Thin Layer C:hromatography of Toxicologically Significant Substances on Silica Gel-Coated Glass Plates and Polyester Sheets P a u l Schweda Ofice of the Chief Meai'cal Examiner, Baltimore, Md., and the Maryland Medical Legal Foundation, Inc., Baltimore, Md. THIS STUDY compares the chromatographic properties of Eastman Chromagram Sheets for TLC (Distillation Products Industries, Rochester, N. Y.)with data on silica gel-coated glass plates and investi,gates the applicability of the sheets to special problems. Toxicologically significant substances, including new drugs, were tested in three representative mobile phases which are routinely used for the analysis of biological specimens anij autopsy material. R , values of drugs on Chromagram sheets have not been previously published. Data of comparison of the two media were lacking, and no information was found in the literature about applicability, dilferences, or performance of a given solvent system chromatographed in tank type containers and sandwich devices on plates and/or polyester sheets. Ten drugs in each group are presented in this paper. Toxicologically significant substances were selected in each group which were approximately evmly distributed over the running distance of 15 cm. To control the efficiency of the media, pairs were included which in the lower, middle, and upper ranges run closely together in plate systems. EXPERIMENTAL
Reagents and Materials. All reagents were of reagent grade; organic solvents were redistilled. Pure drug samples, obtained through the courtesy of the manufacturers and without further purifil:ation, were dissolved in EtOH to give 0.5 solutions. Silica Gel G for TLC, E. Merck A. G., Darmstadt, Germany, was distributed by Brinkmann Instruments Inc., Westbiiry, N. Y. Silica Gel G (binder) for TLC, Research Specialties Co., Richmond, Calif., 20 >: 20 cm glass plates were coated with 250-mp layers of Silica Gel G with the following applicators: Desaga thin layer spres.der with alignment tray (Brinkmann) and Unoplan applicai or with pneumatic alignment base (Consolidated Laboratories, Inc., Chicago, Ill.). Channel plates (250-mp pregrooved glass plates, Kontes Glass Co., Vineland, N. J.) were coated with the Desaga apparatus. Eastman Chromagram Sheets (Type K 301 R2) silica gel without fluorescent indicator, batch No. 3005,3007,3009, and Eastman Chromagram sheets (Type K 301 R) silica gel with fluorescent indicator, batch No. 806, were used. Apparatus. Two types of containers were used. Tank types were rectangular glass jars, 22 X 11 X 23 cm, glass covered and sealed with starch-glycerine paste. The jars were lined on all sides i o a height of 18 cm with filter paper. Sandwich apparatus included the Eastman Kodak Chromagram developing apparatus and the Chromaflex apparatus, Kontes Glass Co., for channel plates. Application capillaries were "Microcaps" disposable micropipets, Drummond Scientific Co., Broomall, Pa., in 25X, lOX, and 5X sizes. Chromatographic Procedures. Glass plates were coated with 250-mp silica gel layers according to Stahl (I). The
z
(1) E. Stahl, "Thin-Layw Chromatography," Academic Press, New York, 1965, pp. 5-14.
coated plates and sheets were air-dried, activated for 30 minutes at 110" C (channel plates were activated for 60 minutes at 90" C), and desiccated. The samples were applied with Microcaps 3 cm from the bottom edge of the plates or sheets, 2 cm apart. The side edges were stripped 0.5 cm wide, One plate and one sheet spotted in the same sequence were developed in the same tank and saturated with the solvent for a minimum of 6 hours. After the solvent front rose to the 15-cm mark on either of the materials, the plates and sheets were removed and the lower solvent-front was marked. The dried plates and sheets were inspected under ultraviolet light (Mineralite UVS-12, Ultraviolet Products, San Gabriel, Calif.) and stained. Spray reagent for System 1 was 0.1 s-diphenylcarbazone (w/v) in 95 alcohol. The plate was sprayed to a slight rose color, allowed to dry for 5 minutes, and thereafter sprayed with 0.33 mercuric nitrate in 0.04N nitric acid (2). For Systems 2 and 3 the spray reagent was potassium iodoplatinate (3). Barbital, codeine, and Compazine were used as reference substances in the respective systems and R,'s were measured by the spot limits method. Standard Conditions. All experiments were conducted at a temperature of 21.5" 1.5" C. Activated plates and sheets were developed in the same tank, containing 300 ml of mobile phase or a sandwich apparatus containing 140 ml of mobile phase (Kodak apparatus) or 50 ml (Kontes apparatus), respectively. The planimetering of spot sizes (where done) was carried out on the still wet media to reduce distortions by diffusion. Ten substances in each group on 10 plates and 10 sheets were analyzed. Runs where the reference standard did not come within R, i: 0.05 of the average were rejected and were replaced by a tank in which three sets (1 plate and 1 sheet) were developed. Chromatographic Systems. System 1, consisting of chloroform:n-butanol:ammonia(25 (70:40:5), separates in a short time and shows a good spread of important acidic substances. The ammonia concentration affects R, values. The center beaker with ammonia, as reported in the original paper (2), does not affect running rates but prolongs the lifetime of the jar. System 2, consisting of benzene :dioxane :ethanol :ammonia (25 (50 :40 :5 :5 ) , was used for the analysis of basic substances with special emphasis on narcotics (3). Redistilled dioxane is stable (free of peroxides) when kept under nitrogen and refrigerated. The system is comparatively sensitive to temperature changes and vulnerable to Tiselius effects (4). System 3 consisted of chloroform :cyclohexane :diethylamine, (50 :40 :lo). This water-free system is reported in the literature in different compositions for a systematic analysis of alkaloids ( 5 ) and is closely related to the im-
z
z z
x),
z),
(2) J. Lehman and V. Karamustafaoagly, Scand. J . Clin. & Lab. Invest., 14, 544 (1962). (3) J. Cochin and J. W. Daly, Experientia, 18,294 (1962). (4) A. Tiselius, Endeauour, 1 1 , 5 (1952). ( 5 ) T. Waldi, K. Schnackerz, and F. Munter, J . Chromalog., 6, 61 (1961). VOL 39, NO. 8, JULY 1967
1019
??????????
0 3 0 0 0 - o - o w
ixai aaS
w
m
+
++I
m
m
-oCC)--ow
k
2
.
.
++++
03'3"O
.
w o o
I
+
+++ I +
++
+
1020
ANALYTICAL CHEMISTRY
+ +
-0-
+
ornww-mo
o-
w-ow-N-
" 0
+
7"
I I
I I++
o o w o - 3 -
m m o o - 0
+
0-
+ I +
-bl-bwmo
I I I + l +
I
-I +
ow---o
1 1 1 1
+
00-
Table 11. Solvent Mobilities on Silica Gel Layers in Three Different Solvent Systems Running distance = 15 cm Tank tyve containers Chromagram SiO? plates Chromagram plain fluor. ind. Rzlative Relative Relative cmlminute mobility cm/minute mobility cm/minute mobility cm/minute mobility cm/minute
+
System 1 System 2 System 3
0.21 0.33 0.32
13.64 1.oo
0.97
0.17
0.52
0.26 0.29
0.79 0.88
0.11 0.16 0.16
portant group of formamide systems used in paper chromatography (@. RESULTS AND DISCUSSION
The means of 10 acceptable R f determinations of each substance in the specific sysi.em are listed in Table I together with data on spot sizes and detection limits. The average values, expressed as R f X 100, me derived from tank type containers and sandwich devices. The column of running rates is followed by columns of reference R,’s ( R f ) r ,which were found experimentally. The differences between these experimental findings and the theoretical value ( Rf ) x / ( R f )are r marked A. Silica Gel-Coated Glass Plates and Chromagram Sheets Silica Gel (without Flluorescent Indicator) Used in Tank Type Containers. In d l three systems the standards on Chromagram sheets run faster, and are not as evenly spaced as on plates; R,’s on sheets and plates are not comparable within permissible limits. Because spot dimensions are important for resolution, substances in different ranges were planimetered and are litited in Table 1. Because the double spray for System 1 widens the spots by diffusion, the thinner layers (Chromagrams) are handicapped and the findings in that group are omitted. The spot sizes on plates increase with the distance from the start and are about three times larger near the front. The spot sizes on Chromagrams are approximately the same over the entire range and larger than those on plates. The minimum detectable amounts are listed in Table I. The sheet:; in this column compare favorably with plates except in System 2. Chromagram Sheets Silica Gel (without Fluorescent Indicator) and Silica Gel (with Fluorescent Indicator) Used in Tank Type Containers. R,’s on fluorescent indicator sheets (lead-manganese-activated calcium silicate indicator) are listed in Table I. The running rates are more comparable to the plain sheets, but the reproducibility of R f 0.05 is frequently exceeded. The incorporation of the indicator slows the R f values at EL constant rate. Micrograms can be detected under ultravicdet light on indicator sheets, No conclusive information can be derived solely from such observations, and sometimes difficulties arose with the subsequent extraction of the spots. Where fluorescent or absorptive characteristics are adequate, the Chromagram fluorescent indicator sheets can be recommended. Silica Gel-Coated Glass Plates and Chromagram Sheet Silica Gel (without Fluoirescent Indicator) Used in Sandwich Developing Chambers. Besides tank type containers, another type of developing chamber (S-chamber) is used in the sandwich technique ( I , 7). R,’s on Chromagram sheets and (6) T. Waldi, Arch. Pharm ,292,206 (1959).
0.33 0.48 0.48
0.21
0.64
0.26 0.79 Not applicable
mobility
0.12
0.36
0.18 0.54 Not applicable
silica gel-coated glass plates in sandwich chambers are listed in Table I. SYSTEM 1. All barbiturates run with the solvent front and only nonbarbiturates have measurable running rates. This cannot be explained by differences in chamber saturation. The system cannot be used with plates or sheets for the detection of barbiturates in sandwich chambers. SYSTEM 2. The substances are differently distributed and expanded over the entire running distance, with a notable inversion of cocaine and Librium on both media. R f values in tank and sandwich type chambers are in most instances no longer comparable within permissible limits. The reproducibility is good and it is somewhat better for plates than in the tank containers. A decline in reproducibility is noticeable for Chromagrams. SYSTEM 3. This system (and modifications thereof) cannot be used in sandwich chambers because of solvent demixing, which also occurs in IO-cm runs and in 10 X 20 sizes. Although partly successful in a few cases with sheets, the system is unreliable in this application and therefore marked as not applicable in Table I. In all three systems used, the solvent mobilities in sandwich devices were slower, contrary to reports in the literature (I, 7). Solvent Mobilities on Silica Gel-Coated Glass Plates and Chromagram Sheets. The solvent mobilities on sheets and plates in all systems over a running distance of 15 cm are listed in Table 11, together with mobilities relative to the fastest running system (0.33 cm per minute in System 2 on plates = 1). The figures would be considerably shorter for 10-cm runs. The solvent mobilities on Chromagram sheets (Type K 301 R) are approximately 10 to 20% less than those on glass plates. The mobilities of the solvents on Chromagram sheet (Type K 301 R) are 20 to 4 0 z less than those on Chromagrams sheets (Type K 301 R2), and correspondingly lower compared with glass plates. These longer runs on sheets are not a serious disadvantage, because of the short running time in TLC. Indicator sheets, however, have running times up to twice that on plates in the same system, Solvent mobilities in sandwich runs compared with tank containers are the same (System 1 on plates) or considerably less, approximately 20% in System 2 on plates, and approximately 40 % on Chromagram sheets (Type K 301 R2). By using the reported solvent systems in well-saturated gas atmospheres under standard conditions, solvent mobilities in tank containers are equal to or faster than those in sandwich devices. Activated and unactivated layers showed no differences in R f values within the permissible limits. The spots on activated layers were generally more compact, better defined, (7)K. Randerath, “Thin-Layer Chromatography,” Academic Press, New York, 1963, pp. 21-9. VOL. 39, NO. 8, JULY 1967
1021
and more reproducible. In 10-cm runs all R/’s were well within f 0.05 of the reported running rates on 15-cm runs, but the separation on the latter was better. Chromagram layers are hard, flexible, and durable in contrast to coated plates. The application devices may touch the layer on sheets repeatedly without leaving perforation marks. The lesser absorptivity of the thinner layer and the necessity of applying smaller aliquots prolonged the application time. The omission of any handling of finely powdered silica gel may be considered safer. Both types of Chromagram sheets were resistant to solvents used in chromatography. Water-rich solvent systems did not cause peeling or softening of the layers. Aggressive spray reagents (concentrated acids) and exposure to chlorine gas in tanks (up to 30 minutes) had no detrimental effects.
ACKNOWLEDGMENT
The author is indebted to Henry C. Freimuth for his advice and critical review of the manuscript, and acknowledges the help of James C. Schlaffer in the experimental work.
RECEIVED for review December 19, 1966. Accepted May 8, 1967. Part of this study was presented at the 746th Meeting 01 the ACS Washington Section, conducted jointly with the ACS Maryland Section, May 6th, 1966, at the University of Maryland, College Park, Md. Work supported by funds from Grant GM-11699 of the National Institutes of Health, U. S. Public Health Service, to the Maryland Medical-Legal Foundation, Inc., Baltimore, Md.
Calcium Electrode Method for Measuring Dissociation and Solubility of Calcium Sulfate Dihydrate F. S. Nakayama and B. A. Rasnick U.S. Water Conservation Laboratory, Phoenix, Ariz. 85040 MANYCA SALTS form both undissociated and dissociated species in solution. For example, when CaS04.2Hz0dissolves in water, three distinct species are present: the Ca+z and SOa-2 ions and undissociated calcium sulfate, CaS04; in sulfuric acid the complex Ca(HS04)+1is also present. The usual chemical techniques for Ca analysis give the total solution Ca and not the individual species. Thus, in order to study the dissociation of Ca salts, indirect methods involving either colligative or electrical properties are used, which require involved instrumentation and theoretical assumptions. Recently, Ca membrane electrodes became available, which provided an opportunity to measure Ca ion activity in solutions at various ionic strengths and made possible the estimation of the dissociation and solubility parameters based on the activity of the dissociated ion rather than on the total concentration. A successful determination of Ca activity in sea water was reported by Thompson and Ross ( I ) . Ross has also described the operational theory of the Ca electrode (2).
Calibration curves of CaC1, were run in water and in different concentrations of NaCl to simulate the ionic conditions anticipated for the CaS04-NaCl and CaS04-NanS04. The electrode was calibrated empirically to avoid any uncertainty in the value of Eoca in the Nernstian equation. Linear calibration curves were obtained for the plot of emf us. the log of the Ca activity. Triplicate measurements at 25” f 1” C were made for the saturated CaS04 solutions for each of the ionic strengths used. The computation of the various solution parameters for CaS04 from the Ca activity measurements is as follows. The Ca electrode measures the activity of the Ca+2in solution and is not responsive to the undissociated and complexed Ca in solution. For a completely dissociated Ca salt (Ca+*) = yca+z [Ca+r] where ( ) refers to the activity, [ 3 denotes the concentration, and yca+z is the ionic activity coefficient of Ca. For a partially dissociated Ca salt, however, the relation must be modified as follows: (Ca+2) =
EXPERIMENTAL
Apparatus. Ca activity (Ca+3 was measured with the Orion Ca membrane electrode in conjunction with a saturated KCI, Hg-Hg2Clz reference electrode and a Corning Model 12 pH-millivoltmeter Solutions. Saturated solutions of CaSOa were prepared by dissolving CaS04.2H20in deionized distilled water and in NaCl and NatS04 solutions of various concentrations up to 0.1M. Procedure. The Ca electrode was calibrated with standard CaC12 solutions, as CaClz is completely dissociated (3). (1) M. E. Thompson and J. W. Ross, Jr., Science, 154, 1643 (1966). (2) J. W. Ross,Jr., Science, in press (1967). (3) W. J. Hamer, Ed., “The Structure of Electrolyte Solutions,” Wiley, New York, 1959, p. 26.
1022
ANALYTICAL CHEMISTRY
ayca+z [Ca+%
(2)
where a is the degree of dissociation of the dissolved salt; [&+ZIT is the total calcium in solution as determined by the versenate, or equivalent, method; (Caf2) is the activity of the salt in solution as measured by the Ca electrode. The product ayc.+z can be calculated from experimentally determined values of (Ca+2) and [Ca+2]~.a and ycS+z are not independent of one another, being related to the ionic strength via the concentration of ions in solution. In the calculation procedure a! and 7 C . t z were determined by an iterative process from measured values of (Ca+2) and [Ca+2]~.An arbitrary value between 0 and 1 was selected for yca+z and a was estimated using Equation 2. From the calculated a,the concentration [Ca+*] was determined using the relation [Ca+Z] = a[Ca+2IT. Subsequently, the ionic strength, p = Zzi2 ci/2, and the activity coefficient, ycn+_2, from the relation log yca+z = - A z 2 d i / ( l B a d d , were calculated, The necessary constants were taken from
+
