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 and 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, long 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
the enzymatic hydrolysis of lactose is not a measure of lactose hydrolysis, because it fails to account for the lactose converted to the galactosyl oligosaccharides. The extent of lactose hydrolysis can be best measured by determining the residual lactose. Chemical methods are not capable of measuring the residual lactose and detecting the oligosaccharides. I n addition, the presence of the oligosaccharides interferes in the monose assay and the monose values obtained by chemical assay must be accepted with some dou,bt. The over-all result, in such cases, is low hydrolysis values. A need exists for a rapid and accurate method for measuring residual lactose. Such a method could be used as a plantlaboratory control procedure for folloiving the hydrolysis of large batches of whey used in the manuficture of animal feeds. Toward this objective, a quantitative horizontal paper chromatographic method has been developed which separates lactose from the monoses and galactosyl oligosaccharides within 4 hours, as compared to 18 to 20 hours by the descending technique. EXPERIMENTAL
The paper chromatographic chamber is shown in Figure 1. Schleicher and Schull S o . 589 White Ribbon filter paper, 12 X 13.5 em., is used. A 1.5-em. tab, folded a t a right angle to the paper, is inserted into the trough containing the developing solvent is), butanol-pyridine-water (9-5-8) (Figure 2). The solvent is prepared
at 22" C. in a temperature-controlled room. After separation of the two phases, the water-rich layer is discarded. A portion of the developing solvent is also placed in the bottom of the chamber in order to keep the chamber saturated with solvent vapors. A 1/16-inch black neoprene gasket is cemented to the flanged surface of the chamber. A glass plate cover is clamped to the flange with Castaloy clamps to keep the chamber airtight and minimize the escape of solvent vapors. The stainless steel chamber offers a number of advantages such as compactness, ease of handling, greater control over solvent evaporation, and less danger of breakage. Seven sugar solutions are introduced along a line 2.5 em. from the tab end of the filter paper strip. Four standard solutions containing, respectively, 3, 6, 9, and 12 y per 0.5 pl. of lactose and monoses (galactose and glucose) are applied alternately to the paper in 0.5-pl. volumes by a Gilmont ultramicroburet. Unknown solutions are applied a t appropriate dilutions alternately with the knoivns. I n the technique reported here galactose and glucose are not separated but move as a unit and are measured as total monoses. After spotting, the filter paper strip is placed in the chamber, ivhich is in a 60" C. oven, and the solvent irrigates the paper, 60 minutes to a development. The chamber's being in the oven provides for an equal heating of the chamber and eliminates the formation of any condensate. Two 60-minute solvent developments are required for the monose determination and four 60minute developments for lactose. I n each instance, the chromatogram is dried for 5 minutes in the 60" C. oven after each solvent development.
The sugar spots are made visible by dipping the chromatogram into a benzidine solution n-hich contains 3 parts of 0.14y0 benzidine in butanol and 1 part of 50y0aqueous citric acid and then heating for 10 minutes a t 105' C. ( 6 ) . The maximum densities of the sugar spots are then measured with a Photovolt electron transmission denqitonieter, using a 1-mm. diameter aperture and a S o . 440 filter purchased with the instrument. A standard curve is prepared by plotting the logarithm of the known concentrations against the densities. The concentrations of the monoses and residual lactose in the hydrolyzate are then calculated by referring to the standard curve. RESULTS
For evaluating this procedure. a number of knoivn lactose and nionose solutions were prepared and run as unknowns. The results of the monose analysis are shown in Table I. The standard monose solutions contain equal n-eights of galactose and glucose. Three known "unknon.n" monose solutions were prepared and analyzed: (1) equal weights of galactose and glucose; ( 2 ) 1 part of galactose and 3 parts of glucose; (3) 2 parts of galactose and 3 parts of glucose, a solution representative of the monose content of lactose hydrolyzates which contain galactosyl oligosaccharides. I n each analysis the results of three chromatograms are averaged and reported, to lessen the error of the analysis. The first solution assayed 923 mg., or 2.67, deviation from the theoretical value of 900 mg.; the second, 2013 mg., or 0.5% devia-
SOLVENT TROUGH, 1"STAINLESS STEEL SANITARY TUBING ENDS PLUG WELDED
A=TROUGH SUPPORT, TYPE 304 STAINLESS STEEL 0.062"THICK, SPOT WELDED, BRACKET SAME ON TH SIDES OF CHAMBER
i''
D SUPPORT, 5 LONG TERIAL AND MOUNTING SAME AS A 285" DIAMETER NOTCHES, $'APART
CHAMBER MATERIAL= I PIECE TYPE 304 STAINLESS STEEL,O.O52"THlCK
L D A L L FOUR CORNERS
Figure 1.
1444
Stainless steel chamber and solvent trough for horizontal paper chromatography
ANALYTICAL CHEMISTRY
tion from 2008 mg.; the third, 495 mg., or 1.0% deviation from 500 mg. Two lactose solutions were prepared and analyzed (Table 11). The average of three chromatograms gave a result
of 993 mg. for the first sample, or 3.4% deviation from the theoretical value of 960 mg. The second sample assayed 1017 mg., or 1.7% deviation from the 1000 mg. theoretical value.
Lactose hydrolyzates were analyzed and the results compared with the values obtained by using the descending paper chromatographic method of McFarren and coworkers (5) (Table 111). The agreement between the two methods for the hydrolysates analyzed was within 5% for both lactose and monoses. Figure 3, A , is a quantitative lactose descending paper chromatogram of a lactose hydrolyzate (18 to 20 hours' solvent development time). Figure 3, B, is a chromatogram of the same hydrolyzate using the horizontal paper chromatographic procedure to assay for lactose (4 hours' solvent development time). DISCUSSION
Figure 2. Horizontal p a p e r chromatographic chamber assembly Showing manner in which Schleicher and Schiill No, 589 White Ribbon filter paper, 12 X 13.5 cm., is supported in a horizontal position within chamber by glass rods
Horizontal Paper Chromatographic Analysis of Known GalactoseGlucose Mixtures Theoretical Monoses Calculated Monoses, Mg. in Sample, (Chromatogram) %Deviation sugars Mg. in Mixture 1 2 3 Av. from Theory 1 part galalactase 1part glucose 900 920 870 980 923 2.6
Toble I.
1 part g$actase 3 parts glucose 2 parts galactose 3 parts glucose
2008
2040
1920
2080
2013
0.5
500
525
480
480
495
1.0
Table II. Horizontal Paper Chromatographic Analysis of Known Lactose Solutions Theoretical Calculated Lactose, Mg. (Chromatogram) % Deviation Lactose, Av. from Theory Mg. 1 2 3 960 980 1030 970 993 3.4 1000 1030 990 1030 1017 1.7
Hyd. A A B
Table 111. Analysis of Lactose Hvdrolvzates Descending Horizontal Paper % Deviation from Paper Chromatography, Mg. Descending Paper Chrom. ( 6 ) , (Chromatogram) Chromatography Sugar Mg. 1 2 3 Av. Value Lactose 528 570 535 530 545 3.2 Monoses 478 525 525 445 498 4.2 Lactose 84 86 78 84 83 1.1
Solvent. Alarge number of solvent systems tested gave a rapid separation of the monoses from lactose. However, they failed t o separate lactose from the oligosaccharides within the short-time solvent development required for its adoption as a plantlaboratory control assay. The solvent systems were appraised for their ability to separate lactose from the oligosaccharides in this short development period by chromatographing the oligosaccharides and lactose separately and as a mixture. The two oligosaccharides, lactobiose (a galactoseglucose disaccharide) and galactobiose (a galactose - galactose disaccharide), which occupy a position directly above lactose on a conventional descending paper chromatogram, were eluted off paper chromatograms of a lactose h y drolyzate. At 22" C., the solvent system butanolpyridine-water (9-5-8) required 105 minutes to travel 10 cm. from the origin. Chromatograms containing four spots at the origin were prepared. The first spot contained the eluted disaccharides, lactobiose and galactobiose, the second lactose, the third galactose and glucose, and the fourth a mixture of the first three. These chromatograms were then subjected to multiple solvent d e velopments of 120 minutes' duration. While four developments did not separate lactose from the oligosaccharides, they indicated that such a separation could be achieved by more developments. However, increasing the number of solvent developments also increased the total development time and this was undesirable. Chromatography at Elevated Temperatures. A number of reports in the literature showed improved separation of sugars by solvent development at a n elevated temperature. T u and Ward (11) were able to separate pairs of disaccharides by using a heated charcoal column and 3% ethanol as the elutrient. Hough and associates VOL. 29, NO. 10, OCTOBER 1957
1445
(4)separated a number of sugars on paper chroinatograms at 37' C., which were difficnlt to separate at room temperat,ure. Counsel1 and coworkers (3) found that a t elevated temperatures the rate of flow of the mobile phase is greatly increased, R, values of the sugars are increased, and if precise and constant control is maintained and equilibrium in the chromatographic apparatus is established, the degree of separation is improved. It appeared, therefore, that chromatography a t elevated temperature might effect separation of lactose from the oligosaccharides. Chromatograms containing lactose, lactohiose, galactohiose, galactose, and glucose were prepared and subjected to multiple solvent developments of 60 minutes' duration at temperatures of 45", 50°, 55", 60", and 65" C., using the solvent systern hutanol-pyridine-water (9-5-8). One, two, three, and four solvent developments were carried out a t each temperature. At 60" C. the desired separation between lactose and the oligosaccharides was achieved when the chromatograms received four 60-minute solvent developments (Figure 4). Chromatographing a t 65" C..also gave good results, but there was no added advantage in using this temperature. Chromatography at 60" C. as compared to room temperature not only increased the solvent flow rate, hut, of greater importance, it improved the degree of separation, Alcock and Cannel1 (1) have published the results of their studies dealing with the paper chromatography of sugars a t elevated temperatures, which help to explain the successful separations reported here. They found that the Ri values of a number of sugars studied varied with the temperature. The speed of development of the chromatograms was also influenced by temperature changes, since physical characteristics such as density, viscosity, and surface tension of the solvent were affected and hence the "diffusion coefficient" of the paper. Color Development. The separation of galactose and glucose was not necessary, as the sensitivity of the benzidine reagent employed is the same for glucose and galactose (10). Individual solutions of the two hexoses, each containing 3.3, 6.7, and 10.0 y per 0.5 pl., were chromatographed and then developed with the benzidinecitric acid reagent for 10 minutes at 105' C. The densities of both sugars a t each concentration were in good agreement. These results were confirmed when 5, 7.5, and 15 y of galactose and glucose, respectively, were spotted and the color development of the spots was effected without prior solvent development. Little, or no,
1446
ANALYTICAL CHEMISTRY
A
B
Figure 3. Quantitative lactose p a p e r chromatograms of same lactose hydrolyzate A . Descending technique 20 hours at 22' C. ( 5 ) B. Horizontal technique, four 60-minute solvent developments at BOD C. K. Knowns applied a t concentrations (left t o right, respectively) of 15,10, 7.5, and 5 y per 2 PI. on descending chromatogram and 12,9,6, and 3 y per 0.5 41. on horizontal chromatogram U. Lactose hpdrolyaate applied at three dilutions in 2.~1. volumes on descending chromatogram and three dilutions in 0.5-PI. volumes on horizontal chromatogram
Butanol-pyridine-water (9-5-8) as developing solvent A . Lactobiose and gdactohiose C. Galmtose and glucose B . Lactose D. Mixture of all 1. 1 solvent development, 60 minutes 2. 2 solvent developments, 60 minutes each 3. 3 solvent developments, 60 minutes each 4. 4 solvent developments, 60 minutes each
differences in the density of the spots of the same concentration were noted. Density Values. A 10-minute heat treatment a t 105' C. was chosen because i t gave t h e highest density readings for lactose a n d t h e monoses when 5-, lo-, a n d 15-minute heating periods were tested. A No. 440 filter is used to read the spot densities, because it gives maximum absorption and the highest density readings of a number of filters tested. ACKNOWLEDGMENT
The author extends his sincerest
appreciation to Wesley Bucek for his chromatographic assistance. LITERATURE CITED
(1) Alcock, Margaret, Cannell, J. S., Nature 177, 327 (1956). (2) Aronson. M.. Arch. Biochem. and Biophys. 39,370(1952). (3) Counsell, J. I., Hough, L., Kadman, W. H., Research 4, 143 (1951). (4) Hough, L., Jones, J. K. N., Wadman, W.H., J . Chem. SOC.1950, 1702. (5) McFarren, E. F., Brand, K., Rutkowski, H. R., AXAL. CHEX. 23,1146 (1951). ( 6 ) Opienska-Blaut, H. J., Xadecka-
.,
Borkowska, I., Borkowski, h'ature 169, 798 (1952).
T.,
(7) Pazur, J. H., Science 117, 355 (1953). (8) Porter, W. L., Hoban, Nancy ANAL. CHEM.26, 1846 (1954). (9) Roberts, H. R., McFarren, E. F., J. Dairy Sci. 36, 620 (1953). (10) Trucco, R. E., Verdier, P., Rega, A,, Biochem. et Biophys. Acta 15, 582 (1954). (11) Tu, C. C., Ward, K., Jr., J . Am. Chem. SOC.77, 4938 (1955). (12) Wallenfels, K., Naturwissenschaften 38, 306 (1951). \ - - - - I -
RECEIVED for review December 18, 1956. Accepted April 4, 1957. Division of Agricultural and Food Chemistry, 130th Meeting, ACS, Atlantic City, N. J., September 1956.
Rapid Procedure for Separating C, to C, Volatile Fatty Acids by Horizontal Paper Chromatography at Elevated Temperature HENRY R. ROBERTS and WESLEY BUCEK Research laboratories Division, National Dairy Products Corp., Oakdale, l o n g Island,
b Acetic, propionic, butyric, valeric, and caproic acids are separated as their ethylamine salts in 1 hour on paper chromatograms b y using watersaturated butanol at 50" C. A horizontal solvent development using 12 X 17 cm. strips of Whatman No. 1 filter paper separates the acids as round compact spots. The acids appear as stable purple spots on a yellow background when the chromatogram i s dipped into a chlorophenol red indicator solution. Preliminary studies indicate that this rapid procedure could be made quantitative b y either the area or maximum spot density method.
D
of a rapid horizontal paper chromatographic procedure for the determination of lactose in lactose hydrolyzates a t a n elevated temperature (8)led to the application of this technique to separation of the C B to Cs volatile fatty acids. Horizontal solvent development a t an elevated temperature not only increases the solvent f l o ~rate, but, of greater importance, i t achieves extremely rapid separation of coniponents a t a great reduction in developing time. The use of rectangular strips of filter paper, rathcr than filter paper disks, in combination with a horizontal solvent flow as first described by Meredith and Sammons (7) permits separation of the components as round compact
spots rather than circular bands. The round spots lend themselves to quantitation by measuring either the color intensity or the area of the spots. Aside from the work of Hough, Jones, and K a d m a n ( 6 ) , Counsell and associates ( S ) , and Alcock and Cannel1 ( I ) , little has been done with paper chromatography a t elevated temperatures because of the difficulty of maintaining adequate equilibrium a t these temperatures in conventional descending and ascending chromatographic apparatus. Horizontal paper chromatography provides a way of resolving this problem. EXPERIMENTAL
EVELOPVEKT
Khatman S o . 1 filter paper, 12 x 17 cm., is used. The origin is 2.5 cni. from one end. d 1.5-cm. tab, folded a t a right angle to the paper, is inserted into the solvent trough. The opposite end of the paper iq serrated by using pinking shears, and :I 1.0-em. tali is folded a t a
F I L T E R PAPER I
TROUGH
G L A S S ROD
Figure 1. Position of filter paper during horizontal solvent development
N. Y. right angle to the paper to permit easy flow of solvent off the paper strip. The chromatographic chamber and the manner in which the filter paper strip is supported in a horizontal position (Figure 1) have been described (8). The Cp to Cg volatile fatty acids are separated as their ethylamine salts ( 5 ) . Aqueous solutions of the acids are adjusted to p H 8.0, by using 33y0 aqueous ethylamine. The ethylamine salts are applied to the paper in 0.5-pl. volumes by either a Gilmont ultraniicroburet or a platinum loop. The developing solvent, 1-butanol saturated with water, is prepared at 22' C. in a temperature-controlled room. The atmosphere within the chamber is made alkaline by making the water-rich layer 0 . 1 s with respect to ethylamine. This layer is placed in the bottom of the chamber. After spotting, the filter paper strip is placed in the chamber, which is in an oven, and solvent development occurs a t one of two temperatures, 50" or 60" C. The oven provides equal heating of the chamber and eliminates the formation of condensate. The lower fatty acids from acetic to caproic are separated in 1 hour a t 50" or 60" C., compared to 20 hours by the conventional descending technique. Following solvent development, the chromatogram is air-dried for 1 hour and then dipped into a 0.2% solution of chlorophenol red in 95% ethyl alcohol. The ethylamine salts of the acids appear as purple spots against a yellow background. These spots ale very stable and even after a month are still visible. If the Chromatograms are to VOL. 29, NO. 10, OCTOBER 1957
1447
,
