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Passion. Power. Productivity.

Carbohydrate Analysis by IC and HPLC

High Performance Liquid Chro-matography (HPLC) is an important tool to identify and quantify carbohy-drates in food and beverage samples, providing key metrics of product quality and related properties, contamination, or adulteration. HPLC plays important roles in quality control, nutritional labeling, authenticity testing, and production processes monitoring, for example, tracking the fermentation of alcoholic beverages.

Separation and detection in high-concentration carbohydrate mixtures, as found in the food and beverage industry, are made challeng-ing by the wide variety of carbohydrate molecules and intricacy of carbohydrate

mixtures existing in nature. Selection of the optimal HPLC approach depends on the sample matrix, carbohydrate concentration, selectivity, and sensitiv-ity required.

HPLC on aminopropyl-bonded silica or polymer-based metal-loaded cation-exchange resins, in conjunction with refractive index (RI) or low- wavelength UV detection, provide simple isocratic methods. In most cases, HPLC on metal-loaded cation-exchange resins with RI detection (HPLC-RI) is used to determine simple mono- and disaccharides in the g/L range. However, some sample matrices require better resolution of sugars from sugar alcohols, organic acids, and sodium chloride.1

High-performance anion- exchange chromatography with pulsed amperometric detection (HPAE-PAD) and specialized CarboPac® columns solve these chromatographic and selectivity issues, while also allowing the determination of alcohols, glycols, and aldehydes. HPAE-PAD can separate sugars, sugar alcohols, oligo-, and polysaccharides with very high resolution, without derivatization or pre-concentration. This approach provides quantification to picomolar levels.2

Dionex offers HPLC-RI and HPLC-PAD solutions optimized for a wide variety of research and monitoring applications.

2

HPLC-RI for Mono- and Disaccharides RI is the next most widely-used

detection method for carbohydrates, as other alternatives such as fluorescence and UV-Vis detectors require pre-column derivatization of sugars. RI allows direct determination and quantification of sugars present in the percent range of most foods.

Metal-loaded cation-exchange columns provide a simple, non-destruc-tive method to separate carbohydrates using a deionized water mobile phase, which is compatible with RI detection. These columns separate compounds using a combination of size exclusion and ligand exchange mechanisms.

For oligosaccharide separations using metal loaded columns, size exclusion is the primary separation mechanism. For monosaccharides, ligand exchange dominates. This mechanism involves the binding of hydroxyl groups in the sugars with the fixed counter-ion of the resin. Ligand exchange is affected by the nature of the counterion (Pb2+, Ca2+, etc.) and by the spatial orientation of the carbohydrate’s hydroxyl groups.

Figure 1 shows the good area repeatability of HPLC-RI, with RSD of less than 0.8% for 100 consecutive injections of a sugar standard (c = 10 g/L each, injection volume: 20 μL). Carbohydrates often occur as major components. Levels of individual sugars up to 5% may be quantified (Figures 2 and 3).

HPAE-PAD TechniquesThe different selectivity of

ion-exchange CarboPac columns with respect to metal-loaded columns can help to better separate carbohydrates and other species in complex matrices. Carbohydrates are separated by anion exchange chromatography at high pH, and detected by pulsed electrochemical detection. Figure 2. Analysis of glucose, fructose, and sucrose in fruit juices.

Figure 1. One-hundred consecutive chromatograms of a high-concentration carbohydrate standard.

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Column: Aminex HPX87CEluent: WaterTemperature: 80 °CFlow Rate: 0.5 mL/minInj. Volume: 20 μLDetection: RI

Peaks: 1. Sucrose 2. Glucose 3. Fructose

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Column: Aminex HPX87CEluent: WaterTemperature: 80 °CFlow Rate: 0.5 mL/minInj. Volume: 20 μLDetection: RI

Peaks: 1. Sucrose 2. Glucose 3. Fructose

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Peaks: 1. Sucrose 2. Glucose 3. Fructose

Apple Juice

Orange Juice

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At high pH values, carbohydrates are deprotonated. The resulting anionic species can be separated by anion-exchange mechanisms, typically using aqueous mobile phases such as NaOH or KOH. For oligosaccharide separations, the mobile phase also contains sodium acetate. Concentration gradients of sodium acetate facilitate the elution of oligosaccharides.

High-pH eluents require the use of polymeric columns. Dionex CarboPac columns provide the basis for optimized carbohydrate separations using these conditions.

Innovative Resin TechnologyThe CarboPac PA20 column uses

Dionex pellicular resin technology for improved chromatographic resolution, peak shape, and efficiency for the six common monosaccharides. CarboPac PA20 columns are packed with a hydrophobic, polymeric, pellicular anion exchange resin that is stable over pH 0–14. This unique pH-stability allows the use of eluent compositions that are conducive to oxidation of carbohydrates at gold electrodes.

The MicroBead™ latex particle was optimized to further improve column performance by imparting a unique chromatographic selectivity. This selectivity results in a significantly improved resolution between the previously-problematic analytes galactose and glucosamine.

Mono- and Disaccharide Separations Using HPAE-PAD

Mono- and disaccharides important in food analysis are typically separated at eluent concentrations lower than 100 mmol/L NaOH. Coffee sugars, such as mannitol, arabinose, galactose, glucose, xylose, mannose, and fructose, can be separated with 2 mmol/L sodium hydroxide (Figure 4) using waveform A, which is described in Dionex Techni-cal Note 21.3 For outstanding inter-run consistency, this analysis can be run using automatically-generated potassium hydroxide eluent on a Reagent-Free™ IC (RFIC™) system with Eluent Generation (RFIC-EG™ system).

Figure 3. Analysis of glucose, fructose, and sucrose in wine.

Figure 4. Separation of coffee sugars using the CarboPac PA20 column.

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Column: Shodex Sugar SC1011Eluent: WaterTemperature: 80 °CFlow Rate: 1.0 mL/minInj. Volume: 20 μLDetection: RI

Peaks: 1. Glucose

Peaks: 1. Sucrose 2. Glucose 3. Fructose

Red Wine

White Wine

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Column: CarboPac PA20Eluent: 2 mM NaOH, isocraticFlow Rate: 0.5 mL/minDetection: Pulsed electrochemical detection, Au electrodeWaveform: Waveform A

Peaks: 1. Mannitol 2. Arabinose 3. Galactose 4. Glucose 5. Xylose 6. Mannose 7. Fructose

nC

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Figure 5. Separation of sugar alcohols, mono- and disaccharides.

Figure 6. Inulin profile using the CarboPac PA200 column.

The analysis of well-resolved sugars can be made faster by increasing the hydroxide concentration. Mono- and disaccharides important in dietary fiber analysis require higher concentrations of sodium hydroxide for timely elution and are readily eluted in less than 12 min with 52 mmol/L sodium hydroxide (Figure 5). This technique provides good resolution between the sugar alcohols and sugars in a single isocratic run.

Predictable, High-Resolution Separation of Oligosaccharides

There is a significant and increasing demand for reproducible, fast, and simple methods to profile oligosaccharides and homologous sugar series such as inulins, amylopectins, and maltooligosaccharides in the food industry. Most HPLC approaches proposed for these applications are limited by insufficient specificity and high limits of detection.

The CarboPac PA200 is a nonporous, high-efficiency, polymeric anion-exchange column that provides the highest resolution available for oligosaccharide mapping and analysis through PAD. The resin consists of 5.5 μm nonporous beads covered with a fine layer of functionalized MicroBead latex particles. This pellicular resin structure permits excellent mass transfer, result-ing in high-resolution chromatography and rapid re-equilibration after gradient elution. The 3 × 250 mm column format provides fast separations. The recom-mended flow rate of 0.5 mL/min results in significant savings in eluent consumption.

Linear Polysaccharide ProfilingInulin and fructo-oligosaccharides

(FOS) are increasingly used as functional food ingredients. Chain-length distribution profiles of commercial products such as those derived from inulin can be

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Peaks: 1. Glycerol 2. Xylitol 3. Sorbitol 4. Mannitol 5. Glucose 6. Fructose 7. Sucrose 8. Lactose

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Columns: CarboPac PA200 (3 × 250 mm)Gradient: 120–320 mM NaOAc in 100 mM NaOH over 40 minFlow Rate: PA200: 0.5 mL/minDetection: Pulsed amperometry, Waveform A, gold electrodeSamples: Inulin from chicory (Sigma)

determined using HPAE-PAD with gradient elution (Figure 6).

Commercial food ingredient products derived from the lower-molec-ular-weight fractions of inulin (DP3-20) can be determined by AOAC Method 997.08, an enzymatic preparation fol-lowed by HPLC. However, Dionex

has developed a more direct HPAE-PAD method that allows commercially available FOS and inulin products to be identified and quantified directly in a variety of foods: Application Note 150, Determination of Plant-Derived Neutral Oligo- and Polysaccharides Using the CarboPac PA200.4

5

AmylopectinsHPAE-PAD with gradient

elution has been used for structural studies on starch-derived materials such as amylopectins, since the chain length distribution is an important parameter for characterizing the molecular struc-ture. These distributions can be used as fingerprints for the amylopectin source (Figure 7).

Systems for Carbohydrate AnalysisDionex offers configurable sys-

tems to support carbohydrate analysis, from robust basic systems to dual-pump models that support parallel, tandem, and other high-productivity LC tech-niques.

Optimized configurations for HPAE-PAD methods include the ICS-3000 basic and dual systems described in the tables to the right. The ICS-3000 dual configuration with autosampler sharing supports one pump performing carbohydrate analysis, while the other with an optional ED or CD detector is available for other ion-exchange determinations for food and beverage applications (e.g., amino acids, organic acids, inorganic anions and cations, biogenic amines).

For RI detection, Dionex features the UltiMate® 3000 basic and x2 Dual systems, detailed in the tables below. The x2 configuration with autosampler sharing supports one pump performing carbohydrate analysis with RI detec-tion, while the other is available for other gradient HPLC applications with UV detection (e.g., vitamins, organic acids, PAHs, pesticides).

Figure 7. Amylopectins separated on the CarboPac PA200 column.

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Column: CarboPac PA200 and guardEluent: Sodium acetate gradient in 100 mM Sodium hydroxide 70 to 300 mM in 30 minFlow Rate: 0.5 mL/minInj. volume: 5 μL from 10 μL loopTemperature: 30 °CDetection: Pulsed amperometry, gold electrodeSample: Red Hook Amber Ale 1:50 dilutionWaveform A: Quadruple potential

ICS-3000 Standard SyStem for Carbohydrate analySIS by hPae-Pad

Part number description

061706 SP Gradient Pump with degasser

062629 EO Eluent Organizer (includes four, 2-L eluent bottles)

063493 EO Regulator Accessory and holder

061790 DC module with one temperature zone and one injection valve, micro bore

061718 ED Amperometric Detector (without cell and working electrode)

061756 ED Cell with reference electrode and spacer block

061749 ED Au working electrode, with gasket and polishing kit

061360 Chromeleon® CHM-1 (including one timebase)

PC OptiPlex 745 MT, standard model with 17” TFT, Windows XP Professional

ICS-3000 dual IC SyStem for food & beverage aPPlICatIonSPart number description

061710 DP Dual Pump - gradient/isocratic with degasser

062629 EO Eluent Organizer (includes four, 2-L eluent bottles)

063493 EO Regulator Accessory and holder

061793 DC module with two temperature zones and two injection valves, microbore

061718 ED Amperometric Detector (without cell and working electrode)

061756 ED cell with reference electrode and spacer block

061749 ED Au working electrode, with gasket and polishing kit

061714 EG Eluent Generator module

058900 EluGen® II KOH cartridge

060477 CR-ATC Continuously Regenerated Anion Trap Column

063353 EG/DP vacuum degas conversion kit

063104 AS simultaneous injection with no injection valves

061364 Chromeleon CHM-1 (includes 2 timebases)

060728 Chromeleon Server option: PDA licence (3D data acquisition)

PC OptiPlex 745 MT, standard model with 17” TFT, Windows XP Prof.

6

ultImate 3000 Standard SyStem for Carbohydrate analySIS by hPlC-rI

Part number description

5035.9250 SRD-3200 Solvent Rack with two degasser channels

5035.0010 ISO-3100A isocratic analytical pump

5035.0600 UltiMate 3000 Manual Injection Valve analytical/micro, with mounting kit and 20 μL sample loop

5722.0000 TCC-3000 Thermostatted Column Compartment

5060.0030 RI 101 Refractive Index Detector

5960.0067 Chromeleon CHM-1 (includes one timebase)

PC OptiPlex 745 MT, standard model with 17” TFT, Windows XP Professional

ultImate 3000 x2 dual-gradIent hPlC SyStem for food & beverage aPPlICatIonS

Part number description

5035.9230 Solvent Rack SRD-3600 with six degasser channels

5035.0014 x2 Dual-Gradient Analytical Pump DGP-3600A

5822.0020 Analytical in-line split loop thermostatted autosampler WPS-3000TSL

5722.0010 Thermostatted Column Compartment TCC-3100 1x2P-6P with 2-position 6-port switching valve

6037.0004 Parallel Operation Capillary Kit, Dual-Gradient Analytical

5080.0020 Photodiode Array Detector PDA-3000, without flow cell

6080.0210 Absorbance Cell for PDA-3000, 13 μL, SST, 10 mm path

5060.0030 RI 101 Refractive Index- Detector

5960.0068 Chromeleon CHM-2 for two UltiMate 3000 LC systems

5960.0020 Chromeleon Server option: 3-D Data Acquisition

PC OptiPlex 745 MT, Standard Model with 17” TFT, Windows XP Professional

MicroBead, Reagent-Free, RFIC and RFIC-EG are trademarks and Chromeleon, UltiMate, EluGen, and CarboPac are registered trademarks of Dionex Corporation in the U.S. and other countries.

Aminex is trademark of BioRad Corporation. Shodex is trademark of Showa, Ltd.

LPN 1971 8M 09/07©2007 Dionex Corporation

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References1. De Vries, J. W.; Nelson, A. L., Food

Technology 1994, July, pp. 76–77.

2. Dionex Corporation. Technical Note 20: Analysis of Carbohydrates by High-Performance Anion-Exchange Chromatography with Pulsed Ampero-metric Detection (HPAE-PAD). 2004.

3. Dionex Corporation. Technical Note 21: Optimal settings for pulsed amperometric detection of carbo-hydrates using the Dionex ED40 Electrochemical Detector. 1998.

4. Dionex Corporation. Application Update 150: Determination of Plant-Derived Neutral Oligo- and Poly-saccharides Using the CarboPac™ PA200. 2005.