Sugar analysis according to the EU Regulation using amperometric detection and Metrosep Carb 2-150/4.0
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1 Sugar analysis according to the EU Regulation using amperometric detection and Metrosep Carb 2-150/4.0 Branch Food and beverages Bottle head Adsorber tube for Dosing Unit Keywords IC; ; 858; Metrosep Carb 2-150/4.0; ; glucose; galactose; lactose; fructose; sucrose; maltose; ; PAD Mode; Inline Ultrafiltration; EU law; Regulation (EU) No 1169/2011; 90/496/EEC-nutrition labelling for foodstuffs; branch 7 Summary The new EU Regulation No. 116/2011 defines the rules for nutrition labelling. The directive should assist action in the area of nutrition education in order to increase awareness of nutrition contents. The declaration for the carbohydrate concentration should be made in g/100 g food. The definition for carbohydrates includes all monosaccharides and disaccharides present in food. Polyols are excluded from this definition and should be declared separately. The Application Work describes the separation and determination of the sugars which are generally present in food: glucose, galactose, fructose, lactose, sucrose and maltose. The sugar determination was made in jam and honey samples. Using the Carb 2 column for analysis it was important to include the sample degasser in the sample path. Otherwise a peak from an oxygen disturbance influenced the lactose determination. Samples Strawberry jam (commercially available) Blackberry jam (homemade) Honey Instruments 930 Compact IC Flex Oven/Deg Professional Sample Processor IC Amperometric Detector IC equipment Wall-Jet cell: Carb (Au, Pd) Metrosep Carb 2-150/ Metrosep Carb 2 Guard/ Metrosep CO3 Trap 1-100/ IC equipment: Inline ultrafiltration PP Eluent bottle, 2L for eluent prep Reagents Sodium hydroxide, NaOH, suprapur 30%, CAS , Fluka ML-F D-(+)-Glucose, C 6H 12O 6, puriss., ACS reagent, anhydrous, 99.0 %(HPLC), CAS , Fluka D-(+)-Galactose, C 6H 12O 6, CAS , Sigma-Aldrich G0625 D-(-)Fructose, C 6H 12O 6, for microbiology 99.0%, CAS , Fluka D-Lactose monohydrate, BioUltra, 99.5%, CAS , Fluka Sucrose, C 12H 22O 11, BioXtra, 99.5%, CAS , Sigma-Aldrich 7903 D-(+)-Maltose monohydrate, C 12H 22O 11 x H 2O, BioXtra, 99.0%, CAS , Sigma-Aldrich M9171 Sodium acetate anhydrous, NaCH 3CO 2, puriss. p.a 99.0%, CAS , Fluka Ultrapure water, resistivity >18 MΩ cm (25 C), type I grade (ASTM D1193) Solutions Eluent c(naoh) = 5 mmol/l sodium hydroxide in carbon dioxide free ultrapure water (resistivity >18 M) + c (NaCH 3CO 2) = 2 mmol/l Page 1 of 8
2 Standard solutions Concentration of glucose, galactose, sucrose, fructose, maltose, and lactose standards in the MagIC Net standard table in [mg/l]. β [mg/l] Sucrose Galactose Glucose Fructose Std Std Std Std Std Std Std β [mg/l] Lactose Maltose Std Std Std Std Std Std Std Sample preparation The jam and honey samples ( g) were weight in 100 ml flasks and then additionally diluted 1:50 in ultrapure water. The sample weight and the last dilution factor were written in the MagIC Net sample table. The final concentration was finally calculated according to formula in the user defined results. The diluted samples were analyzed directly using Inline Ultrafiltration before injection. Analysis The samples and standards were injected with the 858 Professional Sample Processor and Inline Ultrafiltration for sample preparation. Parameters Flow Mode E 1 E 2 E 3 t 1 t 2 t ml/min PAD 0.05 V 0.55 V -0.1 V 300 ms 50 ms 200 ms Measurement duration 100 ms Range 200 µa Channel current Column temperature 40 C Detector temperature 35 C Sample Loop 20 µl Calculation Automatic integration with MagIC Net 3.0 software using peak area. Results N=4, concentration in g/100 g sample Sugar Sucrose Glucose Fructose Maltose Comments Mean conc RSD [%] Mean conc RSD [%] Mean conc RSD [%] Mean conc RSD [%] Strawberry jam Blackberry jam Honey The results show that the commercially produced jam sample contained much more sucrose than the home made jam. The high content of glucose and fructose in the blackberry jam indicates that for this product more fruit was used instead to sweetening it with sucrose. For the eluent preparation, a 30% NaOH solution was used because of lower viscosity of this solution (compared to 50% NaOH). The carbonate from the eluent has to be removed by a carbonate trap column. This avoids additional loading of carbonate onto the column during the daily analysis, which would lead to a strong peak shift. The carbonate trap column was installed between the inline filter and the pulsation absorber. Page 2 of 8
3 A new Carb 2 column was prerinsed with the eluent for 5 h. An older column needs sometimes regeneration. This can be seen when the retention times get shorter as in the shown standard chromatogram. Furthermore, the separation between galactose and glucose can disappear. The column regeneration with a concentrated sodium hydroxide solution removes the carbonate from the column (condition see below). Therefore a used column has to be preconditioned prior to analysis. Column regeneration: Rinse the column, precolumn and the trap column for 13 h with a 300 mmol/l NaOH solution with 1 ml/min at 40 C. The trap column might be regenerated after 2 weeks of usage. Column conditioning: Rinse the columns with the eluent for minimum 12 h to remove the excess of the NaOH. Depending on the sample load, the columns can be regenerated periodically. Date 30 th of April, 2015 Author Gabriele Zierfels Competence Center Ion Chromatography Metrohm International Headquarters Appendix Calibration curves, result chromatograms with peak table. Page 3 of 8
4 Appendix Calibration curves Page 4 of 8
5 Page 5 of 8
6 Page 6 of 8
7 Standard 3 with 1 mg/l sugars Commercially bought strawberry jam sample additionally diluted (1:50); Inline Ultrafiltration Page 7 of 8
8 Homemade blackberry jam sample additionally diluted (1:50); Inline Ultrafiltration Honey sample diluted additionally (1:50); Inline Ultrafiltration Page 8 of 8
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