Principle of HPLC ( Liquid Chromatography ) : Pharmaceutical Guidelines

Principle of HPLC ( Liquid Chromatography )

Learn how High Performance Liquid Chromatography works and how chromatographic column separates the compounds from a mixture.
High performance liquid chromatography is an effective type of column chromatography which is widely used in pharmaceuticals. It is very useful to determine the assay and related substances in drug substances. In general HPLC is used to separate the components of a mixed drug substance.

In HPLC chromatography, column plays a significant role in separation of different compounds because it contains stationary phase. Stationary phase is a bad of polar or non polar particles according to the type of column. Polar and non polar columns are used according to the nature of the sample to be analyzed.

Related: HPLC Column Performance Evaluation and Column Care

HPLC flow chart

Mechanical pumps are used to pump the mobile phase into the system and injector introduces the sample into the mobile phase which enters in column at a constant flow rate.

Related: Guideline for Preparation of Mobile Phase

When a mixture of compound enters in a column, it separates the compounds on the basis of their polarity. If stationary phase is non polar then it attracts the non polar compounds and a polar compound elutes first then a non polar compound and if stationary phase is polar then non polar compound elutes first.

Related: Resolution Factor, Tailing Factor, Theoretical Plates and Capacity Factor in HPLC

HPLC system

Detectors are used to determine the separated compounds by ultra violet absorption. The light absorption depends upon the concentration of compound in mobile phase. These response signals are recorded by the computer software in the form of peaks and purity of compound is calculated by peak area of different compounds. Computer system and software should be validated.

Also see: Calibration of HPLC

Ankur Choudhary is India's first professional pharmaceutical blogger, author and founder of Pharmaceutical Guidelines, a widely-read pharmaceutical blog since 2008. Sign-up for the free email updates for your daily dose of pharmaceutical tips.
.moc.enilediugamrahp@ofni :liamENeed Help: Ask Question

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  1. Anonymous15/7/13 13:46

    Thanks very informative.... :)

  2. Anonymous16/7/13 15:46


  3. Anonymous16/7/13 15:47


  4. Thank's a lot Choudhary; Tell me hoe to expand capability of HPLC for amino acids analysis



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