High performance liquid - PowerPoint Presentation

Slide1

High performance liquid chromatography (HPLC)

Dr. J. Rajesh

Asso

. Prof

Dept of Chemistry

MSEC, Kilakarai

Slide2

A

f

orm

of

separate,

column identify,

chromatography to and quantify the

compounds.Developed in 1970s.

used

analytical

T

h

e

m

o

s

t

w

i

de

ly

separation

technique.

Slide3

Chromatography

C

h

rom

atography is a

technique whichseparates components in a

mixture due tothe differing component

time taken for each to travel through

awhen

carried through itstationary phase by a mobile

phase.

Slide4

Basically, all chromatographic systems consists of two

phases.

M

obile phase - li

quid or gaseous and flows

over or through the stationary phaseStationary phase

- solid, liquid or a solid/liquid mixture which is

immobilized

Slide5

Some chromatography terms

Analyte

S

ub

stance that is to

be separated during chromatography

Immobilized phaseStationary phase which is immobilized on the support particles or on the inner wall of the column tubing

Slide6

Mobile phase

Phase

which

moves in a definite direction. (liquid/gas/fluid).

Consists of the sample being separated/ analyzed and the solvent that moves the sample through the

column.EffluentMobile phase leaving the

column.Some chromatography terms

Slide7

Different types of

chromatography

methodsPaper

chromatographyLiquid chromatographyGas

chromatographyHigh performance liquid chromatography

Slide8

High performance liquid

chromatography

HPLC

is an extension of conventional liquid chromatography.Powerful

tool in analytical techniquesColumns are tightly packed, and

the eluent is forced through the column under high pressure(up to 5,000 psi) by a pump.

Slide9

Allows to

use

a very smaller

particle sizefor the column packing material which gives a much greater surface area

for interactions between the stationary phase and the molecules flowing through it.

Allows a much better separation of

the components of the mixture.

Slide10

HPLC Technique

Utilizes liquid

mobile phase

to separate the mixture

Analytes are first dissolved in a solvent then through the column under high

pressure of up to 400 atmMixture is resolved into its components

in the column

Slide11

The total

separation

time is often

5 or 10minutes rather than hours or even days required for some

separations by gravity flow with the larger systems.

Slide12

Components of HPLC

Pump

Injector

Column

DetectorRecorder or data

system

Slide13

A Flow Scheme for HPLC

Slide14

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Pump

A

pump

forces the mobile phase through the

column at a much greater velocity than gravity-flow columns.The pump can be pneumatic, syringe-

type, reciprocating, or hydraulic amplifier.

Slide16

Pump (cont.)

a

re

used for

Pneumatic pumps preoperative purposes.

The most widely used pump today is the multihead

pump with two or more reciprocating pistons.

Slide17

Pumps are designed

in

order

to maintain a stable flow rate, avoiding pulsations even

when the composition of the mobile phase variesflow range – 0.01-10 ml/min

Slide18

HPLC Pump

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Injectors

Inject the liquid

sample

within range of

0.1- 100 ml of volume under high pressureProduce minimum band broadening

Produce possible flow disturbancesVolume must

be small (0.1-500 uL)

Slide20

A model injector

Slide21

Injector

Slide22

Columns

steel

or heavy-

S

mooth-bore s

tainless walled glass tubing.

Hundreds of packed columns differing in size and packing are available from manufacture.

Slide23

Columns

E.g.

Column packing

vary in

size (3 to 20 um) with the smaller particles used mostly for analytical separations and the larger ones for

preparative separation.The most common material used for

column packing is silica gel.

Slide24

HPLC columns

Slide25

Slide26

Detector

HPLC

detectors

monitor the elute as it leaves the

columnProduce an electronic signal proportional to the concentration of each separated component

Slide27

Detector

Crucial in trace

analysis

High sensitivityFast responseSimplifies quantitation

Insensitive to

changes in type of solvent, flow rate and temp.

Slide28

The most

widely used detection

methods

SpectrophotometersFluorometersElectrochemical detectors

Mass spectrometerRefractive index detector

Slide29

Detectors used in HPLC

Type

Principle

D

etection limit

CommentsSpectro- photom

eterMeasure absorbance of light<1 ngAnalyte must absorb UV or

visible lightFluorometersMeasures f

luorescen

cepg to ng

Analyte

must

fluoresce

Electro-

Electrochemically

pg

to

ng

Useful

for

chemical

measures

catecholamine

detectors

oxidized/

reduce

s

analyte

Slide30

Detectors used in HPLC

Type

Principle

D

etection limit

CommentsMassDetects

ions afterfg to ngAnalyte mustspectrometerseparation by

be converted

mass-to-charge

to ionizedration

form

Refractometer

Measure change in refractive

index

1

µg

Detection of

most

compounds

but

relatively low

sensitivity

Slide31

Depending on the relative polarity of the solvent and

stationary

phase,

there are two variants in use in

HPLC1.Normal phase HPLCUtilize

polar adsorbent surface and non- polar eluentPolar substance in the mixture sticks to polar adsorbent than

non-polarNon-polar ones will pass more

quickly through the column

Slide32

surf

a

c

e and

2. Reversed phase HPLCUtilize

non-polar adsorb

ent polar eluentAttraction between non-polar compound in the mixture and non-polar adsorbent

Slide33

2. Reversed phase HPLC

(cont.)

Polar molecules will

travel through the column more quickly because there is strong attraction between polar solvent

and polar molecules when pass through the columnReversed phase HPLC is

the most commonly used form of HPLC

Slide34

Solvents used in mobile

phase

hexane, heptane, cyclohexane,

carbon tetrachloride, benzene, toluene, diethyl ether, chloroform etc.Adsorbents used in

stationary phasesilica gel, alumina, celite, cellulose powder, ion-exchange, cellulose, starch

Slide35

Retention time

The

time taken

for a particular

compound to travel through the column to the detectorFrom the time at

which the sample is injected to the point at which the display shows a maximum peak height for that

compound.

Slide36

Slide37

Types of chromatic separation

Adsorption

Chromatography

Ion- exchange ChromatographySize Exclusion Chromatography

Slide38

Adsorption Chromatography

Competition

for

adsorption sites occurs

between the molecules of the mixture to be separated and the molecules of the mobile phase

Mobile phase can be either a single solvent or two or more solvents depend on the analytes to

be desorbedSpeed of migration of the component along the column depend on adsorptive affinity

Slide39

Ion- exchange Chromatography

Molecules can

be

separated by their ionic

charges in a process known as Ion- exchange Chromatography.Ion-exchange

resins are used as the column packing materials.This method

is used for separation of ionic species, such as amino acids.

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Known as gel permeation chromatography or gel filtration

chromatography.

Packing material with

very small pore is used.Precisely controlled pore size materials in the column

Large molecules, such as polymers are physically prevented from passing through the column

Size Exclusion Chromatography

Slide42

Applications

HPLC

is

used for

Chemistr

y and biochemistry

researchanalyzing complex mixtures,Purifying chemical compounds

Quality control to ensure the purity of raw materials

Analyzing air and water pollutants,

Slide43

Applications (cont.)

M

o

n

itoring materials th

at may jeopardize occupational safety or

healthMonitoring pesticide levels in

the environment.To survey food and drug products,To

identify confiscated narcotics

To determine the amount of such chemical compounds found in new drugs in pharmaceutics

Slide44

H

P

LC

as co

mpared with t

he classical

techniqueSmall diameter, reusable stain

less steel columnsColumn packing with very small particles

Control flow of mobile phase

Precise sample introduction

Slide45

wi

th t

h

e

classicalHPLC as

compared

technique (cont.)Good pumping systemSpecial continuous flow detectors- can handle small flow rates and detect very small amounts

Rapid analysisHigh resolution

Slide46

Disadvantages of HPLC

Cost

Complexity

Low

sensitivity for some compoundsIrreversibly

adsorbed compounds not detectedCo-elution difficult to detect

Slide47

Summary

The

modern

da

y technique

is greatlyenhanced in terms of selectivity,

resolution, through miniaturization and the use of very elaborate stationary phases.Therefore HPLC is widely used for separation of molecules in biological, pharmaceutical, food, environmental and industrial process

Slide48

References:

Harold

Varley

practical clinical chemistry

http://scimedia.comhttp://www.forumsci.co.il/HPLC

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THANK YOU!