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Chromatography is usually introduced as a technique for separating and/or

Identifying the components in a mixture. The basic principle is that components in a mixture have different tendencies to adsorb onto a surface or dissolve in a separate and purify the intermediates and products in various syntheses. solvent. It is a powerful method in industry, where it is used on a large scale to Chromatography may be preparative or analytical. The purpose of preparative use (and is thus a form of purification). Analytical chromatography is done normally with smaller amounts of material and is for measuring the relative proportions of analytes in a mixture. The two are not mutually exclusive. High performance liquid Chromatography HPLC is an abbreviation for High Performance Liquid Chromatography (It has also been referred to as High Pressure LC) HPLC is a separation technique that involves the injection of a small volume of liquid sample into a tube packed with tiny particles (3 to 5 micron (m) in diameter called the stationary phase) where individual components of the sample are moved down the packed tube (column) with a liquid (mobile phase) forced separated from one another by the column packing that involves various particles. through the column by high pressure delivered by a pump. These components are chemical and/or physical interactions between their molecules and the packing

chromatography is to separate the components of a mixture for more advanced

This is the chromatogram resulting from the injection of a small volume of liquid extracted from a vitamin E capsule that was dissolved in an organic solvent. Modern HPLC separations usually require 10-to 30-minutes each

These separated components are detected at the exit of this tube (column) by a detector is called a liquid chromatogram Instrumentation:

flow-through device (detector) that measures their amount. An output from this

Flow diagram :Recycling Preparative HPLC

Solvent reservoir Degasser Pump Rheodyne Precolumn Main column Detector Recorder Recycle valve Fraction Collector Siphon Waste

(1) (2)

RESOVOIR: brown bottles are used for the protection of light sensitive eluents Degasser : degasser is operated by a vacuum pump in order to detecting noise

eliminate dissolved gasses in the mobile phase resulting in low Pump: The role of the pump is to force a liquid (called the mobile phase) through the liquid chromatograph at a specific flow rate, are in the 1-to 2-mL/min range. During the chromatographic expressed in milliliters per min (mL/min).Normal flow rates in HPLC experiment, a pump can deliver a constant mobile phase composition



(isocratic) or an increasing mobile phase composition (gradient).

Injector: The injector serves to introduce the liquid sample into the flow stream of the mobile phase. The manual sample injector is an 6 way valve making it possible to directly inject an sample into the sample

loop using a syringe from the needle port provided in the valve shaft. also be able to withstand the high pressures of the liquid system. An auto sampler is the automatic version for when the user has many samples to analyze or when manual injection is not practical (5)

Typical sample volumes are 5-to 20-microliters (L).The injector must

PreColumn: The small column placed between the pump and the main column it is also called as the guard column. It removes particles in the mobile phase pre saturates the mobile phase with the stationary phase Or with dissolved substrate to prevent a loss of stationery phase or dissolution of the column and chemically absorbs substances that might interfere with the separation.


stationary phase separates the sample components of interest using

Column: Considered the heart of the chromatograph the columns

various physical and chemical parameters. The small particles inside

the column are what cause the high back pressure at normal flow rates. The pump must push hard to move the mobile phase through the column and this resistance causes a high pressure within the (7) chromatograph.

Detector: The detector can see (detect) the individual molecules that come out (elute) from the column. In preparative hplc we have two detectors ,UV Detector: its light source is deuterium lamp, wavelength is 195-370 nm,its digital display unit is absorbance.

method is deflection,its an universal detector .its digital display is The identification(ID) of individual compounds in the sample: RIU { REFRACTIVE INDEX UNIT }

RI DETECTOR : its light source is led{light emitting diode} ,measuring

The most common parameter for compound ID is its retention time(the time it takes for that specific compound to elute from the column after injection)depending on the detector used, compound ID is also based on the chemical structure, molecular weight or some other molecular parameter

Separation Modes of HPLC There are four major separation modes that are used to separate most compounds: 1.Reversed-phase chromatography 2.Normal-phase chromatography 4.Size exclusion chromatography Reversed-Phase Chromatography (RPC) 3.Ion exchange chromatography

Reversed phase HPLC (RP-HPLC) has a non-polar stationary phase and an aqueous, moderately polar mobile phase. One common stationary R is a straight chain alkyl group such as C18H37 or C8H17. With such phase is a silica which has been surface-modified with RMe2SiCl, where stationary phases, retention time is longer for molecules which are less

Normal phase:

polar, while polar molecules elute more readily (early in the analysis)

The solid packing material adsorbent may be silica or alumina, which is more polar than the solvent { or the mobile phase) ,it has the stronger affinity for the polar molecules than the non polar ones, so the non polar , the faster substances moves through the column. polar molecules eluant comes out first, as the solvent becomes more Ion Exchange Chromatography For solutions of ions, select a column chromatography.

that is packed with an ion exchange resin. This is called as ion exchange Size exclusion chromatography: Molecules diffuse into pores of a porous medium ,depending on their size relative to the pore size, molecules are separated.

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