remove chloral hydrate and trichloroethanol components with 2 minutes of preheating and is strong enough to produce a good color with trichloroethanol at 440 mp. Different concentrations of alkali can be used for optimal determination of these substances individually. DISCUSSION
Although the mechanism of the Fujiwara reaction is not yet understood: it has been observed by the authors and other investigators (4) to be specific for compounds containing at least three halogen atoms on one carbon atom. It is not clear whether the two absorption maxima given by each compound represent competing reactions, or two peaks of a single complex. However, with trichloroethanol the absorption of the two bands can be varied independently by changing the heating time or alkali concentration. It is always advisable to run standards with a set of unknowns and not to consider the extinction coefficients as constants. As shown in the experimental section,
the conditions of the procedures were chosen as a compromise designed to simplify measuring all three compounds. If only chloral hydrate or trichloroacetic acid were to be determined, a heating time of 6 to 8 minutes would be preferable, because it would give a greater absorption at 370 mp than the 4-minute time prescribed in the method. HOKever, the 440-mp absorption of trichloroethanol decreases in intensity after 3.5 minutes of heating. The absorption of trichloroacetic acid and chloral hydrate is strictly additive; however, that of chloral hydrate and trichloroethanol is not. This error is less marked at low concentrations of chloral hydrate and trichloroethanol; its cause is unknown. I n practice, the estimate of chloral hydrate in a mixed sample will be on the low side. Interest here is in accurate measurement of the metabolites which have been formed in experiments in vitro, and the remaining chloral hydrate concentration is of secondary importance. The amount of chloral hydrate remaining is far greater than that of the metabolites formed,
and this greatly reduces the significance of the error. To obtain more accurately the Concentration of chloral hydrate in a mixture of the three compounds, readings of the crimson color a t 540 mp may be made in Procedure A. Then chloral hydrate concentration is given by the difference after absorption due to trichloroacetic acid is subtracted; this may be calculated from Procedure B. LITERATURE CITED
(1) Butler, T. C., J . Pharmacol. Esptl. Therap. 92,49 (1948). (2) Glazko, A. J., Dill, W. A., Wolf, L. M.,Kaeenko, A,, Zbid., 121, 119 (1 9.57). ,. \ - - -
(3) Marshall, E. K., Jr., Owens, A. H., Jr., Bull. Johns Hopkins Hosp. 95, 1 (19W). (4) Ross, J. H., J . Biol. Chem. 58, 641 ( 1923). ( 5 ) Seto, T. A,, Schultze, Ll. 0.) ASAL. CHEST. 28,1625 (1956).
RECEIVED for review November 22, 1957. Accepted June 18, 1958. Taken from the thesis to be submitted by Paul J. Friedman in partial fulfillment of the requirements for the degree of doctor of medicine, Yale University.
Separation of Glycerol from a Polyhydric Alcohol Mixture by Nonionic Exclusion RA
T. CLARK
Forest Products laboratory, Forest Service, U. S. Department of Agriculture, Madison, Wis.
,Column chromatography with an ion exchange resin was used to separate glycerol from a mixture of polyhydric alcohols that resulted from the hydrogenolysis of glucose or sorbitol. Other fractions obtained included sorbitol and erythritol, each with traces of xylitol, and a mixed fraction of ethylene glycol and propylene glycol. The method may be useful for commercial application.
T
separation of simple mixtures of polyhydric alcohols by selective extraction with acetone or other solvents, or by codistillation with chloroform or hydrocarbons, has been reported ( 5 ) . These separations, however, are slow and incomplete. The separation of glycerol from triethylene glycol and other nonionic materials, by passage of their solution through columns of ion exchange resin, has been described by Wheaton and Baumann (9). The separation has been explained on the basis of the extent and rate a t M-hich individual solute constituents of a
mixture are distributed between the solvent (water) inside the resin particles, and that outside, during passage through the resin column. Distribution constants, K d ,for a number of nonionic materials and resins have been reported. This method was used to investigate the separation of a mixture of polyhydric alcohols resulting from the hydrogenolysis of glucose or sorbitol ( 2 ) . The mixture, after removal of traces of ionic impurities by ion exchange, had the following analysis.
HE
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ANALYTICAL CHEMISTRY
70 Sorbitol Xylitol Erythritol Glycerol 1,2-Propanediol Ethylene glycol Water (approximately)
8 87 0 69 2 90 11 60 7 65 4 34 60 00
Traces of ethanol, methanol, mannitol, and other compounds were also present. After glycols were removed by distillation a t reduced pressure, glycerol could not be satisfactorily separated from the mixture by distillation a t a pressure
of 0.5 mm. of mercury, without decomposition of the nondistillable sugar alcohols. The steam distillation and extraction methods cited were unsatisfactory for separating the glycerol from the sugar alcohols. I n the resin column separation, the extent and rate of distribution of a solute between the water phases depend upon such factors as temperature, and the type, ionic form, particle size, and cross linkage of a resin. Other factors determining effectiveness of separations are column-flow rate, column height, and concentration and volume of column feed (9). The effects of several of these variables were studied in a series of experiments to determine the applicability of the method to the large scale separation of a glycerol fraction from the hydrogenolysis mixture, and to permit recycle of sugar alcohols in further hydrogenolysis to glycerol. As reported by Wheaton and Baumann (9) Dowex Type 50 H + resin was used for the experiments on the basis of distribution constants because the best
I
I
I
I
18.0
Table 1. Effect of Charge Size on Separation for Dowex 50-x12 Resin at Flow of 0.10 Gallon per Minute per Square Foot
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4.0
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3% 6% 12% Glycerol, % On glycerol fraction 100 80.3 71.8 Recovered in glycerol fraction 100 8 5 . 5 80.3 Volume of all poly01 fraction eluate, yo of initial charge 1040 670 350 Height equivalent of theoretical plate, in, 0 . 71 0.84 2.18
8
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480
560
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VOLUME COL L EC TED IC.c: 1
Figure 1. Resolution of glucose hydrogenation product (charge = 3% of resin volume)
- Refractive index 0
A
X
Sorbitol Xylitol Erythritol Glycerol Propylene glycol Ethylene glycol
separation of glycerol and ethylene glycol was indicated. Resin of 100- to 200-mesh particle size tvas chosen as a compromise between rapid rate of equilibration and reasonable flow rates. The effects of two cross linkages, 50-xS and 50-x12; two flow rates, 0.10 and 0.15 gallon per minute per square foot of column cross sectional area (equal to 2 and 3 ml. per minute flow); and of three charge volumes, equal to 3, 6, and 12% of the volume of the column bulk resin were investigated. The general effects of these, and of column height, have been s h o m upon less complex mixtures (8, 3). The aqueous solution of hydrogenolysis products of the composition as stated n-as used in these experiments. Experimental. To obtain capacity, the column used was a glass tube, 2.5 cm. (inside diameter) by 240 cm. long, and had a sintered glass disk and capillary stopcock a t the bottom. It m s filled to a height of 204 em. with the ion exchange resin. rlir pockets were avoided by thoroughly mixing the resin with excess water, allowing i t t o settle until bubble free, and pouring the slurry into the column. After settling a few hours, the volume of the resin bulk was adjusted to 1000 cc. Temperature was maintained a t 60' C. by circulating water from an external reservoir through a full-length water jacket. Demineralized, deaerated water for the separations was supplied through a steam-jacketed preheater, corked into the top of the column. A fraction collector was provided which collected 200 volumetrically measured fractions of up to 10 cc. each. I n operation, the water above the resin was drained to the resin level,
2,7-naphthalenedisulfonic acid).
The paper chromatography did not resolve individual hexitols or pentitols. These were expressed as sorbitol or xylitol, respectively. Height equivalent of a theoretical plate (centimeters) rvas calculated from elution curves by the use of a formula suggested by Simpson and others (8).
and the measured volume of hydrogenolysis product introduced into the space above the resin. A flow was started from the bottom of the column a t the predetermined rate, and collection of fractions began. When the level of the charge reached the top of the resin, a flow of preheated demineralized water was started, and followed the polyhydric alcohols into the column. Analysis. Sorbit 01, xyl it 01, eryt hritol, and glycerol were separated by a paper chromatography method using butyl alcohol-pyridine-water, as developed a t the Forest Products Laboratory (8). This was followed by periodic acid oxidation of each fraction. The periodate used was determined by iodometric titration. The volumetric oxidation method was adapted from Siggia (7). Presence of 1,2-propanediol was determined by a colorimetric method in which its periodic acid oxidation product, acetaldehyde, formed a colored complex with pphenylphenol (1, 3). Ethylene glycol was determined by the difference between total formaldehyde formed by periodic acid oxidation of the sample, and the sum of formaldehyde calculated from all polyols other than ethylene glycol. Total formaldehyde was determined by a colorimetric method using chromotropic acid (4) (4,5-dihydroxy-
5,000
3,000
2,000
1,500
where h Ti,
column height liquid volume t o the peak of maximum concentration = interstitial volume of resin bed (38y0 of resin bulk volume) and W = half n-idth of elution curve a t ordinate of l/e of peak concentration. = =
RESULTS
When refractire index and analytical values for fractions from a separation run were plotted against volume collected, curves such as in Figure 1 resulted. These represent a separation which used Dowex 5O-xl2 resin a t a rate of 0.10 gallon per minute per square foot Tvhen the volume of the sample charged t o the column (30 cc.) equaled 3% of its bulk-resin yolume. Three major fractions resulted that contained chiefly sorbitol, glycerol, and the tivo glycols. The use of this resin and this flow rate was found to result in slightly im-
1,200
1,000
800
700
625
50
$2 40 85 30
$5
20
9 10
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20
30
40
50
60
70 WAVE
Figure 2.
80
I00
90
LENGTH
IN
I10
I20
130
I40
150
160
MICRONS
Infrared spectra of glycerol VOL. 30, NO. 10, OCTOBER 1958
1677
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
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. LITERATURE CITED
(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.
(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).
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.
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 chroma tog ra p hic sep a ration 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 porphyrins (1, 4, in addition to coproporphyrins 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
