Enzymatic method with gravimetric correction?

At the ICUMSA meeting in Atlanta (2004) a method GS4-6 entitled "The Determination of the Apparent Total Sucrose coming from Sucrose, Glucose and Fructose in Molasses by an Enzymatic Method" was presented as a draft. An initiative to use an enzymatic method as an alternative to "copper methods" is recognizable from this draft. If main components, such as Sucrose or "Apparent Sucrose" (AS), are analyzed by an enzymatic method in a usual way, the results may be disappointing. The total variation of results is mainly determined by  a) the variation of the sample volume,  b) the total volume in the cuvette and  c) the photometer reading. Some minor variations have to be added. I predict a disappointing result ("Reproducibility") of a collaborative test, if normal enzymatic pipettes will be used and no other precautions will be taken. The "Reproducibility" determines the tolerable difference between corresponding results from two laboratories. If results, differing up to 1 % AS, are regarded as equal, the preconditions for an enzymatic determination are as follows: R = 1 % AS;   sR= 0.36 % AS        assumption of 40 % AS in the sample RSDR = 0.63 % (on mean). It will be difficult to get an RSDR (C.V.R) ≤ 0.63 % with a lot of participating laboratories, since a repeatability figure of RSDr = 1.6 % was found by Allan and Bush with a standard pipetting technique [Proc. Austral. Soc. Sug. Cane Technol., 1983, pp.195-198, Table III]. In the ICUMSA Montreal Report (1978) a poor repeatability standard deviation, found for an enzymatic sucrose method (sr = 1.1 % sucrose), was discussed by John Dutton (pp. 104-105) and studies for improvement were recommended (p. 120). Without any changes a collaborative test for AS will again end up with a disappointing result. In a paper published in 1982 it was possible to get a value of RSDr = 0.38 % with a modified enzymatic method, but nobody picked up this variant, based on a special pipetting technique. The question arises if it is possible to lower the error of a standard pipetting technique by a gravimetric correction via analytical balance (resolution 0.1 mg). ICUMSA already has a polarimetric method GS2/3-1 with gravimetric correction, which has successfully passed a collaborative test. Why not try to introduce a gravimetric correction for an enzymatic method instead of using unconventional pipettes?

The volume of sample solution v = weight1/density1. The deviation from v = weight1 is negligible. Density1 is a constant value near 1.0000, dependent on the pre-dilution of the sample (0.9987 at 20°C for 0.05 % D.M. in the sample solution). The total volume V (mL) is calculated as follows: V = v + 0.1 + weight2/density2 + 0.02 + 0.02. A mean value of density2 with sufficient accuracy could be determined with an accurate 10 mL pipette and an analytical balance. For determination of AS a relative method with sucrose calibration is strongly recommended and therefore a variation of density2 is less important. β-fructosidase+buffer1 (0.1 mL) was not included to weight2, because this would cause a further complication of the procedure. The variation of this small additional volume has low influence on the variation of the total volume. It is possible to check a possible advantage of a gravimetric correction without any enzymes, if water and potassium dichromate solution are used, similar to the paper published in 1982.

2004-09-23       G. Pollach