Metrohm Solutions for determining carbohydrates in essential and non-essential foodstuffs using ion chromatography

1. Determining carbohydrates in essential and non-essential
foodstuffs using ion chromatography
Alfred Steinbach and Andrea Wille
Herisau, Switzerland
This article describes a straightforward ion chromatographic method that uses isocratic elution and
pulsed amperometric detection (PAD) to sensitively determine water-soluble polyols and sugar alcohols
as well as mono-, di-, and oligosaccharides in essential and non-essential foodstuffs. While carbohydrate
determination of most foodstuffs requires only minimal sample preparation such as dilution and
filtration, samples with strongly interfering matrices such as protein-containing dairy products have to
be dialysed before injection.
Carbohydrates or sacccharides, DNA. Because of the widespread use drawbacks, high-performance
also called sugars, are the most and importance of carbohydrates, anion-exchange chromatography
abundant organic molecules their determination is of considerable has increasingly been used for
found in the biosphere. They interest in biological, environmental, carbohydrate determination. In
include monosaccharides (e.g., clinical, and medical research. strongly alkaline mobile phases,
glucose, fructose, xylose, or Additionally, quality control of sugar anions are separated on a
mannose), disaccharides (e.g., foodstuffs assures consumers’ positively charged strong anion-
sucrose, lactose, and maltose), top-quality carbohydrate supply. exchange resin. Subtle differences
trisaccharides, oligosaccharides and The most commonly applied in pKa values of the hydroxyl groups
polysaccharides. Carbohydrates analytical techniques for of the carbohydrates allow for an
are synthesized in plants during determining carbohydrates are efficient separation of low-molecular
photosynthesis using carbon 1
H-nuclear magnetic resonance saccharides. However, sensitive
dioxide, water, and the energy from (NMR), fourier transform infrared and straightforward detection
sunlight. They are one of the three spectroscopy (FT-IR), polyacrylamide of the separated carbohydrates
macronutrients (proteins and fats gel electrophoresis (PAGE), mass has long been a challenge. As a
being the other two) that provide the spectrometry (MS) together with result of missing chromophores
human body with energy. Complex gas and liquid chromatography. and fluorophores, ultraviolet and
polysaccharides are structural While spectroscopic methods fluorescence detectors cannot be
elements in the cell walls of bacteria suffer from high instrumentation used and refractive index detection
and plants and are stored for food costs and need highly skilled suffers from poor sensitivity and
or structural support. In addition, operators, gas chromatographic the fact that gradient elution is not
ribose and deoxyribose form part of methods require time-consuming applicable. Several post-column
the structural framework of RNA and derivatizations. In view of these derivatization reactions have been
A Determining carbohydrates in foodstuffs using IC separation science — volume 3 issue 6 www.sepscience.com

2. Figure 1
samples. Experimental
Materials and Methods Sample preparation of malt extracts
Instrumentation: is straightforward and only involves a
Chromatographic equipment: 871 1:100 dilution. Afterwards the sample
Advanced Bioscan, 838 Advanced IC can be directly injected.
Sample Processor, 833 Advanced IC
Liquid Handling Pump Unit, Results
833 Advanced IC Liquid Handling Glucose, fructose, and sucrose
Dialysis Unit, 818 Advanced IC Pump. are primarily responsible for the
Instrument control, data acquisition, foods` sweetness. As the latter two
and processing were performed by sugars are not present at detectable
Metrodata IC Net software (Metrohm concentrations [Figure 1], malt
AG). extracts are only perceived half as
Reagents and eluents: sweet as mainly sucrose-containing
Carbohydrates were reagent grade products. Apart from glucose
and purchased from Fluka (Sigma and maltose the investigated
Aldrich, Buchs, Switzerland). All malt extract contains several
standard solutions and eluents were maltooligosaccharides.
prepared with deionized water with a The observed saccharide profile of
specific resistance higher than the investigated sample corresponds
18 M Ω•cm. to the general malt composition.
Figure 1: PAD carbohydrate chromatogram of a malt extract sample containing 593 mg/L glucose, 5642 mg/L maltose,
Example 1 – Carbohydrate Example 2 – Carbohydrate content
1029 mg/L maltotriose, 291 mg/L maltotetraose, 164 mg/L maltopentaose, 327 mg/L maltohexaose and 97 mg/L
maltoheptaose. Before analysis, the malt extract sample was diluted 1:100.
determination in malt extract in dairy products
Viscous or dried malt extracts are In contrast to the straightforward
proposed for the spectrophotometric of any reaction products from the obtained from germinated barley sample preparation of soluble
detection of carbohydrates. However, electrode surface. The third, negative and contain naturally present sugar constituents in malt extracts
the improved sensitivity of these potential (E3) reduces any surface enzymes, particularly amylase, which presented above, the analysis of
methods is offset by the labour- oxide on the electrode surface. The convert starch to water-extractable protein-containing samples such
intensive and often error-prone entire three-stage process typically sugars. Malt extracts excel in their as dairy products can pose severe
derivatization procedure. lasts one second and is continuously high physiological and nutritional problems when directly injected.
Because carbohydrates are repeated to prevent electrode fouling. values. Malt is added as a nutritional Precipitation of the proteins fouls
electrochemically active, amperometric Apart from carbohydrates, PAD has supplement to the diets of infants the column und ultimately destroys
detection overcomes the mentioned proven to be effective for amino and elderly persons. Furthermore, it. This can be prevented by the
shortcomings. A triple-step potential sugars, amino acids, biogenic amines, it is a very important intermediate integration of upstream precipitation
waveform referred to as pulsed sulfur-containing species, alcohols, and ingredient in infant and pet foods, procedures such as Carrez
amperometric detection (PAD) is some antibiotics. variety and party breads, instant precipitation. However, apart from
applied. First a positive potential (E1) is Several applications demonstrate coffee, beverages, ice creams, the labour-intensive preparation,
used to determine the target analytes, the potential of ion chromatography pharmaceuticals, etc. Carrez precipitation suffers from
followed by a second, more positive followed by pulsed amperometric coprecipitation, inclusions, and
potential (E2) for oxidative removal detection in food and beverage enhanced sugar decomposition.
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3. Figure 2
Figure 2: Schematic diagram showing (a) inline dialysis cell (b) coupling to the sample changer and IC unit, and (c) spiral-flow dialysis cell.
In contrast, carbohydrate solution is the same as that in carbohydrates migrate from the 95 and 105% indicate quantitative
separation from high-molecular the donor solution. This patented sample stream into the acceptor permeability of the membrane for
proteins can be comfortably stopped-flow procedure takes about solution, the high-molecular protein the target carbohydrates and thus
achieved via stopped-flow dialysis. 14 minutes and can be directly constituents stay on the sample the applicability of the dialysis
This technique is based on the coupled to an IC setup (Figure 2). As side of the membrane. Finally, the technique.
selective diffusion of molecules the dialysis is performed during the acceptor solution is transferred to the
or ions from one liquid (donor recording of the previous sample`s sample loop. Carbohydrate content in various
or sample solution) to another chromatogram, the overall analysis food and beverage products – An
(acceptor solution) via a membrane. time is not prolonged. Results overview
The driving force for the transfer is Figure 3 shows the chromatogram Carbohydrate analysis offers very
the concentration gradient across the Experimental of a fruit yoghurt dialysate with the far-reaching application possibilities
membrane. The molecular separation The carbohydrate content in fruit following peaks: the polyol inositol, for the analysis of beverages, foods,
thresholds are generally determined yoghurt was determined according the sugar alcohol sorbitol and the and sweets (Table 1). Unlike the
by the thickness and porosity of the to the following procedure: Before mono- and disaccharides glucose, above-mentioned protein matrix,
membrane. dialysis, 10 g yoghurt is dissolved in galactose, fructose, lactose, and most foodstuff samples only require
Unlike in dynamic dialysis, where 1 L ultrapure water and the resulting sucrose. Repetitive analyses showed sample preparation methods such
two solutions continuously pass solution diluted 1:10 (v/v). 10 mL no trending in peak areas, peak as comminution, dilution, extraction,
through the dialysis module, in of this solution is passed along the heights or retention times, which sonication, and/or filtration.
equilibrium dialysis, at least one dialysis membrane by the peristaltic suggests that sample proteins did Table 1 gives an overview of the
solution is temporarily stopped until pump, while the acceptor solution not pass the membrane. In contrast, determination of various polyols,
the concentration in the acceptor remains at rest. Whereas dialysable carbohydrate recovery rates between sugar alcohols as well as mono-, di-,
A Determining carbohydrates in foodstuffs using IC separation science — volume 3 issue 6 www.sepscience.com

4. Table 1 Figure 3
Figure 3: PAD carbohydrate chromatogram of a fruit yoghurt dialyzate containing 0.2 mg/g inositol, 0.8 mg/g sorbitol,
21.0 mg/g glucose, 7.4 mg/g galactose, 16.1 mg/g fructose, 26.5 mg/g lactose, and 77.0 mg/g sucrose.
Determination of mono-and disaccharides in http://www.metrohm.com/com/Applications
milk products by high-performance anion- (search for 8.000.6018EN).
exchange chromatography with pulsed
Table 1: Selection of various sugar determinations in different food matrices amperometric detection, Anal. Chem. 2003;
485: 43–49.
and oligosaccharides in different gold working electrode can be used 2. Meyer A., Raba C., and Fischer K., Ion-
foodstuffs. to determine various carbohydrates pair RP-HPLC determination of sugars,
The presented setup also facilitates in different food matrices. Whereas aminosugars, and uronic acids after
carbohydrate analysis in plant most samples require no sample derivatization with p-aminobenzoic acid,
extracts, blood, urine, pharmaceutical preparation other than extraction, Anal. Chem. 2001; 73: 2377–2382.
products, explosives, or biofuels. comminution, dilution, or filtration, 3. P. Deepak, Carbohydrate analysis in food
However, these determinations are carbohydrate determination in products and beverages, Food&Pack, 40
beyond the scope of this article. difficult matrices can be easily solved (August 2002).
by using Metrohm’s proven dialysis 4. Takematsu M.M., Carreira W., Batista F.F.,
Conclusion technique. Zanuni L., and Sertek P.A., Quantification of
High-performance anion-exchange carbohydrates and uronic acids in eucalyptus
chromatography with pulsed References wood species by ion chromatography with
amperometric detection using a 1. Cataldi T.R.I., Angelotti M., and Bianco G., PAD using a gold electrode, Pittcon 2008,
A Determining carbohydrates in foodstuffs using IC separation science — volume 3 issue 6 www.sepscience.com