knowledge of the, anion composition. Where discrepancies exist between the direct group determination and the number of anionic sites, attention is directed to the possibility of unsuspected anionic groups or of configurations t h a t shield a group from interaction with the C P C . Other polyanions known to form precipitates with CPC are the tissue polysaccharides chondroitin sulfate, hyaluronic acid, and heparin; the
bacterial polysaccharides coded B-1973 and B-1459 by the G.S. Department of Agriculture; the carboxylic acids pectin, algin, and carboxymethylcellulose. A single sample of sodium carboxymethylcellulose was determined by this procedure to have a D.S. (degree of substitution) of 0.73 compared to a D.S. of 0.80 when analyzed by the manufacturer (Hercules Powder Co.) using his conventional technique for carbosymethyl groups.
LITERATURE CITED
(1) Graham, H. D., Thomas, L. B., J . Food Scz. 27 ( l ) ,98 (1962). ( 2 ) Scott, J. E., Chem. Ind. (London) 1955,
168.
Marine Colloids, Inc. Rockland. Maine
LEOXARD STOLOFF~ JOHN BLETHEN
Present address, U. S.Food and Drug Administration, Bureau of Scientific Research, Boston, Mass.
Determination of Total Carbon in Water by CombustionGas Chromatography SIR: The need for a rapid sensitive method for the determination of carbonaceous matter in aqueous solutions led to the development of a combust'ongas chromatographic method. Wet oxidation techniques generally used for this determination suffer from incomplete oxidation of the organic matter and interferences by salts (1, gj. A combination dry combustion-infrared method for the determination of organic substances in mater has been reported by Van Hall, Safranko, and Stenger ( 5 ) , and a combustion-gas chromatographic method fcr the determination of carbon in hydrogen peroxide has been reported by Nelsen and Groennings ( 3 ) . A combination dry oxidation and gas chromatographic method was chosen because of the near universal applicability of dry oxidation to organic compounds and the speed, simplicity, and sensitivity of the chromatographic measurement of the CO,. In the
Table I. Recoveries by CombustionChromatographic Method
Sample, wt. % in water6 Methyl alcohol, 0.79 Methvl alcoho1,"O 079 Butyl alcohol, 0 81 Dioxane, 1.03 Sucrose, 1.10 Benzene, 0.180h
Trichloroethylene,
C, wt. %
Added Found 0 296
Recovery,
97,
0 306
103
0 0296 0 029
98
0 525 0 561
0.463
0 528 0 508 0.400
101 91 86
0.166
0.161
97
O.llb 0 020 0 027 135 Distilled water 0.000 0 004 Sample size 10 pl. in all caaes. Saturated solutions at 25' C. Concentrations calculated from solubility data. Q
2194
ANALYTICAL CHEMISTRY
method described here the gas chromatograph replaces the adsorption train in the usual dry oxidation determination of carbon in organic samples. Instead of heating a weighed sample in a boat, we chose dynamic sample introduction in which the liquid sample is injected from a syringe onto a hot copper oxide bed that is continuously swept by the chromatographic carrier gas, helium. Sundberg and hIaresh (4) have demonstrated quantitative oxidation of a wide variety of compounds with hot copper oxide in a helium atmosphere. A method is described that permits determination of as low as 0.004 wt. % carbon in a 10-pl. sample with adequate reliability for many applications. The analysis rate is about 10 to 12 samples per hour.
was injected. The chromatogram (Figure 1) exhibited two peaks, the sharp COn peak followed by the tailing H 2 0 peak. The chromatograph was calibrated with standard mixtures of alcohols in water. A plot of the chromatographic peak area us. the wt. yoC in the standard yielded a linear calibration curve. Standardization by peak area instead of peak height was required because some compounds, such as sucrose, gave lower, broader peaks than the more volatile compounds. The peak broadening was attributed to carbonization and slower reaction with the copper oxide. RESULTS A N D DISCUSSION
Recovery data for known mixtures are summarized in Table I. Although the recoveries for some compounds were
EXPERIMENTAL
Apparatus. The apparatus consists of a copper oxide combustor (Perkin-Elmer part No. 154-0185) attached to a gas chromatograph (1st stage of Perkin-Elmer Model 1%). The combustion chamber was a stainless steel tube (0.25-inch o.d., 0.18inch i d . , 6 inches long) packed with cupric oxide wire (Fisher C-474). The power supply and temperature read-out for the combustor were provided by a Perkin-Elmer power supply (part No. 154-0187). The insulated combustion chamber was heated to 750" C. The sample injection port was a 1'4-inch Swagelok tee equipped with a silicone rubber septum and attached directly to the vertically mounted copper oxide combustor. The chromatographic column was a stainless steel tube (3/16-inch i d . , 79 inches long) packed with 20% Carbowax 1500 on Celite. .i helium flow rate of 100 cc. per minute was employed and the column and detector were maintained a t 110" C. Procedure. After the equipment was brought to the proper temperature and a steady base line was obtained on the recorder, a 10-p1. sample
gt 8[
x4
j4
cy
s 7 E
E 6-
i
5-
5
4-
IY
x4(or x64) means that the,reol peok height is 4(or64) times that shown
L
Q
c
i3
3-
2-
'k,
OO
Y
I Time after Sample Inject, min Figure 1. Chromatogram of 1 .OO vol. yomethyl alcohol in water (0.30wt. C); 1 0-PI. sample injected
70
not quantitative, they were adequate for our application. Refinements in the design of the combustion chamber may be necessary if improved recovery is required. Design criteria for similar combustion chambers are discussed in detail in references ( S ) and ( 5 ) . The relative standard deviation was 3.1y0 at the 0.3 wt. 7, carbon level and 8.27, a t the 0.004 wt. yocarbon level. S o interference is expected from halogens, sulfur, or nitrogen in the organic compounds, or from ionic species in the
solutions. Halogens, SOs, and NOz produced on oxidation of the sample would be irreversibly adsorbed on the chromatographic column or separated from the CO, by the column and would appear a t the same place as the water peak. LlTERATURE CITED
(1) Bertram, F. W., Carlisle, 0. T.,
Murray, J. E., Warren, G. W., Connell, C. H., ANAL.CHEM.30, 1482 (1958). (2) Moore, W. A,, Kroner, R. C., Ruchhoft, C. C., Ibid., 21, 953 (1949).
(3) Nelsen, F. M., Groennings, S., Zbzd., 35f 660 (1963). (4) Sundberg, 0. E., Maresh, C., Ibid., 32, 274 (1960). (5) Van Hall, C. E., Safranko, J., Stenger, V. A., Ibid., 35, 315 (1963).
D. L WEST
Savannah River Laboratory E. I. du Pont de Nemours & Co. Aiken, S. C. The information contained in this paper was developed during the course of work under contract AT(O7-2)-1 with the U. S. Atomic Energy Commission.
Study of Two Crystalline Forms of 6-Chloro-2-Methoxy9-Thiolacridine SIR: Supplementing our previous researches ( S ) , we have investigated the two crystalline forms of the organic complexing reagent 6-chloro-2-methoxy9-thiolacridine. Thiolacridine was reported by Das Gupta ( 2 ) to occur in two different forms: upon recrystallization from 2yo sodium hydroxide solution, thiolacridine slowly precipitated as orange needles; from 9570 ethyl alcohol, it precipitated as deep red crystals in the form of flat plates. The red crystalline form readily dissolved in sodium hydroxide solution and was crystallized from this solution in the form of orange needles. Solution and recrystallization of these orange needles from 957, ethyl alcohol, or heating them on a steam bath, transformed them to the deep red form. As a possible explanation, Das Gupta suggested that one form of thiolacridine was thioenolic and t,he other thioketonic. Both crystalline forms have a melting point of 245" C. R e have found t h a t an equal mixt'ure of the two crystalline forms has the same melting point as the separate forms. This indicated that the two forms were identical at 245" C. The red crystals had a molecular weight of 264 (calculated value of 276) by the Rast method with camphor as the solvent. Calcd. for the red crystals of 6-chloro-2-methoxy-9-thiolacridine (CiJIioOK'SCl): C = 60.98, H = 3.66, S = 5.08, C1 = 12.86. Found: C = 60.84, H = 3.63, N= 4.98, C1= 13.05. X-ray powder diffraction patterns taken at 23" C. were obtained for the two forms (Tables I and 11). The x-ray patterns were run with a Philips x-ray diffraction unit, a copper x-ray tube, and a nickel filter, with the following settings: scanning speed = 1' per minute; kilovolts = 40; milliamps = 20; geiger tube if1525; scale factor = 4 ; multiplier = 1 ; time constant = 4; divergent slit = 1"; scatter slit = 0.006'; and
receiving slit = 1". The two forms have entirely different diffraction patterns and hence different crystalline structures. The pattern for the orange form was more complicated, indicating a more complicated crystalline structure. Thermo!ysis curves established that the orange crystals contained one molecule of water for each molecule of thiolacridine. When the orange needles were heated overnight a t 100" C. they were converted to the red plates. Water began to come off a t 75" C. and a t 100' C., conversion to the red crystalline form was complete. This form was anhydrous on the basis of its thermolysis curve and chemical analysis. Infrared curves were next taken on the two crystalline forms with the KBr pellet technique in a further attempt to obtain evidence of a thiol or thione structure. The preparation mixtures contained 4 to 5 mg. of sample per 400 mg. of KBr. A KBr pellet was run as a blank; it contained no water. Our results are similar in nature to those obtained by Ayres and McCrory ( I ) , who investigated 2,3-quinoxalinedithiol. A thiol would be expected to show an absorption band associated with the S-H stretching frequency at 2500 to 2600 cm.-l; no such band appeared in either spectrum. The thione form should show the stretching frequency of the N-H a t about 3400 cm.-'; a strong band appeared at this frequency in both the orange and the red forms of thiolacridine. The thione form should produce strong absorptions in the 1470 to 1500 ern -l region, which has been assigned to the thioureide structure (4); strong absorptions appeared in this region in both infrared spectra. No definite assignment to these latter absorption bands can be made because of the lack of data on acridine derivatives and the complexity of the infrared spectra in this region. The spectra of
Table I. X-Ray Diffraction Pattern of Orange Form of Thiolacridine 213
d
8.8 10.2 10.8 16.0 18.0 19 5 20 9 22.0 22.6 23.7 24.0 25.0 25 3 25.7 26.5 27.4 30.3 32.1 32.5 34.1 35.0 36.1 39.1 40.6 42.9 46 4
10.4 8.66 8.18 5.53 4.94 4 56 4 25 4 04 3 94 3 76 3.71 3 56 3 52 3 46 3 36 3.25 2.95 2.79 2.75 2.63 2.57 2.49 2.31 2 22 2 11 1 96
Rel. intensity 11 100 23 18 17 18 11 65 18 36 24 32 __
.
20 35 45 97 20 ~. 18 14 12 18 20 11 __
14 14 38
Table (I. X-Ray Diffraction Pattern of Red Form of Thiolacridine 28
d
Rel. intensity
8.1 ii.7 13.0 16.9 20.6 21.9 23.4 23.7 24.8 26.4 27.6 28.0 28.7 33.4
10.89 7.58 6.78 5.24 4.31 4.05 3.80 3.76 3.59 3.37 3.23 3.18 3.11 2.68
88 __ 25 15 18 100 15 11 19 98 92 14 14 18 12
VOL. 36, NO. 1 1 , OCTOBER 1964
2195
