This document summarizes a method for analyzing carbohydrates using capillary electrophoresis with direct UV detection. The method utilizes a high pH background electrolyte without the need for derivatization. Separations can be performed on a variety of sample matrices using a PA 800 plus instrument with DAD detection at 270 nm. Examples show the separation of cyclodextrins, glucose and fructose in soft drinks, and sugar syrup process samples. The method is described as fast, easy to apply, and allows both quantitative and qualitative analysis of reducing and non-reducing carbohydrates and polyols.

1. TM
Easy Method to Analyze Carbohydrates using Capillary Electrophoresis and direct UV Detection
François de l’Escaille, Jean-Bernard Falmagne
ANALIS s.a. • R&D Diag • Zoning Industriel de Rhisnes • rue de Néverlée, 11 • 5020 Suarlée (Namur) • Belgium • Tel: + 32 81 25 50 50 • Fax: + 32 81 23 12 37 • ceofix@analis.be • http://www.analis.com/ceofix
1. Introduction :
Carbohydrates are important elements in living organism as energetic reservoir and as part of We obtain optimal performance using PA 800 plus capillary electrophoresis instrument Different matrices were tested including soda drink, food products, fermentation broth, cell
structural components. They play an essential role in several biological processes. Carbohydrates (Beckman Coulter, Brea, CA, US), equipped with a DAD (Diode Array Detector). The detector culture media, pharmaceutical perfusion liquid, plant extracts, serum, urine etc…
can be present as monosaccharide and disaccharides, but also as oligosaccharides and is set at 270 nm with a bandwidth of 10 nm and the cartridge should be maintained at 18°C.
polysaccharides. After the separation a special rinse step and reconditioning of the capillary is performed (See 3. Examples of Separation :
As the carbohydrates do not absorb in the UV, specific detection techniques need to be instruction manual, Patent pending). 3.1. Analysis of Cyclodextrine
used. Traditionally with CE the mono- or oligosaccharide are converted to primary amines A conditioning of the capillary, as described in the instruction manual, is performed every day; Complex carbohydrate such as non-derivatized
by reductive amination followed by the reaction with a fluorogenic reagents such as APTS and before a new sequence. The BGE on the instrument will be acidified by carbonation and cyclodextrine may be easily analysed with the
(8-aminopyrene-1,3,6- trisulfonate). APTS derivatized saccharides bear negative charge which for this reason a buffer set is good for maximum 25 runs. CEofix™ Carbo kit, see figure 5.
allows a charge to mass separation by CE. The APTS moiety is fluorogenic and is detected by Due to the photoreaction in the detection window, the sample plug is optimized. For the same
the LIF (Laser Induced Fluorescent) detector. reason the calibration curve has one order of magnitude to avoid saturation. 3.2. Analysis of Glucose and Fructose in soft
Stella Rovio (1,2) and co-workers demonstrated recently the possibility to analyse saccharides drinks
using a high pH background electrolyte and direct UV detection. This method is easy to use for 2.3. Results and Specifications Sucrose is used in many drinks, and it is important to Fig. 5 : Separation of alpha-, beta-, and gamma-
the analysis of mono- and disaccharide. It does not need special reagents and is cost effective. cyclodextrine.
verify if it is not hydrolysed by measuring the presence
Furthermore this method may detect reductive and non-reductive sugars as well as polyols. of glucose and fructose at a level of 0.1%. Figure 6
With the CEofix™ Carbo kit, the method described by Rovio has been fully optimized and shows 0.1mM of glucose and fructose in presence
adapted by introducing dynamic double coating (3) of the capillary wall. of 0.1M sucrose.
2. Principle and Method : 3.3. Analysis of process samples
2.1. Principle Sugar Syrups may be analysed, just by dilution with
Carbohydrates have a pKa between 12 and 13. water. To obtain optimal results for all analytes it may
In CE the separation is based on charge to mass ratio, be needed to inject different dilutions of the same
Fig. 2 : Example of separation of carbohydrate and Fig. 3 : Calibration curve for Glucose between 80 ppm sample (Figure 7).
therefore a BGE (Back Ground Electrolyte) with a pH maltitol using the CEofix™ Carbo kit. and 800 ppm or between 0.44 and 4.44 mM Fig. 6 : Analysis of 0.1% Glucose and Fructose, at
Conclusion :
above 12 is needed. When the analyte passes the (sqrt(0.44) = 0.67 and sqrt(4.44) = 2.10). 0.1mM, in presence of high concentration of sucrose.
detection window a reaction takes place under the N=12 Galactose Galactose Fructose Arabinose Xylose
Mean M.T. 14.75 15.51 16.41 16.81 18.73
influence of the Deuterium lamp and the high pH of Fig. 1 : 3D Spectral View of a separation on %RSD 1.46 1.56 1.71 1.68 1.92 This method of analysing carbohydrates and polyols
sodium hydroxide. According to Cédric Sarazin (4) a 5 carbohydrates mixture (galactose, glucose, fructose, Mean P.A. 26046 21963 20095 18806 11287 is fast and easy to apply. Furthermore it allows
%RSD 1.45 1.76 3.76 3.50 3.39
photo oxidation takes place with the formation of UV arabinose, and xylose) using direct UV detection. quantitative and qualitative separation of reducing
The spectra show a maximum absorbance at 270 nm. Table 1 :Typical repeatability in Migration Time (M.T.) and
absorbing unsaturated compounds (malonaldehyde or Peak Area (P.A.) at 2mM for each analyte. and non-reducing sugars but also polyols. However
related conjugated carbonylated derivatives). This compound is unstable. it is important to understand the mechanism of
The configuration needed to obtain the photoreaction includes a DAD (Diode Array Detector) A variety of monosaccharide’s (such as arabinose, detection based on the photoreaction during the time
and the selection of the absorbance set 270 nm (Figure 1). For optimal detection, the lamp glucose, mannose, galactose, fructose, fucose...); the sample passes the detection window.
intensity and the residence time of the carbohydrate in the detection window are important disaccharides (such as saccharose, maltose,
parameters. lactose…); amino glycan’s (such as N-Acetyl-
Glucosamine who co-migrate with Galactose, Fig. 7 : Analysis of Sugar Syrups, at two different
2.2. Method N-Acetyl-Galactosamine,...) polyols (such as sorbitol,
dilution of the same sample, to allow quantification
of small amounts of carbohydrates. The peaks
The BGE (back ground electrolyte) is composed of NaOH (130 mM) and Na2HPO4.2H20 (36 mM) maltitol,...) may be analysed (Figure 2). before the Galactose peaks are mainly composed of
Fig. 4 : Test mix containing Galactose, Glucose, Fructose, oligosaccharides.
at a pH value of 12.7. A rinse is performed with BGE containing a polycation, followed by a rinse The different analytes do not react the same way, Arabinose and Xylose at 2 mM.
step with BGE containing a polyanion. With these two steps a dynamic double coating is applied for this reason the calibration curve will be different Bibliographie:
inside the capillary. The analytes migrate after the EOF (Electro Osmotic Flow) depending on and also the LOD, which vary between 0.05 and 0.1 mM. Figure 3 show the calibration curve (1) Stella Rovio, Jari Yli-Kauhaluoma, and Heli Sirén (2007). Determination of neutral carbohydrates by CZE with direct UV detection.
their charge to mass ratio. As an example sucrose will migrate before glucose and fructose. for glucose, between 0.4 and 4 mM. By using the square root of the concentration we obtain Electrophoresis 28, 3129 -3135.
(2) Stella Rovio, Helena Simolin, Krista Koljonen, and Heli Sirén (2008). Determination of monosaccharide composition in plant fiber materials
The background electrolyte will generate a current around 58 µA when using a fused silica a linear regression with R > 0.99.
2
by capillary zone electrophoresis. J. Chromatogr. A 1185, 139 – 144
capillary (Polymicro, Phoenix, AZ, US) of 25 µ internal diameter and 60.2 cm total length Very good repeatability is obtained, the RSD on Migration Time are below 2% and the RSD on (3) US Patent 5,611,903
(4) Cédric Sarazin, Nathalie Delaunay, Christine Costanza, Véronique Eudes, Jean-Maurice Mallet, and Pierre Gareil (2011). New avenue for mid
(50 cm to the detector). Longer capillaries may been used to increase resolution, and short end Area below 3% (Figure 4 and Table 1). UV-range detection of underivatized carbohydrates and aminoacids in capillary electrophoresis. Anal. Chem.83, 7381-7387.
injection to reduce analysis time. The voltage is set at 30 kV during separation, and a 1 minute R&D-Poster-CePharm-Easy-method-carbohydrate-0912
ramping time is used.