ICUMSA News n°33 – 1998

Message from The President

Those who have read the just published “History of ICUMSA, the first 100 years” would be well acquainted with the achievements of our distinguished forbears. I find it an inspiration to be re-acquainted with the achievements of such dedicated workers as Alexander Herzfeld, Prinsen Geerligs, Frederick Zerban, Lewis Eynon, George Meade, Frederick Bates and more recently Ferdinand Schneider, to name just a few. As we approach our Centenary meeting, besides attending to the business of the moment we need to discuss our future directions. At the time of my assuming the Presidency in 1986 I valued very much the discussion at Cannes of what we should do next. Most of the things which were put forward as ideas then have now been achieved.

Currently a supplement to the Methods book is now available. It comprises 8 new Methods, one new Specification and Standard, 10 revised Methods and 3 revised Specifications and Standards and 2 rewritten Methods, together with new introductory pages.

It is priced as follows: Supplement only, £20 surface mail or £25 airmail; Supplement in ICUMSA ring binder, £30 surface mail or £35 airmail. Orders or enquiries please to: ICUMSA Publications Dept. Fax +44 1493 751 807; e-mail jdutton@compuserve,com.

I will be stepping down at the end of the Berlin Session and I would think the new President will also value whatever guidance the members can give on future directions. The challenges of the next 100 years will surely be worthy of our talents. With a spirit of international co-operation and 100 years of peace we can hope that the achievements will far exceed those of the first 100 years. The achievements of the last twelve years are due to the generosity of members giving so freely of their ideas, their time and technical skills. I have found the experience very rewarding and I look forward to keeping in touch with members in my forthcoming retirement.

Subject 5: Dry substance

Information on the Karl Fischer ring test

by Prof. G.Vaccari (Italy), Referee

In ICUMSA News No. 25 (September 1977) I reported that it was possible to organise a ring-test of the modern Karl Fischer method for the analysis of cane and beet molasses, as well as cane raw sugar.

At that time the statistical evaluation of the data was not ready. Now, thanks to the invaluable cooperation of Mary An Godshall, Referee for Subject 3 “Method Format, Collaborative Testing and Statistical Treatment of Data”, and of Margaret Nemeth of the Monsanto Company, the necessary statistical calculations have been carried out and it is now possible to draw the relevant conclusions. These conclusions, as well as the collected data, will form an integral part of the Report to be presented in Berlin and has been published in detail in the March issue (pp. 186–192) of Zuckerindustrie. They may be summarized as follows:

Cane and beet molasses: The data showed quite good repeatability and reproducibility, especially for cane molasses; it is suggested that this method is suitable for adoption as an Official method for cane and beet molasses.

Raw sugar: The method is not yet suitable for raw sugar but shows potential for improvements; a Tentative status for raw sugars is therefore proposed with further studies to improve the method for application to such samples.

Progress Report for Subject 2

Oligosaccharides and polysaccharides

by Dr. Klaus Thielecke (Germany), Referee

During the working period there were some changes in the Associate Referees. S. Vercelotti resigned and the following Associate Referees were appointed: C. Bobart, J.V. Hormaza, F.F. van Leeuven, R. Lemmes, H.M. Mochtar, S.P. Phadnis and S.P. Shukla. The Referee hopes to get active support from the new Associate Referees.

The most important points resulting from the last Session relating to this Subject were, in the Referees opinion:

1. The development of enzymatic methods for dextran (and levan).

2. Collaborative testing of enzymatic methods for a -galactosides (raffinose) in beet processing samples.

3. Collaborative testing of anion-chromatographic determination of raffinose in molasses.

Some of the Associate Referees commented on further work in this Subject as requested in the Referee’s circulars. Hormaza showed interest in elucidating the role of oligosaccharides and polysaccharides in cane processing and cane sugar quality. The further development of chromatographic methods, such as the HPAEC method, and of immunological methods seemed important to her and steps in this direction were announced. Some criticism of the FZB dextran method was communicated by Clarke and Godshall.

In the opinion of Clarke and Godshall the enzymatic FZB test has no advantages for cane factory routine control in respect of time, accuracy, precision or cost of analysis. The method is said to contain several complicated steps, and to require numerous reagents. The description of the procedure they did not find fully clear in all details. Clarke recommended the re-evaluation of other enzymatic tests published since the early 1970s. The inventor of the FZB test responded that indeed some minor details of the test could still be optimised (more specific enzyme and colour reagent). But is was the opinion of Čopícová that she could not imagine a simple method for dextran and levan in the presence of sucrose having sufficient accuracy and precision, without certain costs and time. Therefore, not all points of the above criticism of the FZB method seemed relevant to her.

Erdem and Leblebici informed the Referee that there had been frostdamaged beet in Turkey in the 1995/96 campain. They tried to determine dextran using the polarimetric method reported by Jadhav.

However, they failed to get reasonable results because they could not get an exact description of the original procedure with all the necessary details. The dextran haze test with a -amylase and ion exchanger addition was found to be inapplicable, even though they modified the method by changing the quantity of reagents and included barium chloride as the clarifying agent. As a result, the FZB test seemed quite promising to these Turkish colleagues and a test was envisaged.

Schäffler informed the Referee of some supplementary details for the high-performance anion-chromatographic determination of raffinose in beet molasses. This gave sharper results than the HPAEC/PAD version published in 1994, which initially aimed at the quantification of invert sugar and sucrose. Because of the generally low concentration of raffinose, Schäffler proposed separate standard and sample solutions for the quantification of raffinose and sucrose in beet molasses. For calibration of the HPAEC system for raffinose determination Erdem and Süral recommended the raffinose and lactose preparations of Sigma Chemical Corporation.

Collaborative tests were carried out on:

1. The determination of total α-galactosides and raffinose in beet processing samples by an enzymatic method (ICUMSA GS8/1/2/3/4-19, Tentative).

2. The determination of raffinose in beet molasses by HPAEC (modified SMRI method)

After starting the HPAEC test on molasses samples, Morel du Boil proposed to modify some of the method details given by Schäffler in 1994. The main points were to reduce standard concentrations (raffinose, lactose) to the level of actual raffinose concentration in molasses and to raise the eluent alkalinity.

The Referee has received nine valid HPAEC trials and 8 valid enzymatic trials from a total of 10 participating laboratories. For the HPAEC method three of the valid double determinations were identified as outliers. The Horwitz values for the remaining five samples were found between 1.3 and 1.9, the r/R ratio between 0.32 and 0.79. In the opinion of the Referee this method is suited for Official adoption.

On the enzymatic Raffinose method one laboratory produced four double determinations which were Cochran outliers. From the rest of the results Horwitz values from 1.74 to 2.58 and r/R ratios from 0.15 to 0.58 were calculated (5 samples). The acceptance of this method which can easily be used in any laboratory should be discussed with the aim of Official adoption at the Berlin session.

Progress Report for Subject 9:

Chromatographic Techniques for Non-sugars

by Dr. Paul Bourlet (France), Referee

During this Session a collaborative study has been carried out using an HPLC procedure to determine betaine in beet molasses. At first only one kind of cation exchange column (Ca++ form) was to be tested. This method, already in use in the Braunschweig Institute and at IRIS was first proposed to the Associate Referees.

Because it was not possible to find enough laboratories using this type of column, the test was extended to all laboratories willing to take part in the study, in order to see if a more general procedure might be feasible.

The test was at first planned as a preliminary collaborative study, so that only four samples of beet molasses were analysed as blind duplicates. Twelve laboratories participated in the study. Six laboratories used the recommended cation exchange column in the divalent form (Ca++), five laboratories employed a cation exchange column in the monovalent form (three in the sodium form and two in the potassium form) and one laboratory used a completely different type of separation on a direct phase HPLC column with -NH2 bonding.

Initially we intended to plan the study for both beet brei and molasses. Due to lack of time, it was not possible to evaluate a freeze-drying method that would have made it possible to include beet brei in the study, so that only beet molasses samples were tested.

With cation exchange columns in the calcium form, glutamine when present, is eluted near to the peak of betaine and thus makes it difficult to determine betaine. Although glutamine is not present in beet molasses, two procedures were investigated to eliminate this compound in beet brei and thin juice. The first was proposed by Thielecke. De-amination of glutamine is achieved with L-asparaginase. The second, used in the Referees laboratory, carries out deamination using formaldehyde. Both methods take less than one hour to complete.

Turning to the preliminary collaborative study, the raw data were statistically evaluated by the Referee for Subject 3. Mary An Godshall stated that there was no difference between results obtained with the columns in the divalent calcium form and the other columns tested in the study. The results obtained from the six laboratories using the recommended column were scattered throughout the data set for all laboratories and did not segregate into one group, which would have happened if there had been a difference between the columns.

The results are thus acceptable for a collaborative study and the different types of columns can be expected to give equivalent results. Only four samples were used in the study, however. Nonetheless, because the results were quite good and because blind duplicates were used, thus obtaining a true measure of within laboratory repeatability, she concluded that this study had met the goals of a successful collaborative study.

A small drawback concerns the quite narrow concentration range studied (5–7 %). However, it is the opinion of the Referee that the method will perform as well at lower concentrations.

This method will therefore be presented for adoption during the forthcoming ICUMSA Session.

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