HPLC by Ekta

8/4/2019 HPLC by Ekta

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Contents:-

Introduction

Principle

Elution technique Instrumentation

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Introduction:

The technique of HPLC is so called because of its

improved performance when compared to classical

column chromatography.

It is also called as high pressure liquid

chromatography since high pressure is used when

compared to classical column chromatography.

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PARAMETER CLASSICAL COLUMNCHROMATOGRAPHY

HPLC

particle size Large60-200

Small3-20

Column length 0.5 -5m 5 -50 cmOperating pressure Low(


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Preparative scale: In this sample load is high.So the collected samples are reused. E.g.

Separation of few gm. of mixtures by HPLC.

Analytical scale: In this recovery of thesamples for normally not done, since the sampleused isvery low. E.g. g quantities.

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Principle of separation of hplc Principle: partition co-efficient

When 2 immiscible liquids are present, a mixture of solutes

will be distributed according to their partition co-efficient.

The component which is more soluble in stationary phasetravels slower & which is more soluble in mobile phase

travels faster.

The stationary phase as such cannot be a liquid. Hence a

solid support is used over which a thin film or coating ofliquid is made which act as stationary phase.

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Normal phase mode:Stationary phasepolar

Mobile phase- non polar

Reverse phase mode:Stationary phase- non polar

Mobile phasepolar

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Elution technique: Isocratic separation: In this technique, the same

mobile phase combination is used throughout theprocess of separation. The same polarity or elutionstrength is maintained throughout the process.

Gradient separation: In this technique, a mobile

phase combination of lower polarity or elutionstrength is used followed bygradually increasing thepolarityor elution strength.

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INSTRUMENTAL REQUIREMENTS Solvent Reservoir

Tubing

Pumps Injector system

Guard column

Analytical column

Detectors

Recorders & integrators9


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SOLVENT RESERVOIR

It should be inert to mobile phase.

Made up ofstainless steel or glass.

Capacity: More than 500ml.

Mobile phase flow rate: 1-2 ml/min

Degassing: In some cases, aqueous solvents and someorganic solvents are degassed prior to use. This is done toprevent formation of gas bubble in the detector.

It is done by various method:-

- stirring of the mobile phase under vacuum

- By sparging with helium gas

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TUBING: The nature of the tubing used to connect all parts of

the system deserves some attention. The tubing should be

- inert

- have the ability to withstand pressure- able to carry sufficient volume.

PUMPS:These are used to pass the mobile phases at high

pressure of about 1000 to 3000 psi into the column,

which is required cause of high resistance to the flow

offered due to the less particle size of the stationary

phase. 14


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Ideal requirement: Produce very high pressure 5000-10000psi.

Produce pulse free output.

Flow rate should be control & reproducible.

Flow rate of mobile phase should be in the range

of0.1-10ml/min. All material of construction should be corrosion

free like Stainless steel, Teflon.

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(a) Reciprocating pump:

Characterized by filling cycle and pumping cycle.

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WORKING:

Contains reciprocating piston that moves back and

forth in hydraulic chamber. By the movement of piston solvent flow into the

column under high pressure.

When piston moves backward inlet valve openwhile exit valve closes. This result in mobile phasebeing drawn into the main chamber (cylinder).

When the piston moves to the front the inlet valve

closes and the exit valve opens. The reduction in volume in main chamber due to

forward motion of piston result in mobile phasemoving out of the exit valve under high pressure.

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Advantage:- Verysmall volume can handle.

- Very high pressure-1000psi.- Gradient process can be applied.

- Flow rate not depend on viscosity and back pressureof the solvent.

Disadvantage:- Produce pulse flow.

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(b)Syringe pump: In which all mobile phase is contained

in the pump.

The piston inside the chamber isactuated by screw feed drive connected

to the motor so volume displaced iscontrolled.

Advantage:

- Cheap.

- Produce pulseless flow.Disadvantage:

- For very limited solvent

capacity.

- Gradient process can not be applied. 20


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(2) Constant Pressure/ Pneumatic pump Mobile phase is held in collapsible containerwhich

is flexible and pressurized by gas. Since the mobilephase is directly in contact with gas so it may be

dissolved. The pneumatic amplifier pump( Haskell pump) is a

modification of this pump, the gas pressure is

applied to large piston which is connected to thesmall piston in contact with mobile phase.

Advantage

- Cheap.

- Produce pulse less flow. 21


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Disadvantage:

- For verylimited solvent capacity.- Gradient process can not be applied.

- Flow rate depend on viscosity and back pressure

of the solvent.- Limited pressure obtained.

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INJECTOR SYSTEM:

These are the devices available for injection ofthe sample into the column.

Ideal requirement:

Sample should be injected in a narrow plug.

Size of sample should bevariable.

Should be reproducible.

System must able to inject against a high pressurewithout sample loss, when system to be

automated.

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Two types are :

(1) Septum injector: Septum comes into direct

contact with mobile phase,so it must be constructed of

inert material like Teflonand must be withstandpressure up to 1000-1500 psiwithout any leakage.

Disadvantage:

- Poor reproducibility

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( ) L i j t


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(2) Loop injector

Advantage

- Can be automated.

- Variable volume loop are available.

- Very accurate. Disadvantage

- Sample loop must be changed in order to change the

volume injected. 25


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Guard Column:

Guard column has very small quantity of

adsorbent and improves the life of the analytical

column. It also acts as a prefilter to remove particulate

matter, if any, and other material.

Guard column has the same material as that of theanalytical columns.

They do not have any contribution in the

separation.

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Analytical Columns: Analytical column is that important part of HPLC technique

which decides the efficiency of separation.

There are several stationary phases available depending

upon the technique or mode of separation used.

Column material :

The columns are made up of either Stainless steel, glass,

polyethylene and PEEK( poly ether ether ketone).

Column length: Varies from 5cm to 30cm Column diameter: Ranges from 2mm to 50mm

Particle size : From 1 to 20

Particle nature: Spherical, uniform sized, porous materials are

used. 27


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Functional group:The functional group present in the stationary

phase depends on type of chromatographic separation.

In normal phase mode it contains the silanolgroups(hydroxyl group).

In reverse phase mode it contains the followinggroups :

C18 Octa Decyl Silane (ODS) column

C8 Octyl columnC4 Butyl column

CN Nitrile column

NH2

Amino column 28


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DETECTORs: Ideal requirement :

High sensitivity

Reproducible response & give response to all

analyte.

Response should be linear over wide range of

concentration. Should not be sensitize to flow rate fluctuation or

temp. change or change in composition.

Capable of withstand at high pressure.

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Parameter related to performance of detectors

Noise : It is the variation in output due to fluctuationin power supply.

Types of Noise:

1. Short term: appear as fuzz on recorder andaffects the minimum determinable quantities.

2.Long term: appear peaks &valleys on the baseline

and causes difficulty in locating small peaks.3. Drift: steadyupward &downward movement of

baseline causing the recorder to go off scale

during a long chromatographic run.30


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Types of detectors

(1) Differential detectors or bulk property detectors

They provides a differential measurement of a bulk property that

is possessed by both the solute and the mobile phase.

They are nonspecific and respond to a wide range of compounds.

Ex:- Refractive index detectors.

(2) Selective detectors or solute property detectors

They measure a property of the sample which is not possessed bythe mobile phase.

Ex:- U.V. & fluorescence detectors.

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(1) UV visible detector:

Useful for aromatic compound.

Measure nanogram quantity of drug.

Based on beers law.

Classified as fixed & variable detector.

Fixed wave length detector:

Provide appropriate wave length.

Line sources & monochromator is filter, is used at254nm.

Used for quantitative purpose.

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Variable wavelength detector

Continuous source & monochromator is grating or prism is

used. Used for scanning purpose.

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Advantage:- Nondestructive.

- Insensitive to change in solvent flow rate & temp. Disadvantage:

- No uniformity of response for different

component.

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photodiode array


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photodiode arraydetector

It is a type of multichannel UVdetector.

Principle Here polychromatic light from the

source is passed via flow cell &diffracted from a grating, such thateach photodiode array receive a

narrow wavelength band ofradiation. This will discharge the capacitor

formed in p-n junction, thecapacitor charge lost is integratedby pre amplifier circuit whichproduce voltage proportional toradiation intensity.

Thisvoltage is converted to signalwhich is recorded as peaks onchromatogram. 36


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Advantage:

- High sensitivity & speed.

- It is a stable to change in temp.& flow rate.

- Highly suitable for gradient analysis.

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(2) Fluorescence detector

Used for quantitative purpose. Increase sensitivity is due to dependence of

fluorescent intensity on the intensity of the

exciting light.

F = 2.3 Io abc Advantage:

- Very sensitive & selective.

Disadvantage:- Response is narrow and non linear for broadconcentration range.

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(3) Refractive indexdetector

When the solute elute out ofcolumn the change in R.I.occur so signals appear onchromatogram.

It is based on Fresnel law. Advantage

- Universal detector- Used for low range of

concentration. Disadvantage

- Depend on the temp.- Not used for gradient

analysis.40

REFERENCES


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REFERENCES

1. Pharmaceutical Analysis Modern Methods,Part B, by JAMES W. MUNSON.

2. Textbook of Pharmaceutical Analysis,

3rd edition, by DR. S. RAVISHANKAR.

3. http://hplc.chem.shu.edu/new/hplcbook/detector5. Instrumental method of analysis (seventh edition )

Willard merritt and dean settle page no 593 600

6. Instrumental method of chemical analysis

B.K.Sharma page no-, 58,59.

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http://hplc.chem.shu.edu/new/hplcbook/detectorhttp://hplc.chem.shu.edu/new/hplcbook/detectorhttp://hplc.chem.shu.edu/new/hplcbook/detectorhttp://hplc.chem.shu.edu/new/hplcbook/detectorhttp://hplc.chem.shu.edu/new/hplcbook/detectorhttp://hplc.chem.shu.edu/new/hplcbook/detectorhttp://hplc.chem.shu.edu/new/hplcbook/detectorhttp://hplc.chem.shu.edu/new/hplcbook/detectorhttp://hplc.chem.shu.edu/new/hplcbook/detectorhttp://hplc.chem.shu.edu/new/hplcbook/detector


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HPLC by Ekta