high performance liquid chromatography (HPLC)

Prepared & presented by: Ekta A. Sharma M.Pharm ( QA) Roll no: 10 Guided by: Dr. N.J.Shah M.Pharm, Ph.D. Principal of DDPC Dharmaj Degree pharmacy college, Dharmaj.
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Contents:

Introduction Principle Elution technique Instrumentation

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.

COMPARISION OF CLASSICAL COLUMN CHROMATOGRAPHY WITH HPLC:

PARAMETER CLASSICAL COLUMN HPLC CHROMATOGRAPHY

Large 60-200 Column length 0.5 -5m Operating pressure Low(<20 psi) Flow rate Low to very low Low to medium (gm. Sample load or mg) <500 theoretical Resolving power plates per meter Scale of operation Preparative scale particle size

Small 3-20 5 -50 cm High(500-3000 psi) Medium to high Low to very low (g) >100000 plates per meter Analytical and preparative scale
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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 the

samples for normally not done, since the sample used is very low. E.g. g quantities.

 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 phase travels 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 of liquid is made which act as stationary phase.

 Normal phase mode:

Stationary phasepolar Mobile phase- non polar

Reverse phase mode:

Stationary phase- non polar Mobile phase- polar

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

mobile phase combination is used throughout the process of separation. The same polarity or elution strength is maintained throughout the process. Gradient separation: In this technique, a mobile phase combination of lower polarity or elution strength is used followed by gradually increasing the polarity or elution strength.
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INSTRUMENTAL REQUIREMENTS

Solvent Reservoir Tubing Pumps Injector system Guard column Analytical column Detectors Recorders & integrators
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SOLVENT RESERVOIR
It should be inert to mobile phase. Made up of stainless steel or glass. Capacity: More than 500ml. Mobile phase flow rate: 1-2 ml/min

Degassing: In some cases, aqueous solvents and some

organic solvents are degassed prior to use. This is done to prevent 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 14 phase.

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 of 0.1-10ml/min. All material of construction should be corrosion free like Stainless steel, Teflon.
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 Two types :

1. constant flow rate. 2. constant pressure.

1)

Constant flow rate:

(a) Reciprocating pump (b) Syringe drive pump

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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 open while exit valve closes. This result in mobile phase being 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 phase moving out of the exit valve under high pressure.
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 Advantage:
of Very small volume can handle. Very high pressure-1000psi. Gradient process can be applied. Flow rate not depend on viscosity and back pressure 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 is actuated by screw feed drive connected to the motor so volume displaced is controlled. Advantage: - Cheap. - Produce pulseless flow. Disadvantage: - For very limited solvent capacity. - Gradient process can not be applied.
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(2) Constant Pressure/ Pneumatic pump

Mobile phase is held in collapsible container which is flexible and pressurized by gas. Since the mobile phase 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 the small piston in contact with mobile phase.
Advantage

- Cheap. - Produce pulse less flow.

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 Disadvantage:

- For very limited 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 of the sample into the column. Ideal requirement:

Sample should be injected in a narrow plug. Size of sample should be variable. Should be reproducible. System must able to inject against a high pressure without 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 Teflon and must be withstand pressure up to 1000-1500 psi without any leakage. Disadvantage: - Poor reproducibility

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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

 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 the analytical 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.
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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 silanol groups(hydroxyl group). In reverse phase mode it contains the following groups : C18 Octa Decyl Silane (ODS) column C8 Octyl column C4 Butyl column CN Nitrile column NH2 Amino column
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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 fluctuation

in power supply. Types of Noise: 1. Short term: appear as fuzz on recorder and affects the minimum determinable quantities. 2. Long term: appear peaks &valleys on the baseline and causes difficulty in locating small peaks. 3. Drift: steady upward &downward movement of baseline causing the recorder to go off scale during a long chromatographic run.
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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 by the 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 at 254nm. 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 It is a type of multichannel UV

detector

detector. Principle Here polychromatic light from the source is passed via flow cell & diffracted from a grating, such that each photodiode array receive a narrow wavelength band of radiation. This will discharge the capacitor formed in p-n junction, the capacitor charge lost is integrated by pre amplifier circuit which produce voltage proportional to radiation intensity. This voltage is converted to signal which is recorded as peaks on chromatogram.

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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 broad concentration range.

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(3) Refractive index

detector

When the solute elute out of

column the change in R.I. occur so signals appear on chromatogram. 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.
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REFERENCES
1. 2. 3. 5.

Pharmaceutical Analysis Modern Methods, Part B, by JAMES W. MUNSON. Textbook of Pharmaceutical Analysis, 3rd edition, by DR. S. RAVISHANKAR.

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

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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