HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Sanjeet

High performance liquid chromatography is highly improved column chromatography used in separation, purification and characterization of non-volatile compounds.

Ø It has three important parts: Sampler, Pumps and Detector

Major modes of separation

Ø Reversed-phase chromatography

Ø Normal-phase and adsorption chromatography.

Ø Ion Exchange Chromatography

Ø Size Exclusion Chromatography

Typical non-volatile compounds that can be analysed in HPLC are

Ø Pharmaceuticals

Ø Proteins

Ø Organic chemicals

Ø Heavy hydrocarbons

Ø Natural product

Main ways to interpret a Chromatography

Ø Peak height (Purification)

Ø Peak area (Quantitative assessment)

Applications

1.    HPLC is optimum for the separation of chemical and biological compounds that are non-volatile, such as:

a)    Salts

b)   Pharmaceuticals – Aspirin

c)    Proteins

d)   Organic chemicals - Polystyrene

e)    Natural hydrocarbons- asphalt 

f)     Thermally unstable- TNT

2.    Identification and quantification of the sample components using the determination of peak height and peak area.

3.    Determination of trace pharmaceutical compounds using very sensitive detectors.

Factors

1.    Reproducibility

Ø Retention in HPLC is temperature-dependent.

Ø If temperature varies, then it is difficult to assign “peaks” to specific compounds in the chromatogram.

2.    Solubility

Ø Certain chemical compounds may have low solubility in the HPLC mobile phase.

3.    Stability

Ø The temperature needs to be much lower down to 4 °C.

Columns

1)   Usually stainless steel.

2)   Length 10-30 cm

3)   Packing are 3, 5 or 10 µm particle size

4)   Normally packed under 6000 psi

Detectors

1)   Filter instrument

2)   Variable wavelength

3)   Diode array detector