to ensure that the method results in nitrosamine production by operating the source at 60 O C . However, because of the low volatility of the nitrosamines, condensation in the source quickly resulted in high backgrounds a t this temperature. Therefore measurements were made at a source temperature of 200 "C. The resulting mass spectra were those of the amine portion of the nitrosamine, although they appeared at the GC retention time of the authentic nitrosamine. In this work, other nitrosamines have not been synthesized directly for absolute yield studies. However, comparison of the measured ion currents from the respective nitrosamines generated in situ with that of diethylamine (for which the sensitivities should be similar) suggests that the efficiency of generation for all amines tested should be well over 90%.
b
a
I
I
.20
.40 .60 .SO nanornoles i n j e c t e d
1
-1
0
10 g k
-2
-3
LITERATURE CITED
-4
2 s 04
Figure 1. (a)Calibration curve for generation of diethyl nitrosamine. Generated standard (- - -). Gravimetrically prepared standard (-). Gravimetric standard on unpacked precolumn (b) Yiekl vs. solution pH for 50 ng of generated diethyl nitrosamine (-e-).
report (7) demonstrates, and we have confirmed, that at ion source temperatures much above 100 "C thermal decomposition is evident, resulting in loss of NO and much variability in the parent peak intensity. During our work, it was possible
(1) G. Hawksworth and M. J. Hill, Biochem. J., 122, 28 (1970). (2) N. P. Sen, J . Chromatogr., 51, 301 (1970). (3) D. H. Fine, D. Lieb, and F. Rufeh, J . Chromatogr., 82, 291 (1973). (4) D. J. Freed and A. M. Mujsce, Anal. Chem., 49, 139 (1977). (5) A. I. Vogel, "A Textbook of Practical Organic Chemistry", 3rd ed. John Wiley & Sons, New York, N.Y., 1966, p 426. (6) S. Siggia, "Quantitative Organic Analysis via Functional Groups", John Wlley and Sons, New York, N.Y., 1963, Chap. 26. (7) G. Schroll, R. G. Cooks,P. Klemmenses, and S A .Lawesson, Ark. Kemi, 28, 413 (1967).
RECEIVED for review April 18,1977. Accepted June 13,1977.
Determination of Intact Oxazepam by Electron Capture Gas Chromatography after an Extractive Alkylation Reaction
'
Jorgen Vessman, * Margareta Johansson, Per Magnusson, and Signhild Stromberg AB KABI, Research Department, Analytical Chemistry, Fack,
S-172 87 Stockholm,
Oxazepam was converted to an N,,03-dlmethyl derivative in an extractive alkyiatlon reaction. The derivative was quantltated by electron capture gas chromatography using iorazepam as an Internal standard. Concentrations down to 1 ng/mL could be determlned. At the 25 ng/mL level 98.4 f 3.2 % were recovered. Serum samples taken after adminlstratlon of diazepam or clorarepate contalned measurable concentrations of oxazepam after 2 to 4 h. The dialkyl derivatives of the two benrodlareplnes, especially that of lorazepam, are converted to Isomeric derlvatlves with hlgh concentratlons of the quaternary ammonium hydroxide In the organic phase. The condltions which glve quantltatlve formation without Isomerization are discussed.
The 1,Cbenzodiazepines are a widely used class of drugs. The compounds have been assayed in biological fluids by various techniques, but gas chromatography (1) and polarography (2) are the principal ones. The bioanalytical field has been reviewed (3). The gas chromatographic techniques used in the early sixties were based on the product of hydrolysis, e.g. aminochlorobenzophenone (4).The sensitivity of electron capture detection was good, but the selectivity could be doubtful in some cases because of the interferences of metabolites yielding 'Present address AB HAESSLE, Fack, S-43120Molndal, Sweden.
Sweden
the same hydrolysis product. Direct determinations for diazepam and medazepam were introduced by de Silva (5). Oxazepam cannot be determined directly as it undergoes ring contraction when injected into the gas chromatographic column. The product of the rearrangement, a quinazoline derivative, has a shorter retention time than that of the unchanged benzodiazepine, but the gas chromatographic properties of the compound make analysis difficult below about 50 ng/mL. The structure of oxazepam favors ring contraction. However, alkylation at the N1 position or absence of the 3-hydroxy group or derivatization of this group will give thermally stable derivatives (6). The present paper describes a procedure for dialkylation of oxazepam by an extractive alkylation procedure and its subsequent determination by electron capture gas chromatography.
EXPERIMENTAL Apparatus. A Varian Model 1400 gas chromatograph with a scandium type electron capture detector was used with a 1.5 m X 1.8 mm glass column, filled with 3 % OV-225 on Chromosorb G (100-120 mesh, acid washed and silanized). The column temperature was kept at 265 "C after conditioning with gas flow for 2 h at 290 "C. The detector temperature was 300 "C. The nitrogen flow rate was 30 mL/min. The mass spectra with electron impact ionization were run in an LKB 9000 gas chromatograph mass spectrometer with an ionization energy of 70 eV. The gas chromatographic column was as indicated above, but used with a helium flow of 20 mL/min at 250 "C. The mass spectra with chemical ionization were run in an LKB 2091 instrument with ANALYTICAL CHEMISTRY, VOL. 4 9 , NO. 11, SEPTEMBER 1977
1545
contraction or hydrolysis. The structure of oxazepam favors ring contraction, which has also been shown to occur with the analogue lorazepam and with 3-hydroxynitrazepam (8). For structures see Figure 1. Substitution at the N1 position will prevent this reaction, cf. 3-hydroxydiazepam. I
\
R
R
N, -Desmethyldiazepam
3-Hydroxydiazepam H
m Figure 1. Structures of the compounds involved. R = H oxazepam, R = CI lorazepam. (I) Benzodiazepine, (11) quinazollne-carboxaldehyde obtained after thermal rearrangement, (111) benzophenone obtained after
hydrolysis, (IV) N,,03-dimethylated benzodiazeplne
isobutane at 0.5 mm Hg and an ionization energy of 300 eV. Reagents. Methylene chloride and toluene, both of analytical quality, Merck, Darmstadt, German Federal Republic, were used. Methyl iodide was analytical quality, redistilled. Oxazepam was obtained from AB KABI, Stockholm, Sweden. Lorazepam was obtained from Wyeth Laboratories Inc., Radnor, Philadelphia, Pa. Tetrahexylammoniumhydrogen sulfate (THA) was obtained from AB HAESSLE, Molndal, Sweden. Standard Solution: A solution containing 500 ng/mL of oxazepam in methylene chloride was prepared. Internal Standard Solution. A solution containing 500 ng/mL of lorazepam in methylene chloride was used. Tetrahexylammonium Hydrogen Sulfate. THA, 0.1 M; 4.51 g are dissolved in and diluted to 1 L with 0.1 M sodium hydroxide. Methyl Iodide. 4 M; 14.2 g (6.2 mL) of methyl iodide are diluted to 25 mL with methylene chloride. Saturated Silver Sulfate Solution. A small amount of Ag2S04 (-25 mg) is shaken with -20 mL of water and heated on a boiling water bath. Prepared fresh each time it is used. All centrifuge tubes were washed with chromium trioxide in acetic acid containing 6% (w/v) sulfuric acid (7) and then rinsed with ethanol. Procedure for Determination of Intact Oxazepam in Serum. (1) A dilution of serum ( 5 2 mL) corresponding to 5 to 50 ng of oxazepam is diluted with 4 mL of a phosphate buffer, pH 7.4. Then 50 pL of internal standard solution are added and the compounds are extracted into 8 mL of methylene chloride for 15 min. (2) After centrifugation the organic layer is filtered through silanized glass wool in a Pasteur pipet into a 12-mL centrifuge tube and then evaporated to dryness. (3) The tubes are cooled and 1.5 mL of 4 M methyl iodide and 0.5 mL of 0.01 M THA (pH 13) are added. The mixture is shaken for 5 min. (4) The organic layer is filtered through silanized glass wool into a centrifuge tube (10 mL) with a tapered bottom section with a volume of about 200 pL. The solvents are evaporated. (5) One mL of hot, saturated silver sulfate solution is added to the residue which is dissolved by vortexing for 10 s on a Vibromixer. (6) One-tenth mL of toluene is added and vibrated in a Vibromixer for 10 s. (7) One to two pL of the toluene layer are injected into the gas chromatograph. A standard curve is prepared from the peak area ratio vs. the weight ratio obtained from the analysis of known amounts of oxazepam and lorazepam. 0,15,30, and 60 pL of the oxazepam standard solution, (Le., 7, 5, 15, and 30 ng) and 50 pL of the lorazepam standard solution (= 25 ng) are added to four centrifuge tubes together with 0.2 to 0.5 mL of a blank serum. RESULTS AND DISCUSSION The aim of this study was to make possible the gas chromatographic determination of oxazepam without ring 1546
ANALYTICAL CHEMISTRY, VOL. 49, NO. 11, SEPTEMBER 1977
I
R
Nitrazepam R
z
-
0
Absence of the hydroxyl group in position 3 also excludes ring contraction, cf. N-desmethyl diazepam (nordiazepam) and nitrazepam. Simple silylation of the 3-hydroxy group giving thermally stable derivatives has recently been described by de Silva (6). The introduction of alkyl groups on the N1 nitrogen can be effected easily by extractive alkylation as shown by Ehrsson and Tilly for nitrazepam (9) and by de Silva e t al. for clonazepam (10). One alkyl group was introduced into these benzodiazepines. The presence of the hydroxyl group in position 3 of oxazepam may, however, also give rise to 0-alkylation. This would indeed be favorable as compounds like, for instance, 3-hydroxydiazepam have rather poor gas chromatographic properties. By extractive alkylation, more than one alkyl group can be introduced as has been clearly demonstrated by Ervik and Gustavii (11)by the introduction of four alkyl groups into chlorothalidon. Initial experiments in extractive alkylation with oxazepam and lorazepam in particular showed that the formation of single derivatives was not readily accomplished. Instead, a mixture of two derivatives in varying proportions was obtained with each of the benzodiazepines. The yields in the studies of effects of pH, quaternary ammonium ion, and alkyl iodide concentration were determined with microgram amounts of the two benzodiazepines using a hydrocarbon, hexatriacontane, as reference substance and flame ionization detection. Identification of the Alkylated Derivatives. Two products were obtained from each benzodiazepine. The derivative formed first (derivative 1)had the longer retention time (See Table I). I t was identified as a dimethylated derivative by gas chromatography and mass spectrometry. By comparison with 0-methylated 3-hydroxydiazepam, available as a reference substance, it was established that both N,- and 03-methylation had occurred with oxazepam. The second derivative appearing in the reaction mixture (derivative 2) and having a shorter retention time (See Table I), was shown by gas chromatography-mass spectrometry (electron ionization and chemical ionization) to have the same molecular weight as derivative 1. The electron ionization mass spectra of derivatives 1and 2 were almost identical. All derivative 1compounds exhibited the expected molecular ion M+. The dimethyl derivatives of oxazepam showed m l e 271 as the base peak which is the same as reported for N-methyloxazepam (or 3-hydroxydiazepam) (8). The corresponding base peak in the dimethyl lorazepam derivative was m / e 305.
Table I. Relative Retention of Oxazepam and Lorazepam Alkyl Derivatives Oxazepam Lorazepam Rearrangement product 0.66 0.85 Methyl derivative la 2.02 2.76 Methyl derivative 2 0.98 1.20 Ethyl derivative 1 1.62 2.47 Ethyl derivative 2 0.84 1.13 Hexatriacontane 1.02 1.00 Diazepamb Temazepam (3-hydroxydiazepam) 3.73 Diazepam has a For structure see Figure 1,IV. retention time of 4.5 min on a column of 1%OV-225 on Chromosorb G at 265 "C.
Extractive Alkylation. The extractive alkylation process can be described as a two-step reaction, in which the anion, X- of an acid HX (e.g., oxazepam), is first extracted as an ion pair with a suitable quaternary ammonium compound, Q', into an organic phase where the alkylation then takes place. X- +
Q'
QXorg t
~ f cQXorg
RIo,
+
RXorg + QIoE
Step 1, extraction (1) Step 2, alkylation (2)
The concentration of QX in the organic phase, QXorgis governed by the partition ratio expressed as DQX = EQX[Q+1
(3)
where E Qis~the extraction constant for the ion pair (12). It is seen from this equation that the partition of the ion pair into the organic phase can be regulated by both the concentration of the quaternary ammonium compound in the aqueous phase, [&+I, and its chemical nature. The more lipophilic the counter ion, Q+, the easier the ion pair will partition into the organic layer and the more rapidly will the alkylation reaction proceed (13). In this study it was necessary to use tetrahexylammonium (THA) as the counterion, as tetrabutylammonium required too long a reaction time. If the ion pair and the undissociated acid simultaneously are partitioned to the organic phase, this will decrease the reaction rate as the ion pair is a considerably more reactive species in solvents like methylene chloride (13, 14). In ion pair extraction this coextraction of the acid can be treated as a side reaction with the use of CY coefficients as discussed in detail by Modin and Schill (12). The conditional extraction constant, EQ$, is related to the true extraction constant, E,,, in the following way if the undissociated acid is extracted simultaneously. EQX'
=
E Q X x -I t
1
a H + (l + k d H X )
(4)
K HX
where k d H X is the partition coefficient for the acid HX with the apparent dissociation constant KHX'. The pKa values of oxazepam have been reported to be 1.7 and 11.6 and those of lorazepam 1.3 and 11.5 (15). The site of deprotonation has been discussed by Hagel and Debesis (16).After comparisons with structurally related compounds, these authors concluded that the slightly acidic hydrogen on the amide function (NJ is responsible for the value around 11.5 and the azomethine nitrogen for the value around 1.5. The amide anion is thus the ion pair forming form. From distribution data in a previous study (17),k d for oxazepam (CH2C12) was calculated to be 100. This means that at pH = pk = 11.5, is only '/loo of EQX. A pH of pk + 2 would, however, give negligible influence from the acid. It was found in practice that the yields of the dialkylated benzodiazepines increased up to about pH 13.
Higher pH values favored the formation of derivative 2 from derivative 1, especially for lorazepam. The tendency to form the rearranged dimethylated product was most apparent with lorazepam, the more lipophilic compound, and was clearly influenced by the THA concentration and the pH of the aqueous phase. The isomerization was negligible when pH was 13 and the concentration of THA 0.01 M. This indicates that the formation of derivative 2, the isomer, is dependent on the amount of THA hydroxide present in the organic phase. (THA'),, t (OH-)aQ (THA' OH-),, (5) The extraction constant for this ion pair has been determined by Nordgren and Modin (18) to be -10, which gives a concentration of 5 x or more in the organic phase in a typical experiment. In experiments with the dimethyl derivative 1of lorazepam (prepared by the analytical procedure) in methylene chloride without any alkylating agent present and with an aqueous phase of 0.05 M THA at pH 13, it was found that derivative 1 completely disappeared in 10 min with the simultaneous appearance of derivative 2. With oxazepam this isomerization process was considerably slower. After 3 h more than 75% of the dimethyl derivative 2 had formed. These findings verified that the isomerization was dependent on the presence of the hydroxide ion pair in the organic phase. Isomerization could also occur when the reaction mixture was evaporated to dryness, as during this operation the concentration of the THA hydroxide increased as well as the temperature, resulting in unreliable yields. This could be prevented by shaking the reaction mixture before evaporation with an acidic aqueous phase. However, under the conditions used in the method, this acid washing step could be omitted. Methyl iodide was used as alkylating agent. T o obtain constant yield from lorazepam and oxazepam, the concentration of the iodide in methylene chloride had to be 1 and 4 M, respectively. The reaction was complete in less than 5 min at room temperature. Ethyl iodide required 15 min with otherwise equal conditions. The dimethyl derivatives 1were very rapidly formed. None of the N1-monoalkylated derivatives (e.g., 3-hydroxydiazepam) were observed under the experimental conditions employed. It was also found that 3-hydroxydiazepam did not react a t all under the experimental conditions. 3-Hydroxydiazepam (N1-methylated oxazepam) does not form an ion pair and is therefore not a probable intermediate during the course of the reaction from oxazepam to the dialkylated derivative. Both alkyl groups are therefore most probably introduced into oxazepam simultaneously. Of the two benzodiazepines it was always possible to convert lorazepam derivative 1 into 2, whereas oxazepam was most easily retained as derivative 1. Conditions which yielded only derivative 1 were therefore chosen in the procedure. Gas ChromatographicProperties. The gas chromatographic properties of the alkylated products were excellent. Compared with the rearrangement product, the adsorption phenomena are eliminated. The high column temperature used is a drawback, however, as the stationary phase is depleted from the support, which shows up in the form of tailing and, finally, adsorption effects. The life time of the column was two to three weeks. A gas chromatogram is shown in Figure 2. The relative retention times for the various derivatives, including the rearrangement products, are given in Table I. It can be seen that the ethyl derivatives have shorter retention times than the corresponding methyl compounds. 3Hydroxydiazepam does not undergo alkylation and will not interfere as such. ANALYTICAL CHEMISTRY, VOL. 49, NO. 11, SEPTEMBER 1977
1547
LOR 1
f
A
LOR 1
1
I
5 5
15 25
m i n 20
15
10
5
0
Flgure 2. Gas chromatogram of derivatlves from a serum extract. (A) Dlmethyloxazepam (=OX 1) from a sample containing 197 ng/mL of
oxazepam. (B) Serum blank.
(LOR
1 = dimethyllorazepam)
The detectability of the dimethyl derivative 1 of oxazepam expressed as MDQ (Minimum Detectable Quantity) was 7 X mol/s in the electron capture detector used (17).This corresponds to -60 pg injected onto the column (retention time 7 min). Elimination of Interference from THA. The presence of residual amounts of the quaternary ammonium iodide caused interferences such as broad solvent peaks already noticeable with the flame ionization detector. Ehrsson removed these by treating the mixture with silver sulfate (19). This approach was utilized here with the modification of heating the silver sulfate solution on a boiling water bath. This decreased the influence of the tailing solvent front considerably. Interferences. When oxazepam is assayed in samples obtained after the administration of diazepam,the metabolites N-desmethyldiazepam and 3-hydroxydiazepam are present in addition to oxazepam. Under the experimental conditions the N-demethylated compound yielded diazepam, whereas the hydroxylated one was found to be unaffected. This metabolite has a longer retention time (Table I). No interference is therefore involved. Under the present conditions the method cannot measure diazepam and N-desmethyldiazepam simultaneously with oxazepam. By the use of another alkyl halide, preferably n-butyl iodide (cf. 20), it would be possible to determine diazepam, N-desmethyldiazepam, and oxazepam in the same sample.
Quantitative Applications to Serum Samples, Recovery and Precision. The alkylation procedure was applied to methylene chloride extracts from human serum samples. The conditions for the extraction of oxazepam from serum were as outlined in the hydrolysis procedure for oxazepam (17). Under the present conditions quantitative analysis was possible down to about 1 ng/mL. The relative recovery at 25 ng/mL was 98.4 f 3.2%. It was found that standard curves from plain aqueous solution and from serum were linear but did not coincide. The standard curves were therefore always prepared with the addition of 0.2 to 0.5 mL of blank serum. Experiments with 14C oxazepam showed that the difference was due to a slight loss (about 5% for 2 mL of serum) of oxazepam in the extraction step when serum was present. The decrease in recovery depended on the amount of serum. Most probably this 1548
ANALYTICAL CHEMISTRY, VOL. 49, NO. 11, SEPTEMBER 1977
TIME Hours
Flgure 3. Serum levels of oxazepam after single dose administratlon of diazepam and clorazepate. 0 = Dlazepam (5 mg p.0.). A = Ndesmethyldiazepam (after clorazepate 10 mg p.0.). 0 = Oxazepam after diazepam dose. A = Oxazepam after clorazepate dose. Diazepam and N-desmethyldiazepam analyzed accordlng to Wretlind et al. (20)
loss was due to incomplete recovery of the organic phase from the interface formed in the presence of the serum sample, an effect that the internal standard apparently did not fully compensate for. The alkylation reaction was found to be more than 99% complete with reference to the standard compound, N1,03dimethyloxazepam. The alkylation reaction was complete within 2 min for extracts, both from water and serum. Twenty-one samples analyzed with the hydrolytic procedure (17) one month before were analyzed with the present method. A plot of the oxazepam concentrations obtained with the two methods gave a regression line with a slope of 0.90 and a regression coefficient of 0.97. The good agreement between the two methods is not surprising as oxazepam in samples after administration of oxazepam does not give rise to interfering metabolites in the hydrolysis procedure (17). It was therefore more interesting to see if the method was sensitive enough to detect oxazepam in serum after the administration of diazepam or clorazepate. These two drugs have oxazepam as the major metabolite. The hydrolytic procedure cannot be applied to samples containing these compounds as the same benzophenone is formed from N-desmethyldiazepam (clorazepate) and oxazepam. Serum samples taken in comparative studies of diazepam and clorazepate with oxazepam (20) were analyzed for the presence of oxazepam. As can be seen in Figure 3, the administration of 10 mg of clorazepate (N-desmethyldiazepam precursor) rapidly produced measurable concentrations of oxazepam with a peak value after about 4 h. The administration of 5 mg of diazepam produced a slower increase in the concentration of oxazepam, probably due to the fact that in this case two metabolic steps are necessary for the formation of oxazepam. In monitoring drug levels after administration of oxazepam, the hydrolysis procedure is somewhat easier to handle in practice. However, the alkylation procedure is more rapid and can be used where the hydrolysis method gives rise to interferences. The present method can also be useful for the determination of lorazepam.
ACKNOWLEDGMENT We thank Ftagnar Ryhage for the chemical ionization mass
spectra. The gift of Temazepam and Nordiazepam from J. A. F. de Silva, Hoffmann La Roche Inc., as well as I4C oxazepam and N1,03-dimethyloxazepam from Hans W. Ruelius
and K*Agersborg’ Jr” wyeth acknowledged.
are gratefdy
LITERATURE CITED D. M. Halley, J. chromatogr., 98, 527 (1974). M. A. Brooks and J. A. F. de Silva, Talanta, 22, 849 (1975). J. M. Clifford and W. Franklln Smyth, Ana/yst(London),99, 241 (1974). J. A. F. de Silva, M. A. Schwarz, V. Stefanovic, J. Kaplan, and L. DArconte, Anal. Chem., 36, 2099 (1964). (5) J. A. F. de Silva and C. V. Pugllsl, Anal. Chem., 42, 1725 (1975). (6) J. A. F. de Sihra, I. Behersky, C. V. Pugllsl. M. A. Brooks, and R. E. Welnfeld, Anal. Chem., 48, 10 (1976). (7) J. Vessman, S.Stromberg, and G. Ritz, Acta pharm. Suec. 7 , 363 (1970). (8) A. Frlgerio, K. M. Baker, and G. Bebedere, Anal. Chem., 45, 1846 (1973). (9) H. Ehrsson and A. Tilly, Anal. Lett., 6, 197 (1973).
(1) (2) (3) (4)
(10) J. A. F. de Silva, C. V. Pugllsl, and N. Munno, J. fharm. Sci., 63, 520 ( 1974). (11) M. Ervlk and K. Gustavli, Anal. Chem., 46, 39 (1974). (12) R. Modln and G. Schlll, Acta fharm. Suec., 4, 301 (1967). (13) H. Ehrsson, Acta fharm. Suec., 8, 113 (1971). (14) A. Brandstrom, “Preparative Ion Pair Extraction”, Apotekarsocleteten and Hassle Lakemedel, Stockholm, 1974, p 93. (15) J. Benett, W. Franklin Smyth, and I. E. DavMson, J . Pharm. Pharmacol., 25, 387 (1973). (16) R. 8. Hagel and E. M. Debesls, Anal. Chim. Acta, 78, 439 (1975). (17) J. Vessman, G. Freij, and S. Stromberg, Acta pharm. Suec., 9, 447 (1972). (18) T. Nordgren and R. Modin, Acta fharm. Suec., 12, 407 (1975). (19) H. Ehrsson, Anal. Chem., 46, 922 (1974). (20) M. Wretllnd, A. Pllbrant, A. Sundwall, and J. Vessman, “The Pharmacoklnetlc Profile of Oxazepam”, Acta Pharmacol. Toxicol. Suppl., 28 (1977).
for review February 11, 1977* Accepted June 13, 1977.
Ion-Exchangers for Gas-Solid Chromatography Roland F. Hirsch”’ Chemistry Department, Seton Hall University, South Orange, New Jersey 07079
Courtenay S. G. Phllllps Merton College, Oxford, England
Lightly-sulfonated porous polymers are efflclent and selective packlngs for gas-solid chromatography. They are easy to prepare and do not show the talled peaks observed when conventional macroreticuiar ion-exchange reslns are used in GSC. The degree of sulfonatlon can be varled, and wlth It the extent of speciflclty for compounds formlng complexes wlth the metal counterlon on the packlng. Reactlons catalyzed by the packlngs were also observed.
Ion-exchange materials are finding increased use in gas chromatography because they offer a great range of potential selectivity through variation of the ionic form of material (1-5). Two difficulties have prevented more widespread use of ion exchangers in GC: their chromatographic separation efficiencies are poor, and they are such strong adsorbants that many substances cannot be eluted without raising the temperature above the decompostion point of the ion exchanger or of the sample. One solution to these problems has been to prepare bound-monolayer cation exchangers, in which the ion-exchange group is covalently bound to a porous silica support (5). Impressive separations of cis-trans isomers of olefins were obtained with these packings. However, their synthesis is time-consuming and requires special handling techniques for some of the reagents, and the matrix must be deactivated by silanization after the ion exchanger has been prepared. We wish to report on the use of lightly-sulfonated porous polymers as ion exchangers for gas-solid chromatography. These materials, which have previously found application in liquid chromatography (6-8), allow efficient separations a t
moderate temperatures. They are easy to prepare and require no special treatment prior to use. The extent of sulfonation of the polymer determines the degree of enhancement of retention of specifically-adsorbed substances; hence it is possible to tailor the packing to the needs of a particular separation problem by choosing the proper synthesis conditions.
EXPERIMENTAL All chromatographic experiments were carried out in PyeUnicam Series 104 gas chromatographs with flame ionization detectors. Nitrogen was the carrier gas. Glass columns (1/4-inch 0.d.) contained the packing materials. Commercial ion-exchangeresins were washed with 2-propanol, water, 1 M NaOH, 1 M HC1, and water, dried at 110 “C for at least 4 h, and sized into the 40-60 mesh range using standard screens. The resins were converted into the silver form by washing with 1M AgN03, to the sodium form by washing with 1 M NaOH, to the nickel form by washing with 0.2 M Ni(N0J2, and to the cadmium form by washing with 0.5 M CdC12,followed in all cases by washes with water, and drying at 110 “C for at least 4 h. Porapak Q (80-100mesh) was sulfonated by suspending a 10-g portion of the porous polymer in about 50 mL of concentrated sulfuric acid (6, 9). The mixture was swirled vigorously for the prescribed time, and then about 50 mL of 50% aqueous sulfuric acid was added to quench the reaction. The resin was washed with dilute sulfuric acid and water. It was then converted to the desired metal ion forms in the same way as already described for the commercial ion exchangers. The degree of sulfonation was determined by controlling the temperature and duration of the sulfonation step, as discussed below. It was measured by titration of a portion of the washed resin with sodium hydroxide solution. The silver ion content of the silver form was determined by atomic absorption spectrometry of an acid-digested portion of the resin.
RESULTS AND DISCUSSION ‘On sabbatical leave at the Inorganic Chemistry Laboratory, Oxford,
1975-76.
For use in gas chromatography, an ion-exchange resin must have a permanent pore structure, as there is nothing present ANALYTICAL CHEMISTRY, VOL. 49, NO. 11, SEPTEMBER 1977
1549
