Colorimetric Determination of Pectic Substances ELIZABETH A. MCCOMB AND R. M. MCCREADY Western Regional Research Laboratory, Albany, Calif. 7 ROWIKG
interest in the role of pectic substances in controlling texture of fruiti and vegetables during processing and storage has focused attention on simpler methods for their determination. Usually complete characterization of the pectic substances before and after a particular change is necessary. Occasionally, the problem may be resolved by determining the total quantity of pectic substances in solution. Methods that have been uaed include weight of alcohol precipitate ( I ) , titration of acid carbox! Is plus saponification of methyl esters ( 5 ) ,weight of calcium pectate (a), decarboxylation by heating in concentrated mineral acids ( 8 ) , and optical rotation (7'). Some of these methods require considerable purification of the pectic substance, some require drying, some are applicable only to macro amounts, and some are not specific. Stark (9)applied Dische's ( S , 4 )carbazole reaction to determine micro quantities of pectic substances in cotton. A critical study of the variables of this simple quantitative colorimetric method was made. Pectic substances from various sources were analyzed for anhydrouronic acid (AUh) by the modified procedure and the results are compared with those obtained by decarboxylation, titration, and calculation from the calcium contents of calcium pectates. REAGENTS
Ethyl alcohol, purified. Reflux 1 liter of 95% reagent grade ethyl alcohol with 4 grams of zinc dust and 4 ml. of 1 to 1 sulfuric acid for 24 hours. Distill, using glass apparatus. Add 4 grams each of zinc dust and potassium hydroxide to 1 liter of the diptilled alcohol and redistill. Carbazole reagent. Dissolve 0.150 gram of reagent grade carbazole (recrystallized from toluene if necessary) in 100 ml. of purified ethyl alcohol. The solution of carbazole is slow and requires stirring. Sulfuric acid, reagent grade, concentrated. Galacturonic acid monohydrate, reagent grade. Check the purity by titrating 0.5 gram with 0.1 N sodium hydroxide to pH 8.0. The theoretical equivalent weight of the monohydrate is 212.
mined. The curves were identical in shape except for intensities and the absorption maxima in all cases occurred a t wave lengths near 525 mp. This indicates that, regardless of the degree of polymerization of the galacturonide or the presence of methyl ester groups, the color measured by this procedure is the same. Holzman et al. (6) determined the spectra of the color produced when sulfuric acid, hexoses, and carbazole are heated together. The position of the maxima was near 545 mp. Although these absorption maxima are separated by 20 mp, hexose sugars in concentrations of threefold excess interfere slightly with the quantitative uronic acid color reaction under these conditions. An Evelyn photoelectric colorimeter equipped with the 520 mp filter was used for measuring per rent transmittance. Duplicate analyses were always made, and the results reported are average values. Effect of Sulfuric Acid Concentration. To 12 ml. of various concentrations of sulfuric acid were added 2 ml. of solution containing 40 micrograms of galacturonic acid hydrate. The mixture was heated for 10 minutes in a boiling water bath, cooled to 20" C., and 1 ml. of 0.15% carbazole reagent was added. After 25 Ilt 5 minutes a t room temperature (about 25' C.), the values shown in Table I were obtained. The sulfuric acid concentration is most critical and precautions should be taken to measure it accurately and store it in stoppered, dust-free containers. Maximum transmittance was obtained with 12 ml. of 95Vo sulfuric acid to give a final concentration of 87$ sulfuric acid in the mixture before addition of the carbazole reagent.
Table I.
Effect of Sulfuric Acid concentration
Concentration of Sulfuric Acid",
%
82
84
86 87 91
Transmittance,
%
80 63 55 51 51
a Concentration of sulfuric acid in sulfuric acid-galacturonic acid mixture brfore addition of carbazole reagent.
METHOD
De-esterify the sugar-free solution of pectic substance of about 0.1% concentration by holding in 0.05 N sodium hydroxide for 30 minutes a t 25' to 30' C. and dilute to 0.002%. Measure 12.0 ml. of concentrated sulfuric acid into a 25 X 200-mm. culture tube. Cool tube and contents to about 3" C. in an ice bath and add 2 mi. of solution containing 5 to 80 micrograms of de-esterified galacturonide. Cover the mouth of the tube with a 5-ml. beaker and mix the contents thoroughly. Replace thr tube in the ice bath and cool to below 5' C. Heat the tube and contents for 10 minutes in a boiling-water bath. Cool to about 20' C., add 1 ml. of 0.15% carbazole reagent, mix thoroughly, and allow to stand a t room temperature for 25 f 5 minutes Determine the intensity of the color, using light of wave lmgth 520 mp. Analyze the samples in sequence, so that the time and temperature from the addition of the carbazole to the color determination are as reproducible as possible. Plot the log of the transmittance against the concentration over the range of 10 to 60 niicrograms of galacturonic acid hydrate. A straight line results, passing through 90 to 95Y transmittance a t zero concentration. Use a standard curve to obtain the concentration of anhydrouronic acid in the samples, and to control daily variations; include a galacturonic acid hydrate standard with each series. The reproducibility falls within about 27,. VARIABLES AND LIMITATIONS
Absorption Spectrum. The absorption spectrum of the galacturonic acid-carbazole solution was previously examined by Dische (S) and by Stark (9). The absorption spectra for the color produced from galacturonic acid, pectic acid, polygalacturonide methyl ester, and methyl galacturonide methyl ester were deter-
Effect of Time of Heating. Test solutions containing 12 ml. of 95% sulfuric acid and 40 micrograms of galacturonic acid hydrate in 2 ml. of solution were heated in a boiling-water bath for 5 , 10, 20, and 30 minutes, and cooled to 20' C. To each tube n a s added 1 ml. of 0.15y0 carbazole reagent. After 25 f 5 minutes a t room temperature (about 25" C.), the transmittances of the colored solutions were determined. The intermediate compounds required for development of the color with carbazole are formed in less than 5 minutes and are stable for heating periods up to 30 minutes or more. Results with polygalaeturonic acid were similar. This property may be useful in analyzing a reries of samples, since after the heating period, the color can be developed by the addition of carbazole reagent several days later, if desired. Effect of Carbazole Concentration and Time of Color Development. With all other factors constant as described, the amount of carbazole per tube (15-ml. final volume of colored solution) was varied and the course of the development of the color followed. The values appear in Figure 1. With 0.5 mg. of carbazole per test, the color development proceeds for an hour or more and is stahle for another hour or more. Fading of the color is proportionately slow. When 1 mg. of carbazole is used per test, the maximum intensity is reached in 30 minutes and remains CORstant for about 15 minutes. Fading is considerably more rapid. K i t h 1.5 mg. of carbazole, the intensity is a t its maximum near 20 minutes, remains constant for 10 more minutes, and then the color fades rapidly. With 4 mg. of carbazole the color develops very rapidly, is constant for only a few minutes, and fades quickly. For the convenience of rapid color development and reasonable stability, we chose 1.5 mg. of carbazole per test. Effect of Methyl Ester Groups. Methyl ester groups might
1630
V O L U M E 24, NO. 10, O C T O B E R 1 9 5 2 Table 11.
'
1631
Effect of Methyl Ester Groups
Recoverpa, Substance Untreated De-esterified Methyl galacturonide meth31 ester monohydrate 78 99 Polygalacturonide methvl ester 88 100 Galacturonic acid monohydrate 100 100 Methyl galacturonide dihydrate 101 101 Polygalacturonic acid 100 100 Compared with the theoretical amount present. By holding for 30 minutes at 2.5' C. in 0.05 A' sodium hydroxide.
i
of the carbazole method was applied to tests with 40 micrograms of galacturonic acid hydrate (33.2 micrograms of anhydouronic acid) in the presence of sugars and organic acids most likely to be present in fruit and vegetable extracts. The results are presented in Table 111. Dische ( 3 , 4) found that the glucuronic acid-sulfuric acid-carbazole reaction gave a more intense color than galacturonic acid. Mannuronic (alginic) acid gave only a slight carbazole reaction. These uronic acids or their derivatives are unlikely to cause interference, since they do not normally occur in pectin extracts. In determining anhydrouronic acid, arabinose interferes slight1 in fifteenfold but not in threefold concentration. Glucose andr fructose interfere slightly in threefold, but not in concentration equal to anhydrouronic acid. Malic acid does not interfere in twelvefold but depresses the color slightly in twenty-fourfold concentration. Oxalic acid does not interfere in twelvefold and citric acid depresses the color slightly in sixfold, and considerably in twelvefold concentration, as compared with anhydrouronic acid. Ethylenediaminetetraacetic acid tetrasodium salt (Versene) did not interfere in sixfold concentration.
Table IV. Comparison of Methods for Determining Anhydrouronic Acid Content of Pectic Substances
I
I
I
i
i
I
l
l
I
10
20
30
40
50
60
70
80
90
1
100
1 ll0
Substance Analyzed Grapefruit pectin Raspberry pectin Apple pectin Apricot pectin Tomato pectin Carrot pectin Grapefruit pectic acid Polygalacturonide methyl
120
TIME IN MINUTES Figure 1. Effect of Carbazole Concentration on Course of Color Development in Uronic-Sulfuric Acid System W i t h 0.5, 1.0, and 1.5 mg. of carbazole per test
prevent the formation of the necessary intermediate compound in the sulfuric acid-galacturonide mixture required for later development of a color with carbazole; therefore, methyl galacturonide methyl ester and polygalacturonide methyl ester were analyzed before and after de-esterification by holding for 30 minutes a t 25' C. in 0.05 N sodium hydroxide. Galarturonic acid, methyl galacturonide, and polygalacturonic acid were analyzed before and after treatment in 0.05 1V sodium hydroxide as controls. The data are shown in Table 11. Both compounds containing methyl ester groups yielded low anhydrouronic acid values. .4fter saponification, the results were brought into agreement. The alkaline treatment of galacturonic acid, methyl galacturonide, and polygalacturonic acid did not affect the results. It is necessary to de-esterify pectic substances before carbazole analysis. Specificity. The utility of most colorimetric methods depends upon their simplicity and specificity. The proposed modification
Table 111. Effect of Interfering Substances Substance Added to 33.2 ~Iicrogranisof AUA" Xothing
.4mount, 11icrograms
Transmittance,
...
49
-4rabinose
100
49
Arabinose
500
47
Fructose
100
48
c7
Glucose
100
47
JIalic acid
400
49
Malic acid
800
50
Citric acid
200
51
Citric acid
400
53
Oxalic acid
400
49
Versene
200
49
:z
a Galacturonic arid hydrate, 40 X = 33.2 micrograms of anhydroL I 1 uronic acid, AUA. Versene is the trade name of ethylenediaminetetraacetic acid tetrasodium salt.
*
Anhydrouronic Acida. % Calculated from calcium Carbon TitraCarpectate dioxide tion bazole
57
..
47 94
63 94
50
93
eater .. 91 9.. .i 91 .~ .. Galacturonic acid hydrate .. 81 83 83 Methyl galacturonidee methyl ester hydrate .. 70 73 72 hloisture- and ash-free basis. Galacturonic acid hydrate, 83%, .IU.4 theory. Methyl galacturonide methyl ester hydrate, 73.3%, AUA, theory.
COMPARISON O F VARIOUS METHODS OF ANALYSES FOR PECTIC S U B S T A 3 C E S
The inconstancy of composition of pectic substances makes difficult any comparisons of quantitative methods except those based upon the anhydrouronic acid content. Partially purified air-dried samples of pectic substances were analyzed for moisture, ash, and anhydrouronic acid. The four methods employed were: the modified carbazole method as proposed, with galacturonic acid hydrate as the standard; the liberation of carbon dioxide upon heating in 19% hydrochloric acid (8); the titrimetric determination of t'otal carboxyl groups (free acid, esters, and those neutralized); and calculrttion of anhydrouronic acid from the calcium analysis of the calcium pectat,e obtained by the Car&-Haynes method ( 2 ) . The carbazole and carbon dioxide method yields results easily expressed in terms of anhydrouronic acid. The sum of the titration of free acidity, saponification of methyl ester groups, and alkalinity of the ash is used to calculate an equivalent weight. This value divided into 176 X 100 yields the per cent anhydrouronic acid. The calcium content of the Carr6-Haynes calcium pectate was determined, and assuming all of the calcium associated with carboxyl groups of pectate, the anhydrouronic acid content of the original was calculated as f o l l o w : found X wt. of calcium pectate X 90.5 7; AUA = V-o calcium10.25 X weight of original sample where 90.5
=
7anhydrouronic acid in pure calcium pectate
and 10.25 = yo calcium in pure calcium pectate The results are summarized in Table IV, and are averages of two or more analyses by each method.
ANALYTICAL CHEMISTRY
1632 The results obtained by the carbon dioxide, titration, and carbazole methods agree fairly well. The anhydrouronic acid values calculated from the calcium analysis of the Carr6Haynes calcium pectate yielded results that were about 10% higher (except for the sample of tomato pectin) than the results from the carbon dioxide or carbazole method. This may be explained by the possibility that all of the calcium fails to associate only with carboxyl groups of pectate. Calcium pectates obtained by the Carr6Haynes procedure from different plant sources are not of constant composition and the calcium analyses of the calcium pectates cannot be used to calculate reliable anhydrouronic acid contents. Therefore, this method has little to recommend it for the quantitative determination of pectic substances The carbazole method was standardized n.ith galacturonic arid hydrate vihose purity was determined by titration. The results of the analyses of pectin are slightly lower than those obtained by the titration method. The presence of acidic constituents, salts, or acetyl groups in pectic substances lead to high anhydrouronic acid values by titration. The precision of the colorimetric carbazole method is about 2%; however, the results in Table IV are ahout 4% higher than those with the carbon dioxide method.
ACKNOWLEDGMENT
The authors wish to express their appreciation to L. R. Leinbach for the carbon dioxide analyses and to Stanley Friedlander for determining the absorption spectra of the uronic acid-sulfuric acid-carbazole mixtures. LITERATURE CITED
(1) Assoc. Official Agr. Chemists, “Official and Tentative Methods of Analysis,” 6th ed., 1945. (2) C a d , M. H., and Haynes, Dorothy, Biochem. J., 16, 80 (1922). (3) Dische, Z., J . BioZ. Chen~.,167, 189 (1947). (4) Ibid., 183, 489 (1950). (5) Hinton, C. L., “Fruit Pectins, Their Chemical Behavior and
Jelling Properties,” New York, Chemical Publishing Co., 1940. (6) Holsman, G., RIacAllister, R. V., and Niemann, Carl, J . Bid. Chem., 171,27 (1947). (7) BIcCready, R. M , , Shepherd, ,4.D., Swenson, H. A , Erlandsen, R. F., and Maclay, IT. D., ANAL.CHEX., 23, 975 (1951). (8) RIcCready, R. hl., Swenson, H. A , and Maclay, K . D., IND ENG.C H E M . , A N A L . ED., 18, 290 (1946). (9) Stark, S. hi., Jr., A s . 4 ~ CHEM., . 22, 1158 (1950). RECEIVED for review March 11, 1952. Accepted June 27, 1962. hleution of products by specific manufacturers does not Imply t h a t they are endorsed or recommended by the Department of Agriculilire over others of a similar nature not mentioned.
Determination of Titanium in Titanium Metal J . M.THOMPSOX Pigments Department, Chemical Dicision, E. I . du Pont de ,Vemours & Co., Inc., Newport, Del.
is described for the determination of titanium in A METHOD high-purity titanium metal. This test was developed by the
Pigments Department, Chemical Division Laboratories of E . I. du Pont de Nemours & Co., Inc., and has been used extensively over the past 6 years. The method is based on the work of Knecht and Hibbert ( 2 ) , who employed a standard solution of ferric salt for titrimetric estimation of titanium. Modification included the use of thiocyanate added directly to the test solution instead of employment as an outside indicator (3, 4 ) . The method was modified and adapted to the determination of titanium in titanium pigments by J. W. Stillman of the Du Pont Chemical Department and C. R. Wicker of this laboratory. Further changes utilizing a twostage reduction were made and applied t o the determination of titanium in ores and pigments by E. N. Kramer and E. D. Lewis of this laboratory. In the instant method for determination of titanium in commercially pure metal, interfering elements are not normally present. Chromium, molybdenum, tungsten, and vanadium may be present in some alloys and must be removed. A method for removal is outlined. Table I indicates the reproducibility of the method. The results listed were obtained by two laboratories on unknown samples of commercial sponge. Each value listed is the average of two determinations. Inspection of the data shows that a difference greater than 0.2% between laboratories occurred on only one sample of the 24 and this maximum deviation was 0.23%. The difficulty in obtaining representative samples of ductile titanium sponge is recognized. Closer agreement will result when improved methods of sample preparation become available. APPARATUS AND REAGENTS FOR DETERMINATION OF TITANIUM
Erlenmeyer flasks, 300-ml., borosilicate glass Tuttle covers Potassium bisulfate, fused, pure Sulfuric acid, concentrated Meker burners
Hydrochloric acid, 50% by volume Florence flask, 1000 ml. Cylinder of carbon dioxide with regulating valve Amalgamated zinc shot Amalgamated 20-mesh zinc Modified Jones reductor Flow meter for measuring carbon dioxide Glass wool Flask tongs Hydrochloric acid, 10% by volume Standard 0.1 N ferric alum solution Potassium thiocyanate or sodium thiocyanate solution, 400 grams per liter PROCEDURE FOR DETERMISATION O F TITANIUM
1. Accurately weigh about 0.2 gram of the sample and transfer to a 300-ml. borosilicate glass flask which contains 15 to 25 grams of potassium bisulfate and 10 drops of concentrated sulfuric acid. 2. Place a Tuttle cover in the neck of the flask and heat over a Meker burner, gently a t first, and then over the full flame unqil the bisulfate has fused and no undecomposed sample remains. This operation will require about 30 minutes. During the fusion, pick up the flask frequently with a pair of tongs and whirl to mix the contents. 3. Remove the flask from the flame with the tongs and by means of a rotary motion cause the melt to solidify in a uniform layer on the side and bottom of the flask. 4. When the contents of the flask have cooled, add 100 ml. of 50y0 (by volume) hydrochloric acid and dissolve the fusion by cafeful heating. Avoid violent boiling. D. Add 60 to 80 grams of amalgamated zinc shot. 6. Place on a hot plate and bring to a boil. Boil gently for 10 minutes. 7 . For the initial sample in a series, prepare the reductor assembly by first drawing the liquid level in the reductor reservoir to about 0.25 to 0.5 inch above the top of the zinc column. Set the 1-liter Florence flask in place in the reductor assembly. Connect the carbon dioxide supply, turn it on, regulate the flow to 1 or 2 liters per minute, and allow it to run for 2 minutes to flush all the air out of the flask before it is used. 8. Drain the acid remaining in the reductor down to the level of the top of the zinc column. 9. Pour the reduced solution from the Erlenmeyer flask into the reductor. Give the zinc shot a preliminary rinse by shaking
