ANALYTICAL CHEMISTRY
1964 The apparatus must be airtight, and the ground-glass joint presented a problem, but it was found that a thick paste of graphite and Dow Corning high vacuum grease worked very well. For best results the solvents must be pure, and the samples completely dry. PROCEDURE
\Veighed samples, 5 t o 15 mg. of the reference and unknown compounds, are placed in bulbs A and R, respectively, with 0.10 to 0.30 ml. of solvent to put each into solution. The amount of solvent required will depend upon the solubility of the compound, but it should be kept :it a minimum. The weights of the samples should be somewhat proportional to their molecular weights. S e s t , 1 ml. of the pure solvent is placed in bulb C, which is then attached to D by means of the ground-glass joint and a fresh supply of the graphite rease. A heavy Figure 1. Apparatus rubber band is pyaced around the hulbs to hold the joint firmly in place, and a water pump vacuum is gradually applied through stopcock B until there is a slight evaporation of the solvent. Ordinary stopcock grease is suitable for the stopcock, but it should be held in place with a clamp of some type. The stopcock is turned off, and the ahole apparatus is immersed in a constant temperature bath with the pipets up. A temperature of 40’ C., used for the
determinations listed, has several advantages; it prevents condensation within the apparatus, speeds up the procedure, and helps to keep difficultly soluble compounds in solution. \Then all of the solvent from bulb C has been absorbed, and this usually occurs within 48 hours, the first reading is made. The apparatus is removed from the water bath, dried, and turned slowly t o a horizontal position, allowing the solutions to run into their respective pipets. The pipets are pointed down and jarred slightly to remove any bubbles, then allowed to drain for 6 to 10 minutes before the solution volumes are read. Bt the end of this period the isopiestic point usually will have been reached, but the apparatus should be replaced in the bath in its original condition for 24 hours as a check. When the second reading is taken, the volumes should have remained constant, indicating that the end point has been reached. If the sample tends to come out of solution upon cooling, or the bath temperature is high, it may be necessary to replace the apparatus in the bath before the volumes are read. The molecular weight, Mu,of the unknown is calculated from the formula Mu = Wu X V X .%!/Vu X W , where Wu is the weight of the unknown, Vu the volume of the unknown, Vthe volume of the reference solution, W the weight of the reference solution, and M its molecular m i g h t . ACKNOWLEDGMENT
The author is indebted to Claire Johnston and Ed. Meyers for their technical assistance, and to Hurshel Hill for making the apparatus. LITERATURE CITED
(1) Clark, E. P., A N ~ LCHEN., . 13, 820 (1941). (2) Signer, R.,Ann., 478, 246 (1930). . ~ A L CHEM., . 24, 1063 (1952). (3) White, I,. If..and Morris, R. T., RECEIVED for review April 1, 1954.
Accepted July 2, 1954.
Silica Qel Microcolumn for Chromatographic Resolution of Cortical Steroids MAX L. SWEAT’ Department o f Pharmacology, University o f Utah College of Medicine, Salt Lake City, Utdh
A silica gel microcolumn is described for the chromatographic analysis of pure mixtures of several steroids in amounts as small as 0.25 y . The column is for use in conjunction with the fluorescent and phenylhydrazine analytical techniques for the determination of steroids.
T
H E application of column chromatography to the resolution of mixtures of corticosteroids in blood and biological fluids has been delayed owing to the lack of methods suficiently sensitive to detect and analyze minute concentrations of these substances. The recent development of the fluorescent method of Sweat, ( 4 ) which analyzes corticosterone, l’i-iiydroxycortivosteronc, and 4-pregnene-11-8, 17-a, 20-p, 2l-tetrol-3-one, and the phenylhydrazine method of Porter and Silkier ( 3 ) which is relatively specific for thr 17-, 21-hydroxy-20-ketone group of +teroids, has made such a Ptudy poPaihle. The present report descrihes a silica gel microcolumn which, \\.hen used in conjunction with the fluorescent and phenylhydrazine analytical techniques, makes possible the analysis of pure mixtures of several steroids in amounts as small as 0.25 -,. The purification and preparation of blood extracts for chromatography and the application of the combined fluorescentsilica gel method for the evaluation of corticosteroids in man and laboratory animals will be described elsewhere. 1 Present address, Department of Physiology, Western Reserve University School of Medicine, Cleveland 6, Ohio.
APPARATUS
Two sizes of chromatographic columns are employed: one having an over-all length of 230 mm. is used for chromatographic analysis of 1 to 10 y of steroids; the other, 126 mm. in length, is used for 0.25 to 1 y. Water jackets are necessary to keep the adsorbent cool. Stopcocks regulate the rate of “packing” in the column. Too rapid packing or too high temperature cause small vapor bubbles to form in the silica gel. Both columns are illustrated in Figure 1. REAGENTS
Adsorbent. A mixture of 250 ml. of 95% ethyl alcohol, 750 ml. of chloroform, and 200 grams of silica gel (Davison Chemical Corp., Baltimore, Md., silica gel No. 922-08-08-226TT200) is allowed to stand several hours. The solvent mixture IS filtered under gentle suction on a Buchner funnel. The silica gel is washed successively with 400 ml. of 3 to 1 ethyl alcohol-chloroform and 1 liter of 95% ethyl alcohol. The partially dried gel is now transferred to a 4-liter beaker containing 2 liters of distilled water. The contents are stirred in a circular motion to concentrate the black particles in the center and bottom of thc beaker. The major fraction of the silica gel is decanted while it is in motion. The process of stirring and decanting is repeated approximately eight times in order to remove most of the black particles. An additional twenty washings with distilled water are necessary to free the silica gel of a water-soluble gray-brown pigment. The silica gel is partially dried on a Buchner funnel, spread on a clean surface to complete drying, and activated in a muffle furnace at 500” C. for 3 hours. Ethyl Alcohol. Absolute ethyl alcohol is distilled from a waterfree system; the first and the last 10% of the distillate are dis-
V O L U M E 26, NO. 1 2 , D E C E M B E R 1 9 5 4
1965
carded. The distilled ethyl alcohol is stored in the dark in bottles with close-fitting glass stoppers. Chloroform. Mallinckrodt analytical reagent grade chloroform, preserved with 0.75% ethyl alcohol, and supplied in glass carboys, is distilled from an all-glass, water-free distillation apparatus fitted with a fractionating column of approximately 2.0 theoretical plates. The chloroform employed in chromatography represented the first 90% of the distillate.
of the silica gel. Filter paper, glass wool, sand, or cotton should not be used in the preparation of the column, because they contain extractives which interfere with the phenylhydrazine and fluorescent tests. Cleaning the Column. Three 10-ml. quantities of ethyl alcohol in chloroform (1 to 10) and three 10-ml. quantities of chloroform are added to the column in succession and collected in 50-ml. round-bottomed flasks labeled A to F, respectively (Table I). Addition of Sample and Development of Column. The sample contained in a 50-ml. round-bottomed flask is dissolved in 0.5 to 1.0 ml. of chloroform and transferred to the column. TR-o additional 0.1-ml. quantities of chloroform are used to wash the flask and complete the transfer of the sample. The quantitative transfers are accomplished by holding the round-bottomed flask vertically and removing the small quantities of liquid from the bottom curvature of the flask by means of a fine capillary pipet fitted with a rubbci, bulb. When the upper surface of the chloroform solution has reached the upper surface of the silica gel, an addit,ional 10 ml. of chloroform are added to the column. The combined t4lumtJ of these four additions are labeled "fraction 1." Additional chloroform and ethyl alcohol-chloroform eluents are added to the column, collected as effluents in 50-ml. roundbottomed flasks, arid labeled in the order shown in Table I. Analysis of Fractions. The ethyl alcohol-chloroform effluents are evaporated in vacuo at 50' C. Absolute ethyl alcohol (0.5 ml.) is added and the flasks are tightly stoppered. After 1 hour or longer, 0.1 ml. of the ethyl alcohol solution of the steroid is transfcxrred to optically marked 10 X 75 mm. borosilicate glass tuhes for fluorescent analysis ( I j . The remainder is tat,ively transferred to a 7 5 X 85 mm. t,uhe for phenylhy%!t$;i analysis ( 2 ) . Standardization of Column. Different batches of chloroform, ethyl alcohol, and silica gel may vary in chromatographic properties and hence the chromatographic patt>ernmay not a l m y s be exactlj- the same as that shown in Table 11. However, adjustment of the system may be made by increasing or decreasing the concentration of ethyl alcohol to cause the elution of corticost,eroriei n fractions 5 to 10. Preparation of 126-Mm. Column. An amount of -30, +40 powdered glass sufficient to form a 5-nim. layer is added to the column and washed into place with a small amount of chloroform. Silica gel (0.1 gram) is int,roduced into t.he column in a manner simil,Lt, to t h t used in the preparation of the 230-mm. column. The compoPition of the solvents and the order of their use for the 126-mm. column are listed in Table 111. Only corticosterone and 17-hydroxycorticosteronr have been demonstrated to be successfully resolved hy this column, as the fluorescent test is the only test sufficiently seni;itive to measure cortical steroids in quantities less t,han 0.25 y. The quantitative resolution and the recovery of a mixture of 0.25 y each of corticosterone and 17-hydroxycorticosterone are shown in Table IV. The analyses of the frizct'ions collected from the small columns are carried out, in exactly the same maiiner i3.s the fractions collected from the
----I5 m m 0 D " ' d / ~ - - -----IOrnrn0.D. -5rnmO.D,
Fritted Glass Disk
Zrnrn Thick
16mm
m m bore stopcock Precision Ground)
1
;[ -1 br,
i1/2 rnm bwe stopccek Precision Ground1
45. Revel
Figure 1. Silica Gel 3Iicrocolumn
Powdered Glass. Small chips of clean borodicate glass are moistened with ethyl alcohol and ground t.o a fine powder in it large mortar. The dried powdered glass is shaken through a series of 20-, 30-, 40-, and 50-mesh sieves. The partirles rejected bj- the 20-mesh and retained by the 30-mesh screens are designated as "-20, $30." The other fractions are likewiwe designated according to the sicw> sizw ivhicli retain and reject the gl:i+ p:trticles. PROCEDURE
Packing 230-Mm. Column. V 3 h the stopcock of the column open and cold tap water running through the cooling jacket, enough powdered glass (-30, f40) is added to the column to form a layer 2 to 3 mm. above the fritted glass disk. The Dowdered glass is I\ ashed into-place with-a small quantity of chloroform. Two mlditional and approximatel\ equalamounts of powdered g l a s ( - 3 0 , +40 and -40, +50)arr added in s u c c e s s i o n . The chloroform is allowed to drain anti the stopcock is closed. A susilension of 0.5 gram of activ:ited d i c a gel and 5 ml. of chloroform is poured into thr column. Two additional 2-ml. qumtities of chloroform aw used to rinse the beaker anti the sides of the column. Tht. stol)cock remains closed until the silica gel has settled completely; the chloraform is nouruii off until its upper level coiiicsitles with the upper level
Table I. Resolution of Corticosteroids on Silica Gel \Iicrocolumn
Cleaning of column
.
.....
Volume Eluent, % of Elnent, E t h y l Alcohol hll in Chloroform 10.0 10 10.0 10 10.0 10 0.0 10 0.0 10 0.0
iddltion sample O* nevelopment of column
Effluent Fraction Nuinbei
't
,loid R ~ w d r i v l
.I
B
c
1-1
1,;
I
0 0 0 0
0 0 0 0
1y 7
10
OB
J
1.0 1.0
0 1Q 7
U.J
J
1
IV
4
. I,
L0
1
5 10.0 I T4, . . . . . . J-i-pregnene-118,17-n, 20-8.21-tetrol-X-onr ( E / ( ,c 10 10.0 a Dotted lines indicate fraction in \vliii.li respective steroids peak. Bars indicate series of effluent fractions in which each of t h e steroids is eluted. b Analyzed by plienylhydrazine method. C Analyzed by fluorescence method. * R = Reichstein's designation. + K = Kendall's designation.
_
_
_
~
~~~.
~
~~
1966
ANALYTICAL CHEMISTRY
10% ethyl alcohol in chloroform and three 10-ml. volumes of chloroform, the background is reduced to a sufficiently Fluorometer Reading low level to add the sample and proceed Volume Composition, 17-Hydroxyof % ETOH Background Corticocorticowith the analysis. Solvent, in Fraction (blank sterone, stprone, The results obtained from the chrohll. Cliloroforni Sumber column) 1Y 1 Y Cleaning of 14.0 matographic analysis of a mixture of column 10.0 0.25 y each of corticosterone and 173.5 2.0 hydroxycorticosterone o n t h e s m a l l 1. .5 1.0 chromatographic column are shown in Table 111. The pattern is essentially the Addition of ( 5 y each uf corticosterone 1/5 sample analyzed by fluorescence and 17-hydrosycorticosterone) sample same as with chromatography of corticoReadings above Background sterone and 17-hydroxycorticosterone on Development 10 0.0 10 of column 0.0 1. d 0 the larger columns. The recovery of B 0.0 1.0 0 10 0.5 1.5 1.0 the two steroids from this column is of 5 0.5 2.5 2.0 relatively high order. 5 1.0 0.5 7.0 5 0.5 1.0 25.0 The sequence of elution of six cortico5 1.0 0.0 13.5 7 1.0 8 0,5 ?,? steroids is shown in Table I. It is seen 5 1.0 9 0.0 D.J that of the steroids listed, clean separa5 1.0 10 0.5 4.0 5 1 0 11 0.0 44.0 tions are possible with the exception of 5 12 4.0 0.0 22.0 4 , (1 13 0.0 9 0 corticosterone and li-hydroxy-ll-dehy'? 10.0 14 0 .i 0.0 drocorticosterone and of 17-hydroxy-1110 0 10 15 1. o 0.0 dehydrocorticosterone and li-hydroxyTotal 67 75 Standard 1 y 70 80 corticosterone. Data not included in Recovery, yo 9 4 . 5 95 0 the table have demonstrated that the latter two steroids m a r be more effectivel) separated by introducing a 2% large columns, \qith the exception that 0.2-ml. instead of 0.5-ml. ethyl alcohol in chloroform eluent between fractions 10 and 11. quantities of absolute eth; 1 alcohol are added to the f l action Corticosterone and 17-hydro.,y-l l-dehrdrocorticosterone have flasks. not been successfully separated on the silica gel column RESULTS
Table 11. Resolution of Corticosterone and 17-Hydroxycorticosterone on 230-Jlm. Column
Table I1 lists data which show the elution pattern obtained from the chromatography of a mixture of 5 y of corticosterone and 5 'r of 17-hydroxycorticosterone. Corticosterone first appears in detectable quantities in fraction 5 . The largest quantity of corticosterone is eluted in fraction 6. Successively smaller amounts follow in fractions 7 , 8, and 9, and a minimum detectable amount is eluted in fraction 10. Traces of 17-hydroxvcorticosterone are eluted in fraction 10. The amounts eluted reach a maximum in fraction 11. The quantities in the subsequent fractions diminish gradually to fraction 14, where only traces of the steroid are found. The recoveries of standard mixtures are of high order (Table 11). The data listed in Table I1 are typical for the micro silica gel chromatographic method. It will be noted in Table I1 (fractions ,4 to F) that silica gel purified as described above still retains background impurity which is eluted with 10% ethyl alcohol in chloroform. After treatment with three 10-ml. volumes of
Table IV.
Recoveries of Steroids from Chromatographic Columns
Steroid Cliromatopaphed CorticoPterone 17-Hydroxycorticosterone
Microcolumn Ultramicrocolumn No. of Range of % Xo. of Range of q samples recovery samples recovery 25 90-101 6 97-104 Av. 96 101 25 93-99 6 96-100 Ar. 95 97
DISCUSSION
The development of a micromethod to resolve and quantitatively analyze individual steroid components of the adrenal secretion is a step forward in solving some of the basic physiological problems of adrenal physiology. The silica gel chromatographic micromethod has been successfully applied to the analysis of human (6,6), dog, and rat (7) blood. The advantage of the silica gel method is that it effectively resolves microgram Table 111. Resolution of Corticosterone and 17-Hydroxycorticosteroneon 126quantities of the major steroid comMm. Column ponents in blood from each other and Amount Composition Fluorometrr Reading from substances causing background of of Solvent. 17-Hydroxy-Solvent, % E T O H in rraction Blank Corticocorticoc o n t a m i n a n t s . When the silica gel hI1. Chloroforni Sumber column sterone sterone column is used in conjunction with the Cleaning of 4.0 10.0 3 2.0 10.0 column j: 1.5 fluorometric procedure ( I ) , mixtures as 2.0 0.0 C 1 small as 0.25 y each of corticosterone 2.0 0.0 D 1 and 17-hydroxycorticosterone may be Addition of 0.25 y B 1/2 iample analyzed b y fluorescence sample 0.25 y F analyzed without appreciable loss of Readings above Background steroids. Development 4.0 0 2 0 of sample 2 0 0 1 5 0 The use of silica gel for the analysis 2.0 1.0 1 2.5 1 f30 1.0 11.0 of minute amounts of corticosteroids is 2.0 1.0 1.5 3.0 a reliable procedure, providing sufficient 2.0 4.0 1.0 13.0 2 0 4.0 0.5 4.0 attention is given certain details. Traces 4.0 2.0 0.5 2.0 of moisture in either the elution mixtures Total 16.5 19.0 0.25 or the silica gel change the polarity of y standard 17 20.0 the chromatographic column and cause Recovery, % 97.3 95.0 erratic elution of the steroids. Traces
V O L U M E 2 6 , NO. 1 2 , D E C E M B E R 1 9 5 4 of ethyl alcohol in the sample to be analyzed, in the silica gel, or in the chloroform used in fractions 0 t o 2 are likely to interfere with the effectiveness of the system in resolving steroids. If the stopcock remains open during the packing of the column or if the silica gel is allowed to become too warm during the process of chromatography, small vapor bubbles tend to form in the column and impede the flow of solvents. I n the chromatographic analysis of the above six steroids, cortisone and corticosterone have the greatest tendency to overlap. However, the quantitative analysis of corticosterone by fluoresence is not influenced by the presence of cortisone as the latter steroid has a.low index of fluoresence with the sulfuric acid technique. 17-Hydroxycorticosterone may also be measured in the presence of cortisone without appreciable error. Separation of desoxycorticosterone, 17-hydroxyl1-desoxycorticosterone, and 4-pregnene1 1-p, 17-01, 20-8, 21-tetrol-3-one from each other and from cortisone, corticosterone, and 17-hydroxycorticosterone appear to present no particular problems. Morris and Williams ( 1 ) and Selson and Samuels ( 2 ) have reported the use of column chromatography in conjunction with their methods for evaluating corticosteroids in blood. These methods, however, require relatively large quantities of blood for analysis. The Xelson and Samuels method requires 20 to 30 ml. of blood. I t does not analyze for cortirosterone. The Morris and Williams method requires 50 ml. of blood and two separate chroniatographic columns €0 analyze for corticosterone and 17-hydroxycorticosterone. Resolution and analysis of the
1967 two steroids is accomplished by the present method with only 5 ml. of blood (2 ml. of plasma) on a single column. ACKNOWLEDGMENT
The author is indebted to George Sayers for advice and friendly criticism throughout the period of this investigation, to Gordon L. Farrell for the phenylhydrazine analyses, and to Ella Sandberg for her valuable technical assistance. The corticosterone, li-hydroxy-11-desoxycorticosterone and 4-pregnene-1 1-8,17-a, 20-8, 21-tetrol-3-one were supplied through the kindness of Leonard R. Axelrod, William J. Haines. Dan A. McGinty, and Tadelis Reichstein. LITERATURE CITED
(1) llorris, C. J. 0. R., and Williams, D. C., Biochem. J . , 54, 470 (1953). ~, (2) Nelson, D. H., and Samuels, L. T., J . Clin. Endocrinol. and Metabolzsm, 12, 519 (1952). (3) Porter, C . C., and Silber, R. H., J . Bzol. Chem., 185, 201 (1950). 26, 773 (1954). (4) Sweat, 31. L., ANAL.CHEM., (5j Sweat, AI. L., manuscript in preparation.
( 6 ) Sweat, AI. L., hbbott, W. E., Jeffries, W. Li., and Bliss, E. L.. Federation PTOC., 12, 141 (1953). (7) Sweat, M.L., and Farrell, G. L., J . Clin. Endocrinol. and Metabolism,12, 968 (1952). RECEIVED for review M a y 7, 1954. Accepted July 31, 1954. Investigation supported by a research grant (h-331) from t h e National l n s t i t u t e of Arthritis a n d Metabolic Diseases of the National Institutes of Health, Public Health Service.
Some Errors in the Determination of Calcium in Aged Blood Serum Eliminated by Flame Photometry P. S. CHEN, JR.,
and
T. Y . TORIBARA
School o f M e d i c i n e and Dentistry, University of Rochester, Rochester,
Errors in the measurement of the calcium in aged blood serum by precipitation as calcium oxalate, followed by titration of the oxalate, are manifold. The principal sources are the nonspecificity of the oxidizing agent, the incompleteness of calcium precipitation, and the contamination of the precipitate by oxalates other than calcium. The direct measurement of the calcium by flame photometry of acidified solutions from which protein has been removed eliminates most of the errors.
C
ALCIUM in blood serum has probably been determined most frequently by the Clark-Collip ( 4 ) modification of the Kramer-Tisdall ( 5 ) method, which is based on the precipitation of calcium oxalate by the addition of ammonium oxalate to serum. A thorough coverage of the variable results obtained by other investigators has been made by Sendroy (8) in the introduction of a paper in which he reports the results of a very detailed study of many of the sources of error. He shows that identical results are obtained whether precipitation of calcium by oxalate was carried out on dilute serum, ashed serum, or the trichloroacetic acid filtrate of serum (protein removed) after appropriate corrections have been made for the calcium content of the ashing reagents, filter paper, and trichloroacetic acid. His studies were carried out on human serum from blood freshly drawn from ambulant dispensary patients. A direct measurement of the calcium by the flame photometric procedure ( 3 ) has again shown that calcium may be precipitated completely as the oxalate from fresh serum. Analyses on aged serum samples, however, gave variable and erratic results. For
N. Y.
this reason a calcium balance was made on a number of aged serum samples, and a typical balance of a sample of calf serum which had been stored in a refrigerator for several weeks is shown in Table I. The trichloroacetic acid filtrate of serum contains all of the serum calcium, and all of this calcium can be precipitated by osalate by raising the p H to 5 . There was no detectable difference in the calcium content of the supernatant liquid when the protein was separated by centrifugation or by filtration through a Whatman No. 42 paper. S o error was introduced by the use of ashless filter paper in the preparation of protein-free filtrate-. The presence of trichloroacetic acid (Eastman white label) in the amounts used increased the emission a t 620 mp less than 1 yo. DISCUSSION
The inconsistencies in the results obtained with protein present may be attributed to the age and condition of the serum. In freshly drawn serum, the calcium may be completely precipitated
Table I.
Typical Calcium Balance Determined on Aged Calf Serum
Component .4nalyzed Serum, direct dilution, I t o 100 Serum, oxalate ppt. Serum after p p t n . by oxalate TrichlAroacetic acid filtrate, direct Trichloroacetic acid filtrate, oxalate p p t . Filtrate direct reading a f t e r pptn. Proteins, pptd. by T C A , washed twice with T C A
Calcium, hIg./100 hll 12.3 11.1 1.2 12.0
12.0 0.008
0.08
