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A SIMPLE WAY TO UNDERSTAND THE HPLCINSTRUMENTATION

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Also called HIGH PRESSURE

LIQUID CHROMATOGRAPHY

Used by chemists to separate

compounds that are dissolved in solution

The mobile phase is a liquid solvent

containing the sample as a mixture ofsolutes

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Parts of highperformance

liquidchromatography

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Invented by Greenley and Volpe

Serves as the site from which the mobile

phase is pumped into the liquids

chromatography systems column.

A reservoir for mixing and holdingliquids

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PUMP

RECIPROCATING

PUMP

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Used to deliver a mobile phase solvent at a uniform rateat pressures that are typically from 500 to 5,000 psi on

traditional and from 6,000 to 10,000 psi on modern pumps.

The flow rate should be constant because most pumps

are flow sensitive and errors in quantity will result to

changes on flow rate.

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3 MAJOR TYPES OF

PUMP1) Screw-driven Syringe Type -produce a pulse free

delivery whose flow rate is readily controlled.

2) Reciprocating pump most widely used type ofpump. It is consist of a small cylindrical chamber

that is filled then emptied by the back and forth

motion of a piston.

3) Pneumatic pump - consists of a collapsible solventcontainer housed in a vessel that can be pressurized by a

compressed gas. They are simple, inexpensive and pulse

free.

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INJECTOR

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The function of the injector is to place the sample

into the high-pressure flow in as narrow volume as

possible so that the sample enters the column ashomogeneous, low-volume plug.

The shortest possible length of the tubing should beused to minimize the spreading of the injected volume

during the transport to the column

The needles to be used should have smooth or blunt

tip not sharp or tip with metal burrs

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SEPARATION COLUMN

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When components pass through the column, the

separation of sample components are achieved.

Are commonly filled with silica gel because its

particle shape, surface properties and pore

structure helps to get a good separation.

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TYPES OF COLUMN ACCORDING TO

FUNCTION

1) NORMAL PHASE- the retention is governed by the interaction of the

polar parts of the stationary phase and solute.

2) REVERSE PHASE- the packing material is relatively non-polar and the

solvent is polar with respect to the sample.

3) SIZE EXCLUSION - In this column type, molecules are separated

according to size. Small molecules penetrate into the pores within the

packing while larger molecules only partially penetrate the pores. The

large molecules elute before the smaller molecules.

4) ION EXCHANGE - the sample components are separated based upon

attractive ionic forces between molecules carrying charged groups of

opposite charge to those charges on the stationary phase.

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DETECTOR

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Equipped with flow-through cell which

was applied by the group of Tiselius in

Sweden in 1940.

Have high sensitivities often allowing thedetection of nanograms of material.

Modern models are very flexible to allow

rapid conversion from one mobile phase to

another and from one mode to another.

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1) PARTITION CHROMATOGRAPHY

Most widely used type of HPLC in which the stationary phase isa second liquid that is immiscible with the liquid mobile phase.

Based on a thin film formed on the surface of a solid support by

a liquid stationary phase.

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2 SUBDIVISIONS OF

PARTITION

CHROMATOGRAPHY

1)LIQUID-LIQUID CHROMATOGRAPHY

The stationary phase is a solvent that is held

in place by adsorption on the surface of packing particles.

2) LIQUID-BONDED PHASE CHROMATOGRAPHY

The stationary phase is an organic species that is

attached to the surface of the packing particles by chemical bonds.

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2) ADSORPTION CHROMATOGRAPHY

Also called Normal Phase HPLC

Probably one of the oldest types of chromatography

around.

It utilizes a mobile liquid or gaseous phase that is

adsorbed onto the surface of a stationary solid phase.

The balance between the mobile and stationary phaseaccounts for the separation of different solutes .

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3) ION EXCHANGE CHROMATOGRAPHY

Use of a resin (the stationary solid phase) is used to covalently attach

anions

or cations

onto it.

Solute ions of the opposite charge in the mobile liquid phase are attracted

to the resin by electrostatic forces.

The retention is based on the attraction between solute ions and charged

sites bound to the stationary phase.

Ions of the same charge are excluded.

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4) MOLECULAR OR SIZE EXCLUSION CHROMATOGRAPHY

Also known as gel permeation or gel filtration chromatography

Liquid phase passes through a porous gel which separates the molecules

according to its size.

Powerful technique that is particularly applicable to high-molecular weight

species.

This type of chromatography lacks an attractive interaction between the

stationary phase and solute.

The pores are normally small and exclude the larger solute molecules, but

allows smaller molecules to enter the gel, causing them to flow through a larger

volume. This causes the larger molecules to pass through the column at a faster

rate than the smaller ones.

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5) AFFINITY CHROMATOGRAPHY

This is the most selective type of chromatography.

Relies on the property of biologically active substances to

form stable, specific, and reversible complexes.

.It utilizes the specific interaction between one kind of

solute molecule and a second molecule that is immobilized

on a stationary phase.

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6) ISOCRATIC FLOW AND GRADIENT ELUTION

ISOCRATICconstant composition

Coined by Csaba Horvath who happens to be one of the pioneers of HPLC

GRADIENT ELUTION - separation in which the mobile phase

composition is changed during the separation process.

Gradient elution decreases the retention of the later-eluting componentssothat they elute faster, giving narrower (and taller) peaks for most components

In gradient elution, the elution order may change as the dimensions or flow

rate change.

In isocratic elution, the selectivity does not change if the column dimensions

(length and inner diameter) change - that is, the peaks elute in the same

order.