Determination of sugars in spirit drinks of viti-vinicultural origin
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1 OIV-MA-BS-11 Detemination of sugas in spiit dinks of viti-vinicultual oigin Type II method Intoduction Spiit dinks of viti-vinicultual oigin may be sweetened by vaious compounds, and in cetain legislations the concentations of sweetene ae subject to minimum o maximum levels. 1. Scope This method is suitable fo the detemination of the glucose, fuctose, and sucose contents of spiit dinks of viti-vinicultual oigin. It is not suitable fo spiit dinks containing daiy poducts o eggs. 2. Nomative efeences ISO 3696:1897 Wates fo analytical use - Specifications and test methods. 3. Pinciple High pefomance liquid chomatogaphy (HPLC) to detemine the glucose, fuctose, and sucose concentations. This method is descibed as an example. It uses an alkylamine stationay phase and diffeential efactomety detection. Othe columns/detectos may be used, fo example anion exchange esins as the stationay phase. 4. eagents and Mateials 4.1 Glucose (CAS ), at least 99 % pue. 4.2 Fuctose (CAS ), at least 99 % pue. OIV-MA-BS-11 :
2 4.3 Sucose (CAS ), at least 99 % pue. 4.4 Pue acetonitile (CAS ) fo HPLC analysis. Acetonitile is a highly flammable liquid. It is toxic by inhalation, in contact with skin and if swallowed. It is iitating to eyes. 4.5 Distilled o deminealised wate, pefeably mico-filteed. 4.6 Solvents (example) The elution solvent is pepaed befoehand by mixing: 75 pats by volume of acetonitile (4.4), 25 pats by volume of distilled o deminealised wate (4.5). Pass helium though at a slow ate fo 5-10 minutes pio to use to degas. If the wate being used has not been mico-filteed, it is advisable to pass the solvent though a filte fo oganic solvents with a poe size less than o equal to 0.45 µm. 4.7 Ethanol, absolute (CAS ). 4.8 Ethanol solution (5 %, v/v). 4.9 Pepaation of stock standad solution (20 g/l) Weigh 2 g each of the sugas to be analysed (4.1 to 4.3), tansfe them without loss to a 100 ml volumetic flask. Adjust to 100 ml with a 5 % vol. alcohol solution (4.8), shake and stoe at aound +4 C. Pepae a new stock solution once a week if necessay Pepaation of woking standad solutions (2.5, 5.0, 7.5, 10.0 and 20.0 g/l) Dilute the stock solution, 20 g/l, (4.9) appopiately with a 5% vol. alcohol solution (4.8) to give five woking standads of 2.5, 5.0, 7.5, 10.0 and 20.0 g/l. Filte with a filte of a poe size less than o equal to 0.45 µm (5.3.). OIV-MA-BS-11 :
3 5. Appaatus and Equipment (as an example - othe systems that povide equivalent pefomance can be used) Standad laboatoy appaatus, A gade volumetic glasswae and, in paticula, the following: 5.1. HPLC system capable of achieving baseline esolution of all of the sugas High-pefomance liquid chomatogaph with a six-way injection valve fitted with a 10 µl loop o any othe device, whethe automatic o manual, fo the eliable injection of mico-volumes Pumping system enabling one to achieve and maintain a constant o pogammed ate of flow with geat pecision Diffeential efactomete Computational integato o ecode, the pefomance of which is compatible with the est of the set-up Pe-column: It is ecommended that a suitable pe-column is attached to the analytical column Column (example): Mateial: stainless steel o glass Intenal diamete: 2-5 mm Length: mm (depending on the packing paticle size), fo example 250 mm if the paticles ae 5 µm in diamete Stationay phase: coss-linked silica with adicals containing the alkylamine functional goup, maximum paticle size 5 µm Chomatogaphy conditions (example): Elution solvent (4.6), flow ate: 1 ml/minute Detection: Diffeential efactomety To make cetain that the detecto is pefectly stable, it may be advisable to switch it on a few hous befoe use. The efeence cell must be filled OIV-MA-BS-11 :
4 with the elution solvent Analytical balance accuate to 0.1 mg Filtation equipment fo small volumes using a 0.45 µm membane. 6. Sample stoage On eceipt, samples ae to be stoed at oom tempeatue pio to analysis. 7. Pocedue 7.1. PAT A: Sample Pepaation Shake the sample Filte the sample though a filte with a poe size less than o equal to 0.45 µm (5.3) PAT B: HPLC Detemination Inject 10 µl of the standad solutions (4.10) and samples (7.1.2.). Pefom the analysis unde suitable chomatogaphy conditions, fo example those descibed above Should any peak of a sample have a geate aea (o height) than the coesponding peak in the most concentated standad, then the sample should be diluted with distilled o deminealised wate and e-analysed. 8. Calculation Compae the two chomatogams obtained fo the standad solution and spiit. Identify the peaks by thei etention times. Measue thei aeas (o heights) to calculate the concentations by the extenal standad method. Take into account any dilutions made to the sample. The final esult by convention is the sum of sucose, glucose, and fuctose, in g/l. 9.. Method pefomance chaacteistics (Pecision) The following data wee obtained in 2000 fom an intenational method- OIV-MA-BS-11 :
5 pefomance study caied out on a vaiety of spiit dinks, following intenationally-ageed pocedues. Key to the tables below: nlt Numbe of laboatoies (2 esults pe laboatoy), nl Numbe of laboatoies to calculate pecision values, epeatability limit epeatability standad deviation SD epeatability standad deviation expessed in % of the level epoducibility limit S epoducibility standad deviation SD epoducibility standad deviation expessed in % of the level PSD SD pedicted with the Howitz fomula Ho Hoat value = SD / PSD 9.1 Glucose SD S SD Ho Liqueu ,4 5,4 1,9 2,1 13 4,8 5,2 1,8 Liqueu ,2 9,7 3,5 3, ,8 9.2 Fuctose SD S SD Ho Liqueu ,2 1,2 1,3 8,5 3,0 3,5 1,2 Liqueu ,6 2,3 2,5 22 7,7 8,3 2,9 OIV-MA-BS-11 :
6 9.3 Sacchaose SD S SD Ho Liqueu ,2 2,4 24 8,7 5,0 1,9 Liqueu ,3 1, ,0 2,1 Liqueu ,0 2, ,1 1,3 Pastis ,2 0,1 0,8 2,2 0,8 7,3 1,9 Ouzo ,1 0,8 3,1 2,6 0,9 3,8 1,1 Kisch ,1 2,2 2,1 12 4,2 4,0 1,4 9.4 Suces totaux SD S SD Ho Liqueu ,7 3,1 0, ,2 1,8 Liqueu ,6 1, ,9 1,3 Liqueu ,0 2, ,1 1,3 Pastis ,4 0,1 1,2 2,2 0,8 7,3 1,8 Ouzo ,1 0,8 3,1 2,6 0,9 3,8 1,1 Kisch ,1 2,2 2,1 12 4,2 4,0 1,4 10. Bibliogaphy. Wittkowski, A. Betand, P. Beeton, C. Guillou, POJECT SMT4-CT , Validation of analytical methods of analysis fo spiit dinks. EPOT NO. 02/09 - WOKSTEAM 10. P. Beeton, S. Hasnip, A. Betand,. Wittkowski, C. Guillou, Analytical methods fo the detemination of spiit dinks, Tends in Analytical Chemisty, Vol. 22, No. 1, 19-25, OIV-MA-BS-11 :
7 OIV-MA-BS-11 :
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