Paper Chromatographic Separation and Complexometric Titration of Trace Amounts of Strontium and Calcium in Biological Material PIERRE A. DUMONT laboratory o f Zoophysiology, University of louvain, louvain, Belgium
b A specific, sensitive, and simple method of simultaneous determination of calcium and strontium in biological material is described. Samples are burned in a closed atmosphere of oxygen, then taken up in dilute HNOa. An aliquot is submitted to ascending paper chromatography. Calcium and strontium are separated from each other, then recovered b y calcination. Both elements are estimated b y complexometric microtitration with EDTA, using calcein as indicator. Amounts of 25 X 10-9M calcium and strontium are quantitatively recovered to within 1 to 2y0 and can be determined with a coefficient of variation of about 5%. For larger quantities, the reproducibility ranges from 1 to 4%.
0
to the great similarity in the chemical properties of strontium and calcium, the quantitative determination of one of these elements in the presence of the other, more explicitly that of strontium in the presence of a large excess of calcium, is a difficult problem. Methods of neutron activation (S), x-ray fluorescence (6), and emission spectrography analysis (5, 8, I I ) , have been described. They are highly specific but require complex equipment and laborious preliminary standardizations. An easier approach is flame spectrophotometry. Unfortunately, interference by external and internal factors is a very serious and frequent cause of errors and requires, among other necessary precautions, either partial purification of the samples (4) or standard solutions for each type of unknown to be analyzed. As a rule, all the previous methods are suitable for routine analysis of the same sample but they become exceedingly time-consuming with samples from different sources and of variable composition. In the course of studies of calcium and strontium metabolism in animals, the author has tried to develop a method for simultaneous determination of both these elements which would be characterized by high specificity, be free from any interference by foreign ions present in the samples, and possess WING
a large sensitivity range in order to determine values for the Ca-Sr ratio far removed from unity. Basically the procedure includes preliminary separation of calcium and strontium by paper chromatography, followed by calcination of the corresponding spots and microtitration of the two metals by EDTA. Amounts of 1 Mg. of calcium and 2 pg. of strontium can be determined. The applicability of the method to biological preparations has been tested on crab shell. EXPERIMENTAL
Preparation of Samples. Organic materials-fragments of crab shellsare incinerated in an atmosphere of oxygen, according to the Schoniger procedure (7). Thomas-Schoniger combustion flasks (300-ml.. Arthur H. Thomas Co.. Philadelphia, Pa.) are used for 10- to'20-mg. samples. The combustion takes place in the presence of 10 ml. of l N "0,. Oxygen, analytical grade, is rendered dust-free by filtration through a column of cotton wool. Chromatographic Technique. Calcium and strontium are separated by ascending paper chromatography. The chromatograph tank is a n airtight glass-walled chamber (40 X 25 X 40 cm.). I t s internal parts-Le., the container for solvent (35 X 23 X 5 cm.) and the frame for the support of the sheets-are made of Perspeu. The top of each sheet is held pressed between two flat bars of Perspex, secured about their ends with elastic bands. The opposite upper edges of the frame are slotted to receive the bars but in such a way that two positions can be adopted: an upper position (shallower slots) or saturation position, and a lower position (deep slots) or development position. cHROM-4TOGRAPHIC PAPER. Schleicher and Schuell filter paper S o . 20431, is used. To eliminate the residual metallic impurities, the sheets must be thoroughly washed with the same solvent as that used for the development. After this treatment, they are dried for several hours in warm air and stored a t 20' C. in a dust-proof cabinet. SOLVENT. Methanol-HC1-water (v./v.), 80:5: 15 ( 2 ) . This one-phase
solvent system is prepared with calciumfree reagents and stored in light-tight polyethylene bottles in a cold room. ApPLIC.4TION O F SAMPLE. The Spots are placed 3 cm. from the lower edge of the paper and a t intervals of 7 cm. They are marked with the aid of an aluminum template as described by Block, Durrum, and Zweig ( 2 ) . The samples are applied with 5- or 10-pl. micropipets, the tips of which have been siliconized (Kimax micropipets, Class A, Owens-Illinois Glass Co., Toledo, Ohio), placed a t right angles to the paper. DEVELOPMENT.After a 12 - hour period of saturation, the solvent is allowed to travel 25 t o 30 em. beyond the point of application. During this period of 4 to 5 hours, the room temperature is kept a t 20" =t2" C. DRYING.The development is stopped by drying the chromatogram with a fan-type electric heater and, once the bulk of the solvent has been eliminated, with the help of an infrared lamp. Overheating must be avoided, as it makes the sheets crisp and difficult t o handle. It is important to continue the drying until the sheet no longer contains traces of residual acid, inasmuch as these traces interfere with the reaction used for detecting the alkaline earths and decrease its sensitivity. DETECTION OF SPOTS. Spots of calcium and strontium are made visible by spraying the chromatograni with a solution of 1% 8-quinolinol in alcohol, according to the method of Sommer ( I O ) . After drying, the paper is suspended for 5 minutes in an atmosphere of ammonia and then examined in ultraviolet light. Limit of Detection. Sr, 2 X 10-9M; Ca, 1 X lO-9M. The spots are lightly ringed with pencil, allowing a margin of 5 mm. for security. Recovery of Calcium and Strontium. The spotted areas corresponding t o calcium and strontium are cut o u t just inside the pencil line, folded with the aid of forceps, and conveyed to 4-ml. porcelain crucibles (Staatlich b 3/0, Berlin, Germany). Pieces of paper of identical size, cut out from the adjoining region, are treated in the same way and serve as blanks. The crucibles are ignited in a closed electric furnace a t 410" to 430" C. until the contents are reduced to a fine white ash, removed from the oven, and VOL. 33, NO. 4, APRIL 1961
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allowed to cool under cover. Half a milliliter of 0.1N "03 is added to each crucible. Temperature exceeding 500" C. causes a loss of material, partly by volatilization and partly by the formation of alkaline earth silicates. Complexometric Microtitration by EDTA. Calcium and strontium a r e titrated directly in t h e crucibles with 0.01M E D T A , according t o t h e procedure of Socolar and Salach (9). For the sake of reproducibility, i t is essential that the volumes of the reagents used by these authors be accurately transferred. EQUIPMENT. Scholander-type ultramicroburet (Misco, Berkeley, Calif.), one division of which corresponds t o 3.89 X lop2pl.
Table 1.
Ca
a
Calcium and Strontium Determination by Method of Socolar and Salach Av. Taken, No. of -4v. Found, Std. Dev., Difference, x 10-9~ Trials x 1O-pM % x 10-9~ 250.0 125.0 48.5 24.95 A250.0 B 236.5 A 120.0 R 113.3 A 50.0 B 47.3 A 25.0 B 23.7
Sra
Microscopic illuminator fitted with a blue filter (Parra-Mantois VB 7, France). REAGENTS.Calcein indicator. Stock solution, 2% (w./v.) in 1N KaOH, stored a t 4" C. in the dark. Working % (m./v.) in metalsolution, 6 X free water. The reagent is obtained from the Siegfried Co., Zofingue, Switzerland, and from the G. Frederick Smith Chemical Co., Columbus, Ohio. Standard EDTA solution (0.01iV). Disodium EDT-4 2-hydrate (Nerck, Germany) (3.7210 grams) is dissolved in metal-free water and the solution is made up to exactly 1 liter. It is standardized against CaC03. Calcium and strontium standard solutions are prepared with analytical
5 5 5 14
250.0 125.2 48.0 24.7 238.0 225.0 106.0 103.0 36.0 37.8 14.0 15.4
0 $0.2 -0.5 -0.25 - 1 2 . 0 iz 5 . 0 * -11.5 4.7 -14.0 & 2 . 4 -io.S 2.i -14.0 f 0.93 -9.5 f0.6 -11.0 f 0.85 - 8 . 3 It 0 . 8
1.15 2.0 2.9 4.8 2.1 2.1 2.3 2.05 2.6 1.6 5.9 5.0
* *
A and B refer to different batches of reagents. Std. dev.
Table II. Calcium and Strontium Determination in Mixtures Crab Shell Standard Amounts Unknown Level, x lO-9M and 25 150 Unknown Increment Ca Sr Ca Sr Ca Sr Ca Sr Taken, x 10-PM 24.95
22.8
150
..
150
523"
180"
526 522 522 524 528 52 1 522 524 524 0.5 +0.2
182 180 182 172 172 179 179 170 177 2.75 -1.7
Found, x lO-9M 25.4 24.6 25.4 24.2 24.2 23.3 27.1 26.25 22.9 25.0 25.4 25.0 22.9 23.75 Av. 24.7 Std. dev. %b 5 . 0 Av. diff., yo - 1
22.9 24.2 22.9 23.35 23.35 22.922.5 21.3 20.85 23.35 23.35 22.9 21.7 22.5 22.7 4.0 -0.44
156 158 142 144 148 146 151 155 150 4 0
158 153 148 158 148 150 150 148 152 2.8 +1.3
226.0 233.5 230.0 227.0 227.0 229.5 228.0 231.0 229.0 1.1
81.1 83.5 77.9 80.3 83.1 83.5 83.5 82.3 81.9 2.5
...
...
Sum of added increments; 98 x lO-gM Sr and 294 x lO-gM Ca, and of corresponding average values initially present, 81.9 x 10-9 Af Sr and 229 X 10-9 M Ca.
grade CaC03 (Merck) and SrC03 (British Drug Houses, England), previously dried a t 104" C. for 24 hours. Deionized water. Metal-free water is prepared by passing glass-distilled water over an ion exchange resin cartridge (Deeminac A-6, Crystal Research Lab., Inc., Hartford, Conn.) All other chemicals are of reagent grade quality. RECOMMENDED PROCEDURE
A convenient adaptation of the method to routine analysis includes the following steps.
Processing of Paper. The filter paper is cut t o t h e desired size (40 X 28 cm.), and marked with three spots. T h e sheet is introduced in a first tank (washing tank), and allowed to hang in contact with the solvent until t h e front has arrived approximately 1 cm. from t h e top edge (approximately 12 hours). Metallic ions, contaminating the paper, accumulate in this region and no longer interfere with t h e further chromatographic process. Finally the sheet is dried and stored hanging vertically. Chromatographic Separation. On two spots are applied definite volumes (5 to 10 pl.) of the solution to be analyzed and on the third a n equal volume of a standard calcium and strontium solution, the concentration of which is of the same range as the unknown. The sheet is transferred to the second chromatogram tank (analysis tank). After saturation, the chromatogram is developed, until the front of the solvent arrives a t 4 to 5 em. from the top edge. Finally, the spots of Ca and Sr are detected and ringed. Quantitative Estimation. = 0.389 ( N - N o ) Ca, (mole Sr, (mole 10-9) = 0.389 ( N - N o ) T
+
where N = number of diTisions of microburet (1 division = 3.89 X lop2 pl,). Subscript o refers to the blank value given by the paper. T is an experimental term determined by analyzing the spot of strontium standard and subtracting the obtained value from the actual one. It varies from one batch of reagents to another, between 5 and 20 x 10-9 mole. Titration of the spot of calcium standard allows the titer of 0.01M EDTA and the calcium recovery to be controlled. Finally a volume of unknown solution, equal to that applied on the chromatogram, is titrated under the same conditions. The observed value is compared to the sum of Ca, and Srz, and constitutes a check of recovery for t h e over-all procedure. RESULTS AND DISCUSSION
5
Jy
b
Std. dev., yo =
566
3
ANALYTICAL CHEMISTRY
x 100.
Applicability of Socolar-Salach Procedure to Strontium Determinations. A preliminary investigation was undertaken to determine the feasibility
of extending the Socolar-Salach method (9) of calcium determination to strontium. Direct titrations of standard samplcs of calcium and strontium mere carried out in parallel. The experimental data are presented in Table I. Both in accuracy and precision, the r t w l t s of calcium estimations obtained are in very close agreement with those of the above-mentioned authors. Strontium can be titrated to an end point as sharp as that observed with calcium. The values of the standard deviation are of the same range. However, a systematic negative error is apparent. This error has been proved to be independent of the absolute amount of strontium and of the presence of calcium. Its value has been found identical whether determined from standard amounts of strontium submitted or not to the chromatographic process or from knonn increments of strontium added to samples of Eriocheir shell. If the standardized conditions of the method are respected, the error varies but slightly from one batch of reagents to another. Its origin appears to be linked to the indicator. Concentrations of calcein (working solution) lying between 4 and 8 X 10-370 give the lowest values. Final results are corrected for the error by applying the correction term, T. For highest accuracy, i t is advisable to determine T by analyzing standard amounts of strontium, as close as possible to the unknown. Simultaneous Determinations of Calcium and Strontium in Mixtures. T h e results obtained by applying t h e over-all procedure, which includes t h e chromatographic separation a n d t h e subsequent recovery of calcium and strontium, are shown in Table 11. Analyses were carried out on a standa r d mixture of pure calcium and strontium salts, and on mineralization products of shells of crabs, Eriocheir sinensis, kept after their molt in an artificial medium equimolar in strontium and calcium. I n this last case, a check was made of the recovery of calcium and strontium, introduced in known amounts in a preanalgzed sample. Both metals were quantitatively rworered, and no influence of alkaline earth complesing anionic material, frequently present in biological samples, could be detected. As described, the method includes two steps which contribute to remove the possible interfering anions-Le., incineration of the organic part of the sample and subsequent chromatographic separation of the combustion products. The present results are indicative of their effectiveness. Specificity of Process. As reported by Socolar and Salach (9), the complexometric titration does not differcn-
Table 111.
SrCln
Ra of Calcium and Strontium in Media of Different Compositions Compositiono Mmoles Ca Sr 10 10 50 100 200
++ CaC12 ++ CaClz CaCIp + CaClz CaClp
5000 30 30 10 100 500
CaClp SrClp KaC1 KaCl NaCl FeCL
++ + + + nigcip
a
b
10 10
0.55 A 0.02b 0.55 =k 0.02 0.56 A 0.025 0.56 zk 0.02 0.56 zk 0.02
0.36 =k 0.03b 0.36 =t 0.02 0.37 f 0.025 0.39 f 0.025 0.40 =k 0.03
0.52 zk 0.52 zk 0.63 zk 0.54 zk 0.52 5
0.34 =t 0.01 0.36 =t 0.01 0.39 =t 0.01 0.38 i.0.02 0.37 =t 0.02
0.02 0.025 0.02 0.02 0.02
5-111. Pamples applied.
Std. dev.
Recovery of Calcium and Strontium in Presence of Ferric and Magnesium ionsa Solution Ab Solution Bc Ca Sr Ca 6 r 150.0 142.0 142.0 Taken, X lO-QM 150.0 151.5 140.1 148.6 140.6 Found, pmole X 1.6 2.2 2.5 0.9 Std. dev., c;C $1.0 -1.3 -0.9 -1.0 Av. difference, yo 4 4 4 4 No. of trials
Table IV.
a
5-111.samples applied. Contains 30 mmoles CaC12,28.4mmoles SrC12, 10 mmoles MgC12. Same as solution A, except for 10 mmoles FeC13, instead of MgC12.
ACKNOWLEDGMENT
tiate among calcium, strontium, and barium. I n addition, high excesses of magnesium and iron interfere, especially by masking the end point. The preliminary chromatographic procedure allows a clear-cut separation of all these metals. With starting spots of 30 to 150 X 10-9LV of their chloride, the following average values of RF (at 20’ i 2’ C.) have been observed :
The author expresses gratitude t o H. J. Koch, head of the Department of Zoophysiology, for his helpful suggestions and continuous interest during the course of the work. Thanks are also due to R. Lontie, who kindly discussed the final manuscript. The technical assistance of J. Evans and E. van den Borren is greatly appreciated.
Ba+2O.l8 A O.Oz5 Sr+20.36 & 0.02 Ca+20.53 A 0.02 Mg+20.74 0.02 Fe+3 0.79 0.03
(1) Block, R. J., Durrum, E. L., Zweig, G., “Manual of Paper Chromatogra-
*
Std. dev. Separation of the calcium and strontium spots is not hampered by the presence of a 500-fold excess of calcium or by large variations of the KaCl content of the medium. Neither magnesium nor ferric ions interfere (Table 111). Finally i t was proved that neither ion decreased the recovery of calcium and strontium during the over-all procedure (Table IV). Thus, the combination of the characteristics of the chromatographic and titrimetric procedures leads to a high specificity. Furthermore, an advantage inherent to paper chromatography is that the extent of separation can be directly viewed at the time of the development.
LITERATURE CITED
phy and Paper Electrophoresis,” Academic Press, Xew Yorlr, 1955. (2) Dusing, TV., Kunze, H., Z. anal.
Chem. 162, 346 (1958). (3) Harrison, G. E., Nafure 182, 792 (Sept. 20, 1968). (4) Harrison, G. E.,Ravmond, &‘., J . Nuclear Energy 1, 290 (i955). ( 5 ) Hodges, R. M . , MacDonald, h-.S., Nusbaum, R., Steams, R., Ezmirlian, F., Spain, P., Machrthur, C., J. Biol. Chem. 185, 519 (1950). (6) Roberts, TT. M. B., Kuture 183, 887 (March 28, 1959). (7) Schoniger, TT’., Jlakrochim. Acta 1, 123 (1955). (8) Shalimoff, G. V., Conway, J. G., Pitzer, A. E., A p p l . Spectroscopy 12, 120 (1958). (9) Socolar, S. J., Salach, J. I., ANAL. CHEM.31, 473 (1959). (10) Somnier, G.,2. anal. Chem. 151, 336 (1956). (11) Turekian, K. K.,Kulp, J. L., Science 124,405 (1956).
RECEIVEDfor review May 2, 1960. Accepted December 7, 1960. VOL. 33, NO. 4, APRIL 1961
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