High performance liquid chromatography-(HPLC)

(HPLC)-High performance liquid chromatography

·        HPLC is a product of the scientific effort towards optimization of the conventional column chromatography.

·      This method uses an extremely high pressure. The flow rate therefore is high and the experimental time is shortened considerably.

·      This technique is suitable for both analytical and preparation purposes.

·      The technique may be used with small amounts of sample (pico gram)

·         It is popular for the separation of polar compounds such as drug metabolites, which, in general are poorly resolved by other techniques.

·         Thus, it may apply the principle of adsorption, partition, ion-exchange, exclusion, and affinity chromatography.

Working principle

·       It is a technique for separation identification and quantification of components in a mixture.

·        It is especially suitable for compounds which are not easily volatilized, thermally unstable and have high molecular weights.

·         The liquid phase is pumped at a constant rate to the column packed with the stationary phase.

·         Before entering the column the analysis sample is injected into the carrier stream.

·         On reaching the column the sample components are selectively retained on the basis of physico-chemical interactions between the analyte molecules and the stationary phase.

·         The mobile phase moving at the steady rate elutes the component based on the operating conditions.

·         Detection techniques are employed for detection and quantification of the eluted components.

INSTRUMENTATION

Application

·         Pharmaceutical applications.

·         Environmental applications.

·         Applications in forensics.

·         Food and flavor.

·         Application in clinical tests.

Advantage

·         It offers a quick, automated and highly accurate method to identify certain chemical components in a sample

·         It is highly efficient and uses a pump rather than gravity, to force a liquid solvent through a solid adsorbent material

·         It is accurate and highly reproducible.

·         HPLC run cans can be performed with minimal training.

Disadvantage

·         HPLC is costly, complex and doesn’t work for all samples

·         HPLC does have low sensitivity for certain compounds, and some cannot be detected as they are irreversibly adsorbed.

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