the tetraalkyltetrazenes in Figures 6 and
7. Because the reproducibility of either peak height or area measurements with amount of the tetraalkyltetrazenes was not so good as desired, the resolved solute fractions were analyzed by ultraviolet absorption techniques (Table I). More recently this problem was considered in greater detail because peak height and area data are often used to indicate amount of eluted components (2). I n Figures 8 and 9, peak height and area measurements for a sequence of experiments performed in random order were plotted as a function of amount of pure tetramethyl- and tetraethyltetrazene. The peak heights (Figure 8) were not directly proportional to amount as fiist believed; instead the nonlinear relationship persisted even though the tetramethyltetraaene was diluted t o 9.159% by weight with hexane. However, in Figure 9, a linear relationship was obtained between area measurements
and amount of tetraalkyltetrazenes even though the amount was varied by a factor of 100. Recently, Whatmough ( 5 ) observed similar phenomena in the gas-solid chromatographic separation of methane in air. For these experiments, the recorded chiomatograms became increasingly skewed as the sample size increased from 0.005 to 0.050 ml. With the larger samples, the relative change in peak height was rapidly diminished (Figure 8). This flooding condition often prevailed in preparative applications which required the separation of large amounts of material. Factors that should be considered in preparative rvork include: the collection time of the solute as related to the recorded chromatograms; the time required for the collection of each component; and possible condensation and subsequent contamination of the solute in the collection system. For problems similar t o those described, the characteristic elution time must be defined
as the time from the introduction of the sample to the point of its initial emergence as recorded by the detector response on the chromatogram. In addition, it was demonstrated that area measurements are a more valid criteria of amount of component than are peak height measurements. REFERENCES
(1) Jones, W. L., Kieselbach, Richard, ANAL.CHEM.30, 1591 (1958). ( 2 ) Karr, C., Brown, P. M., Estep, P. A,, Humphrey, G. L., Fuel 37, 227-35
(1958).
(3) Keulemans, A. I. M., "Gas Chroma-
tography," pp. 31-2, Reinhold, New
York, 1957. (4)McBride, W. R., Kruse, H. W., J. A m . Chem. SOC.79, 572-6 (1957). (5) Whatmough, P., Nature 182, 863-4
(1958).
(6) Zimmer, H., hudrieth, L. F., Zimmer, M., Rowe, R. A . , J. A m . Chem. SOC.
7 7 , i90-3 (1935). RECEIVED for review November 14, 1958. Accepted March 24, 1959. Division of Analytical Chemistry, 134th Meeting, ACS, Chicago, Ill., September 1958.
Colorimetric Determination of MethylcelI uI ose with Diphenyla mi ne GRACE KANZAKI and EUGENE Y. BERGER New York University Research Service, Goldwater Memorial Hospita 1, Welfare Island, New York 7 7, N. Y.
Methylcellulose may be determined in concentrations of 10 to 100 y per ml. by the intensity of color developed on heating with an acid solution of diphenylamine. The color has a maximum absorbance a t 640 mp and color development obeys Beer's law. Amounts of methylcellulose solution and diphenylamine reagent, the temperature, and duration of heating may b e varied within limits to obtain the desired extinction coefficients without affecting the reproducibility among multiple samples of the same concentration. The reaction is sensitive to temperature and for a given set of conditions there must b e a uniform bath temperature within 0.1 "C. during heating. The precision of the method is to k 2%.
has been determined by the anthrone reaction (13). Carbohydrate has been determined by this relatively simple procedure by the intensity of bluegreen color developed when a sulfuric acid solution of anthrone is added t o a solution of carbohydrate ( 7 ) . The heat required for the color development
is supplied by that evolved when concentrated acid is added to an aqueous solution. The method has a precision of to =t5% a t best (2, 14) and further improvement centers around control of the evolved heat. There are several procedures to control this variable, such as layering the acid under the aqueous solution and then mixing (5,10, 12) or making the addition in the cold and subsequently heating in a bath (9, 11, 14-16). The use of diphenylamine for the determination of methylcellulose has one advantage over the anthrone method. When a glacial acetic-hydrochloric acid solution of diphenylamine is added t o an aqueous solution, the heat evolved is insufficient to produce a color change (6). This report describes a procedure for the determination of methylcellulose with diphenylamine.
ETHYLCELLULOSE
REAGENTS AND APPARATUS
Methylcellulose solution. A standard solution of 0.4% is prepared by adding distilled water a t 80" t o 90' C. to 4 grams of methylcellulose and stirring t o disperse the methylcellulose. On cooling, the solution clarifies and is diluted t o 1 liter. Reagents.
Subsequent aqueous dilutions of 20, 40, 60, and 80 y per ml. are made. Diphenylamine reagent is prepared by dissolving 3.75 grams of colorless diphenylamine crystals in 150 ml. of glacial acetic acid and adding 90 ml. of concentrated hydrochloric acid (1, 8 ) . This is sufficient for 45 determinations and is prepared fresh prior to use. Zinc sulfate reagent, 100 grams of zinc sulfate seDtihvdrate. in 1 liter of 0.25N sulfuric acid. " Sodium hvdroxide. 0.75iV, Apparatus. Test 'tubes, borosilicate glass, 18 X 200 mm. This length allows the reactants t o sit deeply in the bath. Syringe for delivery of diphenylamine reagent, 10-ml. size, mounted in a holder so that a stop limits the nithdrawal of the plunger a t 5 ml. Glass tears. glass marbles, or flatheaded glass stoppers with the shank smaller than the inside diameter of the 18 X 200 mm. tubes t o cover tubes during heating. An oil bath which in the ranne of 105" to 110" C. will control the"temperature within 0.1 " C. The tubes containing the reactants are inserted and removed from the bath simultaneously using a rack sufficient for 40 tubes. Cuvettes, 18 x 150 mm. rimless borosilicate glass test tubes. VOL. 31, NO. 8, AUGUST
1959
1383
Spectrophotometer (Coleman llodel 6A). PROCEDURE
Five milliliters of dipheiij laniine reagent is added to 2 ml. of a n unknown solution (containing 20 to 80 y of methylcellulose per ml.) in an 18 X 200 mm. test tube. The reactants are miued, and the tubes are covered with glass tears, and heated at 108" C. for 30 minutes in a n oil bath. The rack of tubes is removed and the tubes are immediately cooled for approximately 10 minutes in cold water. The mixture is transferred to cuvettes and the color intensity is read in a spectrophotometer a t 640 mp against a similarly heated blank of 2 ml. of water and 5 ml. uf diphenylamine reagent. The heated tliphenylamine blank should not read less than 90% transmittance (usual reading is 95To?,) against distilled water. Tranqniittance less than 90% usually indicates deterioration of the diphenylamine crystals. With each heating. a set of standards of 20, 40. 60, and 80 y of niethylccl-
Table 1.
Application of Beer's Law to Color Development
lulose per nil. is included to calculate the concentration of the unknown. If protein is present, it is removed by the Somogyi zinc precipitant ( I ? ) prior to the addition of diphenylamine reagent. One milliliter of zinc reagent is added to a n appropriate dilution of methylcellulose to yield a concentration of 20 to 80 y per ml. in the final volume of precipitating mixture (10 to 20 ml.). Protein is then precipitated by the addition of 1 ml. of 0.75X sodium hydroxide. The mixture is let stand a minimum of 15 minutes and is then centrifuged (filter paper should not be used because it contributes a blank to the diphenylamine reaction). Two milliliters of the supernatant solution is added to 5 ml. of diphenylamine reagent.
The ratio of the volume of diphenylamine reagent to volume of methylcellulose solution map be varied, as well as the temperature of the bath and duration of heating. The absorbance will differ for each set of conditions. Ten sets of conditions are presented where the absorbance per microgram increases from 0.7 to 28.8 X lo3 (Table
EVALUATION A N D DISCUSSION
The color developed by the interact,ion of methylcellulose and diphenylimine has a maximum absorbance a t 640 mp (Figure 1). Color development in the concentration range of 10 to 100 y per ml. obeys Beer's law. For concentrations from 10 to 100 y per ml., absorbance per niirrograni per milliliter of standard solution varies =kl% froin the mean (Table I).
(Tv-o milliliters of methylcellulose in concentrations of 10 to 100 -, per ml. hratetl for 30 minutes a t 108" C. with 5 ml. of diphenylamine reagent dhsorhtlncr ,it
I 50
7
63
120
90
150
161
Time, minutes
Figure 2. Absorbance as function of duration of heating, temperature constant 2 ml. of 10,20, or 4 0 ' per ml. of methylcellulose heated a t 108.3' C. with 5 ml. of diphenylamine reagent for 10 to 1 8 0 minutes. Standards and blanks read against distilled water and appropriate blank subtracted prior to calculation of absorbance per y
640 nip)
Methylcellulose Concn
-~b~~lxmce Dev from -,,'nil, mean
~
~
-,/I11
x 103 x 10
10 20
20 40 40
60 60 80 80
90 1 88 8
173 9 177 2 347 354 320
523 710 694 878
103
9 01 8 88 8 TO
8 86
8 68
8 85
8 67
x
103
-0 21 4 0 08 -0 10 $0 06 -0 12 +O 05 -0 13 -0 08 $0.08
8 72 8.88 8.68 -0.12 8 78 -0.02 100 100 886 8.86 +0.06 Mean 8 80 +0.09' Av. dev. a9 7; of mean * l . 05 Sum of deviations disregarding sign divided by number of observations.
Table ii.
B:ttli Temp., O
c.
89.6
100.4 108.3
Duration
-
102
I10
106
Figure 1 . Absorbance as function of wave length 2 ml. of 4 0 y per ml. of methylcellulose heated 30 minutes a t 109.7' C.
Figure 3. Absorbance as function of temperature, duration of heating constant 2 ml. of 40 to 120 y per ml. of methylcellulose heated for 30 minutes with 5 ml. of diphenylamine reagent a t temperatures from 9 0 . 4 ' to 110.5' C.
5
5 10 5 12
15 6
10 5 5
.4bsorbance Mean No. of per y -4naly- Absorbance ses x 103 X lo3 0.70 20 224 20
10
10 10 10 10 10 19 20
321 333 372 411 422 466
502 355 576
4.02 8.33 9.30 10.28 10.56 11.64 12.54 17.75 28.81
20 2 20 2 120 Variance ( u 2 )divided by mean squared where uz = [Zz2/A' - 27 [.V/(S - l)]. Compared to 2 ml. methylcellulose d u t i o n plus 5 ml. reagent heated 30 minutes at 108.3' 60
1384
98
Ternperoture ' C
~~ethYlce1111lose Diphenylamine Reagent, M1.
of Heating, Concn., Amount, Min. *{/mi. ml. > 30 320 2 30 80 1 30 40 2 2 1 2
*
94
Absorbance and Reproducibility of Varying Volume of Reactants, Heating Time, and Temperature
2
a
1
95
ANALYTICAL CHEMISTRY
Av. Deviation, 5%
of Mean 1.59 1.00 2.61 1.24 2.00
0.088
0.204 0.583
0.99 3.47
2.415
1.28
0.244
2.10 2.15
c.
2 0.513 0.171
0.188
0,731 0.719
F d o *
FZ%
2.51
4.80 3.52 5.35
1.19
4.85 .
..
2.86
1.08 11.85 1.20
3.59 3.53
5135 5.35 5.35 5.35 4.86 4.80
11). When 10 ml. of diphenylamine reagent is heated with 2 ml. of 40 y per ml. methylcellulose solution, the absorbance is 357, higher than' that obtained Kith 5 ml. of reagent; the absorbance per microgram per milliliter of final solution increases 2.3fold. The use of 15 ml. of reagent does not further increase the absorbance per microgram per milliliter of the original solution. The combination of 2 ml. of methylccllulose and 5 ml. of diphenylamine reagent heated for 30 minutes a t 108.3' C. was selected because of the convenient volumes and concentrations in the experiments where the method mas applied (3, 4). The reproducibility among samples of the same concentration run as separate sets with different combinations of reactants, time of heating, and temperature mas evaluated and compared by the F test with the reproducibility of 2 ml. of methylcellulose heated with 5 ml. of diphenylamine reagent for 30 minutes a t 108.3' C. (Table 11). Varying volumes of reactants, heating time, and temperature does not alter the reproducibility among multiple samples, except for the use of 1 ml. of methylcellulose and 6 ml. of reagent. Here the variability among samples of the same concentration is greater than 2 ml. of methylcellulose and 5 nil. of reagent (Frat,,> F z q 0 ) . With temperature constant (108.S0C.),
the absorbance increases rapidly during the first hour of heating; a t 30 minutes, the absorbance is increasing a t the rate of 3.8% per minute (Figure 2). Further heating produces relatively small increases in absorbance only after 2 hours. Reproducibility among multiple samples of the same concentration was not improved by a 1- or 2-hour heating us. a 30-minute heating (Table 11). With time constant (30 minutes), there is a curvilinear increase in absorbance with bath temperature (Figure 3). The absorbance per microgram increases tenfold between 90' and 110' C.. and a t 108" C. there is a 8.7% increase in absorbance per degree rise in temperature. The temperature of the bath, as such, is not crucial, because a set of standards is included in each heating, but for reproducibility within a given heating, all tubes must be exposed to the same conditions for 30 minutes. These conditions are established by adequate stirring and by maintenance of uniform bath temperature within 0.1' C. The precision of the method is indicated by the average deviation of the absorbance per microgram as a per cent of the mean (calculated as in Table I). The per cent average deviations for 158 sets of standards Ivere summated and the mean of the series was +2.1%.
LITERATURE CITED
(1) Alving, A. S., Rubin, J., Miller, B. F., J . Biol. Chem. 127, 609 (1939).
(2) Berger, E. Y., unpublished observations.
(3) Berger, E. Y., Kanzaki, G., Homer, M. A., Steele, J. M., Am. J . Physiol. 196, 74 11959). (4) Berger,' E. Y., Steele, J. Id.,J . Gen. Physiol. 41, 1135 (1958). (5) Bridges, R. R., ANAL.CHEW24, 2004 (1952). (6) Dische, Z., Mikrochemie 7, 33 (1929). ( 7 ) Dreywood, Roman, ISD. ENG.CHEM., ASAL. ED. 18. 499 11946). (8) Harrison, €1: E., Proc. doc. Erptl. Biol. M e d . 49, 111 (1942). (9) Koehler, L. H., ASAL. CHEM.24, 1576 (1952). (10) Loewus, F. A., Zbid., 24, 219 (1952). (11) McCready, R. M., Guggolz, Jack, Silvera. Vernon. On-ens. H. S.. Zbid.., 22., 1156 (1950). ' (12) hlorse, E. E., Zbid., 19, 1012 (1947). (13) Sanisel, E. P., DeLap, R. A,, Ibad., 23, 1795 (1951). (14) Scott, T. A., Jr., Melvin, E. H., Ibid., 25, 1656 (1953). (15) Seifter, S., Dayton, S., Kovic, B., Muntwyler, E., Arch. Biochem. 2 5 , 191 (1950). (16) Shetlar, M. R., ANAL. CHEM..24, 1844 (1952). (17) Somogyi, M,, J . Biol. Chem. 86, 655 (1930). RECEIVED for review January 16, 1959. Accepted March 31, 1959. From the Department of Medicine, Sew York University College of Medicine. Research supported in part by a grant (A-311) from the Kational Institute of Arthritis and Metabolic Diseases, Public Health Service.
Stoichiometry of Chlorite-Aldehyde Reactions Analytical Procedures HERBERT
F. LAUNER and
YOSHIO TOMIMATSU
Western Regional Research Laborafory, Albany 7 0, Calif.
b Sodium chlorite showed no fixed stoichiometric relationship to aldehyde groups in reactions with aldoses, benzaldehyde, and dextran dialdehyde, not because of overoxidation but presumably because the unstable chlorine intermediates react with chlorite to varying extents. Stoichiometric ratios varied with aldehyde, kind and concentration of buffer, reaction rate, and other factors. Phosphate buffer 0.5M yielded ratios of 2.63 to 3.77, limiting its usefulness to known aldehydes or to those whose oxidation rates resemble those of known aldehydes. Phosphate buffer (3M) yielded ratios ranging from 2.56 to 2.79, permitting the use of a mean value in the analysis of unknown aldehydes including polysaccharides, with an uncertainty near
=t2.6% a t 0.06 mM aldehyde. Analytical procedures, determinations of ratio-time curves, and residual aldehyde concentrations are discussed.
I
attempt to develop an acidic micromethod for the determination of aldehyde groups in alkali-sensitive polymeric carbohydrates, glucose was initially used as a model substance. The results of studies of the reaction between phosphate-buff ered sodium chlorite (h'aCIOz) and glucose, from the practical analytical (5) and fundamental kinetic (6) standpoints have been described. Chlorite was adaptable to a precision of a few per cent a t 5 y of glucose per ml. with a corresponding precision to 0.6 y per ml. K AK
Using acetate-buffered chlorite a t a higher concentration, Launer, n'ilson, and Flynn ( 7 ) studied various procedures for determining glucose and, to a limited extent, cellobiose, melibiose, maltose, and lactose, and developed both photometric and volumetric methods. Stitt, Friedlander, Lewis, and Young (10) used a photometric procedure for applying chlorite to the determination of glucose in the presence of an excess of a ketose and fructose, and discussed the theoretical and quantitative aspects of the reactions involved. I n no study was overoxidation of the aldehyde group past the carboxyl stage observed. This conclusion appears warranted, although reagent decomposition during and after oxidation complicated the results. It is also in VOL. 31, NO. 8, AUGUST 1959
1385
