HPLC Analysis of Sugars

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1 HPLC Analysis of Sugars Pre-Lab Exercise: 1) Read about HPLC, sugars and the experiment and its background. 2) Prepare a flowchart as appropriate for the lab exercise. 3) Note the various sugar concentrations found in fruit juices (theoretical values). This is required so that you may determine the dilution required for your sample. Introduction: Carbohydrates are the most abundant and widely distributed food component. Carbohydrates can be found in both plant and animal tissue as well as in microorganisms. It is found in many forms and at many different concentrations. Carbohydrates include a) monosaccharide, such as xylose, arabinose, glucose and fructose b) oligosaccharides or polymers of monosaccharides such as a disaccharide like sucrose and c) polysaccharides which contain more than 10 units in the glycosidic chain. An example of a polysaccharide is starch. While it is possible to separate simple sugars using a C18 column (Martin-Villa et. Al., 1982) this will not always give complete separation of some of the monosaccharides (Fig. 1). The more polar NH 2 column is better able to separate these sugars (Fig. 2). Suppliers of HPLC columns also offer columns to separate simple carbohydrates based on gel permeation and normal phase chromatography. Cation exchange chromatography can be used by immobilizing specific metal cations on a column and separating mono- and di saccharides based on their affinity for the immobilized metals (Olechno et al., 1987). 1 P age

2 While chromatographic techniques by nature separate sample components, it is sometimes expedient to use a clean-up step prior to injecting the sample. This clean-up is designed to partially remove unwanted co-eluting or late eluting compounds from the sample. Solid phase extraction is one common way to remove excipients from a sample. It is very important to consistently follow recommended 2 P age

3 procedures for the preparation and use of solid phase extraction tubes (i.e., Sep Pak C18). Failure to use this clean-up procedure properly could result in incomplete or variable recovery of your analyte(s) of interest. Experimental: Reagents and supplies: Solvents: Deionized water, methanol. Acetonitrile (HPLC grade) Reagents: fructose, glucose and sucrose Equipment: Column: NH 2 (25cm x 4mm) Mobile phase: Acetonitrile: water (75:25) - premixed Flow rate: 1.25 ml/min Injector Loop: 20µL Detector: UV at 193nm OR refractive index at 40 o C Run time: 12 minutes C-18 Sep-Pak 0.45µm syringe filters NOTE: All solvents, reagents and samples must be HPLC grade and filtered through 0.45µm filter before use. The acetonitrile/water mixture must be must be thoroughly degassed before use; it may be necessary to degas this solvent every 5-6 hours (or have a continuous flow of helium passing through the mobile phase). Procedure: 1. Preparation of standards: A mixed Stock Standard of fructose (10mg/mL), glucose (100mg/mL) and sucrose (100mg/mL) is prepared to 100mL using deionized water. Working standards: If the solvent mixture is not identical for each of the working standards the analyst will observe fluctuations in the retention times of the three sugars. To avoid this, use the following table to prepare the working standards. Add the recommended volumes of the standard to the 10.0 ml volumetric flasks. Make to volume using Acetonitrile: water (75:25) mobile phase. 3 P age

4 Table 1: Preparation of Working Standards Working standard Volume of stock standard (ml) Fructose Concentration (mg/ml) Glucose Sucrose # # # # Sample Preparation and Extraction: Select one of the samples provided. Checking the expected content (from your prelab exercise) of simple sugars, estimate the required dilution so that your sample contains approximately 0.6 mg of fructose/ml or 6.0mg sucrose/ml. 3. Sample Clean-up: The C-18 Sep-Pak is prepared by pretreating it with 2mL of acetonitrile followed by 5mL of deionized water. Do not let the Sep-Pak cartridge go dry. Apply 5-10 ml of supernatant from the previous step. Discard the first 2mL (this may have been diluted by residual water in the cartridge). Collect 4-6 ml of the eluate. 4. Sample dilution and solvent adjustment: Perform 1:10 dilution by pipetting 1mL of eluate into a 10mL volumetric flask. Make to volume using the acetonitrile: water mobile phase. Filter the sample using a 0.45µm syringe filter. Inject under the same HPLC conditions as used for the standards. 5. Report: A) Identify and record the retention times for the three sugars found in the standards. 4 P age

5 B) Using the area counts from the standard chromatograms draw standard curves for the three sugars (fructose, glucose and sucrose). Use excel, minitab etc. to draw these standard curves. Remember that you should have 3 curves, one for each sugar (you are plotting Concentrations (X-axis) vs Area Counts (y-axis). C) Using retention times and comparison to standards identify sugars present in the sample. D) Using the standard curves prepared determine the concentrations of the various sugars present in the sample. Remember to take into consideration your dilution factor. References: Martin-Villa, C., Vidal-Valverde, C. and Rojas-Hidalgo, E HPLC determination of carbohydrates in raw and cooked vegetables. J. Food Sci. 47: OLechno, J.D., Carter, S. R., Edwards, W.T., and Gillen, D. G Developments in chromatographic determination of carbohydrates. American Lab. Shaw, P.E. and Wilson, C.W Separation of fructose, glucose, and sucrose in fruit by HPLC using UV detection at 190nm. J. Sci. Food Agric. 34: Wilson, A.M., Work, T.M., Bushway, A.A. and Bushway, R.J HPLC determination of fructose, glucose and sucrose in potatoes. J. Food Sci. 46: Last updated May, P age

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