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.