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  • Food And Beverage

    09 September 2016

    Carbohydrate Analysis by IC and HPLC

    High Performance Liquid Chromatography (HPLC) is an important tool to identify and quantify carbohydrates 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 challenging 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 sensitivity required.
    HPLC on aminopropyl-bonded silica or polymer-based metal-loaded cation-exchange resins, in conjunction with refractive index (RI) or lowwavelength 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 monoand disaccharides in the g/L range.
    However, some sample matrices require better resolution of sugars from sugar alcohols, organic acids, and sodium chloride.
    High-performance anionexchange 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.
    Dionex offers HPLC-RI and HPLC-PAD solutions optimized for a wide variety of research and monitoring applications.

    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-destructive 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 Techniques

    The 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.
    At high pH values, carbohydrates are deprotonated. The resulting anionic species can be separated by anionexchange 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 Technology

    The 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 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 Technical 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).
    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, resulting in high-resolution chromatography and rapid re-equilibration after gradient elution. The 3 × 250 mm column format provides fast separations. The recommended flow rate of 0.5 mL/min results in significant savings in eluent consumption.

    Linear Polysaccharide Profiling

    Inulin 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 determined using HPAE-PAD with gradient elution (Figure 6).
    Commercial food ingredient products derived from the lower-molecular-weight fractions of inulin (DP3-20) can be determined by AOAC Method 997.08, an enzymatic preparation followed 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

    Amylopectins

    HPAE-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 structure. These distributions can be used as fingerprints for the amylopectin source (Figure 7).

    Systems for Carbohydrate Analysis

    Dionex offers configurable systems to support carbohydrate analysis, from robust basic systems to dual-pump models that support parallel, tandem, and other high-productivity LC techniques.
    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 detection, while the other is available for other gradient HPLC applications with UV detection (e.g., vitamins, organic acids, PAHs, pesticides).
     

    ICS-3000 Standard System for Carbohydrate Analysis by
    HPAE-PAD

    Part Number Description
    079819 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)
    AAA-061756 ED Cell with reference electrode and spacer block
    079850 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 Applications

    Part Number Description
    079823 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
    079830 ED Amperometric Detector (without cell and working electrode)
    AAA-061756 ED cell with reference electrode and spacer block
    079850 ED Au working electrode, with gasket and polishing kit
    079827 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.

     
     

    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

     
     

    References:

    1. 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 Amperometric
    Detection (HPAE-PAD). 2004.
    3. Dionex Corporation. Technical Note
    21: Optimal settings for pulsed
    amperometric detection of carbohydrates
    using the Dionex ED40
    Electrochemical Detector. 1998.
    4. Dionex Corporation. Application
    Update 150: Determination of Plant-
    Derived Neutral Oligo- and Polysaccharides
    Using the CarboPac™
    PA200. 2005.

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