL.

4) Repeat the same at least for two or more time to avoid instrumental error

5) Measure the % FI of different standard and sample solutions

6) Plot the graph between the concentration vs FI and determine the concentration of

unknown sample by extrapolating the found FI.

Observation:

Sr No. Conc. (μg/mL) % FI

1 0

2 0.5

3 1

4 1.5

5 2

6 2.5

7 unknown

90
80
70
60
50
% FI 40
30
20
10
0
0 0.5 1 1.5 2 2.5

CONC (μg/mL)

Concentration of unknown sample =……… μg/mL

Sample solution dilutes by 10 times, so the conc. = ………. ˟ 10 = ………. μg/mL

Result:

The concentration of quinine sulphate in the given sample was found to be ……. mg/mL

MCQ`s

1) The process of raising electrons in the atoms or molecules to higher energy level is

called?

a) Partition

b) Transition

c) Excitation

d) Derivatization

2) ……….. is the general term applied to the absorption and emission of radiant energy

a) Fluorescence

b) Phosphorescence

c) Luminescence

d) Affinity Chromatography

3) Re-emission as radiation of all or a part of radiant energy is known as ……...

a) Phosphorescence

b) Fluorescence

c) Adsorption

d) Absorption

4) An increase in the intensity of the light incident on the sample produces proportional

……… in the fluorescence intensity

a) Decrease

b) Increase
 c) Alternation

d) Conjugation

5) Electron donating groups like -NH2, -OH, etc. often ………. Fluorescence.

a) Increase

b) Decrease

c) Nullify

d) Withdraw

Questions

1. Define Fluorescence. Enlist the factor affecting it.

Ans: Fluorescence is the emission of visible light by a substance that has absorbed

light of a different wavelength. The emitted photon has a longer wavelength.

2. Enlist types of Luminescence?

Ans:

i. Photoluminescence

a. Fluorescence

b. Phosphorescence

ii. Chemiluminescence

iii. Bioluminescence

iv. Triboluminescnce

v. Cathodluminescence

vi. Thermoluminescence

3. Define Phosphorescence?

Ans: It is related to fluorescence in emitting a photon; however a phosphorescent

material does not immediately re-emit the radiation it absorbs.

4. Enlist the factor affecting Fluorescence.

Ans:

i. Temperature

ii. pH

iii. Dissolved Oxygen

iv. Solvent

v. Adsorption

vi. Conjugation

5. Define Photoluminescence

Ans: The absorption of light is due to the absorption of photon (light) this

luminescence is called as Photoluminescence.

 Experiment No. 7

Aim:

Study of quenching of fluorescence

Requirements:

Apparatus: Volumetric flask, Beaker, Pipette

Chemicals: Quinine sulphate, 0.1N H2SO4, Distilled water, Potassium Iodide

Instrument: fluorometric

Principle:

Quenching is the decrease in fluorescence intensity due to specific effects or presence of

certain ions, constituents of the solution itself. These effects may be due to various factors

like Concentration, pH, presence of specific chemical substances, temperature, viscosity, etc.

Types of Quenching

Self-quenching

Chemical quenching

Static quenching

Collisional quenching

Procedure:

Preparation of Potassium Iodide solution:

Dissolve 100 mg KI in 100 mL 0.1N H2SO4

Take 10 mL of above solution and dilute to 100 mL with 0.1 N H2SO4 (100 μg/mL).

For final conc. Again add 1 mL of above solution and dilute to 100 mL with 0.1 N H2SO4 (10

μg/mL)

Preparation of Standard Quinine sulphate solution

1. Weigh accurately 100 mg of Quinine sulphate powdered drug

2. Dissolve in 100 mL of 0.1 N H2SO4 to get Stock solution (1000 μg/mL)

3. Take 10 mL of stock solution and dilute to 100 mL with 0.1 N H 2SO4 (100 μg/mL).

4. For final conc. Again add 1 mL of above solution and dilute to 100 mL with 0.1 N

H2SO4 (10 μg/mL)

5. In six 10 mL volumetric flask take 1 mL of standard quinine solution.

6. Add 1,2,3,4 and 5 mL of KI solution in each flask and make up the volume to 10 mL.

7. Set the FI to 0% by using 0.1 N H2SO4 as blank and 100 % by using standard solution

(without KI)

Figure 3 : Typical spectrum depicting quenching of fluorescence intensity of quinine

sulfate by potassium iodide
Observation Table:

Sr No. Volume of KI (mL) %FI

1 0

2 1

3 2

4 3

5 4

6 5

Result:

A decrease in fluorescence intensity shall be observed for quinine solution with the addition

of potassium iodide solution.

MCQ`s

1) ………… is reduction of fluorescence intensity by the presence of substances in the

sample other than the analyte

a) Quenching

b) Filtering

c) Excitation

d) Derivatization

2) Fluorescent compounds have characteristic ……….

a) Excitation

b) Emission

c) A and B

d) None of above

3) Fluorescence is ………. In molecules that possess rigid structure

 a) Increases

b) Decreases

c) Remain unaffected

d) None of above

4) Static quenching occurs mainly because of ……….

a) Colloidal effect

b) Collisional effect

c) Complex formation

d) Chemical changes

5) When conc. increases, fluorescence intensity does not increase proportionally. This

phenomenon is called as …………

a) Self-Quenching

b) Chemical Quenching

c) Static Quenching

d) Collisional Quenching

Questions

1. Define Quenching.

Ans: In materials science, quenching is the rapid cooling of a work piece in water,

oil or air to obtain certain material properties. A type of heat treating, quenching

prevents undesired low-temperature processes, such as phase transformations, from

occurring.

2. Enlist types of Quenching

Ans:

i. Self or Concentration Quenching

ii. Collisional
 iii. Static
iv. Chemical
3. Enlist Detectors used in Fluorimetry.

Ans:

i. Photovoltaic Cell
ii. Phototube
4. What is the effect of viscosity in Fluorescent intensity?

Ans: Increase in viscosity leads to decreased collisions of molecules which lead to

enhancement of fluorescence intensity & vice versa.

5. Give the instruments that measure fluorescence?

Ans:

i. Filter monochromator
ii. Spectrofluorometer
 Experiment No. 8

Aim:

Determination of sodium by flame photometry

Requirement:

Apparatus: Volumetric flask, beaker, pipette

Chemicals: Sodium Chloride, Distilled water

Instrument: Flame Photometer

Principle:

When a solution of metallic salt is sprayed on the flame, fine droplets are formed. Due to the

thermal energy of the flame, the solvent in the droplets evaporate, leaving behind fine

residue, which are converted to neutral atoms. These neutral atoms are converted to excited

state atoms by the thermal energy of the flame. As the excited state is not stable, these excited

atoms return to ground state, with the emission of radiation of specific wavelength. The

wavelength of the radiation emitted is characteristic of the element and is used to identify the

element. The intensity of the radiation emitted depends upon the conc. of the element

analysed.

Liquid sample formation of droplets fine residue formation of

Neutral

atoms

λ & Intensity of emitted Emission of radiation Excitation of radiation

radiation measured of specific wavelength by thermal energy

Procedure:

1) Weigh accurately 100 mg of NaCl and dissolve in 100 mL of distilled water

2) Take 10 mL of the solution and dilute to 100 mL with distilled water

3) Prepare a series of standard solutions of 10, 20, 30, 40 and 50 μg/mL concentrations.

4) Switch On the Instrument and select the sodium filter

5)
Set the gas in the flame in order to get non-luminous flame and air pressure at 0.4 to 0.5

kg/cm3

6) Atomize the Flame intensity to 0 % using distilled water with the knob.

7) Atomize the Flame intensity to 100 % using 50 μg/mL standard solutions (highest conc.).

8) Measure the percent flame intensity of all the standard solutions (10, 20, 30, 40 and 50

μg/mL concentrations)

9) Plot the graph of conc. of solution and % Flame intensity

10) From the graph, mark the % flame intensity of the unknown sample and by extrapolating,

determine the corresponding conc.

Dilutions:

100 mg dissolve in 100 mL water = 1000 μg/mL

10 mL diluted to100 mL water = 100 μg/mL

1 mL of 100 μg/mL diluted to 100 mL =10 μg/mL

2 mL of 100 μg/mL diluted to 100 mL = 20 μg/mL

3 mL of 100 μg/mL diluted to 100 mL = 30 μg/mL

4 mL of 100 μg/mL diluted to 100 mL = 40 μg/mL

5 mL of 100 μg/mL diluted to 100 mL = 50 μg/mL

Observation Table:

Sr No. Conc (μg/mL) FI (%)

1 0
 2 10

3 20

4 30

5 40

6 50

7 unknown

Result:

The conc. of sodium ion in the given sample of NaCl solution was found to be ……. μg/mL

by Flame photometry

MCQ`s

1) To get higher temperature in the burner, in flame photometer, the following combination

is used …….

Hydrogen/Air

Hydrogen/Oxygen

Acetylene/Air

Acetylene/Oxygen

2) Which of the following element is most easily detected by flame photometry?

Lithium

Calcium

Sodium

Beryllium

3) Which of the following is not the requirement of a good flame in flame photometer?

Liquid sample must be evaporated to form solid residue

Solid residue must decompose to form atoms

 Atoms must be produced such that they have the ability to get excited to higher states

Atoms must be produced such that they are in stable states

4) Which of the following is the principle of flame emission photometers?

Radiation is absorbed by non-excited atoms in vapour state and are excited to higher

states

Medium absorbs radiation and transmitted radiation is measured

Colour and wavelength of the flame is measured

Only wavelength of the flame is measured

5) Which is application of flame photometry?

To determine functional group

To study of chemical structure

To assay of drug

To estimate metallic ions like sodium, potassium, etc.

Questions

1. Define Flame Photometry.

Ans: It is the measurement of the intensity of light emitted at a particular wavelength

from the atoms that are excited thermally in flame.

2. Describe the steps involved in Flame Photometry.

Ans:

3. Enlist the components of Flame Photometer.

Ans:

i. Fuel gas Pressure Regulator

 ii. The atomizer

iii. Sample cell

iv. The Nebulizer-burner System

v. Optical System

vi. Photo sensitive detector

vii. Recording or read out device

4. Describe the function of flame in Flame Photometer.

Ans:

To convert the constituent of liquid sample into vapour state

To decompose the constituent into atoms or simple molecules:

To electronically excite a fraction of the resulting atomic or molecular species.

5. Enlist methods of Atomization

Ans:

i. Flame atomization

ii. Electro Thermal Atomization.

 Experiment No. 9

Aim:

Determination of potassium by flame photometry

Requirement:

Apparatus: Volumetric flask, beaker, pipette

Chemicals: Potassium Chloride, Distilled water

Instrument: Flame Photometer

Principle:

When a solution of metallic salt is sprayed on the flame, fine droplets are formed. Due to the

thermal energy of the flame, the solvent in the droplets evaporate, leaving behind fine

residue, which are converted to neutral atoms. These neutral atoms are converted to excited

state atoms by the thermal energy of the flame. As the excited state is not stable, these excited

atoms return to ground state, with the emission of radiation of specific wavelength. The

wavelength of the radiation emitted is characteristic of the element and is used to identify the

element. The intensity of the radiation emitted depends upon the conc. of the element

analysed.

Liquid sample formation of droplets fine residue formation of

Neutral

atoms

λ & Intensity of emitted Emission of radiation Excitation of radiation

radiation measured of specific wavelength by thermal

Procedure:

1) Weigh accurately 100 mg of KCl and dissolve in 100 mL of distilled water

2) Take 10 mL of the solution and dilute to 100 mL with distilled water

3) Prepare a series of standard solutions of 10, 20, 30, 40 and 50 μg/mL concentrations.

4) Switch On the Instrument and select the potassium filter,

5)
Set the gas in the flame in order to get non-luminous flame and air prtessure at 0.4 to 0.5

kg/cm3

6) Atomize the Flame intensity to 0 % using distilled water with the knob.

7) Atomize the Flame intensity to 100 % using 50 μg/mL standard solution (highest conc.).

8) Measure the percent flame intensity of all the standard solutions (10, 20, 30, 40 and 50

μg/mL concentrations)

9) Plot the graph of conc. of solution and % Flame intensity

10) From the graph, mark the % flame intensity of the unknown sample and by extrapolating,

determine the corresponding conc.

Dilutions:

100 mg dissolve in 100 mL water = 1000 μg/mL

10 mL diluted to100 mL water = 100 μg/mL

1 mL of 100 μg/mL diluted to 100 mL =10 μg/mL

2 mL of 100 μg/mL diluted to 100 mL = 20 μg/mL

3 mL of 100 μg/mL diluted to 100 mL = 30 μg/mL

4 mL of 100 μg/mL diluted to 100 mL = 40 μg/mL

5 mL of 100 μg/mL diluted to 100 mL = 50 μg/mL

Observation Table:

Sr No. Conc (μg/mL) FI (%)

1 0
 2 10

3 20

4 30

5 40

6 50

7 unknown

Result:

The conc. of potassium ion in the given sample of KCl solution was found to be ……. μg/mL

by Flame photometry

MCQ`s

1) Which of the following is the principle of Flame emission photometers?

Radiation is absorbed by non-excited atoms in vapour state and are excited to higher

states

Medium absorbs radiation and transmitted radiation is measured

Colour and wavelength of the flame is measured

Only wavelength of the flame is measured

2) Which is not application of flame photometry?

To estimate sodium, magnesium, calcium

Assay of metformin.

Used to determine magnesium and calcium in cement.

To detected metallic ions in sample

3) Choose correct sequence of flame photometry?

Sample residue→excited atoms→Return in ground state→Emission of radiation

Sample residue → ground state → excited state → Emission of radiation

 Emission of radiation → excited state →ground state → Sample residue

Sample residue → ground state → excited state → Emission of radiation

4) Which of the following is not a detector used in Flame emission photometers?

Photronic cell

Photovoltaic cell

Photoemissive tube

Chromatogram

5) If Hydrogen and air are used in burner how many °C temp. is produced?

1900°C

2200°C

2300°C

2100°C

Questions

1. Enlist detectors used in Flame Photometry.

Ans:

i. Photovoltaic Cells or Barrier-Layer Cell

ii. Phototubes or Photoemissive tubes

iii. Photomultiplier tubes.

2. Give advantages of Flame Photometry

Ans:

i. Very simple method of analysis

ii. Quick

iii. Convenient

iv. Selective & Sensitive for analysis

 v. Low Concentrations of metals can be determined.

3. Enlist the fuel gas used in Flame Emission Spectroscopy

Ans:

i. Propane

ii. Hydrogen

iii. Acetylene

4. Define Interferences.

Ans:

Differences between the sample & the standard solutions can lead to systemic errors

are called as interferences.

5. Enlist Interferences in Flame Emission Spectroscopy

Ans:

i. Spectral Interferences

ii. Background Interferences

iii. Chemical Interferences

iv. Cation Interferences

v. Anion Interferences

vi. Oxide Formation Interferences

vii. Foreign Elements Interferences

viii. Salts & Acids Interferences

ix. Matrix Interferences

 Experiment No. 10

Aim:

Determination of Chlorides and Sulphated by Nepheloturbidimtery

Requirement:

Chloride standard solution, 0.1N Silver Nitrate, Sulphate Standard solution, Dilute nitric acid,

Barium Chloride Solution, Dilute acetic acid, Distilled water.

Principle:

Nephelometry: It is the measurement of scattered light as function of concentration of

suspended particles. The intensity of scattered light is proportional to the concentration. The

intensity of scattered light is normally measured at 90º. It can also be measured at any

convenient angle like 45º, 60º,135º etc.

Turbidimetry: It is measurement of transmitted light as function of concentration of

suspended particles. The intensity of transmitted light is measured at 180º. The intensity of

transmitted light as a function of concentration i.e. when concentration is more, it is less and

when concentration is less, It is more.

Procedure:

Determination of Chloride:

Preparation of Standard Opalescence:

1) Take 10 mL of Chloride standard solution (25 ppm) into Nessler cylinder.

2) Add 10 mL of Dilute HNO3 and make up the volume to 50 mL with distilled water.

3) Add 1 mL of 0.1 N AgNO3 solution and stir immediately to produce the turbidity.

4) Allow it to stand for 5 minutes protected from light.

Preparation of Sample Opalescence:

1) Pipette out 10 mL of sample solution into Nessler cylinder.

2) Add 10 mL of dilute HNO3 and make up the volume to 50 mL with distilled water
3) Add 1 mL of 0.1 N AgNO3 and stir immediately.

4) Allow the resulting solution to stand for 5 minutes protected from light.

Measurement of Nepheloturbidimetric unit (NTU)

1) Switch on the instrument

2) Allow it to stabilize for 10 minutes.

3) Place distilled water and adjust reading to 0 %

4) Discard the water and take standard opalescence and adjust the reading to 100 %.

5) Run sample opalescence and compare the % NTU with Standard.

Determination of Sulphate:

Preparation of Standard Opalescence:

1) Pipette out 1.5 mL of ethanolic standard sulphate solution into Nessler cylinder.

2) To this add, 1 mL of 25 % w/v barium chloride solution.

3) Mix and allow to stand for 1 minute.

4) To this add, 15 mL of (10 ppm) standard sulfate solution and 0.15 mL of 5 M acetic acid.

5) Stir immediately with glass rod and allow to stand for 5 minutes.

Preparation of Sample Turbidity:

1) Pipette out 1.5 mL of ethanolic standard sulphate solution into Nessler cylinder.

2) To this add, 1 mL of 25 % w/v barium chloride solution.

3) Mix and allow to stand for 1 minute.

4) To this add, 15 mL of sample solution.

5) Add 0.15 mL of 5 M acetic acid.

6) Make up the volume to 50 mL with distilled water.

7) Stir immediately with glass rod and allow to stand for 5 minutes.

Measurement of Nepheloturbidimetric Unit (NTU):

1) Switch on the instrument

2) Allow it to stabilize for 10 minutes.

3) Place distilled water and adjust reading to 0 %

4) Discard the water and take standard opalescence and adjust the reading to 100 %.

5) Run sample opalescence and compare the % NTU with Standard.

Observation Table:

For Chloride:

Sr. Preparation NTU Range Meter Reading Total NTU x Meter

No. Reading

1. Standard 1000

2. Standard

For Sulphates:

Sr. Preparation NTU Range Meter Reading Total NTU x Meter

No. Reading

1. Standard 1000

2. Standard

Result:

As the NTU range x meter reading of the sample found less than that of the standard solution,

therefore the given sample passes/fails the limit test for sulphate. Similarly, as the NTU range

x meter reading of the sample found less than that of the standard solution, therefore the

given sample passes/fails the limit test for chloride.

MCQ`s

1) Smaller particles undergo scattering to give rise a symmetrical pattern of secondary rays

in space having maximum intensity of …………

1) 90

2) 180

3) 60

4) 45

2) Phosphorous can be determined by turbidimetry using which reagent

1) Nessler reagent

2) Strychnine Molybedate reagent

3) Barium sulphate

4) Silver chloride

3) Turbidity is the official assay method for ………

1) Vitamin B12

2) Vitamin A

3) Vitamin B1

4) Vitamin B6

4) Nepheloturbidimtery method is ……… times better than HPLC method

1) 50 times

2) 100 times

3) 80 times

4) 90 times

5) Turbidity of macromolecules depends on ………

1) Number of molecules
2) Concentration

3) Molecular weight

4) Polarity of Molecules

Questions

1. Define Quenching.

Ans: In materials science, quenching is the rapid cooling of a work piece in water,

oil or air to obtain certain material properties. A type of heat treating, quenching

prevents undesired low-temperature processes, such as phase transformations, from

occurring.

2. Enlist types of Quenching

Ans:

i. Self or Concentration Quenching

ii. Collisional

iii. Static

iv. Chemical

3. Enlist Detectors used in Fluorimetry.

Ans:

i. Photovoltaic Cell

ii. Phototube

4. What is the effect of viscosity in Fluorescent intensity?

Ans: Increase in viscosity leads to decreased collisions of molecules which lead to

enhancement of fluorescence intensity & vice versa.

5. Give the instruments that measure fluorescence?

Ans:

i. Filter monochromator
ii. Spectrofluorometer
 Experiment No. 11

AIM- To perform the paper chromatography for the separation of amino acids present in the

given sample.

THEORY

The chromatographic techniques used to separate out mixtures of different substances into

their discrete components. All forms of chromatographic techniques work on the same

principle. They all have basic requirements of stationary phase (a solid or a liquid supported

on a solid) and a mobile phase (a liquid or a gas). The mobile phase tides through the

stationary phase and carries the components of the mixture with it. Different components

travel at different rates based on their affinities toward stationary phase and mobile phase. In

paper chromatography, the stationary phase is a very uniform adsorbent paper and the mobile

phase is a suitable liquid solvent or mixture of solvents.

Retention (or) retardation factor (Rf)

Retention factor value (Rf Value) is defined the ratio of the distance travelled by the solute to

the distance travelled by solvent. The distance travelled relative to the solvent is called the Rf

value.

Rf = Distance travelled by solute / Distance travelled by solvents

REQUIREMENTS

Apparatus: Glass beakers, Whatmann filter paper, Petridishes, Measuring cylinder,

developing chamber and Capillary tubes etc.

Chemicals: n-butanol, Glacial acetic acid, Distilled water (4:1:5), Amino acids (Tryptophan

and threonine), Ninhydrin reagents etc.

PROCEDURE

Solvents system and methods preparation – For solvents system and methods preparation

n-butanol and water are taken in ratios of 4:5 in a flask and allow it to saturate for 24 hours,
by using the separating funnel separate out the n - butanol and water. The saturated n -

butanol and glacial acetic acid both are taken in the 4:1 ratio which can be used as a solvent

system (or) mobile phase.

Paper chromatography - For paper chromatography method is quite simple as compared to

other methods of chromatography. The chromatography paper is cut into rectangular strips

and marks a line on the paper with pencil at about 2 cm from the bottom. With the help of

capillary tube, the samples are applied at different points on the starting line and place the

chromatography paper in the developing chamber, which contains the mobile phase. While

placing the paper, it is important that the solvent level should not reach the starting line or the

sample spots and paper shouldn’t touch the walls of the developing chamber. After sometime

the solvent rises up the paper or the stationary phase by capillary action and dissolves the

sample. The components of the sample move along with the solvent in upward direction.

Checkered if the solvent has reached near the top level of chromatography paper, then the

paper is removed when it reaches the top and marked the level with pencil. This level (or)

height is called the “solvent front”. By using UV light, Ninhydrin or iodine vapors examined

the different spots of varied colors.

RESULTS

For paper chromatography

The distance moved by tryptophan and threonine is …… cm and ….. cm respectively, and

the solvent is ……cm

Rf value of tryptophan is ……

Rf value of threonine is ……..

Rf value of unknown mixture is ….. & …..

MCQ’s

1. Which is not development technique of paper chromatography?

a. HPLC

b. Ascending
 c. Descending

d. Two dimensional

2. Which type of mobile phase (MP) is used in paper chromatography?

a. The MP which gives Rf value range between 0.8-1

b. The MP which gives Rf value range between 0.6.

c. The MP which gives Rf value range between 0.1-0.3

d. The MP which gives Rf value range between 0.2-0.8

3. When there are too complex mixture are separated which development technique of paper

Chromatography is used?

a. Ascending

b. Two dimensional

c. Descending

d. Radiation

4. How much time is essential to saturate of chamber?

a. 24 hour

b. 6 hour

c. 12 hour

d. 10 hour

5. Which force is control for the separation of the components in descending paper

chromatography?
 a. Partition

b. Adsorption

c. Gravity

d. All of the above

Questions
1. What do you mean by paper chromatography?
Ans. Paper chromatography is applicable as a separation technique where separation
of a mixture of compound is mainly achieved by a flow of solvent on a
chromatographic paper and stationary phase has bound or absorbed water present with
the cellulose of the chromatographic paper and the mobile phase is an organic solvent
which is immiscible with stationary phase.
2. What is a principle of paper chromatography?
Ans. Paper chromatography is mainly work on principal of the adsorption &
partition. The mechanism of separation in paper chromatography is mainly partition
in Type. So, when a drop of the solute is treating with the solvent on the paper, the
more strong components back while less strong components are move forward.
3. Define Rf value with their formula.
Ans. The relative rate of the movement of solvent and solute is expressed by a term
Rf. It is defined as the ratio of the distance travelled by the compound at
its maximum.
Rf = Distance travelled by solute / Distance travelled by solvents

In many cases it has been observed that the solvent front os run off the end of the
chromatogram. Rx value is the ratio of the distance travelled by a substance to the
distance travelled by a reference standard.

4. Enlist factors, which effects on RF value.

Ans. Following factors are generally effect on Rf Value - The temperature of
environment, Purity of solvent used viscosity of solvent, The type of paper, speed of
paper, ash content, pH of the mobile phase, Chemical reaction if any during
development.
5. Give chromatographic development stages.
Ans. There are mainly three technique are developed

- Ascending
- Descending
- Radial
- Two dimensions
 Experiment No. 12

AIM: To perform the separation and analysis of some sugars by using thin layer

chromatography (TLC)

THEORY

The inordinate consideration has been given just too some comparatively rapid techniques of

analysis. Thin layer chromatography (T.L.C.) is previously accepted and established as a

laboratory tool for routine analytical work. Its low cost effective, ease, and rapidity along

with its capacity for separating and identifying small quantities of compound mixtures make

the technique important tool for research. The components with less affinity towards the

stationary phases are travels rapidly. The major objective of this investigation was to adapt a

method for separation, identification, and approximation of different sugars in beet

processing liquors, thick juice from storage, and beet storage samples.

REQUIREMENTS

Apparatus: Glass beakers, measuring cylinder, developing chamber glass plates etc.

Chemicals: Chloroform, acetic acid, and water, 85% Orthophospheric acid, diphenylamine,

aniline acetone etc.

PROCEDURE

For the preformation of TLC practical be prepared following things firstly as Glass plates are

precoated with 0.25 mm dry silica gel. Solvent system which consists of a mixture of

chloroform, acetic acid, and water (3:3.5:0.5) by volume, respectively and spraying agent is
made from 1 gram diphenylamine and 1 ml of aniline in 100 ml acetone. This mixture is

further mixed with 85% orthophosphoric acid prior to use (10: 1 v/v, respectively).

Sample is applied and then the plate is dried in air for approximately 30 minutes. The solvent

system irrigated within the ascending direction in a tight container. The solvent is allowed to

move upward about 12.5 cm, this usually requires 90 minutes. The plate is removed from the

tank. Then it is kept for drying in air for about 30 minutes. The plate is placed back in the

same developing solvent and let the solvent to move in the same direction to the same

distance of 12.5 cm. It takes 45minutes and plates should then be dried for approximately 30

minutes.

RESULTS

The distance moved by glucose .… and …… is …… cm and ….. cm respectively and the

solvent is ……cm.

Rf value of glucose is ……

Rf value of fructose is ……..

MCQ’s

1. The components with less affinity towards the stationary phases is

a. Travels slowly

b. Travels rapidly

c. A and B

d. None of the above

2. The mobile phase is flow by

a. Partition coefficient

b. Temperature
 c. Capillary action

d. All of the above

3. Which verdict is false about

a. It is less time consuming.

b. It is very sample method

c. It is very costly.

d. Applied to allow type of sample.

4. What is the principle of Thin Layer Chromatography?

a. Absorption

b. Adsorption

c. Partition

d. A and B

5. In TLC following which is not factor for choosing adsorbent?

a. Temperature.

b. Nature of the adsorbent

c. Solubility of the adsorbent

d. Chemical nature

Questions
1. What is a principle of thin layer Chromatography (TLC)
Ans. The principle of TLC is separation is adsorption. When the mixture contains one
or more components is spotted on a TLC of absorbent coated on a chromatographic
plate and introduce into mobile phase, which are continuing distributing. The mobile
phase is flow by capillary action. The components with more affinity travel slow and
that of lesser affinity travel faster. TLC is method of analysis in which the stationary
phase is spread as a thin layer on a rigid supporting plate. The mobile phase a liquid is
allowed to migrate across the stationary of plate. TLC is a solid- liquid form of
Chromatography, where the stationary phase is polar and mobile phase is non polar.
 2. Give characteristics of thin layer Chromatography (TLC)
Ans. - It’s a simple and rapid technique
- Low cost technique and takes less time
- Applied for all types of mixture samples
- Detection is easy and not tedious
- More efficiency of separation
- Corrosive spray reagents can be used without any damage to plate

3. Why activation of TLC plate is necessary?

Ans. Plate is also allowed to saturate with these solvent vapour if required by
exposing the plate to solvent vapour is closed chamber. Then the plate is dipped in
developing to effect separation of analytical mixture. Horizontal, sandwich, two-dimensional
techniques are also use in TLC.

4. What is a purpose of TLC?

Ans. Thin - layer chromatography (TLC) is a chromatography technique used to
separate non-volatile mixtures. Thin-layer chromatography can be used to monitor the
progress of a reaction, identify compounds present in a given mixture, and determine the
purity of a substance.
5. Why silica gel is used in TLC?
Ans. Silica gel is by far the most widely used adsorbent and remains the dominant
stationary phase for TLC. The surface of silica gel with the highest concentration of
geminal and associated silanols is favored most for the chromatography of basic
compounds because these silanols are less acidic.
 Experiment No. 13

AIM - To separate the plant pigments by using column chromatography.

THEORY

The plants leaves contain a different colored pigments generally falling into two categories,

carotenoids and chlorophylls. Carotenoid is a part of a larger collection of plant - derived

compounds called terpenes and they are tetraterpenes (eight isoprene units). Chlorophylls A

and B are the pigments that make plants look green. Lycopene, the compound responsible for

the red coloring of watermelon, tomatoes and β -carotene, the compound that causes carrots

and apricots to be orange, are examples of carotenoids. Spinach leaves contain chlorophyll a

and b and β -carotene as major pigments as well as smaller amounts of other pigments.

Chlorophyll A and B are same in structure and may not be able to be resolved in this process.

REQUIREMENTS

Chemicals: Petroleum ether (hexane) and acetone etc.

Apparatus: Round bottom flask, chromatography column pipettes, glass beakers, measuring

cylinder etc.

PROCEDURE

Extraction of the pigments – For Extraction of the pigments takes about 5 grams of leaves

and dried and placed it in a mortar and then pigments are extracted by grinding the leaves

with a pestle with about 5-10 ml of in the ratio 80:20 mixture (v/v), petroleum ether (hexane)

and acetone after it liquid decanted into a 50 ml round bottom flask. A quick filtration is must

require.

Preparation of the column – For Preparation of the column wet pack method is used. The

chromatography column made with plastic tip with frit, the one-way stopcock, and the plastic
funnel. The column is filled with enough alumina to get the required height. The dry alumina

is poured into a beaker and hexane (pet ether) is added. The mixture is swirled and then

poured into the column. The column is tapped gently, so air is not trapped as the alumina

settles. Then it is added with a small amount of sand after the alumina has been settled. The

column should not contain air bubbles and should be homogeneous. Then the solvent level is

allowed to drop to the level of the alumina sand intersection.

Running the column - Using a long pipette, some of the pigment mixture is added directly

onto the sand. Then it is added enough to fill the sand layer with color. Then the stopcock is

opened and let the liquid level falls to the top of the alumina. Gently add petroleum ether to

fill the sand layer. Then the stopcock is opened and let the liquid level fall to the top of the

alumina. These steps are repeated at least three times or until all the colored compounds are

in the alumina. Now the column is filled with petroleum ether.

Kindly do not ever let the column run dry, then the open stopcock to allow a drip rate of

around 1 drop per second. First the yellow-orange β-carotene is eluted. As the yellow-orange

colored product is eluted, it was collected in a test tube. When the β-carotene has been

eluted, the elutions of the chlorophylls are eluted by using a more polar solvent. Let the

solvent level fall to the top of the alumina. Gently the column was filled with either pure

acetone or a petroleum ether acetone combination by using 70% petroleum ether: 30 %

acetone combination, it might be able to separate chlorophylls A and B and then the

chlorophylls collected in the separate test tube.

RESULTS

The Rf values of the above plant pigments are ___________.

MCQ’s
1. . What do you mean eluent?

a. is a liquid solution that is a result from Elution

b. is a liquid solution

c. is a solvent that used for separation of absorbed material from

stationary phase

d. None of the above

2. In column chromatography which compound are hold by stationary phase?

a. Nom polar compound

b. Polar compound

c. Both A and B

d. None of the above

3. Chromatogram is a _______________

a. Solute concentration vs. Elution time

b. Solute concentration vs Elution volume

c. Both A and B

d. None of the above

4. Which force is involved in the column chromatography?

a. Hydrogen bonding

b. London force

c. Electric static force

d. All of the above

5. In which Chromatography stationary phase is more polar than mobile phase?

a. Reversed chromatography

b. Ion exchange
 c. Liquid Chromatography

d. None of the above

Questions
1. What is principle of column chromatography?
Ans. The principle behind column chromatography is adsorption, in which a
mixture of components dissolved in the mobile phase is introduced in to
the column and the components move depending on their relative affinities. The
choice of the solvent depends on the solubility characteristics of the mixture.
2. Enlist steps involved in column chromatography?
Ans. - Preparation / Packing of an adsorbent column
- Selection of Solvent system / Mobile phase
- Application of samples
- Sample elution
- Collection and analysis of fraction
3. What are the kinds of column chromatography?
Ans. Five distinct chromatographic methods that are use columns are
gas chromatography (GC), liquid chromatography (LC), Ion exchange
chromatography (SEC) and Chiral Chromatography.

4. Why is Rf useful?
Ans. Rf values can be used to identify unknown chemicals if they can be compared to
a range of reference substances. The Rf value for a particular substance is always the
same if the same solvent and stationary phase are used.

5. What do you mean by chromatography?

Ans. Chromatography is based on the main principle where molecules applied in
mixture onto the surface or into the solid, and fluid stationary phase is separating
from each other while moving with the aid of a mobile phase.
 Experiment No. 14

AIM - To perform assay of Azithromycin by HPLC in tablet dosage form.

THEORY

As an antibiotic azithromycin is administered in different dosage forms for the treatment

infections. Few of them infection symptoms includes as intestinal infections, middle ear

infections, throat infection, pneumonia, traveler’s diarrhea, and overall for respiratory tract

infections. The molecular weight of azithromycin is 748.996 g/mol and molecular formula is

C38 H72 N2 O12.

REQUIREMENTS

Chemicals: Azithromycin tablets, dipotassium hydrogen phosphate, phosphoric acid,

acetonitrile, water etc.

Apparatus: Volumetric flask, mortar pestle, weighing balance, filter paper, HPLC

instrument

HPLC column [C18 column (15 mc x 4.6 mm, 5 μm)] etc.

PROCEDURE

Buffer solution - Dissolve 1.16 gm of dipotassium hydrogen phosphate in water and dilute to

100 ml with water. Adjust pH to 6.5 with phosphoric acid.

Mobile phase - Mix buffer: acetonitrile: water in the ratio of 10:35:55

Diluent - Mix acetonitrile: water in the ratio of 40:60

Standard solution - Weigh 20 mg Azithromycin (API) in 100 ml volumetric flask containing

about 50 ml of diluent, mix well and make up the volume up to 100 ml by diluent.
Sample solution - Triturate azithromycin tablets in a mortar pestle. Weight 20 mg of

powdered sample in a 100 ml volumetric flask containing about 50 ml of diluent, shake

strongly to dissolve complete for about 10 -15 minutes, make up the volume up to 100 ml by

diluent. Filter sample solution by 0.45 μm filter paper.

Chromatographic conditions – Kindly maintain the chromatographic conditions are as -

Column size - 18 column (15 mc x 4.6 mm, 5 μm), Column temperature - 70°C, Mobile

phase flow rate - 1 ml/min, Detection wavelength - 215 nm, Injection volume - 100 μl, Run

the HPLC only with mobile phase at least for 10 minutes for conditioning of column.

At the present inject standard solution six times by taking solution from volumetric flask each

time and record the response/area. Similarly inject filtered sample solution six times by

taking solution from volumetric flask each time and record the response/area.

Observation table -

Sr. No. Standard Area Sample Area

Avg.

Calculation –
RESULTS

The content (mg/tab) of market brand tablet of Azithromycin was found to be ____.

MCQ’s

1. Which of the following sentence is not applicable of HPLC?

a. Ion-exchange chromatography of protein

b. Pre-concentration of trace components

c. Identification of polysaccharide

d. Ligand-exchange chromatography

2. Which of the following types of liquid chromatography uses immobilized

biochemical as a stationary phase?

a. Affinity chromatography

b. Gel permeation chromatography

c. Exclusion chromatography

d. Ion exchange chromatography

3. In reversed phase HPLC, we used ______________.

a. Non-polar solvent / polar column

b. Polar solvent / polar column

c. Polar solvent/ Non polar column

d. All of the above

4. The main purpose of the derivatization in HPLC is ________.

a. To decrease the retention time

b. To increase the resolution

c. To increase the detectability of a compound

d. All of the above

 5. In HPLC degassing of the mobile phase can be done by all of the following except

a. Reverse Osmosis

b. Vacuum Pumping

c. Distillation

d. Sparging

Questions
1. What is principle of HPLC?
Ans. High performance liquid chromatography (HPLC) is an analytical technique
which useful for separation, identification or quantification each component in a
mixture. The mixture is separated using the basic principle of column chromatography
and then identified and quantified.
2. Which types detectors is used in HPLC?
Ans. Following common detectors used in HPLC are as UV-Vis Detectors, Refractive
Index Detector, Fluorescence Detectors, Evaporative Light Scattering Detector, and
Conductivity Detector.
3. Which typescolumn employed in HPLC?
Ans. Normal Phase HPLC Columns
Reverse Phase HPLC Columns
Ion Exchange HPLC Columns
Size Exclusion HPLC Columns
4. What are troubleshooting errors in HPLC?
Ans. Pumping system is common problem occurs and some of the more common
indications are erratic retention times, noisy baselines, or spikes in the
chromatogram. Leaks at pump fittings or seals will result in poor chromatogram.
5. Gives process for preparation of standard and sample solution in HPLC
Ans. Standard solution - Weigh 20 mg sample in 100 ml volumetric flask containing
about 50 ml of diluent, mix well and make up the volume up to 100 ml by diluent.
Sample solution - Triturate the sample in a mortar pestle and weight 20 mg of
powdered sample in a 100 ml volumetric flask containing about 50 ml of diluent,
shake strongly to dissolve complete for about 10 -15 minutes, make up the volume up to 100
ml by diluent. Filter sample solution by 0.45 μm filter paper.
 Experiment No. 15

AIM – Introduction of Gas Chromatography

THEORY

This technique is generally used to separate the gases in gaseous solutions. The more mutual

technique is gas - liquid chromatography in which the stationary phase is a porous solid

covered with an absorbing the liquid. Gas-liquid chromatography (GLC) is commonly used

for separation of wide variety of organic compounds. The basic requirements for GLC are

that the sample may be volatile and that it not decomposes in all vaporization process.

Meanwhile the vaporization occurs in an inert atmosphere and decomposition of sample is

generally not a problem in which.

A basic chromatograph consists of the following:

 An injector for introduction and vaporization of the sample.

 A separating column consisting with metal tubing packed with a solid material coated

with the stationary absorbing liquid.

 A carrier gas, N2 or He to sweep of sample throughout column.

 A flow control equipment for the maintenance a constant flow of carrier gas

throughout column.

 A detector for the measurement of quantity of a separated component.

 A hot air ovens and heaters for the column, detector and injector temperature control.

 An integrator and strip chart recorder combination to provide the maintain record of

these analysis.
The separation of different mixtures into its components depends upon the solubility

differences of sample vapour in a liquid (stationary phase), and generally stationary phase is

coated in a thin layer on the solid particles of large surface area and then packed uniformly

into the column. A constant flow of an inert carrier gas badges through the column and

transports solute molecules in the gas phase. The column is enclosed by an oven for precise

temperature control.

A sample of the analyte is introduced by syringe injection into the heated injector tube, where

it is vaporized and mixed with the carrier gas. As the sample vapor/carrier gas mixture flows

onto the column, the analyte partitions between the gas and liquid phases according to the

analyte component's solubility in the liquid at the column operating temperature. This

equilibrium partitioning continues as the sample is moved through the column by the carrier

gas. The rate at which the sample travels through the column is determined by the sample

solubility in the stationary phase, the carrier gas flow rate, and the temperature gradient

(temperature program) applied. Each component travels at a characteristic rate, and if the

column has sufficient length and resolving power, the sample will be completely separated by

the time it reaches the detector and identified by comparing the "retention time".

INSTRUMENTATIONS
 Figure – Instrumentation of Gas Chromatography

1. Carrier gas in a high-pressure cylinder with attendant pressure regulators and flow

meters - In Gas Chromatography Helium, N2, H, and Argon are used as carrier gases in

high-pressure cylinder. Helium is preferred for thermal conductivity detectors because of

its high thermal conductivity relative to that of most organic vapors. N2 is preferable

when a large consumption of carrier gas is employed. Carrier gas from the tank passes

through a toggle valve, a flow meter, (1-1000 ml/min), capillary restrictors, and a

pressure gauge (1-4 atm). Flow rate is adjusted by means of a needle valve mounted on

the base of the flow meter and controlled by capillary restrictors. The operating

efficiency of the gas chromatograph is directly dependent on the maintenance of constant

gas flow.

2. Sample injection system – In sample injection system liquid samples are injected by a

micro syringe with a needle inserted through a self-scaling, silicon-rubber septum into a

heated metal block by a resistance heater. Gaseous samples are injected by a gas-tight

syringe or through a by-pass loop and valves. Typical sample volumes range from 0.1 to

0.2 ml.

3. The column separation - The heart of the gas chromatography is the column which is

made of metals bent in U shape or coiled into an open spiral or a flat pancake shape and

copper is very useful up to 2500 . Swege lock fittings make column insertion easy.

Several sizes of columns are used depending upon the requirements.

4. Liquid phases - An infinite variety of liquid phases are available limited only by their

volatility, thermal stability and ability to wet the support. No single phase will serve for

all separation problems at all temperatures.

 - Non-Polar – Paraffin, squalane, silicone greases, apiezon L, silicone gum

rubber. These materials separate the components in order of their boiling

points.

- Intermediate Polarity – These materials contain a polar or polarizable group

on a long non-polar skeleton which can dissolve both polar and non-polar

solutes. For example. Diethyl hexyl phthalate is used for the separation of high

boiling alcohols.

- Polar – Carbo waxes – Liquid phases with a large proportion of polar groups.

Separation of polar and non-polar substances.

- Hydrogen bonding – Polar liquid phases with high hydrogen bonding e.g.

Glycol.

- Specific purpose phases – Relying on a chemical reaction with solute to

achieve separations. e.g AgNO3 in glycol separates unsaturated hydrocarbons.

5. Supports - The structure and surface characteristics of the support materials they are

very important parameters, which determines the efficiency of the support and the

degree of separation respectively. The support should be inert but capable of

immobilizing a large volume of liquid phase as a thin film over its surface. The surface

area should be large to ensure the rapid attainment of equilibrium between stationary and

mobile phases. Support should be strong enough to resist breakdown in handling and be

capable of packed into a uniform bed. Diatomaceous earth, Kieselguhr treated with Na

2CO 3 for 9000 C causes the particle fusion into coarser aggregates. Glass beads with a

low surface area and low porosity can be used to coat up to 3% stationary phases. Porous

polymer beads differing in the degree of cross-linking of styrene with alkyl-vinyl

benzene are also used which are stable up to 2500.

6. Detector - Detectors sense the arrival of the separated components and provide a signal.

These are either concentration-dependent or mass dependent. The detector should be

close to the column exit and the correct temperature to prevent decomposition.

7. Recorder - The recorder should be generally 10 mv (full scale) fitted with a fast

response pen (1 sec or less). The recorder should be connected with a series of good

quality resistances connected across the input to attenuate the large signals. An integrator

may be a good addition.

APPLICATIONS

1. Gas Chromatography is useful to compute the content of a chemical product, for

example as promising the quality excellence of products in the industry and for

measuring and identification of toxic substances in soil, air or water.

2. Gas chromatography is also valuable for analysis of:

(a) Air borne contaminants

(b) Recital performance improving drugs in urine samples

(c) Oil spills

(d) Essential oils in perfumes

3. Gas chromatography is very precise if used correctly and for measurement pico-

moles of a substance or PPB concentrations of gaseous samples.

4. Gas Chromatography also used widely in the forensic science as disciplines as diverse

as solid drug dose qualitative and quantitative analysis, paint chip analysis, and

toxicology cases, employ gas chromatography to crime-scene evidence.

MCQ’s

1. Which method is useful for separation of thermally stable and volatile substance?
 a. Gas Chromatography.

b. Nuclear Magnetic Resonance Spectroscopy

c. Mass Spectroscopy

d. High Performance Liquid Chromatography

2. Which one liquid used in GLC?

a. Kieselguhr

b. Diatomaceous earth

c. A and B

d. None of the above

3. . Which one solid used in GSC?

a. Alumina

b. Carbon

c. Granular silica

d. All of the above

4. Following which sentence is true for gas Chromatography?

a. It is work on principal of the adsorption and partitions.

b. When stationary phase is liquid it is called a GSC

c. It has gaseous stationary phase.

d. It is not used to volatile substance.

5. Which of the following is not a feature of carrier gas used in gas chromatography?

a. It must be chemically inert

b. It should be suitable for the detector employed

c. It should not be completely pure

d. It should be cheap
Questions
1. What is principle of Gas Chromatography?
Ans. The principle of gas chromatography is the sample solution injected into the
instrument enters a gas (stream) which transport the sample into a separation
tube is known as the "column", for that Helium or nitrogen is used as the so-called
carrier gas. The various components and mixture of components are separated
inside the column.
2. Give applications of Gas Chromatography.
Ans. Gas Chromatography is useful to compute the content of a chemical product;
Gas chromatography is also valuable for analysis of: Air borne contaminants, Recital
performance improving drugs in urine samples, oil spills and essential oils in
perfumes. Gas chromatography also used correctly and for measurement pico-
moles of a substance Gas Chromatography widely applicable in the forensic science
as disciplines as diverse as solid drug dose qualitative and quantitative analysis, paint
chip analysis, and toxicology cases, employ gas chromatography to crime-scene
evidence.

3. What are the types of gas chromatography?

Ans. The two types of gas chromatography are encountered: gas
solid chromatography (GSC) and gas-liquid chromatography (GLC). The Gas Solid
Chromatography is based upon a solid stationary phase on which retention of analytes
is the consequence of physical adsorption.

4. What is mobile phase and stationary phase in gas chromatography?

Ans. In gas chromatography (GC), the mobile phase is an inert gas such as helium.
The mobile phase carries the sample mixture through what is referred to as
a stationary phase. The stationary phase is a usually chemical that can selectively
attract components in a sample mixture.

5. What is a purpose of gas chromatography?

Ans. The main purpose of the gas chromatography technique is to separate out the
compounds that possess: High volatility, Low molecular weights and Thermal
stability.
 List of solvents

Molecu
Polar Boiling p Melting p solubility
lar density
Solvent ity oint oint in H2O1
formul (g/mL)
index (o C) (o C) (g/100g)
a
cyclohexane C6H12 0.006 80.7 6.6 0.779 0.005
pentane C5H12 0.009 36.1 -129.7 0.626 0.0039
hexane C6H14 0.009 69 -95 0.655 0.0014
heptane C7H16 0.012 98 -90.6 0.684 0.0003
carbon tetrachlorid
CCl4 0.052 76.7 -22.4 1.594 0.08
e
carbon disulfide CS2 0.065 46.3 -111.6 1.263 0.2
p-xylene C8H10 0.074 138.3 13.3 0.861 0.02
toluene C7H8 0.099 110.6 -93 0.867 0.05
benzene C6H6 0.111 80.1 5.5 0.879 0.18
ether C4H10O 0.117 34.6 -116.3 0.713 7.5
methyl t-
butyl ether C5H12O 0.124 55.2 -109 0.741 4.8
(MTBE)
diethylamine C4H11N 0.145 56.3 -48 0.706 M
dioxane C4H8O2 0.164 101.1 11.8 1.033 M
N,N-
C8H11N 0.179 194.2 2.4 0.956 0.14
dimethylaniline
chlorobenzene C6H5Cl 0.188 132 -45.6 1.106 0.05
anisole C7H8O 0.198 153.7 -37.5 0.996 0.10
tetrahydrofuran (T
C4H8O 0.207 66 -108.4 0.886 30
HF)
ethyl acetate C4H8O2 0.228 77 -83.6 0.894 8.7
C9H10O
ethyl benzoate 0.228 213 -34.6 1.047 0.07
2
dimethoxyethane C4H10O
0.231 85 -58 0.868 M
(glyme) 2
C6H14O
diglyme 0.244 162 -64 0.945 M
3
methyl acetate C3H6O2 0.253 56.9 -98.1 0.933 24.4
chloroform CHCl3 0.259 61.2 -63.5 1.498 0.8
3-pentanone C5H12O 0.265 101.7 -39.8 0.814 3.4
C2H4Cl
1,1-dichloroethane 0.269 57.3 -97.0 1.176 0.5
2
di-n-butyl C16H22
0.272 340 -35 1.049 0.0011
phthalate O4
cyclohexanone C6H10O 0.281 155.6 -16.4 0.948 2.3
pyridine C5H5N 0.302 115.5 -42 0.982 M
C10H10
dimethylphthalate 0.309 283.8 1 1.190 0.43
O4
methylene chloride CH2Cl2 0.309 39.8 -96.7 1.326 1.32
2-pentanone C5H10O 0.321 102.3 -76.9 0.809 4.3
 2-butanone C4H8O 0.327 79.6 -86.3 0.805 25.6
C2H4Cl
1,2-dichloroethane 0.327 83.5 -35.4 1.235 0.87
2
benzonitrile C7H5N 0.333 205 -13 0.996 0.2
acetone C3H6O 0.355 56.2 -94.3 0.786 M
dimethylformamid C3H7N
0.386 153 -61 0.944 M
e (DMF) O
t-butyl alcohol C4H10O 0.389 82.2 25.5 0.786 M
aniline C6H7N 0.420 184.4 -6.0 1.022 3.4
dimethylsulfoxide C2H6O
0.444 189 18.4 1.092 M
(DMSO) S
acetonitrile C2H3N 0.460 81.6 -46 0.786 M
3-pentanol C5H12O 0.463 115.3 -8 0.821 5.1
2-pentanol C5H12O 0.488 119.0 -50 0.810 4.5
2-butanol C4H10O 0.506 99.5 -114.7 0.808 18.1
cyclohexanol C6H12O 0.509 161.1 25.2 0.962 4.2
1-octanol C8H18O 0.537 194.4 -15 0.827 0.096
2-propanol C3H8O 0.546 82.4 -88.5 0.785 M
1-heptanol C7H16O 0.549 176.4 -35 0.819 0.17
i-butanol C4H10O 0.552 107.9 -108.2 0.803 8.5
1-hexanol C6H14O 0.559 158 -46.7 0.814 0.59
1-pentanol C5H12O 0.568 138.0 -78.2 0.814 2.2
acetyl acetone C5H8O2 0.571 140.4 -23 0.975 16
C6H10O
ethyl acetoacetate 0.577 180.4 -80 1.028 2.9
3
1-butanol C4H10O 0.586 117.6 -89.5 0.81 7.7
benzyl alcohol C7H8O 0.608 205.4 -15.3 1.042 3.5
1-propanol C3H8O 0.617 97 -126 0.803 M
acetic acid C2H4O2 0.648 118 16.6 1.049 M
C2H7N
2-aminoethanol 0.651 170.9 10.5 1.018 M
O
ethanol C2H6O 0.654 78.5 -114.1 0.789 M
C4H10O
diethylene glycol 0.713 245 -10 1.118 M
3
methanol CH4O 0.762 64.6 -98 0.791 M
ethylene glycol C2H6O2 0.790 197 -13 1.115 M
glycerin C3H8O3 0.812 290 17.8 1.261 M
water, heavy D2O 0.991 101.3 4 1.107 M
water H2O 1.000 100.00 0.00 0.998 M