Application Notes
Fatty Acids by HPLC/ELSD
The determination of fatty acids by reversed phase HPLC with ELSD detection was found to be superior to that by UV detection. Analytical-bore columns with flow splitters were found to produce equivalent results when narrow-bore columns were not available
Evaporative Light Scatting Detection Simplifies Fatty Acid Analysis, Alltech Application Note 0045E, June 5, 2000.
Fatty acids cannot easily be determined by GC due to poor resolution and sample decomposition. Derivatization to allow their determination by reversed phase HPLC with UV detection is time consuming and complicated. HPLC separation of fatty acids with detection by evaporative light scattering does not require post-column derivatization as in the case of UV detection among other advantages.
A mixture of six underivatized fatty acids (linolenic, myristic, linoleic, palmitic, oleic and stearic acid) ranging from 15-80mg/L was separated by gradient elution in around 20 minutes by reversed phase HPLC on an Alltima C18-LL, 5µm, 250 x 2.1mm column (Part No. 88389) using water/acetonitrile and detected by evaporative light scattering using an Alltech Model 500 (Evaporative Light Scattering Detector.) The UV detection at 210 nn showed almost no peaks.
A mixture of the same fatty acids at around 600mg/L separated using an analytical-bore column (Alltima C18-LL, 5µm, 250 x 4.6mm, Part No. 88099) with a flow splitter produced better sensitivity than without one by allowing the drift tube temperature to be reduced thereby reducing sample evaporation.
In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser.
Key Analyte(s)
linoleic acid
linolenic acid
myristic acid
oleic acid
palmitic acidPalmitic acid is a naturally occurring fatty acid also produced commercially from palm and coconut oil. It is used is soaps, cosmetics and food additives. It is most often measured using HPLC, GC/FID and GC/MS.
stearic acidStearic acid, prepared by hydrogenation of cottonseed oil, is used in manufacturing soaps and greases. It is most often measured using HPLC, GC/FID and GC/MS.
Equipment
Alltech Alltima Reversed-Phase ColumnsAlltech Alltima Reversed Phase columns are a line of HPLC columns in the Alltech HP family. The Alltech Alltima family are high quality general purpose columns. They are made with spherical silica, polymerically bonded and double endcapped for long life
Technique
HPLCHigh Performance Liquid Chromatography (HPLC)is a technique for separating components in a sample on the basis of interactions between the component, a liquid mobile phase and a solid stationary phase.
Source
Alltech Associates, Inc.Alltech Associates, Inc., one of the leading suppliers of HPLC columns, manufactures and distributes HPLC, GC and IC columns, accessories and consumables and maintains a large searchable database of application notes and chromatograms on its website.
