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Anal. Chem. 1989, 61, 728-732
spectrochromatograms. These possibilities form the subject of continuing work in the authors’ laboratories.
LITERATURE CITED Westerberg, A. W. Anal. Chem. 1989, 4 7 , 1770-1777. Gladney, H. M.; Dowden, B. T.; Swalen, J. D. Anal. Chem . 1969, 4 1 , 883-888. Anderson A. H.; Gibb, J. C.; Llttlewood, A. B. J. Chromatogr. Sci. 1970, 8, 640-846. Roberts, S . M.; Wilkinson, D. H.; Walker, L. R. Anal. Chem. 1870. 42. 886-893. Grushka, E.; Myers, M. N.; GMdings, J. C. Anal. Chem. 1970. 4 2 , 21-26. Lindberg, W.; Ohman, J.; Wold, S . Anal. Chem. 1986, 5 8 , 299-303. Fell, A. F.; Scott, H. P.; Gill, R.; Moffat, A. C. J. Chromatogr. 1983, 273, 3-17. Byline, A.; Sybilska, D.; Grabowski. 2. R.; Koszewski, J. J. Chromatogr. 1973, 83, 257-262. Wrlght, A. G.; BerrMge, J. C.; Fell A. F. Chromatographla 1987, 2 4 , 533-539. .-..- ..
Marr, J. G. D.; Clark, B. J.; Fell, A. F. Anal. R o c . 1988, 2 5 , 150-154. Seaton, G. G. R.; Marr, J. G. D.; Clark, B. J.; Fell, A. F. Anal. Proc. 1986, 2 3 , 424-426. Seaton, G. G. R.; Fell A. F. Chromatographla 1887, 2 4 , 208-216. Sanchez, E.; Ramos, L. S.; Kowalski, B. R. J. Chromatogr. 1987, 385, 151-162. Vandeginste, 8. M. J. Chemom. 1987, 7 , 57-71. Wightman, R. M.; Scott, R. L.; Reiliey, C. N.; Murray, R. W.; and Burnett, J. M. Anal. Chem. 1974. 4 6 , 1492-1499. Denton, M. S.; De Angelis, T. P.; Yacynych, A. M.; Heineman, W. R.; Gilbert, T. W. Anal. Chem. 1876, 48, 20-24. Klatt, L. N. J. C h r m t o g r . Sci. 1979, 17, 225-235. Fogarty, M. P.; Shelley, D.C.; Warner, I . M. ttRC CC, J . ResoM. Chromtogr. Chromtogr. Commun. 1981, 4 , 561-588. Shelly, D. C., Fogarty, M. P. and Warner, I . M. HRC CC,J . Hlgh Resolut. Chromtogr. Chromatagr. Commun. 1981, 4 , 616-626. Clark, B. J.; Fell, A. F.; Westerlund, D. J. Chrornatogr. 1984, 288, 261-273. Fasanmade, A. A. Ph.D. Thesis, Heriot-Watt University, 1985. Fasanmade, A. A.; Fell, A. F.; Scott, H. P. Anal. Chlrn. Acta 1986, 787, 233-240. Kirmse, D. W.; Westerberg, A. W. Anal. Chem. 1972, 4 3 , 1035-1039.
(24) Malczewski, M. L.; Grushka, E. J. Chromtogr. Scl. 1981, 79, 187- 194. (25) Osten, D. W.; Kowalski. B. R. Anal. Chem. 1984, 5 6 , 991-995. (26) Vandeglnste. 8.; Essers. R.; Bosman, T.; Reljnen, J.; Kateman, G. Anal. Chem. 1985. 57. 971-985. (27) Malinowskl, E. R.; Howery. D.G. I n Factor Analysis In Chemistry; Wiley: New York, 1980. (28) Colller, G. L.; Singleton, F. J. Appl. Chem. WS6. 6 . 495-510. (29) Collier, G. L.; Panting, A. C. M. Spectrochlm. Acta 1950, 74, 104-118. (30) Singleton, F.; Collier, J. L. Brit. Pat. 760729 18 December 1953. Institut&nof (31) French, C. S.; Chwch, A. B.; Eppley. R. W. I n Ca+ Washlngtoo Yearbwk ; Cemegie Institutkn of Washlngton: Washing ton, E€, 1953; Vol. 52, pp 182-183. (32) Glese, A. T.; French, C. S. Appl. Spectrosc. 1955, 9(2). 78-96. (33) Savltzky, A. Anal. Chem. 1961, 33, 25A-48A. (34) Money. J. R. Anal. Chem. 1968, 4 0 , 905-914. (35) Fasanmade, A. A.; Fell, A. F. Anewst (London) 1985, 770, 1117-1 124. (36) Kambara, T.; Saitoh, K.; Ohzekl, K. Anal. Chem. W67, 39, 409-410. (37) Berman, A. B.; Frank, Y. A.; Yanovskll, C. A. Zavod. Lab. 1968, 3 4 , 272-274. (38) Essigman, G. M.; Catslmpoolas. N. J. Chromatop. 1975, 703, 7-13. (39) Savitzky. A.; Golay, M. J. E. Anal. Chem. 1984, 36,1627-1639. (40) Grushka, E.; Monacelli, 0. C. Anal. Chem. 1972, 4 4 , 484-489. (41) Baker, P. B.; Gough, T. A. J. chromefogr. S d . 1961, 79, 483-489. (42) Fell, A. F.; Scott, H. P.; Gill. R.; Moffat. A. C. J. Chromtogr. 1983, 282, 123-140. (43) Fasanmade, A. A.; Fell A. F. J. pherm. Pharmecol. 1985, 37(Suppl). 128 p. (44) Fell, A. F. UV Specfrom. Group Bull. W60, 8, 5-31. (45) Fell, A. F. Roc. Anal. Oh. Chem. Soc.1978, 75, 280-267. (46) Talsky, G.; w r i n g , L.; Kreuzer, H. Angew. Chem. Int. Ed. Engl. 1978, 77, 785-799. (47) Horne, D. S. and Parker, T. 0..Blochim. Blophys. Acta 1980. 625, 18-97 .(48) Zek D. T.; Owen, J. A.; Marks, G. S. J. Chromtogr. 1880. 789, 209-216. (49) Fell, A. F.; Clark, B. J.; Scott, H. P. J. Chromatogr. 1984, 316, 423-444.
RECEIVED for review June 23, 1988. Accepted December 9, 1988.
High-Performance Liquid Chromatography of Quaternary Ammonium Compounds on a Polystyrene-Divinylbenzene Column B. M. Van Liedekerke, H.J. Nelis, W. E. Lambert, and A. P. De Leenheer* Laboratoria uoor Medische Biochemie en uoor Klinische Analyse, Rijksuniuersiteit Gent, Harelbekestraat 72, B-9000 Gent, Belgium
Thls report derscrfbes a new Uquld chromatographic approach to the separation of quaternary ammonium compounds. The key feature of the method Is the use of a polymeric poiystyrene-divinylbenzene column that udke most conventional octadecyisiilca reversed phase materials, yleids very symmetrical peaks. Wlh thiazine dyes as model anaiytes R is shown that the peak shape and retention are strongly pH dependent, whereas the resolutlon between closely related analogues Is Improved by the addition of a iipophiiic phosphoric acid derivative. Other quaternary ammonlum compounds of practical Interest, inciudlng muscle relaxants and an antidote against cholinesterase Inhlbitors, were also successfully chromatographed in this system.
Reversed-phase chromatography of quaternary ammonium compounds is common but problematic (1-12). A close in-
* To whom correspondence should be addressed.
spection of various published chromatograms, often displayed at low chart speed to mask peak asymmetry, reveals that these compounds mostly elute from octadecylsilicacolumns as badly tailing peaks. The addition of ion pairing agents and/or quaternary amines to the mobile phase does generally not eliminate this unwanted phenomenon. For years we have had a similar experience with the chromatography of quaternary thiazine dyes, a class of compounds widely used as part of Romanowsky stains in hematology and histology (13). In our original work (13)an “old” Spherisorb ODS reversed-phase column was used, i.e. one that had been repeatedly loaded with extracts of biological materials. Although the resolution between individual compounds was similar on a subsequently used new Spherisorb ODS column, the original satisfactory peak symmetry could not be reproduced, neither on Spherisorb ODS nor on other silica-based reversed-phase materials. This failure was presumably attributable to a gradual but uncontrollable deactivation of active sites on the old column, caused by the repeated injection of biological extracts.
0003-2700/89/0361-0728$01.50/00 1989 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 61, NO. 7, APRIL 1, 1989
I
R1
R2
R3
I
H
H
H
H
II
CH3
H
H
H
111
CH3
CH3
H
H
IV
H
CH3
CH3
H
V
CH3
CH3
CH3
H
VI
CH,
CH,
CH3
CH3
R4
Tailing of basic compounds in general on alkyl-bonded phase columns has been repeatedly observed (14,15). Jansson (16) solved this problem by modifying a silica column with aqueous ion-pairing reagents to confer hydrophobic properties onto the stationary phase. Polymeric columns have already proven their superiority to octadecylsilica for the analysis of other compounds, including some pharmaceuticals (I7). In our hands,the substitution of an octadecylsilica by a polymeric polystyrene-divinylbenzene column was found to afford a considerable improvement in the peak shape of thiazine dyes. In addition, two isomers that could previously not be resolved were now near base line separated. Our method could also be extended to the chromatography of other quaternary ammonium compounds of practical interest, e.g. muscle relaxants. This report is concerned with a study of the basic features of this new chromatographic system, including the effect of eluent parameters on retention, resolution, and peak symmetry, using thiazine dyes as model analytes.
EXPERIMENTAL SECTION Chemicals and Reagents. Thionin (I), azure C chloride (II), azure A chloride (III), azure B chloride, azure B bromide, azure B tetrafluoroborate (V), and methylene blue (VI) (structural formulas are shown in Figure 1)were purchased from Sigma (St. Louis, MO). Azure A tetrafluoroborate (asymmetrical dimethylthionin) (111) and symmetrical dimethylthionin chloride (IV) were gifts from D. Wittekind (Freiburg, FRG). These compounds had been isolated as impurities from deteriorated azure B preparations by preparative column chromatography. The identity of both isomers was confirmed by using absorption spectrometry, fast atom bombardment (FAB) mass spectrometry,
720
liquid chromatography, and comparison with authentic azure A from Sigma. The absorption spectra of 111, IV,and authentic azure A all displayed a maximum at 630 nm (in ethanol), suggesting the overall azure structure for I11 and IV. FAB mass spectra, obtained in a 3-nitrobenzyl alcohol matrix, conclusively demonstrated that I11 and IV had the same molecular weight (base peak at m / z 256 in both spectra) indicative of dimethylthionins. The base peaks of other azure homologues were observed at m / z 242 (monomethylthionin,azure C, 11)and m/z 270 (trimethylthionin, azure B, V), respectively. Since both dimethylthionins exhibit different retention characteristics in reversed-phase liquid chromatography (see below), they obviously possess different structures and, hence, have to be isomers. One of them (111)is coeluting with authentic azure A (asymmetrical dimethylthionin). Therefore, strong evidence suggests that IV is symmetrical dimethylthionin, for which however no reference component is available. Neostigmine bromide came from Federa (Brussels,Belgium), N-methylphenazone and edrophonium chloride from Sigma (St. Louis, MO), and contrathion from Specia (Paris, France). Pipenzolate bromide, mepenzolate bromide, and pyridostigmine bromide were donated by P. De Moerloose (Gent, Belgium). The structural formulas of miscellaneous quaternary ammonium compounds are presented in Figure 2. Phosphoric acid 85% was supplied by UCB (Braine L'Alleud, Belgium), acetonitrile (HPLC grade) by Fisher Scientific (Fair Lown, NJ), and tetramethylammonium hydroxide (TMAH) (20% (v/v) in methanol) by Janssen Chimica (Beerse, Belgium). Bis(2-ethylhexy1)phosphoric acid (BEHP), containing up to 50% mono(2-ethylhexy1)phosphoric acid was obtained from Fluka AG (Buchs, Switzerland). Apparatus. The liquid chromatograph used was equipped with an LKB 2150 pump (LKB, Bromma, Sweden), a Varichrom variable wavelength detector (Varian Associates, Walnut Creek, CA) set at 650 nm for the thiazines and at 210-270 nm for other compounds, a Spectra-Physics 4270 integrator (Spectra-Physics, San Jose, CA), and an N 60 sample valve with a 50-pL loop (Valco Instrument Co., Houston, TX). A differential refractometer R 403 (Waters Associates, Milford, MA) was used for the determination of breakthrough curves. Fast atom bombardment mass spectra were obtained on a VG70SEQ instrument (VG Analytical, Manchester, UK) with a secondary ion accelerating voltage of 8 kV. The primary atom beam of xenon was produced by using a saddle field ion gun (Ion Tech) operated with a tube current of 1 mA at an energy of 8 kV. The FAB mass spectra were obtained by averaging five scans and matrix (3-nitrobenzylalcohol) background subtraction. Chromatographic Conditions. The liquid chromatographic columns used were a 5-pm Spherisorb ODS, 15 X 0.46 cm (Phase Sep., Queensferry, UK), a 5-pm Zorbax ODS, 15 X 0.46 cm (DuPont, Wilmington, DE), a 5-pm Hypersil ODS, 15 X 0.46 cm (Shandon, Sewickley, PA), a 10-pm Vydac 201 TP 54, 25 X 0.46 cm (the Separations group, Hesperia, CA), a 10-pm Hamilton
N
h
111
I II Flgure 2. Structural formulas of miscellaneous quaternary ammonium compounds: I , pyridostigmine; I I,contrathion; I II, N-methylphenazone; IV, edrophonlum; V, neostigmine; V I , mepenzolate (R = -CH3) and pipenzolate (R = -C2H5).
730
ANALYTICAL CHEMISTRY, VOL. 61, NO. 7, APRIL 1, 1989
Table I. Comparison of the Asymmetry Factor (AS) of Azure B Obtained on Different Reversed-Phase Columns eluent
column
AS
A. Octadecylsilica HpO-CH3CN(44:56 (v/v)) 0.0024M BEHP 0.0799 M TMAH Hypersil ODS cf. Spherisorb ODS Zorbax ODS cf. Spherisorb ODS Vydac 201 TP 54 H,O-CH,CN (63:37 (v/v)) 0.0090 M BEHP 0.1143 M TMAH
Spherisorb ODS
u 0
4 MI
B. PRP-1 (Polystyrene-Divinylbenzene) HpO-CH&N (32~68' (v/v)) 0.124M TMAH HpO-CHBCN (3268(v/v)) 0.0027 M BEHP 0.124M TMAH
w 0
4 YIW
Figwe 3. Separation of thiazine dyes on Spherkorb ODs (15 X 0.46 cm): eluent (A) H,O-CH,CN (5545 (v/v)), containing 0.1 M TMAH, pH 2.5; (6)H&-CH,CN (5050 (v/v)) containing 0.09 M TMAH and 0.002M BEHP, pH 2.5. Flow rate was 1 mL/min and detection was at 650 nm. Peak identification was as follows: 1, azure C; 2, azure A; 3, symmetrical dimethylthionln; 4, azure B.
PRP-1, 25 X 0.41 cm and 15 X 0.41 cm (Hamilton, Bonaduz, Switzerland). Mobile phases consisted of mixtures of water-tetramethylammonium hydroxide (TMAH), variable percentages of acetonitrile, and sufficient phosphoric acid to adjust the pH of the aqueous component to 2.5. For the chromatographyof the thiazine dyes, the definitive eluent was water-acetonitrile (68:32 (v/v)), containing 0.124 M TMAH and 0.0013 M BEHP. The eluent for the other quaternary ammonium compounds contained no BEHP and required lower or higher concentrations of acetonitrile and TMAH for proper retention adjustment. The flow rate was 1.0 mL/min and the temperature was ambient. Possible adsorption of BEHP or TMAH onto the column packing material was investigated by determining breakthrough curves using a refractive index detector. The polystyrene-divinylbenzene column was first washed with acetonitrile. The adsorption was evaluated by means of the mobile phase volume passed through the column until there was a detection of the migration front of the TMAH or BEHP, respectively, by refractive index. Before the mobile phase with TMAH was passed, the column was preequilibrated with the eluent of the same composition but without TMAH. The same procedure was followed for BEHP by using a preequilibration eluent with TMAH but without BEHP. Analytes. The compounds were dissolved in absolute ethanol or water and diluted with the aqueous component of the mobile phase.
RESULTS AND DISCUSSION Chromatography of Thiazines on Octadecylsilica's. The performance of a new Spherisorb ODS column, not previously "contaminated" with biological extracts and using the originally developed eluent combination (13), for the chromatography of thiazine dyes is illustrated in Figure 3A. Despite the presence of a tetramethylammonium derivative in the eluent, all peaks exhibited considerable tailing (asymmetry factor of azure B as high as 5.3). This contrasts with the positive effect noted for the tetraalkylammonium compounds in connection with the chromatography of amines (16). Addition of ion-pairing agents, e.g. sodium heptanesulfonate or sodium bromide only affected the overall retention but neither the peak shape nor the resolution of the thiazines. Of various additives tested only BEHP displayed an interesting effect in that it improved the resolution between three previously coeluting impurities (Figure 3B), with a concomitant increase in overall retention. We may presume that the amount of mono(2-ethylhexy1)phosphoric acid present in the BEHP plays a role in the separation of the thiazines. Although BEHP is an effective ion-pairing agent for basic compounds ( I @ , this phenomenon can hardly be rationalized in terms of
0
4
u
8 YIN
0
4
8
5.3 4.3 3.3 2.4
1.6 1.0
u MIN
0
4
8 YlN
Figwe 4. Influence of BEHP concentration on the resolution between thiazine dyes: column, PRP-1 (25 X 0.41 cm); eluent (A) H,O-CH&N (81:19 (v/v)) containing 0.124 M TMAH; (6) H,O-CH,CN (74325.5 (v/v)) containing 0.124 M TMAH and 0.0013 M BEHP; (C) H,O-CH&N (68:32 (v/v)) containing 0.124 M TMAH and 0.0027 M BEHP. Flow rate was 1 mL/mln and detection at 650 nm. Peak identification was as follows: 1 to 4 same as in Figure 3; 5, methylene blue.
ion-pairing mechanisms because a t the eluent pH of 2.5, BEHP (pK, = 3.22) (18) predominantly occurs in its acidic form. Unfortunately, BEHP left the peak shape of the thiazines unaffected. Other octadecylsilica columns (Zorbax ODs, Hypersil ODs, and Vydac 201TP) did not perform differently from Spherisorb ODS, despite their substantially different physicochemical properties (non-end-capped material, totally endcapped material, and material with a polymeric C-18 phase, respectively). As evidenced from the asymmetry factors of the azure B peak, all columns tested yielded severely tailing peaks (Table IA). Chromatography of Thiazines on a Polymeric PRP-1 Column. The original mobile phases developed for the chromatography of thiazines on Spherisorb ODS (13)proved directly applicable to PRP-1 columns. The latter afforded a considerable improvement in peak symmetry (Table IB), regardless of the age or the degree of "contamination" of the columns. Both an intensively used old 15 X 0.41 cm and a new 25 X 0.41 cm column displayed equal performance in this respect. Bower (19) stated that the use of an organic modifier, e.g. acetonitrile causes swelling of the microporous structure of polymeric columns resulting in improved peak shape. However, even in the presence of acetonitrile peak symmetry could still be considerably improved by adding BEHP to the eluent (Table IB). The addition of BEHP increased the overall retention and yielded a near base line separation of azure A and symmetrical dimethylthionin (Figure 4C), the two positional isomers that remained unresolved on ODS column^, even in the presence of BEHP. Capacity ratios of all com-
ANALYTICAL CHEMISTRY, VOL. 61, NO. 7, APRIL 1, 1989
731
Table 11. Relationship between Concentration of BEHP and the Capacity Factor (k')of Azure B on a PRP-1 Polystyrene-DivinylbenzeneColumn'
BEHP concn,
BEHP concn, mM
k'
mM
k'
0.0 1.3 2.7
1.0 3.4 5.5
4.1 5.4
6.5 12.2 16.9
8.2
'Regression equation: y = 1 . 9 6 ~+ 0.47; r = 0.984.
"
. .g z z
0
1
-> 2
3
Ml
tlUtBc V & l @
Flgure 6. Structures of (A) BEHP and symmetrical dimethyithionin and (B) BEHP and azure A (asymmetrical dimethyithionin), suggesting possible sites of alkyl-alkyl Interactions.
Flgure 5. Breakthrough curve of BEHP on PRP-1 (25 X 0.41 cm). Eluent, H,O-CH&N (6832 (v/v)) containing 0.124 M TMAH and 0.0027 M BEHP. Flow rate was 0.2 mL/min and detection was by refractive Index. Key: 1, dead volume; 2, first breakthrough: 3, second breakthrough. 0
pounds of interest in the standard eluent containing BEHP were 1.4 (thionin), 2.2 (azure C), 2.7 (azure A or asymmetrical dimethylthionin), 4.0 (symmetrical dimethylthionin), 5.5 (azure B), and 6.6 (methylene blue). Elution takes place according to the degree of methylation (0 to 4 methyl groups) of the two amino group substituents (cf. Figure 1). A linear relationship was observed between the capacity ratio (k') of azure B and the concentration of BEHP (Table 11). At the same time the (Y value (selectivity factor) for the pair azure A/symmetxical dimethylthionin increased from 1.00 (no BEHP) to 1.08 (1.3 mM BEHP) to 1.48 (2.7 mM BEHP) (Figure 4A-C). This improvement in resolution might be rationalized in terms of the affinity of the methylated amino groups for the alkyl chains of BEHP. As evidenced from the breakthrough curve (Figure 5) the lipophilic and poorly water soluble BEHP is adsorbed onto the column, thus supposedly favoring alkyl-alkyl interactions of azure with BEHP rather than a direct interaction with the polymeric hydrophobic sorbent itself. In this theory the symmetrical dimethylthionin having methylamino groups on both ends of the molecule would interact more readily with the bifurcated BEHP molecule (Figure 6A), as opposed to the azure A which only possesses a dimethylamino group on one side (Figure 6B). This is consistent with the elution order (azure A < symmetrical dimethylthionin) of both compounds. This hypothesis gained some support from the calculation of the interatomic distances in BEHP and symmetrical dimethylthionin (20). The total distance between the methylamino groups in the structure of symmetrical dimethylthionin (15.1 A) approximates the distance between a butyl group on one end and an ethyl group on the other end of the BEHP molecule (16.1 A). A key factor governing peak symmetry of thiazines, both in the absence and the presence of BEHP, was the pH of the eluent (Figure 7). The plot of the peak asymmetry factor (AS) versus pH shows a minimum a t pH 2.5 (AS = 1.00) (BEHP present) or 1.6 (no BEHP present). At higher pH the peak shape progressively deteriorates (maximum AS = 3-3.4). This pH effect is obviously linked to the degree of protonation of the azure molecule. At low pH, e.g. 2.5, all molecules
1.o1 2.5
1
3:5
/I
. 4:s
0
5.5
6.0
PH Flgure 7. Plot of k'values of azure B and peak asymmetry factor (AS) versus pH: 0, AS value obtained in the standard eluent without BEHP W, AS value obtained In the standard eluent with BEHP 0 and 0, corresponding k' values.
conceivably contain three positive charges (the pK, of NJVdimethylaniline is 5.15). The uniformity in the nature of the protonated species probably also entails a uniform interaction with the stationary phase, thus precluding tailiig. In contrast, in a weak acidic medium mixtures of di- and triprotonated species will be present. This relative heterogeneity supposedly underlies a less consistent interaction pattern. An alkaline pH would lead to a monoprotonated species but no experiments were carried out in this pH region, because of solubility problems. Retention is likewise pH dependent (Figure 7). With no BEHP present, the retention increase as a function of pH is explained by the progressive (partial) suppression of the ionization of the dimethyl- and monomethylamino groups (see above). However, in the presence of BEHP an increase in pH initially causes the opposite phenomenon, i.e. a decrease in
732
ANALYTICAL CHEMISTRY, VOL. 61, NO.
7,APRIL 1, 1989
Table 111. Asymmetry Factors (AS), Plate Counts (N),and Capacity Ratios (k?Obtained for Miscellaneous Quaternary Ammonium Compounds on a PRP-1 Column Eluted with a Solvent Mixture Containing Variable Concentrations of TMAH (0.124-0.182 M) and Acetonitrile but no BEHP, pH 2.5
structure Azure B pyridostigmine
Figure 1 V Figure 2 I contrathion Figure 2 I1 N-methylphenazone Figure 2 I11 edrophonium Figure 2 IV neostigmine Figure 2 V mepenzolate Figure 2 VI pipenzolate Figure 2 VI
% CH&N
32 5 0 15 5 5 25 25
AS
N
k'
1.0 2143 4.8 1.2 1767 1.6 1.0 4986 0.4 1.0 2575 0.8 1.7 1205 5.2 1.4 1364 8.6 1.3 757 2.8 3.6 1.3 590 -
retention. Eventually, at pH 4.2-4.3 a near-plateau is reached. Although there is no conclusive experimental proof, this effect might be rationalized in terms of the degree of ionization of BEHP in conjunction with its adsorption on the stationary phase. At low pH the predominating undissociated form of BEHP will be more readily adsorbed onto the polymeric packing material than ita ionized counterpart and, hence, favor the kind of hydrophobic BEHP-azure interaction suggested above. Likewise at higher pH less adsorption will result in weaker interaction and hence lower retention. The influence of TMAH on retention was less pronounced. Increasing concentrations of TMAH (0.045, 0.091, and 0.18 M, respectively) were associated with decreasing k 'values (3.4, 1.7, and 1.2, respectively), but no linear relationship between both parameters became apparent). Although adsorption of tetraalkylammonium ions on C18reversed-phase columns has been reported (21),this does not occur on the PRP-1 polymeric column: there was no delayed breakthrough of TMAH (breakthrough curve not shown). Chromatography of Other Quaternary Ammonium Compounds on a PRP-1 Column. Aside from thiazine dyes the PRP-1 column also proved effective for the chromatography of other quaternary ammonium compounds, including muscle relaxants, an antidote against cholinesterase inhibitors, and a phenazine dye. Asymmetry factors, capacity ratios, and theoretical plate counts (N)obtained for the different compounds tested are given in Table 111. Some polar compounds, e.g. contrathion, exhibited poor retentivity, even in a purely aqueous mobile phase. All quaternary ammonium derivatives could be chromatographed a8 reasonably symmetrical peaks. However, from the results in Table I11 some differentiation can be made. The lowest AS factors and the highest N values were obtained for pyridinium-type compounds, i.e. pyridostigmine (I),contrathion (11), and N-methylphenazone (111) (Figure 2) as well as for azure B. Compounds of the N phenyl-N-trialkyl type, e.g. edrophonium (IV) and neostigmine (V), gave intermediate results, whereas aliphatic derivatives lacking a ring nitrogen and N-phenyl substitution (mepenzolate and pipenzolate (VI)) yielded particularly low theoretical plate counts. Applications. The present chromatographic system is now routinely used for the quality control of Romanowsky stains. Of particular interest is the finding that the anionic component of the stain, eosin Y, can be determined along with the cationic azure B on the same column using a similar mobile phase. The system would also lend itself to the differentiation of commonly used muscle relaxants, as shown in Figure 8. In general, the good peak shape, the column stability, and the remarkable reproducibility between runs (even after long periods of column storage) appear to make this approach
I
I
0
I
I
8
I
I
I
16
MIN
Figure 8. Separation of muscle relaxants on PRP-1 (25 X 0.41 cm). Eluent was H,O-CH,CN (955 (v/v)) containing 0.173 M TMAH. Flow rate was 1 mL/min and detection was at 220 nm. Peak identification was as follows: 1, pyridostigmine bromide; 2, edrophonium chloride; 3, neostigmine bromide.
attractive as a basis for the quantitative determination of quaternary ammonium compounds, e.g. muscle relaxants, in biological materials.
ACKNOWLEDGMENT The authors are indebted to Professor Dr. H. Wittekind (Freiburg/Br, FRG) for his continuous advice and valuable suggestions as well as for the gift of azure samples. B.V.L. and H.J.N. acknowledge their positions of Research Associates from the Belgian Foundation for Scientific Research (NFWO). The authors are grateful for the generous help of M. Claeys (UIA, Antwerp, Belgium) who took the FAB mass spectra. LITERATURE CITED ( 1 ) Van Der Maeden, F. P. B.; Van Rens, P. T.; Buytenhuys, F. A,; Buurman, E. J. Chromatog. 1977, 742, 715-723. (2) Greving, J. E.; Bouman. H.; Jonkman, J. H. G.; Westenberg. H. G. M.; De Leeuw. R. A. J. Chromatogr. 1979, 786, 683-690. (3) De Ruyter, M. G. M.; Cronnelly, R.; Castagnoli, N. J. Chromatogr. 1980, 183, 193-201. (4) Crommen, J. J. Chromatogr. 1980, 193,225-234. (5) Damon. C. E.; Pettitt, B. C. J. Chromatogr. 1980, 795,243-249. (6) Abidi, S. L. J. Chromatogr. 1881, 273, 463-474. (7) Abdl, S. L. J . ChrOfMtOgr. 1885, 324, 209-230. (8) Shih, M. L.; Smith, J. R.; Eliin, R. I . Anal. Letf. 1888. 19,1137-1151. (9) De Schutter, J. A.; Van Den Bossche, W.; De Moerloose, P. J. Chromatogr. 1988. 366. 321-328. (10) TharassaBlock, C.; Chabenat, C.; Boucly, P.; Marchand, J. J. Chromatogr. 1987, 427. 407-411. (11) MatSunaga. H.; Suehiro. T.; Saita. T.; Nakano. Y.; Morl, M.; Takata, K.; Oda. K. J . ChrOmatOgr. 1987, 422, 353-355. (12) Yturralde, 0.; Lee, R.-Y.; Benet, L. 2.; Fleckenstein, L.; Lln, E. T. L. J . Llq. ChfOmafOgr. 1987, IO(70), 2231-2246. (13) Nelis, H. J. C. F.; De Leenheer, A. P. Clin. C h h . Acta 1986, 756, 247-258. (14) Sokolowski, A.; Wahiund, K.-G. J . Chromatogr. 1980, 189,299-316. (15) Chan Leach, D.;Stadallus, M. A.; Berus, J. S.; Snyder, L. R. LC-GC 1988, 6 , 494-499. (16) Jansson, S.; Johansson, M. J. Chromato@r. 1987, 395. 495-501. (17) Cope, M. J.; Davldson, I. E. Analyst 1987, 772, 417-421. (18) Woogewljs, G.; Massart. D. L. Anal. Chim. Acta 1979, 706, 271-277. (19) BOWWS,L. D.;Ped@, S. J . C h r M a t q r . 1986, 371, 243-251. (20) Handbook of Chemlstrv and Physlcs; Weast, R. C., Ed.; CRC Press: Boca Raton, FL, 1975; p F-214: (21) Del Rey, M. E.; Vera-Avila, L. E. J. Llq. Chromafogr. 1987, 70, 2911-2929.
RECEIVEDfor review September 30,1988. Accepted December 13,1988. Financial support for the mass spectrometer by the Belgian National Fund for Medical Research (FGWO) (Grant No. 3.0089.87) and the Belgian Government (Grant No. 87/92-102) is gratefully accepted.
