proved concentrations from the 50-x8 resin and 0.15 gallon per minute per square foot rate used in preliminary runs. Resolution was not significantly affected by either resin or flow rate used. Trailing of constituents into adjacent fractions was more pronounced with 50-x8 resin and the 0.15 gallon per minute flow rate. When the column charge was 3% of the resin volume, a pure glycerol fraction was obtained. I n Figure 2 the infrared spectra of this glycerol(I1) are compared with that of C.S.P.glycerol(1). Resolution of other fractions was incomplete, but 68% of the erythritol charged was obtained in a fraction of
LITERATURE CITED
(4) Lambert, M.,Neish, iz. C., Can. J . Research 28 (3), 83 (1950). ( 5 ) Metayer, G. L., Ann. Chzni. (12) 2, 790 (1947). (6) Moore, W E., Effland, .!L J , Clark, I. T., unpublished manuscript. ( 7 ) Siggia, Sidney, “Quantitative Organic Analyses via Functional Groups,’’ p. 8, Wiley, New York, 1949. (8) Simpson, D. W.,Wheaton, R. M., Chem. Eng. Proyr. 50,45 (1954‘. (9) Wheaton, R. M.,Baumann, W. C., Ann. N . Y . Acad. Sci. 57, 159-76 (1953).
(1) Barker, S. B., Summerson, W. H., J . Biol. Chem. 138, 535 (1941). (2) Clark. L. T.. Ind. Ena. Chem. 50. . ,1125. (1958).. ’ (3) Feigl, Fritz, “Spot Tests,” p. 250, Elsevier, Amsterdam, 1954.
RECEIVED for review September 11, 1957. Accepted April 24, 1958. Division of Analytical Chemistry, 132nd Meeting, ACS, New York, N. Y., September 1957. Work supported in part by the Ordnance Corps.
64% purity. The sorbitol fraction, containing small amounts of xylitol and erythritol, was available for recycling in the hydrogenolysis process. Resolution improved with diminishing size of the charge used. This is illustrated in Table I, which shows recovery and purity of glycerol fractions from the 3, 6, and 12% separations.
Colorimetric Method for Determination of Urinary Porphyrins T. C. CHU and EDITH JU-HWA CHU Immaculate Hearf College, 10s Angeles, Calif.
A method for the colorimetric determination of urinary porphyrins is based on the esterification and subsequent chromatog ra p hic separation of porp hyrins on a Hyflo Super-Cel column. The calibration data for esters of coproporphyrin I (from 0.6 to 110 y per ml. of eluent), and uroporphyrin 1 (from 0.7 to 530 y) are given. They are convenient working ranges for the determination. Examples and the precision of the method are illustrated and discussed.
D
of porphyrins in urine samples has been complicated by the presence of less known in addition to coproporphyrins (1, 4, porphyrins and uroporphyrins. Their porperties are so similar that the conventional methods of solvent extraction or chromatographic elution of the esters are not always satisfactory. For instance, one of the minor porphyrins, a pentacarboxylic porphyrin (band 2, Figure I ) , which is found in pathological as well as normal urines, has the same hydrochloric acid-number (10) as coproporphyrins ( 2 ) . Therefore the determination of coproporphyrins by ether and dilute hydrochloric acid extraction would give a higher result. On the other hand, the usual chromatographic elution of esters would wash down the narrowly separated minors with the neighboring main zone. The determination is further complicated by the presence of porphobilinogen and other ETERMINATION
1678
ANALYTICAL
CHEMISTRY
chromogens. Although conversion of chromogens or porphobilinogen to porphyrins prior to the determination (3, 9) has been reported, the results are still unsatisfactory (5, 6 , 8, 11). Also a slight change of p H would change the results (2). As a practical procedure for the routine analysis and preparative work for porphyrins, the method reported is based on the easy separation of porphyrin esters on a Hyflo column ( I ) , and the simple colorimetric determination with a Spectronic 20 colorimeter. The instrument has been calibrated with pure porphyrin esters as standards. Special attention has been paid to the determination of coproporphyrins and uroporphyrins as a matter of general clinical interest. The method is applicable to normal and pathological urine samples. PROCEDURE
Colorimeter Calibration. A Bausch & Lomb Spectronic 20 colorimeter was used for t h e determination. A sample of chromatographically separated uroporphyrin I methyl ester (UI), melting point 295” C. ( I ) , and pure coproporphyrin I methyl ester (CI) of melting point 254” C. prepared from uroporphyrin I methyl ester were used as standards. The Bausch 8: Lomb inch test tubes were used as sample tubes. The calibiation data are listed in Tables I and 11. For eluents of lower or higher concentrations than those listed, other calibrations were made a t different n-ave length scales (Table 111) to cover a wider range for the routine analysis. When larger
columns are used, as in preparative M ork, the concentrated eluents may be properly diluted before the determination. Separation of Porphyrins. Only preformed porphyrins were further considered. A 24-hour sample of freshly collected urine from a normal subject, or a 100-ml. portion from a pathological case FT as used. T h e size of a sample may be reduced n-hen there is higher concentration of porphyrins. It was acidified t o p H 3.5 with 10% hydrochloric acid and 2 t o 3 grams of talc was added with thorough shaking. After decantation, the supernatant liquid was shaken with another 1 to 2 grams of talc. It was filtered through a small Biichner funnel with suction. The filtrate was shaken again with another gram of talc and filtered through the same funnel. The combined talc adsorbent was washed and finely divided for quick drying in a dark ventilated hood, Then the adsorbed porphyrins were esterified overnight with 10 ml. of methanol-sulfuric acid (20 to 1 by volume). The talc 11 as removed by filtering through a sintered glass filter, and washed several times with methanol-sulfuric acid until the washing was free from any red fluorescence. The combined filtrate was diluted with 2 volumes of water, neutralized to Congo red with a saturated solution of sodium acetate, and repeatedly extracted with small volumes of ethyl acetate. The ethyl acetate solution was washed with water and transferred to an evaporating dish for drying in a hood. h‘leanwhile a chromatographic column of Hyflo Super-Cel was prepared. An ordinary chromatographic tube (1.8 cm. in diameter
packed with 14 cm. of Hyflo) was used for the separation of about 0.5 mg. of crude porphyrin esters. The details of working with such a column have been described (1). The mixture of porphyrin esters was dissolved in about 1 ml. of chloroform and thoroughly mixed with 0.2 to 0.3 gram of Hyflo. After drying in air, i t was evenly introduced onto the packed column, followed b y another layer of plain Hyflo. It was developed b y a mixture (1 to 2 b y volume) of chloroform (U.S.P.) and light petroleum ether (boiling point 30" to 60" C.). Within a few minutes, when the solvent front almost reached the bottom of the column, the suction was stopped. Each fluorescent zone was carefully dug out with a spatula under ultraviolet light (Mineralight 3660) to avoid any possible contamination of a nearby nonfluorescent band as might appear in some cases. The general appearance of typical Hyflo chromatograms are shown in Figure 1. The first or lowest zone contains methyl esters of coproporphyrins. whereas the fifth zone contains those of uroporphyrins. The rest are esters of less known porphyrins. After drying in air, each segment was repacked in a small Allihn filter tube, and eluted with chloroform directly into a colorimeter tiihe for the determination. Determination of Porphyrins. T h e wave length scale of t h e colorimeter was s P t at 500 mp for the average porphyrin concentration. A blank of chloroform n a s adjusted to 100% transmittance. Then the sample tube was introduced and the per cent transmittance, T, was read. The unit concentration of the first eluent-Le.. esters of coproporphyrins-was read from Table I, which was compiled according to the nianual of methods and calibrations for the instrument. 8imilarly, the concentration of uroporphyrins n-as determined n-ith reference to Table 11. If other porphyrins are to he determined. the fourth zone, containing heptacarbouylic porphyrin mainly of the type I11 (I), may be estimated as uroporphyrin I methyl ester, and the second and third zones containing the respective penta- and hexacarboxylic porphyrins as coproporphyrin I methyl ester. liecause of their respectively closer allsorption mauima. There are still some faint zones of unknown nature above the fifth zone lyhich are not considered here.
!F 9 k ! 0
E
D
C
0
0
0
0
5
5
5
5
5
4
4
4
4
4
3
3
3
3
3
2 I
2
2
2
I
I
I
2 I
Figure 1.
F
Typical Hyflo chromatograms of urinary porphyrin esters
A. Normal urine 6. Intermittent acute porphyria C. D.
E. F. 1.
2, 5. 0.
Cutanea tarda Mixed type porphyria Congenital porphyria Lead poisoning case Coproporphyrins 3, 4. Penta-, hexa-, heptacarboxylic porphyrins, respectively Uroporphyrins Residue and faint bands of unknown nature
Calibration Data for Coproporphyrin I Methyl Ester
Table 1.
[Units y/mL (chloroform). Kave length 500 mp] T 0 10 20 30 40 ~.
50 60 70
80
1
78 51 38 28 21 15 10
2
m
3
70 48 36 26 19 14 9 6
74 50 37 27 20 15 10 6
4
67 47 35 26 19 14 9 6
64 45 34 25 18 13 9 5
Example. Transmittance reading T on scale
40, under 7 = 23 -,/ml.
Table 11.
T 0 10 20 30 40 50 60 70 80
5
6
7
8
9
110 62 44 33 24 18 13 8
101 59 43 32 24 17 12 8
94 57 41 31 23 17 12 8
88 55 40 30 22 16 11 7
83 53 39 29 22 16
= 47.
67 44 31 23 17 12 8
Calibration Data for Uroporphyrin I Methyl Ester
64 42 30 22 16 12 8 5
5
11 7
Concentration found across from
[Units -//ml, (chloroform). Wave length 500 m p ] 1 2 3 4 5 6 7 61 41 30 22 16 11 8
Table 111.
EXPERIMENTAL RESULTS AND DISCUSSION
Normal Urine. T h e preformed porphyrins of a 24-hour urine sample of 1170 nil. were determined as esters (Figure 1, A ) . T h e eluent of 4.5 ml. of t h e first zone had a transmittance reading of T = 61%. As seen in Table I , t h e total content of coproporphyrins in t h e urine sample as esters was 4.5 X 15, or 68 y. Crystals were separated from chloroform-methanol as long needles, melting point 240-245" C. The eluent of 3 ml. of the faint fifth zone (the instrument requires a minimum of 3 ml.), had a T =
B
A
58 40 29 21 15
11 7
92 53 37 27 20 14 10 7
55 38 28 20 15 11 7
80 49 35 25 19 13 9 6
85 51 36 26 19 14 10 6
8
9
75 47 33 24 18 13 9 6
71 45 32 24 17 13 9 5
0.6
Calibration Data for Porphyrin Esters
A. CI, Wave length 405 me T y/ml.
17 5.0
24 4.0
28 3.6
11
16 4.0
20 3.5
32 3.2
37 2.8
41 2.5
80
46 2.2
52 1.8
65 1.2
44 1.8
53 1.4
1.0
73 0.7
40 130
50 95
60 65
70 40
B. VI, TVave length 405 mp
T y/mL
4.8
24 3.1
34 2.5
38 2.1
63
C. UI, Wave length 625 m p
T y/d.
8 530
15 340
20 270
25 220
30 185
35 155 ~
~~~
~
VOL 30, NO. 10, OCTOBER 1958
1679
53%, when the wave length scale was set a t 405 mp. From Table 111, B, the total uroporphyrin content was 4.2 y. It crystallized in fine needles, melting point 280-285" C. The fainter second, third, and fourth zones were also eluted out, and checked by paper chromatography (1) with markers of knomn quality. The first zone, coproporphyrins consisted of about 70% I isomer and 30% I11 isomer. The second. third, and fourth zones behaved exactly like esters of penta-, hem-, and heptacarboxylic porphyrins, respectively, isolated from pathological urines. The fifth zone uroporphyrins consisted of 80% uroporphyrin I methyl ester and 20% of the I11 isomer. Porphyria Urine. Two consecutive 24-hour urine samples of 1135 and 2100 ml. were collected from a patient xx-ith intermittent acute porphyria. Both samples showed t h e presence of porphobilinogen (8). Both were found to contain 42 y of coproporphyrins per 100 ml. of sample. The amount of uroporphyrins was 24 y per 100 ml. in the first sample and 48 y per 100 ml. in the second sample. Apparently the daily fluctuation of porphyrin level was considerable. The porphyrins n-ere obtained in crystalline forms. Methyl esters of coproporphyrins containing 20% of I and 80% of I11 isomers, melted a t 155-163" C., and those of uroporphyrins containing 50% each of I and I11 isomers melted a t 268-276' C. The chromatogram is shown in Figure 1,B. This case has indicated that a n abnormal porphyrin pattern of a urine sample can be detected before other manifestations of porphyria symptoms, which is useful in clinical diagnosis.
The findings from other porphyria cases, given in Figure 1 as references, were determined in the same way.
Recovery Experiment. A mixture of 285 y of uroporphyrin ester, melting point 2817-289" C., and 105 y of coproporphyrin I methyl ester melting point 254" C., was hydrolyzed overnight with 25y0 hydrochloric acid to free porphyrins, and then immediately introduced into 250 ml. of the deporphyrinated filtrate of the normal urine sample. A recovery of 270 y of uroester, melting point 286-290" C., and 101 y of coproporphyrin I methyl ester melting point 253-254"C., was obtained. It amounted to about 95 to 96% over-all recovery. For comparison, porphyrin mixtures of the same composition mere directly chromatographed on calcium carbonate and magnesium oxide columns with benzene-chloroform (10 to 6), and benzene-methanol (100 to 4) as the respective developers (5'). In both instances, a longer period was needed for the development, and the separation of porphyrin esters was incomplete. The loss due t o chromatography alone was over 10%. From the fraction of the coproporphyrin of the magnesium oxide chromatogram two kinds of crystals were observed under a microscope, mainly long needles of melting point 235-240" C., and some hairlike crystals melting point 275282" C. By paper chromatography it was confirmed that the coproporphyrin fraction y a s contaminated m-ith uroporphyrin. With these calibration data and chromatographic patterns, determinations of practically all kinds of porphyrins in urine samples, even without any authentic porphyrin standard, may be
made. The crystalline products obtained can be saved for further identification and experiment,ation. ACKNOWLEDGMENT
The authors are grateful to H. L. Mason of the Mayo Clinic, Rochester, Minn., R. I f . Halpern of Beverly Hills, Calif., and G. R. Kingsley of Veterans Administration Los Angeles, Calif., for samples B, E, and F listed in Figure 1
1.
LITERATURE CITED
(1) Chu, T. C., Chu, E. J.-H., J . Bid. Chem. 227, 506 (1957). ( 2 ) Chu, T. C., Chu, E. J.-H., unpublished
data. (3) Eriksen, L., Scand. J . Clin. & Lab. Invest. 4, 55 (1952). (4) Falk, J. E., "Ciba Foundation Symposium on Porphyrin Biosynthesis and 3letabolism," p. 63, Little, Brown, Boston, 1955. (5) Formijne, P., Poulie, N. J., Ibid., p. 3Afi - --. (6) Markovitz, hf., J . Lab. & Clin. Med. 50, 367 (1957). ( 7 ) Kicholas, R. E. H., Biochem. J. 48, 311 (1951). (8) Schn-artz, S., Vet. Administration Tech. Bull. TB 10-94 (1953). (9) Watson, C. J., Pimenta de Mello, R., Schwartz, S., Ha-vkinson, V. E., Bossenmaier. I.. J . Lab. & Clin. Med. 37, 831 (195i). ' (10) Willstatter, R., Mieg, IT., Ann. Chem. 350, l(1906). (11) Zondag, H. A, Van Kampen, E. J., Clin. Chiin. Acta 1, 133 (1956).
RECEIVED for review February 26, 1958. Accepted June 6, 1958. Work supported by research grant, il-lOOO(C7), from the Kational Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Public Health Service.
Determination of Sulfur Oxides in Stack Gases EDWIN 8. SEIDMAN
Shell Oil Co., Wilmingfon, Calif.
b Sulfur trioxide in stack gases as low as 0.001 % gas volume may be determined in the presence of as much as 0.370 gas volume sulfur dioxide. Ammonia and/or nitrogen oxides do not interfere. Sulfur trioxide is absorbed quantitatively in an 8Oy0isopropyl alcohol solution, which inhibits oxidation of sulfur dioxide. The sulfate i s titrated with 0.01N barium chloride in a solution of 80% isopropyl alcohol, using Thorin indicator. The end point i s sharp and reproducible and the titration is rapid. A modification of this procedure also has been used for the determination of total sulfur oxide. 1680 *
ANALYTICAL CHEMISTRY
I
rapid and accurate methods for the determination of sulfur dioxide and trioxide content of combustion gases has been stimulated by atmospheric pollution studies in several major cities. The standard Anierican Petroleum Institute methods used by the petroleum industry (1, 2 ) are subject to interference from ammonia and nitrogen oxides, both of which are known to be present, for example, in catalytic cracking flue gases. The API methods require absorption of the gas sample in a known amount of standard base. After absorpI~TEREST IK
tion, excess base is titrated and total sulfur oxides are obtained by difference. The sulfur trioxide is then precipitated as benzidine sulfate, redissolved, and titrated. Sulfur dioxide is determined by the difference. Even when no interfering substances are present. several difficulties have been encountered in applying the API methods to refinery stack gases. First, the methods involve small differences between large numbers. I n addition, the colorimetric titration end point is frequently obscured by dark-colored oxidation products from the inhibitors present in the absorber solution. (Ex-
