HPLC

Presented byANU BALA

OUTLINE

• INTRODUCTION• TYPES OF CHROMATOGRAPHY• RP LIQUID CHROMATOGRAPHY• INSTRUMENTATION• CHROMATOGRAM• METHOD DEVELPOMENT LAYOUT• ADVANTAGES OF HPLC

INTRODUCTION• High performance liquid chromatography• A form of liquid chromatography used to

separate compounds that are dissolved in solution.

• HPLC instruments consist of a reservoir of mobile phase, a pump, an injector, a column, and a detector

• Compounds are separated by injecting a sample mixture onto the column

• Separation is based on Partition of compounds towards stationary and mobile phase

A. Based on modes of chromatography

1. Normal phase mode

2.Reverse phase mode

B. Based on principle of separation

1. Adsorption chromatography

2. Ion exchange chromatography

3. Partition chromatography

4. Size exclusion

C. Based on elution technique

1. Isocratic separation

2. Gradient separation

TYPES OF CHROMATOGRAPHY

D. Based on the scale of operation

1. Analytical HPLC

2. Preparative HPLC

E. Based on the type of analysis

1. Qualitative analysis

2. Quantitative analysis

RP LIQUID CHROMATOGRAPHYStationary Phase• Is non polar.• The stationary solid surface is coated with a 2nd liquid (the

Stationary Phase) which is immiscible with mobile phase

Mobile PhaseA polar mobile phase(ACN, MeOH, WATER + BUFFER)

Principle Is based upon the principle of partitioning coefficient .It is the ratio of concentration of analyte in stationary phase and concentration of analyte in mobile phase

Reverse phase chromatography (RP)

Non Polar Polar

INSTRUMENTATION

HPLC Pump

Column

Bonded Phases

• C-2 Ethyl Silyl -Si-CH2-CH3

•CN Cyanopropyl Silyl -Si-(CH2)3-CN

• C-18 Octadecyl Silyl -Si-(CH2)17-CH3

•C-8 Octyl Silyl -Si-(CH2)7-CH3

The longer the alkyl chains, the longer the retention time in a reversed phase column.

Silica Stationery Phase support

Injector

Detector

Detector is a device used in liquid and gas chromatography to visualize components of the mixture being eluted off the chromatography column

There are two general types of detectors1 Destructive detector2 Non destructive detector

UV Detector

Photodiode Array Detectors

CHROMATOGRAM

BAD CHROMATOGRAM

METHOD DEVELOPMENT

PHYSICOCHEMICAL PROPERTIES (i.e. Structure, Solubility, Nature, components)

SAMPLE PREPARATION(i.e. Concentration, Dilutions)

SELECTION OF HPLC PARAMETERS1. Mobile phase : Selection of solvents and combinations Selection of Buffers Isocratic / Gradient Flow rate2. Column: NP/RP Temperature3. Detection: Selection of wavelength4. Chromatography: Parameters of Chromatogram

METHOD DEVELOPMENT

PRE METHOD VALIDATION1. Selection of Upper and Lower Limit of Quantitation2. Impurity profiling3. Accuracy and Precision4. Linearity5. Selectivity (LOD, LOQ)6. Stability

Validation

Advantages of HPLC

High sensitivity

High performance

Rapid process and hence time saving

Can be used for qualitative as well as quantitative estimation

Can be used for both analytical and preparative purpose

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