In vitro HPLC (high performance liquid chromatography)

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> In vitro HPLC (high performance liquid chromatography)

Explanation

HPLC is a chromatographic separation method that can be used to separate chemical substances as well as to identify, quantify and purify them using specific standards. The analyzed sample is pumped with a running medium (mobile phase) over a stationary phase in a separation column (2-30cm length, Ø2-5mm). Depending on the two phases, the components of a mixture of substances interact differently with the stationary phase and are thus eluted at different retention times. Depending on the type of the sample, detection is performed by UV/VIS detector, fluorescence detector, diode array detector (DAD) or light scattering detector. The velocity is determined by the re-dissolution into the mobile phase. There are two distinct methods: normal phase HPLC (NP) with polar stationary phase (e.g. silica gel) and non-polar mobile phase, where polar molecules are retained on the column, and reversed phase HPLC (RP). While using non-polar stationary phase (silanes, substituted with non-polar long-chain hydrocarbons, e.g. C18), the elution strength decreases with increasing polarity.

A chemical compound can only be identified to a limited extent by HPLC by comparing the retention time of the unknown substance with that of a defined reference compound (external standard method) or by following mass spectroscopic (MS) analysis.

 

Proof

• Organic compounds e.g. proteins, vitamins, sugars, ingredients of cosmetic formulas

 

Suitability

• Analysis and chemical purification of soluble pollutants (e.g. those adhering to particulate matter) as well as protecting active compounds (e.g. antioxidants)

 

Literature

• Gaby Aced, Hermann J. Möckel: Liquidchromatographie – Apparative, theoretische und methodische Grundlagen der HPLC, VCH, Weinheim 1991, DOI:10.1002/jobm.3620320603
• Heinz Engelhardt (Hrsg.): Practice of High Performance Liquid Chromatography. Applications, Equipment and Quantitative Analysis. Springer, Berlin u. a. 1986
• Lloyd R. Snyder, Joseph J. Kirkland, John W. Dolan: Introduction to Modern Liquid Chromatography. 3rd Edition. John Wiley & Sons, Hoboken NJ 2010, DOI:10.1007/s13361-010-0021-8
• Henk Lingeman, Willy J. M. Underberg (Hrsg.): Detection-Oriented Derivatization Techniques in Liquid Chromatography (= Chromatographic Science. Bd. 48). Marcel Dekker Inc., New York NY u. a. 1990