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FORENSIC SCIENCE

PAPER No.5: Forensic Chemistry & Explosives MODULE No. 5 : Chromatographic Methods of Chemical Analysis

Subject FORENSIC SCIENCE

Paper No and Title PAPER: 5, Forensic Chemistry & Explosives

Module No and Title MODULE No. 5, Chromatographic methods of chemical analysis

Module Tag FSC_P5_M5

TABLE OF CONTENTS

1. Learning Outcomes 2. Introduction: Chromatography 3. Principle of Chromatography 4. Classification of Chromatographic Techniques

4.1 Adsorption Chromatography 4.2 Partition Chromatography

5. Common Chromatographic Techniques 6. Paper Chromatography 7. Thin Layer Chromatography 8. Column Chromatography 9. Gas Chromatography 10. High Performance Liquid Chromatography 11. Summary

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1. Learning Outcomes

After studying this module, you shall be able to know

• Basic principle of chromatography • Various techniques of chromatography • Application of different techniques of chromatography in forensic analysis

2. Introduction: Chromatography

The term Chromatography is actually a combination of two greek words i.e. chroma which means "color" & graphein which literally means "to write". It is the term that is collectively used to refer all the laboratory techniques which are employed to separate the different mixture components. However, the credit of development of this separation technique goes to Mikhail Tswett, a Russian Botanist. In 1903, he was succeeded in separating plant pigment’s mixture by using a calcium carbonate column. With the application this technique he firstly discovered that instead of being a single compound, chlorophyll is a mixture of different compounds. Till date several modifications have been made which resulted in the development of various modern chromatographic techniques, the majority of which can be automated and adapted to deal with large or minute quantities of different substances for their purification and separation.

3. Principle of Chromatography

Chromatography relies on the basic concept of differential solubilities and affinities of the different mixture components in different phases. The mixture that is to be separate out is dissolved in a suitable solvent which is called the mobile phase, which carries it through the second component of this technique i.e. stationary phase. Choice of phases is purely dependent on the nature of the sample to be analyzed. Component, having higher affinity toward stationary phase, will obviously pass through it in a longer. The separation of various components of a mixture depends on their differential affinities towards stationary phase.

3.1 Distribution of components of a mixture between phases

The distribution of components of a mixture (analytes) between phases is a simple reaction. When an analyte is in a state of equilibrium between the two phases:

Amobile Astationary

The equilibrium constant which is also known as partition coefficient, denoted by k, is defined as the analyte’s molar concentration in stationary phase divided by the analyte’s molar concentration in mobile phase.

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Retention time (tR) is the time lap between injection of sample & an analyte reaching a detector connected at the end of the column. Each analyte of the sample will have its own characteristic retention time. However, tM is used to denote the time taken by the mobile phase to pass the column length.

4. Classification of Chromatographic Techniques

Based on the principle of separation, chromatographic techniques are chiefly classified into following two categories:

4.1 Adsorption Chromatography

In this technique, the different components of the sample get separated based on their greater differential affinities to adsorb to the solid stationary phase in comparison to the mobile phase. This principle is applicable when mobile phase is liquid & stationary phase is solid. Thus, the component that has higher affinity for the matrix of stationary phase will more effectively compete for the binding sites, and thereby causing displacement of all those components that have relatively lesser affinities.

4.2 Partition Chromatography

The first type of chromatography that was developed by chemists is partition chromatography. It employs a solvent retained on the surface of a solid supporting "inert" matrix. Analyte molecules partition between a liquid mobile and stationary phase. In this method, analytes get separated based on their differential polarities. In this technique, the separation of sample molecules takes place on the basis of relative differences in their partition and dissolution in different phases. The components having higher affinity towards the mobile phase are separated quickly in comparison to those having higher affinity towards stationary phase. Here both mobile & stationary phases are either liquid in nature or may have liquid as stationary phase & gas as mobile phase. The liquid exist as thin layer on a solid surface acts as stationary phase. The analytes that are polar in nature, diffuse and get retained into a stationary water layer associated with the polar stationary

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phase. The stronger the interactions between the polar analyte & the polar stationary phase as compared to the mobile phase, the longer will be the time of elution.

5. Common Chromatographic Techniques

The common chromatographic techniques that are employed in forensic analysis are:

6. Paper Chromatography

Paper chromatography is a chromatographic technique which is employed in the separation & identification of mixtures, especially pigments. This can also be employed in primary or secondary colors in experiments of ink. These days, TLC has largely replaced this technique; but still it is a powerful analytical tool.

6.1 Principle

Principle involved in paper chromatography is similar to those that is involved in other chromatographic techniques. Here, stationary phase is a high quality paper & a developing solvent works as the mobile phase that migrates through this stationary phase, carrying the samples to be analyzed with it. The different sample components of the sample get separate based on their relative affinity towards the stationary phase versus their relative solubility in the mobile phase.

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6.2 Procedure

The sample under analysis is placed on a filter paper, one end of which is immersed in a solvent. The solvent diffuses up the paper, thereby dissolving and separating the various sample components.

6.3 Rƒ value

The Retention Factor (Rƒ) may be defined as the ratio of the distance traveled by the sample component to the distance that is traveled by the solvent. Rƒ values are generally expressed as a fraction up to two decimal places. If Rƒ value of any sample comes out to be zero, this means that the sample is immobile & is insoluble in the mobile phase. If Rƒ value = 1 then the solute has no affinity for the stationary phase & travels with the solvent front.

6.4 Types of Paper Chromatography

6.4.1. Descending Paper Chromatography-In this type, the upper part of the paper contains the mobile phase and the development of chromatogram is done by the downward migration of the solvent.

6.4.2. Ascending Paper Chromatography- In this type, the lower part of the paper contains the mobile phase and the development of chromatogram is done by the upward migration of the solvent.

6.4.3. Ascending-Descending Paper Chromatography-It involves the characteristics of both the above mentioned techniques. The upper part of the paper which carries ascending chromatography is turned over a rod and letting the paper to perform descending chromatography after that.

6.4.4. Radial Paper/ Circular Chromatography- Here the sample is placed at the center of a circular filter paper. After drying the spot the filter paper tied over a Petri-dish in such a manner so that Wick of the paper is dipped in the solvent present in that petri-dish. The solvent moves upwards through the wick and the component of the mixture get separated forming concentrated circular zone.

6.5 Applications

Paper chromatography is helpful in analyzing the purity of various compounds and in the identification of toxic substances. Paper chromatography often requires sample in smaller quantities and give quick results. This technique is also quite handy in the analysis of different dyes.

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7. Thin Layer Chromatography

Thin layer chromatography (TLC) is a chromatographic technique that is employed in separating a mixture of non-volatile substances. TLC is performed on a sheet of plastic, aluminium foil, glass, etc. which is coated with a thin layer of adsorbent material, generally cellulose, aluminium oxide or silica gel. This adsorbent layer functions as the stationary phase.

After applying the sample on the plate, the mobile phase made up of one or more solvents, through capillary action, is drawn up the plate. As different analytes have their own characteristic speed of migration, they get separated on this basis.

7.1 Plate preparation

Generally the TLC plates are commercially available, with a wide range of particle size for improving the results obtained. Such plates are made up of adsorbent material like silica gel, mixed with small quantity of inert binder commonly water and calcium sulfate. This mixture is coated in the form of an even layer on an un-reactive carrier sheet, usually plastic, aluminum foil or glass. The plate so prepared is dried & activated by heating it in an oven at a temperature about 110 °C for 30 minutes. Adsorbent layer thickness generally ranges from 0.1 – 0.25 mm for analytical purposes & from 0.5 – 2.0 mm for preparative TLC.

7.2 Procedure

The procedure adopted for carrying out TLC is as follows:

• About 1.5 cms from the bottom edge, a small spot of the sample under analysis is applied.

• A mobile phase in appropriate amount is taken into a glass beaker. • The plate is placed in the chamber in such a manner that none of the spot(s) touch the

eluent surface. The solvent front migrates in the upward direction through capillary action, and consequently separates the sample.

Fig 1: Development of a TLC plate, a purple spot separates into a blue & red spot.

The mobile phase & solute competes for the binding sites present on the stationary phase leading to the separation of various components of a mixture. For instance, the normal silica gel is polar in nature & thus, has a greater affinity towards the polar compounds. In a

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consequence of this, the compounds those are less polar in nature moves higher up the plate which results in a comparative higher Rf value.

7.3 Analysis

As the separated chemicals may be colorless, a number of methods can be employed to visualize the spots:

• Blacklight (366 nm) could be used to detect analytes that are fluorescent in nature like quinine

• Fluorescent compounds like manganese-activated zinc silicate could be added to the stationary phase at the time of plate preparation which allows spot fluoresce under ultraviolet-C light (254 nm). The plate thus will fluoresce under UV light, but analyte spots will interrupt this fluorescence.

Rf value of each spot is obtained by dividing the distance travelled by a component to the distance travelled by the solvent front. These values are totally dependent on the factors like nature of TLC plate, solvent used, etc.

7.4 Applications

TLC can be used to monitor a reaction’s progress, in identifying the different compounds of a mixture and in determining a substance’s purity. Specific examples of these applications may be as follows: detection of toxic materials including pesticides or insecticides, analyzing the dye composition of fibers & inks, identification of medicinal plants and their constituents having toxic effects, etc.

Fig 2: Showing Separation of Black Ink on a TLC Plate

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8. Column Chromatography

Column chromatography is a c technique chromatographic technique employed in purifying and identifying the individual chemical compounds from a mixture. The biggest advantage of this technique is that it is much economic the stationary phase used in it are easily disposable.

8.1 Column preparation

The column used is a tube made up of glass with a diameter from 5 mm to 50 mm & a height of 5 cm to 1 m with a tap and some kind of a filter at the bottom of the tube. The methods that are generally used in column preparation are: the dry method and the wet method.

• In the dry method, the column is filled up with dry powder that constitutes the stationary phase, after this the mobile phase is made to pass through it until that powder becomes completely wet.

• For the wet method, slurry is made by mixing up the powder of stationary phase and the eluent outside the column. The slurry is then carefully packed in the column. Proper care should be taken so that bubbles are not formed during this packing.

All the sample components show differential affinity towards the stationary phase & thus elute out of the column at different times along with the moving mobile phase. During this process the eluent is gathered in a series of fractions. Fraction collector automatically collects these fractions. The chemical composition of each fraction is analyzed by any of the following method, e.g. UV absorption, fluorescence, etc.

8.2 Stationary phase

In column chromatography, the stationary phase is a solid. Silica is the most common, followed by alumina. Generally the stationary phases are micro-porous and finely ground gels or powders. This is so done for increasing their surface area. The ratio between the weight of analyte and stationary phase that can be applied onto the column is also very important. This ratio ranges from 1:20 to 1:100, for silica column.

8.3 Mobile phase

A single or a mixture of solvents could be used as mobile phases. The nature of the compound under analysis affects the choice of the mobile phase. A correct choice of mobile phase can give accurate results with a higher degree of resolution.

All sample components show differential affinity towards the stationary phase & thus elute out of the column at different times along with the moving mobile phase. Gravitational pull is the only force that works on a simple laboratory column. But in modern versions a much faster flow rates are achieved by employing compressed gases like argon, nitrogen, etc or by making use of a pump to push the mobile phase through the column.

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In flash column chromatography, the grain size of the adsorbent material that makes up stationary phase is finer in comparison to the gravity column chromatography.

8.4 Resolution Calculation

The eluent that pass out through the column are collected as fraction by a fraction collector attached at the end of the column. But before this process of fraction collection, each fraction is made to pass through a detector most commonly a spectrometer so as to make a qualitative as well as quantitative analysis.

8.5 Applications

It is useful in both the qualitative and quantitative analysis of various suspicious substances found at the scene of crime. It helps in identifying the nature and type of a substance which help in linking the criminal with the crime scene.

9. Gas Chromatography

Gas chromatography (GC) is a popular analytical technique employed in separating & analyzing the volatile compounds.

9.1 Procedure

In GC, a carrier gas constitutes the mobile phase, usually an un-reactive gas like nitrogen or an inert gas like helium. A microscopic layer of liquid present on the metal or glass beads constitutes the stationary phase.

The gaseous compounds under analysis interact with the stationary phase which causes each component to elute out of the column at a different time which is called the retention time of that component.

On the basis of basic principle, GC is similar to other previously mentioned chromatographic techniques, but has various marked differences. Firstly, in GC the mobile phase is a gas & the stationary phase is liquid whereas in column chromatography liquid acts as mobile phase and a solid as stationary phase. Hence "Gas–liquid chromatography" is the full name of this technique. Secondly, there is an oven which contains column through which the mobile phase passes to control the temperature of the gas whereas there is no such temperature control in column chromatography.

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9.2 Procedure

In a GC analysis, a known quantity of liquid or gaseous analyte is injected into the column through a micro syringe. As the mobile phase migrates down the column, the various sample components get adsorbed on the stationary phase based on their relative affinities. This rate of migration not only depends upon the nature of stationary & mobile phase but also on the sample under analysis. Since all the sample components show differential affinity towards the stationary phase & thus elute out of the column at different times along with the moving mobile phase. Fraction collector automatically collects these fractions. The chemical composition of each fraction is analyzed by any of the following method like UV absorption, fluorescence, etc.

9.3 Analysis

Analysis can be done on the following two grounds:

9.3.1 Qualitative Analysis

Chromatographic data generally is presented in the form of a graph having retention time on x-axis and detector response on y-axis, such a graph is known as chromatogram. Qualitative analysis is made that is based on retention time i.e. different sample components can be identified. The peak patterns remain constant for a given component if the operating conditions are kept constant. In modern applications, GC is connected to a mass spectrometer which acts as a detector to compliment gas chromatography (GC-MS).

9.3.2 Quantitative Analysis

In a chromatogram, the peak area represents the amount of a particular sample component. The concentration of each component of the analyte in the sample is calculated by determining the peak area.

9.4 Applications

This chromatographic technique is employed in the qualitative and quantitative analysis of various suspicious substances- like analysis of body fluids, fibers, etc. found at the scene of crime. It helps in identifying the nature and type of a substance which help in linking the criminal with the crime scene.

Gas chromatography – mass spectrometry is a popular method for analysis of ignitable liquids in arson cases.

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10. High Performance Liquid Chromatography

HPLC is a chromatographic technique that not only separate different mixture components, but also identify & quantify them. The main component of this instrument is the pump that helps in passing a pressurized mobile phase containing the sample mixture to pass through the column filled with stationary phase. Each component of the sample reacts differently towards the stationary phase, causing the different components to flow out at different rates which lead to the separation of the components of that sample.

Fig 3: A modern self-contained HPLC

The stationary phase is a solid granular material made up of materials like silica, polymers, etc., having a size range of 2–50 micrometers. The various components of a mixture get separated based on the grounds of their differential affinity towards stationary phase. The pressurized mobile phase could be a mixture of solvents like water, acetonitrile or methanol. Its temperature and composition are the chief influencing factors of this technique.

HPLC has marked differences from its traditional low pressure version as here operational pressures are markedly higher (50–350 bar), contrary to this traditional version was totally dependent on the gravitational pull for the movement of solvent through the column. As HPLC is capable to analyze even the minute sample size, the diameter of column may vary between 2.1–4.6 mm and the length between 30–250 mm. Along with this the average particle size of HPLC are also comparatively smaller ranging from 2–50 micrometer. The high resolving power of HPLC makes it a popular analytical technique.

10.1 Operation

The sample under analysis is introduced in the column along with a stream of mobile phase. The different mixture components elute out the column at different rates on the basis of their relative affinity towards the stationary phase which in turn depends upon the chemical nature of each component, as well as the nature and composition of the mobile and stationary phase. The

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time taken by an analyte to come out of the column is called its retention time which is an identifying feature of that particular analyte.

10.2 Parameters

10.2.1 Internal diameter

The internal diameter of the column is a significant parameter of HPLC that not only affects the separation but also the sensitivity of the instrument. Indirectly it controls the amount of analyte that can be packed in the column.

10.2.2 Particle size

The smaller the particle size more will be its surface area and the better will be the separation that it will provide, but the pressure that is required for optimum linear velocity increases by the inverse of the particle diameter squared. Thus, changing to particles to half of their size, will double the performance, but the increased pressure must be increased by a factor of 4.

10.2.3 Pump pressure

The performance of pumps is measured based on their ability to yield a reproducible and consistent flow rate. It may achieve a pressure as high as about 400 atmospheres. Various improvements have been made to modern HPLC systems to make them work at even higher pressures, and consequently much smaller particle sizes ranging about <2 μm. These are called "Ultra HPLC" systems.

10.3 Applications High Performance Liquid Chromatography is an important forensic technique, often used in cases involving drug trafficking. It is used in detecting performance enhancing drugs from urine samples and separating the components of a different biological sample.

14. Summary

• The term Chromatography is actually a combination of two greek words i.e. chroma which means "color" & graphein which literally means "to write". It is the term that is collectively used to refer all the laboratory techniques which are employed in the separation of different components of mixtures.

• Chromatography relies on the basic concept of differential solubilities and affinities of various mixture components in different phases.

• The mixture that is to be separate out is dissolved in a suitable solvent which is known as mobile phase, which carries it through the second component of this technique i.e. stationary phase.

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• In adsorption chromatography, the different components of the sample get separated based on their greater differential affinities to adsorb to the solid stationary phase in comparison to the mobile phase. This principle is applicable when mobile phase is liquid and stationary phase is solid.

• The first type of chromatography that was developed by chemists is partition chromatography. It employs a solvent retained on the surface of a solid supporting "inert" matrix. Analyte molecules partition between a liquid mobile and stationary phase. In this method, analytes get separated on the basis of their differential polarities.

• The common chromatographic techniques that are employed in forensic analysis are Paper Chromatography, TLC, Column Chromatography, Gas Chromatography and HPLC. However, all these chromatographic techniques work on the same basic principle.

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