stressed that the signal level for the 4-bromobiphenyl is lower here because the measurements are made at 37.5 Hz instead of 25 Hz as in the phase method. The precision of quantitative measurements by the phase resolution method are given in Table VI. The per cent relative standard deviation expected is of the same order as the per cent error in the analysis of the binary mixtures. The principal sources of random noise in this experiment are bubbling noise in the liquid nitrogen cool-
ant and electronic noise from the amplifier's. Of these two, the bubbling noise is especially bad at the low frequencies of operation. Received for review December 28, 1973. Accepted April 8, 1974. Research was carried out as a part of a study on the phosphorimetric analysis of drugs in blood and urine, supported by U S . Public Health Service Grant No. GM11373-11.
Pulsed Source Time Resolved Phosphorimetry for the Quantitative and Qualitative Analysis of Drugs K. F. Harbaugh,' C. M. O'Donnell,2 and J. D. Winefordner3 D e p a r t m e n t of Chemistry. University of Florida, Gainesville, Fla. 326 7 7
Pulsed source time resolved phosphorimetry is used to qualitatively identify and quantitatively measure synthetic mixtures of drugs. No physical separations are needed but rather tempciral resolution is sufficient to resolve mixtures of phosphors. The time resolved phosphorimeter consisted of a pulsed xenon flash lamp source, an emission monochromator with photomultiplier tube detector, and a signal averager-recorder readout system. Because phosphorescence lifetimes of organic molecules vary considerably with structure and with environment, whereas the phosphorescence emission spectra of organic molecules are quite similar, temporal resolution is a far more selective method of measurement than spectral resolution. The drugs studied in this work were morphine, ethylmorphine, codeine, quinine, procaine, phenobarbital, amobarbital, cocaine, amphetamine, and rnethamphetamine.
Temporal resolution in phosphorimetry was first used by Kiers, Britt, and Wentworth ( I ) in 1957, when they showed that a binary mixture of structurally similar compounds could be resolved by judicious selection of excitation and emission wavelengths and by choice of the delay before the observation time and after termination of the exciting radiation. Initial time resolution studies made use of a spectral continuum source with mechanical shutters for termination of exciting radiation. Kiers et ul. ( I ) used a modified Becquerel type rotating disk, St. John and Winefordner ( 2 ) a manual guillotine shutter, and Hollifield and Winefordner ( 3 ) a single disk mechanical phosphoroscope. Winefordner ( 4 ) was first to suggest the advantages of pulsed source, gated detector instrumentation for time resolved analytical phosphorimetry, and O'Haver and Wine-
fordner (5) put pulsed source phosphorimetry on a sound theoretical basis. Fisher and Winefordner (6) described a pulsed source, gated detector phosphorimeter and compared the system to conventional phosphorimeters. Later, O'Donnell, Harbaugh, Fisher, and Winefordner (7) modified this system and demonstrated the utility of time resolved phosphorimetry for the quantitative measurement of synthetic mixtures of halogenated biphenyls. They also suggested the use of time resolved phosphorimetry for qualitative identification of phosphorescent molecules. Since that time, however, no other analytical uses of pulsed source time resolved phosphorimetry have been shown. The use of drugs for both legitimate and illicit purposes is extremely widespread. The qualitative identification of drugs and the estimation of drug levels in biological materials for therapeutic purposes is needed for the effective use and/or control of drugs. Because conventional phosphorimetry as well as fluorimetry have already proved to be sensitive analytical methods for measurement of drugs which phosphoresce ( 8 - E ) , the application of time resolved phosphorimetry to the selective determination of drugs should greatly extend the usefulness of luminescence spectrometry to drug assay. In the present study, a pulsed source, gated detector, time resolved phosphorimeter is used for the qualitative and quantitative measurement of selected drugs in serial solvents. The advantages and limitations of this technique are illustrated for this application. The feasibility of drug analysis in real samples is indicated.
EXPERIMENTAL Apparatus. The pulsed source phosphorimeter used in these studies was i d e n t i c a l t o t h e one described previously b y O'Donnell, Harbaugh, a n d Winefordner (7). A l l phosphorescence mea-
Present address, Celanese F i b e r Co., Charlotte, N.C. Present address, D e p a r t m e n t o f Chemistry, Colorado State University, F o r t Collins, Colo. A u t h o r t o w h o m r e p r i n t requests should be sent. (1) R . J. Kiers. R . D. Britt, Jr., and W. E. Wentworth, Anal. Chem.. 29, 202 (1957) ( 2 ) P.A. St. John and J. D. Winefordner, Anal. Chem.. 39, 500 (1967). ( 3 ) H . C. Hollifield and J . D. Wlnefordner. Chem. Insfrum.. 1 , 341 (1969). ( 4 ) J. D. Winefordner, Accounts Chem. Res . 2, 361 (1969)
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ANALYTICAL CHEMISTRY, VOL. 46, NO. 9, AUGUST 1974
T. C. O'Haver and J. D. Winefordner, Anal. Chem.. 3 8 , 6 0 2 ( 1 9 6 6 ) . R. P. Fisher and J. D.Winefordner, Anal. Chem. 44, 948 ( 1 9 7 2 ) . C. M. O'Donnell, K . F. Harbaugh, R. P. Fisher, and J. D. Winefordner. Anal. Chem.. 45. 609 (1973) J D Winefordner and M. Tin, Anal C h m Acta 32, 64 (1965) /bid 31. 239 11964) W. J. McCarthy, P. A.St. John, and J. D. Winefordner, "Phosphorimetry as a Means of Chemical Analysis," in "Fluorescence Assay in Biology and Medicine," S. Udenfriend, Ed.. Academic Press, New York, N.Y., 1970. H. C. Hollifield and J. D. Winefordner. Talanta. 12, 860 (1965) C. J Miles and G . H. Schenk, Ana/. Chem , 45, 130 (1973)
(p
Table I. Comparison of Curve Fit and Graphical Methods for Determination of Phosphorescencea Intensities at t = 0 ( P o )and Lifetimes (7)
OCH,
Figure of merit
Po
OH
OH
Codeine
Morphine
Method
[arbitrary units]
C u r v e fit Graphical
55.4 53.9
SP,/PO
r(sec)
s1/7
0.11
0.81 0.83
0,035 0.041
0.12
a T h e measured species was 0.092 mg/ml of quinine in chloroform; 8 sweeps was used for calculations.
-
OC,H, Cocoino
I
OH
(415)
Pkonobarbitol
Amobarbital
Ethylmorphine
Amobarbital (400) Wo thamphe tamine
(395)
Meperidine
Phenobarbital
Amphotomino
(390)
CH Mepe ridine
(390)
Amphetamine
Procaine
OH I HC-C
0
H
400
60 0
So0 Wavelength ( n m )
-N-CH,
Figure 2. Phosphorescence emission spectra of drugs at 77 "K studied by t i m e resolved phosphorimetry. Solvent used was c h l o r o f o r m . Peak phosphorescence emission wavelengths are given in parentheses
CH? Methamphetamine
Quinine
0 H H H I 1 H C-C+-C-O-CH,
I
I
I
I
kCH, C-O-C-C6Hj
I
HC-C-CH H H
I
II
0
H Cocaine
Figure 1. C h e m i c a l structures of d r u g s studied by t i m e resolved phosphorimetry
surements were performed with a stationary quartz tubular sample cell (5-mm 0.d. x 3-mm i.d.). Reagents. The drugs used in this study were reagent grade; all drugs were ordered in accordance with BNDD regulations (Applied Science Labs, Inc., State College, Pa.), and were used as received. The structures of the drugs used in this work are illustrated in Figure 1. Phosphorimetric quality anhydrous ethanol was prepared from 95% US1 ethanol according to a modification of a procedure de-
scribed by Lund and Bjerrum (13).The chloroform was certified ACS Spectroanalyzed (Fisher Scientific Company, Fair Lawn, N.J.) and was distilled once on a 6-ft glass bead-packed column to reduce the phosphorescence background (chloroform results in a reproducible snowed matrix suitable for phosphorimetric studies). The ethyl ether, anhydrous (Mallinckrodt Chemical Works. St. Louis, Mo.j and cyclohexane (Matheson Coleman and Bell, Los Angeles, Calif.) were used as received. The EPA was a commercially-prepared mixture (American Instrument Company, Silver Spring, Md.). Procedure. Methods of data handling have been described previously (6, 7 ) . In addition to the graphical methods described elsewhere ( 7 ) ,data points of the exponential decay of single component solutions in this work were fitted to a curve of the form 3 = yo@" by a calculator (Wang 600 Series, Wang Laboratories, Inc., Tewksbury, Mass. j to determine the initial phosphorescence intensity, P o ~ o )and , the phosphorescence lifetime, r ( - l / b ) ; this curve fit method was used for calculations of lifetimes and for estimation of PO'Sfor standard solutions, while the graphical method described previously (6, 7) was used to determine the PO'Sfor each component of a multicomponent curve (see Table I for comparison of methods).
RESULTS AND DISCUSSION Quantitative Analysis. In t i m e resolved phosphorimetry, i t is first necessary t o m e a s u r e t h e spectra and the (13) H . L u n d
and J. Bjerrum, B e r . Dent Chem. G e s . . 6 4 6 , 210
(1931)
A N A L Y T I C A L C H E M I S T R Y , VOL. 46, NO. 9, AUGUST 1974
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Table 11. Phosphorescence Lifetimes of Drugs in Various Solvents Lifetime, Drug
Emission peak, nm
Meperidine Amphetamine Methamphetamine Amobarbital Cocaine Phenobarbital Procaine Quinine Ethylmorphine Morphine Codeine
390 390 395 400 415 385 445 515 510 510 515
E PAa I . .
5.9 0.0015 2.2 1.8 b 1.0 0.043 0.028 0.040
(in sec)
EA5
CFa
...
0.55 0.52 0.55 0.0013 0.82 0.77 1.45 0.85 0.033 0.014 0.028
7.5 9.6 0.0016 2.4 1.65 2.9 1.1 0.040 0,020 0,039
b
T
CHa
DEE"
...
... b b b
... b b b b b 0,041
b b 0.0016 2.2 1.7 b b b b 0.041
" Solvents used are: EPA = 5 : 5 : 2 volume ratio of ether:isopentane:ethanol.E A = anhydrous ethanol. CF = chloroform. CH = cyclohexane. D E E = diethyl ether. Drug was a t least partially insoluble in the solvent, and so was not run.
*
Table 111. Relative Phosphorescence Signals of Selected D r u g s in Various Solvents Log (Po) values Drug
Amphetamine Methamphetamine Amobarbital Cocaine Phenobarbital Procaine Quinine Ethylmorphine Morphine Codeine
EA"
CF"
DEE"
EPAn
3.0 2.8 6.0 4.6 3.0 4.0 3.95 3.7 3.7 3.8
4.0 4.0 6.0 5.3 4.6 4.0 4.5 3.5 2.0 3.7
... ...
3.5
5.6 4.0 5.0
6.0 4.0 4.6
...
...
...
,..
4.0 3.3 4.0 3.5
... , , . ...
a Solvents used are: EA = ethanol. C F = chloroform. D E E = diethyl ether. EPA = 5 .,rJ : 2 volume ratio of ether:isopentane:ethanol. Pa = relative phosphorescence signal obtained by extrapolation of phosphorescence decays t o time, t = 0.
phosphorescence lifetime of individual species that may be determined in the multicomponent mixture; from these data, the drugs which can be spectrally-resolved, those which can be temporally-resolved, and those which can be resolved by a combination of these techniques can be readily chosen. The phosphorescence emission spectra of the drugs studied here in chloroform solvent are given
in Figure 2. Because phosphorescence spectra do not change appreciably with changes in solvent, the phosphorescence emission peaks measured in chloroform are representative of the peaks obtained with other solvents. The phosphorescence lifetimes on the other hand vary significantly with a change in solvent as illustrated in Table 11. A third important consideration in time resolution studies is the relative phosphorescence signals of the components being measured. For the best results (greatest signal-to-noise ratios), the phosphorescence signals of the components being measured should be about the same, or the species with the lower signal will be lost in the noise on the observed signal. The logarithm of the relative initial phosphorescence signals (PO)of various drug binary mixtures are shown in Table 111. The analytical results of several synthetic binary mixtures determined by time resolved phosphorimetry are given in Table IV. Quinine, morphine, ethylmorphine, and codeine exhibit severe overlap of emission spectra, and all combinations of these four drugs are temporallyresolvable except for the mixture of codeine and ethyl morphine. The most precise results should be obtained in ethanol, because the lifetime differences are comparable to those in chloroform and greater than in EPA, and the relative phosphorescence signals for morphine are higher in ethanol than in chloroform. From the results in Table 111, several important aspects
Table IV. Quantitative Analysis of Synthetic D r u g Mixtures by Pulsed Source, T i m e Resolved P h o s p h o r i m e t r y Mixture
Solventa
Slopeb
Drug present, mg ml
Concn found, mg ml
EA
0.95
0 067
0.068
+1.6
EA EA
0.97 0.95
0,023 0.11
0,022 0 12
-4.3 +9.0
EA
0.96
0.052
0.042
- 20
EA
0.95
0.11
0.16
+45.
EA
...
d
d
d
EA
0.95
0.16
0.16
C
EA CF
c
CF
0.99 1.00 1.05
0.062 0.038 0,021
0.058 0.042 0.018
Emission wdvelength, nm
Morphine
Rrl error,
r6
510
Codeine MorDhine 510
Ethylmorphine Morphine 510
Codeine + Ethylmorphine Morphine
0
510
Quinine Morphine Cocaine
-7.0
+lo. -14.
Solvent abbreviations given in Tables I 1 and 111. Slopes of analytical curve !log-log plot) over concentration range of interest. Error of slopes is *0.05 or less. I n this mixture, spectral as well as temporal resolutioi was utilized, i.e., the emission measurement for cocaine was a t 415 nm and for morphine a t 510 nm. Since the lifetimes of codeine and ethylmorphine are nearly identical, it is impossible t o resolve them. a
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A N A L Y T I C A L C H E M I S T R Y , VOL. 46, NO. 9. AUGUST 1974
of pulsed source time resolved phosphorimetry are apparent. Morphine, codeine, and ethylmorphine have only slightly different phosphorescence lifetimes, but morphine can still be temporally-resolved from either or both of the other two. Because ethyl morphine and codeine are metabolites of morphine, in vivo, this measurement illustrates the possibility of analysis of drugs and metabolites that would be difficult to separate by physical means. Time resolution of a short- and long-lived species is illustrated by the analysis of morphine and quinine. This particular combination also has a potential practical application because quinine is used as a diluent for heroin. Morphine, being the immediate metabolic product of heroin if adequately separated from the urine mixture, could be determined in the presence of quinine, assuming the quinine is also extracted along with the morphine. Spectral overlap of amobarbital and phenobarbital prevent spectral isolation; time resolution can be applied to the determination of this mixture. However, spectral and temporal overlap ‘of amphetamine and methamphetamine do not allow either/or both spectral and/or time resolution of this mixture. Cocaine and phenobarbital similarly do not show a sufficient difference in phosphorescence lifetimes (see Table 11) to allow temporal resolution of a mixture of these species. No results are given here for real street samples. However, time resolved phosphorimetry should save considerable time for a number of specific analyses, such as some of the ones mentioned above. Most street drug analyses involve simply a qualitative analysis via TLC and/or colorimetric methods. Quantitative analyses of drugs of abuse in biological fluids and in street samples generally involve extensive “clean-up” procedures (14-18), as well (14) D . Sohn, J Chromatogr. S c i . . 10, 294 (1972) (15) A. Romano, J . Chromatogr S c i . . 10, 342 (1932)
as one or more separation steps (14-18), and finally some sort of measurement step including gas chromatography (16-18), fluorimetry (16, 18), and absorption spectrophotometry (16, 18). The greater selectivity of time resolved phosphorimetry could certainly enable the analyst to omit or shorten some of the steps prior to the final measurement. Qualitative Analysis. Because excitation and emission spectra and phosphorescence lifetimes of a molecule in a given environment are characteristic of that molecule, and because these parameters may change to a different degree for different molecules with a change in environmental conditions, time resolved phosphorimetry could be a useful technique for molecular identification. From an examination of the data in Table I and Figure 2, it is obvious that, of these drugs, any single (phosphorescing) component sample could be identified qualitatively uia phosphorescence excitation and emission spectra and lifetime, except for the binary mixtures of meperidine and any of the amphetamines and ethyl morphine and codeine. (It could well be, however, that with additional measurements of lifetime of these species in other solvents, these combinations would be resolvable and therefore identifiable.) Therefore, qualitative analysis could be accomplished on multicomponent samples as long as the species are spectrally- and/or temporally-resolvable. Received for review November 21, 1973. Accepted March 19, 1974. Research was carried out as a part of a study on the phosphorimetric analysis of drugs in blood and urine, supported by U.S. Public Health Service Grant No. GM11373-11. (16) H . E. Sine, M . J. McKenna, M . R . Law, and M. H . Murray, J Chrornatogr. S c i . 10, 297 (1972) (17) L. Kazak and E. C. Knoblock, Anal. Chem.. 35, 1448 (1963). (18) “ A Bibliography of References on the Analysis of Drugs of Abuse,’ J. Chromatog S C I . 10, 352 (1972)
Kinetics of Chromatographic Processes with Application of the Steepest Descent Approximation Method Kuang-Pang Li,’ David L. Duewer, and Richard S. Juvet, Jr.* Department of Chemistry, Arizona State University, Tempe, Ariz. 8528 1
This paper extends earlier fundamental studies of the mechanism of chromatographic processes to include, in addition to simple partitioning, the interaction of the solute with the stationary phase through either physical or chemical reaction. The steepest descent approximation method is applied in overcoming the inherent mathematical difficulties introduced by these additional considerations. It is evident from computer simulation and solution of the appropriate differential equations that, not only is retention a function of the stability of the association product formed between the solute and the liquid phase, but also the elution peak profile is a function of the rates of formation and dissociation of the association product formed on the column. This work id:,
’ Present address, Department of Chemistry, University of FlorGainesville, Fla. 32601.
- T o whom correspondence concerning this contribution should he addressed.
suggests the direct evaluation of the rate of solvolysis or the rate of solute complex formation from peak profile studies. The often observed shift in base line following peak elution may also be explained solely on rate considerations and need not involve adsorption or irreversible chemical reactions on the column.
The theory of chromatography is well established and numerous authors have contributed to the description of on-column phenomena. Early theoretical studies, such as the pioneering work of Lapidus and Amundson ( I ) , van Deemter e t al. (2), and Giddings and Eyring ( 3 ) ,empha(1) L. Lapidus and N. R . Amundson. J. Pbvs. Chem.. 56.984 11952) (2) J J van Deemter, F J Zuiderweg, and A Klinkenberg, Chem Eng S o , 5, 271 (1956) (3) J C Giddings and H Eyring, J Phys Chem 59. 416 (1955)
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