GAS CHROMATOGRAPHY

Gas Chromatography

Gas chromatography was developed in 1941 by AJP Martin and RLM Synge as a purely analytical method. But in 1952, RLM Synge got Nobel Prize for the discovery of gas chromatography.

Gas chromatography is used to separate and analyze mixtures of gases and volatile liquids or solids in their gaseous state.

Gas chromatography is basically a separation technique in which the compounds of vaporized sample are separated as a consequence of partition between mobile gaseous phase and a stationary phase held in a column.

Based on the nature of stationary phase, GC is of two types.

1. Gas Solid Chromatography                        2. Gas Liquid Chromatography

1. Gas Solid Chromatography:

In GSC, the stationary phase consists of an active solid absorbance such as granular silica, carbons, aluminium oxide

2. Gas Liquid Chromatography:

In GLC, separation occurs by partition between a mobile gas phase and a thin layer of non-volatile liquid coated on an inert support. The most common inert support is diatomaceous earth.

WORKING PRINCIPLE

The carrier gas passes through a flow regulator for the adjustment of flow rate of gases and enters into the sample injector. A little amount of the sample is introduced into the sample injector with the help of syringe (hypothermal). The sample injector is maintained at the high temperature than the boiling point of the highest boiling point of the sample in order to ensure rapid vaporization of liquid sample.

            The carrier gas entering the sample injector carries the vaporized sample to the thermostated column. The components of the sample pass through the column and are distributed between the stationary phase and mobile phase and pass down the column at different rates. The compounds (sample components) get partitioned between the stationary phase and the gaseous mobile phase and hence separated because of differences in their partition coefficient.

The carrier gas with the separated components now enters the detector which measures the changes in composition of carrier gas as it passes through it.

As each component reaches the detector, a peak is provided by the recorder. The separated components of the sample in the carrier gas register a series of signals which appears as succession of peaks above a baseline on the recorded chromatogram.

Identification of chromatogram is done by the use of peak called retention volume.

Retention volume: Retention volume is the volume of carrier gas that passes out of the column to the time peak maximum is obtained.

Figure 1: Retention time graph

V_R = t_R - F_c

Where                         V_R = Retention volume

t_R = retention time

F_c = flow rate of the carrier gas

Retention time: Retention time is the time from the point of the injection of sample to the time of emergence of separated components from the column.

The retention time and retention volume are specific for individual components.

BASIC COMPONENTS OF GAS CHROMATOGRAPHY

1.       A higher pressured cylinder containing carrier gas
2.       Flow regulator
3.       Sample injector
4.       Column
5.       Thermostated column
6.       Detector
7.       Recorder and data handling device

Figure 2: Optical Diagram of Gas Chromatography

Figure 3: Diagrammatic representation of Gas Chromatography

1. A HIGHER PRESSURED CYLINDER CONTAINING CARRIER GAS

Normally nitrogen and helium are the most commonly used carrier gases. The most common carrier gases are helium, hydrogen, nitrogen, argon and carbon dioxide. The most important requirement of carrier gas used in

a.       It should be inert.

b.      It should be available at the low cost because large quantities are used.

c.       It should allow the detector to respond in an adequate manner.

Helium or nitrogen gas fulfills all the above requirements and hence these are used most in carrier gas. For most analytical purposes, hydrogen gas is not used as carrier gas because of its explosion hazard and its reactivity toward unsaturated compounds.

2. FLOW REGULATOR

A simple flow meter is soap bubble flow meter. Soap bubbles are generated in the burette by squeezing a rubber bulb the connecting with the burettes are filled with the soap solution. The time required for soap bubble to move between two graduation on the burette can be measured which directly gives the flow rate of carrier gas. If flow rate of gas is slow, the eluted peaks will be broad. And if flow rate of gas is fast, eluted peak will not be resolved i.e. flow rate greatly influences column efficiency.

3. SAMPLE INJECTION

The sampling part or sample injection system is a small box which is electrically maintained (temperature control).

The amount of sample required for gas chromatography depends upon:

•        Nature and concentration of solutes present in the sample.
•       The size of the column and
•        Sensitivity of the detector

Generally a small amount of sample is loaded. The usual range is from 0.1-50 ml and for the solid samples milligram of fraction. For the solid sample, it is first dissolved in suitable solvents such as ether and methanol and injected as solution.                                                                                                                             

Devices by which required amount of sample that can be introduced into the carrier gas stream are:

1. By syringe
2. By ampule
3. By valve

A. Syringe method: Syringe technique is most commonly used technique for introducing gas and liquid sample into the carrier gas. Hypodermic syringes are available in many calibrated size which is inserted into the column through a replaceable rubber septum.

B. Ampule method: Viscous liquid and solid sample are weighed in a thin walled glass ampoules and are introduced into the carrier gas and then crushed. But this method isn’t commonly used.

C. Valve method: Valve methods are used for introducing the samples which are especially convenient to gaseous sample. It has two stop cocks (a pair of identical dual stop cocks). First stop cock no.1 is turned 90° and measured amount of the sample is filled in the reservoir. Then, it is turned to its original position. Stop cock no. 2 is then turned to 90° where a major quantity of sample is flushed into the column.

4. COLUMNS

Columns are made up from variety of material such as stainless steel, Cu, glass or plastic and may be coiled in U-shaped or W-shaped. Metal columns though expensive are prepared because they are stout, inert and possess good thermal properties.

Glass column are fragile and difficult to coil, so they are not commonly used.

Types of Column

      A. Partition column

      B. Adsorption column

A. Partition Column: Partition column is used in gas liquid chromatography. The partition columns are packed with inner support carrying a non-volatile liquid phase. Support material consists of celite, firebricks or glass bead. Liquids commonly used are silicon oil and grease, apieson oil and grease, squalance (C₃₀H₆₂).

Methods of Packing Column in GLC: The required amount of liquid is dissolved in a volatile solvent and then desired amount of inert solid support is mixed with it in an open container and volatile solvent is removed by evaporation.

The solid with its liquid coating has the appearance of free flowing sand which can be packed into a long tube with tapping or vibrating to promote even packing.

B. Adsorption column: They are used in gas solid chromatography. Several adsorbing material such as silica gel, activated carbon or aluminium oxide can be used in gas solid chromatography.

Method of Packing Column in GSC: While packing the column, one end is closed with glass wool plug and as adsorbent material is introduced into the column through the other end of column by tapping the column thoroughly.

5. THERMAL COMPARTMENT

The column is never operated at room temperature in case of gas chromatography. The temperature of column is controlled by using air baths or using vapor jacket or by using electrically heated blocks. The temperature of the column should not be as high as to vaporize the stationary phase. Normally a temperature equal to or slightly greater than the average boiling point of sample gives good elution period. If sample containing components with a broad boiling point is to be fractionated then it is desirable to increase the temperature of the column as the separation proceeds.

6. DETECTOR

The function of detector is to measure the small amount of separated components present in the gas steam leaving the column. Based on the physical properties of gases, detectors are of various types. For example;

•       Electron capture detector
•      Thermal conducting
•       Ionization detector
•       Flame ionization detector
•       Gas density detector

7. RECORDER AND DATA HANDLING DEVICES

The output from the detector is fed to a recorder which provides a chromatogram. So, recorder records or traces out a series of peak forming chromatogram. Most gas chromatography is collected to a mass spectrophotometer i.e. capable of identifying the angle represented by the peak for quantitative estimation; we can calculate the peak area by applying geometrical method. The area of the peak is the direct measure of concentration of each compound present in the sample. The peak area is the product of the peak height and the peak width at half peak height.

APPLICATIONS OF GAS CHROMATOGRAPHY

•     Gas chromatography can be used for analysis and separation of petroleum products, fatty acids, steroids, rubber and rubber products.
•    In cosmetic and perfume industries, gas chromatography is helpful in determining the composition of various cosmetics and to check the quality of ingredients used for preparing cosmetics.
•      An important use of gas chromatography is determination of elements such as carbon, nitrogen, oxygen and sulfur in inorganic metallic sample.
•       GSC can be used to analyze pesticide residues in soil sample, milk sample and different food products like honey etc.

AFFINITIY CHROMATOGRAPHY

Affinity Chromatography

The attracting force exerted in different degrees between atom which causes them to enter into it and remain in combination is the type of adsorption chromatography in which there is high degree of specificity in the interaction between adsorbent and the compound to be separated. The technique requires that material to be separated is capable of binding reversibility to a specific ligand that is attached to an insoluble matrix.

M                    +                      L                     ®                    ML

  (Macromolecules)                        (Ligand)                                  (Complex)

Affinity chromatography was originally devloped for purification of enzymes but extended to nucleotides, nucleic acid and immunoglobulins. In case of the purification of ligand attached to adsorbent is usually a powerful inhibitor which will bind only one enzyme in a complex mixture.

Figure 1: Binding of ligand and macromoleuclar complex (Matrix)

An ideal matrix for affinity chromatography must possess following characteristics:

1.   It must be stable during binding of macromolecules and in subsequent elution.

2.   It must be at the most interactive with a specific compound and only weakly with other macromolecules to minimize non specific adsorption.

3.   It must contain suitable and sufficient chemical group to which ligand may attched and it must be stable under condition of attachment.

4.   It should exhibit good flow properties i.e. the matrix has to be macroporous to allow large molecules assess to the binding site and need to have good flow properties.

5.   It has to be devoid of non-specific adsorption sites but must contain functional group to which ligand can be attached.

The most common matrixes that are used in Affinity chromatography:

·         Dextran

·         Porous glass

·         Polyacrylamide

·         Agarose

·         Cellulose

The ligand is covalently attached to the supporting matrix so that the chromatography materials can be designed for a specific purification task. The chemical nature of ligand is determined by biological specificity of the compound to reseparate it. For e.g. in case of enzyme purification, the ligand attached to matrix is usually a powerful inhibiton which will bind only one enzyme in a complex mixture.

While selecting ligand one should consider that the selective ligand will bind one particular compound (compund to be separated). Ligand should be specific.

It is essential that the ligand  should possess the suitable chemical group that will be involed in the binding of ligand to the macromolecules to be separated. Ligand should possess functional group that will bind with matrix. The most common such groups are amine groups, carboxyllic groups, phenolic groups and alcoholic groups.

Ligand	Affinity
1. Nucleotides 5¢ AMP 2; 5¢ AMP	NAD+ dependent dehydrogenase, some kinase NADP+ dependent dehydrogenase
2. Fatty acids	Fatty acid-binding proteins
3. Proteins A and G	Immunoglobulins
4. Soyabean lectin	Glycoprotein containing N-acetyl -µ or b D galactopyrabosyl residues
5. Phenylboronate	Glycoproteins
6. Poly (A)	RNA containing poly (U) sequences
7. Lysine	RNA

Spacer arms   ®        1,6 diaminohexane

®        6 amino hexanoic acid

®        1,4 bis (2,3 epoxypropoxy) butane

Spacer arm is nearly inserted  between matrix and ligand so that large molecule can get assess the binding sites. The spacer arm positions the ligand at the certain distance from the matrix. In  practice spacer arms of 2-10 C atoms has to be found optimum.

The principle of affinity chromatography.

Figure 2: Binding of specific molecules in the affinity resin

When the complex mixture contains specific compound to be purified is added to the ligand contained in chromatographic column only specific compound bound to ligand. All the washed and bound compound can be away and subsequently purified can be recovered.

Purification of acetylcholine esterase by Affinity chromatography

Acetiylcholineesterase is membrane bound enzyme which are extracted fron mice brain to rat brain.

Steps:

1. Preparation of an affinity column /  material

The most common method of attachment of ligand to the matrix involves treatment of matrix with cyanogen bromide activated sepharose 48. Cyanogen bromide is toxic and causes explossion which is impure.

2. Addition of Spacer arm.

The spacer arm is introduced next by reacting the activated sepharose with diaminobutanne then lengthen by addition of succinic anhydride so that the ligand 3 amino N methyl pyrimide ion can be linked to the activated spacer arm.

The affinity material prepared is N-methyl 3 amino pyridine agarose.

Figure 3: Coupling of Ligand

3. Packing of column

Column of 10 cm x 1 cm size is used. After liquid attachment , the affinity material ions packed into the column. Method of packing of column is similar to that of column chromatography.

4. Sample Application

It is done with micropipette at the top of affinity column.

5. Binding

The ligand attached to the sepharose i.e. 3 amino N methyl pyridine ion is strong inhibitor of acetylcholine esterase so when crude (impure) extract is added to the column, the acetylcholine esterase molecule specifically bind to the ligand.

6. Washing

The other proteins present in the extract donot bind to affinity material and are removed from the column by washing. The washing is achieved by running the solution contaning the protein through the column followed by elution buffer until protein is no longer detected in the eluent. NaCl 1 mol/L in the triton is used as elution buffer.

7. Elution

The bound enzyme i.e. acetylcholine esterase is eluted from the column by incorporating an inhibitor becamethomium bromide in the elution buffer which has an even higher affinity for acetylcholine esterase than that shown by ligand.

8. Reactivation of Acetylcholine esterase

Reactivation means the removal of bound acetylcholine esterase with demethomium bromide. This is carried out by adding the cation exchange resin, amberlite G-120 which strongly binds the decathomium bromide. Thus pure form of acetylcholine bromide can be obtained. After this ion exchange chromatography is run.

                                                           

Application

1.      It has been used in purify a wide range of enzyme and proteins including Ig. For e.g.: DNA related enzyme can be purified by employing affinity chromatography . Hepain agrose is used  to purify the hepain binding domain of platelet i.e thrombrspondin can be purified from blood platelets using affinity chromatrography an heprose agqrose.

2.      Messenger RNA are routively isolated by using poly(u)sepharose 4B

3.      Clinical application: It includes immuno assay. Affinity chromatrography are widely used in theapentic drug monitoring and allergy testing.

4.      It has also been applied in concentration of dillute protein solution and storage of unstable protein in the immobilized form.

Figure 3: Specific example of affinity chromatography, Antigen-antibody reaction

GEL FILTRATION

Gel Chromatograpphy

Gel filtration has also been called by different name: Gel permeation, Exclusion chromatography or Molecular sieve chromatography.

Gel chromatography is technique in which separation is based upon molecules, size and shape of the species in the sample.

Theory:

The chromatographic media used in this technique are porous, polymeric organic compounds with molecular sieving properties. These are cross linked polymers which swell considerably in water forming a gel of a three dimensional net work of pores. The size of pore is determined by degree of cross linking of polymeric chains. Different solutes in a mixture get separated on the basis of their molecular size and shape during their passage through a column packed with the swollen gel particles. The terms ‘exclusion chromatography’, ‘gel filtration’ and ‘molecular si’ve' chromatography are used for this separation process. The large molecules in sample are unable to penetrate through the pores into the gel beads. Obviously the volume of the solvent accessible to large molecules is very much less (V_o), whereas small molecules which can freely penetrate into the gel have access to solvent inside (V_i) as well as outside (V_o) the spherical beads.

A sample containing a mixture of large and small molecular weight substances is applied onto the column. Solvent usually a buffer is used as an eluent. As components of the sample travel down the column, the compounds whose molecular size exceeds the fractioation of range of the gel, are unable to enter into gel particles and hence remain completely excluded from beads and travel through the interstitial spaces. Molecules of smaller compounds however diffuse into the gel matrix through the pores and get distributed between the mobile phase inside as well as outside the gel particles. These thus followed a longer path than the larger molecules and hence their movement down the column is retarded. Consequently different components in the sample get separated from each other with larger molecules getting eluted first followed by smaller molecules.

For a given type of gel, the distribution coefficient, K_d which represents fraction of the liquid within the gel particles accessible to molecules of a given substance will predominately depend upon molecular size of that compound. Very large molecules, due to their exclusion, have no access to the mobile phase within the gel hence have a K_d value of 0, whereas smaller molecules for which the inner mobile phase is completely accessible have K_d value of 1. For the molecules of the intermediate size, the K_d value have varies within this range (0-1). This difference in K_d values of various compounds in the sample accounts for their separation during gel filtration. The relationship of K_d with elution volume V_e is given by:

Ve=Vo + Kd. Vi

Where:           

V_e:          is the elution volume and represents volume of the mobile phase required to elute the compound from the column.

V_o:                      is void volume or the volume of the mobile phase outside the gel particles

V_i:          is the volume of the mobile phase present inside the gel particles and can be calculated  from the relationship: Vi=a. Wr where a= dry weight of the gel and Wr = water regain. Values of Wr for different types of gels are given as:

K_d:          is the fraction of Vi accessible to a particular compound. Tranformation of Equation

Kd = ( Ve-Vo) / Vi

As can be noted, for very large molecules which are completely excluded from the gel V_e = V_o and so Kd = 0, but small molecules which can freely diffuse into and out of the gel beads V_e =V_o + V_i and so K_d = 1.

Figure 1: Elution from gel filtration

Gel Matrix

In gel chromatography, the granulated or beaded gel material is called as the packing material. The solute which are disturbuted throughout the entire gel phase is called stationary phase and the liquid flow through the bed is called as mobile phase. The solid support or gels that are used in gel chromatography are group of polymeric organic componuds that possess a three dimentional network of pore that confers the gel properties upon them. This gel have a tendency to swell in a suitable solvent and as a result of swelling of gel. The space between polymer chain increases in size. For a gel there will be a critical size of molecule that can just penetrate the interior. Molecules larger than the pores of gel are completely excluded from the region (gel). Smaller molecules can enter the gel and larger molecules can exclude the gel.

Examples of Gel

a.       one of the most widely used gel is crosslinked dextrans which is sold under the name Sephadex.

b.      Polyacrylamide cross linked dextran, called Biogel P

c.       Agarose (Sepharose or Biogel A)

d.      Polystyrene (Biobeads)

Properties of Gel

·         A good gel shoould be inert and doesn’t react with molecules to be fractionated.

·         It should be chemically stable.

·         Its particle size distribution should be controlled. For e.g. for ordinary lab work, powder gel of particle size of 70 micron in daimeter is quite effective (0.007 mm).

·         It should be high mechanical rigidity

Table 1: Molecular weight fractionating range of Sephadex and Sepharose

Gel type	Fractionation range (MW)		Bed Vol. (ml/gm dry material)	Dry bead diameter (mm)
	Peptides and globular proteins	Dextrans
Sephadex G-10	-700	-700	2-3	40-120
Sephadex G-5	-1500	-1500	2.5-3.5	40-120
Sephadex G-25 (Fine)	1000-5000	100-5000	4-6	20-80
Sephadex G-50 (Fine)	1500-30000	500-10000	9-11	20-80
Sephadex G-75	3000-80000	1000-50000	12-15	40-120
Sephadex G-100	4000-150000	1000-100000	15-20	40-120
Sephadex G-150	5000-150000	1000-150000	20-30	40-120
Sephadex G-200 (Super fine)	5000-250000	1000-200000		10-40
Sephacryl S-200	5000-2.5X105	1X103-8X104		40-105*
Sephacryl S-300	1X104-1.5X106	1X103-7.5X105		40-105*
Sepharose 2 B	7X104-40X106	1X105-20X106		60-200*
Sepharose 4 B	6X104-20X106	3X104-5X104		60-140*
Sepharose 6 B	1X104-4X106	1X104-1X106		45-165*

Steps involved in Gel Chromatography

1. Selection of a column

The column consists of a straight glass tube with a bed support at the bottom. The bed support allows only the liquid to pass through without disturbing the bed material. Column of 100 cm length and 10-20 cm in height  are sufficient for lab work in many cases.

2. Gel Preparation

There are two main methods of preparation of the gel.

       I.            First, the powder gel is mixed with excess of solvent to be used as eluent. It is then allowed to swell and left as such till the equilibrium condition is achieved. This procedure takes longer time.

    II.            Second, t.he powder gel is mixed with excess of solvent and the slurry so obtained is warmed to about 100°C for about 30 mins in a water bath. By warming, bacteria and fungus if present in suspension is also killed and dissolved air is also removed. As a result, the gel swells in few hours. The slurry is cooled before packing.

3 . Packing of the column

The method for packing of the column with gel depends upon type of gel to be used. Soft gel like sephadex are packed carefully while hard gel donot require much precaution.

There are generally  2 methods

1.      The column is first filled with eluent and then the slurry of the powder gel is poured  into the column through a funnel attached to the top of the column. the entire amount of slurry should be added on one step. Packing in many steps should be avoided as it gives uneven packing.

2.      It is most employed method. The gel is allowed to swell in the solvent. It is prepared by warming the slurry and allowed to cool before packing. After swelling , the gel is allowed to settle and the supernatant liquid is off to about half the volume of sedimented gel. This is again mixed with the solvent with constant stirring to make a slurry of the gel. The slurry is then carefully poured into the column in one step with the help of glass rod.

Figure 2: Gel filtration of different cellular inclusions

4. Application of sample

Sample can be loaded at the top of gel surface with the help of the pipette or syringe. The sample of 1-2% of total bed volume is sufficient. In group separation, sample of 25 -30 % of the total bed volume is requried.

5. Elution method

Then the sample solution is allowed to pass down through gel bed. Small volume of the eluent is added by means of pipette having a bent tip. And the last traces of sample is washed with eluent. Single solvent is used for elution. E.g.: sodium chloride, H₂O, organic solvents are used as elution buffer.

6. Collection and analysis of eluate

Each fraction of eluate is collected by keeping the flow rate 1 ml/min.

7. Analysis

It can be done by spectrophotometric methods or colorimetric methods. For the separation of polysaccharide, each fraction collected can be identified by paper chromatography.

Application

•     It is used in separation of sugars, salts, polypeptides, amino acids, proteins, lipids,  polystyene and silicon polymers.
•       The main appliction of gel chromatography is in purification of biological macromolecules, viruses, proteins, enzymes, hormones, antibodies, nucleic acids and polysaccharides by the of appropriate gel. Sephadex G75 is used for purifying macromolecules such as various species of RNA viruses. Sephadex G15 have been used in separation of maltose and glucose.
•      It is used for solution concentration. Solution of high relative molecular mass substances can be concentrated by the use of sephadex G-25. Water and low relative molecular mass substances are adsorbed by the swelling gel whereas high relative molecualr mass substances remain in the solution. Then the gel is removed by centrifugation leaving high molecular mass substances in the solution increasing the concentration.
•           Plasma protein fractions can be quantitatively determined in the diagnosis of certain human disease such as Hyperglobulinema.