BS ISO 27105:2016 pdf download

BS ISO 27105:2016 pdf download.Milk and cheese — Determination of hen’s egg white lysozyme content by high performance liquid chromatography
1 Scope
This International Standard specifies a method for the quantitative determination of hen’s egg white lysozyme content in milk and cheese.
The method is suitable for measuring low levels of hen’s egg white lysozyme with a quantification limit of 10 mg/kg.
2 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
2.1 hen’s egg white lysozyme content
mass fraction of substance determined by the procedure specified
Note 1 to entry: The lysozyme content is expressed as milligram per kilogram.
3 Principle
Casein and denatured whey proteins from milk and cheese are precipitated isoelectrically at pH 4,3 (cheese) or at pH 2,2 (milk). Acid-soluble hen’s egg white lysozyme is then determined by reversed-phase high-performance liquid chromatography (HPLC) and fluorescence detection. The lysozyme peak can be verified by LC/MS (see Annex A).
4 Reagents and reference substances
Use only reagents of recognized analytical grade, unless otherwise specified, and only distilled water or water of equivalent purity.
4.1 Reagents and materials
4.1.1 Sodium chloride solution, c(NaCl) = 1 mol/l.
Dissolve 58,44 g of sodium chloride in 1 l water.
4.1.2 Hydrochloric acid, c(HCl) = 1 mol/l.
Dissolve 4,0 ml of hydrochloric acid with mass fraction 37 % in a 50 ml one-mark volumetric flask.
Dilute to the mark with water.
4.1.3 Sodium hydroxide, c(NaOH) = 1 mol/l.
Dissolve 2,6 ml of sodium hydroxide with a mass fraction of 50 % in a 50 ml one-mark volumetric flask.Dilute to the mark with water.
4.1.4 Trifluoracetic acid (CF 3 COOH), analytical grade.
5 Apparatus
Usual laboratory equipment and, in particular, the following.
5.1 pH-meter, accurate to 0,1 unit.
5.2 Fluted filter, diameter 15 cm 2) .
5.3 Membrane filter, pore size 0,22 μm 3) .
5.4 Balance, capable of weighing to the nearest 100 mg, with a readability of 10 mg.
5.5 Analytical balance, capable of weighing to the nearest 0,1 mg, with a readability of 0,01 mg.
5.6 Magnetic stirrer.
5.7 Homogenizer 4) , capable of spinning at a rotational frequency of 3 000 rpm to 3 500 rpm.
5.8 HPLC equipment.
5.8.1 Gradient pumping system, capable of operating with a speed of 1,0 ml/min.
5.8.2 Manual or automatic injector, capable of injecting amounts of 50 μl.
5.8.3 Column heater, capable of maintaining a column temperature of 45 °C ± 2 °C.
5.8.4 Column, reversed phase 5) , 5 µm, 250 mm × 4,6 mm
6 Sampling
Sampling is not part of the method specified in this International Standard. A recommended sampling method is given in ISO 707|IDF 50. [1] It is important that the laboratory receives a sample, which is representative and has not been damaged or changed during transport or storage.