(8) (9)
(IO) (11)
(12) . . (13)
(14) (15) (16) (17) (18) (19) (20) (21) (22)
Stiuctwes”, Abstracts, Division of Computers in Chemistry, 172nd National Meeting, American Chemical Society, S a n Francisco, Calif., August 1976. H. 9. Woodruff and M. E. Munk, J . Org. Chem., 42, 1761 (1977). H.9. Woodruff and M. E. Munk, Res.lDev., 28 (E), 34 (1977). H. J. Reich, M. Jautela!, M. T. Messe. F. J. Weigert, and J. D. Roberts, J . A m . Chem. Soc.. 91. 7445 (1969). G. C. Levy and G. L. Nelson, “Carbon-13 Nuclear Magnetic Resonance for Organic Chemists”, Wiley-Interscience, New York, N.Y., 1972. C. L. Wilkins. R. C. Williams. T. R. Brunner. and P.J. McCombie. J . Am. Chem. Soc., 96, 4182 (1974). T. R. Brunner, R. C. Williams, C. L. Wilkins, and P. J. McCombie, Anal. Chem.. . . .. . ., 46. . ., 1798 . . .- 11974) T. R. Brunner, C . L: Wilkhs. R. C. Williams, and P. J. McCombie, Anal. Chem.. 47. 662 (1975). C. L. Wilkins and’T. L.’Isenhour, Anal. Chem., 47, 1849 (1975). H. 9. Woodruff, G. L. Ritter, S. R. Lowry, and T. L. Isenhour, Appl. Spectrosc., 30, 213 (1976). J. 9. Justice and T. L. Isenhour, Anal. Chem., 46, 223 (1974). T. Wittstruck and K. I.H. Williams, J . Org. Chem., 38, 1542 (1973). H. Eggert and C. Djerassi, J . Org. Chem., 38, 3788 (1973). J. W. ApSimon. H. Beierbeck, and J. K. Saunders, Can. J . Chem., 51, 3874 (1973). D.Leibfri!z and J. D. Roberts, J . Am. Chem. SOC.,95, 4996 (1973). N. S.Bhacca, D. D. Giannini, W. S. Jankowski. and M. E. Wolff, J . Am. Chem. Soc., 95, 8421 (1973).
(23) E. Breitmaier and W. Voelter, “l3C NMR Spectroscopy: Methods and Applications”, Verlag Chemie. Weinheim/Bergatr., Germany, 1974. (24) R. 0. Duda and P. E. Hart. “Pattern Classification and Scene Analysis”, Wiley-Interscience, New York, N.Y., 1973. (25) T. M. Cover and P.E. Hart, I€€€ Trans. Info. Theory, 1113, 21 (1967). (26) H. 9. Woodruff, S. R. LOW, G. L. R i e r , and T. L. Isenhour, AM/. Chem., 47, 2027 (1975). (27) D. J. Rogers and T. T. Tanimoto, Science, 132, 1115 (1960). (28) H.9. Woodruff, S. R. Lowry, and T. L. Isenhour, Appl. Spectrosc., 28, 226 (1975). (29) J. Franzen, Chromatographia, 7, 518 (1974). (30) S. R. LOW, H. 9. Woodruff, G. L. R i e r , and T. L. Isenhour, AM/. Chem., 47, 1126 (1975). (3 1) L. J. Sobberg, C. L. Wilkins, S.L. Kaberline, T. F. Lam, and T. R . Brunner, J . Am. Chem. SOC.98, 7139 (1976). (32) H. Rotter and K. Varmuza, Org. Mass Spectrom., 10, 874 (1975). (33) N. A. 9. Gray, Anal. Chem., 46, 2265 (1976). (34) S.R. LOW and T. L. Isenhour, J. Chem. Inf. &mp. Sci., 15, 212 (1975). (35) M. Sjostrom and U. Edlund, J . Magn Reson.. 25, 285 (1977).
RECEIVED for review August 3,1977. Accepted August 3,1977. Financial support by the National Institute of General Medical Sciences (GM21703) is gratefully acknowledged.
lodine-Amine Charge-Transfer Complexes as Spectrophotometric Detectants in High Pressure Liquid Chromatography C. Randall Clark,” Charles M. Darling, Jen-Lee Chan, and Alfred C. Nichols School of Pharmacy, Auburn University, Auburn, Alabama 36830
Iodlne-amine charge-transfer complexes are demonstrated to enhance the UV detectability of N,Ndlmethylbenzylamlne. The complexation reactlon is rapid, reaching equlllbrlum In less than 7 s. The molecular ratio of reactants In the complex Is 1:l 1,:amIne. Maximum charge transfer band Intenshy which reflects the total amount of complex present was observed at a 1 O : l 1odlne:amlne ratio. An HPLC analysls Is described lor N,N-dlmethylbenzylamlne which Includes the direct chromatography of the free amine, the “In line” formatlon of the complex, and the detectlon of the amine in the charge transfer complex form. The use of the complex allows for a 20-fold Increase In peak area over the same concentration of uncomplexed N , N-dlmethylbenzylamlne. This procedure appears to be of general appllcablllty to all types of amines.
A charge-transfer complex can be described as a molecular complex formed by the weak interaction of an electron donor and an electron acceptor. Charge-transfer complexes usually involve simple integral ratios of the components and the enthalpy of formation is usually only a few kcal/mol. The rates of formation and decomposition into the components are so high that the reactions appear to be instantaneous by normal techniques. In most cases, the complex has absorption peaks in its electronic absorption spectrum which are not common to either component. Charge-transfer complexes of organic materials with iodine have long been used as a method of visualization of thin-layer and paper chromatography. Iodine has been described as a sacrificial a-acceptor and amines as increvalent n-donor. Yada et al. ( I ) have shown that iodine-aliphatic amine complexes show two characteristic absorption bands in the 430-410 and 280-230 nm regions. Taha and co-workers (2) 2080
ANALYTICAL CHEMISTRY, VOL. 49, NO. 13, NOVEMBER 1977
reDorted a considerL,,; increase in the JV bani intensity of many alkaloids via charge transfer complexation with a-acceptors such as iodine. The increase in absorptivity (up to 100 times the value of the uncomplexed alkaloid) was much greater for weak UV absorbers such as the tropine alkaloids, ephedrine, codeine, and spartane. The UV analysis of other pharmaceuticals utilizing iodine charge-transfer complexation has been reported ( 3 ) . The highly efficient separation powers of high pressure liquid chromatography have been demonstrated in many areas of analytical chemistry. The most common method of detection in HPLC is spectrophotometric; therefore, the detection limits of a compound are directly related to its absorptivity. Molecules having high natural absorptivity can be detected in low concentrations by HPLC. However, compounds with low absorptivity values must be derivatized (4) with a strong chromaphoric group in order to achieve good detectability. The use of derivatizing reagents to produce molecules of high absorptivity has been applied to many classes of compounds (5-7). These derivatization procedures generally link chromaphore to substrate through a covalent bond. The formation of covalently linked amine derivatives depends greatly on the degree of nitrogen substitution. Primary and secondary amines serve as substrates for such reactions but tertiary amines are inert to these procedures. Therefore, the use of covalent chromophores cannot be applied to all types of aliphatic amines. Regardless of degree of substitution, all aliphatic amines possess the pair of n electrons on nitrogen and should serve equally well as donors in charge transfer complexes. This paper reports the results of our spectrophotometric studies on amine-iodine charge-transfer complexes and our initial efforts a t the use of tertiary amine-iodine complexes as spectrophotometric detectants in
HPLC utilizing N,N-dimethylbenzylamine as a model compound. EXPERIMENTAL Apparatus a n d Materials. Ultraviolet absorption spectra were recorded using a Hitachi Model 60 or a Perkin-Elmer Model 200 spectrophotometer. The liquid chromatograph consisted of a Waters ALC-202 modified by the iodine delivery system described below. N,N-Dimethylbenzylamine was obtained from Aldrich Chemical Co., Milwaukee, Wis. Resublimed analytical reagent grade iodine was used in preparing the required iodine solutions. Equilibration Time. The time required for the chargetransfer complexation process to reach equilibrium was measured by UV spectrophotometry. A 5 X M solution of N,N-dimethylbenzylamine in dichloromethane was added to a 1-cmcuvette and placed in the sample holder of the spectrophotometer. The solution was referenced against dichloromethane. The recorder was set to run at 24.0 cm/min and the absorbance measured at M iodine solution in 254 nm. A 0.05-mL sample of a 5 X dichloromethane was added to the sample cuvette and the change in absorbance plotted as a function of time on the recorder. Molecular Ratio of Reactants in Complex. The Job method of continuous variation (8) was employed to determine the molecular ratio of reactants in the complex. Solutions of N , N-dimethylbenzylamine (5.0 X lo4 M) and iodine (5.0 X lo4 M) were prepared in 1,2-dichloromethane. A series of 10-mL quantities of mixtures of these solutions was made consisting of complementary proportions of the two solutions (010, 1:9, ..., 5 5 , ...,91,100) in 10-mLvolumetric flasks. Absorbance was measured at 254 nm. Molar Ratio of Reactants Required for Maximum Absorbance. The iodine-amine ratio required for maximum charge-transfer band intensity was determined as follows. A solution of amine ( 5 X M) in dichloromethane was mixed in equal volumes with iodine solutions of varying concentration and the absorbance of the resulting charge-transfer complex recorded. The absorbance obtained was plotted against the ratio of iodine to amine molar concentration. Iodine Delivery System. The iodine solution was introduced into the HPLC system by installing a '/ls-inch Swagelok stainless steel union tee in the line between the column and the photocell. The tee junction was connected to a 500-mL stainless steel reservoir by means of l/le-inch stainless steel tubing. The upper outlet of the reservoir cylinder was connected to a two-stage regulator attached to a nitrogen cylinder. The flow of iodine solution into the system was regulated by altering the pressure of nitrogen gas over the solution in the reservoir. A nitrogen pressure of approximately 40 psi was sufficient to deliver 0.4 mL/min of iodine solution at a column flow rate of 2.0 mL/min. The flow rate from the reservoir was determined by the following procedure: With the reservoir line disconnected from the system and the tee connection plugged, the HPLC pump was set at the desired flow rate. This rate was confirmed by measuring the volume per minute of effluent from the system. The reservoir was then connected to the tee joint and the nitrogen gas pressure over the iodine solution was adjusted to achieve the desired increase in total effluent volume per minute. Chromatographic Studies. The HPLC was equipped with a '/4-inch 0.d. by 30 cm micro-bond CN column (Waters catalogue No. 34042). The system was operated at a flow rate of 2.0 mL/min of spectrophotometric grade dichloromethane producing an inlet pressure of 1500 psi. In all runs 5 WLof a dichloromethanesolution of amine was injected using a 25-bL syringe. The chromatography was carried out at ambient temperature without thermostating. The iodine delivery system was operated at a flow rate of 0.1 mL/min of 3.1 X M iodine in dichloromethane. RESULTS AND DISCUSSION The initial phase of this investigational work involved a study of the ease of formation, the molar ratio of reactants, and the spectrophotometric properties of iodine-amine charge-transfer complexes utilizing NJV-dimethylbenzylamine as a model compound. These studies were designed to determine the extent to which these complexes, reported to have
Ijl
%I
i
Flgure 1. Rate of equilibration of the iodine-N,Ndimethylbenzylamine charge-transfer complex
very high molar absorptivities, might be employed as chromophoric derivatives for HPLC. T h e rate of formation of the iodine-N,N-dimethylbenzylamine charge-transfer complex was rapid with maximum charge-transfer band intensity achieved in less than 7 s (Figure 1). The established equilibrium maintained a constant absorption for a period of several minutes. Other work (3) has noted variations in absorbance reading €or iodine-amine complexes for u p t o 1 h after formation. However, such variation was not observed in this study. The frequency and intensity of the absorption bands for many charge-transfer complexes have been shown t o be solvent sensitive (9). Furthermore, the association constants ( K m )of iodine-amine charge-transfer complexes are known t o be solvent dependent (2). For maximum utility in HPLC the iodine-amine complex should have an intense UV absorption at 254 nm (most common wavelength used in HPLC detectors). The results of our studies indicate dichloromethane and 1,Z-dichloroethane obtain maximum absorbance of iodine-amine charge-transfer complexes. Gomaa and Taha (IO) found 1,2-dichloroethane to produce the greatest enhancement of the absorptivity of iodine-alkaloid chargetransfer complexes. Furthermore, many solvents possessing n-electrons can form charge transfer complexes with iodine and such solvents competitively inhibit the formation of iodine-amine complexes (9, p 127). For example, a solvent consisting of 5 % methanol in dichloromethane inhibited the complexation of iodine and N,N-dimethylbenzylamine. The stoichiometric ratio of iodine and amine in the charge transfer complex was determined by the J o b method of continuous variation (8). The absorbance of solutions of varying iodine-amine ratios were determined in dichloromethane. The results of this study (Figure 2) confirm a charge-transfer complex resulting from the interaction of one molecule of iodine (12) and one molecule of amine. In compounds with 2 nitrogen atoms, H 1:2 iodine-amine ratio has been reported (3). Numerous methods for the evaluation of the equilibrium constant for acceptor-donor complexes (Km) and the molar absorptivity are available (9). The majority of these methods require charge-transfer complex formation in solutions containing excess donor (amine). The use of charge transfer complexes as amine detectants requires maximum amine complexation. Therefore, instead of measuring the molar absorptivity of the complex, the iodine-amine ratio required for maximum charge-transfer band intensity was determined. ANALYTICAL CHEMISTRY, VOL. 49, NO. 13, NOVEMBER 1977
* 2081
Flgure 2. Continuous variation plot obtained for the iodine-N,N-dimethyibenzyiamine charge-transfer complex
J ‘L h
-
L
A
-l%E
y
’.
TES
Figure 4. High pressure liquid chromatography of N ,Ndimethylbenzylamine. Curve A, N,Ndimethylbenzylamlne; Curve B, N , Ndimethylbenzylamine plus 0.1 mL/min of 3.1 X M iodine in dichloromethane
y 5
5
,
,
,
,
,
,
,
,
,
,
I5
.i
:c
25
IO
35
LO
LS
5s
.j
Yj.ii
M‘.O
[ “ I t dI
Flgure 3. Charge-transfer band Intensity as a function of the iodlne (acceptor)-N,N-dimethylbenzylamine (donor) molar ratio Maximum absorbance was observed in solutions of excess acceptor (iodine) and a plot (Figure 3) of the iodine-amine ratio vs. absorbance revealed the relationship to be linear up to a ratio of 1O:l. Mixtures of iodine and amine formed in the determined proportions should allow for maximum sensitivity. Thus, these initial experiments indicate that iodine-amine charge-transfer complexes present some unique advantages over conventional amine derivatives as detectants in HPLC. The effects of solvent on the equilibrium constant and molar absorptivity of iodineamine charge-transfer complexes greatly limits the choice of the mobile phase for chromatography. The solvent must be one that promotes maximum absorption of UV radiation by the complex in the 254-nm range. Using dichloromethane as the mobile phase solvent, N,N-dimethylbenzylamine was found to exhibit good chromatographic properties on a micro-bond CN column. A linear plot of peak area vs. concentration was obtained in this system. The addition of iodine to the HPLC system was attempted by three methods: (1)the direct injection of the iodine-amine complex; (2) the use of an iodine solution as the mobile phase; (3) infusion of an iodine solution into the mobile phase stream at a point between the column and the photocell detector. The injection of the preformed complex was not successful since the peak areas obtained corresponded to the concentration of amine, not complex. Thus, the complex did not survive the chromatographic process. Competition by solvent or stationary phase components are apparently strong enough to overcome the weak interactions of charge-transfer bonds. The retention of iodine by the column which resulted from 2082
ANALYTICAL CHEMISTRY, VOL. 49, NO. 13, NOVEMBER 1977
using iodine in the mobile phase further indicates an interaction between iodine and the column material. The addition of the iodine solution into the HPLC system at a point in the flow stream beyond the chromatographic column allows for the separation process to take place using the free amine and the detection of the amine as the iodine-amine charge-transfer complex. The equilibration time for the formation of the complex is rapid enough to permit iodine infusion at this point. Thus, no special equipment or techniques need be applied for the chromatography of the charge-transfer complex. The addition of the iodine solution was achieved by the iodine delivery system previously described. The developed methodology was applied to the HPLC analysis of N,N-dimethylbenzylamine. A plot of peak area vs. concentration was determined in the absence of iodine and in the presence of a molar excess of iodine. The plots were linear and a 20-fold increase in peak area was observed for the charge-transfer complex. Although other research (9, p 171) has indicated a deviation from the Beer-Lambert relationship when the concentrations of donor and acceptor differ in magnitude, the graphical data maintained a linear relationship. This may be attributed to maximum complex formation in the presence of a 10-fold excess of iodine. A sample of the chromatography obtained is shown in Figure 4. Since the chromatography is achieved with the free amine and derivatized in this post-column procedure, the retention time is the same for the free amine and the iodine-amine complex. The iodine infusion rate must be maintained at a low level to prevent serious chromatographic zone broadening. A t low rates of iodine flow (0.1 to 0.2 mL/min), no serious zone broadening effects were noted; however, at higher rates of iodine flow, the zones became substantially broadened. Although the iodine delivery system has no effect on the chromatographic separation process, the constant infusion of relatively large volumes of a second solution following the chromatography column can produce effects similar to chromatographic band spreading. This preliminary investigation indicates that this technique can be successfully used to enhance the detection of amines through the formation of the iodine-amine charge-transfer complex. However, the effects of solvent on the properties of the charge-transfer complex places some severe restrictions on the choice of chromatographic mobile phase.
ACKNOWLEDGMENT The technical assistance of John Gunnels and Tommy Hardwick in this project is gratefully acknowledged. LITERATURE CITED (1) H. Yada, J. Tanaka, and S . Nagakura, Bull. Chem. SOC. Jpn., 33, 1660 (1960). (2) A. M. Taha, A. K. S. Ahmad, C. S . bmaa, and H. M. ECFatatry, J . pharrn. Sci.. 63, 1853 (1974). (3) H. S . 1. Tan, E. D. Geriach, and A. S . Dimattio, J . Pharm. Sci., 66, 766 (1977). (4) H. Jupiiie, A m . L a b . , 8, 85 (1976).
(5) C. R. Clark, J. D. Teague, M. M. Wells, and J. H. Eiiis, Anal. Chem., 4g, 912 (1977). (6) N. E. Hoffman and J. C. Liao, Anal. Chem., 48, 1104 (1976). (7) F. A. Fltzpatrick, M. A. Wynaida, and D. G. Kaiser, Anal. Chem., 49, 1032 (1977). (8) J. Rose, “Advanced Physico-Chemical Experiments”, Pitman, London, England, 1964, p 54. (9) R. Foster, “Organic Charge-Transfer Complexes”, Academic Press, New York, N.Y., 1969, p 62. (10) C. Gomaa and A. Taha, J. Pharm. Sci., 64, 1398 (1975).
RECEIVED for review July 7, 1977. Accepted August 29, 1977.
Ion Electrode Measurements of Complement and Antibody Levels Using Marker-Loaded Sheep Red Blood Cell Ghosts Paul D’Orazlo and G. A. Rechnltz’ Deparfment of Chemistry, State University of New York, Buffalo, New York 14214
We Introduce a new method, using Ion-selectlve membrane electrodes, for the measurement of Immunoagents. The method Involves the use of marker loaded, sensltlzed sheep red blood cell ghosts to produce potentlal changes In response to varylng levels of antlbody or complement. The release of marker Ion from the cell ghosts, as monitored with the membrane electrode, Is shown to be a sensltlve measure of the concentrationsof antlbody or complement under controlled condltlons.
Recent advances in the development of bio-selective electrode systems have focused primarily upon the use of immobilized enzymes at electrode surfaces, but it has also been pointed out ( I ) that electrodes suitable for immunomeasurements might be feasible if the selective action of antibody-antigen (Ab-Ag) interactions could be quantitated and coupled to an appropriate indicator electrode. We now report on a new approach to this problem through the use of vesicles, such as sheep red blood cell ghosts (SRBC ghosts), to release an electroactive marker ion in response to the immunoreaction. By using a very large number (>lo6)of SRBC ghosts a t the electrode, the electrode response can be related to the amount of immunoreactive material present; at the same time, selectivity is achieved through the action of “complement” which functions as a catalyst and amplifier in the lysis of the marker-loaded vesicles. It will be seen that this system, although unfamiliar to most analytical chemists and seemingly complicated in the abstract, is readily capable of yielding analytical measurements of either antibody or complement levels with relatively inexpensive equipment and commercially available components. Much work, accurately described in several reviews, has dealt with the use of complement in quantitative immunology as well as the resolution and study of the specific actions of the several complement components (2-5). The reader is referred to these reviews for information concerning theory of complement action and specific details of complement fixation procedures. T h e ability t o produce lysis of erythrocytes is the phenomenon which has been exploited to determine the level of complement in biological fluids. The recognition and destruction of a sensitized erythrocyte, one which has bound to it its specific antibody, permits a quantitation, under optimal
conditions, of the level of complement or of the anti-erythrocyte species present. In general terms, Figure 1 illustrates the sequence of events that permits a quick screening of test serum for any antibody of interest (Abl). Unfixed complement, e.g., that which has not been inactivated by the Abl-&, complex in reaction system 1, is capable of producing lysis of sensitized red blood cells. The extent of oxyhemoglobin release as determined by visual inspection can be related to the presence of the antibody of interest. Alternatively, the system can be made quantitative hy assaying the released oxyhemoglobin optically at 541 nm. Red blood cells are useful as indicators for the determination of complement fixation reagents. However, the use of spectrophotometry in conjunction with red blood cells has the disadvantage that both lysed and unlysed cells must be separated from the reaction mixture before any assay of hemoglobin can be attempted. It would therefore be useful to devise a procedure whereby the release of marker could be measured without any need for separations and with a minimum of sample handling. It may also be desirable to follow the course of the complement fixation reaction by a continuous monitoring of marker release as a function of time. This leads to a consideration of a novel indicator for the complement fixation reaction, e.g., red blood cell ghosts. It has been known that erythrocytes can be re-formed after osmotic lysis under the proper conditions of osmolarity, temperature and pH, in order to trap any solutes present at the time of re-forming (6-8). Cells can be lysed in hypotonic solutions containing low salt concentrations and subsequently re-formed by raising the ionic strength of the lysing medium to isotonicity with the solute or solutes that are to be entrapped in the re-formed cells. Obviously, the limited permeability of blood cells to many electrolytes and nonelectrolytes (6) could be quite advantageous for purposes of complement fixation since release of marker could be attributed to complement mediated lysis only. Humphries and McConnell (7) have used mammalian erythrocyte ghosts loaded with the spin label tempocholine chloride to follow complement fixation reactions. The present work describes the use of sheep erythrocyte ghosts loaded with the cation trimethylphenylammonium (TMPA’), which can be conveniently measured using an ion selective membrane electrode. Figure 2 illustrates the system used in this study. Sheep erythrocyte ghosts containing TMPA4+have binding sites of ANALYTICAL CHEMISTRY, VOL. 49, NO. 13, NOVEMBER 1977
2083
