5.1. Preparation of standard VC solution (solution A)
5.1.1. Concentrated standard VC solution at approximately 2 000 mg/kg
Accurately weigh to the nearest 0.1 mg a suitable glass vessel and place in it a quantity (e.g. 50 ml) of DMA (3.2). Re-weigh. Add to the DMA a quantity (e.g. 0.1 g) of VC (3.1) in liquid or gas form, injecting it slowly onto the DMA. The VC may also be added by bubbling it into the DMA, provided that a device is used which will prevent loss of DMA. Reweigh to the nearest 0.1 mg. Wait two hours to allow equilibrium to be attained. If an internal standard is to be employed, add internal standard so that the concentration of the internal standard in the concentrated standard VC solution is the same as in the internal standard solution prepared under point 3.3. Keep the standard solution in a refrigerator.
5.1.2. Preparation of dilute standard VC solution
Take a weighed amount of concentrated standard solution of VC (5.1.1) and dilute, to a known volume or a known weight, with DMA (3.2) or with internal standard solution (3.3). The concentration of the resultant dilute standard solution (solution A) is expressed as mg/litre or mg/kg respectively.
5.1.3. Preparation of the response curve with solution A
NB:
The curve must comprise at least seven pairs of points.
The repeatability of the responses(1) must be lower than 0.002 mg VC/litre or kg of DMA.
The curve must be calculated from these points by the least squares technique, i.e., the regression line must be calculated using the following equation:
y = a1x + ao
where:
and:
where:
=
the height or area of peaks in any single determination;
=
the corresponding concentration on the regression line;
=
number of determinations carried out (n ≥ 14).
The curve must be linear, i.e., the standard deviation (s) of the differences between the measured responses (yi) and the corresponding value of the responses calculated from the regression line (zi) divided by the mean value (y) of all the measured responses shall not exceed 0·07.
This shall be calculated from:
where:
and:
where:
=
each individual measured response;
=
the corresponding value of the response (yi) on the calculated regression line;
=
≥ 14.
Prepare two series of at least seven phials (4.4). Add to each phial volumes of dilute standard VC solution (5.1.2) and DMA (3.2) or internal standard solution in DMA (3.3) such that the final VC concentration of the duplicate solutions will be approximately equal to 0, 0·005, 0·010, 0·020, 0·030, 0·040, 0·050, etc., mg/litre or mg/kg of DMA and that each phial contains the same total volume of solution. The quantity of dilute standard VC solution (5.1.2) must be such that the ratio between the total volume (μl) of added VC solution and quantity (g or ml) of DMA, or internal standard solution (3.3) does not exceed five. Seal the phials and proceed as described under points 5.4.2, 5.4.3 and 5.4.5. Construct a graph in which the ordinate values show the areas (or heights) of the VC peaks of the duplicate solutions, or the ratio of these areas (or heights) to those of the relevant internal standard peaks, and the abscissa values show the VC concentrations of the duplicate solutions.
See recommendation ISO DIS 5725: 1977.