corresponds to n = 6.2, n = 6.2, (3 = 2 x 6.2/53.52, and y = 0.01/2 are substituted in the above expression for f7&*
Experiment 2. pH-Composition Relation of a Borate Buffer. I n ordei t o obtain t h e pH us. z relationship for a borate buffer prepared from two stock solutions of 0 . 2 5 boric acid and 0 . 2 s sodium hydroxide, a sampling titration was performed in which boric acid was titrated with sodium hydroxide semicontinuously . A sample n a s withdran-n from t h e titration vessel after addition of each titrant increment and its pH measured in a pH meter. As the interest in that particular work was in the p H range from 9 to 9.5, it n a s not necessary to start sanipling at a sodium hydroxide volume fraction equal to 0. Instead, the initial mixture had an initial sodium hydroxide volume fraction of zo = 0.2 and initial volume of v0 = 2.5 ml. The ratio y n a s macle equal to 'Izwith a = 1 ml. and b = 2 ml. and the initial pH was found to be equal to 8.58. Values of fn corresponding to the
different, samples were calculated using the simple relation Equation 13; hence, the exact composition of the buffer corresponding to each experimentally measured pH value was deduced as illustrated in the following table.
n p s
1 to
x =
f, =
1
2
3 1 5 6
7 8
8 i5 8 92 9.07
9.21 9 36 9 54 9 74
10 05
1 1 1 1 1 1 1 1
085 136 190 250 316 389 470
563
0 0 0 0 0 0 0 0
233 267 300 333 367 400 433
z>o c 11,
467
2: z 01
NOMENCLATURE
a
= volume of each titrant increment b = volume of one sample d = b - a f = continuous sampling coefficient f n = semicontinuous sampling coefficient vi. r = integer numbers
@
y $
A
number of samples or titrant increments = any physical or chemical property -for example color intensity or PH = volume of samples withdrawn = volume of titrant required during sampling = volume of titrant required to produce titrant volume fraction equal to z in bulk solution if no samples were 11ithdran-n = 210 - b = volume of solution to be titrated a t beginning of sampling titration = volume a t any time during sampling process = final volume of solution in titration vessel = partial volume of titrant in vessel = volume fraction of titrant in vessel = fn,f when p and y are the same for the semicontinuous and continuous process = s / r o or nb/ao = t / s = a / b = constant = v,/d = first finite difference =
RECEIVEDfor review August 29, 1956. iiccepted May 10, 1957.
Evaluation of Quantitative Sugar Analysis by Paper Chromatography ROBERT H. QUICK' The lnstitufe of Paper Chemistry, Applefon, Wis.
As p a r t of a study on the hemicellulose removed during a neutral sulfite semichemical cook of aspenwood, i t was necessary to characterize the hemicellulose more completely. Known sugar solutions of D-galactose, D-glucose, D-mannose, t-arabinose, Dxylose, D-ribose, and 1-rhamnose can b e separated on a single chromatogram and determined with an accuracy 10% b y using the periowithin date oxidation method. A laboratory method for the quantitative determination of six unknown sugars and a reference sugar on a single paper chromatogram has been devised.
*
I
s A recent study of the hemicelluloses removed during a neutral sulfite semichemical cook ( S ) , a chemical characterization of the carbohydrate material was necessary. This was effected by using a slight modification of the quantitative method of sugar analyPresent address, Pulp Division, Weyerhawser Tiinher Co., Longview, Wash.
sis suggested by Hirst and Jones ( 1 ) . The accuracy and. precision of this niethod were exaininecl and evaluated for the conditions used in the study. PERIODATE OXIDATION
OF SUGARS
First the periodate oxidation of known sugars \vas studied, to determine the accuracy with which D-galactose, Dglucose. D-mannose, L-arabinose, D-XSlose, D-ribose, and L-rhaninoce could be determined.
A 11-eighed sugar sample was transferred quantitatively into a 10-ml. volumetric flask and diluted n i t h carbon dioxide-free distilled nater. The pipet t o be used \\-as checked b y neighing the delivery of distilled nater a t a knon-n temperature. A 1-ml. aliquot of the sugar solution was delivered to the reaction flask and 2 ml. of carbon dioxide-free distilled water and 1 ml. of 0.25-11 sodium metaperiodate, c.P., were added to the solution. The reaction tube (38 X 200 mm. test tube) was stoppered with a 25-ml. Erlenmeyer flask and kept in a boiling water bath for 20 minutes.
Subsequently, the reaction tubc was cooled under t a p water and 0.2 nil. of t'echnical grade ethylene d;\-col as added. The mixture JTas ailon-ed to stand 5 minutes and then was titrated Ti-ith about 0.00-1.Ysodium hydroxide. Methyl red u-as used as the indicator. Phenolphthalein TKE an unsatisfactory indicator. The data collected (Table I) shon that the sugars studied n-ere determined n ith accuracies of 96 to 1027, ivith good precision and are a confirmation of the work done by Hirst and Jones ( I ) . There ic no apparent reason for the relatirely poor recovery of D-galactose. as all sugars used in this study n ere high , quality C.P. chemicalq. CHROMATOGRAPHIC ANALYSIS OF SUGARS
The quantitative determination of sugars from paper chromatograms is more difficult than the oxidation of the simple sugars from a known solution The problem is complicated by the separation and elution errors as well as by the oxidation and titration errors. It is very easy to separate D-galactose VOL. 29, NO. 10, OCTOBER 1957
1439
from D-mannose, D-xylose, and D-ribose; but it is difficult to separate D-galactose from D-glucose and D-mannose from Larabinose on the same chromatogram. A method for separating and determining these sugars and L-rhamnose on ape chromatogram seemed most appropriate for studying the spent liquor hydrolyzates a t various intervals during a neutral sulfite seniichemical cook. Chromatograms for quantitative determination of sugars were prepared by spotting a (3.5-inch sugar band 2.5 inches from the end of a 7.75 X 24 inch piece of Khatman S o . 1 filter paper. Experience shon-ed that the most satisfactory results were obtained n-hen a minimum of 2 to 3 mg. of each sugar was applied to the chromatograni. The chromatogram was placed in a developing tank and developed n i t h either a solution of 9 parts of ethyl acetate, 2 parts of acetic acid. and 2 part? of water b y volume (9:2:2); or of 10 part5 of 1-butanol, 3 parts of pyridine, and 3 parts of n-ater by volume (10 :3 :3 ) . The cliromatograms 11-ere deyeloped by the descending f l o ~method a t room temperature. Satisfactory separations of all sugars were effected using the folloir-ing procedure (solwnts in parentheses) : 12 hours (9:2:2). dry: 24 hours (9:2:2), dry; 12 hour; (10:3:3). dry; 24 hours ( 10 : 3 : 3), dry,
Table l.
Recovered, Mg. 2.65 3.18 2.96 3.02 11.32 3.24 3.11
D-Galactose D-Glucose D-Mannose L--irabinose D-Syloae L-Rhamnose D-Ribose
II.
The 9: 2 :2 solvent separates L-arabinose from D-mannose, while the 10:3 : 3 solvent separates D-glucose from D-galactose. A longer time in either system will aid the separations but Jvill cause the loss of the rapidly migrating L-rhamnose from the paper. The intermediate drying seemed to give sharper separations of the sugar bands. The final drying period should be a t least 24 hours, so that as much solvent as possible can be removed from the paper to avoid spurious results. The sugar bands were located by cutting longitudinal strips from each side of the chromatogram and spraying n-ith aniline hydrogen phthalate. These sprayed strips were dried in a n oven a t 105’ C. for about 5 minutes. After drying, the sugar spots xere easily detected. These sprayed strips were attached to the chromatogram; n i t h these strips as guides. the sugar bands were located on the unsprayed portion of the chromatogram. The horizontal sugar bands were cut from the chromatograni and the sugar wis eluted n ith distilled water from each piece of paper contaiiiing a sugar band. The sharpness of sugar separations and the detection of the sugar b y the de-
Quantitative Estimation of Simple Sugars with Periodate
Sugar
Table
DISCUSSION
Knorn, 11g.
Recovered,
Precision,
2.76 3.20 2.9G 3.07 11.36 3.17 3.13
96.0 99.4 100.0 98.5 99.6 102.2 99.4
Mg. 0.01 0.02 0.00 0.01 0.01 0.00 0.01
Comparison of Cutoff and Complete Elution Methods of Determining Sugar in a Paper Chromatogram
Standard Deviations Complete elution Cutoff method method
Detected, Mg.
Sugar
a
7c
9.6 3.2 D-Galactose 17.3 1.9 n-Glucose D-Mannose 5.7 1.6 5.6 1.9 L-Arabinose D-Sylose 49.6 0.6 4.8 0.5 L-Rhamnose Standard deviation calculated from four determinations
Table Ill.
0.66 0.53 0.38 0.16 0.13 0.22
Quantitative Chromatographic Sugar Study
(Strips eluted by the complete elution method) Recovered, Recovered, KnoR n, Sugar D-Galactose D-Glucose D-Mannose arabinose
D-XylOse L-Rhamnose
1440
Rlg. 24 2 19 7 13 7 23 5 414 1 9 5
ANALYTICAL CHEMISTRY
AIg 26 7 20 3 12 8 25 1 430 4 10 3
70
90.6 97.0 107.0 93.6 96.2 92.2
Standard
Dev., Mg. 1.5 1.2 1.2 0.7 17.1
0.2
scribed method was checked by spraying a previously marked chromatogram. The results were good unless there was a serious “dip” or “dome” in the sugar band. The dipping and doming were minimized b y intermittent drying, but irregularities due to the variability of the paper may occur. Preliminary studies of the hydrolyzed spent liquor from the neutral sulfite semichemical cook shoned that the D-xylose present represents about 82% of all the sugars present. Thus, it is difficult to keep the D-xylose titration in the optimum range of 20 to 30 ml. of sodium hydroxide and a t the same time obtain a satisfactory titration for the other sugars. The problem was alleviated but not solved by using 0.0025.1sodium hydroxide instead of 0.005?;, and increasing the amount of sugar aq niuch as possible without hindering the sugar separations. I n addition, the blank titration was reduced sodium hyfrom 1 to 3 ml. of 0.005-1droxide to 0.1 to 1.0 ml. of 0.0025-1- sodium hydroxide, b ~ eluting the sugar from the paper strips directly into the reaction flask rrith distilled water instead of eluting the sugar to the bottom of the strip and then cutting off that portion of the paper strip which contained the sugar. The blank titration of ethylene glycol and distilled water m s less than 0.05 ml. of 0.0025s sodium hydroxide. The sugars ivere eluted from the paper strips n-ith distilled water b y the niicroscope slide and Petri dish method used by Lea ( 2 ) . Lea eluted the sugar to the bottom of the paper strip, cut off that portion of the strip containing the sugar, and placed it in the reaction flask. This method gave rise to high blanks, which )$-ereprobably due to the oxidation of cellulose. Even n h e n the blanks were corrected for the area of paper transferred to the reaction flask, the serious errors persisted. The error becomes especially great \Then only small quantities of sugar are determined. This method was modified by eluting the sugars directly into the reaction flask, The elutions were carried out a t room temperature under a bell jar for 2.5 hours. The eluted strips were checked for complete sugar removal b y spraying the eluted piece of paper ivith aniline hydrogen phthalate. After the strip was dried, i t was observed under ultraviolet light and no sugar spots were detected. The quantity of sugar detected was calculated by the reference sugar method using Dribose as the reference sugar. These data recorded in Table I1 indicate t h a t the complete elution method yields a lower and more constant standard deviation. A known sugar solution which contained D-galactose, D-glucose, D-mannose, L-arabinose, D-xylose, L-rhamnose,
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 and the area of each spot is taken as changing the solvent systems. 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
