HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

 HPLC

The technique of HPLC was developed by Kirkland and Huber 1969. HPLC is a chromatographic technique and is used in biochemistry and analytical chemistry to identify and to purify the individual components of the mixture. HPLC is a type of liquid chromatography using high pressure pump operating at pressures upto 30,000 Pascals.

In HPLC, separation can be done in column containing stationary phase and one end of the column is attached to a source of pressurized liquid mobile phase. With HPLC adsorption and partition, exclusion and ion-exchange column separation are possible with great success. HPLC utilizes different types of stationary phases contained in a column.

Some of the example of stationary phase used in HPLC;

1. Partition HPLC

·         Octadecysilane

·         Alkylamine

2. Adsorption HPLC

·         Silicon or alumina

3. Exclusion HPLC

·         Agarose, polyvinylchloride, polystyrene divinyl benzene

4. Ion-exchange HPLC

·         Strong acid cation exchanger

·         Strong basic anion exchanger

·         Weak acid cation exchanger

WORKING PRINCIPLE OF HPLC

As in other forms of liquid chromatography, separation in High Performance Liquid Chromatography or High Pressure Liquid Chromatography (HPLC) is also obtained on the basis of partitioning, adsorption, ion exchange or molecular sieving phenomenon. The conventional column chromatography suffers two major drawbacks: it is generally a time consuming process and quality of resolution is poor. This is mainly because of the fact that in conventional column chromatography the mobile phase percolates through the column under the force of gravity or by small pressure applied by peristaltic pump. This account for the slow flow rate which in addition to extending the time required for elution of the sample creates the problem of peak broadening through diffusion phenomenon resulting in poor resolution. In general, resolution of individual components can be improved by decreasing the particle size of the stationary phase. However, in conventional column chromatography this is not feasible because the use of the fine gel materials will further lower the permeability of the column contributing to the decreasing the flow rate thereby providing greater time for band broadening. The resistance to flow of mobile phase can be overcome by the use of high pressure. In recent years the stationary phases of smaller particle size which can withstand high pressures, have been developed which has facilitated the development of a new chromatographic technique called HPLC. It gives faster and superior resolution with sharp and compact peaks.

Samples to be separated and analyzed are introduced in a small volume into the stream of mobile phase passing through the column.

The components of the sample move through the column at different velocities of specific physical or chemical interaction with stationary phase.

The time at which a specific analytic elutes (emergence from the column is called retention time). The retention under particular condition is considered as identifying characteristics of the given analyte.

INSTRUMENTATION

1. Solvent delivery system which include pump

2. Choice of Mobile phase

3. Sample injection system

4. Column

5. Detector

6. Recorder and data handling device

Figure 1: Diagrammatic representation of HPLC

1. SOLVENT DELIVERY SYSTEM

The main feature of HPLC is pumping system to deliver mobile phase. The pump can deliver mobile phase. The pump can deliver solvent from the reservoir to detector through the pump. The pump can deliver solvent at a pressure upto 10,000 Pascals with a flow rate over 50 ml/min

I.       Gas displacement pump-utilizes constant volume

II.     Reciprotecting pump-utilizes constant volume

III.    Syringe pump-utilizes constant volume

2. CHOICE OF MOBILE PHASE

It is very important in HPLC. Polar solvents are most often used. Optimum separation condition can be obtained by making use of mixture of 2 solvents and is gradient elution by gradient elution technique.

·         Coater, methanol and acetonitile

·         Hexane, pentane, iso-octane and tetrahydro solution

                                     Hexane,        peptone,   iso-octane and  tetrahydro solution

Figure 2: Mixing Chamber

·         It is also essential that gas should be degassed.

·         Degassing can be done by several methods

·         Washing the solvent or by stirring it vigorously with or magnetic stirrer

3. SAMPLING INJECTOR

Sample is introduced into flowing steam of solvent with a sample injector. The sample can be introduced by syringe method or by microwave sampling method.

Syringe method: A syringe method is used to project sample through an inert septum directly into mobile phase.

Microwave method: This method enables the sample to be introduced into pressurized column with or without interpretation of the mobile phase.

The sample is loaded into an external loop in the valve and introduce into the mobile phase by rotation of the valve. In microwave system, we can load the sample from 2-100 ml.

4. COLUMN

The columns are made from stainless tubing which stands high pressure and the dimension of the column is 10-30 cm length and 4-5 mm in diameter. Straight columns are usually porous and rigid particles and the diameter of the particle either > 20 mm or < 20 mm.

PROCEDURE FOR COLUMN PACKING:

Procedure depends mainly on the mechanical strength of the packing materials and particles size. Particles of diameter > 20 mm can usually be dried packed whereas particle with diameter < 20 mm is usually suspended in a suitable solvent and slurry is packed.

5. DETECTOR

Detectors are the devices which continuously monitor the changes in the composition of effluent coming out of the column. In HPLC, mostly used detectors are refractive index detector or UV detector or flourimetry detector.

6. RECORDER AND DATA ANALYZING DEVICES

The signals from detectors are recorded as deviation from the base line (Other same as gas chromatography).

APPLICATIONS OF HPLC

1.   A variety of inorganic anions can be separated by making use of anion exchanger and aqueous eluent containing sodium carbonate.

2.   For the separation of cations, sulphonated resins can be used along with HCl as eluent.

3.  HPLC has been successfully applied for the separation of all classes of steroid hormones.

4.  HPLC has been applied at several different levels in Pharmacy and Pharmacology.

·         To control the drug stability

·         Quantity  of drug determination from pharmaceutical dosage forms, ex. Paracetamol determination in panadol tablet

·         Quantity of drug determination from biological fluids, ex: blood glucose level

      E.g. A no. of antibiotics have been analyzed and separated by HPLC.

5.   HPLC technique has been used for analysis of psychotropic drug in body fluids.

6.   Application in forensic chemistry: E.g. A number of adducting drug such as LHD, heroine and poisons has been analyzed by HPLC.

7.   HPLC can be used for separation of coal and petroleum products.

8.   Vitamins A, D and E have been separated on silica column using HPLC.

9.   Clinical test: - Monitoring of hepatic cirrhosis patient through aquaporin 2 in the urine.

10. Food and essence manufacture:  

·         Sweetener analysis in the fruit juice.

·         Preservative analysis in sausage.

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