Easy MEthod to Analyze Carbohydrates using CE and direct UV Detection
1
Carbohydrates are important elements in living organism as energetic reservoir and as part of structural components. They play an essential role in several biological processes. Carbohydrates can be present as monosaccharide and disaccharides, but also as oligosaccharides and polysaccharides. As the carbohydrates do not absorb in the UV, specific detection techniques need to be used. Traditionally with CE the mono- or oligosaccharide are converted to primary amines by reductive amination followed by the reaction with a fluorogenic reagents such as APTS (8-aminopyrene-1,3,6- trisulfonate). APTS derivatized saccharides bear negative charge which allows a charge to mass separation by CE. The APTS moiety is fluorogenic and is detected by the LIF (Laser Induced Fluorescent) detector. Stella Rovio (1,2) and co-workers demonstrated recently the possibility to analyse saccharides using a high pH background electrolyte and direct UV detection. This method is easy to use for the analysis of mono- and disaccharide. It does not need special reagents and is cost effective. Furthermore this method may detect reductive and non-reductive sugars as well as polyols. With the CEofix™ Carbo kit, the method described by Rovio has been fully optimized and adapted by introducing dynamic double coating (3) of the capillary wall. 2.1. Principle Carbohydrates have a pKa between 12 and 13. In CE the separation is based on charge to mass ratio, therefore a BGE (Back Ground Electrolyte) with a pH above 12 is needed. When the analyte passes the detection window a reaction takes place under the influence of the Deuterium lamp and the high pH of sodium hydroxide. According to Cédric Sarazin (4) a photo oxidation takes place with the formation of UV absorbing unsaturated compounds (malonaldehyde or related conjugated carbonylated derivatives). This compound is unstable. The configuration needed to obtain the photoreaction includes a DAD (Diode Array Detector) and the selection of the absorbance set 270 nm (Figure 1). For optimal detection, the lamp intensity and the residence time of the carbohydrate in the detection window are important parameters. 2.2. Method The BGE (back ground electrolyte) is composed of NaOH (130 mM) and Na 2 HPO 4 .2H 2 0 (36 mM) at a pH value of 12.7. A rinse is performed with BGE containing a polycation, followed by a rinse step with BGE containing a polyanion. With these two steps a dynamic double coating is applied inside the capillary. The analytes migrate after the EOF (Electro Osmotic Flow) depending on their charge to mass ratio. As an example sucrose will migrate before glucose and fructose. The background electrolyte will generate a current around 58 µA when using a fused silica capillary (Polymicro, Phoenix, AZ, US) of 25 µ internal diameter and 60.2 cm total length (50 cm to the detector). Longer capillaries may been used to increase resolution, and short end injection to reduce analysis time. The voltage is set at 30 kV during separation, and a 1 minute ramping time is used. We obtain optimal performance using PA 800 plus capillary electrophoresis instrument (Beckman Coulter, Brea, CA, US), equipped with a DAD (Diode Array Detector). The detector is set at 270 nm with a bandwidth of 10 nm and the cartridge should be maintained at 18°C. After the separation a special rinse step and reconditioning of the capillary is performed (See instruction manual, Patent pending). A conditioning of the capillary, as described in the instruction manual, is performed every day; and before a new sequence. The BGE on the instrument will be acidified by carbonation and for this reason a buffer set is good for maximum 25 runs. Due to the photoreaction in the detection window, the sample plug is optimized. For the same reason the calibration curve has one order of magnitude to avoid saturation. 2.3. Results and Specifications A variety of monosaccharide’s (such as arabinose, glucose, mannose, galactose, fructose, fucose...); disaccharides (such as saccharose, maltose, lactose…); amino glycan’s (such as N-Acetyl- Glucosamine who co-migrate with Galactose, N-Acetyl-Galactosamine,...) polyols (such as sorbitol, maltitol,...) may be analysed (Figure 2). The different analytes do not react the same way, for this reason the calibration curve will be different and also the LOD, which vary between 0.05 and 0.1 mM. Figure 3 show the calibration curve for glucose, between 0.4 and 4 mM. By using the square root of the concentration we obtain a linear regression with R 2 > 0.99. Very good repeatability is obtained, the RSD on Migration Time are below 2% and the RSD on Area below 3% (Figure 4 and Table 1). Different matrices were tested including soda drink, food products, fermentation broth, cell culture media, pharmaceutical perfusion liquid, plant extracts, serum, urine etc… 3.1. Analysis of Cyclodextrine Complex carbohydrate such as non-derivatized cyclodextrine may be easily analysed with the CEofix™ Carbo kit, see figure 5. 3.2. Analysis of Glucose and Fructose in soft drinks Sucrose is used in many drinks, and it is important to verify if it is not hydrolysed by measuring the presence of glucose and fructose at a level of 0.1%. Figure 6 shows 0.1mM of glucose and fructose in presence of 0.1M sucrose. 3.3. Analysis of process samples Sugar Syrups may be analysed, just by dilution with water. To obtain optimal results for all analytes it may be needed to inject different dilutions of the same sample (Figure 7). This method of analysing carbohydrates and polyols is fast and easy to apply. Furthermore it allows quantitative and qualitative separation of reducing and non-reducing sugars but also polyols. However it is important to understand the mechanism of detection based on the photoreaction during the time the sample passes the detection window. 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 • [email protected] • http://www.analis.com/ceofix 1. Introduction : Conclusion : 3. Examples of Separation : 2. Principle and Method : N=12 Galactose Galactose Fructose Arabinose Xylose Mean M.T. 14.75 15.51 16.41 16.81 18.73 %RSD 1.46 1.56 1.71 1.68 1.92 Mean P.A. 26046 21963 20095 18806 11287 %RSD 1.45 1.76 3.76 3.50 3.39 R&D-Poster-CePharm-Easy-method-carbohydrate-0912 TM Fig. 1 : 3D Spectral View of a separation on 5 carbohydrates mixture (galactose, glucose, fructose, arabinose, and xylose) using direct UV detection. The spectra show a maximum absorbance at 270 nm. Fig. 2 : Example of separation of carbohydrate and maltitol using the CEofix™ Carbo kit. Fig. 3 : Calibration curve for Glucose between 80 ppm and 800 ppm or between 0.44 and 4.44 mM (sqrt(0.44) = 0.67 and sqrt(4.44) = 2.10). Fig. 4 : Test mix containing Galactose, Glucose, Fructose, Arabinose and Xylose at 2 mM. Fig. 5 : Separation of alpha-, beta-, and gamma- cyclodextrine. Fig. 6 : Analysis of 0.1% Glucose and Fructose, at 0.1mM, in presence of high concentration of sucrose. Fig. 7 : Analysis of Sugar Syrups, at two different dilution of the same sample, to allow quantification of small amounts of carbohydrates. The peaks before the Galactose peaks are mainly composed of oligosaccharides. Table 1 :Typical repeatability in Migration Time (M.T.) and Peak Area (P.A.) at 2mM for each analyte. Bibliographie: (1) Stella Rovio, Jari Yli-Kauhaluoma, and Heli Sirén (2007). Determination of neutral carbohydrates by CZE with direct UV detection. Electrophoresis 28, 3129 -3135. (2) Stella Rovio, Helena Simolin, Krista Koljonen, and Heli Sirén (2008). Determination of monosaccharide composition in plant fiber materials by capillary zone electrophoresis. J. Chromatogr. A 1185, 139 – 144 (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 UV-range detection of underivatized carbohydrates and aminoacids in capillary electrophoresis. Anal. Chem.83, 7381-7387.
Transcript of Easy MEthod to Analyze Carbohydrates using CE and direct UV Detection
Carbohydrates are important elements in living organism as energetic reservoir and as part of structural components. They play an essential role in several biological processes. Carbohydrates can be present as monosaccharide and disaccharides, but also as oligosaccharides and polysaccharides.
As the carbohydrates do not absorb in the UV, specific detection techniques need to be used. Traditionally with CE the mono- or oligosaccharide are converted to primary amines by reductive amination followed by the reaction with a fluorogenic reagents such as APTS (8-aminopyrene-1,3,6- trisulfonate). APTS derivatized saccharides bear negative charge which allows a charge to mass separation by CE. The APTS moiety is fluorogenic and is detected by the LIF (Laser Induced Fluorescent) detector.
Stella Rovio (1,2) and co-workers demonstrated recently the possibility to analyse saccharides using a high pH background electrolyte and direct UV detection. This method is easy to use for the analysis of mono- and disaccharide. It does not need special reagents and is cost effective. Furthermore this method may detect reductive and non-reductive sugars as well as polyols.
With the CEofix™ Carbo kit, the method described by Rovio has been fully optimized and adapted by introducing dynamic double coating (3) of the capillary wall.
2.1. PrincipleCarbohydrates have a pKa between 12 and 13. In CE the separation is based on charge to mass ratio, therefore a BGE (Back Ground Electrolyte) with a pH above 12 is needed. When the analyte passes the detection window a reaction takes place under the influence of the Deuterium lamp and the high pH of sodium hydroxide. According to Cédric Sarazin (4) a photo oxidation takes place with the formation of UV absorbing unsaturated compounds (malonaldehyde or related conjugated carbonylated derivatives). This compound is unstable.
The configuration needed to obtain the photoreaction includes a DAD (Diode Array Detector) and the selection of the absorbance set 270 nm (Figure 1). For optimal detection, the lamp intensity and the residence time of the carbohydrate in the detection window are important parameters.
2.2. MethodThe BGE (back ground electrolyte) is composed of NaOH (130 mM) and Na2HPO4.2H20 (36 mM) at a pH value of 12.7. A rinse is performed with BGE containing a polycation, followed by a rinse step with BGE containing a polyanion. With these two steps a dynamic double coating is applied inside the capillary. The analytes migrate after the EOF (Electro Osmotic Flow) depending on their charge to mass ratio. As an example sucrose will migrate before glucose and fructose.
The background electrolyte will generate a current around 58 µA when using a fused silica capillary (Polymicro, Phoenix, AZ, US) of 25 µ internal diameter and 60.2 cm total length (50 cm to the detector). Longer capillaries may been used to increase resolution, and short end injection to reduce analysis time. The voltage is set at 30 kV during separation, and a 1 minute ramping time is used.
We obtain optimal performance using PA 800 plus capillary electrophoresis instrument (Beckman Coulter, Brea, CA, US), equipped with a DAD (Diode Array Detector). The detector is set at 270 nm with a bandwidth of 10 nm and the cartridge should be maintained at 18°C. After the separation a special rinse step and reconditioning of the capillary is performed (See instruction manual, Patent pending).
A conditioning of the capillary, as described in the instruction manual, is performed every day; and before a new sequence. The BGE on the instrument will be acidified by carbonation and for this reason a buffer set is good for maximum 25 runs.
Due to the photoreaction in the detection window, the sample plug is optimized. For the same reason the calibration curve has one order of magnitude to avoid saturation.
2.3. Results and Specifications
A variety of monosaccharide’s (such as arabinose, glucose, mannose, galactose, fructose, fucose...); disaccharides (such as saccharose, maltose, lactose…); amino glycan’s (such as N-Acetyl-Glucosamine who co-migrate with Galactose, N-Acetyl-Galactosamine,...) polyols (such as sorbitol, maltitol,...) may be analysed (Figure 2).
The different analytes do not react the same way, for this reason the calibration curve will be different and also the LOD, which vary between 0.05 and 0.1 mM. Figure 3 show the calibration curve for glucose, between 0.4 and 4 mM. By using the square root of the concentration we obtain a linear regression with R2 > 0.99.
Very good repeatability is obtained, the RSD on Migration Time are below 2% and the RSD on Area below 3% (Figure 4 and Table 1).
Different matrices were tested including soda drink, food products, fermentation broth, cell culture media, pharmaceutical perfusion liquid, plant extracts, serum, urine etc…
3.1. Analysis of CyclodextrineComplex carbohydrate such as non-derivatized cyclodextrine may be easily analysed with the CEofix™ Carbo kit, see figure 5.
3.2. Analysis of Glucose and Fructose in soft drinksSucrose is used in many drinks, and it is important to verify if it is not hydrolysed by measuring the presence of glucose and fructose at a level of 0.1%. Figure 6 shows 0.1mM of glucose and fructose in presence of 0.1M sucrose.
3.3. Analysis of process samplesSugar Syrups may be analysed, just by dilution with water. To obtain optimal results for all analytes it may be needed to inject different dilutions of the same sample (Figure 7).
This method of analysing carbohydrates and polyols is fast and easy to apply. Furthermore it allows quantitative and qualitative separation of reducing and non-reducing sugars but also polyols. However it is important to understand the mechanism of detection based on the photoreaction during the time the sample passes the detection window.
Easy Method to Analyze Carbohydrates using Capillary Electrophoresis and direct UV DetectionFrançois 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 • [email protected] • http://www.analis.com/ceofix
1. Introduction :
Conclusion :
3. Examples of Separation :
2. Principle and Method :
N=12 Galactose Galactose Fructose Arabinose XyloseMean M.T. 14.75 15.51 16.41 16.81 18.73
%RSD 1.46 1.56 1.71 1.68 1.92
Mean P.A. 26046 21963 20095 18806 11287
%RSD 1.45 1.76 3.76 3.50 3.39
R&D-Poster-CePharm-Easy-method-carbohydrate-0912
TM
Fig. 1 : 3D Spectral View of a separation on 5 carbohydrates mixture (galactose, glucose, fructose, arabinose, and xylose) using direct UV detection. The spectra show a maximum absorbance at 270 nm.
Fig. 2 : Example of separation of carbohydrate and maltitol using the CEofix™ Carbo kit.
Fig. 3 : Calibration curve for Glucose between 80 ppm and 800 ppm or between 0.44 and 4.44 mM (sqrt(0.44) = 0.67 and sqrt(4.44) = 2.10).
Fig. 4 : Test mix containing Galactose, Glucose, Fructose, Arabinose and Xylose at 2 mM.
Fig. 5 : Separation of alpha-, beta-, and gamma-cyclodextrine.
Fig. 6 : Analysis of 0.1% Glucose and Fructose, at 0.1mM, in presence of high concentration of sucrose.
Fig. 7 : Analysis of Sugar Syrups, at two different dilution of the same sample, to allow quantification of small amounts of carbohydrates. The peaks before the Galactose peaks are mainly composed of oligosaccharides.
Table 1 :Typical repeatability in Migration Time (M.T.) and Peak Area (P.A.) at 2mM for each analyte.
Bibliographie:
(1) Stella Rovio, Jari Yli-Kauhaluoma, and Heli Sirén (2007). Determination of neutral carbohydrates by CZE with direct UV detection. Electrophoresis 28, 3129 -3135.
(2) Stella Rovio, Helena Simolin, Krista Koljonen, and Heli Sirén (2008). Determination of monosaccharide composition in plant fiber materials by capillary zone electrophoresis. J. Chromatogr. A 1185, 139 – 144
(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 UV-range detection of underivatized carbohydrates and aminoacids in capillary electrophoresis. Anal. Chem.83, 7381-7387.
