1982_chorn_the direct determination of

8/6/2019 1982_Chorn_The Direct Determination Of

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-. June 1982

THE DIRECT DETERMINATION OF LACTIC ACID IN CANEMOLASSES BY GAS-LIQUID CHROMATOGRAPHYBy T. A. CHORN

Huletts Sugar Limited, Mount EdgecombeAbstract

A method f or the direc t determination of lactic acid inand accu rate. Th e method overcomes the lengthyange separation of lactic acid, currently in use.

e quantification of lactic acid in molasses is impo rtantLactic acid can be formed during pro-of sucrose, glucose and

9 and also by microbiological attack by bacteria5~ 6. Therefore a simple rapid and accurate

timisa tion of sucrose yields.There have been numerous papers devoted to the deter-

s~ 9 lo. At present, at Hulettse separation followed by a colorimetriction. Th e accuracy of the ion-ex chang e method isle and the concentration of lactic acid is reported

Th e purpo se of this investigation was to de termine lacticolasses, (afte r derivatis ation of the lactic acid pre-columns. Various procedures have been published

l2 and these differ frommethod in terms of the sample preparationInitial research into a direct gas chromatographic methodg,13 where they first mention the use of bis (trimethyl-a derivatising agent. Work don e

197614 showedintroduction of trifluoroacetic acid improved thehich vastly improve the precision of this method

:-performing the analysis by the internal standard method.introduction of ace tonitrile as the solvent for th e deri-vatisation step which ensures rapid quantitative derivati-sation and also improves integration of the peaks sinceacetonitrile shows no serious tailing, andperforming the analysis on a capillary column whichhas a greater ability to se parate components as com paredto a packed column.

. All reagents used were of an alytica l grade.(a) Bis (trimethylsilyl) trifluoroacetamide, (BSTFA);Ohio Valley Speciality Chemical Inc.(b) Acetonitrile; BDH Chemicals Ltd.(c) Trifluoroacetic acid; BDH Chemicals Ltd.

(d) Lithium lactate; BDH Chemicals Ltd., and(e) Heptanoic acid; Sigma Chemical Company.

Gas-liquid chromatography was performed on a fusedsilica, wall coated open tubular column, 0,31 mm x15 m coated with SE-54 as stationary hase using a Hew-lett Packard 5840 gas chromatograp! ith flame ioni-sation detection. Samp le injection was in the split mode.3 . Initial Recovery Trial

In order to evaluate whether quantification was possibleby gas-liquid chrom atography , a num ber of syntheticsamples were analysed using the internal standard hep-tanoic acid. Prior to injection the sample requiredderivatisation of the carboxyl and hydroxyl groups. Th egeneral derivatisation procedure is outlined in theAppendix.4 . Results

Two synthetic samples were analysed in duplicate usingfour c alibration standards. Tab le 1 lists the amount oflactic acid added to the samples and the calculatedamoun t of lac tic acid recovered .TABLE 1Analysis of two synthetic lactic acid samples

Samples lA, 1B and 2A, 2B refer to duplicate determi-nations of the same sample. As the agreem ent betweenthe expected and calculated percen tage of lactic acid isexcellent, the quantification of lactic acid in m olasseiappeared feasible provided that no interferencesoccurred.

Sample1A1B2A2B

5 . Analysis of final molasses sanlp les taken from the Dar-null, Felixton and Mount Edgecombe MilisThe general sample preparation procedure adopted forthese molasses samples is outlined in the Appendix. Allthe molasses samples were analysed in duplicate using sixcalibration standard s. Also, a syn thetic sample was pre-pared to provide an external check on the recovery ofthe method . Th e results are listed in Table 2, whichshows excellent agreement on duplicate samples and alsoexcellent recoveries on the synthetic sample.These results show the method to be precise. T o showthat the method is also accurate we decided to spike amolasses samp le with varying amo unts of lactic acidwhich would then give an internal check on the accu-racy of the method. This contrasts with the synthetic

Lactic acid added (%)0,240,240,250,25

Lactic acid calculated (%)


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Proceedings of The Sourh African Sugar Technologists' Association- une 1982s a m ~ l e n T able 2 which is an external check on the TABLE 4acc;racy of the meth,od.

TABLE 2Analysis of final molasses samples

The results are listed in Table 3. Samples FX lA andFXlB refer to the original molasses sample, whereassamples FX2A and FX2B and FX3A and FX3B referto the original molasses sample to which was added 0,03and 0,06 percent by mass of lactic acid respectively.

Analysis of final molasses composites by gas-liquid chromatography(g 1 c) and ion-exchange procedures

Lactic acid calculated (%)-

-Sample-

L ADL BFX AFX BME AME BSynthetic 1ASynthetic 1B

Lactic acid added (%)------0,180,18

TABLE 3Internal accuracy check results by the ion-exchange method. The synthetic sampleswere only used for the analysis by gas-liquid chroma-Sample / Lactic acid added (%) 1 Lactic acid caealculated (%) tography.

Sample

ME AME BEM AEM BAK AAK BFX AFX BDL ADL B

Synthetic 1ASynthetic 1BSynthetic 2ASynthetic 2B

From Table 3 it can be concluded that the method isprecise and accurate since the calculated percentagesagree with the theoretical percentages.

6. Further Quantitative DataAs the method had produced reliable data, it could nowbe used on a routine basis. Lactic acid in molasses wasdetermined for all five Huletts mills and these resultscompared with the present ion-exchange colorimetricmethod in use. The samples used were final molassesmonthly composite samples.The results for December 1981 are listed in Table 4.Samples A and B refer to duplicate determinations foreach mill's sample.

Lactic acid (%)

For gas-liquid chromatography each sample was deter-mined in duplicate whereas they were determined once

BY g l c

able 4 highlights two points :( 1 ) The ion-exchange method consistently overestimateslactic acid in molasses, probably because thismethod is not specific to lactic acid as is an analysisby gas-liquid chromatography, and(2) the excellent agreement between duplicate samplesanalysed by gas-liquid chromatography.Further quantitative data is presented in the Appendix,in Tables 5, 6, 7, and 8.

By ion-exchange

DiscussionClearly the method developed bor the quantification oflactic acid is precise and accurate. Sample preparation timehas been kept to a minimum thus reducing analyst time. Ifthe analysis is performed on an automatic gas chromato-graph with data reduction facilities, then the total analysttime required for a batch of samples (5 here) is approxi-mately three hours. This is a significant improvement onthe ion-exchange procedure currently in use in this labora-tory.Figure 1 shows the chromatogram of a standard preparedtls described. As BSTFA will derivatise all compounds withLactic acid

0,30

0,17Ieptanoic acid 0,360,260,220,320,30Lactic acid added (%)FIGURE I Chromatogram of lactic acid and heptanoic acid.


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of The South African Sugar Technologists' Association- une 1982

Therefore, when analysing

It was shown that the response to varying concentrationsf lactic acid was linear by a plo t ofarea lactic acid mass lactic acidarea heptanoic acid mass heptanoic acid

ConclusionsAccurate quantitative results are possible for samples

- This lower limit can be de-atising larger aliquots of sam ple.Th e use of packed columns is not possible for this ana-s because the analysis required high resolution. For thisTh e choice of solvent was found to be important. Pyri-

Th e analys is of mixed juice is, in my opinion , not feasiblef the much lower concentrationsf lactic acid present. A number of modifications ar e en-d fo r this analysis and this will be the subject of a

AcknowledgementsThe author wishes to thank the following people for their

:S . Rangasamy and M. Archary for preparing samplesfor analysis,J. B. Alexander, D. W. Francis, A. E. Dixon and Miss

P. J. Bennett for their useful comments on thestyle and content of the paper, andStaff of the Analytical Department, Huletts Researchand Development, for providing comparative ana-lyses by the ion-exchange method.

Appendix1. Chromatographic conditions

The following program was used for the the samples. Initialtemperature was 95C for eight minutes, thereafter program-med at 30C per minute to 270C with an analysis cycle of30 minutes. The injection port temperature was 250C andthat of the flame ionisation detector was 270C.2. General derivatisation procedure

All samples were derivatised in the following manner. Eachsample (5 p,l) was dissolved in acetonitrile (0,l ml) and to thissolution was added BSTFA (0,5 ml) and trifluoroacetic acid(50 pl). The resulting mixture was heated at 80C for tenminutes, cooled and chromatographed.3. Sample preparation procedure

Molasses samples were prepared as outlined below. To themolasses sample (10 g) was added heptano ic acid (0,045 g) andwater (5 g). Owing to the insolubility of heptanoic acid, asmall quantity of concentrated sodium hydroxide solution(ca. five drops) was added and the resulting solution mixed.Five p l was withdrawn in duplicate an d derivatised as in (2)above. Three calibration standards plus two synthetic sampleswere also prepared with concentra tions bracketing the molassessamples.

TABLE 5Lactic acid in molasses- eptember,1981

TABLE 6Lactic acid in malaises - October, 1981

Sample

ME AME BEM AEM BAK AAK BFX AFX BDL ADL B

TABLE 7Lactic acid in molasses- November, 1981

Lactic acid (%)

Sample

ME AM E BEM AEM BAK AAK BFX AFX BDL ADL B

-y g l c

0,41

TABLE 8Lactic acid in molasses- January, 1982

By ion-exchange-

0,290,250,450,300,28

Lactic acid (%)

SampleME AME BEM AEM BAK AAK BFX AFX BDL ADL B

BY g I c

0.230,24

By ion-exchange0,290,190,260,270,31

Lactic acid (%)

Sample7 ME AME BEM AEM BAK AAK B

FX AFX BDL ADL B

B y g l c

0,13

Lactic acid (%)BY g l c / By ion-erchange

0,350,270,250,300,28

---By ion-exchange0,310,210,220,290,29


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54 Proceedings of The South African Sugar Technologists' Association - une 19824. Further quantitative data

Data for a l l f i ve Hule t t s Mi l l s for the months Sep tember ,1981, October, 1981, N o v e m b e r , 1 9 8 1 and J a n u a r y , 1982 arelisted in t ab l e s 5, 6 , 7 and 8. Comparisons are made w i t h theion-exchange method.REFERENCES1. Boelhouwer, C., Boon, E. F., Butter, J. A. and Waterman, H. I.(1956). The alkaline decomposition of sucrose. J. Appl. Chem.,6: 310-316.2 Ramaiah, N. A., Agarwal, S. K. D., and Kumar, M. B. (1962).Quantitative investigations on the production of organic acids duringalkaline destruction of reducing sugars - a mechanism for caramelisa-tion. ISSCT Proc 11:932-940.

3. Bichsel, S. E. (1965). The chemical destruction of sucrose, fructoseand glucose in hot alkaline process juices and liquors. J. ASSBT,13(5): 406-414.4. McMaster, L. and Ravno, A. B. (1975). Sucrose loss in diffusionwith reference to themophilic bacteria and lactic acid. SASTAProc 49: 1-4.

5. McMaster, L. and Ravno, A. B. (1934). The occurrence of lacticacid and associated micro-organisms in cane sugar processmg.ISSCT Proc 15: 1-15.6. Tilbury, R. H. (1975). Lactic acid bacteria in beverages and food.Edited by Carr, J. G., Cutting, C. V., and W hiting, G. C . Pg. 177-191.7. Olson, R. F. (1964). A rapid method for the routine factory analysisof lactic acid. J. ASSBT, 13(1): 59-61.8. Kiely, M. and O'Drisceoil, P. (1971). Lactic and volatile acids inmolasses. Int Sugar J, 73: 135-1 39.9. Bruijn, J. and Vanis, M. (1972). The determination of nitrogen freeorganic acids in Natal cane molasses. SASTA Proc 46: 64-67.

10. Oldfield, J. F. T. and Shore, M. (1970). A routine high precisionmethod for the determination of lactic acid. Int Sugar J, 72: 3-4.11. Oldfield, J. F.T., Parslow, R. and Shore, M. (1970). The measure-ment of lactic acid in beet sugar processing. Int Sugar J. , 7 2: 35-40.12. Kiely, M. and O'Drisceoil, P. (1971). Lactic acid and volatileacids in beet sugar manufacture. Int Sugar J , 73: 196-197.13. Reinefeld, Von E., Bliesener, K. -M., Rexilius, L. and Reinefeld, A.(1973). Scnellmethode zur gaschromatographischen milchsaure-bestelling in melassen. Zucker, 26 (4): 186-188.14. Schaffler, K. J., Morel du Boil, P. a nd de Gaye, D. (1976). HulettsResearch and D evelopment Quarterly Reports.

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