Analytical Appkations of
L
I 4
. 0
m. Neil Purdie and Kathy A. Swallows Department of Chemistry Oklahoma State University Stillwater, OK 74078-0447
Chiroptical methods are optical methods that can differentiate between two enantiomers. They include polarimetry, optical rotatory dispersion (ORD), and circular dichroism (CD). Detection is based on the interaction between a chiral center in the analyte and the incident polarized electromagnetic radiation. Early applications dealt primarily with elucidation of molecular structures, particularly of natural products for which a technique capable of confirming or establishing the absolute stereochemistry was critical. Only recently has significant attention been given to the application of these techniques to analytical chemistry. Although many requirements must be considered when analytical methods 0003-2700/89/0361-077A/501 S0,O
@ 1989 American Cnemica. Soc erv
Polarimetry, Optical Rotatory Dispersion, and Circular Dichroism
or procedures are being developed, the sometimes mutually exclusive properties of analytical selectivity and breadth of application are particularly important. Analytical selectivity depends on the structural oroDerties of the analvte and the abilit; of the chosen detector to
REPORT differentiate the analyte from a potentially large number of interferences. One approach to analyzing a complex mixture is to eliminate the problem of interferences altogether by performing a total separation. Nonselective detectors of hrkad general applirability such as refractive index or U\' absorptiun detectors ran then he used. It is seldom essential to identify and quantitate every component of a mixture, huu,ever, u,hich is helpful because total separation often is not possible. At the other ANA-YTCA-
ChEMISTRV.
extreme, the ideal selective detector would operate well without any sample separation at all and despite the interferences. Reality is compromise. How many molecular properties are necessary to achieve an acceptable level of selectivitv? If onlv one oronertv is necessary, separation is essential unless a more sophisticated procedure that is either time or phase sensitive is used. If three or more properties are necessary, the number of potential analytes is greatly diminished. The optimum number appears to he two. Thus of the chiroptical methods, we strongly advocate the use of CD, which measures both rotation and absorbance simultaneously. This REPORT will describe the use of chiroptical phenomena for analvtical detection. narticularlv for system; for which separation is not a prerequisite to the identification pro. cess. L
.
I
,.
ChirOiJticai phenomena Numerous excellent treatises on the physical phenomena of chirality and VOL 61. NO 2. JAhUARY 15. 1989
77A
REPORT the manifestation of its interaction with polarized light are available (1-3). For chemical analysis, an elementary understanding of the nature of the interactions and their relationships as well as the dependence of the experimentally measured parameters on the concentration of the optically active species ( 4 ) is sufficient. Polarimetry and ORD both determine the extent to which a beam of linearly polarized light is rotated on transmission through the medium containing the chiral sample. The two techniques are entirely equivalent for nonabsorbing chiral species and differ only in that ORD yields a spectral response whereas polarimetric measurements usually are restricted to a limited number of preselected wavelengths. The degree of rotation is dependent on the rotatory strength of the chiral center, the concentration of the chirophore, and the path length. The older literature (1-3) uses obtuse terms and unusual concentration units when dealing with solution media, such as
[O]= lO-’M[a] and [a] = 100 a/(c’d)
(1)
where a,[a], and [a]are the rotation, specific rotation, and molar rotation, respectively; M is the molar mass; d is the sample path length; and c’ is the solute concentration, expressed either as a percent or as g/dL. Combining these equations results in an equation that is totally analogous to the BeerLambert law, namely a = [aldc’. IUPAC recommends retaining the concentration unit g/dL because of the wealth of information in the literature; we disagree with this recommendation, however, and have chosen to use moles per liter in our work to make the linear relationship equivalent to the absorbance-concentration relationship. The units of [a]therefore are degree cm2/
dmol. (This is mentioned only in case someone compares data from our work with data on the same systems taken from the older literature, where they will find a difference in the order of magnitude reported for [a].)Experimental values for a are usually on the order of millidegrees (mol unless laser sources are used, in which case microdegrees can be measured. In the absence of absorption, the plain ORD spectrum changes monotonically with wavelength. This change can be either positive or negative (see Figure la). For chiral media that absorb the polarized light beam, anomalous rotations in the ORD spectrum are produced if the chiral center and the chromophore are structurally adjacent to each other in an arrangement called a chirophore. This anomalous behavior is referred to as the Cotton effect (5) and is limited to the wavelength range of the absorption band, where it is seen superimposed on the monotonically changing plain curve. The anomaly takes the form of a sigmoidal curve with peak and trough extrema whose wavelength values are roughly bisected at an intermediate crossover point a t which the rotation is zero, as shown in Figure lb. In the simplest sense, where a single Cotton effect exists, the height between the extrema can be used for quantitative measurements. ORD has not been extensively pursued as a worthwhile method for analytical work because of a lack of specificity in differentiation and because of the uncertainty in defining the baseline, which is the undeveloped part of the plain curve under the Cotton band. CD is the most sophisticated of the three chiroptical methods in that the rotation and absorbance measurements are made simultaneously. Linearly polarized light consists of two beams of circularly polarized light propagating in phase but in opposite
rotational senses. In chiral media the beams are phase differentiated because they “see” two different refractive indexes and consequently travel a t different speeds, a phenomenon that produces the rotation effect. If a chromophore is present, differential absorption also occurs that causes the transmitted beam to become elliptically polarized, as shown in Figure 2. The long axis of the ellipse is rotated from the original plane of polarization by the angle a,as before, and the ellipticity is defined as = tan-’ (OA/OB). Both terms are wavelength dependent and combine to produce the CD spectrum. The perimeter of the ellipse at each wavelength is the trace of the diagonal of the parallelogram whose adjacent sides are the two absorption vectors. Therefore is directly related to the absorbance difference, represented as the difference in vector lengths. Mathematically, the relationship is given by the expression
*
*
= a(cL
- fR)/A,
or Z‘’ = a A d A (2)
where 6 is the molar absorptivity; EL and cR are the molar absorptivities for left and right circularly polarized light, respectively; and A is the wavelength a t which the ellipticity is measured. In the application of CD to analytical problems, the two physical properties of chirality and absorption provide the information necessary for qualitative and quantitative determinations, respectively. Because the change in molar absorptivity, Af, is defined as (LLfd,CD spectra can have positive and
Flgure 2. Production of elliptically polarized light in CD.
Flgure 1. Typical chiroptical curves. (a) Plain OR0 curve, (b) ORD CYNB With a single Cmton effeot, acd (c)CD curve with a single conon C U N ~and a positive maximum. The sdium Diine is the u6uBi wavelength for polarimetric m8asuremems.
78A
ANALYTICAL CHEMISTRY, VOL. 61, NO. 2, JANUARY 15, 1989
OP is the direction Of polarization of the incident beam. OL and OR are the absorption vectors for left and right oirouiariv ooiarlzed beams. and a is the angle of rotation
Good chemistrv begins with contaminihfree
sample containers.
That’s why I-Chem~1 ~s th rep different Protocols”to prepare containers. One for glass bottles and jars. One for polyethylene and Nalgene pro ucts. And a third one for vials. In fact,our vials are prepared at a totally separate facility in a chemical free environment. To give you that extra i . assurance,a ll I-Chem containers come with heavy duty ,030 Teflon”lined closures.Vialsand septum bottles are supplied with our quality TeflonMicone septa. __ We put our containers to the test before you do. Using trace analpcal techniques I such as graphite firnace AA, ICP and GC/MS, we profile each batch of containers before they go to your lab. Pick up the phone and call I-Chem todayyou’ll fmd yourselftalkmgto other ks+ch - emists.Chemists who understand what it takes to do good chemistry I CHEM Chemists in the container business. ~ i n t h e c o n f r u. n rBusinrss” r
4 .
* -
_ . I -
I
REPORT
a
Polarized
Figure 3. A conventional CD in strum en.^ LCP and RCP are the iefl and right circularly pOlarized light beams, respectively
negative variations from the baseline as well as wavelengths where Af = 0 (crossover points), as shown in Figure IC.Because there is no CD signal at wavelengths where there is no absorption by the analyte, the baseline is easily defined. Thus CD is superior to ORD for analytical applications. In the earliest CD instruments the experimental parameter measured was in fact the ellipticity of the transmitted beam. Modern instruments, however, are modified absorption spectrophotometers that measure the differences in the absorbances of the two beams as a function of wavelength. The ordinate on the CD spectrum is still reported as an ellipticity, however, in deference to the historical development of the chiroptical methods. Because absorption is being measured, the data conform with the Beer-Lambert law. In CD the proportionality constant is OM, the molar ellipticity, which is related to Ac by the equation = 3300 Af. Once again, hecause we have defined the chiroptical property relative to mole/L and not to mg/dL, the orders of magnitude of our data and the literature data for the same system will differ. The necessity of a chiral center in the analyte molecule makes CD a much more selective method than straightforward absorption spectrophotometry. Broad featureless bands in absorption spectra are often separated into more than one Cotton hand in a CD spectrum, and the bands are frequently of opposite polarity, enhancing the identification capabilities of CD over absorption. In addition, because the direct correlation is between the difference in the molar absorptivity, Af, and the molar ellipticity, OM, for a BOA
given analyte, the strongest CD signals are not necessarily associated with strong absorbers. This is especially useful at wavelengths longer than 240 nm, where molar absorbances are small compared with the strong bands observed in the far UV, yet CD hands are sufficiently large. Analyte concentrations are typically 10-4 M or less for wavelengths longer than 240 nm. CD signals in the far UV can he very large, but the signal-to-noise quality is poor whenever strong absorbers are present. Because Af is much smaller than the average c value, the CD signal is actually a very small millivolt quantity riding on top of a relatively large value. Despite the large difference in signal size, detection limits are 0.1 fig/mL for analytes with OM values of -200 m o l mohcm at the band maxima. This can be improved by introducing fluorescence detection (61,which is, in fact, commercially available. (The use of fluorescence, however, introduces the need for a third structural requirement in the analyte molecule, which effectively decreases the applicability of CD.) Instrument operating conditions and solution concentration variables are chosen to give the optimum ratio of CD to the total absorption, hearing in mind that a mixture may contain several absorbing species. When fluorescence is the detector of choice, more serious interferences from the emissions of CD-passive fluorophores can he expected, conceivably canceling any gains made in lowering the level of detection. To predict if an analyte will he optically active and thus can be determined by CD, the presence of chirality and absorbance must be confirmed. (One
ANALYTICAL CHEMISTRY, VOL. 61, NO. 2, JANUARY 15, 1989
must remember, however, that although the molecular structure may suggest that chirality is present, the substance may only he available as a racemic mixture and therefore undetectable.) Although these requirements may seem to make CD too selective for practical analytical use, the applicability of CD can he increased by adding the missing molecular property by in situ derivatization. One can make an achiral absorbing analyte CD active by reacting it with a chiral partner, preferably one that is nonabsorhing, and a chromophore can be introduced in a way that either does or does not affect the overall chirality of the molecule. These CD-induction reactions should not he considered exclusively as possible pre- or postcolumn derivatization reactions in chromatographic applications using CD detection. They are instead intended to he so specific that they can be used for analysis of unseparated mixtures. A block diagram of a conventional CD instrument is shown in Figure 3. The small signal intensity requires that the incident power he very large; a 500-W Xe lamp is usually used. This source must he water cooled and oxygen must be removed from the instrument to reduce the production of ozone, which is detrimental to the optics. The volume of the typical I-cm pathlength cell is about 3.5 mL; smaller path length cells, which may require focusing of the incident beam, are available for analyses that require smaller volumes.
LC detection Chiroptical detectors used in liquid
a
Intearated ChromatoaraDhv. A Shimadzu Advance
x
c
1'1 1---Im n r I a--a
I
More than just components. With Shimadzu matched modules you can configure your HPLC system just right: From isocratic to full gradient from analytical to biocompatible to preparative systems.
More than just svstem control. Taking you way beyond mere 'dialogue",the new CR501 data processor and our 'Integrated Chromatography"concept provide for true interlocking of chromatographic and data processing parameters. Circle t i 4 6
@
More than just sample handling. Our new SlL-9A autosampler and new fraction collector handle
and "prepare" your preparative or analytical samples, the latter even with pre-column derivatization, dilution and spiking. Circle t i 4 7
More than just simple separation. Our application specific HPLC systems and new line of columns combined with innovative software provide a choice of tools for the demanding chromatographer. cimie#14a
@
More than just reliable hardware. We provide complete after-sales support from seven regional offices. And our technical staff speaks HPLC fluently. Now you can see how it all works together. The advantage of the synergism of Shimadzu will give you truly integrated chromatography. Call or write: Shimadzu Scientific Instruments, Inc. 7102 Riverwood Drive, Columbia, MD 21046 (301) 381-1227
Synerssm a W: Fulb aummaredgredlenf spmm LC-64. For demo, Circle X149: For h f q circle X150
-a
REPORT
.he ANALYTICAL CHEMISTRY Library :me. . . a handsome and convenient vay to keep your ANALYTICAL :HEMISTRY issues protected and easy o reach. iach speciallydesigned library case iolds u p to seven issues-one-half rear's subscription of ANALYTICAL :HEMISTRY. Designed to keep your \NALYTICAL CHEMISTRY issues at lour fingertips, the ANALYTICAL :HEMISTRY Library Case is perfect for iome, office. or library. The Library Case is available in two ;tyles: the slipcase or the binder. Both ityles are covered in attractive navy eatherette with embossed gold lettering tnd both are reasonably priced. You lay only $6.95 for the slipcase and 68.50 for the binder. 3rder your ANALYTICAL CHEMISTRY Abrary Case today! Your Satisfaction is inconditionally guaranteed or your noney will be immediately refunded. Simply fill in the coupon below and mail n with your payment .1111111111111
Dlease send me: -ANALYTICAL
CHEMISTRY
Library Slipcase(s) at $6.95 each -ANALYTICAL
CHEMISTRY
Library Binder($ at $8.50 each I am enclosing a check or money order for $ ' (Make payable to Jesse Jones Industries. Add $2.50 per case lor orders outside the US.) Name IAddress
ICitylStatelZip 1 'Payment must acwmpany all Orders and 1 must be made in U.S. currency only. Orders I from Outside the U.S. add $2.50 per case. Mail to: Jesse Jonas Industries. PO. Box I 5120ANALY-C, Philadelphia PA 19141
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 82 A
-
ANALYTICAL CHEMISTRY, VOL.
chromatography (LC) are primarily single-wavelength detectors. Because of the constraints of both signal size and small elution volumes, lasers are the most suitable light sources for these detectors. Yeung and eo-workers have described both polarimetric and CD detectors for LC (6, 7). Stopped-flow CD spectral detection for LC has been described both by development scientists from JASCO, Inc. (8) and by Westwood, Games, and Sheen (9).We are unaware of any real-time, on-thefly, full-spectrum CD detection in chromatography, although it is not beyond development if the need could ever be justified. Because chiroptical detectors respond to anything that is chiral, they are particularly useful in studying substances of natural origins, and their use can complement the more common chromatographic detectors in studying complex mixtures. The majority of the applications developed to date have involved laboratory preparations; the number of real samples investigated is very small. If total separation of a mixture is possible, polarimetry is the chiroptical detector of choice. It has been used, for example, to identify and quantitate structurally related carbohydrates in a common mixture (IO).Polarimetric instrumentation is comparatively inexpensive both to purchase and to operate, and the detector responds equally well to absorbing and nonabsorbing analytes. Furthermore, the effectiveness of a polarimetric detector has heen demonstrated in both the direct mode, where the rotation caused by the analyte is measured ( 7 ) ,and in the indirect mode, where the change in the measured background rotation for an optically active mobile phase is used to quantitate the analyte (11). In the latter example the analyte itself need not even be chiral. Limiting CD detection to a singlewavelength measurement reduces it to no more than a very expensive polarimeter with a much narrower range of application because the nonabsorbing chiral compounds are now transparent to the detector. If, however, separation is not complete, then the differentiation capability of the full-range CD detector becomes necessary.' As the need arises, convenient instrumentation for fast-scan measurements may become available. Early attempts to develop such devices have been described in the literature ( I 2 , 1 3 )but , a lack of demand is prohahly responsible for their absence from the laboratory. Enantiomeric differentiation Enantiomeric differentiation is a two10. 2, JANUARY 15, 1989
level problem. If the identity of the substance is known and only one isomer is present, then the sign of the rotation easily estahlishes its stereochemical identity, in which case polarimetric detection is enough. If both enantiomers are present, which is the rule rather than the exception, the analysis takes on a different dimension: the determination of the enantiomeric excess (EE) or optical purity. There are two general chromatogrbphic approaches to enantiomeric difierentiation. In the first approach the enantiomers are converted into diastereoisomers, which have different retention properties and thus can he physically separated. The derivatization to diastereoisomers can he done in either a precolumn reaction or in situ on the stationary phase of the column. For precolumn derivatization, any one of the well-known diastereoisomerizing reagents can be used (e.g., optically active amines for the separation of acid enantiomers). A convenient derivatization reaction cannot always be found, however. The in situ derivatization can be done only if the stationary phase is chiral; a variety of chiral stationary phases are available ( 1 4 , 1 5 ) . The real thrust of this work, however, is to find effective and convenient ways to enhance optical purities. The actual determination of the EE is of secondary importance. A chiroptical detector is required to establish the elution order, but once this is done it can be replaced hy a general-purpose detector. Polarimetry is the best choice for determination of enantiomeric enrichment at the exploratory level where eluted volumes are small. When chromatographic procedures are developed to the point where large-scale separations are possible, CD is the better detector, because differences in the full spectrum of the analyte compared with that of the standard signify the presence of a coeluted chiral interference. The second chromatographic approach is to determine the EE without first preparing diastereoisomers. Measuring the EE or optical purity without either a derivatization step or chiral stationary phase separation is actually easier than might be imagined. Because nonderivatized racemic mixtures will coelute from conventional LC columns, neither a conventional detector nor a chiroptical detector alone is adequate to determine the EE. If the detectors are placed in series, however, a quantitative distinction can he made. Data from either an absorbance or RI detector provide the sum of the concentrations of the two isomers and the signal from the chiroptical detector-
L
\And
Name.
Send me information on Radiometer's G Titration systems 0 pH- and ION meters 0 Electrodes 0 Conductivity meters
after
.I
C
Company:
Dept.:
Address. Postal Code:
Town
CountrylState: Tel. NO.:
i i y o ~ a o n o t i i k e i o c umyour t magame. send a photocopy instead
~ihenyouneedlobesure
R A D I O M E T E R A N A L Y T I C A L A/S KROGSHOIVEI 4 9 . DK-2880 BAGSVAERD . DENMARK .%e us SIthe Prmburgh Ccnfwonce '89, Bonh numbers 2500, 2515, 2515, 2517. CIRCLE 137 ON READER SERVICE CARD
ENVIRONMENTAL SCIENTISTS AND ENGINEERS,..
ORDER YOUR COPIES OF THE MOST REQUESTED SPECIAL REPORTS FROM ES&T Introducing three series madp available by ES&T cmm'ng maim areas in m'ronmental science. You'll aunt to order all three!
Cancer Risk Assessment 12m
T
ius spzcial report explorer the m o u s sieniik topis of pubbc ioniern to reguhis c h s m d s in the environment. Among the iopis yuu'll h J .1 !ere1 i o help you mvlage thc risk anJ :ommumcaie he rerults oi risk dsvismcnt are: * Being more realistic about chemical carcinogenesis *Exposure assessment Cancer dose-response extrapolations *physiological p h c o l i n e t i c modeling *Amd more.. .
Wastewater Treatment 48m
U -
pdatc your knowledge oi the laicbt procerrer for rhc ueauneni uf wastenater with itus in-depth repori. You'U Cud tupics ruth as thne . . Removig particles in water and wastewatu Removing dissolved orgauc contaminants from water * Removing dissolved inorganic contaminants from water *Anaerobic wastewater treatment *Tolame a few.. .
-
Computer Usage in Engineering (npP)
U
dKe the Jf the COmpUlCr 10 bridge h e gap betwen enpneers and p u b k decisionmaken uith thx npon ihat bnefs vou on ihe mnpuier basics and then discusses new trends and applications, such as artilicii intelligence, and their potential impact on environmental engineering. You'll a h see how personal computers might change the practice of environmental engineering in the future, and much more.
1
IO ORDER,wnd check or money order with name, addre,,: teephone numkr, and
quanun of ea;h
xns, IJ
American Cbcrmcal Soncty P.0 Box 17136 West End Slauun Washington, DC 20037 (202) 872.4363
04 A
ANALYTICAL CHEMISTRY, VOL.
REPORT which is either the rotational difference, (a(+) - +)), for the polarimetric detector or the difference in ellipticity, (%'(+) - %'(-I), for CD-provides information from which to calculate the concentration difference (16, 17).The concentration of each isomer is then readily obtained from the simultaneous solution of these equations. In many situations where CD is the detector of choice, its selectivity is so great that it can he used as a standalone detector and will provide the concentration difference information without separation (28).This is especially important whenever eluted volumes are small, because of the small CD signal. An aliquot is injected onto a conventional column, and the total concentration of both enantiomers is measured using absorption detection. The concentration difference is calculated simultaneously from the CD spectrum of another aliquot of the unseparated mixture. The EE is then calculated, as described earlier. Whichever method is used to determine the EE, the quality of the results depends entirely on the optical purity of the standard materials. These can never be considered to be either 100% pure or of equivalent purity. The absolute purity argument is somewhat moot in that the instrumental or separation methods are limited by their resolution capabilities. Theoretically, for spectroscopic analyses it is necessary to have only one of the isomers for instrument calibration provided that diastereoisomerization is not a prerequisite to the determination, as it is in NMR. In chiroptical methods it would be nice to have both isomers of equivalent optical purity as an internal check of the calibration, but this is an unlikely if not impossible expectation. Reports of enantiomeric ratio determinations should dispel the idea that the determination is made against an absolute standard of purity and emphasize the fact that the ratio is relative to the purity of the best available standard reference material only. Detection limits The detection limits obtained with chiroptical detectors are equivalent to those obtained with absorbance detectors for bulk measurements. In conventional chromatographic systems, nanogram detection limits are commonplace; in state-of-the-art detector development, picogram or even femtogram limits have been reported (7). The ability to detect such small quantities is of critical importance only when the physical size of the sample is limited, as is the case in microbore LC, where the eluted volumes are exceed0. 2, JANUARY 15, 1989
ingly small. If the sample size is not limited, the simple alternative is to scale up the experiment. Chiroptical methods are not yet competitive within the lowest of these achievable detection ranges, showing a typical cutoff in the nanogram-per-milliliter range, unless fluorescence is used for signal enhancement or laser sources are used (6, 7). When sample sizes are not a prohlem and a typical working sample volume is a few milliliters, detection limits on the order of micrograms per milliliter are readily achieved using CD. Analytical applicatlons The answer to the question of how selective chiroptical methods really are appears to be that polarimetry and ORD have little to no potential as selective detectors. They do have specific redeeming applications, but they are truly functional only when all interferences have been removed. CD is in the same category as long as its use is limited to single-wavelength chromatographic detection. The applications described here therefore are primarily those utilizing the selectivity capabilities of full-spectrum CD analyses in which neither separation nor derivatization steps have been performed. Some instances in which derivatization is necessary are included. Our experience shows that the most useful wavelength range is from 240 to 400 nm, which encompasses the transitions from the aromatic ring and unsaturated ketone chromophores. Few suhstances are CD active in the visible range, and at wavelengths shorter than 240 nm, signal-to-noise ratios are significantly decreased because of the extremely intense absorption bands. In addition, CD bands are observed to be broad and featureless and usually of only one sign, resulting in spectra that are the same as the corresponding absorption spectra. Forensic science. The use of chiroptical detectors in forensic science generally is limited to drug analysis. Many abused substances are of natural origin; some are synthetic. If used in LC, the function of the detectors is to either establish or confirm the stereochemistry of the material, which is significant in states where laws defining controlled substances fail to address the chirality variable. Because in some states only natural sources of cocaine are controlled, a racemic laboratory preparation, although still physiologically active, is not included on the controlled substances list. The presence of L-cocaine chirality must often be verified for conviction. This can be done polarimetrically after the drug has been completely separat-
ed from all of the other optically active components added as bulk materials (19). The absence of rotation for the proper eluted fraction is clear evidence that the material is racemic and therefore synthetic. Because such separations are difficult, distinction is often made using the subjective microcrystalline tests instead. Using CD, however, L-cocaine can be confirmed objectively in alcohol extracts of the confiscated materials without separation (20). Other controlled substances, including LSD (21),opiates, (22),cannabinoids, (23), and phenethylamines (241, have been conveniently identified and quantitated in the same way. Potential interferences are reduced to include only those that are also CD active. Thus although some of the extracts are intensely colored, the only effect this has on the analysis is to significantly reduce the signal-to-noise ratio and therefore increase the detection limit. When the extract contains more than one CD-active analyte (22,23),the concentration of each is determined by mathematically fitting the spectrum of the mixture using the s u m of weighted contributions from the standard refer-
ence spectra for each of the components. Determination of poisons is another area in which CD can be of value in a forensic context. Colchicine, strychnine, brucine, and curare alkaloids all produce excellent CD spectra when dissolved in dilute HCI (IS),and each of these can be identified without a separation step. The presence of these substances can be confirmed by a simple solvent extraction of the material ingested by the victim or examination of stomach contents. Phencyclidine (PCP) and its analogs and the barbiturates are inherently achiral and seemingly not detectable by CD unless chirality is induced, for example, by forming a molecular association complex with structurally organized media such as the cyclodextrin sugars. The intensity of the induced signal generally is quite small-typcally one-tenth of the inherent molar ellipticity of a structurally equivalent molecule-which naturally raises the detection limit. 0-cyclodextrin (O-CD) can be used to determine both the PCP group and the barbiturates (25) because the sugar does not absorb in the range of the chromophores in the ana-
I
lytes. The CD signal is proportional to the concentration of the complex only, and the formation constant for its molecular complex with o-CD must be obtained to determine the analytical concentration of the target material. Great care must be exercised when analyzing mixtures where CD activitv is induced because of the potential fo; competing association reactions. Thus far little has been done in the analysis of body fluids, although this area has tremendous potential. How effective CD might be depends on both the problems with interferences and the detection limits. The most significant interferences in body fluids would be from proteins, which have strong CD signals in the wavelength range 180-220 nm. This is outside the favorable range of 220-400 nm, so protein interferences are less of a difficulty than might have been expected. Solvent partition, volume reduction, and derivatization are commonly used in preparing a biological sample to increase the analyte concentration and enhance the detection limit. The detec~~~~~~~~~. tion limit fur CD should be comparable to that obtained by LC with UV detection, but for full-spectrum analysis ~
II
R A D I O M E T E R A N A L Y T I C A L A/S KROCSHOIVEJ49 DK-28x0 EACSVAERD DENMARK 8w us at the Plbburgh Conhnnce '89, Bwfh numbers 2500,25i3,2515,2517. CIRCLE 138 ON READER SERVICE CARD ANALYTICAL CHEMISTRY. VOL. 61, NO. 2, JANUARY 15. 1989
asA
REPORT larger original samples may be necessary. In preliminary urinalysis studies good results have been obtained for tetracycline (4) and morphine. Complications from CD-active metabolites may require that the specimen be separated using conventional LC before the full spectrum is run. Clinical chemistry. This area is a natural continuation of the analysis of biological specimens discussed above, but here the emphasis is on metabolic disorders. LC-polarimetry can be used to study sugars in urine (26) as well as cholest&oland its ester derivatives in buman serum (27). All of these compounds are present in relatively large concentrations and have strong rotational strengths. Derivatization with a fluorophore (e.g., dansylation) can be used to imorove the detection limit for amino acids (28),whereas enzyme and hormone levels are orobablv at the limit of polarimetric detection. Other potential analytes include the sterones, some of which have been studied using ORD (29) and CD (30).If these compounds are present in sufficient quantity, they could conceivably be determined in urine. Because of a general lack of chromophores among the sugars, amino acids, and sterols, the application of CD to their detection is limited unless a derivatization reaction is used to introduce the necessary chromophore. Color induction is already a standard procedure in clinical chemistry when absorption is the analytical method of choice. Althouah these same color induction reactions may beexpected to produce a CD-active derivative. this is not generally the case, and specific deriiatization reactions that yield CD-active products are necessary. Cholesterol, for example, contains an isolated double bondand isinherently CDactivewitha maximum around 200 nm-too close to the waveleneth limit of the instrumentation to be of practical use in analysis. We have found. however. that chloroform extracts of human gallstones produce several strong Cotton hands in the 30&600 nm range, as shown in Figure 4. We have attributed these bands to some molecular complexation interaction between cholesterol and bilirubin (which absorbs in this range but is CD inactive) because the intensities of the bands vary directly with the darkness of the stones. This is supported by the reported CD induction reaction of bilirubin with albumin (31). This result led us to a fuller study of serum cholesterol. There is considerable concern in the clinical chemistry community about the lack of accuracy and reproducibility among laboratories reporting blood cholesterol levels and 86 A
Figure 4. CD spectrum of the chloroform extract of a human gallstone. the distribution of the total cholesterol among the various high (HDL), low (LDL), and very low (VLDL) density lipoproteins. Automated procedures are commercially available that are based on absorption and give results for total cholesterol directly and HDL cholesterol levels after the selective precipitation of the low-density lipid fractions. VLDL is estimated to he onefifth of the total triglyceride (TGL) level. LDL, recognized as the principal culprit responsible for the formation of arteriosclerotic deposits, cannot be determined directly and is calculated instead from the mass balance relationship [LDL] = [total] - (HDL] - 0.2[TGL]. Of course the errors in each of the three independent measurements are cumulative, which is the source of the poor precision in the analyses. Using a novel chromogenic chemical reaction and full-spectrum CD detection, we have successfully measured the total low-density lipid (VLDL LDL) fraction directly (32). HDL levels are also obtained from the same experiment from data taken at a different band maximum, as shown in Figure 5. Except for the earliest preliminary experi-
+
ments in which i t was confirmed that the 525-nm Cotton band was caused exclusively by low-density fractions, the customary low-density lipid precipitation step is not a part of the routine analvsis. The selectivitv of the CD detector is so great that no spectral distortions are observed. even in the presence of hemolysis or extreme hyperlipidemia (TGL levels greater than 400 mgIdL). Ketosteroids are amenable to direct CD detection (30),which brings up the interesting question of whether the method might be useful for the detection and determination of anabolic steroids in urine. Preliminary studies have been made on three anabolics: methandrostenolone. methandriol. and stanozolol(32). The first, a ketosteroid, has a CD spectrum similar to testosterone but the bands are displaced to longer wavelengths and differentiation is. in fact, possible. Methandriol is a structural analog of cholesterol and combines with the reagent used in the cholesterol test to produce a colored product that has a completely different CD spectrum from that shown in Figure 5. Its presence in urine should thus be detectable after sample concentration. Stanozolol is CD inactive and does not react with the "cholesterol reagent." A review of the chemistrv of the compound may suggest suiiable reagents for color induction. Thus far no definitive tests have been made to determine the limits of detection for these substances using CD. Pharmaceutical chemistry. Chiroptical detectors can be used in pharmaceutical applications for quality control and fordetermination of optical purity or EE. Polarimetry and ORD applications as before are limited tu LC detection, for example, the neomycins (33).amino acids (281. . .. and 17-ketosteioids (29). CD. however. has been successfullv applied to direct determination of nu"merous products, including opiates; quinine and quinidine (easily distin-
Flgure 5. CD spectrum of the colored product of the cholesterol reaction. GUNS (a) represents me total lipid cholesterol. whereas the shaded area Is the spectrum after addition of the L M precipitatingagent and is thus represenlaliveof the H M fraction only.
ANALYTiCAL CHEMISTRY, VOL. 61, NO. 2, JANUARY 15, 1989
rigure 8. CD spectra of (a) penicillin-\/,
(Dj
cepnaiosporin,
(8.07rnglrnL) in aqueous pH 5.4 buffer. guishable from each other because the Cotton bands are of opposite polarity); reserpine; tetracyclines; penicillins and cephalosporins (whose CD spectra bear no mutual similarities [see Figure 61 in comparison with the absorption spectra); and vitamins ( 4 ) . Common additives such as aspirin, caffeine, and most simple sugars are noninterfering (4). This list is not especially long, partly
ana IC) Deniciiiin-v
DrOth
method of detection. A second group of pharmaceuticals, including atropine, meperidine, dilantin, the barbitals, and the diazepams ( 4 ) are achiral. These drugs are either inherently achiral (e.g., dilantin), or they exist as exact racemic mixtures e(& ,.! atropine), but each group can be assayed using P-CD-induced CD if they contain a suitable chromophore. Racemic mixtures can be included despite the fact that the enantiomers may interact differently with &CD, and what is actually recorded is the net induced signal. This differing interaction is, of course, part of the reason that enantiomers can be partially separated on the chiral cyclodextrin stationary phases (15).
because the point clearly has been made that the task of using CD for quality control, without chromatographic separation, is elementary and particularly reliable. The method is easily extended to each new application because its role is simply that of a detector, and extraction procedures and workup for any given sample are the same as they are for any other
h k begins with an excellent WeNieW of the evolution of computer graphisand will introduce you to the four general types of software packages currently available 0 xientitic word processing packages packager that allow graphics entry of chemical structures but do not allow substructure searching or interfacing with other systems 0 packager that allow graphics structure entry and substructure searching and interfacingwRh Other systems 0 packages designed to act as front ends to molecular modeling systems. This volume continues with discussions and cornpadsons of various W a r e packages for the chemist Information managers. information scientists, systems analysts. bench chemists. physical chemists. and PC enthusiasts will find this book a vital. necessaiy reference. Wendy A. Warr. Edtor AeS Sympsium Series No. 341 176 paqfs (1987)Clothbound 1C 87-3575 ISBN 08412-144-8 US R Canada 544.95 mort 553.95
The need for two detectors in the determination of EE or optical purity was discussed earlier. Some early examples combined UV or RI with polarimetric detection, and cocaine and codeine U9),epinephrine (34), and Dand L-penicillamine (35) were investigated in this manner. We have successfully used UV and CD in series for prepared mixtures of (R)-and (SI-nicotine in which solu-
-when you need to be sure.
im American Chemical Society. Distribution Office Dept. 58 1155 Sixteenth Si., N.W., Washington, DE 20036
ANALYTICAL CHEMISTRY, VOL. 61, NO. 2, JANUARY 15, 1989
87A
REPORT
, ,jura 7. (a)UV spectra of an S-(-)-nicotine standard, (b) UV spectra of a smokeless tobacco 2-propanol extract, (c)CD spacrrum of the nicotine standard, and (d) CD spectrum of the tobacco extract. tions of the natural isomer were deliberately spiked with aliquots of the other (18). Subsequently, leaf extracts were spiked with the unnatural isomer and the EE was determined using conventional LC. The total nicotine concentration is measured by LC using an absorbance detector, and the CD spectrum of either an aliquot of the unseparated mixture or the eluate from a conventional LC column gives the data from which to calculate the concentration difference. The concentration of each is obtained by the simple solution of the simultaneous equations. The limit to the measurement of enantiomeric excess is on the order of fl% with one exception: It is exact for the racemic mixture because the net CD signal will be zero. All of this discussion presumes that the identity of the compound is known. Agriculture and food science. The use of LC with combined UV and either polarimetric or CD detection for the identification of enantiomers of the pyrethroid insecticides (17) is an excellent illustration of the potential difficulties in attempting to make a full analysis of stereoisomers in a complex mixture extracted from a natural source. The analytical focus usually is on the commercially important constituent(s), and the other ingredients are looked upon as undesirable interferences that complicate the assay. Because they are very complex, plant and food extracts are taken through a progression of simplifying separations that usually terminate with a definitive GC 88A
-
or LC step. Even then, a complete analysis is not guaranteed. For example, the CD study of pyrethrins also included an investigation of the Arnaryllidacene alkaloids in both the single-wavelength and stopped-flow detection modes (9), and even then the metabolites associated with a number of the peaks could not be identified. Sugars have been separated from prepared laboratory mixtures and from urine extracts by conventional LC and identified using either RI or UV detectors in series with a polarimeter, with and without laser illumination (I6,26). Sensitivity can be enhanced by first complexing the sugars with molybdate ion (36).Although the complex is colored, the possibility of changing the detection system from polarimetry to CD was not explored. Ketoses can be determined directly using CD ( 3 3 , but patience is necessary to ensure that the time-dependent mutarotation process is complete. After demonstrating the extreme selectivity of the full-spectrum CD detector for moderately complex systems, we applied it to the study of plant extracts. There was plenty of information to suggest that it would function very well as an LC detector, because the polarimeter does (9, 17), but how would it perform without sample separation? Two things must be borne in mind in this context. First, the extract will contain many compounds, and many of these could be CD active. But unless the product of the molar ellipticity, OM, and the molar concentration exceeds the
ANALYTICAL CHEMISTRY, VOL. 61, NO. 2, JANUARY 15, 1989
minimum detection limit of the instrument, the spectrum of that analyte will not be observed. This reinforces the selectivity capabilities of the technique even further. and CD then focuses on the commercially important component. Second, there are no good standard reference materials for most biological samples, so the results can only be compared with data reported from other analytical methods. Most often a result is considered to be acceptable if it falls within broadly defined limits. The study of nicotine in tobacco (388) illustrates these points. Many nicotine analogs and other organic bases are extracted along with the parent suhstance bur are present in such relative. ly low percentages that the CD spectrmn for the extract is an exact replica of the spectrum for pure nicotine, as shown in Figure 7. The estimated assay of 1-290 nicotine by weight of dry leaf is within the realm of the accepted figures for Nicotinia tabocum, and the direct CD method of analysis is considered to be satisfactory. The identity of the counterion in the salt form would not interfere with the CD detection, and the method would be a convenient alternative for the assay of nicotine in agricultural insecticides. Other equally successful studies have involved assays of hops (humulone) ( 4 ) ; belladonna (atropine) ( 4 ) ; marihuana (THC and CBD) (23);opium (morphine, codeine, thebaine, noscapine) (22);Rauwolfia serpentina (reserpine) (39); fermentation broths (penicil1in.V) (40); cattle feed (aureo.
mycin (18) (Figure 8); and fresh vegetables, fruit, and spices (vitamins C and D) (18). Standard separation procedures were used in every case; in some instances aqueous and organic phases were investigated separately. The assay of food glycosides is a relatively unexplored area with some interesting possibilities for the application of chiroptical detectors. Structurally these compounds meet the requirements for CD activity by having the chiral center in the sugar moiety and an aromatic chromophore in close ji?xtaposition; the connection between the two parts is through either carbon (cyanogenics), nitrogen (nucleosides and nucleotides), oxygen (saponins and flavones), or sulfur (glucosinolates). The magnitude of the CD signal will depend on how adjacent the nearest chiral center on the sugar is to the chromophore, and it may become necessary to enhance the signal for the weakest cases. Polarimetric work on neomycins was mentioned earlier (33), and an accurate CD assay of riboflavin was included in a recent general investigation of the vitamins (18). A cursory review of the literature indicates that the levels of riboflavin in meat and fresh vegetables are comparable to those in dispensary doses of vitamins already investigated, and CD detection without chromatographic separation should be possible.
References (1) Crabbe, P. ORD and CD in Chemistry and BLochemrstrv: An Introduction: Academic Press: Ne; York, 1972. r2, Charney.E. TheMolerulorBosisofOpr ~ a Acriciiy; l Wile).:New York, 1Y7Y. (3, Mason. S. F. Q. Reu. Chcm. Sot.. 1961, IZ "9n~ ,~-..
(4) Purdie, N. Prog. Anal. At. Speetrosc. 1987,10,345. (5) Cotton. A. ComDt. Rend. 1895.. 120.. 989-1044. (6) Synovec,R. E.; Yeung, E. S. J. Chromotogr. 1986.3,68,85. (7) Svnovec. R. E.; Yeung, E. S. Anal. Chchem. 1986,58,1237A. (8) Takakuwa, T.; Kurosu, Y.; Sakayanagi, N.; Kaneuchi, F.; Takeuchi, N.; Wada, A,; Senda, M. J. Liq. Chromatogr. 1987, 10,
Figure 8. CD spectra of antibiotics in cattle feed. (a) Chiortelracvciine in 1 0 M In OH5 6 buffer and (b) aureomycm In pH 5 6 buffer exwact 01 canieleed crumbles.
(16) Boehme, W. Chromatogr. Newsl. 1980, 8.38. (171 Meinard. C.; Bruneau. l'., l'erronneti, J. J . Chromarogr. 198b349,109. r l 8 1 Purdie. N.; Swalli,ws, K . A , , u m u b -
lished results.
(19) Palma, R. J.; Young, J. M.; Espenscheid, M. W. Anal. Letters 1985,IB(BS), 641.
(20) Bowen, J. M.; Purdie, N. Anal. Chem.
1981,53,2237. (21) Bowen, J. M.; McMorrow, H. A,; Purdie, N. J. Forensic Sci. 1982,27,822. (22) Han, S. M.; Purdie, N. Anal. Chem. 1986,58,113. (23) Han, S.M.;Purdie, N. Anal. Chem. 1985,57,1068. (24) Bowen, J. M.; Crone, T. A,; Head, V. L.; McMorrow, H. A,; Kennedy, R. K.; Purdie, N. J. Forensic Sci. 1981, 26, 664. (25)Han, S.M.; Purdie, N. Anal. Chem. 1984,56,2822,2825. (26) Kuo, J. C.; Yeung, E. S. J. Chromatogr. 1981,223,321. (27) Kuo, J. C.; Yeung, E. S. J. Chromatogr. 1982,229,293.
(28) Reitsma, B. H.; Yeung, E. S. Anal, Chem. 1987,59,1059. (29) Gergely, A.; Szasz, G. Acta Pharm. Hung. 1983.53.280. (30) Gergely, A.; Szasz, G.; Soos, J.; Fresenius, 2.Z.Anal. Chem. 1986,323,157. (31) Berglof, J.; Grahnen, A,; Gjoholm, I. Clin. Chem. Acta 1975,62,169. (32) Murphy, L.H.; Purdie, N., unpub-
lished results.
(33) Gergely, A,; Papp, 0.; Szasz, G.; Vamos, J.; Bacsa, G. Acta Phorm. Hung. 1980,50,98. (34) Scott, B. S.; Dunn, D. L. J. Chromatogr. 1985,319,419, (35) DiCesare, J. L.; Ettre, L. S.J. Chromatogr. 1962.251,l. (36) Dokladolova,J.; Upton, R. P. J. Assoc. Off. Anal. Chem. 1973,56,1382. (37) Hayward, L. D.; Angyal, S. J. Carbohydr. Res. 1977,53,13. (38) Atkinson, W. M.; Han; S. M.; Purdie, N. Anal. Chem. 1984,56,1947. (39) Swallows. K.A.: Purdie. N. Pharm.
Res., in press.
(40) Purdie, N.; Swallows, K.A. Anal, Chem. 1987,59,1349.
97FO
(9) Westwood, S.A,; Gs L. J. Chromi (10) DiCesare,
(I? 191 1121
1311.
(14) Pirkle, W. H.; Finn, J. M. In Agvmmetric Synthesis; Morrison, J. D., Ed.; A:ademic Press: New York, 1983,pp. 87124. (15) Armstrong, D. W. J. Liq. Chromatogr. 1984.7.353.
Neil Purdie (right) is professor and head of the Chemistry Department at Oklahoma State University. He obtained a B.Sc. degree and a Ph.D. from the University of Glasglow, Scotland, and joined the faculty a t Oklahoma State in 1965. His research interests include relaxation kinetic studies of metal-ligand complexation reactions and analytical detection using chiroptical methods. He is also involved in the development of matrix materials for the controlled release of pharmaceutical products and agrichemicals. Kathy A. Swallows (left) isafourth-yearPh.D.studentinchemistry at Oklahoma State University. She obtained a B.S. degree from Central State University in Edmond, OK. Her research interests include the development of controlled release materials and the measurement of optical purities. ANALYTICAL CHEMISTRY, VOL. 61, NO. 2. JANUARY 15,
1989
89A
