BS ISO 26462:2010 pdf free download Milk — Determination of lactose content — Enzymatic method using difference in pH
Dissolve 30 g of nitric acid (HNOg) with a mass fraction, w(HNOa)= 69 %,30 g of hydrochloric acid (HCl) witha mass fraction, w(HCI)=37%,30 g of sodium fluoride (NaF), and 1 g of octyiphenoxypolyethoxyethanol in a1 000 ml one-mark volumetric flask (5.6).Make up to the mark with water and mix.
The strong regeneration solution can be kept for 1 year if stored in non-corroding material at roomtemperature.
5 Apparatus
Usual laboratory equipment and, in particular,the following.
5.1Analytical balance, capable of weighing to the nearest 1 mg.
5.2Micropipettes, capacity 20 ul, ISO 7550[5], with positive displacement.5.3Water bath, capable of maintaining a temperature of 38 C± 1 °℃.
5.4One-mark volumetric flasks, capacities 100 ml and 1 000 ml, ISO 1042[2] class A.5.5Differential pH apparatus, shown schematically in Figure A.1.
The differential pH apparatus consists of peristaltic pumps to circulate liquids, a mixing chamber, two glasscapillary flow-through electrodes(E1 and E2), and an electronic system for measurement.
5.6One-mark volumetric flasks,capacity 1 000 ml and of material capable of storing the extremelycorrosive strong regenerating solution (4.6).
6 Sampling
Sampling is not part of the method specified in this International Standard. A recommended sampling methodis given in lso 707lIDF 50[1].
lt is important that the laboratory receive a truly representative sample which has not been damaged orchanged during transport or storage.
7 Preparation of test sample
Warm the test sample to 38 C in the water bath (5.3) while mixing.Cool the sample to 20 C, beforepreparing the test portion.
8Procedure
8.1General
Since the various types of differential pH apparatus (5.5) available differ in design and handling, the operatorshall carefully follow the instrument manufacturer’s instructions for setting up, cailibration, and operation of theinstrument. Switch the instrument on and allow its operating conditions to stabilize.
lf the time between two consecutive measurements is 5 min or more, renew the buffer solution (4.1) in themixing chamber of the apparatus.
8.2Blank determination
Using a micropipette (5.2), add 20 ul of glucokinase enzyme solution (4.2.1) into the mixing chamber of thedifferential pH apparatus (5.5).
Dilute the enzyme solution with buffer solution (4.1) to a total volume of 1 200 ul and mix.
Fill the flow-through electrodes, E1 and E2(see Figure A.1), of the pH apparatus (5.5) with the buffer andglucokinase mixture obtained. Measure the offset differential pH,D1, between the two electrodes. Thedifference between the electrodes shall be within 0 mpH ± 150 mpH, where mpH is a milli-pH unit.
Using another micropipette,add 20 ul of β-galactosidase enzyme solution (4.2.2) to the buffer andglucokinase mixture in the mixing chamber and mix. Only fill E2 with the mixture of buffer, glucokinase, andβ-galactosidase.Again, measure the offset differential pH, D2, between the two electrodes.
Calculate the numerical value of the difference in pH for the blank, ADo, using Equation (1):
(1)
where
Dris the numerical value of the differential pH between the electrodes both filled with the buffer and
glucokinase mixture;
Dis the numerical value of the differential pH between electrode E1 filled with the bufferiglucokinase
mixture and electrode E2 with the buffer/glucokinase/3-galactosidase mixture.
The difference, ADo, shall be in the range -20 mpH units to 4 mpH units,while the difference between twoconsecutive differential measurements shall be u 1,o mpH unit.
lf these results are not obtained, check the buffer solution and repeat the above procedure. If the results stilldo not fulfil requirement(s), clean the electrodes (see 8.7) and restart the blank determination specified in thefirst four paragraphs of this subclause.
8.3Calibration
8.3.1 Calibration solution pH difference
Add,with one micropipette (5.2),20 ul of lactose standard solution (4.3) and,with another,20 ul ofglucokinase enzyme solution (4.2.1) to the mixing chamber of the differential pH apparatus (5.5). Dilute withbuffer solution (4.1) to a total volume of 1 200 ul. Fill both E1 and E2 with the mixture of buffer, lactosestandard, and glucokinase obtained.Measure the offset differential pH, D3, between the two electrodes.
Using a micropipette, add 20 ul of β-galactosidase enzyme solution (4.2.2) to the mixture of buffer,lactosestandard, and glucokinase in the mixing chamber and mix.Fill E2 with the mixture of buffer, lactose standard,glucokinase,and β-galactosidase.After completion of the enzymatic reaction, measure the offset differentialpH,D4, between the two electrodes.
Calculate the difference in pH for the calibration solution, △Dc, using Equation (2):