chromatography (with silica gel) the paraffinic-naphthenic fraction can readily be segregated from the aromaticsulfur rompound fraction, but it is clifficult to separate all the sulfur compounds froin the aromatics. The distance bet\\ een the thiophene and aromatic line< in Figure 1 indicates that this separation is possible. Further experimentation, a t temperatures other than 84" C., with hydrogen sulfide, and dimethyl, diethyl, and diisopropyl tliwlfide indicated that the nitrile column muld resolve these compounds from other materials in their respective boiling ranges. Satisfactory resolutions can he made a t about 30' C. for hydrogen sulfide, whereas temperatures of about 90" to 95" C'. nould be desirable for the disulfides. For comparatire purposes, isopropyl alcohol was tried in the nitrile column a t the same conditions at which isopropyl mercaptan was run, and it was found that the alcohol had a retention volume of 549 nil. as compared with 117 mI. obtained nith the mercaptan. Other alcohols yielded correspondingly
large retention yolumes. The nitrile column has definite possibilities in determining alcohols and perhaps other oxygenated compounds in presence of hydrocarbons. The selection of lvhite oil as the sol\-ent for volatility delineation was not a wise choice because many of the peaks Fere unsymmetrical and excessively high retention volumes were obtained for materials boiling above 126" C.; consequently, compounds examined in this study were limited to those boiling below that temperature. Even though the correlations shown in Figure 1 are based upon only two or three points, the data indicate that &p'iminodipropionitrile is an excellent solvent to resolve a number of sulfur compounds of the same boiling range. Its resolving ability is due to its marked electron-donating properties, since all of the electrophilic compounds tested were found to have high retention volumes. Several other solvents should be better than the white oil. Squalane should afford better volatility differentiation, and dinonyl phthalate (6) or tricresyl
phosphate (1) should offer possibilities in resolving the sulfides from the primary normal mercaptans. ACKNOWLEDGMENT
The author thanks the Humble Oil & Refining Co. for permission to publish this paper, Marjorie T. Walker for her experimental assistance, and W. C. Jones, Jr., for his suggestions concerning gas-liquid partition chromatographic techniques. LITERATURE CITED
(1) hmberg, C. H., Can. J. Chem. 36, 590 (1958). (2) Coleman, H. J., Thompson, C. J., Ward, C. C., Rall, .4. T., As.4~.CHEW 30, 1592 (1958). (3) Desty, D. H., Whyman, B. H. F., Zbid.,29, 320-9 (1957). (4) Karchmer, J. H., Zbid, 30, 80 (1958). (.5 ,) Rvce. S. -4..Brvce. W. -4..Zbid.. 29.325 (1957). (6) Spencer, C.'F., Bauman, F., Johnson, J. F., Zbid., 30, 1473 (1958). 1
"
RECEIVEDfor review January 19, 1959. Accepted .4pril 20, 1959.
Preparative Aspects of Gas-Liquid Chroma tog raphic Separation Quantitative Determination of Tetraalkyltetrazenes EVERETT M. BENS and WILLIAM R. McBRlDE Chemistry Division, U. S. Naval Ordnance Tesf Station, China lake, Calif.
bA
method for the gas-liquid chromatographic separation and determination of some tetraalkyltetrazenes, empirical formulas C4H12N4 to C16HaN4 was used to analyze three-, six-, and ten-component mixtures of tetraalkyltetrazenes, which were formed after oxidation of mixtures of 1 ,1 -dialkylhydrazines in acid solution. The quantitative separation of known mixtures of tetramethyl-, 1,I -diethyl-4,4dimethyl-, and tetraethyltetrazene and their recovery as pure compounds were substantiated by ultraviolet absorption techniques. The relationship between area and peak height measurements with amount of tetraalkyltetrazene introduced on the column is discussed; special consideration is given to conditions of flooding as it relates to preparative work.
A
the coupling mechanisni of the ionic intermediates (R2N+= NH) formed in the oxidation of the LTHOUGH
1,l-dialkylhydrazines (R2T\"H2) in acid quantitative media has been studied (4, data for separation of mixtures of tetraalkyltetrazenes (R2Sii+=NNR2) thus produced had not been obtained. The quantitative separation and analysis of mixtures of tetraalkyltetrazenes were desirable to clarify the dimerization mechanism of the ionic intermediates. Separation by distillation was difficult, because these materials may decompose below their boiling points. The development of a gasliquid chromatographic method provided a means for the quantitative separation and determination of these tetraalkyltetrazenes. Ultraviolet analysis techniques, rather than peak height or area measurements, were used to determine the distribution of tetraalkyltetrazenes formed in the reactions. APPARATUS A N D REAGENTS
CHROMBTOGRAPH. A Perkin-Elmer Model 154C Vapor Fractometer was
modified by the replacement of the sample collection line with a 1/8-inch asbestos-covered copper tubing whose orifice was closed with sintered stainless steel to permit greater dispersion of the gas bubbles. The detector response was recorded on a 5-mv. Leeds & Northrup Speedomax G recorder having I-second response and a chart speed of inch per minute. PARTITION COLUMXS.Perkin-Elmer 2-meter K and F columns used for initial work were not so satisfactory as a poly(ethy1ene glycol) column (loyo by weight on 20 to 35 mesh C-22 firebrick), 43 inches in length. The poly(ethy1ene glycol) (average molecular weight 400 from the Gemex Chemical Co.) ri-as dissolved in sufficient n-pentane t o cover the firebrick; the solvent mas then carefully evaporated under vacuum with agitation t o provide even coating of the support. The 1/4-inch diameter stainless steel tubing was mechanically vibrated to ensure proper packing while being filled and then bent to the proper shape. Helium carrier-gas flom- rate of about 80 ml. per minute \Tas obtained a t 2 p.s.i.g.; the VOL. 31, NO. 8, AUGUST 1959
1379
column temperature was maintained a t 70" C. MICROSYRINGE.A 0.050-ml. Hamilton hfiniature KO. 705 inicrosyringe mas calibrated with water. A mean of six determinations of 0.0100-1ii1. samples was 0.01001 gram with a standard deviation of 0.00032 gram or an error of 3.2%. Data on 16 samples of 0.0050 to 0.0300 ml. gave a standard deviation of 0.00034 gram from the linear regression line, SPECTROPHOTOMETER. All absorliancc measurements were made a t room teniperature with a Cary Model 11 11s recording spectrophotometer using 1-cni. quartz cells. REAGENTS. Most 1,l-dialkylhr-drazines used in the preparation of tetraalkyltetrazenes were available from Westvaco Chlor Alkali Division, Food Machinery & Chemical Corp., or Conimercial Solvents Corp., or n-ere prepared by the reduction of the corresponding nitrosodialkylamine (6). Pure tetraalkvltetrazenes were p r e p a r d as described (4). All reagent grade chemicals were used without further purification.
IO0
was made to collect the solute in these later experiments,
,
r 32 TMT
90
RESULTS A N D DISCUSSION
Several tetraalkyltrtrazenes have been separated and the solutes recovered quantitatively by gaq-liquid chromatographic techniques. The relationship hetn een the recorded chromatographic elution ciii\c+ obtained ttt TO" C. and the solute distribution for 0.010-ml. samples of tetraniethyltetrazene (TMT), 1 , l - diethyl - 4>4 - diriiethyltetrazene (DEDMT), and tetraeth? ltetrazene (TET) is slionn 111 Figures I , 2 , and 3. The eluted niatriial collected a t time intervals of 0.5, 0.5, and 1.0 minute, respectively, and the quantity of tetraalkyltetrazene measured by ultraviolet analysis. In these experiinents, the recover)' was 8.81 mg. (theory 8.90 nig.) for tctraniethyltetrazene, 8.22 nig. (theory 8.77 mg.) for l,l-dieth\~l-~,.l-ttil!ieth\-ltctrazene,and 8.30 mg: (theory 8.6; mg.) for tetraethyltetrazene; these tetmalkyltetra-
80
70
5
60
0 !J A
50 L
L Y
40
-J
30
PO
10
8
PROCEDURE
Ifixtures of tetraalkyltetrazenes n-ere prepared by neutralization of acid solutions containing the ionic intermediates formed by the oxidation of two or more 1,l-dialkplhydrazines 11-ith potassium bromate a t about 0" C. The tetraalkyltetrazenes were estracted with n-pentane and concentrated by partial evaporation of the n-pentane at temperatures up to 80" C.
A 0.010-ml. sample of the tetraalbyltetrazenes was introduced through the septum of the instrument with a microsyringe, and the recorder deflection attenuated to provide maximum peak height and area measurements. The solute fractions were collected in 20 ml. of absolute ethyl alcohol for subsequent absorbance measurements, For the determination of solute distribution, the collection tubes were changed a t specified time intervals, while in other experiments they were changed when the recorder trace indicated the appearance of another component. The solute fractions mere transferred to a 25-1111. voluinetric flask and diluted to volume. The concentration of tetraalkyltetrazencs was determined by comparison of the absorbance of the sample with a standard linear calibration curve obtained from the absorbance of pure compounds a t known concentrations. The purity of the resolved tetrazenes was established by the ratio of the absorliance in basic aqueous solution a t 277 and 248 mp. If the absorbance was low in the aqueous solution, preference was given to the measurements made in absolute ethyl alcohol. The relationship between peak height and area measurements with the amount of tetramethyltetrasene and tetraethyltetrazene was determined by varjing the sample size introduced on the column from 0.005 to 0.050 nd. No attempt 1380
ANALYTICAL CHEMISTRY
Figure 2. Solute distribution of 1 , l -diethyl4,4-dimethyltetrazene
'Mi
u
*i
it \\as repeatedly demonstrated that losses were within the error expected due to pipetting so that the losses in these experiments must be due to the numerous changes of solute collectors. I n this discussion, the elution time is that from the introduction of the sample through the septum in the instrunient to the initial appearance of the sample component on the recorded elution curve ( I ) . The time of the niaxiniuni peak height for these niaterials is dependent upon the amount of sample introduced in the column. For example, in the case of tetraethyltetrazene, 0.005 ml. took 15.7 minutes and 0.050 nil. took 26.9 minutes from its introduction to the time of maximum elution; yet the time of its initial appearance was 12.2 and 12.3 minutes, respectively.
..I0.010-ml. sample of a synthetic mixture containing tetramethyltetrazene, l,l-diethy1-4,4-diniethyltetrazeneJand tetraethyltetrazene was resolved into its components as indicated in Figure 4. The first peak, due to air and n-pentane, is a t sensitivity 1, while the other peaks are a t the attenuation indicated. The ultraviolet absorbance of the original mixture and that of the separated fractions are given in Figure 5. Points on the mixture curve correspond to the sun1 of the absorbance for each of the fractions a t that wave length. Purity of the resolved components was estimated by coniparison of the ratios of the abqorbance of the basic aqueous solutions of the pure compounds with that of the separated fractions a t 277 and 248 nip. These ratios were 1.364, 1.019. and 0.604 for the pure compounds
'O
i
60
-
1
z
$'
I
501 Y
0
32
.. I
Figure 3.
Solute distribution of tetraethyltetrazene
in basic aqueous solution as contrasted with 1.39, 1.09, and 0.66 for the isolated fractions. However, in the latter case, the effect of the 10% ethyl alcohol on the absorbance was sufficient to change the ratio from 0.604 to 0.650. The ultraviolet analysis indicated 40.7% tetramethyltetrazene (theory 41.9%),
32.7%
l,l-diethyl-4,4-dimethyltet-
razene (theory 29.8%), and 29.4% tetraethyltetrazene (theory 28.3%). The total of 102.8% is within the error expected from pipet calibration, although there was some evidence of contamination of the second and third fractions. Attempts to collect the solute in distilled water and 50% ethyl alcohol were unsatisfactory because of incomplete retention of components. The data for the separation and determination of the tetraalkyltetrazenes
which were formed after oxidation of mixtures of 1,l-dialkylhydrazines are given in Table I. From experiments 1 to 7, the effect of mole ratio of the reactants 1,l-dimethylhydrazine and 1,l-diethylhydrazine on the relative amounts of tetramethyltetrazene, 1,ldiethyl-4,4-dimethyltetrazene, and tetraethyltetrazene could be determined. The elution times for all components were within the error of reading the chart paper, regardless of attenuation, which was varied by a factor of 4 in some cases; the peak heights and areas obtained from this data indicate a nonlinear relationship of peak height but linear relation of area with concentration as measured by ultraviolet absorption over the range studied. Other experiments relating peak height or area to quantity will be discussed
Chromatographic Separation and Analysis of Tetraalkyltetrazenes
Table I.
111
2 2/1
Elution time, min. Attenuation Peak height Aread Analyses Ethyl alcohol, mg. Aqueous, mg.
3 3 32 21 9 52 6
3.2 32 32.6 110.8
1 413 1 338
2,450 2.473
Elution time, min. Attenuation Peak height Aread rlnalyses Ethyl nlcoliol, mg. Aqueous, mg.
6 3 16 49 3 285 9
6.4 32 21.3 272.6
3 475
3.100 3.150
1
Mole Ratio ( D l I H / I l I < H )
later. In experiments 9 and 10, the data for the separation of six and ten tetraalkgltetrazenes formed by the oxidation of three and four 1,l-dialkylhydrazines in equal mole ratios, respectively, are given. The chromatographs for these experiments are presented in Figures 6 and 7. I n Figure 6, experiment 9, the isomeric pair, tetraethyltetrazene and l,l-dimethyl-4,4-din-propyltetrazene (DMDn-PT) was not completely resolved, although the shoulder is indicative of the presence of t n o compounds. The analysis of this isomeric mixture was obtained by the solution of simultaneous equations based on the absorbance data for the pure compounds. Other compounds not prcviously considered include 1,l-diethyl4,4-di-n-propyltetrazene (DEDn-PT) and tetra-n-propyltetrazenc (Tn-
3 413
Experiment No. 3 4 5Q 6 1/ 2 111 111 3/ 1 Tetramethyltetrazene 3.4 3.3 3.2 3 2 64 16 32 32 33.7 8.9 18.7 19 9 49 05 18.7 50.49 143 3 0.530
(0.595)
1.263 1.313
1.285 1.310
1,l-Diethyl-4,4-dimethyltetrazene 6 3 6 3 6 3 16 16 32 38 7 22 2 36 3 270 8 257 1 220 6
2.675 3.338 2.975 2.713 3.313 3.083 Tetraethyltetrazene 12 3 12.3 12 5 8 8 16 50 0 31 7 12 0 575 0 336 0 312.0
3.288 3 325 6 4 32
I
1 '3
3 4
4 43 1 14 0 0 314 (0 340) G I
251 1
16 32 0 192 0
2 868 2 888
2 450 2 325
18 2
-
9t 1!1
10 c
111
3 3 8 26.8
17 4
3.2 8 11:3,4 8.0
0 474 0 485
(0.275)
0,231
6 3
6.2
8
8
13.2 43 . G
28 8 100 8 1 268
1 263
0.595 (0.658)
Elution time, min. 12 3 12.3 12 2 12 2 12 3 12.1 Attenuation 4 8 8 16 4 8 Peak height 16.6 35 9 2.3 3 18 4 8 est 4 0 est 731 6 Aread 366 2 167.7 109 5 96 4s Analyses Ethyl alcohol, mg. 3 413 1 860 1.700 2 388 1.100 0 729 4 250 (0 740' 0.:340 2 325 Aqueous, mg. (1.220) 3 470 2.175 1 763 4 375 0 730 0 713 ( 0 370) a 1,l-Dialkylhydrazines osidized separately, solutions mixed. and neutralized to form tetraalkyltrtritzeries. * Three reactants in eqiial mole ratio, 1,l-dimethyl-, 1,1-diethyl-, and 1,l-di-n-propylhydrazine. e Four reactants in equal mole ratio, all in * and, in addition, 1,l-di-n-butylhydrazine. ilrea based on peak height at attenuation of 32 for TMT, 16 for DEDMT, and 8 for TET. Width at one half peak height is assumed to be same regardless of attenuation.
VOL. 31, NO. 8 , AUGUST 1959
1381
scribed column or two ne\T columns 42 inches in length containing 15% poly(ethylene glycol) or propylene glycol on 42- to 60-mesh C-22 firebrick. However, equal mole mixtures of the two symmetrical compounds, tetraethyl- and 1,4 - dimethyl - 1,4 - diisopropyltetrazene, were partially separated on the new poly(ethy1ene glycol) column. Although the chromatographic separation of the tetraalkyltetrazenes formed in the oxidation reactions of mixtures of 1,l-dialkylhydrazines was achieved, the problem was complicated by their high boiling points (or low vapor pressures a t ambient temperatures) as contrasted to their decomposition a t temperatures as low as 100" to 200' C. In these examples the empirical formulas of the tetraalkyltetrazenes varied from C4HL2N,to ClJl3~PIT4in going from tetramethyltetrazene to tetra-n-butyltetrazene. The large difference in vapor pressure of these tetraalkyltetrazenes was of great concern in the collection of the solute fractions a t the exit orifice. In experiments 9 and 10. the total recovery of the samples in the time allowed was 86.6 and 73.6%, respec-
l e
U W
4
m
I C
LL
ro 4
05
0 0
3,
220 2 4 0 260 280 300 320 WPVE LENGTH, MILLIMICRONS
200
Figure 5. Ultraviolet analysis of separated fractions of tetraalkyltetrazenes
tively. In spite of the insulation of the exit tube with asbestos, condensation of the higher homologs probably occurred in the exit line. Because the exit line on the Perkin-Elmer Vapor Fractometer is not heated to the temperature of the column, general use of this instrument for preparative work is not recommended without suitable modifications. Others (1) have had similar difficulties and have provided additional heat to the exit lines. The stringent temperature requirement which prevented the increase of temperature to a point where all the components in the mixture would have a reasonably high vapor pressure led to some interesting observations. Preliminary calculations suggested that even with tetraethyltetrazene, 5 to 10 minutes would be required for the volatilization of a 0.0100-ml. sample under the described experimental conditions. For liquid mixtures of higher homologs the differences in elution times might not exceed the time required to vaporize the components in the sample. It was believed that such conditions existed in the separation of several of
20 0-
- -. . 5C
48
46
51
54 56
58
6C
64
62
66
68
70
72
78
8 '
-4
84
80 82
86 88 90
-
2
1
€
E
0
12
I4
6
18
20 22
24
26
28
30
32
34
36
38
40
u 42 44
-1ME. MIN
150 ,52
54
56 I58 160 162
I64 166 I68
I10 I72
,74 ' 7 6 I78 '80 ~
~--
~ a b , 3-~-46
~
30 52
48
ANALYTICAL CHEMISTRY
58
60
62
64
66
68 70 72
16
14
80 8 2
78
84
86 88 90
@Eon-P DEDM
2
PT). In Figure 7, experiment 10, only one isomeric pair, ll1-dimethyl-4,4-din-propyltetrazene and 1,l-di-n-butyl4,4-dimethyltetrazene (Dn-BDMT) , was not resolved. Additional tetraalkyltetmzenes formed in this reaction include 1,l-di-n-butyl-4,4-diethyltetrazene (Dn-BDET), 1,l-di-nbutyl - 4.4 - di - n - propyltetrazene (Dn-BDn-F'T) , and tetra-n-butyltetrazene (Tn-BT). T o determine the column's capability to rcsolve nearly identical isomeric tetraalkyltetrazenes, equal mole mixtures of 1,l-diethyl- and 1-methyl-lisopropylhydrazine were oxidized as described. The products of this reaction contained three isomeric compounds, tetraethyl-, 1,l - diethyl -4methyl - 4 - isopropyl-, and 1,4dimethyl - 1,4 - diisopropyltetmzene, with the mole concentration of the unsymmetrical product twice that of the two symmetrical compounds. S o chromatographic separation of this mixture was apparent on either the above de-
56
54
w
Figure 6. Chromatographic separation of tetraalkyltetrazenes formed from a mixture of three 1 ,I -dialkylhydrazines
1382
48
p~~
5
0
38 40 142 I44 4 6
-
~~
0
I
136
w-
4
6
8
'0
I2
14
16
8
20 22 21 TIUE MIN
26
28
30
32
34
36
18 40 12
44
Figure 7. Chromatographic separation of tetraalkyltetrazenes formed from four 1 , I -dialkylhydrazines
Figure 8. Correlation of peak height to amount of tetramethyl- and tetraethyltetrazene Upper coordinate 9.1 59% tetramethyltetrazene
Figure 9. Correlation of area to amount of tetramethyl- and tetraethyltetrazene Upper and right coordinates tetramethyltetrazene
are
9.159%
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 at 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
