and D-ribose was prepared, applied to paper, and developed. The sugar bands were located and the sugars were eluted from the paper strips by the complete elution method. The eluted sugar solution n as allowed to react Tvith periodate according to the method described for simple sugar solutions. The only differences are that 0.5 ml. of ethylene glycol n-as added instead of 0.2 ml. and that the reaction tubes nere cooled in n ater for 5 minutes prior to its addition. Results of the knon-n sugar chromatographic study are recorded in Table 111.
Each sugar was determined in quadruplicate and the original amount present in the solution was calculated from Dribose, which was the reference sugar. These data show that it was not possible to separate and detect sugars with the accuracy or precision that was obtained with knon-n unchromatographed sugar solutions (Table I), Hon ever, considering all the variables involved, the results are satisfactory. The method described for the separation of seven sugars on a single chromatogram n-as unsatisfactory for the de-
tection of l part of D-galactose in 100 parts of D-ghcOSe. LITERATURE CITED
(1) Hirst, E. L., Jones, J. K. S . ,J . Chern. Soc. 1949, 1639.
(2) Lea, D. C., T a p p i 37, S o . 9, 393
(1954).
( 3 ) Quick, R. H., “Study of Order and Nature of Asoenwood Hemicellulose
Removed d&ing a Xeutral Sulfite Semichemical Cook,” doctor’s dissertation, The Institute of Paper Chemistry, hppleton, Wis., 1955. RECEIVED for revieiT December 5, 1955. ..iccepted May 13, 1957.
Quantitative Determination of Volatile Acids by Paper Chromatography for Application to Sewage Sludge Digestion RAYMOND M. MANGANELLI a7d F?EDERICY
F!.
BROFAZI
Department o f Sanitation, Rutgers Universify, New Brunswick, N. 1.
A paper chromatographic method for
the
separation of the volatile C?to Cg fatty acids has been developed for application to sewage sludge digestion. The acids are separated as their ethylamine salts in a butanolwater-ethylamine solvent system. After color development with chlorophenol red indicator solution, the acids appear as purple spots on a yellow background, stable for at least 3 hours. A straight-line relationship exists between the area of the spot and the concentration of the acid present. A statistical analysis of the method shows that the volatile acids can be separated, identified, and determined with an accuracy within = t 3 to 6% for each acid.
A
ISVLSTIGATIOS has been undertaken to study the volatile fatty acids formed during the anaerobic digestion of carbon-14-tagged stearic acid in sewage sludge. Preliminary to this study, it was necessary to develop a method by nhich these acids could be separated and determined quantitatively. Successful paper chroniatographic separations of the volatile acids as nonvolatile derivatives have been described (2-7, 9. 10, 12). However, niost of these methods are only qualitative in nature. Duncan and Porteous ( d ) , Isherwood and Hanes ( 6 ) , and Reid and Lederer (12) have outlined quantitative procedures which have the following disadvantages. Complex procedures are required and the developed acid spots are unstable.
N
solutions of tlie various acids in tlie concentration raiige of 15 y per 5 b1. to 40 y per 5 p l . are prepared and :idjusted to pH 6.0 to 9.0 u i t h ethylamine. Five-microliter samples of the standards are placed 3.5 cin. from one edge of a sheet of Whatman S o . 1 paper, n hich is 8.5 inches n-ide and 20 inches long. The applied solutions are dried a t room temperature and the sheet% are suspended in the clironiatograpliic PROCEDURE tank TT ith one end dipping into Solution B in the solvent trough. Descending REAGESTS. Solvent system. F i r e chromatography iq employed. A tlehundred milliliters of 1-butanol are 1-elopment time of 20 hours i equilibrated a t 20” C. n i t h an equal for complete separation of the C? to C6 volume of distilled water. The two volatile acids. When development i i phases are d r a n n off separately. The complete. the papers are remored from TI ater layer (lon-er phase) is designated the tank and allon ed to air-dry for 1 to as Solution A. Ten milliliters of 33.33% 2 hours a t room temperature. aqueous ethylamine are added to 490 COLORDETELOPMEST OF CHROUATOnil. of the butanol layer 17-hich is the GRAMS. Sufficient chlorophenol red upper phase of the original equilibrated indicator solution is poured into a shalmixture. This solution (Solution B) is lon tray to yield a liquid layer of apshaken thoroughly and allowed to stand proximately 0.5 inch. After air-drying for 1 hour. The small aqueous (loner) the chroniatogranis are subjected to layer is discarded. rapid immersion in the indicator soluIndicator solution. Two hundred tion and hung up to dry. The ethylmilligrams of chlorophenol red are amine salts of the acids appear as purple dissolved in 100 ml. of 95% ethyl spots on a yellow background. These alcohol. EQUILIBRATION OF CHROMATOGRAPHICspots are stable for a t least 3 hours after immersion in the color indicator. STSTEJI. Sheets of Whatman S o . 1 Q U A N T I T A T I ~ E DETERWKATIOK OF paper are used as a m-ick to liiie the ACID COSCESTRBTIOS. T n enty minchromatographic tank, a rectangular utes after the chromatograms are jar S X 12 X 21 inches in height obdipped, the acid spots are carefully outtained from Research Specialties Co. lined on tracing paper. The outline Solution A is placed in the bottom of of the acid spots is traversed 10 times the tank. The solrent troughs, which n i t h a planimeter, to minimize any erare a t the top of the tank, are filled with rors from a single reading. This area solution B and the system is allowed to measurement is divided by 10 to obtain equilibrate a t 20” C. for 24 hours, the spot area for a single determination. Equilibration is necessary only after the The procedure is repeated three times initial introduction of solvents or when changing the solvent systems. and the area of each spot is taken as PREPARATIOiX AND CHROMATOGRAPHthe average of three planimeter deterI N G OF STANDARD SOLUTIONS.Standard minations. The acid standards are The method described in this report deals n ith tlie paper chroniatographic separation of tlie Cn to Cg volatile fattjacids as their ethylamine salts. This procedure, nhich is a niodification of the iiiethod originally developed by Roberts ( I s ) ,allon s for quantitative determination of the volatile acids.
VOL. 29, NO. 10, OCTOBER 1957
1441
chromatographed on each of four sheets and the area measurements are based on the mean of quadruplicate chromatographic determinations. DETERMINATION O F VOLATILE ACIDS UNKNOWN SAMPLES. Two dilutions of C1 to Ce acid standards must be chromatographed each time a n unknown sample is to be determined. These standards are used to obtain R, values which identify the unknown acids, and standard curves for determining the concentration of the acids. IN
thus allowing accurate measurement of the areas. For the ranges studied, a linear relationship was found to exist between the areas of the acid spots and the concentration of the acids present. Regres sion analysis was applied to the data obtained from acid standards in the concentration range of 15 to 40 y per 5 pl. y
OF
(Table 11). The associated variation ranges from a high of 99.58'30 for acetic acid to a low of 92.15% for valeric acid. The average error of estimate, which is to be interpreted analogous to a standard deviation, ranges from ~t0.6716y per 5 pi. for acetic acid to %2.9190 y per 5 p1. for valeric acid. A second series of acid solutions was
E A C H ACID PER 5 PI.
A"
RESULTS
The chromatographic separation of the C2to Csvolatile acids results in well delineated spots with R, values (Table I) ranging from 0.14 to 0.68 at 20' C. These R, values show that the acids are completely separated on the chromatograms. A typical separation of these acids may be seen in Figure 1. The yellow background offers a strong contrast to the stable purple acid spots,
Table I. R, Values of Ethylamine Salts of Volatile Acids
Acid Acetic Propionic Butyric Valeric Caproic
Table
II.
R, Values at Z O O C. IndividnaI Aoid acids mixtures 0.14 n 14 0.21 0.23 0.33 0.35 0.50 0.52 0.62 0.68
30 MINUTES
Figui 1. Typical chromatograms showing separation of Cz to Cg volatile acids 30 minutes after immersion in color indicator
Statistical Relationship between Spot Area and Concentration of Chromatographed Volatile Acids
,.'.
Sb.
s,.* (Av. Error (Percentage (Av. Error of tbS of Assocd. of Estimate Regression (1 Variation)L in v / 5 ~ 1 . p Caeff.Id Ratiop 712
Estimating Equation" X,' = -1.9469 10.6896Xt 99.58 0.6716 X,' = -2.1186 11.3848X2 97.51 1.7147 XI' = -2.5186 12.5195X3 99.33 0.9729 X,' = -3.8656 14.5248Xz 92.15 2.9190 XI' = -4.5532 12.9739X~ 97.95 1.6260 * Regression equation. Coeff.of correlation X 100. Std. error of estimate. Std. error of regression coeff. a Regression coeff. divided by its std. error (b,.z)/s~,,w All t values are highly significant at 1 % level.
Acid Acetic Propionic Butyric Valeric Caproic
+ + +
++
Table 111.
Acid Acetic
Propionic Butyric Valeric Caproic S'=-
1oos,.*
X,
1442
0,3472 0.9096 0.5030 2.1107 0.9385
30.7880 12.5162 24.8897 6.8522 13.8241
Comporison of Coefficients of Variation with Observed Errors
Range of Concn., (r/5 SI.) 1439 1440 14-44
1540 1543 100
x,- X,' ~
XI N
ANALYTICAL CHEMISTRY
No. of Concn. 6 6 6 6 6
Coeff. of Variation, V* 2.72 7.02
3.92 11.79 6.54
Observed Error, Av. '%b 5.01 3.36 4.68 5.73 3.46
chromatographed and their areas were determined. The concentrations of the acids were predicted from the estimating equations (Table 11). The predicted concentrations were subtracted from the actual concentrations to obtain the observed errors. The average of the observed errors was calculated for each acid of the second series of samples. The errors were expressed as percentages and were compared to the average per cent error (coefficient of variation) for the first series of standard solutions. These results are summarized in Table Ill, whicIi shows that the coefficient of variation ranges from 2.72% for acetic acid to 11.79'% for valeric acid and that the average per cent observed error ranges from 3.36'% for propionic acid to 5.73% for valeric acid. DISCUSSION
The modified Roberts method for the paper chromatographic separation of the Cz to C, volatile acids has advantages over other procedures. No pretreatment of chromatographic sheets is necessary. Acid spots are well defined and very stable. The acids are completely separated as shown by the E , values. The concentration of volatile acids can
be quantitatively determined. The aqsociated variation, ranging from 99.58 to 92.151%~indicates that variations in acid spot areas may be attributed to changes in concentration. Errors in the measurement of spot areas are the probable cause for the deviation of the associated variation from 1 0 0 ~ , A range of t ratios from 6 to 30 means that the linear relationship between spot area and acid concentration could happen a t a maximum by chance less than 1% of the time. Therefore, the relationship betn een area and concentration is very stable and highly significant. To obtain a measure of the accuracy of the method, the errors of the second series of acids were compared to the espected errors from the first series of acid solutions. The evpected error, the coefficient of variation, of the first series is the per cent error within nhich two thirds of the experimental data should fall. The average per cent observed errors of the second series of samples agreed IT ell n ith the coefficient of 1-ariatiori. The coefficient of variation ranged i'ioni 2 to 127,, while the average observed error ranged from 3 t o 6%. Therefore, this method i- accurate in predicting the acid concentrations nithiii 1 3 to 6'3 of the wtual acid concentration. APPLICATION
This method has been used successfully in studying the formation of volatile acids during sewage sludge digestion. The volatile acid fraction is isolated from the sludge by a standard procedure ( I ) . The steam distillate is collected in a known volume of standard sodium
hydroxide. An aliquot of the distillate is back-titrated to phenolphthalein end point with hydrochloric acid. The normality of the volatile acids present is then calculated. The remaining alkaline steam distillate is concentrated by evaporation t o a predetermined volume a t n-hich the volatile acid normality is approximately 0.40. To this concentrated mixture iq added an equimolar amount (to the sodium hydroxide uqed) of aninionium wlfate. The .ample 1. acidified with sulfuric acid to pH 3.0 and made to react n-ith ethylamine until a pH of 8 to 9 is obtained. The sample is diluted to a knorrn volume to ohtain a volatile acid normality of 0.20 or greater. Spots of measurable size are obtained nhen 5 11. of this final mixture are chromatographed and the acid concentrations fall nithiii the ranqe of 15 to 40 y per 5 p1. The concentration of the acids determined on the chroniatograin can be readily related t o the quantity of each acid in the original sludge. The presence of the sodium ion in the sample to be chroinatographed causes a spot retention on the starting line, IT hich prevents quantitative determiiiatioii of acetic acid. The addition of a n equimolar amount of ammonium sulfate conipletely eliminate. the sodium ion interference and permits the quantitative determination of acetic acid. Formic acid aiid acetic acid have the same R , values and are not qeparated from one another. Because no formic acid has been found during sludge digestion (8, 1 1 ) , acetic acid can be quantitatively determined by this method.
ACKNOWLEDGMENT
This investigation was supported by a research grant from the Dorrco Educational Trust, Stamford, Colin. LITERATURE CITED
(1) Am. Public Health Assoc., "Standard Methods for the Examination of Kater, Sewage, and Industrial Wastes," 10th ed., p. 346, S e w I'ork, S . Y . , 1955. ( 2 ) Brown, F., Bzocheni. J . 47, 598 (1950). (3) Brown, F., Hall, L. l'., 'Yature 166, AH (1950) --
(4) DL&XI, R. E. B., Porteolls, .J. w., Analyst 78, 641 (1953). (5) Hiscox, E. R., Berridge, S. J., .Vature 166, 522 (1950); (6) Isherwood, F. A , , Hanes, C. S., Biocheni. J . 5 5 , 824 (1953). (7) Jones, A . R., Dowling, E. J., Skraba, W. J., ana^. CHEM. 2 5 , 394 (1953). (8) Kaplovsky, A. J., Ph.D. thesis, Ruteers Univ., S e w Brunsxick. s.5., 1950. (9) Kennedy, E. P., Barker, H. A, ASAL. CHEM.23, 1033 (1951). (10) Long, A . G., Quayle, J. R., Stedman, R. J., J . Cheni. Soc. 1951, 2197. LI.,Banta, A . P., Ponieroy, (11) Rawn, R.. Proc. Ani. SOC. Czval E l m s . 63; 1673 (1937). (12) Reid, R. L., Ledern, hI.> Riocheni. J . 50,60 (1951). (13) Roberts, H. R., Research Laboratories, Division of rational Dairy Products Corm, Oakdale. L. I.. S . T., private'communication. \ - -
.
3 -
RECEIVEDfor review October 22, 1956. .L\ccepted May 7, 1957. Division of TS'ater, Sewage, and Sanitation Chemistry, 130th Meeting, ACS, Atlantic City, X .J., September 1956. Paper of Journal Series, Xew Jersey Agr. Expt. Sta., Department of Sanitation, Riitgers University, New Brunswick, S .J.
Qua nt ita tive Dete rmination of Lactose and Monoses in Lactose Hydrolyzates Rapid Horizontal Paper Chromatographic Method H E N R Y R. ROBERTS Research laboratories Division, National Dairy Products Corp., Oakdale, l o n g Island,
b A rapid horizontal paper chromatographic technique has been developed for the analysis of lactose hydrolyzates. Chromatographing at 60" C. with the solvent system butanol-pyridine-water (9-5-8), two solvent developments, each o f 60 minutes' duration, separate galactose and glucose (as a unit) from lactose. Four such developments separate lactose from the galactosyl oligosaccharides sufficiently for quantitative treat-
ment. Analysis of known lactose and monose solutions and comparison of lactose hydrolyzates with a descending paper chromatographic method give results with an error well under
5%. om-dimensional descending paper chromatographic procedure for the quantitative determination of lactose, galactose. and glucose was de-
N. Y. veloped by McFarren and his cowvorkers in 1951 (6). Application of this technique to the analysis of lactase-hydrolyzed lactose preparation. shon ed that in addition to galactose. glucosp. and residual lactose. other rugars w r e also formed. Subsequent studies hy a number of investigators (2, 7 , 9,12) showed that these additional sugars werp galactosyl oligosaccharides synthesizrd by the lactase enzyme. The amount of monoses formed during VOL. 29, NO. 10, OCTOBER 1957
1443
