EN ISO 22184:2021 download.Milk and milk products Determination of the sugar contents High performance anion exchange chromatography with pulsed amperometric detection method (HPAEC-PAD).
1 Scope
EN ISO 22184 specifies the quantitative liquid chromatographic determination of specific sugars (galactose, glucose, fructose, sucrose, lactose and maltose) in various milk and milk products, applying arabinose as an internal standard.
The method is applicable to the following dairy matrices: milk, sweetened condensed milk, milk powder, cheese, whey powder, infant formula, milk dessert and yoghurt.
The method does not apply to dairy products containing soy or to the determination of the lactose content in low-lactose milk products at levels below 1 mg/g.
A high performance anion exchange chromatography method in combination with pulsed amperometric detection (HPAEC-PAD) method is appliedl5llll4J. With this method, thirteen different monosaccharides, disaccharides and trisaccharides can be separated: fucose, arabinose, galactose, glucose, fructose, sucrose, lactose, lactulose, maltose, melibiose, trehalose, isomaltulose and maltotriose.
The method is applicable to labelling for the six most important sugars that can be present by nature or by addition in milk and milk products. The method does not apply to sugar contents less than 0,1 %.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content constitutes requirements of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies.
ISO 3696, Water for analytical laboratory use — Spec,fi cation and test methods
3 Terms and definitions
No terms and definitions are listed in this document.
ISO and IEC maintain terminological databases for use in standardization at the oIlowing aOclresses:
S Reagents
Use only reagents of recogrnzed analytical grade and water in accordance with ISO 3696. unless otherwise specified.
5.1 Water, conforming to ISO 3696, grade 3 and grade 1.
52 SodIum hydroxide (NaOH) pellets.
5.3 Aqueous sodium hydroxide solution, substance concentration c I mol/l.
Add to a 1 000 ml volumetric (task 40 g ± I g NaOI-l pellets (5.2). dissolve in about 500 ml of water and, after cooling down, dilute with water to the mark and homogenize.
5.4 Sodium hydroxide solution, mass fraction w(NaOH) 33 % in water.
5.5 SodIum hydroxide solution, mass fraction w(NaOH) =50% In water.
The amount of carbonate and mercury in the reagent should be minimized. Do not shake or stir the solution before use. A suitable commercially available carbonate sodium hydroxide solution may also be used.
5.6 Concentrated hydrochloric acid (HCI), mass fraction of 36% to 38% in water.
5.7 Aqueous hydrochloric acid solution. c = 1 mol/L
Add to a 1 000 ml volumetric flask (62) 500 ml of water followed by 83 ml of concentrated HCI (i6) and, after cooling down, dilute with water to the mark and homogenize.
5.8 Acetonitrlle (H PLC quality).
5.9 Acetonitrile in water, a volume fraction of 5% In water.
Add to a 1 000 ml volumetric flask (.6.2) 50 ml of acetonitrile (5.). dilute with water grade 3 to the mark and homogenize.
5.10 Anhydrous sodium acetate (CH3COONa) (H PLC quality).
5.11 Eluent I (El). aqueous solution of sodium acetate (CH3COONa), c. 1.0 mol/l.
Add to a 1 000 ml volumetric flask (6.2) about 800 ml o’ degassed water grade I (eluent 3.5.13) followed by 82,0 g sodium acetate (5.10). Then dilute the aqueous solution with degassed water (eluent 3. 523) to the mark and homogenize. Store the eluent under an inert atmosphere.
5.12 Eluent 2 (EZ), aqueous solution of carbonate free sodium hydroxide (NaOH). c.0.2 mol/l.
Add to a t 000 ml volumetric flask (6.2) about BOO ml of degassed water grade 1 (eluent 3, 5.13) and purge for 15 mm with helium. Add 16.0 g of sodium hydroxide solution (5.5). Then quickly dilute the aqueous solution with degassed waler grade I (eluent 3,5.13) to the mark, immediately close the bottle and homogenize. Store the eluent under an inert atmosphere.
5.13 Eluent 3 (E3), degassed water grade I, stored under an inert atmosphere.
5.14 fluent 4 (E4), aqueous solution of sodium acetate (CH1COONa). c = 0,025 molfl.
Add to a 1 000 ml volumetric flask () about BOO ml oldegassed water grade I (eluent 3,5,13) followed by 2,05 g of sodium acetate (5,10), Then dilute the aqueous solution with degassed water (eluent 3, £13) to the mark and homogenize, Store the eluent under an inert atmosphere.
5.15 Post column reagent, aqueous solution of sodium hydroxide, c = 0,3 mol/I.
Add to a 1 000 ml volumetric flask (6.2) about 800 ml of degassed water grade 1 (eluent 3. 513). Purge for 15 mm with helium. Add 24,0 g of sodium hydrotide solution (5,5) and quickly fill up to the mark with the degassed water grade 1 (eluent 3. &13). Immediately dose the flask and homogenize. Store the post column reagent under an inert atmosphere.
IMPORTANT — It Is extremely important to remove dissolved carbon dioxide from the eluents and post column reagent prior to use and during use to avoid fast reduction in detector sensitivity. The eluents and post column reagent are maintained under an inert gas during use.
5.16 MIxture of a volume ft-action of 95 % of ethanol (with a volume fraction of 96 % ethanol and 4% of water) and a volume fraction of 5% methanol.
5.17 PotassIum hexacyanofcrrale (II) trihydrate, K4Fe(CN)6-3H20.
5,18 Zinc acetate dihydrate, Zn(CI13COO)2-2H20.
5.19 Glacial acetic acid.
5.20 Carrez reagent I.
Weigh 106 g of K,4Fe(CN)b-3H20 (i12) In a 1 000 ml volumetric flask (62). dIssolve In 800 ml of water (5._i) and dilute with water grade 3 to the mark. Store the Carrez reagent tin the refrigerator.
5.21 Carrez reagent II.
Weigh 220 g of Zn(CH3COO)2’2H20 (Sill) in a 1 000 ml volumetric flask (62). dissolve in 800 ml water. add 30 ml of glacial acetic acid (19) and dilute with water grade 3 to the mark. Store the Carrez reagent II in the refrigerator.
IMPORTANT — Do not use Carrez reagent II with zinc sulfate.
522 Buffer solution of piperazlne.N,N’.bis(Z-ethanesulfonlc acid) (PIPES) (c 1.5 mol/l and pH = 6,9).
Add 22,5 g of the PIPES buffer solution to a 100 nil conical flask and add 20 ml of sodium hydroxide solution (5,3). Adjust the pH to pH = 6,9 with NaOl-l 33 % (5,4) in water. Transfer the PIPES buffer solution quantitatively Into a 50 ml calibrated tube and fill up with water grade 3 till 50 ml. The pFI of the obtained buffer solution shall be within the range of 6.8 to 7,0.
5.23 Arabinose.
5.24 Galactose.
5.25 Glucose.
5.26 Fructose.
5.27 Sucrose.
5.28 Lactose.
5.29 Maltose.
5.30 Internal standard stock solution arabinose.
Weigh, to the nearest mg, approximately 7 g of arabinose (5.23) into a 50 ml volumetric flask (6,2). Add about 30 ml of water grade 3 and dissolve the arabinose. Add 2,5 ml of acetonitrile (5), fill up to the mark with water and homogenize the solution.
5.31 Sugar standard stock solution.
Weigh, to the nearest 0,1 mg, approximately 260 mg of the monosaccharides galactose (5.24), glucose (5.25) and fructose (5.26), and approximately 400 mg of the disaccharide sucrose (5.27), lactose (5.28) and maltose (5.29) into a 500 ml volumetric flask (6,2). Add about 200 ml of water grade 3 and dissolve the sugars. Add 25 ml of acetonitrile (5), fill up to the mark with water grade 3 and homogenize the solution.
5.32 Sugar standard solutions for calibration.
Prepare the different dilutions of the sugar calibration standards as specified in Table 1. Mix the specified volumes of the internal standard stock solution arabinose (520) and sugar standard stock solution (531) in a 200 ml volumetric flask, add about 50 ml of water grade 3 and homogenize. Add 10 ml of acetonitrile (5), fill up to the mark with water and homogenize.