Application Work AW IC ES6-0004-052016_G Fast HPIC-PAD determination of several carbohydrates on food products with Metrosep Carb 2 Branch Food, stimulants, beverages, flavors Keywords IC PAD 930 919 inositol xylitol sorbitol glucose fructose lactose sucrose maltose, Drinks, Food, Vegetables, Meat, Alcohols, Pulsed Amperometric Detection, PAD-IC, Metrosep Carb 2-150/4.0, branch 7 Summary HPIC-PAD (Pulsed Amperometric Detection) technique for Carbohydrates determination in several matrices was studied. This AW describes the use of Metrosep Carb 2 150/4.0 column in combination with a high concentrated NaOH/ CH3COONa eluent. The use of this eluent eliminates the problem of retention time shifting because of carbonation. Reagents Samples • Sodium hydroxide, puriss. 50 – 52%; Food aqueous extracts of several matrices (chocolate, CAS 1310-73-2, Fluka 72064 cookies, chewing gum) • Sodium Acetate anhydrous >99,0 % CAS 127-09-3; Instruments Sigma-Aldrich S2889 930 Compact IC pro 2.930.2460 • Carbohydrates reagent standards Sigma Aldrich 919 Professional Sample Processor 2.919.0020 Analytical grade IC Amperometric Detector 2.850.9110 • Soda lime p.a., with indicator, pellets; CAS 8006-28-8; Sigma-Aldrich 72073 IC equipment Wall-Jet cell: Carb (Au, Pd) 6.5337.010 • Metrosep Carb 2 150/4.0 6.1090.420 Ultrapure water (UPW) resistivity >18.2 MΩ·cm (25 °C), type I grade (ASTM D1193) from Flex 5 ELGA water Metrosep Carb 2 Guard 6.1090.500 system MagIC Net™ 3.1 Professional 6.6059.242 IC equipment for ultrafiltration 6.5330.110 Solutions Adsorbing tube, large and bent 6.1609.000 Eluent Sodium Hydroxide 300.0 mmol.L-1 Sodium Acetate 1.00 mmol.L-1 CO2 free ultrapure water Rinsing Solutions UPW : Methanol (1:1) Standard solutions Sorbitol and Xylitol from 0.5 to 10 mg.L-1 Mono and di-saccharides from 0.5 to 50 mg.L-1 Sample preparation An aqueous extraction of every matrix was performed. For example 1 to 5 g of sample was diluted with 50 to 200 mL of UPW (depending on sample matrix). Heating slightly (<50ºC) Page 1 of 12 Application Work AW IC ES6-0004-052016_G Fast HPIC-PAD determination of several carbohydrates on food products with Metrosep Carb 2 and stirred for 30 min. Extracting solution was centrifuged to separate solids. Depending of sample concentration an additional dilution was done before transferring to sample Metrosep Carb 2 showed a high capacity, stability and vials. robustness working with the high concentrate eluent. Nevertheless, is advisable to rinse high pressure pump with Analysis UPW to avoid pump problems (almost once a week when Every solution was filtrated with 0.2 um inline ultrafiltration previously to injection. working continuously) Eluent Flow 0.5 mL.min-1 In Appendix are showed some chromatograms of several determinations and a calibration example of poli-alcohols and Detector Mode PAD (3 pulse steps) carbohydrates. E1 0.05 V References E2 0.55 V [1] Determination of Lactose in Lactose-free Yogurt, with E3 -0.10 V Pulsed Amperometric Detection Ion Chromatography (PAD-T1 300 ms IC). AW IC ES6-0003-092013_G Gomensoro IC team SPAIN T2 50 ms [2] Application Work AW IC CH6-1105-082012, Sugar T3 200 ms analysis in lactose free products using the amperometric Measurement duration 100 ms detector and Metrosep Carb 1 - 150/4.0 Metrohm AG, Range 200 μA Herisau Channel measurement Current (A) [3] AN-P-046 Lactose in lactose-free dairy products applying Col. Oven temperature 30 °C pulsed amperometric detection after Inline Dialysis, Metrohm Detector temperature AG 35 °C [4] Application Work AW IC CH6-1278-042016 Determination Sample Loop 20 uL of 5 sugars with a highly concentrated eluent on a Metrosep Carb 2 - 250/4.0 Metrohm AG Calculation [5] Application Work AW IC CH6-1241-052015 Automatic integration using peak areas evaluated with MagIC Sugar analysis using amperometric detection and Metrosep NetTM 3.1 Carb 2 150/4.0 Metrohm AG Results Date See appendix with chromatograms below 18th May 2016 Comments In this study was found the combination of high concentration NaOH for eluent with the Metrosep Carb 2 column showed Authors high stability of retention times of carbohydrates almost for 4-GOMENSORO IC TEAM 5 days working with the same eluent bottle. Maria Aguirre It was shown the degassing of water previously to eluent IC Specialist preparation improve the stability of the eluent more days in Javier Espuelas comparison with a non-degassed one. A degassing of 10-15 minutes for a liter of water was assayed with successful Lab Manager / IC Specialist result. Miguel Espinosa It was found the using of plastic bottle (HDPE) for eluent is PM IC preferred to a glass made one due to strong alkaline conditions and certain peak shapes. Using inline ultrafiltration for sample preparation showed 24-48 hours of continuous operability with one 0.2 um filtration membrane (RC) for several matrices such meat and vegetable extracts, honey, candies, juices, pills, and another foods. Sample line cleaning between samples was performed with UPW: Methanol (50:50 %vol.) and UPW. Page 2 of 12 Application Work AW IC ES6-0004-052016_G Fast HPIC-PAD determination of several carbohydrates on food products with Metrosep Carb 2 Appendix Example Chromatograms Fig 1. 10 uL injection of Milk chocolate (diluted 1:4000) Page 3 of 12 Application Work AW IC ES6-0004-052016_G Fast HPIC-PAD determination of several carbohydrates on food products with Metrosep Carb 2 Fig 2. 10 uL injection of Chewing gum (diluted 1:1000) Page 4 of 12 Application Work AW IC ES6-0004-052016_G Fast HPIC-PAD determination of several carbohydrates on food products with Metrosep Carb 2 Fi 3. 20 uL injection of Yogurt (diluted 1:2000) Page 5 of 12 Application Work AW IC ES6-0004-052016_G Fast HPIC-PAD determination of several carbohydrates on food products with Metrosep Carb 2 Fig 4. 20 uL injection of Cookies (diluted 1:2000) Page 6 of 12 Application Work AW IC ES6-0004-052016_G Fast HPIC-PAD determination of several carbohydrates on food products with Metrosep Carb 2 Example of Repeatability Fig 5. Overlay of 10 chromatograms of cookies sample Fig 6. Overlay of 10 chromatograms of yogurt sample Fig 7. Overlay of 10 chromatograms of chocolate sample Page 7 of 12 Application Work AW IC ES6-0004-052016_G Fast HPIC-PAD determination of several carbohydrates on food products with Metrosep Carb 2 Example of Standard Chromatograms 20 uL of injection Fig 8. Standard containing 11 carbohydrates (Poly-alcohols and mono-di saccharides) 10 mg.L-1 Eritritol, Xylitol, Sorbitol, Mannitol, Glucose, Lactitol, Fructose, Maltitol, Lactose, Sucrose, Maltose Fig 9. Standard containing 5 carbohydrates (Mono-di saccharides) 10 mg.L-1 Glucose, Fructose, Lactose, Sucrose, Maltose Page 8 of 12 Application Work AW IC ES6-0004-052016_G Fast HPIC-PAD determination of several carbohydrates on food products with Metrosep Carb 2 Fig 10. Standard containing carbohydrates (5 Poly-alcohols) 10 mg.L-1 Eritritol, Xylitol, Sorbitol, Mannitol, Lactitol, Maltitol Page 9 of 12 Application Work AW IC ES6-0004-052016_G Fast HPIC-PAD determination of several carbohydrates on food products with Metrosep Carb 2 Calibration example Page 10 of 12 Application Work AW IC ES6-0004-052016_G Fast HPIC-PAD determination of several carbohydrates on food products with Metrosep Carb 2 Page 11 of 12 Application Work AW IC ES6-0004-052016_G Fast HPIC-PAD determination of several carbohydrates on food products with Metrosep Carb 2 Page 12 of 12