Page 64 - Modern Derivatization Methods for Separation Sciences

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etc.) and contribute to bitter and sweet tastes. Separation of peptides is mainly performed by HPLC or
CE. However, identification of peaks is, undoubtedly, the most difficult task of peptide analysis due to
the wide range of peptide structures (amino acid composition andsequence).

The following methods have been reported recently; a small hydrophilic peptide in cheese was
analysed, and extracted samples are subject to gel permeation (Sephadex G-25) followed by FMOC
derivatization with the pre-column method and separation on HPLC. The peptide derivatives were
deprotected with piperidine followed by sequencing by the Edman degradation method [96]; small
peptides in wine are identified after separation with Sephadex G-10 followed by post-column OPA
derivatization and photodiode array detection [97].

1.2.2.3—
Fatty Acids

Fatty acids are naturally present in foods and are an important energy source. They are also functional
groups of many surface active agents presently used as food additives. Naturally occurring fatty acids
are the ester compounds of glycerol and higher alcohol, free fatty acids are not commonly found. Fatty
acid analysis has been used to determine fatty acid composition. Highly unsaturated fatty acids such as
linoleic acid and arachidonic acid are known to be essential fatty acids, and the determination of their
composition in foods is important. Fatty acids often need to be quantified as indicators of rancidity,
freshness or adulteration of fats. The analysis of fatty acids in food includes extraction of fat, hydrolysis
of fats to free fatty acids, GC or HPLC determination after derivatization or GC determination after
direct transesterification (methyl esterification) under acid or alkaline catalyst. Traditional fatty acid
analysis is based on the GC method, while HPLC method has been seldom reported.

The analysis of fatty acids with HPLC employs UV detection (215 or 192 nm) or RI detector, however,
the method is not suitable for ppb level trace analysis of food due to the interference of coexistents. As
for the HPLC method with derivatization the post-column ion-pair extraction technique is reported [98].
The method is based on the principle that fatty acids are extracted as ion-pairs with chloroform from the
aqueous acetonitrile mobile phase after the post-column addition of aqueous methylene blue solution.
The chloroform phase containing the ion-pairs is monitored with an absorbance detector at 651 nm.
This method was applied for the analysis of orange juice, margarine and butter. The detection limits
ranged from 26 to 83 ng, depending upon the acid. One problem associated specifically with the system
as set up for fatty acids was the need routinely to backflush the detector cell with acetonitrile to remove
deposits which caused fatty acid adsorption and thus loss of resolution and sensitivity.

This same approach was applied to the direct determination of the artificial sweeteners cyclamate,
saccharin and acesulfame K in diet beverages using the dye methyl violet 2B [99]. An additive, sodium
dioctylsulfosuccinate, was also detected in beverage powder by applying a similar approach [100].

The use of pre-column HPLC method for the analysis of fatty acid with 2-nitrophenylhydrazine
hydrochloride (2-NPH) was recently reported [101,102]. After alkaline hydrolysis of vegetable oils
such as coconut oil, olive oil, and margarine, and fat in milk, yogurt, ice cream, cheese, butter, beef
tallow and lard, and sardine oil, free fatty acids are reacted with 2-NPH and then derivatized to
corresponding fatty acid hydrazides. Each of the derivatives were separated on reversed-phase HPLC
with isocratic elution and detected at VIS 400 nm. This method was able to determine 29 saturated and
mono and polyunsaturated fatty acids (C8:0-C22:6), including cis-trans isomers and double-bond
positional isomers.

For the analysis of fatty acids (C12-C18) in beer, after liquid-liquid extraction with ether/pentane under

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