Ligand Exchange Chromatography of Amphetamine Drugs Cecilia M. de Hernandez’ and Harold F. Walton University of Colorado, Boulder, Colo. 80302 Amphetamine, phenethylamine, and related compounds have been separated by liquid chromatography on columns of cation-exchange resins loaded with ions of copper, nickel, and cadmium. Polymethacrylate resins are much better than those of the polystyrene type, and Cu(ll) was the most effective cation. Eluents were aqueous alcohol solutions containing ammonia, most commonly 0.1M ammonia in 33% ethanol. Metamphetamine was eluted first, then ephedrine, amphetamine, norephedrine, and phenethylamine. Detection and quantitative measurement was easily possible at 0.1 mg/ml, using 0.5-ml samples. However, extraneous peaks appeared at one column volume that seemed to be due to decomposition products.
LIGANDEXCHANGE CHROMATOGRAPHY was introduced in 1961 by Helfferich ( I ) . He used a column of carboxylic cationexchange resin loaded with copper(I1) ions to absorb a 1,3-diarnine from a dilute aqueous solution, and regenerated the column with aqueous ammonia (2, 3). Subsequently, other workers adapted the ligand exchange technique to the chromatographic analysis of mixtures ( 4 , 5 ) . Applications were made to aliphatic amines and diamines, ethanolamines, substituted hydrazines, and aziridines (6). We have extended our study to amphetamine, metamphetamine, and drugs related to phenethylamine. Amphetamine and related drugs are currently detected and determined by thin-layer (7) and gas chromatography (8, 9). A method that depends on oxidation by Ce(1V) followed by ultraviolet spectrometry is described (IO). It is common practice to use ion exchange to separate the drugs from urine ( 7 , 8 ) but ion-exchange chromatography has found little use in separating the drugs from one another, probably because of their very strong absorption on resins having a polystyrene matrix. Amberlite IR-45, a weakly basic anion-exchange resin with a polystyrene matrix, was used to absorb amphetamine, ephedrine, and epinephrine, presumably by nonionic absorption (11). Amphetamine, metamphetamine, ephedrine, epinephrine, and related compounds were separated by saltingout chromatography on polystyrene-type cation and anion exchangers (12). 1
Present address Pedagogical Institute, Caracas, Venezuela.
(1) F. G. Helfferich, Nature, 189,1001(1961). (2) F. G. Helffericn, J . Amer. Chem. Sac., 84, 3237 (1962). (3) F. G. Helfferich, ibid., p 3242. (4) A. G . Hill, R. Sedgeley, and H. F. Walton, Anal. Chim. Acta, 33, 84 (1965). (5) K. Shimomura, L. Dickson, and H. F. Walton, ibid., 37, 102 (1967). (6) H. F. Walton, “Ligand Exchange Chromatography,’’in Advances in Ion Exchange, Vol. 3, J. Marinsky, Ed., Marcel Dekker, New York, N.Y., 1972 (in press). (7) A. M. Heaton and A. G. Blumberg, J. CArornatog., 41, 367 (1969). (8) R. C. Baszlt and L. J. Casarett, ibid., 57,139 (1971). (9) E. Anggard, Int. Symp. Amphetamines Related Cpds., Proc., 1969,191 ; Chem. Abstr., 74,21643e (1971). (10) J. E. Wallace. J. D. Binas. 40, -- , and S . L. Ladd, ANAL.CHEM., ‘ 2207 (1968). ’ ( 1 1 ) M. G . Vincent. E. Kruoski. and L. Fischer. J. Pharm. Sci.. . 46, 85 (1957). ’ (12) J . Halmekoski and A. Vidgren, Farm. Aikak., 75,282 (1966); Cliem. Abstr., 66,22230q (1967). .
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We made tests with the sulfonated polystyrene cationexchange resin, Dowex-50 X8, loaded with nickel ions, with ammonia solutions as eluents. Columns of this material are very effective in separating amino compounds (5, 6). We found, as expected, that amphetamine and metamphetamine were absorbed strongly. Relatively concentrated ammonia was needed for elution, and the substrates emerged as broad bands with considerable tailing and virtually no separation (Figure 1). We therefore turned our attention to carboxylic cation-exchange resins. These are polymers of acrylic and methacrylic acids that contain no aromatic groups except for cross-links introduced as divinylbenzene. They have a high capacity for holding ions, and bind divalent cations so strongly that they are not washed out by the dilute ammonia solutions used as eluents. The cations used were Cu(II), Ni(II), Cd(II), and Zn(11). Of these, Cu(I1) gave the best results. The principal substrates studied were: Phenethylamine Amphetamine Metamphetamine Norephedrine Ephedrine
CsH5CH2CH2NH2 CsHsCH2.CHCH3.NH2 CsHsCH2.CHCH3.NHCH3 C6HjCHOH.CHCHB.NH, CsHsCHOH .CHCH,. NHCH3
Tests were also made with benzylamine, caffeine, epinephrine, and dopamine. The eluents were solutions of ammonia in mixtures of water with methanol, ethanol, and isopropanol. EXPERIMENTAL
Materials. The resin was Bio-Rex 70, 200-400 mesh, from the Bio-Rad Corp., Richmond, Calif. It is a methacrylate polymer manufactured by the Diamond Shamrock Corp., Redwood City, Calif., ground and purified for laboratory use. It was converted to the metal forms by stirring with solutions of the metal-ammonia complexes, then transferring to a wide column and washing with more metalammonia complex, then water. In the chromatographic column, the resin tended to pack slowly and unevenly. It was necessary to add more and more resin to the column until it finally stabilized. The packing and flow characteristics were improved by adding 5 %-lo% by weight of cellulose, Cellex-N (Bio-Rad Corp.), and this was our standard practice. For unknown reasons, the cadmium and zinc forms of the resin were much harder to pack and caused much more flow resistance than the copper and nickel forms. Microscopic examination showed no obvious differences. Under the microscope, the particles of resin had irregular shapes and a fuzzy appearance, indicating a large surface area. This was true of both the copper and the cadmium forms. The microscopic structure of the grains of Bio-Rex 70 may be significant, for we tested a new product, Amberlite IRC-72, obtained from the Rohm and Haas Co. We ground and screened this resin ourselves. The particles looked smoother and more transparent than those of Bio-Rex 70, and this may be connected with the fact that in the column, IRC-72 gave broad bands with bad tailing. Amphetamine sulfate, metamphetamine hydrochloride, ephedrine and norephedrine sulfates, dopamine hydrochloride, and epinephrine were obtained from K and K Laboratories, Plainview, N.Y., in their dl forms and used without
I
Table I. Elution from Copper-Loaded Column (Multiples of bulk column vo1ume)a
0
IO
20
30
40
SOLVENT, m i
Figure 1. Elution curve from column of nickel-Dowex 50, 6mm X 51cm Metamphetamine and amphetamine, each 2.5 mg/ml : eluent, 2M NH3 in 50% ethanol; 12 ml/hr
purification. Phenethylamine (Eastman Kodak Co.) was distilled before use. Amphetamine and metamphetamine bases were used in some of the experiments. They were made from the salts by adding a slight excess of sodium hydroxide to the aqueous solutions, then extracting with chloroform and evaporating the chloroform solvent on a steam bath. A solution of amphetamine in 50% alcohol was also prepared by adding to the sulfate solution an equivalent amount of standard barium hydroxide solution, then centrifuging. Equipment. The columns and fittings, pump, and detector were supplied by Chromatronix, Inc., Berkeley, Calif. Solvents were fed into the columns by a variable-speed pulseless pump, Model CMP-2, and the samples were introduced by a valve, Model SV 8031, with Teflon (Du Pont) slider and 0.5ml sample loop. As a rule, the materials were injected in the same solvent that was used for elution. Detection was done with a two-cell ultraviolet photometer, Model 200, connected to a Leeds and Northrup Speedomax recorder, which measured the absorbance at 254 nm. The solvent passed from the pump through the reference cell of the photometer, then through the sample injection valve and into the column. From the column outlet, the liquid flowed through the sample cell of the photometer and out into a receiver. Sometimes samples of effluent were collected and their metal contents measured by atomic absorption spectrometry, using a Varian Techtron spectrometer. Ultraviolet absorption spectra were scanned in some cases, using a Cary Model 17 spectrophotometer with expanded scale. The columns were 9 mm in internal diameter, except for those used with the nickel-loaded carboxylic and sulfonic resins, which were 6 mm in diameter. The length was 58-60 cm in most experiments. The ionic capacity of the packed columns was not known exactly, but it was at least 1.5 meq of metal ions per milliliter bed volume. (This capacity was measured with columns that had settled under gravity.) RESULTS AND DISCUSSION
Metal Content of Effluents. The normal metal ion contents were: copper, 0.2-0.5 mg/l; nickel, 0.1 mg/l; cadmium, 0.2-0.5 mg/l. They were no higher when an amine was
being eluted than when it was not, except for the few tests made with the catecholamines epinephrine and dopamine. With these two compounds the copper and nickel concentrations rose to 6 mg/l, corresponding to 1 mole of metal to 2-3 moles of base, and the solutions were blue. The absorptivity of the Cu(I1)-dopamine complex was over 5000 1.
IsoAlcohol Ethanol propanol Volume concn 33 % 50% 66% 33% Ammonia, N 0.06 0.10 0.06 0.11 0.06 0.11 Elution volume of: Metamphetamine 1 . 9 1.75 1 . 1 ...... 1.05 Amphetamine 2.85 2.15 1 . 8 1 . 0 1 . 3 1.25 Ephedrine . . . 2.12 . . . . . . . . . 1.25 Norephedrine . . . 2.7 . . . . . . . . . 1.65 Phenethylamine 5.5 3.9 3.1 . . . 1.85 2 . 2 Benzylamine . . . 2.9 . . . . . . . . . ... Caffeine . . . 0.6 . . . . . . . . . ... a Void volume was 0.45 times the bulk volume. Alcohol concentrations are expressed by volume. Table 11. Elution from Cadmium-Loaded Column (Multiples of bulk column volume)&
Ethanol, 55 “C Alcohol and temp. Ethanol, 20 “C Volume concn 33% 50% 7 5 z 33% Ammonia, N 0.09 0.15 0.11 0.09 0.11 Elution volume of: Metamphetamine 1.0 1.0 . . . . . . 1.15 Amphetamine 1.65 1.35 1 . 0 0.95 1.75 Ephedrine 1.2 . . . . . . . . . . . . Norephedrine 2.0 . . . . . . . . . . . . Phenethylamine 2.7 2 . 2 1.45 1.15 2.8 Void volume was 0.7 times the bulk volume. centrations are expressed by volume.
Methanol, 60 “C __33% 50% 0.17 0.17
. . . . . . 1.25 1.05
. . . . . . 1.6 ... 2.0
2.0
Alcohol con-
mole-’ cm-l at 610 nm. These complexes were eluted a t one void column volume and will be the subject of a future report. Except for the catecholamines, one can say that the bases do not remove metal ions from the column. Conversion of Amine Salts to Bases on the Column. It made no difference to the elution volume whether amphetamine, metamphetamine, and ephedrine were injected as the free bases or as the sulfate or chloride salts. An examination of the spectra of the eluents showed that the spectra were those of the free bases, not the salts. It was also found that sulfate or chloride ions appeared in the effluent just after one void column volume, whereas the amine base appeared later. Thus it seems that the amine cations are converted to bases by the excess of ammonia. Their preferential absorption by the resin, compared to ammonia, probably plays a part, for ammonia is a weaker base than amphetamine by a factor of 5 (13). Elution Volumes; Effects of Metal, Solvent, and Temperature. Elution volumes with Bio-Rex 70 are summarized in Tables I, 11, and 111. Each compound was run alone before running mixtures. The volumes are quoted as multiples of the bulk column volume to enable comparisons between short (18-20 cm) and long (58-60 cm) columns. Void volumes were estimated from the first rise in ultraviolet absorption caused by decomposition products (see below) or by an added “marker compound,” C6H50C2H40C2H5, which was presumed not to be absorbed. (13) D. D. Perrin, “Dissociation Constants of Organic Bases in Aqueous Solution,” Butterworth, London, 1965. ANALYTICAL CHEMISTRY, VOL. 44, NO. 6, MAY 1972
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Table 111. Elution from Nickel-Loaded Column, Ethanol-Water (Multiples of bulk column volume)*
z
z
Alcohol volume concn 33 50 Ammonia, N 0.085 0.13 Elution volume of: Metamphetamine 1.4 ... Amphetamine 1.55 1 .o 1.55 ... Ephedrine Norephedrine 1.9 ... Phenethylamine 3.1 1.35 a Void volume was 0.7 times the bulk volume. Alcohol concentrations are expressed by volume.
0
P x
W
u
z a
m K
0 v)
m a
0
I
I
20
40
I
I
I
60 80 100 SOLVENT, m l
1
I
120
140
Figure 3. Elution curve from column of cadmium-Bio-Rex 7 0 , 9 mm X 57.5 cm Each base 2 mg/ml; eluent 0.08M NHI in 33%ethanol; 12 ml/hr Peaks: ( I ) metamphetamine, (2) ephedrine, (3) amphet:rmine, ( 4 ) norephedrine, (5) phenethylamine
0
0
6
0 W
SOLVENT, rnl
Figure 2. Elution curve from column of copper-Bio-Rex 70, 9 m m X 60cm
:
4-
U
m CT
0 v)
Metamphetamine, amphetamine, and ephedrine, each 1.5 mg/ml ; norephedrine and phenethylamine, 2.5 mg/ml; eluent, 0.10M NHa in 33 % ethanol; 12 ml/hr Peaks; ( I ) decomposition products: (2) metamphetamine: ( 3 ) ephedrine and amphetamine (original shows two distinct peaks separated by less than one standard deviation); ( 4 ) norephedrine; (5) phenethylamine
m 2-
a
1
o 1
b,V1(
(1)
SOLVENT, rnl
Figure 4. Elution curve from column of nickel-Bio-Rex 70, 6mmX49cm
Figure 1 shows an elution curve for a polystyrene type resin; Figures 2-5 show curves for the elution of mixtures from columns of Bio-Rex 70. No curve is shown for the zinc-loaded resin; this column showed bad tailing and was tested only briefly. In every case, the elution order was the same. Metamphetamine was eluted first, followed by ephedrine, amphetamine, norephedrine, and phenethylamine. This order is consistent with our earlier experience (6)that elution is hastened by placing alkyl groups on the amine nitrogen and on the carbon atom next to the amine nitrogen. The metal-amine complexes in aqueous solution are destabilized in a corresponding way, according to our preliminary (unpublished) tests. Comparing ephedrine with metamphetamine, and norephedrine with amphetamine, we see that the hydroxyl group of ephedrine and norephedrine strengthens the binding to the column, probably by coordinating with the metal ion. The solvent effect is as expected; the higher the proportion of alcohol the more soluble are the bases, and the more easily they are eluted from the column. Isopropanol is a better solvent than ethanol, and methanol is poorer. Methanolwater solvents behaved in a peculiar way with the copper resin which will be discussed later. Raising the ammonia 892
ANALYTICAL CHEMISTRY, VOL. 44, NO. 6, MAY 1972
Phenethylamine 0.6 mg/ml, other bases 0.2 mg/ml; eluent 0.082M NH, in 33 ethanol; 6 ml/hr Peaks: ( I ) decomposition products, (2) metamphetamine, (3) amphetamine, ( 4 )norephedrine, (5) phenethylamine Photometer sensitivity was doubled between 16 and 31 ml, and ordinate reading 'should be divided by two
concentration lowered the elution volume, and the net elution volume, after subtracting the void volume, was inversely proportional to the ammonia concentration. The effect of temperature, as noted with the cadmium resin (Table 11), was small. The elution volumes for comparable ammonia concentrations were larger at the higher temperature, and the resolving power was somewhat improved. Plate Numbers and Resolution. The 58-cm cadmium resin column whose performance is shown in Figure 3 had a plate number of 150, calculated from the sharper (front) side of the phenethylamine peak. This was the best number ever found for this column. The flow rate was 12 ml/hr., temperature 20 "C. When this column was heated to 5 5 "C, cracks and cavities appeared within the resin bed, yet the
Table IV.
Amphetamine and Metamphetamine Peak Areas. Area (arbitrary Ratio to 10 mg/ml
Concentration,
Flow rate,
mg/ml
ml/hr
10.0 10.0 5.0 5.0 5.0 2.0
6 12 6 6 24 6 24 24
units) area MetMetAmphet. amphet. Amphet. amphet. 219 230 Averages 229 235 10.00 (reference)
-
104 106 110 35 41 19
107 109 121 40 45 20
4.65 4.75 4.95 1.55
4.60 4.70 5.2 1.7 1.95 0.85
1.85 2.0 0.85 1 .o Note: the areas on the recorder chart were multiplied by scale factors to allow for flow rate and the photometer sensitivity.
hydrodynamic resistance increased. A short column, 23 cm long, was easier to pack and gave plate numbers of 45 and 75 at 20 and 55 "C for phenethylamine at 24 ml/hr. Both columns were 9 mm in diameter. Cadmium gave better resolution of ephedrine and amphetamine than other metal ions (see Figure 3) but the packing characteristics of the column were so poor that work with this resin was abandoned in favor of copper- and nickelloaded resins. The nickel-loaded carboxylic resin was tested in a column of 6 mm internal diameter, 51 cm long, a t room temperature only. It separated phenethylamine well from the others, but the separation of metamphetamine, amphetamine, and ephedrine was poor. The plate number for phenethylamine was 90 at 12 ml/hr, 100 at 6 ml/hr. The copper resin gave the sharpest bands. It packed well and the pressure drop was moderate. Typical plate numbers for phenethylamine in a column 60 cm long and 9-mm internal diameter were 400-500 at 12 ml/hr and 300-400 at 24 ml/hr. Peculiarities of the Copper-Loaded Column. Two special features of the copper columns can be seen from Figure 5 and less clearly from Figure 2. The first is the shape of the bands; they were unsymmetrical with the sharper side the back, or trailing side. This may be due to a nonlinear absorption isotherm with the distribution ratio increasing as the solute concentration increases. We thought it might be due to a slow chemical decomposition on the column, but the dissymmetry did not change with flow rate, and the absorption spectra of the effluents from the first and last portions of the peak were not significantly different. The second feature is the preliminary wave that appears at one void column volume. The absorption spectrum of the material eluted in this wave is quite different from those of the five solutes investigated. The spectra of these solutes are similar, with a sharp maximum absorbance at 258.5 nm, satellite peaks at 253, 262, 264, and 268 nm, and a marked minimum at 233 nm. The preliminary wave material showed absorbance increasing with decreasing wavelength below 300 nm, with a more rapid increase below 250 nm. In the case of phenethylamine, this material was produced by atmospheric oxidation, for the preliminary wave was greater the longer the solution of phenethylamine base was exposed to air before injection. For the other bases, the preliminary wave appeared whether or not the sample solutions were left exposed to air, and it appeared whether they were injected as salts or as free bases. The preliminary peak was made up of two or more components; see Figures 2 and 5. Their identity is still a mystery. An even more perplexing phenomenon appeared with the copper column when methanol-water solvents were used.
I
15
I
20
1 25
h
1 60
I 65
I 70
I
I
75
80
I 85
I
90
SOLVENT, m l
Figure 5. Elution curve from column of copper-Bio-Rex 70, 9mm X 60cm Metamphetamine hydrochloride and amphetamine sulfate, each 5.0 mg/ml. Eluent O.1DM NH, in 33 ethanol ; 6 ml/hr Peaks are (in order) decomposition products, metamphetamine, amphetamine. Note break in volume scale
lor
0
2
I 20
I
I
40
60
SOLVENT, m l
Figure 6. Elution curve from column of copper-BioRex 70,9 mm X 57 cm Amphetamine base, 10 mg/ml. 50 methanol; 24 ml/hr
Eluenl 0.18M NH, in
Figure 6 shows the kind of waves that appeared. The material that appeared just after one column volume gave the same featureless spectrum that we found with the other preliminary waves. The main solute wave consisted of two parts, a broad diffuse wave followed by a very sharp peak. The absorption spectra of the materials eluted in these two fractions were very similar. Obviously there is a chemical reaction occurring on the column with methanol, yet it must be a relatively mild reaction that does not affect the ultraviolet-absorbing part of the molecule. We did not investigate it further, since we could get good chromatography with ethanol solvents. However, even with ethanol, the solutes decomposed on the column when the temperature was raised to 60 "C. Therefore we operated the copper-resin column at room temperature. Quantitative Analysis by Peak Area Measurement. A standard solution containing 10.0 mg each of amphetamine sulfate, and metamphetamine hydrochloride per milliliter was injected into the copper column by means of the 0.5-ml samples loop, and the areas of the metamphetamine and amphetamine peaks were measured. Solutions containing 5.0, 2.0, and 1.0 mg/ml were prepared by dilution and also injected. The peak areas were compared, with the results shown in Table IV. ANALYTICAL CHEMISTRY, VOL. 44, NO. 6, MAY 1972
893
Roughly the areas were proportional to concentrations, but at lower concentrations they were smaller than they should have been, taking the 10.0 mg/ml solution as reference. The discrepancy exceeds experimental error and may indicate that the samples were decomposing, and that decomposition was more serious at lower concentrations. It may be significant that the preliminary peak area was 2 4 x of the total amphetamine-metamphetamine peak areas at 2 mg/ml but only 1 3 x a t 10mg/ml. We do not exclude the possibility that air dissolved in the feed solution oxidizes the bases, with copper(I1) acting as catalyst. However, boiling the water beforehand and keeping the feed solution under nitrogen did not seem to reduce the preliminary wave. CONCLUSIONS
Ligand exchange chromatography on metal-loaded carboxylic resin columns gives satisfactory resolution of mixtures of amphetamine bases. A resin loaded with copper(I1) ions gives the best resolution and the sharpest bands, but the Cu (11) seems to promote some decomposition of the substrates that we have been unable to characterize or control. Nevertheless it is possible to detect and determine these bases at concentrations down to 0.1 mg/ml, using 0.5-ml samples. Probably this performance could be improved. From this work and previous work in our laboratory (5, 6), we may make certain generalizations about elution orders in metal ion-loaded columns. Strong bases are more strongly held than weak ones, and primary amines are more strongly held than secondary, tertiary, or heterocyclic amines. The
binding to the metal ion-loaded resin is weakened by: (a) methyl groups or other alkyl groups on the amine nitrogen; (b) methyl groups one carbon atom removed from the amine nitrogen; (c) hydroxyl groups more than two carbon atoms removed; (d) methoxyl groups (mescaline, for example, is less strongly bound than phenethylamine). It appears that binding is weakened by steric hindrance around the amine nitrogen and by hydrophilic substituents. The matrix of the ion-exchange resin has a large effect, as we have noted, and it can change elution orders (5). Relatively few of the drugs and biogenic amines that we have studied are strongly held by metal-loaded carboxylic resins, and the selectivity thus shown can be exploited analytically. Obviously, resolution can be improved and the method can be adapted to modern high-speed liquid chromatographic techniques (14). ACKNOWLEDGMENT
We gratefully acknowledge the help of Vernon Shaw, who was sponsored by the National Science Foundation’s Summer Research Participation Program for College Teachers, and of Jared Baker. Harold Heim, Dean of the University of Colorado School of Pharmacy, gave valuable advice. RECEIVED for review November 8,1971. Accepted January 7, 1972. Work supported by the National Science Foundation, Grant No. GP-25727. (14) J. J. Kirkland, Ed., “Modern Practice of Liquid Chromatography,” Wiley-Interscience, New York, N.Y., 1971,
Gas Chromatographic Analysis of Complex Deuterated and Tritiated Mixtures with Packed Columns Fabrizio Bruner, Paolo Ciccioli, and Antonio Di Corcia Laboratorio Inquinamento Atmosferico del C.N.R. and Istituto di Chimica Analitica dell’ Unioersith, 00185 Roma, Italy High efficiency packed columns of about 100 m in length and 130,000 theoretical plates have been employed to separate isotopic mixtures with tritium, as they are originated by the reaction between propene and HT in the presence of catalysts. Separations have been carried out at easily controllable temperature, such as 0 “C and -78 “C. Quantitative analysis can be made also for deuterated mixtures like C2H6C2H6Dand CaH7D-C3H8.
MANY ATTEMPTS have been made to exploit gas chromatography for the analysis of isotopic compounds, either with packed (1-9), or capillary columns (10-12). Careful thermo-
dynamic calculations on the isotope effect in gas chromatography were also possible because of the high resolving power of capillary columns (11, 12). However, in only one case was the problem of analysis of deuterated and tritiated mixtures completely solved with the almost complete separation of all deuterated and tritiated methanes (13). This technique, involving the use of etched glass capillary columns at liquid nitrogen temperature, is further complicated by the necessity of employing a deactivating gas, to be mixed with the carrier. More recently, some of us (8) succeeded in analyzing deuterated methanes by using high efficiency packed columns ;
(1) K. Wilzbach and P. Riesz, Science, 126,748 (1957). (2) P. L. Gant and K. Yang, J . Amer. Cl7em. SOC.,86,5063 (1964). (3) J. W. Root, K. C. Lee, and F. S . Rowland, Science, 143, 376 (1964). (4) W. A. Van Hook and M. E. Kelly, ANAL.CHEM.,37, 509 (1 965). ( 5 ) J. J. Czubryt and H. D. Gesser, J . Gas Chromatogr., 6 , 41 (1968). (6) F. Bruner and A. Di Corcia, J . Chromatogr., 4,5, 304 (1969). (7) A. Di Corcia and F. Bruner, ibid., 49, 139 (1970). (8) A. Di Corcia, D. Fritz, and F. Bruner, ibid., 53,135 (1970).
(9) R . J. Cvetanovic, F. Duncan, and W. E. Falconer, Cm. J . Chem., 41, 2095 (1963). (10) W. E. Falconer and R. J. Cvetanovic, ANAL. CHEM.,34, 1064 (1962). ( 1 1) F. Bruner, G . P. Cartoni, and A. LibeIti, ihid., 38, 298 (1966). (12) F. Bruner, G. P. Cartoni, and A. Liberti, in “Gas Chromatography 1964,” A. Goldup, Ed.. Institute of Petroleum, London, 1964, p 301. (13) F. Bruner, G. P. Cartoni, and M. Possanzini, ANAL.CHEM., 41?1122(1969).
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