Determination of Acetyl in Pectin and in Acetylated Carbohydrate Polymers Hydroxamic Acid Reaction E. A. McCOMB and R. M. McCREADY Western Utilization Research Branch, Agricultural Research Service, United States Department of Agriculture, Albany 7 0, Calif.
,The reaction of the ester groups in pectin with alkaline hydroxylamine at room temperature produces hydroxamic acids. Pectin hydroxamic acid forms with ferric ions an insoluble complex and acetohydroxamic acid, produced from secondary acetyl groups of pectin, forms a soluble red complex. These reactions, applied to pectic substances, serve as the basis for a specific and rapid colorimetric method for the determination of up to 450 y of acetyl per sample, with an accuracy within about =t2%. These reactions apply also to the quantitative determination of acetyl in acetylated carbohydrate polymers.
S
controversy exists about the presence of acetyl in some pectic substances but it seems now to be generally accepted that pectin from sugar beets (20) and from some fruits (17) may contain about 4% acetyl. A chemical characterization of pectic substances from a particular source material requires a determination of acetyl as well as the determination of the methoxyl content and molecular weight. Several methods have been described for the determination of acetyl esters in pectin (9, 16, 19); however, no single method is both rapid and specific, yet sensitive enough to determine micro amounts of acetyl in milligram quantities of pectic substances. The reaction between esters and hydroxylamine to produce hydroxamic acids has been applied successfully for the analyses of many types of esters (2, 4, 8, lG-14, 24), lactones ( I ) , amides, (2,3, d I ) , anhydrides (6, 7 ) , and nitriles (23). The reaction of the ester groups of pectin with hydroxylamine a t room temperature produces hydroxamic acids. Pectin hydroxamic acid forms with ferric iron an insoluble red complex (18), whereas the acetohydroxamic acid produced from secondary acetyl groups forms a soluble red iron complex. The present work describes the colorimetric determination of 2 to 10 microOME
r
+ 2 n CH,CONHGH i n C H P H
H
Acetylated Pectin
HO
Pectin Hydroxamic Acid
Acetohydroxa mic Acid
500 ml. with chilled reagent grade absolute methanol. Glucose Pentaacetate Standard. Weigh 108.9 mg. of pure crystalline @-D-glucose pentaacetate, dissolve by heating gently in about 5 ml. of ethyl alcohol, and make to 100 ml. with water. Take 2-, 4-, 5-, and 7-ml. aliquots of this solution and make to 50 ml. v i t h water. Five-milliliter aliquots of these diluted aqueous solutions represent 120, 240, 300, and 420 y of acetyl. Prepare a blank using 5 ml. of water in place of REAGENTS the standard solution. Use this blank to set the colorimeter at 100~otransSodium Hydroxide. Dissolve 9.4 mittance. Prepare a standard curve by grams of reagent grade sodium hyplotting the galvanometer readings obdroxide in 100 ml. of water. tained against the micrograms of acetyl Hydroxylamine Hydrochloride. Dison semilog one-cycle paper. Use the solve 3.75 grams of hydroxylamine standard, which is linear from 0 to 500 hydrochloride in 100 ml. of water. y of acetyl per test, to obtain the Ferric Perchlorate. Dissolve 1.93 grams of ferric chloride (FeCh. 6H20) concentration of acetyl in the samples. in 5 ml. of concentrated hydrochlonc acid, add 5 ml. of 70% perchloric acid, METHOD and evaporate the solution almost to dryness. [Smith (22) states that “abPectic Substances. Pipet accusolute ethyl alcohol may be mixed a t rately 2 ml. of a freshly prepared 1 to ordinary temperatures with 72.5% per1 mixture of sodium hydroxide and chloric acid without the least necessary hydroxylamine solution into a 25-ml. apprehension.’’ Nevertheless normal volumetric flask. T o this mixture add precautions should be used in mixing, with agitation 5.0 ml. of the sample handling, and storing perchloric acidsolution estimated t o contain betn-een containing reagents.] Dilute to 100 100 to 450 y of acetyl. ml. with water for use as a stock solution. After 5 minutes or longer, add 5 ml. This solution, when stored in the reof acid methanol solution and mix the frigerator, is stable for a t least 1 month. solution thoroughly, and then make to Add 8.3 ml. of 70% perchloric acid to volume with the ferric perchlorate 60 ml. of the stock ferric perchlorate solution, adding it in small increments, solution. Cool in ice and make to with thorough mixing after each addi500 ml. with chilled reagent grade tion. After 5 minutes remove the absolute methanol. This solution is precipitated pectin hydroxamic acidstable for a t least 1 week a t room temferric complex by filtering the solution perature. through a Whatman No. 12 filter paper Acid Methanol Solution. Chill 35.2 directly into a colorimeter tube. Determl. of 70’% perchloric acid and make to mine the intensity of the color, using a moles (86 to 430 y) of acetyl per milliliter of test solution in about 20 minutes with an accuracy within about *2%, The variables and limitations of the method indicate that it satisfies the requirements for a specific, sensitive, and rapid colorimetric procedure for acetyl in pectic substances and acetylated carbohydrate polymers.
VOL. 29, NO. 5, MAY 1 9 5 7
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wave length of 520 mu. Prepare a sample blank by adding to 5 ml. of sample in a 25-ml. volumetric flask, 1 nil. of the sodium hydroxide solution. Mix thoroughly and allow to stand 2 to 3 minutes; then add 1 ml. of the hydroxylamine hydrochloride solution and proceed as outlined above. Determine the amount of acetyl in the sample from the standard curve. Acetylated Carbohydrate Polymers. Keigh a sample of acetylated carbohydrate polymer estimated to contain 2 t o 11 mg. of acetyl into a 100-ml. beaker. Add a stirring bar and while stirring on a magnetic stirrer add 25.0 ml. of hydroxylamine solution. Add 25.0 ml. of the sodium hydroxide solution dropwise while stirring for 15 minutes. Continue stirring until the carbohl drate polymer is dissolved. Pipet accurately 2 ml. of t h e solution into a 25-1111. volumetric flask. Add 5 ml. each of water and acid-methanol and mix thoroughly. Make t o volume with t h e ferric perchlorate solution, taking care to add it in small increments and t o mix thoroughly after each addition. After 5 minutes, determine the color as previously described. Variables and Limitations. The reaction of pectin acetate jyith alkaline hydroxylamine is shown below. The analytical possibilities of the hydroxamic acids Ivere introduced by Feigl, Anger, and Frehden (6, 7 ) , and since that time an extensive literature on various aspects of the hydroxamic acidferric ion reaction \vas accumulated. The alkaline hydroxylamine solution of Thompson (24) was employed and the other reagents, with minor changes, are those of Goddu, LeBlanc, and Kright (8). The 66% methanol reaction mixture aids in the precipitation of the insoluble pectin hydroxamic acid-ferric complex, in which condition it is removed from the solution prior to the colorimetric determination of the soluble ferric acetohydroxamic acid complex. Although pectic acid has no methyl ester groups to react with hydroxylamine, its ferric salt is insoluble and is removed from the solution. The reaction time of 5 minutes with the alkaline hydroxylamine is in excess of that required for the carbohydrate acetates that were tested and these results are in agreement with the findings of Hestrin (10). The sample was added to the hydroxylamine to ensure the presence of a n excess of the reagent at all times. Formation of hydroxaniic acids a t a reduced rate was found even in acidic conditions and it was not advisable to prepare a blank by adding reagents in a reverse order. Sodium hydroxide, added initially, de-esterified the material so completely that no esters were available for hydroxamic acid formation. A blank prepared in this manner was included with each sample and ueed to adjust the colorimeter to 100% transmittance. Alcoholic solu-
820
ANALYTICAL CHEMISTRY
tions of acid and ferric perchlorate were used to ensure complete precipitation of the pectin hydroxamic acid-ferric complex formed during the reaction. Poor precision ryas obtained unless the acid solution was added separately and prior to the addition of the ferric perchlorate. The maximum color intensity of the ferric acetohydroxamic acid complex was reached after 5 minutes and remained unchanged for a t least 2 hours. The absorption maximum of this complex, determined 11-ith the Cary recording spectrophotometer, was 520 mp and is in agreement with values reported by others ( 1 2 , 24). An Evelyn colorinieter (520-mp filter) was used for the color readings and in accordance with Beer's law, the color density was directly proportional to the concentration in the range u p to 420 y of acetyl per 5 ml. of test sample. The intensity of the color was found to be insensitive to temperature in the range of 20" to 30" C. The success of the proposed procedure depends upon the complete precipitation of ferric pectate and pectin hydroxamic acid-ferric complex and its removal from the solution. Precipitation of polygrtlacturonide hydroxamic acids under these conditions depends in part upon their molecular weights and degree of substitution. Polygalacturonide hydroxamic acids having a degree of polymerization of 10 (niolecular weight, 2000) are soluble in the reaction medium. Katurally occurring pectic substances have much higher molecular weights and a n order of a degree of polymerization of 100 and above; hence, no naturally occurring polygalacturonide esters are likely to be encountered that m-ould fail to precipitate under these reaction conditions. The soluble ferric acetohydroxamic acid complex was not adsorbed or selectively removed from the reaction mixture by filtration under the conditions described for removing the insoluble material. The reaction conditions and the colorimetric procedure outlined in the proposed method were employed to determine the extent of conversion of glucose pentaacetate to acetohydroxamic acid. Compared with a sample of crystalline acetohydroxnmic acid as a standard, the reaction with p-D-glucose pentaacetate proceeded to an extent of 86%. This value is in agreement with that reported by Thompson (24),who used different reaction conditions. Sorbitol hexaacetate, araban diacetate, starch triacetate, and mannogalactan triacetate react with the alkaline hydroxylamine to a n extent of 86% as in the case with the p-D-glucose pentaacetate. The reaction of acetyl esters of carbohydrates with hydroxylamine in the proposed method apparently proceeds to the same extent regardless
of the nature of the carbohydrate moiety. -4lthough acetohydroxamic acid is a stable crystalline compound, i t seemed preferable to use a standard curve prepared with crystalline p-Dglucose pentaacetate so that no further corrections were required. RESULTS
Various pectins \yere analyzed by the proposed methods and compared with the distillation method of Pippen and coworkers (19) and the results are given in Table I.
Table 1. Values Obtained by Proposed Colorimetric and Distillation Method
Raspberry
0.25 0.24 Apricot 136 1 36 Citrus 0 23 0 25 Strawberry 1.47 1.38 Cherry 0.18 0.17 Sugar beet 2.50 2.50 Synthetic pectin acetate 4.80 4.80
0.53 0.53 2.09 2.09 0 32 0 36 1.92 1.89 ... ...
2.82 2.76 5,45 5 36
Comparison of the colorimetric and distillation values for all of these samples analyzed shows that the distillation procedure gives results higher than the colorimetric method. The percentage difference between the methods varies from 11 to 55%. This indicates that the difference is real and due perhaps to the presence of volatile acids incorporated during the isolation of the pectin or possibly formed during the preliminary saponification procedure before distillation (16). The differences are not consistent and cannot be accounted for by assuming a lower degree of conversion of the pectin acetate to acetohydroxamic acid than for the standard. Such conditions would result in a fairly constant difference between the two methods. Duplicate acetyl analysis of the pectin samples determined by the colorimetric method shows good precision and in view of the known deleterious effects of alkali and other recognized deficiencies of available methods for the analysis of acetyl in pectin, the colorimetric method seems to give reliable results for naturally occurring acetyl in the range up to 450 y per test. Analysis of the P-n-glucose pentaace-
tate standaid, arabinose tetraacetate, and sorbitol heuaacetate by direct saponification and by the proposed method are in good agreement and gave calculated values for these pure compounds. Deterniinatioii of acetyl in acetylated c a r b o h y h t e polymers is shon-n in
Table II. Determination of Acetyl in Acetylated Carbohydrate Polymers
Acetate Glucose penta-
Acetyl, Colori- Saponilnetric fieation Calcd. 57 5 56 5 55.1 55
Table 11. The acetates of guar mannogalactan, araban, starch, and pectin gave results in good agreement nith those obtained by saponification. ACKNOWLEDGMENT
The authors thank Mildred Gee for the preparat’ion of the synthetic pectin acetate, sorbitol hexaacetate, acetohydroxamic acid, and the pectic acid methyl ester of low molecular w i g h t ; Glen F. Bailey for the sDectrophotometric determhation of the absdlption maximum, and S. Floy Bracrlin for drafting the reaction diagram.
1
LITERATURE CITED
Guar triAraban di-
41.3
41.7
39.8
Starch tri-
43.9
43.9
44.8
Pectin di-
37.3 35.3 36.6
33. lb
a Containe carbomethoxyl groups which saponify under same conditions as acetyl. b Calculated on basis of loopc anhydrouronic acid.
Abdel-hker, AT., Smith, F., .J. A n ! . Chena. Soc. 73,5859 (1951). Bauer, F. C., Jr., Hirsch, E. F., Arch. Biochem. 20,242 (1949). Berpmann. F.. ANAL.CHEJI.24. 1367 (r952). Buckles. R. E.. Thelen. C. J.. Ibid.. ‘ 22,676 (1950). (5) Diggle, W. M.,Gage, J. C., Analyst 78,473 (1953). (6) Feigl, F., “Laboratory Manual of Spot Tests,” pp. 186-8, Academic Press, Xew York, 1943. ( 7 ) Feigl, F., Anger, V., Frehden, O., Mikrochemze 15, 9 (1934). (8) Goddu, R. F., LeBlanc, pi. F., Wright, C. l f . , ..iu.sr,. C“E\I. 27, 1251 (1955). ,
I
(9) Hengleln, F. A., Vollmert, B., Makromol. Chem 2 , 77 (1948). (10) Hestrin, S.,J . Bzol. Che/tl. 180, 249 (1949). (11) Hill, U.T., I u n Esc,. ( I H E \ I .i\ir~. , ED.18.317 (1946). (12) Ibid., 19,’932(i947). (13) Kaye, 11. -1. G., Kent, P. II-., J . Chem. Soc. 1953, 79. (14) Keenan, -4.G., Can. Chern. Process Ind. 29, 857 (1945). (15) Icertesz, Z. I., “Pectic S~lhstances,” pp. 80-2, 242-3, Interscience, Kew York, 1951. (16) Kertesz, Z. I., Lavin, 11. I., Food Research 19, 627 (1954). (17) XcCready, R. M.$McComh, E. -I., Ibid., 19, 530 (1954). (18) McCready, R. lI., Reeve, R. LI., J . A g r . Food Chem. 3, 260 (1955). (19) Pippen, E. L., McCready, R. lf., OJvens, H. S., ANAL. Cmar. 22, 1457(19501 (20) Pippen, E. L., McCready, R. &I., Owens, H. S., J . .4m Cheni. Soc. 72,813 (1950). (21) Polya, J. G., Tardew, P. I,,, h s a ~ . CHEK 23. 1036 11951). (22) Smith, G. F., Anal. Chzm. Actu 8, 397 (1953). (23) Soloway, S., Lipschitz, h., . 4 s i ~ . CHEW,24,898 (1952). (24) Thompson, A4.R.. 4ustrulzan J . Scz. Research 3A, 128 (1950).
RECEIVEDfor review June 21, 1956. Accepted October 29, 1956. Mention of manufacturers and commercial products does not implv recommendation by the Department of Agriculture over others of a similar natrirr not mentioned.
CompIexometric Titrations Using Azoxine Indicators JAMES S. FRITZ, WILLIAM J. LANE, and ANN SUTTON BYSTROFF Institute for Atomic Research and Deparfmenf o f Chemistry, Iowa State College, Ames, Iowa
b7 - (1
-
- -
naphthylazo) 8 quinolinol5-sulfonic acid and several related compounds are valuable as metal ion indicators in complexometric titrations. Cadmium, cobalt, copper, lead, nickel, rare earths, thorium, yttrium, and zinc can b e accurately titrated in acid solution with 0.05M (ethylenedinitri1o)tetraacetate. In many cases a small amount of copper must b e present in order for the indicator to function properly. Calcium and magnesium do not interfere if the pH is 5.5 or less. With citrate as a masking agent, zinc and other divalent metals can be titrated in the presence of uranium(VI), thorium, or zirconium. The use of tartrate, fluoride, iodide, or thiourea as masking agents in certain cases also increases the selectivity of the method.
C
which form highly colored metal complexes are widely used as indicators for complexometric titrations of metal ions with (ethylenedinitri1o)tetraacetic acid (EDTA), Eriochrome Black T was the first such indicator to gain great popularity. It has been widely used for the titration of calcium and magnesium n-ith EDTA, as ne11 as for the direct titration of zinc, manganese, cadmium, and lead. It cannot be used for direct titration of metals such as copper, cobalt, and nickel because these metals react irreversibly with the indicator. Slight traces of iron and copper interfere with other titrations in which Eriochrome Black T is employed as the indicator. Other indicators have been proposed for use in the titration of certain of the divalent metals. llureuide is reasonably OMPOUXDS
good for copper and calcium, but is poor for zinc, cobalt, and the like. Pyridylazonaphthol (1) serves as a n indicator for some titrations. I t s chief disadvantage is its slow reaction in acid solution. Pyrocatechol Violet has been proposed as an indicator ( 6 ) , but it does not seem to give a good color change for titration of divalent metals. Zincon ( 5 ) has been suggested as a n indicator for EDTA titrations but information regarding its use is thus far sketchy. Aluminum (and probably several other metal ions) can be determined in an acidic water-acetone solution by adding excess EDTA and back titrating with zinc using dithizone indicator (7‘). This procedure works well if the pH is carefully controlled. This paper is concerned with the analytical applications of a new class of VOL. 29, NO. 5, MAY 1957
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