BS EN 14122:2014 pdf free

BS EN 14122:2014 pdf free.Foodstuffs — Determination of vitamin B1 by high performance liquid chromatography.
Thiamin is extracted from food after acid hydrolysis followed by dephosphorylation using an enzymatic treatmentand quantified by HPLC with pre- or post-column derivatization to thiochrome.An external standard is used forquantification.For further information see [1] to [7].
4Reagents
During the analysis, unless otherwise stated, use only reagents of recognized analytical grade and water of at leastgrade 1 according to EN ISO 3696, or double distilled water.
4.1 Methanol, mass fraction w(CH:OH)≥ 99,8 %,HPLC grade.
4.2Acetic acid solution, substance concentration c(CH COOH)= 0,02 mol/1.
4.3lsobutanol, w(C4H1oO)≥ 98 %.
4.4Sodium dihydrogen phosphate, w(NaH2PO4)≥ 99,8%.
4.5 Hydrochloric acid solution, w(HCI)= 36 %.
4.6Hydrochloric acid solution, c(HCI)= 0,1 mol/.
4.7Sulfuric acid solution, c(H,SO4)= 0,05 mol/.
4.8Sodium hydroxide, w(NaOH)≥ 99 %.
4.9Sodium hydroxide solution, mass concentration p(NaOH)= 150 g/l.
4.10 Sodium hydroxide solution, p(NaOH)= 200 g/.
4.11 Potassium hexacyanoferrate lll,w{Ka[Fe(CN)6]≥99 %.
4.12 Potassium hexacyanoferrate lll solution, p{Ka[Fe(CN)6]}= 10 g/l.
4.13 Alkaline potassium hexacyanoferrate l solution (pre-column derivatization), Pp(KIFe(CN)J]}=0,4 g .Dilute 2,0 ml of the potassium hexacyanoferrate lll solution (4.12) to 50 ml with sodium hydroxide solution (4.9)Prepare fresh each day of analysis.
4.14 Alkaline potassium hexacyanoferrate lll solution (post-column derivatization), P(KIFe(CN)J=0.5 glDilute 2,5 ml of the potassium hexacyanoferrate ll solution (4.12) to 50 ml with sodium hydroxide solution (4.10)4.15 Enzyme or enzyme mixture, with the ability to liberate vitamin B; from foods as free thiamin.
NOTE1 For the precision data in Table B.1,Taka-Diastase from Pfaltz and Bauer 1 has been used.For the precision data inTable B.2 and Table B.3 an enzyme mixture of β-amylase from barley and Taka-Diastase from Serva’ have been used.
NOTE2 lf incomplete dephosphorylation occurs, this can be solved by the separate quantification of TMP(Thiamin MonoPhosphate), see [7].
4.16 Sodium acetate solution, c(CH COONa · 3HzO)= 2,5 mol/1.4.17Sodium acetate solution, c(CH COONa· 3HzO)= 0,5 mol/1.4.18 HPLC mobile phases
Examples of appropriate mixtures with volume fractions of e.g.10 % to 50 % methanol (4.1) in water or usingphosphate or acetate buffer are given in Annex A and Annex C. The possibility of using ion pairing agents is alsogiven.
4.19 Phosphate buffer (pH = 3,5),c(KH2PO4)= 9,0 mmol/l.
4.20Tetraethylammoniumchloride, w(CgH2oNCI)≥ 98 %.
4.21Sodium heptanesulfonate, w(CH5NaOgS)≥ 98 %.
4.22Acetate buffer (pH= 4,0),c(CHgCOOH)= 50 mmol/l.4.23Standard substances
4.23.1 Thiamin chloride hydrochloride, w(C12H+zCIN4OS·HCI) 2 99 %.For external calibration, see 6.3.
4.23.2 Thiamin monophosphate chloride, w(Cv2H,zCIN4O4PS)2 98 %.For check of enzymes, see 6.2.2.
4.23.3 Thiamin pyrophosphate chloride (cocarboxylase), w(C12HgCIN4O-PzS)2 98 %.
For check of enzymes, see 6.2.2.4.24 Stock solutions
4.24.1 Thiamin chloride hydrochloride stock solution, p(C12H,zCIN.OS ·HCI) = 0,1 mg/ml.
Dissolve an accurately weighed amount of the thiamin chloride hydrochloride standard substance (4.23.1) in adefined volume of an appropriate solvent, for example 10 mg of vitamin B， standard substance in 100 ml ofhydrochloric acid solution (4.6).This solution can be stored for four weeks at + 4℃.
4.24.2 Thiamin monophosphate stock solution, p(C12H,zCIN4O4PS) ~ 0,1 mg/ml.
Dissove an accurately weighed amount of the thiamin monophosphate chloride (4.23.2) in a defined volume of anappropriate solvent, for example 10 mg of thiamin monophosphate chloride in 100 ml of hydrochloric acid solutio(4.6).This solution can be stored for four weeks at – 20 °℃.
4.24.3 Thiamin pyrophosphate stock solution, p(C12HxgCIN4O-PzS)~ 0,1 mg/ml.
Dissove an accurately weighed amount of the thiamin pyrophosphate chloride (4.23.3) in a defined volume of anappropriate solvent,for example 10 mg of the thiamin pyrophosphate chloride in 100 ml of hydrochloric acidsolution (4.6).BS EN 14122 pdf download.