Abstract
A study of parameters (organic content, additives and pH of the mobile phase) to yield good separation and detection of a series of commercially available free fatty acids ranging from C12:0 (lauric acid) to C18:3 (linolenic acid) using HPLC-CAD is undertaken. Working methods using a C18 silica column were assessed by measuring the experimental limit of detection (LOD) and sensitive ranges with consideration to effects of the Charged Aerosol Detector (CAD) temperature and detector voltage. An isocratic method with high content in acetonitrile and low pH was developed that allowed the CAD detection and quantification of the less volatile fatty acids in the range of from around 1 to 5 ng/uL to over 200 ng/uL. A power fit calibration curve was necessitated since the response of the standards did not display a true linear relationship using linear regression analysis. Conditions or mobile phase additives were not found to increase detection of semi-volatile fatty acids such as lauric acid or myristic acid (C14:0). Column bleed was potentially identified as an unexpected additive that resulted in enhanced peak detection, attributed to the formation or stabilization of bigger aerosol particles. The isocratic method was tested for an olive oil standard using an acetonitrile: 0.01M TFA (96.5:3.5) mobile phase, ion voltage at -300 V, and CAD heater setting of 35°C. Using those conditions, separation and detection of major C16 to C18 fatty acids were achieved although palmitic and oleic acids were not completely resolved. The olive oil analysis showed that relative recovery of the major fatty acid components is consistent and supports the use of HPLC-CAD system for a rapid detection of fatty acids at trace levels.