Short peptides (oligopeptides) are highly valuable products in the food industry because of their digestibility, low allergenic
properties, flavor, textural properties and nutraceutical abilities. Oligopeptides are found in low concentrations in aqueous
streams of organic origin, such as in sugar and starch production and in process streams. Therefore, there is a significant
interest in processes, which can selectively concentrate peptides or peptide fractions. Besides oligopeptides, other organic
components (e.g., sugars, amino acids, carboxylic acids and organic salts) are present in higher concentrations in many process
streams. Triglycine can be seen as a model to study processes for the recovery of peptides. (Figure 1)
Figure 1. Basic structure of a tripeptide. (R1, R2, and R3 are H for triglycine).
To investigate the selectivity of peptide separation processes, multicomponent aqueous feeds are used, with a sugar (fructose)
and an amino acid (glycine) present. To obtain the required data, an analysis method capable of determining the concentrations
of triglycine, glycine and fructose simultaneously is preferred.
There are various ways to analyze amino acids and oligopeptides using HPLC by first derivitizing with, for example, o-phthalate
aldehyde (phthaldialdehyde) (OPA), N-(9-fluoroenyl)methoxy-carbonyl (FMOC)or 4:3-b-Naphthopyrone-4-acetic acid N-hydroxysuccinimidyl
ester (NPA-OSu) (1) and only a few where a derivatization step is not required (2). Most of them use UV–vis or fluorescence
spectroscopy for detection. There are also many methods using HPLC with refractive index detection (RID) (4–7) to analyze
fructose (3). The determination of fatty acids in the presence of carbohydrates by reversed-phase (RP) HPLC after derivatization
with phenacyl bromide8 and as a gas chromatography (GC) method comprising a derivatization of the carbohydrates with alkylchlorosilanes
have also been recorded.8 No method for the simultaneous determination of fructose, glycine and triglycine has been observed.
Table I. Chromatographic parameters.
For most of the HPLC analyses of carbohydrates, columns packed with either cation-exchange resins or amino-modified silica
gel are used. Detection is mostly performed using RID. The cation-exchange columns require pure water or diluted mineral acid
as the mobile phase. The amino modified columns are run with eluents of water and acetonitrile (4).
ELSDs measure the amount of light scattered by analyte particles that have been obtained through nebulization and evaporation.
In general, ELSDs deliver a signal for all compounds that do not evaporate or decompose during evaporation of the mobile phase.
A lot of applications using HPLC with ELS detection have been published in the past few years (9–15).
ELSDs are more universal than refractive index detectors. Furthermore, they are compatible with a much wider range of solvents
and modifiers, and produce stable baselines during gradient elution. Compared with spectroscopic detectors, ELSDs produce
more uniform detection sensitivity for most analytes, regardless of their physical and chemical properties (16).
Chemicals: Reagent-analytical grade sodium dihydrogenphosphate and phosphorous acid (85%) were obtained from Merck (Darmstadt, Germany).
Acetonitrile for HPLC was obtained from Sigma-Aldrich (Seelze, Germany). Reagent-analytical grade d–fructose, glycine (amino
acetic acid) and triglycine (Gly–Gly–Gly) were obtained from Sigma-Aldrich (Steinheim, Germany). Ultra-pure water was obtained
from Milli-Q185 from Millipore (Molsheim, France).
HPLC with UV–vis detection: The chromatographic separation was performed using a Varian Pro Star HPLC system (Varian Nederland, Middelburg, The Netherlands),
consisting of a quaternary pump (Model 240) and a variable UV–vis detector (Model 310). The UV absorbance was monitored at