Analysis of Fructose, Glycine, and Triglycine Using HPLC UV-vis Detection and Evaporative Light-Scattering Detection

Aug 10, 2009

Figure 1. Basic structure of a tripeptide. (R1, R2, and R3 are H for triglycine).
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)

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.

Table I. Chromatographic parameters.
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.

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 190 nm.