Identification and Determination of Nonnitrogenous Organic Acids Of Sugar Cane by Partition Chromatography E. I. ROBERTS and
L. F. MARTIN
Sugarcane Products Division, Southern Regional Research Laboratory, N e w Orleans, La.
~ i i elTec8tt.d s by ion exchange ti,r:itnicvit of the juice. Qualitative rvidcnce n-as obtained for the presence of malic, aconitic, citric, glycolic, and possibly glyoxylic acids. Evidence for the presence of glucose-1-phosphate was also obtained. Partition chromatography, used in the present investigation, was even more effective i n establishing the presence of all of these compounds except the glyoxylic acid and glucose-1-phosphate, and in isolating inesacwiir, and fumaric acids. For the study of variations in the composition of sugar cane in i.dation to its growth and processing characteristics it is essential to obtain quantitative data on the concentrations of all of the organic acids present in different varietal samples. The only misting information on the concentration of these acids consists of the approximate values reported by Yoder ( I ; ) for aconitic, malic, and oxalic acids in one sample of juice and the extensive data that have been obtained recently on the aconitic acid content of Louisiana sugar cane (6) by application of the decarboxylation method of Ambler and Roberts ( I ) . Separation of the acids by liitrtition chroniatography on silica gel columns by Marvel and Rands' (8) modification of the procedure developed by Claborn :ind Patterson ( 4 ) and Neish (9) is particularly suitable for this purpose. -4dnptations of these methods provide both the necessary complete quantitative data on composition and adequate (pantities of individual acids for complete purification and the preparation of characteristic dei,ivatives for positive identification. For the latter purpose, the large columns described by Marvel and Rands (8) were employed, using 10 times the volume of silica gel specified for the standard analytical columns. Samples of 3 grams of total juice solids, equivalent to 20 to 25 ml. of ,juice, suffice for a complete quantitative analysis, while t h e :mount of acids obtainable from 30 liters of juice is adequate for. isolation and identification purposes. An important advance ilt the application of the partition method is the direct application of the sample material to the chromatographic column by the techriique of Wise (16) and Bulen, Varner, and Burrell (S),which eliminates the necessity of preliminary concent.rations by extraction i)r ion exchange through which some of the acids may he lost.
ipplication of partition chromatography on silicic acid columns of large capacity has made it possible to isolate and identify all of the hitherto undetected nonnitrogenous organic acids present in sugar cane juices. 'J'hese included mesaconic and fumaric acids as well as succinic acid, which was not adequate13 identified in earlier work. The method also permits more satisfactory confirmation of the malic, oxalic, citric, and glycollic acids, for which there w-as previous eiidence. Aconitic acid is the predominant compound of this class in the juice and the only one that has been studied extensively as a constituent of sugar cane and i LS products. The method of >lariel and Rands employing their small standard colunlns was applied to the analjsis of all of the nonnitrogenous acids present in cane juice solids. The techniqueof appl3ing an acidified, preadborbed solution of the total ljophilized solids directl) to the columns aioids the necessity of preliniinary separations or concentrations through which some of the acids may be lost. This procedure has been used t o analqze the juice of seieral different varieties of cane and provides a ialuable tool h> which studies may be carried out on the effect of variety, conditions of growth, iiiaturit\, and other factors. on the composition and qualit> of the juice for recoier) of sugar.
T
HE only nonnitrogenous organic, acids present in sugar can^ that have been isolated and defiiiitely identified are aconitic
( d ) , Lvhirh is present in relativelj. largc wncent,rations, and malic, oxalic, and syringic acids (10, 22). The methods available to rarlier xorkers were ineffective for the separation of small amount6 of closely related compounds from thv complex mixture of substances in sugar cane juice solids. Shorey (11) reported evidencc for the presence of glycolic acid based upon tests which a1.e now known not' to be specihr for this compound. I t was not, purified nor were characteristic derivatives prepared, and the quantity of acid reportedly obtained must have included aconitic acid which he confused with glyrolic. Winter (16)obtained qualitative tests on a sirupy preparation which led him to believe that succinic acid is present in sugar cane. The more recent isolation of citric acid by Tanabe ( I S ) provides satisfactory evidence of its presence, although an insufficient quantity for complete purification and preparat ion of characteristic derivatives was obtained by processing 55 liters of juice. Neither succinic nor fumaric acids were detected in the preparations obtained from this quantity of juice by the methods used by Tanabe. Earlier investigation of a single sainple of Louisiana cane juice by Yoder ( I ? ) , which provided the onljquantitative estimates of the amounts of malic and oxalic acids, failed to reved any citric acid by methods that should have been adequate for separating and det,erniining it in concentrations of thc order found by Tanabe. Chromatographic, methods Iiroiide the only mean? of investigating this entire group of trace constituents without resorting to esccssivelj- large samples of rane or juice material. Paper chromatography has been applied recently by Wiggins (1 $) for the tentative identification of a numher of thesr acids. t'reliminary separation and concentration of the anion fraction
EXPERIMENTAL
Methods and Apparatus. Thr procedure.: used were essentially those of Marvel and Rands (8). Columns 18 mm. in diameter, packed to a height of approximately 185 mm. with 21) grams of silicic acid as described by those authors, were used for quantitative analysis. Samples containing t,otal amounts of about 0.1 gram, or 1.5 milliequivalents, of acid? n-ere separated c4Tectively on these columns. Cane juice solids were added to the cdumns tiy the procedure of Bulen ef al. (3). Three grams of the inat,erial were dissolved in dilute sulfuric acid and mixed with 5 to ti grams of silicic acid to produce a dry pow-tler that was transferred quantitatively to the top of the column. Columns 55 mm. in dianicter were used for isolation of the acids; they contained 200 grams of silica gel packed to the same height, as the analybical oolumns, 185 mm. Most of thc acids encountered in sugar cane juice are sparingly soluble in chloroform and were dissoIved in but,$ alrohol and diluted with chloroform for application t o the large columns. Air pressure was wed for displacement. applied above the solvent supply in separatory funnels attached to the tops of the columns. The adsorbent used was blallinckrodt silicic acid, 100-mesll, rspecially prepared for chromatographic analysis. Fisher Scientific Co. n-butyl alcohol was used and found to require no correction for titratable acidity. The chloroform was B. R . Elk & Co. C.S.P. grade. PITO difficulty was encounterrd by esteri815
816
ANALYTICAL CHEMISTRY
fication of any of these acids with n-butyl alcohol, which proved more satisfactory than tert-butyl alcohol used in a preliminary experiment , Preparation of Material for Analysis. Samples of fresh, raw sugar cane juice were available from the grinding of individual varieties of cane under commercial conditions in the 2-foot wide mill train of the experimental Audubon sugar factory a t Louisiana State University. Approximately 2 tons of cane were used in each pilot plant milling and processing experiment as described by Keller and Schaffer ( 7 ) and by Guilbeau et al. (6). Two 1liter samples were drawn promptly from the thoroughly mixed, screened, fresh juice and preserved for chromatographic analysis by immediate lyophilization. The lyophilizer had ade uate capacity for drying the frozen juice by ice sublimation in &out 24 hours, from sample tubes 10 cm. in diameter by 30 cm. long, each of which held 1 liter of juice conveniently. The dry juice solids were subsequently transferred to desiccators containing phosphorus pentoxide for preservation and complete desiccation. This procedure yielded 250 to 300 grams of juice solids from each cane sample in the form of a friable powder that was sufficiently homogeneous and from which subsamples of 3 grams were readily weighed for quantitative chromatographic analysis. The larger amounts of acids required for isolation and identification were obtained from juice that had been clarified by the standard commercial procedure of liming, heating, and settling. The cooled, clarified juice was passed through columns of Permutit-S anion exchanger to collect the total organic and inorganic acids, and the cmcentrated acid fraction was recovered by regeneration with sodium hydroxide. Eight liters of regenerate were obtained from 30 gallons of clarified juice. This solution was adjusted to pH 7.2 and concentrated to 800 ml. under vacuum. Acidification with sulfuric acid to p H 1.5 precipitated some inorganic salts that were removed by filtration, and the filtrate x-as used for the qualitative analyses.
acid. The chloroform extract was concentrated t o a small volume and amounts containing approximately 1 gram of the mixture of chloroform soluble acids were applied to the 55 X 185 mm. columns and developed by elution with 1-liter quantities of chloroform-butanol mixtures in which the percentage of butanol was increased in 5% steps from 0 to 50% by volume. The effluent was collected in 100-ml. fractions and 10-ml. aliquots were titrated with 0.01S sodium hydroxide to determine the fractions in which individual acids were eluted. The fractions comprising the displacement band of each acid were combined, the chromatographic solvents were removed completely by evaporation, and appropriate solvents listed in Table 11 were used for crystallization. Choice of solvents was facilitated by the tentative identification of some of the acids afforded by their peak effluent volumes.
lI O2 l
Table 1. Organic Acids Isolated from Preparations of Juice Solids
Acid Syringic Mesaconic Fumaric Succinic Aconitic Glyqolic Malic Citric Oxalic
Peak E B u e n t Volumes, Liters Total anion Chloroform extract' exchange Ether extract of ether-soluble regenerate of regenerate fraction 0.4 0.4 0.5 1.8 1.8 1.6 2.3 2.5 2.6 3.5 3.6 3.7 4.6
5.9 7.8 9.1 >10.0
4.8 5.8
7.8 >10:0
4.5
... 7.8
...
...
400 600 MILLILITERS OF EFFLUENT
200
eo0
1000
Figure 1. Acids Detected 11y Direct Chromatography Table I1 summarizes data on the identification of the acids. The first acid eluted, with a peak effluent volume of 0.4 liter, was found to be syringic acid by its melting point and that of its methyl ester, verified by niiued melting points with authentic specimens. hlicrocombustion analyses give carbon: 51.20, 54.67%; hydrogen: 5.19, 5.10%; calculated €or CrHloOb: carbon, 55.03%, hydrogen, 5.29%. Mesaconic acid, with a peak effluent volume of 1.7 to 1.9 liters, had not been chromatogrammed previously by this method and was finally crystallized from water. Fumaric acid was also crystallized from water, succinic acid was crystallized from acetone, and malic and citric acids were crystallized from ethyl acetate by addition of petroleum ether (Skellysolve). I t was not possible to crystallize the glycolic acid and the quantities of oxalic acid were insufficient for the purpose, so that the identities of these acids are based on effluent volume
Identification of Organic Acids. An aliquot of the concentrated regenerate solids in the form of salts was evaporated to dryness, and 0.3 gram of the salts was applied to an analytical column by the technique of acidification and mixture with silica gel. This preliminary chromatogram gave evidence of the presence of nine different acids, including those with peak effluent volumes corresponding to glycolic, citric, and oxalic acids, as shown in Table I and Figure 1. This is the total number of acids detectable by direct application of juice solids to the columns without intermediate separation or concentration by ion evchange or solvent extraction. The remainder of the 800 ml. of concentrated filtrate containing the acids was extracted ~ i t h Table 11. Identification of ether for 40 hours in a continuous liquid-liquid Peak hfelting Pi. extractor. The ether extract was evaporated to Effluent Crystallization and Nixed Vol., and Melting Pt., dryness and a preliminary chromatogram was Acid Litera Purification O C made of an aliquot of the mixture of free acids. Syringic 0 4 Water '203-294 Mesaconic 1. B Water 189-202 The second column of Table I shows that eight of (Sealed tube) the nine acids were recovered, only citric acid beFumaric 2 0 Waterand 198-200 snbliination (Sealed tube) ing lost through ether extraction. The mixture Succinic 3 . 7 Acetone 186-188 still contained a preponderant quantity of aconitic Aconitic 4 . 5 Water 189-191 acid, most of which was removed so that sufGlycolic" 5.9 hlalic 7 . 8 Etliyl'acetate, 97-100 ficiently large charges of the othei acids could be pet. ether Citiic 9 . 1 Ethylacetate. 148-152 separated by each partition on the large columns. pet. ether This was accomplished by eutracting the dry acid Oxalicn >10.0 mixture with chloroform in a Soxhlet extractor, Identified by effluent rolumes only. discarding the residue which was largely aconitic
Sugar Cane Acids Derivative Slrtliyl ester
F'henacvl ester
hlelting Pt. and Mixed Melting Pt..
c.
83-84
129-31
Plienacyl ester
197-(18
u-Broniophenacyl
209-1 1
eitrl' .. ~~
. . ,
.
. .
...
V O L U M E 2 6 , NO. 5, M A Y 1 9 5 4
817
Table 111. Organic .4cid C o n t e n t s of Dry Juice Solids of Seven Varieties of Sugar C a n e
Acids
44-101b Plant Tr. 0 03 Tr. 0 03 0 02 0 16 0 17 0 04 1 19 1 64
(Samples processed in pilot plant of BAIC a t L.S.U.) Varieties and T y p e a 44-101 44-154 44-154 44-155 44-155 44-184 Stubble Plant Stubble Plant Stubble Plant Tr. Tr. Tr. Tr. Tr. Tr. 0.02 0 03 0.04 0 02 0 02 0 05 Tr. Tr. Tr. Tr. Tr. Tr. 0.03 0.04 0 01 0 06 0 03 0 05 0 05 0 01 0.04 0.03 0 02 0 04 0 12 0 17 0 22 0.25 0.27 0.18 0 20 0.14 0 02 0 21 0 05 0 21 0 05 0.06 0.03 0 os 0 07 0 43 1 . 1 1 0 90 1.35 1,16 1 45 0 70 1 99 1.76 1.60 2 02 123
Syringic Mesaconic Fumaric Succinic Glycolic Malic Citric Oxalicd Aconitic Total Aconitic, b y decarboxylation on clarified juice 1 31 O.Ij8 1 61 1 35 sirup Aconitic, 70total by chromat. 72 62 67 153 a Present in traces of less t h a n 0.01$ is indicated by "Tr." b All C.P. (Canal Point) varieties except S . C o . 310 (Natal-Coimbatore). C C.P. 43-47from 1951 crop samples; all others are 1952 samples. d Value for oxalic acid is only a n approxiination.
-
-
-
only. Citric acid was not extracted by ether and its isolation was effected by evaporating the aqueous residue to a sirup, extracting with acetone, and chromatographing the solids obtained. Analysis of Sugar Cane Juice Solids. The standardized procedure employing the small columns containing 20 grams of silicic acid was applied to the systematic analysis of the solids obtained from the juice of different varieties of cane. Three grams of material were used for each chromatographic analysis and were adequate to give quantitative values for all but fumaric and syringic acids, which are present in the smallest amounts. Solid samples were dissolved in acid and applied to the columns by the procedure described previously. The schedule of development of the chromatograms was the addition of 100-ml. volumes in chloroform-butanol in which the percentages of butanol increased in 5% steps from 0 to 50%. The effluent was collected in approximately 10-ml. fractions with the aid of an automatic fraction collector and the acid content of each fraction was determined by titrating the entire amount with 0.01.V sodium hydroxide, using phenol red as indicator. Curves of titration versus effluent volume were plotted and a summation was made of the number of equivalents of acid in each effluent peak. From the known identities and molecular weights of the acids corresponding to each effluent peak, the percentage by u-eight of the acids in the total dry solids of the juice was calculated. Results of analyses of several sugar cane varieties are assembled in Table 111. Recovery of Acids. Marvel and Rands (8) reported recoveries of 99.3 and 100.7Oj, for succinic and aconitic acids, respectively, by this method. Rulen, Varner, and Burrell ( 3 ) reported recoveries of 95 to 100% for all the acids of this series except formic and oxalir acids, which %-ere recovered to the extent of 85%. The recovriirq of all the organic acids of sugar cane were found to be essentially qiiantitative except oxalic, which was found to be erratic. Sincr the recoveries of syringic and mesaconic acids have not been rrported, these were determined by the addition of known quantities of these acids to a sample of cane juice solution before prrndsorption. The recovery of syringic acid was 100% and that of mesaconic acid u-as 102.3%, as shown in Table IV. RESULTS AND DISCUSSION
Application of partition chromatography has effected the separation of nine nonnitrogenous acids from sugar cane juice. The methods used provide reasonable assurance that these are all of the constituents of this class contained in the Louisiana juice samples examined. Separation of the acids on the large-capacity silica gel columns has made it possible to obtain the acids from relatively small quantities of juice in sufficient amounts for purification and complete identification. Hitherto undetected or unsuspected arid conFtituents isolated by this means are
-
-
1.34
1 26
69
il
-
44-184 Stubble Tr. 0 05 Tr. 0.05 0 09 0.20 0 17 0 14 1 37 2 07
-
1 52 71
1 53
X,Co. 310
Plant
1 71
72
66
Table IV. Acid Syringic Mesaconic Succinic Fumaric Glycolic Citric Malic
48-103 Plant Tr. 0 03 Tr. 0 02 0 02 0 26 0 15 0 07 1 44 1 99
2 22
3.00
1.61
2.09
64
Added, M g . 1.39 0.71 0.64
Total Found, M g . 1.39 1.368 1.18
...
...
...
, . .
...
69
Recovery of Acids
Present in Juice, J l g . Trace 0.625 0.537 ...
...
43-47C Stubble
... ...
... , . . , . .
Recovery, % 100.00 102.30 100.30 95.68 97,80 95.26 98.40
fumaric and mesaconic. The procedure leads in a straightforward manner to the purification and identification of acids, the presence of which was not anticipated and is often difficult in the case of isolation by paper chromatography. Its principal advantage is that, once the identities of the acids have been established, it provides a fairly rapid and reliable procedure for the systematic analysis of very small samples of dry juice solids in studying variations in juice composition. The use of automatic fraction collecting equipment with the small, analytical silica gel chromatographic columns for routine analysis of numerous samples should be no more time-consuming than their examination by paper chromatography, while the order of accuracy of the quantitative results obtained is believed to be far superior. The time required for a quantitative determination of all of the acids is about 12 hours. The necessary quantities of material can be obtained readily and preserved, and direct addition of samples of acidified preadsorbed solutions of the total solids to the analytical columns eliminates errors inherent in the manipulations necessary for applying microgram quantities of previously concentrated acids to papergrams. The larger samples used for the partition method are more accurately representative of the total material analyzed. Some acids of this class may have unusual solubilities or may be either too weakly or strongly ionized to be separated on silicic acid under the conditions used. This may be true of the glyoxyl acid and glucose-1-phosphate detected on the paper chromatograms of Wiggins (14), but it is more probable that the concentrations of these compounds in Louisiana sugar cane a t the stage of maturity a t which it is harvested are so low as to be below the limit of qualitative detection by this method with the maximum sizes of samples employed. The cane used in these experiments was cut 2 to 4 days prior to milling and ground under commercial conditions. Similar considerations apply to the failure of succinic, fumaric, mesaconic, and syringic acids to appear on the papergrams of Wiggins, who analyzed fresher and presumably more mature cane in Barbados. The aconitic acid content of the tropical cane was not determined quantitatively by paper chromatography, but it is known to be much lower in tropical than in Louisiana cane ( 5 ) ,and concentrations of the other acids roughly
818
ANALYTICAL CHEMISTRY
parallel that of aconitic. Somc of them limy pi acticall) disappear when the plant ripens, although this assumption requires vel ification. It is difficult to explain Tanabe’s (IS) failure to isolate succinic or fumaric acids, which were specifically sought in his examination of juice which yielded appreciable quantities of aconitic acid. 308
I
approximately the same in tliffei,ent varieties. The agreement in composition of this organic acid frwtion of the plant cane and the stubble, or second growth cane of variety Canal Point 44-154 is striking. It is more remarkable in view of the fact that the second growth cane was harvested almost a month earlier than the sample of first year plant cane used. This result may be a coincidence and will require checking hy more extensive analyses of plant and stubble cane juicw. Aconitic acid determinations were made directly on the juice samples in the course of other work on the processing of these sugar cane varieties, rather thaii on the same lyophilized juiw solids prepared for chromatographic. analysis. Agreement of the values obt,ained by the decarbosylation method Kith those determined chromat’ographically are good except in the case of variety Canal Point 44-101, in which aconitic acid and all of the other acids are present in the smallest amounts. I n this case the chromatographic value is probably more accurate, as the decarboxylation method is subject to errors of constant magnitude i n the deterniination of small amounts of carbon dioxide and is less reliable than chromatography for analysis of juice samples cont:tining lrss than 1% aconitic acid nn solids. ACKNOWLE1)GMENT
2000
4000
6000
8000
10000
MILLILITERS O F EFFLUENT
Figure 2.
Effect of Chloroform Extraction on EtherExtractable Acids
Data pertaining to the separation of the acids foi, qualitative identification are assembled in Table I, which sho\vs t,he peak effluent volumes as defined by AIarvel and Rands for the frartionP obtained by chromatography of various concentrated preparations of juice solids. The results show that some of the acids may be lost by certain procedures used for preliminary conrentrations : neverthelcss, concentration is necessary so that rharges of practical amounts of less abundant acids may be applied to tho columns without overloading them with the very large e s w s s of aconitic acid present in these juice samples. All nine acids dtBt,ectable by the direct analyticul technique were collected 11y Permutit-S anion exchanger :ind could be separated from t,he regenerate by applying the totul aridified solids mixture dirertly to the chromatographic c:olunin. \Vheri the organic acids x e r o extracted from the acidified regenerate solution with ether, larger charges could be used but citric acid was lost in this step because of its insolubility in ether. Tlir further chloroform extraction of the ether-extractable acids necessary to eliminate the bulk of the aconitic acid resulted in the loss of glycolic and o d i c ac%l!: in addit,ion to the citric acid, as shown in Figure 2. The aqueous solution remaining after ether extraction readily yiplded ritric acid upon concentrat,ion, extract,ion of the sirupy concentrate with acetone, and chromatographic purification. Suffirient quantities were thus obtained for crystallization and preparation of derivatives. Because of their similarity in solubility properties to aconitic acid, the accumulation of glycolic and oxalic acids presented greater difficulty, but both of these have been independently detected previously by other methods. The excellent agreement of the peak effluent volume found for glycolic acid in this work with that determined by lIarvel and Rands provides satisfactory characterhation of this acid, supported by the paper chroni:~tographicidentification of Wiggins. The properties of oxalic acid place it at the limit of dt‘ectivcness of the partition method of itleiitification but the pi’esence of this acid in sugar cane has been fully estnblished by scveral workers using standard analytical procedures (10, 13, I ? ) . The results of analyses of individual varieties of cane aesenihted in Table 111 are preliminary and itre given to indicate the applicability of the method to projected investigations of the composition of sugar cane juice. These limited data show that’ the vxieties differ in the total concentrations of the acids determinrd but that the relative pi,o~iortionsof individual :irids are
The authors are indebted to C. .A. Fort,, B. A. Smith, and I$-,I?. Guilbeau, of the Sugarcane Products DiviBion, for their assistance in preparing the ion exchanger regenerates and lyophilized juices, and to Lawrence E. Brown, of the Analyt~ic.al.Phy,qical Chemical, and Physical Division, for microandyses. LITER i r w E CITED
dmbler, J. A., and Roberts. E . ,l,,. k < r . . CHRX, 19, 877 (1947). Behr, Arno, Ber., 10, 351 (1877). Bulen, W.A., Varner, J. E., an(l 13urrell. 11. C.,.4xu3.CHEM., 2 4 , 1 8 7 (1952).
Claborn, H. V., and Pattewon, W , I., .I. ASROC.Offir. A y r . Chemists, 31, 134 (1948). Fort, C. .4.,Smith, B. -4.,13l:ic.k. (’. I. .. and Xhrtiti 1,. F., Sugar, 47, h-0. 1 0 , 3 3 (1952). Guilbeau, W. F., Black, C. L., atid AIartin, I,. I?., Sicgar J . , 14. S o . 6. 18 (1951). Rei&, A. G., and Schafer, F. C . , Loriisinn!i State Cniv., I h g . Expt. Sta., Bull. 24 (1951). Xlarvel, C. S.,and Rands, 11. I),, ,TI., .I. .4m, f ’ / i v w , .Sot... 72, 2642 (1950). Seish,A. C., Can. .I. R e s e a d t , 27B, 6 ( 1 949). Payen, -4., Compt. rend., 28, 013 (1849). Shorey, E. C., J . Am. Chem. Soc.. 2 1 . 4 5 (1899). Takei, S., and Imaki. T.. H u l l . 1mt. Phus. (”hvm. Ruscawh (Tokvo), 15, 1055 (1936). Tanabe, T., Rept. Il’uirc’nrr Sicyui. E.rpt. Stu. (Formosa). So, 4, 33 (1937). Wiggins, L. F., Intoii. Sirgar I . , 54,324 (1952). Winter, H., 2. V e r . deict. Z i k h r - I r d , 38, 780 (1888). Wise, W. S., Anal!la/.76, 816 (1951). Yoder, P. A , , I d , Ettu. ( ‘ h p n i . , 3 , 640 (1911). R E C E I V Efor D review October 5 , 103X. . h r e p t e d February 10, 1954. h e sented as part of the Yyiiijiosiiilii on Analytical .\lethods before the Di\-isions of hnalytical Chemistry and Carbohydrate Chemistry at the 124th XIeetinr of the .i\rtxxcas CHi;\rrcar. SOCIETT, (’liicago, I l l . , 19.53. Hefrrences to specific 1)rodrirtri of colir~iir’icial inanufarture are for illustration only and do not constitute endorsrrrirnt by the U . 9. Department of Agririittuw of the 1)rodIlcts na~rird.
Automatic Spectrophotometric Titrations-Correction In the article on “Automatic, Spc,ctropliotonietri(: Titr;itinns” [llalnistadt, H. V., and Gohrbandt, I
