1975_kort_analysis of final molasses

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Proceedings of The South Afvican Sugar Technologists' Association- unelJuly 1975ANALYSIS FINAL MOLASSES FOR SUCROSE AND POL

By M. KORT,,M. MATIC, P. MELLET and D. NUROKSugar Milling R esearch Institute

AbstractFinal molasses from several factories were analysed forsucrose by isotope dilution, gas chromatography and thechemical method and pol was determined after clarificationwith 10 g of dry basic lead acetate. The results were comparedand it was concluded that, while GC is the method of choice,the chemical method, corrected for kestose, is acceptable forroutine purposes. No information on sucrose content of finalmolasses can be obtained from pol readings.Introduction

As a result of an investigation1 in which several methods forsucrose determination in final molasses proposed by ICU MS A2were compared and after an interlaboratory study on therepr~ducib i l i t y ,~he chemical method (modified MackayResearch Institute Method) was recommended for use inSouth A frica.

Since no correction f or trisaccharides present in molasses isapplied to the results obtained by this method, sucrose inmolasses is always higher th an the value determined by isotopedi1ution.l This and the fact that a correction for suspendedsolids in mixed juice was introduced tw o years ago lead to theanomaly that the calculated undetermined losses sometimeshad negative values. It has therefore been suggested that polrather than sucrose in molasses should be used for factorybalance purposes.This investigation was undertaken in order to test the

validity of the suggestion. For this purpose samples of finalmolasses from six selected factories were analysed th rough outlast season for sucrose by chemical method, isotope dilutionand gas chromatography and pol of these samples was deter-

mined under standardised conditions. In addition, the molasseswere analysed for glucose, fructose, kestose and reducingsugars. The investigation was extended to cover the influencewhich dry basic lead acetate has on polarisation of molasses.Experimental 'Composite weekly samples of final molasses, representingthe third week of every month, were collected from DL, JB,ML, TS , U F and UK . These mills represented a fair selectionon a geographical basis as well as in terms of the RS/ashratios of their molasses. Altogether 51 samples were collectedfrom May, 1974 to January, 1975.

The samples were warmed to 50C and thoroughly mixedbefore subsamples were taken for various analyses.Sucrose by chemical method

The Mackay Research Institute inversion method-smodified by Beams and MacGillivrayl was used.Sucrose by isotope dilution

The m ethod described by Bruijn and Carreyett5 was used.The analysis was done in duplicate and the average valuereported.Sucrose by gas chromatographySucrose as a TMS derivative was determined using acapillary column coated with OV17 as described by N uro k a ndR e a r d ~ n . ~he analysis was don e in duplicate and the averagevalue reported.Po lUnless otherwise stated, 50 g of molasses were made up to250 g with water. A 100 ml aliquot was diluted to 200 ml,

TABLE 1Final molasses analysis for sucrose by chemical method, isotope dilution and gas chromatography and Pol.

Mill / Method / May I June I July 1 August 1 September October November 1 December 1 JanuaryDL . . . . . . . .

M L . . . . . .

PolChem.I.D.G.C.

PolChem.I.D.G.C.23,3028,7927,5726,86

PolChem.I.D.G.C.

28,3033,4631,1529,80

TS . . . . . . . .

PolChem.I.D.G.C.

22,2529,5027,6527,7832,2036,7732,223 4 3 5

PolChem.I.D.G.C.

27,55 1 29,OO32,75 1 33,303 1,04 31,4430,78 1 30,49

PolChem.I.D.G.C.

24,2030,4829,1429 , l l26,8530,9031,0830,65

35,8537,5637,4237,15

26,7032,2930,6430,W

28,5532,5930,3930,12

24,4530,5129,6628,9426,7532,1131,1430,78

35,lO38,3136,5137,33

28,4532,9232,2931,29

29,8533,7131,1831,19

22,3029,2928,8228,1928,lO32,4632,6931,99

34,1537,5036,6436,13

27,7032,4731,7230,68

30,30 1 28,1533,70 32,0331,90 1 30,7131,65 / 30,16

21,0529,6128,2527,1728,8033,5032,6531,82 '

34,6036,5735,9535,86

27,6532,2031,4230,40

27,OS30,9030,2629,74

23,OO.30,3028,8628,2729,8034,30

36,OO39,0837,6737,57

31,0533,4532,2631,52

30,2033,1531,5130,76

28,7031,5330,5629,7126,5030,9230,2129,78

31,0834,78

35,4038,Ol36,6536,44

27,5532,8031,0930,69

32,5033,8732,8932,13

33,3731,7033,88

35,7037,9236,1236,OO

33,9529,5532,2634,0933,97

3 1,6032,9731,8331,5032,8535,0834,4734,15

32,51 1 33,19 31,8831,1928,7531,1529,8729,40


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10 0 Proceedings of Th e South African Sugar Technologists' Association- unelJuly 1975TABLE 2

10 g of dr y basic lead acetate were added and the clarifiedsolution was filtered and polarised using a Saccharomat I.Reducing sugars

These were determined by Lane and Eynon titration usingan aliquot of the sample prepared for analysis of sucrose bythe chemical meth0d.l

glucose andSeptember

1,317,84,11,918,760,607,62,o3,817,430,688,73,82,319,600,8912,29 31,320,110,796 33,71,515,250,616 33 o2 214,62

Glucose and Fructose

fructose,August

1,059,14,91,918,180,528,22,73 O16,920,6610,88 21,323,260,869 66 31,519,990,747,64 21,816,070,587 33,42 215,61

kestose.October--1,468,95,3

1,722,630,908,12,73,O19,200,837,34,21,718,351,298,o% I1,619,420,996 22,72,315,310,765,62,52 212,79

These monosaccharides were converted into oximes beforesilylation and were determined by gas chromatography usinga capillary column coated with OV17 as described by Nurokand Reard0n.O

November1,669,34 62,O23,510,997,8! 26,616,980,866,s2,82,316,661,306 93,71,917,330,9810,O6 21,615J10,725,62,52,213,20

December1,577,13,91 816,650,925,91 o5,913,500,908,44,71 319,OO1,216 84,11,716,931,037,o4,')1,816,360,776 23,41 312,45

reducing sugars,July0,957,64,71,616,210,537,92,92,715,760,6211,39,91 224,OO0,757,96 71 217,360,727,93,92,O14,660,595,63 21 313,51

Mill

DL . . . . . . . .

JB . . . . . . . .

M L . . . . . . . .

TS . . . . . . . .U F . . . . . . . .

U K . . . . . . . .

KestoseThis trisaccharide was determined as a TMS derivative bygas chromatography using a two phase system and conditionsdescribed by N ~ r o k . ~ ~ ~

January1,598,O4 s1 319,060,846 82,23 ,l14,990,697,64,71,618,811,308,44,91,718,740,917,85,O1,519,03

Results and discussion

FinalAnalysisKes.Fruct.Gluc.F / GR.S.Kes.Fruct.Gluc.F / GR.S.Kes.Fruct.Gluc.F I GR.S.Kes.Fruct.Gluc.F I GR.S.Kes.Fruct.Gluc.F / GR.S.Kes.Fruct.Gluc.F I GR.S.

The results of sucrose analysis in molasses by the threemethods as well as the pol determinations are reproduced inTable 1. Reducing sugars, glucose, fructose and kestose contentsof these samples are given in Table 2.Although the reproducibilities of isotope dilution and gaschromatographic methods were very similar (Mean Deviationof 51 pairs of duplicates 0,09 % and 0,08 % respectively),sucrose determined bv the former method was on the average

molasses analysis forMay 1 June

chromatographic analysis of the combined "pure" sucroseresidues on which radioactive count was made. In the trisac-charide region (Fig. 1) kestose (ab out 0,4 %) and small amountsof other unidentified compounds were detected, indicatingthat the steps used in the isotope dilution method for purifica-tion of sucrose are not sufficient to completely eliminateoligosaccharides present in molasses. As any impurity remain-ing in sucrose inflates the results of the isotope dilutionmethod, the sucrose values obtained by this method wereobviously too high. The results obtained by gas chromato-graphy are therefore considered to be more accurate and wereaccepted in this work as true sucrose values.

0,888,63,72,317,070,578,O3,42,416,940,649,57,31,320,380,8010,l7,51,420,31

"0,5% higher. The difference in the results obtained by the two , tc in trisaccharide region of ,.pure..methods was shown by statistical analysis to be significant at residues on which radioactive count was performed inthe 99 % level. The reason fo r this discrepancy was found by gas I.D. method. Conditions as in Ref. 7.

0,916 74 21,615,530,568,43,82 217,740,5611,O12,60,923,940,778 37,11 218,610,667,14,o1,814,050,446,94 21,515,71


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Proceedings of The South A frican Sugar T echnologists' Association- unelJi,Perusal of Table 1 confirms the p revious findings1!111 l4 thatthe chemical method overestimates sucrose in molasses. Thedifferences between the results of this method and sucrose asdetermined by gas chromatography are listed in Table 3.However, as kestose which is present in m olasses also producesreducing sugars upon hydrolysis and will be recorded in thechemical method as sucrose, the results have to be correctedfor kestose content of respective samples and these are given

in Table 2. T he correction fac tor is 1,02. Although th e differencefrom true sucrose becomes considerably smaller after thiscorrection, as is apparent from Table 3, it is not completelyeliminated. This is possibly due to the presence in molassesof other substances which produce reducing sugars onhydrolysis. The mean and standa rd deviation of the differencesfor each mill before and after correction are given in Table 4.TABLE 3

Difference between the chemical method (in bold corrected for kestose) andgas chromatographic method of sucrose determination.

TABLE 4Mean difference of various methods from GC results and standarddeviations.

M a y . . . , .J u n e . . . . .J u l y . . . . .A u g u s t . . . .Se pt e m be r . . ,October . . . .November . . .December . . .January . . . .

The concentration of kestose in molasses, as can be seenfrom Table 2, does not vary much from an average value of0,9 %. In view of this a simple correction by subtracting 1,Ofrom the results of the chemical method will give acceptablesucrose figures for routine purposes. It is therefore suggestedthat this procedure be adopted by mill laboratories, wherespecialised equipment required for kestose analysis is lacking.

UF-0,41-0,260,980,251,370,620,710,lO1,510,501,570,571,920,870,930OO

The difference between pol of molasses when 10 g of leadis used for clarification an d sucrose by G C are listed in T able 5and the mea ns of these differences and standar d deviations inTable 4. As expected pol values are lower than sucrose butthe magnitude of the difference and its fluctuations are sur-prisingly high an d illustrate the g reat influence which impuritiespresent in molasses have on pol reading.

UK-1,1509701,360,761,3103720,930,311,630,851,060,330,980,19

TABLE 5Difference between sucrose by gas chromatography and pol when lo g oflead is used for clarification.

D L3,662,761,971,042,811,842,291,221,630,291,790,301,800,111,930,331,820,20

TS------,940,47------,890,59------,080,99

Difference 1 DL

J B1,931,351,721,151,370,831,571,041,100,492,441,522,031,021,140,202,111,25

M L------,221,570,25-0,321,330,700,47-0,201,680,991,790,941,590,714 , 0 9-1,OO1,070,37JB1,710,460,990,424,691,13

Chem.-GC .Corrected chem .- G C . . .GC-Pol . .

That optically active substances affect polarisation of sugarsolutions is well known8, 9. Glucose and fructose, in view oftheir high concentration in final molasses, are of specialinterest in the context of this investigation. Fructose inparticular, being strongly levorotatory, will profoundly influ-ence pol readings. It is therefore of interest to note (Table 2)that fructose and not glucose is more abundant in molassesand that the amounts of these two monosaccharides as wellas their ratio vary over a wide range. Consequently, thecontribution of these substances to pol of molasses shoulddiffer from one sample to another. No correlation could befound however between either the amounts of reducingsugars, fructose, glucose or the FIG ratio and the differencebetween sucrose and pol as determined in this work.

T S2,471,652,521,732,051,291,870,991,160,252,391,071,740,411,470,241,750,42

ML1,150,780,420,792,870,87

U F1,180,500,330,381,370,59

The influence of lead used for clarification of molasses onvarious reducing sugars has also been studied in the past butthe conclusions reached are somewhat contradictory. Thatlead suppresses levorotation of fructose and under certainconditions can even make fructose dextraro tatory seems to bewell established, but to what extent, if at all, reducing sugars

M a y . . . . .J u n e . . . . .J u l y . . . . .August . . . .September . . .October. . . .November . . .December . . .January . . . .

UK1,200,250,550,260,980,58

MeanSt .Dev .MeanSt .Dev .MeanSt.Dev.

I I I 15 10 20 30 40 50

g dry basic lead acetate per 2 lg of molassesFIGURE 2 Influence of lead used in clarification o n p ol of final molasses

D L1,503,231,493,302,842,982,750,471,01

2,190,660,900,912,171,06

UK-1,301,311,741,320,420,150,60-

JB3,565 3 34,914,495,896,125,273,283,14

ML2,353,804,033,893,022,712,112,271,64

TS------,571,341,352,012,690,564 3 7-0,lO0,65

U F-1,302,231,981,261,571,040,301,30


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102 Proceedingsof The South Afvican Sugar Technologists' Association- unelJuIy 1975are precipitated and removed from solution is not quiteclear8? . There is also no indication of exactly what amountof lead should be used for clarification purposes, "minimumquantity required for good clarification" being usuallyspecified.

The effect of lead on pol in molasses is illustrated in Fig. 2in which the pol readings obtained with molasses from severalfactories are plotted against the amount of lead used forclarification. An almost linear increase in polarisation isobtained with the increase of dry basic lead acetate until aplateau is reached. In Fig. 2 this can represent as much as12 pol units. However, both the slope of the curves and theamount of lead required to reach the plateau vary from onesample to another. It is further apparent that the "minimumamount required" fluctuates in these examples between about3 and 6 g of lead which in terms of pol represents nearlyone unit.

The preliminary results with diluted molasses indicate thatthe quantity of fructose removed during clarification increaseswith the amount of lead used while glucose remains largelyunaffected as shown in Table 6. When dilute acetic acid isadded to filtered molasses solution in order to restore levoro-tation of fructoses> the obtained pol readings are dependenton the amount of lead originally used (Table 7).

TABLE 6Removal of fru ctose and glucose during clarification.

TABLE 7Polarisation of molasses after addition of acetic acid.

Lead used inclarification (g) Found in solution %Fructose 1 Glucose

Conclusions

ml of 20%acetic acidadded

It is apparent from the da ta presented that the G C methodof sucrose determination in final molasses should be themethod of choice. However, as mill laboratories are at presentnot equipped to carry out this analysis, the chemical methodcorrected by a subtraction of one from the obtained sucrosevalues, will give acceptable results for routine purposes. Theresulting error will influence a Factory Balance by about 0,2 %on the average.

Horne's dry lead used for clarification (g)-----1 6 7 / 8 1 1 0

While pol determination o n factory products of high puritymay be a reasonab le substitute for sucrose analysis,12 he resultsof this investigation support the view expressed by Clayton13that "the pol . . . of final molasses is prac tically meaningless".The amount and the composition of optically active impuritiesvary to such an extent in final molasses a nd the interaction oflead with these substances is so complex that there is no wayin which sucrose content can be computed from pol readings.It is further clear that if reproduci~blepol readings are tobe achieved the quantity of lead in the clarification stagemust be specified. Ten grams of dry basic lead acetate as usedin this work are proposed for this purpose. Under thesearbitrary conditions an error in a Factory Balance of between0,3 % and 1,5% can be expected.

AcknowledgementsThe authors wish to thank Mrs M. Wolff and Mr T. J.Reardon for technical assistance and iMr S. Baker of Hulett'sR & D for help with statistical analysis.

References1. Beams, W. F. and MacGillivray, A. W. (1971). SASTA Proc.45,114.2. Stachenko, S. (1970). Proc. 15th Session ICUM SA, 61.3. SM RI Annual Report (1972). 9.4. Proc. 15th Session ICUM SA (1970). 76.5. Bruijn, J. and Carreyett, R. A. (1973). SASTA , Proc. 47, 44.6. Nurok, D. and Reardon, T. J. (1975). SASTA, Proc. 49, in press.7. Nurok, D., J. Chrom . Sci., in press.8. Brown, C. A. and Zerban, F. W. (1941). Physical and ChemicalMethods of Sug ar Analysis, 3rd Ed., W iley, New York.9. Spencer-Meade (1963). Cane Sugar Handbook, 9th Ed., Wiley,New York.10. Lab oratory Man ual for Q ueensland Sugar, Mills, 4th E d. (1961).Bureau of Sugar Experiment Stations, Brlsbane.11. Pommez, P. (1974). Proc. 16th Session IC U M S k (Subjects 7 & 8).In press.12. Mahoney, V. C. and Lucas, P. C. (1971). Int. Sug. J., 73, 291.13. Clayton, J. L. (1971). System of Cane Sugar Factory Control, 3rdEd., ISSCT, 12.14. Schaffler, K . J. and Loker, C. (1974). ISSCT Proc. 15, 1380.

1975_kort_analysis of final molasses

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