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Published on January 11, 2008

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The HPLC Determination of Oligosaccharides with Evaporative Light Scattering Detection:  The HPLC Determination of Oligosaccharides with Evaporative Light Scattering Detection Alltech Associates, Inc. 2051 Waukegan Road • Deerfield, IL 60015 Phone: 1-800-ALLTECH • Web Site: www.alltechWEB.com Slide2:  Outline • Introduction • Experimental • Estimating Chain Length Distribution • Fructo-oligosaccharides and Inulin • Malto-oligosaccharides (Corn Syrup, Maltodextrin, Fermented Beverages) • Detection Sensitivity • Partially Hydrolyzed Dextran • Conclusion • References • Acknowledgements Introduction:  Introduction The HPLC analysis of oligo- and polysaccharides is increasingly important in the study of human nutrition. Much research has been done in extending the range of DP (degree of polymerization) resolution over a homologous series [1,2]. Two traditional approaches are problematic [2-4,5,7,8-11]: Methodology Associated Problems Silica-Based Amino Column • Severe baseline instability and RI Detection • Short column lifetime • Incompatibility with gradients • Column pH constraints (2 - 7.5); higher order polysaccharides require alkaline conditions for solubility Anion-Exchange Column • Expensive and Pulsed Amperometric • Difficult to equilibrate Detection • Lacks application versatility Methodology Associated Problems Slide4:  Previous work [8,12] has shown the methodology consisting of the polymeric Prevail Carbohydrate ES column and evaporative light scattering detection to be preferred for the general determination of mono-, di- and trisaccharides and sugar alcohols. The current study applies the methodology to the investigation to oligosaccharides. • Ruggedness and long column lifetime • Ease of use • Sensitivity to the 30ng level using diverse mobile phases • Excellent peak shapes and resolution • Total gradient compatibility • Flat baselines under both isocratic and gradient conditions • pH ruggedness (2 – 13) with volatile modifiers Attributes of Prevail Carbohydrate ES and ELS detection Slide5:  Experimental The following materials and equipment were used for all experiments: Pump: Hitachi/La-Chrom L-7100A Injector: Alltech 580 Autosampler; 20µL Loop (Part No. 580100) Detector(s): Linear 200 UV-Vis at 227nm (Part No. 27410000) Alltech ELSD 2000 (Part No. 600100) Data System: Alltech AllChrom™ at 10Hz (Part No. 272100) Solvent Degasser: Alltech On-Line Degasser (Part No. 590102) Column(s): Alltech Prevail Carbohydrate ES, 5µm, 250 x 4.6mm (Part No. 35101) Slide6:  Sample Preparation: Inulin - 60mg of commercial Inulin (SIGMA-Aldrich) was added to 0.5mL of conc. ammonium hydroxide (SIGMA-Aldrich) and 1mL deionized water The mixture was sonicated for 45 minutes and then syringe filtered (0.45µm; nylon) into a glass autosampler vial for injection. Fructo-oligosaccharides (FOS) - The contents, 500mg, of a GNC Brand Natural FOS capsule was emptied into a vial to which 4mL 1% ammonium hydroxide (SIGMA-Aldrich) was added. The mixture was sonicated for 5 minutes and then syringe filtered (0.45µm; nylon) into a glass autosampler vial. Fermented Beverages - Hacker-Pschorr Weisse (Englewood, CO) and Guiness Extra-Stout (Guiness-Bass Import of Stamford, CT) were degassed and then syringe filtered (0.45µm; nylon) neat into a glass autosampler vial. Slide7:  Onions - 0.5g dried, minced onions were combined with 2mL deionized water, 1mL conc. ammonium hydroxide and then sonicated for 30 minutes. The mixture was then syringe filtered (0.45µm; nylon) into a glass autosampler vial for injection. Corn Syrup Solids - 100mg of 42DE corn syrup solids (courtesy LEAF/Hershey) was dissolved in 3mL deionized water. The mixture was syringe filtered into a glass autosampler vial for injection. Maltodextrin - 100mg of commerical maltodextrin (SIGMA-Aldrich) was dissolved in 2mL deionized water. The mixture was syringe filtered into a glass autosampler vial for injection. Partially Hydrolyzed Dextran - 100mg Dextran (SIGMA-Aldrich) was placed into a 5mL glass vial. 1mL deionized water and 0.5mL conc. ammonium hydroxide were added. The mixture was sonicated for 45 minutes and a 1mL aliquot was syringe filtered into a glass autosampler vial for inection. Slide8:  Estimating Chain Length Distribution A powerful benefit of the Prevail Carbohydrate ES/ELSD methodology is the ability to deliver rugged, reproducible gradient separations. An optimized “screening” gradient for mono-, di-, and oligosaccharides (Figure 1) and sugar alcohols is shown in Figure 2.   • Tool for estimating chain length (DP) distribution • Gives evidence for molecular structure • Maltodextrin is a convenient, full-range DP reference: - Glucose - Maltose (DP2) - Homologous series DP3 – DP22. Benefits of the Screening Gradient for Oligosaccharides Slide9:  Figure 1 Mono-, Di-, and Oligosaccharides and Sugar Alcohols Slide10:  0 Estimating Chain Length Distribution - Screening Gradient for Mono-, Di-, and Oligosaccharides 10009 Column: Prevail Carbohydrate ES, 250 x 4.6mm Mobile Phase: A: Acetonitrile B: 0.04% NH4OH in Water Gradient: Time: 0 25 40 80 %B: 17 27 45 65 Flowrate: 1.0mL/min Column Temp: Ambient Detector: ELSD 2000 Figure 2 1. Iso-erythritol 2. Fructose 3. Sorbitol 4. Mannitol 5. Glucose 6. Inositol 7. Sucrose 8. Maltitol 9. Maltose (DP2) 10. Raffinose (DP3) 11. Maltotriose (DP3) 12. Maltotetraose (DP4) 13. Maltopentaose (DP5) 14. – 22. DP6 – DP14 Fructo-oligosaccharides and Inulin:  Fructo-oligosaccharides and Inulin The utility of the screening gradient may be illustrated by the determin-ation of Inulin and its hydrolysis products [14-18], the fructo-oligosaccharides, Figure 3. Inulin Storage polysaccharide (DP range 3–60) Common in vegetables (artichokes, onions @ 6-8 grams/serving) Dietary fiber Stimulates growth of bifidobacteria - aids in digestion Inhibits growth of pathogenic bacteria Lowers blood serum cholesterol Fructo-oligosaccharides Hydrolysis products of Inulin Short chain oligomers (1 glucose unit, multiple fructose units) Slide12:  Figure 3 Sucrose and Principal Fructo-oligosaccharides DP3 – DP5 Sucrose Ketose Nystose 1-b-Fructofuranosyl-D-nystose Slide13:  The screening gradient run on a commercial sample of fructo-oligosac-charides (Figure 4) showed four peaks whose retention times gave evidence of their DP. The principal fructo-oligosaccharides are well known and peak IDs were confirmed by authentic injection of standards. The screening gradient was then applied to a real sample reconstituted, minced onions (Figure 5) and commercial Inulin (Figures 6,7). * Inferred from position in screening chromatogram ** Confirmed by authentic injection Slide14:  Figure 4 10032 10038 For conditions, please refer to Figure 2, Chrom 10009 Estimation of Chain Length Distribution - Commercial Fructo-oligosaccharides (FOS) 1. Sucrose 2. Kestose (DP3) 3. Nystose (DP4) 4. 1--Fructofuranosyl-D-nystose (DP5) FOS DP Reference Slide15:  10043 Figure 5 Fructo-oligosaccharides in Reconstituted, Minced Onions For conditions, please refer to Figure 2, Chrom 10009 1. Sucrose 2. DP3 3. Kestose (DP3) 4. Nystose (DP4) 5. DP4 6. 1--fructofuranosyl- D-nystose (DP5) 7. DP5 8. DP6 9. DP7 10. DP8 11. DP9 12. DP10 13. DP11 14. DP12 15. DP13 16. DP14 17. DP15 18. DP16 Slide16:  Figure 6 10035 10038 Estimation of Chain Length Distribution - Inulin For conditions, please refer to Figure 2, Chrom 10009 Fructans 1. DP3 2. DP10 3. DP19 4. DP25 Inulin DP Reference Slide17:  Fructans 1. DP3 2. DP10 3. DP19 4. DP25 5. DP40 10039 Figure 7 Optimized Separation - Inulin (DP3 to DP40) Column: Prevail Carbohydrate ES, 250 x 4.6mm Mobile Phase: A: Acetonitrile B: 0.04% NH4OH in Water Gradient: Time: 0 25 40 120 %B: 17 27 45 65 Flowrate: 1.0mL/min Column Temp: Ambient Detector: ELSD 2000 Malto-oligosaccharides:  Malto-oligosaccharides The food and beverage industry makes heavy use of the hydrolysis products of corn starch. Corn syrup is used widely as a food and beverage sweetener while Maltodextrin, less sweet to taste, is used mainly as a texture-, bulking-, moistening-agent. The screening gradient profile for commercial corn syrup solids was established as in Figure 8 and a more optimal gradient separation (DP3 – DP15) is seen in Figure 9. The screening gradient profile for commercial maltodextrin was established as in Figure 10 and a more optimal separation (DP3 – DP22) is shown in Figure 11. Note the appearance and separation of (1,4-)-linked and (1,6-)-linked polymers for each degree of polymerization [3]. Fermented beverages (beers, ales, lagers, stouts, etc.) are typically high in malto-oligosaccharides. Two such profiles appear in Figure 12, using the screening gradient as an aid to peak ID. Slide19:  Figure 8 10037 10038 Estimation of Chain Length Distribution - Corn Syrup 1. Glucose 2. Maltose 3. Maltotriose 4. Maltotetraose 5. Maltopentaose 6. Maltohexaose For conditions, please refer to Figure 2, Chrom 10009 Corn Syrup DP Reference Slide20:  Figure 9 Column: Prevail Carbohydrate ES, 250 x 4.6mm Mobile Phase: A: Acetonitrile B: Water Gradient: Time: 0 15 25 %B: 35 50 50 Flowrate: 1.0mL/min Column Temp: Ambient Detector: ELSD 2000 1. Glucose 2. Maltose 3. Maltotriose (DP3) 4. Maltotetraose (DP4) 5. Maltopentaose (DP5) 6. Maltohexaose (DP6) 7. DP7 8. DP8 9. DP9 10. DP10 11. DP11 12. DP12 13. DP13 14. DP14 15. DP15 9354 Optimized Gradient Separation – Corn Syrup Slide21:  Figure 10 10038 Screening Gradient Reference - Maltodextrin 1. Glucose  2. Maltose (DP2)  3. Maltotriose (DP3)  4. Maltotetraose (DP4)  5. Maltopentaose (DP5) 6. Maltohexaose (DP6) For conditions, please refer to Figure 2, Chrom 10009 Slide22:  Figure 11 1. Glucose (DP1) 2. DP3 (1,6-a; 1,4- a*) 3. DP6 (1,6- a; 1,4- a*) 4. DP13 5. DP18 9534 Column: Prevail Carbohydrate ES, 250 x 4.6mm Mobile Phase: A: Acetonitrile B: Water Gradient: Time: 0 50 60 %B: 35 60 60 Flowrate: 1.0mL/min Column Temp: Ambient Detector: ELSD 2000 Optimized Gradient Separation - Maltodextrin * Linkage assignments found in Koizumi, K., Fukuda, M., J. Chromatogr., 585 (1991) 233-238. Slide23:  Figure 12 10041 Malto-oligosaccharide Profiles – Imported Weiss and Stout 10042 For conditions, please refer to Figure 2, Chrom 10009 Weiss Stout 1. DP2 2.  DP3 3.  DP4 4. DP5 5.  DP6 6.  DP7 7.  DP8 8.  DP9 9.  DP10 10. DP11 11.  DP12 12. DP13 13.  DP14 14.  DP15 15.  DP16 16.  DP17 17.  DP18 18.  DP19 Detection Sensitivity:  Detection Sensitivity With the Prevail Carbohydrate ES column, the ELSD 2000 detector has shown sensitivity to monosaccharides down to LOD=30ng on-column. Using the separation in Figure 11, and based on a peak area % calculation, the sensitivity to maltooligosaccharides DP16 – DP18 was approximately 500ng on-column. Sample Injection: 10µL of 50mg/mL = 500µg on-column Partially Hydrolyzed Dextran:  Partially Hydrolyzed Dextran In polysaccharide structural studies, partial hydrolysis of the poly-saccharides followed by identification of the resulting oligosaccharides is a powerful aid [3,10]. Dextran Mixture of (1,6-a)-linked glucose polymers, called glucans Used clinically in the prevention of thromboembolism by increasing peripheral blood flow [19]. The screening gradient profile for partially hydrolyzed Dextran was established as in Figure 13. From this, the range of glucans that showed a degree of separation appeared to be from DP3 to DP36. Finally, a step gradient (Figure 14) was used for a more optimal determination of Dextran. Slide26:  Figure 13 1. DP4 2. DP10 3. DP22 10044 Estimation of Chain Length Distribution - Partially Hydrolyzed Dextran - (1-6)--D-glucans For conditions, please refer to Figure 2, Chrom 10009 Slide27:  Figure 14 9540 Step Gradient - Partially Hydrolyzed Dextran - (1-6)--D-glucans Column: Prevail Carbohydrate ES, 250 x 4.6mm Mobile Phase: A: Acetonitrile B: Water Gradient: Step (5min. Hold at 40%B, 5%B steps every 5min. to 70%B) Flowrate: 1.0mL/min Column Temp: Ambient Detector: ELSD 2000 Conclusion:  Conclusion The Prevail Carbohydrate ES/ELSD methodology is effective for the determination of oligosaccharides up to DP40 for those samples analyzed. However, a baseline separation of carbohydrate oligomers seems to be limited to a chainlength of DP23 for glucose and/or fructose units. The screening gradient using acetonitrile-water formulations at basic pH is a reliable method for estimating the distribution of polymer chain lengths. References:  References (1) Asp, N. O., Am. J. Clin. Nutr., 61 (1995) 930S – 937S. (2) Churms, S. C., J. Chromatogr. A, 720 (1996) 151. (3) Koizumi, K., Fukuda, M., J. Chromatogr., 585 (1991) 233-238. (4) Koizumi, K., Kubota, Y., Tanimoto, T., and Okada, Y., J. Chromatogr., 464 (1989) 365-373. (5) Herbreteau, B., Lafosse, M., Morin-Allory, L., and Dreux, M., J. Chromatogr., 472 (1989) 209. (6) Lafosse, M., Dreux, M., and Morin-Allory, L., J. Chromatogr., 404 (1987) 95. (7) Ikemoto, N., Lo, L. C., and Nakanishi, K., Angew. Chem. Int. Ed. Engl., 31 (1992) 890. (8) Young, C. S., Cereal Foods World, 47 (1) (2002). (9) Brons, C., and Olieman, C., J. Chromatogr., 259 (1983) 79. (10) Johnson, D. C., LaCourse, W. R., Anal. Chem. 62, (1990) 589A- 597A. (11) A Practical Guide to HPLC Detection, D. Parriott-Editor (Academic Press, Inc., San Diego, 1993), Chapter 2. Slide30:  (12) Young, C. S., A Total Solution for the Determination of Carbohydrates by HPLC, Poster PP052, Alltech Associates, Inc., 2001. (13) Churms, S. C., J. of Chromatogr. A, 720 (1996) 75-91. (14) Niness, K., Cereal Foods World, 44 (2) (1999) 79 – 81. (15) Bouhnik Y., Flourie, B., Riottot, M., Bisetti, N., Gailing, M. F., Guibert, A., Bornet, F., Rambaud, J. C., Nutr. Cancer., 26 (1) (1996) 21 – 29. (16) Briet, F., Achaour, L., Flourie, B., Beaugerie, L., Pellier, P., Franchisseur, C., Bornet, F., Rambaud, J. C., Eur. J. Clin. Nutr. 49 (7) (1995) 501 – 507. (17) Jackson, K. G., Taylor, G. R. J., Clohessy, A. M. Williams, C. M., Br. J. Nutr., 82 (1999) 23 – 30. (18) Yamashita, K., Kawai, K., Itakura, M. Nutr. Res., 4 (1984) 961 – 964. (19) Griffel, M. I., Kaufman BS. Crit. Care Clin., 8 (1992) 235-53. Slide31:  Acknowledgements Alltech Associates, Inc. acknowledges that this technical presentation represents a considerable investment in time and effort on the part of many dedicated professionals. Author(s): Craig S. Young Laboratory Contribution(s): Craig S. Young Editorial Contribution(s): Bob Ziegler Graphics Preparation: Liz Fisher and Julia Poncher www.alltechWEB.com

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