HPLC Analysis of Sugars

Transcription

1 HPLC Analysis of Sugars Pre-Lab Exercise: 1) Read about HPLC, sugars and the experiment and its background. 2) Prepare a flowchart for the lab exercise. 3) Research 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 and they can be found in both plant and animal tissue as well as in microorganisms. Carbohydrate species vary widely in structure and are found in nature in a wide range of concentrations. Carbohydrates include monosaccharide, oligosaccharides and polysaccharides (e.g. xylose, sucrose and starch, repectively). While it is possible to separate monosaccharides using a C 18 column (Martin-Villa et al., 1982) this will not always give a complete separation of these compounds (Fig. 1). Using a more polar column, such as a NH 2 column, a better separation is often possible (Fig. 2). Simple sugars may also be separated by both gel permeation and normal phase chromatography. In addition, cation exchange chromatography (using a column with immobilized specific metal cations) can be used to spearate monoand disaccharides based on their affinity for the immobilized metals (Olechno et al., 1987). 1 P age

2 In order to facilitate separation, it is often useful to perform a clean-up step. This clean-up is designed to partially remove unwanted co-eluting or late eluting compounds from the sample. Solid phase extraction (SPE) using commercially available products (such as Sep Pak C 18 ) is one common method that is used to remove unwanted materials from a sample. If this is done, it is extremely 2 P age

3 important to follow an established procedure so that consistent analyte recovery realized. 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 and degassed 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 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, in order to displace other dissolved gases). 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 0.6mg 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 5-10mL of supernatant from the previous step. Discard the first 2mL (this may have been diluted by residual water in the cartridge). Collect 4-6mL 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 the 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. Missing page numbers. 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 Sept, P age