Determination of Carbohydrates in Beverages and Milk Products by HPAE-PAD and Capillary HPAE-PAD

Transcription

1 Determination of Carbohydrates in Beverages and Milk Products by HPAE-PAD and Capillary HPAE-PAD Terri Christison, Monika Verma, Aaron Kettle, Carl Fisher, Linda Lopez; Thermo Fisher Scientific, Sunnyvale, CA, USA

2 Overview Carbohydrates in beverage samples and milk products were directly determined using capillary format high performance anion-exchange with pulsed amperometric detection (HPAE-PAD) on a capillary reagent-free, high-pressure system., FIGURE. Conven This method eliminates the costly and labor-intensive derivitization used in other methods. Lactose and lactulose determinations are also demonstrated on the new µm resin particle, high-capacity Thermo Scientific Dionex CarboPac SA-µm mm column optimized for fast separations of mono- and di- saccharide sugars. Conven electrod Introduction Monosaccharide and disaccharide determinations are important to the food industry to ensure product formulation and product quality and to report ingredients to immune-and allergy-sensitive individuals. Because carbohydrates are poor chromophores, chemical derivitization is needed for absorption. However, derivitization is costly, labor-intensive and may cause changes in molecular configuration. High Performance Anion-Exchange chromatography with Pulsed Amperometric Detection (HPAE-PAD) is a proven sensitive method to directly and selectively determine carbohydrates. In HPAE-PAD, carbohydrates are ionized in strong base and separated by anion-exchange chromatography. The carbohydrates are detected by PAD with a gold working electrode using a four-potential waveform selective and sensitive for carbohydrates. This sensitivity allows carbohydrate analysis down to pmole concentrations or when the samples are limited. This sensitivity is moderated in beverage samples which contain g/l concentrations by minimizing the flow path combined with moderate dilution. Here we demonstrate the determinations of the different sugars used to sweeten beverages and the fast and easy determination of lactose and lactulose in milk products. This work further demonstrates the versatility of HPAE-PAD, easily optimized for low or high carbohydrate concentrations, and separations on different carbohydrate columns, and High-Pressure IC (HPIC ) and capillary HPIC systems. Experimental Results Sugars in Beverag Beverages are gen available in the man sweetening. The m or lowest cost. The Corn syrup, which i increased sweetnes hydrolyzed to gluco which can cause st Figure show the s µl sample separate FIGURE. Six c Sample Preparation The beverage samples were prepared by dilution and filtration, as appropriate. The milk products ( g/ ml water) were treated with µl each of Carrez I (potassium hexacyanoferrate(iii)) and Carrez II (zinc sulfate) solutions for deproteinization according to AOAC Method 98. and as described in AN 8. The samples were mixed, diluted to ml, centrifuged, and the supernatant filtered and treated with a Thermo Scientific Dionex OnGuard IIA sample preparation to remove anionic contaminants and neutralize the sample. Method Thermo Scientific Dionex HPIC Ion Chromatography systems used: Thermo Scientific Dionex ICS-+ ion chromatography (IC) system, standard and capillary format Dionex ICS-+ IC modules: EG Eluent Generator, DC Detector Chromatography, and DP Dual Pump Thermo Scientific Dionex ICS- Capillary HPIC system Detection: Figures to 7 sho typical capillary ga characteristic of na indicating high fruc of product sweeten fructose and gluco Electrochemical Detector and Electrochemical cell Gold on PTFE Disposable Electrode and ph,-ag/agci Reference Electrode FIGURE. Nativ PTFE Gaskets:.,., or. Autosampler: Thermo Scientific Dionex AS-AP Autosampler Software: Thermo Scientific Dionex Chromeleon Chromatography Data System Determination of Carbohydrates in Beverages and Milk Products by HPAE-PAD and Capillary HPAE-PAD Figure shows the instrument flow diagram for a capillary HPAE-PAD system. The + 7

3 Electrochemical Detector and Electrochemical cell FIGURE. Nativ Gold on PTFE Disposable Electrode and ph,-ag/agci Reference Electrode PTFE Gaskets:.,., or. Autosampler: Thermo Scientific Dionex AS-AP Autosampler 7 Software: Thermo Scientific Dionex Chromeleon Chromatography Data System Figure shows the instrument flow diagram for a capillary HPAE-PAD system. The Dionex ICS-+ system configured for mm or mm format columns has the same flow path but without the CRD and suppressor bypass modules. Glucos Figures and show four-potential waveform for detection of carbohydrates (Figure ) using a gold disposable working electrode (Figure ). This waveform is optimized to create a stable gold oxide layer resulting in highly reproducible responses. FIGURE. Flow diagram for a capillary HPAE-PAD system. H Data & System Management High-Pressure Non-Metallic Capillary Pump CRD Bypass Eluent Generator (OH or H+) FIGURE. HFC in Suppressor Bypass Regen flow in back CR-TC % % Separation Column Waste 9 Degas ED Sample Inject (Autosampler) FIGURE. Four Potential Waveform for carbohydrate determinations. Potential (V vs. Ag/AgCl) ohydrates in Beverages and Milk Products by. Time (s) Start. -. End Potential (V) Integration aron Kettle, Carl Fisher, Linda Lopez; Thermo Fisher Scientific, Sun determined using ometric detection Time (Seconds) FIGURE. Conventional and disposable gold working electrodes. FIGURE 7. Cane Working electrode ed in other Sucros 7 ew µm resin -µm mm gars. Conventional electrode Disposable electrode ood industry to to immune-and hores, chemical abor-intensive Results ometric Detection ermine and separated y PAD with a gold e for Sugars in Beverages Beverages are generally sweetened with the most available and the lowest cost sugar Figure 8 shows th available in the manufacturing region. Native sugars from fruit may be used for sensitivity and req sweetening. The manufacturer often selects the sugar for its product based on availability sweetening. or lowest cost. These sugars are sourced from either corn, Thermo sugar Scientifi cane or sugar beets. c Poster Note PN79_HPLC E_/S Corn syrup, which is primarily glucose, is enzymatically hydrolyzed to create fructose for Au Contact pad

4 food industry to s to immune-and phores, chemical labor-intensive Results erometric Detection etermine e and separated by PAD with a gold ve for mole erated in beverage combined with Sugars in Beverages Beverages are generally sweetened with the most available and the lowest cost sugar available in the manufacturing region. Native sugars from fruit may be used for sweetening. The manufacturer often selects the sugar for its product based on availability or lowest cost. These sugars are sourced from either corn, sugar cane or sugar beets. Corn syrup, which is primarily glucose, is enzymatically hydrolyzed to create fructose for increased sweetness. Beet sugar and cane sugar, which are sucrose, are partially hydrolyzed to glucose and fructose to increase sweetness and to minimize crystallization which can cause storage problems. o sweeten e in milk products. mized for low or ydrate columns, Au Contact pad Figure show the separation of carbohdyrates using capillary format HPAE-PAD,. µl sample separated on. mm i.d. columns at 8 µl/min flow rates. Figure 8 shows sensitivity and re sweetening. FIGURE 8. HFC FIGURE. Six carbohydrate standard. Column: Dionex CarboPac PA with guard,. mm Eluent Source: Thermo Scientific Dionex EGCKOH capillary Eluent: mm KOH Flow Rate:.8 ml/min Inj. Volume:. µl Column Temp.: C Detection: PAD, Au disposable, -Potential Gasket:. PTFE Samples: µm Mixed Standard ropriate. Carrez I for in AN 8. The ant filtered and aration to Peaks: tem, standard and. Fucose. Galactosamine. Glucosamine. Galactose.. Mannose Figures to 7 show the analysis of beverage samples using capillary HPAE-PAD with a typical capillary gasket of. thick. In Figure, the sample had a mixture of sugars, characteristic of native sugars. In Figure, only glucose and fructose are present indicating high fructose corn syrup (HFC) for sweetening. Figure 7 shows a typical profile of product sweetened with cane sugar (predominantly sucrose with equal parts of fructose and glucose). ector Figure 9 shows t SA-µm colum ( mm) and the s facilitates the sep FIGURE 9. Lacto C B Electrode FIGURE. Native sugars in apple cider. A Data System 7 Column: system. The s has the same drates (Figure ) optimized to ses. Dionex CarboPac PA with guard,. mm Eluent: mm KOH Flow Rate:.8 ml/min Inj. Volume:. µl Column Temp.: C Detection: PAD, Au disposable, -Potential Gasket:. PTFE Sample Prep.: -fold dilution Peaks: Data & System Management FIGURE. HFC in a carbonated beverage. Determination of Carbohydrates in Beverages and Milk Products by HPAE-PAD and Capillary HPAE-PAD %,. Void Volume. Galactose.. Mannose.. -- µm. Conclusio Capillary HPA without costly Modifying the analyzing high An RFIC syste thereby elimin Fast determin a simply 8-min Reference. Thermo Fish and Disacch

5 FIGURE. Native sugars in apple cider. 7 Column: Dionex CarboPac PA with guard,. mm Eluent: mm KOH Flow Rate:.8 ml/min Inj. Volume:. µl Column Temp.: C Detection: PAD, Au disposable, -Potential Gasket:. PTFE Sample Prep.: -fold dilution Peaks:. Void Volume -- µm. Galactose... Mannose.. FIGURE. HFC in a carbonated beverage. %, % 9 Column: Dionex CarboPac PA with guard,. mm Eluent: mm KOH Flow Rate:.8 ml/min Inj. Volume:. µl Column Temp.: C Detection: PAD, Au disposable, -Potential Gasket:. PTFE Sample Prep.: -fold dilution, degas Peaks:. 98 µm. 9 Thermo Scientifi c Poster Note PN79_HPLC E_/S

6 FIGURE 7. Cane sugar in coconut water flavored beverage 7 Column: Dionex CarboPac PA with guard,. mm Eluent: mm KOH Flow Rate:.8 ml/min Inj. Volume:. µl Column Temp.: C Detection: PAD, Au disposable, -Potential Gasket:. PTFE Sample Prep.: -fold dilution Peaks:. Void Volume -- µm. Galactose.. 7. Figure 8 shows that using a. gasket versus the. capillary gasket reduces sensitivity and requires less dilution. The resulting analysis suggest the use of HFC for sweetening. FIGURE 8. HFC in a carbonated beverage --. Gasket. %, % Column: Dionex CarboPac PA with guard,. mm Eluent: mm KOH Flow Rate:.8 ml/min Inj. Volume:. µl Column Temp.: C Detection: PAD, Au on PTFE disposable, Four-Potential Carbohydrate waveform Gasket:. PTFE Sample Prep.: -fold dilution, degas Peaks: mg/l % Ratio. Void Volume Figure 9 shows the 8 min separation of lactose and lactulose on the Dionex CarboPac SA-µm column using only mm KOH. This method utilizes the standardbore format ( mm) and the standard. thickness gasket. A high-pressure capable system facilitates the separation. Determination of Carbohydrates in Beverages and Milk Products by HPAE-PAD and Capillary HPAE-PAD

7 FIGURE 9. Lactose and lactulose in raw unpasteurized milk. C B A Instrument: Dionex ICS- + HPIC system Column: Dionex CarboPac SA-µm and guard, mm Eluent Source: Dionex EGC KOH Eluent: mm KOH Flow Rate:. ml/min Inj. Volume: µl Column Temp.: C Detection: PAD, Au on PTFE disposable, Four-potential Gasket:. thick PTFE Ref. Electrode: ph-ag/agcl Sample Prep.: Carrez digestion, centrifuge, filter, Dionex OnGuard IIA Sample: A: -fold diluted raw, unpasteurized milk B: Sample A +. mg/l lactulose C:. mg/l carbohydrate standard Peaks: A B mg/l. Galactose Lactose Lactulose --.8 Conclusion Capillary HPAE-PAD and HPAE-PAD are direct, selective and sensitive methods without costly and labor intensive derivitization Modifying the gasket type affects the sensitivity which can be advantageous when analyzing high carbohydrate concentrations An RFIC system with electrolytically generated eluent requires only adding water, thereby eliminating eluent preparation, increasing reliability, stability, and ease-of-use Fast determinations of lactose and lactulose in milk products were demonstrated using a simply 8-min isocratic separation on the Dionex CarboPac SA column References. Thermo Fisher Scientific Technical Note : Determinations of Monosaccharides and Disaccharides in Beverages by Capillary HPAE-PAD. Sunnyvale, CA,. [Online] ication%&%technical%notes/chromatography/ion%chromatography/ic% and%rfic%systems/tn--determination-monosaccharides- Disaccharides-Beverages-TN7-E.pdf (accessed April 7, ).. Thermo Fisher Scientific Technical Note : Fast Determinations of Lactose and Lactulose in Milk Products Using HPAE-PAD. Sunnyvale, CA, [Online] duct%manuals%&%specifications/chromatography/ion%chromatography/ TN_789_Rev.pdf (accessed April 7, ). 8 Thermo Fisher Scientifi c Inc. All rights reserved. ISO is a trademark of the International Standards Organization. All other trademarks are the property of Thermo Fisher Scientifi c Inc. and its subsidiaries. This information is presented as an example of the capabilities of Thermo Fisher Scientifi c Inc. products. It is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. Specifi cations, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details. Africa + Australia Austria Belgium + 7 Brazil + 7 Canada China (free call domestic) 8 Denmark + 7 Europe-Other + Finland France Germany +9 8 India Italy Japan Korea +8 8 Latin America Middle East + Netherlands New Zealand Norway Thermo Fisher Scientifi c, Sunnyvale, CA USA is ISO 9:8 Certifi ed. Russia/CIS + Singapore Sweden Switzerland Taiwan UK/Ireland + USA PN79_E /S