HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

BY
Abhinav Kumar Bhaskar M.Pharm 1styear Industrial Pharmacy

CONTENT1.

Introduction 2.Principle 3.Stationary phase4.Mobile phase 5.Instrumentation 6.Application

INTRODUCTION:
Chromatography may be defined as a method of separating a mixture of components into individual component through equilibrium distribution between a stationary phase and a mobile phase. Classical liquid column chromatography are generally slow and examination of recovered fraction can be tedious columns percolating through the column under gravity. The technique of high performance liquid chromatography is so called because of its improved performance , where pressure is applied to the column, forcing the mobile phase

PRINCIPLE :

The principle of separation in HPLC is partition. It is known that the resolving power of a chromatographic column increases with column length and the no. of theoretical plate per unit length. As the no. of therotical plate related to the surface area of the stationary phase it follows that the smaller the particle size of the stationary phase, better the resolution.

INSTRUMENTATION
The system consist of : A solvent reservoir and mixing system A high pressure pump A sample injection system A column A detector and recording unit.

Reservoir | Pump | Gradient controller | Mixing chamber | Solvent conditioning column | Injector | Precolumn | Analytical column | Detector |

1.Reservoir -Glass/ stainless steel container capable to 12 liter of mobile phase. -Highest purity sub. -Filtration -Degassing using vaccum , sonication.

High pressure pump:

Its performance directly affects retention time, reproducibility and detector sensitivity. Types of pump depending upon the constant flow or constant pressure control.Gas displacement pump: These offers non pulsating flow but have limited solvent capacity. Reciprocating pump: Type of mechanical pump. This consist of a small chamber in which the solvent is pumped by a motor driven piston.

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Gradient controller

This is an electronic device that synchronizes the operation of the two pumps to provide a mobile phase mix. Of a desired concentration.

Solvent conditioning column

Most HPLC column packing material are prepared from silica gel, which will dissolve in solvent whose pH values are below 2or above 7 This result in a shrinkage of the packing material, giving rise to void spaces in which separated solutes remix or diluted thereby leading to a loss of resolution. To prevent this a small column(5 to 1ocm) packed with HPLC grade silica gel is inserted into the liquid stream after the pump and before the injector. The material in this column is dissolved preferentialy, saturating the mobile phase and

Sample Injection System:

The sample is introduced into the flowing stream of solvent with an injector. Injection method:1. Syringe Injection: Septum injectors allow sample introduction by a high pressure syringe through a self-sealing eastomer septum. 2. Stop flow Injection: The flow of solvent is momentarily stopped and the sample is directly injected on the head of the column.

Precolumn
when stationary phases consist of a thin layer of liquid coated on a solid support, the liquid slowly dissolves in the mobile phase causing degradation of resolution. In this case the precolumn will contain solid support coated with a higher percent of liquid phase than the analytical column to saturate the mobile phase and retard dissolution. Also it protect the column by trapping the particulate matter.

Columns:
Columns are made up of stainless steel and are manufactured so that they can withstand pressures of up to 5.5x107 Pa. Columns of 20 to 50 cm in length and 1 to 4 mm in diameter are generally used. Porous plugs of stainless steel and Teflon are used in the end of the columns to retain the packing material. Columns are thermostatic ovens which employed either jacket method or air circulating method.

Column Packing:
The packing consist of small, rigid particles having a narrow particle-size distribution . Three forms of packing Microspores supports where Microporus ramify through the particles which are generally 5 to 10 m in diameters. Pellicular supports where porous particle are coated onto an inert solid core such as a glass bed of about 40 m in diameter. Bonded phase where the stationary phase is chemically bonded onto an inert support.

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Solvent:
Selection of mobile phase is most important in HPLC. In general, polar materials are separated using partition chromatography while non polar substances are separated using adsorption chromatography. Degassing of solvent necessary by the use of vaccum, sonication method.

*STATIONARY PHASEType 1. Normal phase chromatography stationary-alkyl nitrile /alkylamine. Mobile-hexane

2.Reversed phase chromatography stationary-C8/C18 Hydrocarbon Mobile-water/acetonitrile , water/methanol.

Detector:
Detectors are used depends upon the property of the compounds to be separated. Different detector available areUV detector: This Detector is based upon the light absorption characteristics of the sample. Two types of detector fixed wavelength detector and variable wavelength detector. Refractive index detector: This is a non specific or universal detector. It has low sensitivity and specificity. Conductivity detector: Based upon electrical conductivity, the response is recorded. This detector is used when the sample has conducting

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Flourimetric detector: This detector is based on the flourescent radiation emitted by some class of compounds. This detector has more specificity and sensitivity. Amperometric detector: This detector is based on the reduction or oxidation of the compounds when a potential difference is applied. This is applicable when compounds have functional group which can be either oxidized or reduced.

Recorder:
Recorder are used to record the response obatined from detectors after amplification. The signal from the detectors are recorded as deviated from a base line. Two open recorders are used with instruments having two detectors.

Application:
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Qualitative analysis: It is identification of a compounds. This is done by comprising the retention time of the sample as well as the standard. If there is deviation then they are not the same compound. Checking the impurity of a compound: By comparing the chromatogram of the standard and that of the sample, the purity of the compound can be inferred. If additional peaks are obtained, impurities are present. Multicomponent analysis: Multicomponent analysis can also done easily. Marketed formulations which contain several drugs, can be determined quantitatively for each component. Isolation and identification of drugs or metabolites in urine, plasma, serum etc can be carried out.