EN 1137-1994 pdf download.Fruit and vegetable juices — Enzymatic determination of citric acid (citrate) content — NADH spectrometric method.
6 Apparatus
Usual laboratory apparatus and, in particular, the following:
6.1 Enzyme test pipettes, graduated along the stem
only, with long ungraduated delivery tip.
6.2 Pipettes. with accuracy equivalent to 6.1
(alternative to 6.1) e.g. positive displacement
capillary pipettes.
6.3 Cuvettes, made of glass or plastic, with 10 mm optical path length, and which do not have significant absorption at 334 mm, 340 mm or 365 nm.
6.4 Spectral-line photometer, with mercury lamp and filters for measuring at 334 nm or at 365 nrn.
6.5 Spectrometer, (variable wavelength) for measuring at 340 nm (alternative to 6.4).
7 Procedure
7.1 Preparation of the test sample Normally products shall not be pretreated and their analysis by this method shall be on a volumetric basis, results being expressed per litre of sample. The analysis of concentrated products may also be carried out on a volumetric basis, after dilution to a known relative density. In this case, the relative density shall be indicated. Based on a weighed sample and taking the dilution factor for analysis into account, the results may also be expressed per kilogram of Product. In products with high viscosity andlor very high content of cells (for example pulp), determination on the basis of a weighed test sample is the usual procedure.
Dilute the sample to be examined so that the citric acid concentration is between 0,02 gIl and 0,4 gIl. This solution is used directly for the determination, even if it is coloured. Mix cloudy juices well and dilute them; they, and also very strongly coloured juices, may need to be diluted beyond that required by the citrate content.
7.2 Test procedure
7.2.1 General
The determination shall normally be carried out at constant temperature, between 20°C and 25°C. A constant temperature in the range 25 °C to 37°C may also be used, providing equivalent results are obtained.
The absorption maximum of NADH is at 340 nm. When using a variable wavelength spectrometer. measure at the absorption maximum only. When using a mercury vapour lamp, spectral-line photometer. measure at a wavelength of 334 nm or 365 nm.
Do not use single-mark transfer pipettes for pipetting the solutions. Solutions of enzyme. coenzyme and buffer may be added from suitable automatic pipettes. Enzyme test pipettes (6.1) or their equivalent (6.2) shall be used for pipetting the sample solution.
The determination may also be carried out using a commercially available test-combination kit.
If the substance to be determined is available in a suitably iure form, it is recommended to include it as a standard solution.
7.2.2 Blank test solution Pipette into cuvette 1,00 ml buffer
solution (5.2). 0,1 ml NADH solution (5.3), 2.00 ml water and 0.02 ml MDHILDH enzyme suspension (5.4). Mix, read the absorbance (A1)Blank of the solution against air (no cuvette in light path) after approximately 5 mm.
7.2.3 Test sample solution
Pipette into cuvette 1,00 ml buffer
solution (5.2), 0,1 ml NADH solution (5.3), 0,20 ml of the test sample, 1,80 ml water and 0,02 ml MDH/LDH enzyme suspension (5.4). Mix, read the absorbance Ai)mpic of the solution against air (no cuvette in light path) after approximately 5 mm.
If the initial extinction value is too high (A1 > 1,000) start a new determination, beginning at 7.2.2 and using a reduced concentration of NADH. The capacity of the test is affected by this measure, so that the concentration range of the test sample shall also be reduced.
If the citrate concentration of the samule solution is less than 0,02 gIl, the sample volume to be pipetted into the cuvette can be increased to as much as 2,0 ml. In this case, the volume of water to be added is correspondingly reduced so that both blank and sample cuvettes contain the same total volume. The volume of sample solution (V2) in the calculation (see clause 8) shall then be altered accordingly.
7.2.4 Enzyme reaction and quantification Start the reaction by the addition of 0,02 ml CL enzyme suspension (5.5) each of the solutions 7.2.2 and 7.2.3. Mix, and after the reaction is complete (about 5 mm to 10 mm) read the absorbance (A7) of the solutions against air (no cuvette in light path). Check the completion of reaction by reading after a further 2 Enin.EN 1137-1994 pdf download.