Chapter 31 Chiroptical detectors for HPLC of carbohydrates

Ziad El Rassi (Editor) Carbohydrate Analysis by Modern Chromatography and Electrophoresi s Journal of Chromatography Library, Vol . 6 6 © 2002 Elsevier Science B .V. All rights reserved

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Chiroptical Detectors for HPLC of Carbohydrates NEIL PURDI E Department of Chemistry, Oklahoma State University, Stillwater, OK 74078-0447, USA

31 .1 . INTRODUCTIO N Chiroptical detectors are devices that are based upon one or other of the three physica l phenomena that are peculiar to optically active (chiral) molecules, namely polarimetry , optical rotatory dispersion (ORD) and circular dichroism (CD) . As analytical detectors the devices are very selective . Polarimetric detection is conceptually the simplest consisting of the measuremen t of the direction and magnitude of the angle of rotation of a beam of incident linearl y polarized light on its transmission through a chiral medium . Dispersion of the angl e of rotation with wavelength leads to the production of ORD . If the rotatory dispersio n increases monotonically with decreasing wavelength, in either a positive or a negativ e angular direction, the resultant spectral curve is said to be `plain' (Fig . 31 .1) . I f rotation is intimately coupled to light absorption by the molecule then the dispersio n is `anomalous' . In its simplest complete form, a single anomalous dispersion curve i s sigmoidal in shape over the wavelength range of the absorbance, showing two maxim a with opposite signs at wavelengths that are almost centered around a wavelength where the rotation is zero (Fig . 31 .1) . First explained by Aime Cotton [ 1 ], the anomalous ORD produced by the combination of rotation and absorption, is described as a Cotton effect . A Cotton effect i s essential to CD activity which is, in effect, a differential absorbance measurement . There is no CD analog to a `plain' ORD curve, and outside the range of the absorption band , the CD signal is zero (Fig . 31 .1) . Working either alone or in combination with non-selective detectors such as absorbance, electrochemical, or refractive index (RI), chiroptical detectors can be use d to confirm molecular stereochemistry, to gather structural information such as linkag e isomerism in polymers, and to measure enantiomeric purities of chiral analytes . References pp . 1148—1149



Chapter 3 1

1136 rotation in deg. an d ellipticity in mdeg . (c) (b)

wavelength (nm)

Fig . 31 .1 . Idealized spectra for (a) a positive plain ORD curve ; (b) a single positive anomalous ORD curve ; and (c) a single positive CD curve . Polarimetric measurements are usually made at the Na—D line .

31 .2. HISTORY OF DEVELOPMEN T A historical description of the manifestation, creation, and measurement of polarize d light in its many forms is given in the classical text by Lowry [21 . Briefly, th e phenomenon of polarimetry was first observed by the French astronomer Arago durin g experimental investigations on the transmission of solar radiation through calcit e (CaCO 3 ), just one year before Biot, in 1812, demonstrated that solutions of certai n organic compounds also rotate a beam of incident polarized light . Biot and Fresnel , working independently, observed that the power of a substance to rotate linearl y polarized light increased as the wavelength decreased, now called ORD . In 1846 , Haidinger reported a difference in the absorption of left and right circularly polarize d light on its passage through amethyst quartz crystals, a difference now identified a s CD . The first experimental interpretation of the physical basis for optical activity wa s provided by Pasteur who observed the hemihedrism duality of tartrate crystals . Aqueou s solutions prepared from the separated crystals were observed to rotate a plane polarize d light beam in opposite clockwise directions that were consistent with the orientatio n of the original tetrahedral crystalline facets, and with it the direct connection betwee n macroscopic and microscopic, or molecular, asymmetry was made . The first theoretical model of optical activity was proposed by Drude . In it, charge d particles in a dissymmetric structure were constrained to move in a helical path . Optica l activity is one physical consequence of the interaction between electromagnetic radiation and the helical electronic field . Efforts to combine molecular geometric models (e .g . th e tetrahedral carbon atom) with the physical model of Drude have involved both couple d oscillator and perturbation theories . Quantum theories require so many simplification s and so many assumptions that they are limited to the point where, even yet, there i s still no comprehensive theory that will allow for the predetermination of the sign an d magnitude of molecular optical activity.



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Polarimetry was the first chirality based method to be used as an analytical detector . It was, and sometimes still is, the method of choice for the determination of sucrose i n raw and refined forms, which led to the development of a singular instrument for tha t purpose, i .e . the saccharimeter [3] . Measurements are generally limited to detection a t just one wavelength, such as at the Na–D line, or at most a few wavelengths in th e emission spectrum of a Hg arc lamp . In post World War II years, optical and electroni c technologies had advanced to the point where high quality, full spectrum, ORD an d CD instruments became commercially available . When it was established that absolut e molecular configurations of chiral natural products could not be determined by eithe r method [4,5], an intense interest in their applicability to three dimensional structura l analyses of proteins [6] and carbohydrates [7] developed . These applications are still th e principal uses for CD data, especially from data measured in the IR and far UV spectra l ranges [8] . It is ironic that as early as 1963 Djerrasi [4] suggested that, because of thei r molecular specificity, ORD and CD should see development as analytical detectors . The anticipated development, however, is relatively recent having been motivated in part b y a need in the applied sciences to develop methods with which to determine enantiomeri c purities .

31 .3 . BASES BEHIND CHIROPTICAL DETECTOR S A beam of monochromatic linearly polarized light is the product of the coalition o f two in-phase circularly polarized components whose electric vectors rotate in opposit e angular directions . Passage of the beam through an achiral medium produces no angula r deviation from the direction or plane of incidence . When the propagating medium i s chiral, the existence of two different refractive indices causes one of the beams to b e retarded . The beams are no longer in phase, and, during transmission, an angular rotatio n is observed . The direction of the rotation is determined by the structural properties of th e medium . Since the basis of polarimetry is a difference in refractive indices, the detecto r is akin to refractometry for which the incident light is usually unpolarized . Polarimetri c detection, like RI, is sensitive to temperature changes and to the composition of th e solvent system . Analytical determinations are based upon measured rotations that are interpolate d in the correlation curve for the rotation angle versus the solution concentration for a standard reference material (SRM) . Concentrations, however, are generally quite hig h and sample pathlengths long . Because of the dispersion property, rotations are greate r at shorter wavelengths . All other things being equal, detection in the UV will enhanc e the limit of detection . This fact is quite generally exploited in chromatographic chiropti c detection systems . If it happens that the chiral analyte also absorbs in the UV, rotations associate d with the anomalous ORD are typically ten to several hundred times greater than th e values for plain curves, enhancing detection limits even more . Molar absorbances , however, are also greatest in the UV range, so the intensity of the transmitted ligh t is proportionately much lower, which introduces the need for brighter light sources . References pp . 1148—1149



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The property of an anomalous ORD dispersion that is related to the analyte solution concentration is its amplitude expressed as the difference between the positive an d negative rotation maxima (Fig . 31 . l b) . In order to calculate the amplitude, rotations must be measured at several wavelengths over the absorption band . Accurate determination s are complicated by the fact that ORD spectra are seldom simple and seldom complete , and anomalous dispersions lie on top of regular plain ORD curves, making baselin e correction particularly difficult . In early CD instruments the experimental variable measured was the ellipticity o f the transmitted polarized beam . Modern instruments measure the absorbance differenc e (DA) as a function of wavelength . CD spectropolarimetry therefore, is simply a modified form of absorbance spectrophotometry . It is most effective as a spectra l detector although, so far, in HPLC applications, it has most often been used as a single wavelength UV detector . The UV is the range of specialty because the ubiquitou s chromophores in chiral organics are the aromatic ring and the carbonyl functional group . All the experimental variables that affect an absorption measurement, such as pH an d solvent effects, also affect CD . Data analyses are no different . It is therefore rather eas y for anyone with experience in absorbance spectrophotometry to become acquainted wit h the experimental capabilities of CD . A general problem with all absorbance related U V detectors is the low intensity level of the transmitted light beam . For CD, the signal s are small to begin with, and all components that absorb, both chiral and achiral, wil l adversely affect the signal-to-noise (S/N) ratio of the CD signal . In other words, strong absorbers can be a serious interference to CD detection . In spite of the apparent structural constraints, CD detection is not limited t o only chiral molecules that are unsaturated . Molecules that satisfy just one of thes e prerequisites can sometimes be derivatized and meet both . Derivatizations have eve n been done for molecules that meet neither prerequisite . Complexing an achiral analyt e that absorbs with a host molecule that is chiral is quite commonplace now in C D detection . Reactions that introduce unsaturation or extend conjugation in a chiral analyt e are intended to shift CD bands into the near UV, e .g . for amino acids, peptides, an d carbohydrates, or even all the way into the visible range where interferences from stron g UV absorbers are minimized [9-11] . In the context of LC detection, these are more likely to be options for pre- or post-column derivatizations . Similarities between C D and absorbance extend, of course, to CD and fluorescence [12] and circularly polarize d luminescence [ 13] detection as well . Three prerequisites are essential for fluorescenc e detected circular dichroism (FDCD) detection . Seldom are all three properties inheren t to a single molecule, so one has to resort again to inducing the missing property by a derivatization reaction 114] . The added advantage of fluorescence-CD detection is th e extra sensitivity it brings to the differential measurement . Only polarimetry and CD are really practical as analytical detectors, whether use d directly or in HPLC . They are, in some ways, complementary . Polarimetry will detec t all chiral species while CD detection is more selective, being transparent to all chiral s that do not absorb . Either detector can have an advantage depending upon the particula r application . The lack of specificity in single wavelength polarimetric detection doe s imply that chromatographic separations must be complete . With multiple wavelength detection, discrimination among co-eluted species is possible, if their identities are



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known . Single wavelength CD detection is, in some applications, just an expensiv e polarimeter. There is less of a premium put on complete separations if CD is th e detector, but co-elution of CD active species should be prevented .

31 .4 . INSTRUMENTATION 31 .4.1 . General descriptions 31 .4.1 .1 . Polarimeters

Modern polarimeters do not differ significantly from the original designs used 15 0 years ago . Quality of the components, however, has improved . The instrument layou t (Fig . 31 .2) begins with a beam of unpolarized monochromatic light from the sourc e (LS) which is linearly polarized on its passage through a Rochon polarizing elemen t (P 1) . Rotation of the beam from the incident direction, as it is transmitted through a chiral sample (S), is measured by the angle a that the analyzer (P2) prism is rotated t o restore the null, or crossed polarizers, position . Replacement of Na-lamp sources by high radiance Hg or Xe arc lamps and lasers, and the addition of focusing optics are the majo r changes that have been made to the basic system . Both modifications were dictated by a desire to interrogate the chiral properties of solutions in the very small eluted volume s (1–20 µ.l) that are typical of narrow bore HPLC and capillary electrophoresis column s 115,16] . These applications invariably involve only single channel detection, so tota l separation is essential for accurate work . 31 .4.1 .2 . Spectropolarimeters

This is a generic name for ORD and CD instrumentation . Both are multichanne l instruments . Polarimetric measurements made at just the wavelengths of the principa l emission lines of the Hg arc lamp would constitute a rudimentary ORD instrument . ORD is not commercially available as a stand alone instrument . It is offered as a n accessory to CD instrumentation and an ORD detector for HPLC that operates over a relatively narrow wavelength range (60 nm) has been introduced by Jasco Inc . Extensions to the polarimeter layout that convert it for CD detection are a dispersio n monochromator (M), a linear polarizer (LP) an electro-optic modulator (EOM), o r Pockels cell, and a recorder (R) (Fig . 31 .3) . This is the layout introduced by Grosjea n and Legrand [ 17] and is still the method of choice for all CD instruments . A Rocho n a

1_4c LS

pi

S

P2

Fig . 31 .2 . Block diagram for a polarimeter . LS = unpolarized light source ; P1, P2 = polarizing elements ; S = sample.

References pp . 1148—114 9



Chapter 3 1

1140

LS

M

LP

EOM

S

PMT

LI

R

Fig . 31 .3 . Block diagram for the Grosjean–Legrand CD spectropolarimeter . LS = light source; M = monochromator ; LP = linear polarizer ; EOM = Pockels cell or electro-optic modulator; S = sample ; PMT = detector ; LI = lock-in amplifier; R = recorder. RCP and LCP are the right and left circularly polarized light beams created by the EOM .

prism produces the orthogonal linearly polarized beams, one of which is blocked fro m further propagation . The function of the EOM or Pockels cell is to generate and phase-separate the left (LCP) and right (RCP) circularly polarized beams . The beams are passed simultaneousl y through the sample medium (S) . Absorbance differences, AA = (A 1_cp — A RC )), are measured as a function of wavelength using a high-gain, low-noise photomultiplier tub e (PMT) as detector. The modulation frequency applied to the EOM is usually around 5 0 kHz, and the light exiting the device contains an ac component that is due to the CD . A lock-in on this frequency (LI) discriminates the CD signal from the dc signal du e to the large background absorbance . The threshold value for AA that will produce a n observable CD signal is on the order of 0 .0005 absorbance units, which is close to the noise specifications for most high quality absorption spectrophotometers . To minimiz e the effects of noise and enhance the S/N ratio, CD spectral data are accumulate d by signal averaging over several spectral scans . Data collection, therefore, is a time intensive process . This is not a limitation if CD is used for direct analysis of bul k systems, i .e . in a time-independent mode, but it is for dynamic LC—CD applications .

31 .4 .2. Modifications for chromatographic detection Important parameters to consider in modifying chiroptical detectors for use i n chromatography are first, the very small sample volumes that are involved, and second , the very short time intervals that separate consecutive peaks . Nowadays exceedingl y small volumes can be handled with relative ease and are no longer a major concern . CD signal intensities, like absorbances, correlate with solution concentrations . Expresse d as concentrations, limits of detection are not particularly impressive, say micromola r levels, but at volumes as little as microliters, the limits of detection are actually a t the picomole level and sometimes lower [12,15,16,18] . The engineering priorities are to develop the technology to focus the beam on such small targets while maintainin g the high level of radiance needed for chiroptic detection . Continuous wave lasers ar e an obvious place to start, but source noise and instability are problems to conten d with [181 . As an alternative, one can use pulsed lasers . Because of the added radiance , these are able to stretch the limit of detection to even lower levels . Laser sources are also limited in the number of their output wavelengths . Dye lasers offer the best,



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albeit still very narrow, ranges (ca . 60 nm). Current CD instruments, where the sourc e is laser illumination, really do operate at just a single wavelength . An option for multichannel LC–CD detection does, however, exist [19–211 . Stopped-flow accessorie s for commercial instruments are available which allow part of an eluted fraction to b e taken off-line into a micro-cell placed in the regular sample compartment where dat a are measured in the normal way . The method still requires rapid scanning capabilities . Repeated injections and multiple scans can be averaged to improve the quality of th e signal . On-line LC–CD spectral detectors, where 0& is again on the order of only 6 0 nm, have been described in the literature [19,22,23] . In 1998, Jasco International Co . produced the first off-line dedicated polarimetric detector, the OR-990 detector, and the first CD cum absorbance detector, the CD-1595 Chiral Detector, for HPLC . The CD detector operates in the 220–420 nm range with a 20-nm bandwidth . The illuminating source is a 150-W Hg–Xe lamp . Wavelength dispersion is done usin g a diffraction grating and monochromator . The CD detector doubles as an absorbanc e spectrophotometer. Injections are typically in the microgram range . Minimum detectabl e amounts are on the nanogram scale . The sensitivity of the CD detector is intermediate between polarimetric and absorbance detection . For a relatively simple molecule like flavonone [24], the detection limit is reported to be 200 times higher than the optica l rotation detector, but a factor of four lower than the absorbance detector. Eventually these devices may turn out to be the starting point for the developmen t of CD diode-array detectors . Adjusting the scanning speed for on-line, wide-spectrum , CD measurements is a formidable problem . A major reason for the problem is th e incongruity between the time it takes to accumulate CD data, even for just one spectra l `pass' using the very best currently available diode array technology, and the typica l dispersion time between chromatographic peaks . The situation may very well change a s faster electronic detection devices become available . 31 .4.3 . Calibrations 31 .4 .3 .1 . Instruments

Polarimeters are regularly calibrated against solutions of sucrose . By some chanc e of nature, specific rotations for sucrose solutions and crystalline quartz are equivalent . A typical detector, therefore, is made using two quartz wedges placed in direct contact , one or both of which can be displaced very precisely . Their lateral movement change s the thickness of the quartz traversed in a way that will ultimately compensate for th e rotation produced by the sucrose solution [3] . The calibration correlation is distanc e travelled versus rotational angle . It happens that rotatory dispersions are also equivalent , and are described quantitatively by the same polynomial equation . Therefore, the sam e SRM sucrose solution can be used to calibrate a polarimeter that operates at any othe r wavelength . A corollary of this is that in saccharimetry the source might just as well b e white light which, when it is used, does simplify detection and does improve accuracy . It is usually recommended that DA scales for CD detectors be calibrated at severa l wavelengths . If, however, an application is limited to a relatively narrow range, which i t References pp . 1148—1149



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is in LC detection, then calibration using an SRM that absorbs in that range is sufficient . LC—CD detection is, for the most part, used in the UV so the usual compounds, R-(+) and L-(—)-camphor sulfonic acids or their ammonium salts, D- or L-pantoylactone, and androsterone are satisfactory reference standards . 31 .4.3 .2 . Compounds

Chiroptical measurements on underivatized carbohydrates are regularly made i n the UV wavelength range in order to take advantage of the increased dispersion a t short wavelengths . All molecules are accessible to polarimetric detection, but C D detection is limited to only those molecules that contain the carbonyl-, amide-, N-alkyl , and carboxylate-functional groups [7] . Molar rotations are much greater for reducin g sugars and ketoses . Calibrations for solutions of reference standards for monomer s are sometimes complicated by equilibration reactions, e .g . between anomeric and ope n chain structural forms . Anomers of the same molecule rotate linearly polarized light i n opposite angular directions so, beginning with a pure form of either one, the observe d rotation changes with time until the value for the equilibrium mixture is reached . Calibration curves for equilibrium solutions are non-linear and subject to greater error s in analyses . Derivatization reactions that are used to obtain structural information on monome r linkages and stereochemical arrangements between adjacent groups, e .g . in acycli c polyols, can also be used for analytical calibrations and determinations . If the applicatio n involves LC then the derivatization is most conveniently done post-column . Derivatizing reagents are typically strong absorbers, for example dansyl chloride, which comple x with the sugar to induce anomalous rotations or CD activity, and aromatics wit h extended conjugations, for instance para-substituted benzoates and hydroxycinnamates , 9-anthroates, and polyacenes 18], chosen because their principal polarization axe s coincide with the long molecular axes . In reality, the principal role by far of carbohydrates in HPLC and capillary electophoresis, is not as an analyte, but as a stationary phase with the special application o f providing an avenue to measure enantiomeric purities of chiral molecules of particula r importance to the pharmaceutical and biotechnology industries .

31 .5 . APPLICATIONS Chiroptical detectors have been used for the analysis of samples both in bulk system s and after chromatographic separation . In chromatography, they have been used alon e and in series with non-selective detectors, such as RI and absorbance . The Jasco CD 1595 detector, mentioned earlier in the text, automatically measures CD and absorbanc e simultaneously. Methods are either direct or indirect . In indirect analyses, a chira l solvent or chiral solution is chosen as the mobile phase . The direction and magnitud e of the rotational change from the background value for the mobile phase on the elution of any component, even one that is inherently achiral, is used for its determination [25] . Analyte derivatization reactions, both pre- and post-column, have been added to enhanc e selectivity and sensitivity. Examples for all of these are included in what follows .



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31 .5.1 . Bulk system s Where supplies are obviously unlimited, commercial chiroptic devices have been used for the direct determination of chiral analytes in bulk solutions, and very ofte n without separation [9–11] . For polarimetry, sucrose in cane sugar is an obvious example . Polarimetric detection in bulk systems is not as common as CD, however, because o f the enhanced selectivity associated with the spectral properties of the latter . CD has been used to determine the ketoses D-fructose, D-tagatose, L-sorbose [26,27], as well a s turanose [28], D-ribose, and vitamin C [9,11], and the N-acetyl content of chitosan fro m crustacean shells [29] . In a rather unusual direct application, CD was used to determin e D-fructose in vitreous humor [26] .

31 .5 .2 . Bulk systems with induced chiralit y The function of derivatizing reagents is to provide the prosthetic group that is neede d for, or that simplifies, detection . For polarimetry, optical activity is typically induce d by complexing the analyte with a chiral host . For CD, either optical activity and/o r absorption (fluorescence) are induced by interaction with the appropriate auxiliar y reagent . Sometimes the degree of analytical selectivity one gets exceeds all expectations . A case in point is the L-tartrate enantiomeric analog of the racemic biuret reagent, a strongly basic solution of Cu(II)-D,L-tartrate, coupled with either CD or polarimetri c detection . The biuret test is a standard method for the measurement of total seru m protein . The first illustration of the selectivity capabilities of this reagent/CD detecto r combination was the undisputed identification of the four enantiomers of the ephedrin e diastereomers [9–1 11 . The concentration of the copper tartrate complex is kept i n large excess so that a 1 : 1 drug for tartrate ligand exchange is virtually complete . Prepared binary mixtures were analyzed for several enantiomeric ratios using bot h polarimetry and CD detection on bulk solutions at several wavelengths . Multivariat e regression models were derived using principal component analysis and partial leas t squares algorithms . Imprecisions in the training sets and for subsequent predictions o f enantiomeric ratios on a series of unknowns are on the order of ±0 .5% over the whol e range of composition . The significance of this result to the present chapter is that , by combining multichannel detection with multivariate regression, total and complet e chromatographic separations might no longer be an experimental necessity. Or th e method can be applied directly to overlapping peaks whether they are symmetrical or not . The simple substitution of L-tartrate with D-histidine extended the discriminatory power of the CD detector to a level that approaches analytical specificity for severa l series of analogous peptides and proteins 130–33] . With but one exception, the metho d discriminated among 53 di- and tripeptides [30j ; between the D- and L-enantiomeri c forms of penta- and hexapeptides ; among a series of 17 neuropeptides comprised of dynorphins, enkephalins, and miscellaneous others [31] ; and among human, huma n LysPro, porcine, and bovine insulins [32] . Visible range CD detection (the absorbanc e requirement originates in the colored Cu(II) complexes) coupled with chiral ligan d References pp. 1148—1149



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5 -

PLO 17 ; ICI 174,86 4

x DALDA

- -

D CTAP

-3 – Principal Component # 1 -4 I 1 -11 -6 -1

(9) (10) 4

9

Fig . 31 .4 . Cluster plot of the first and second principal components derived by PCA from spectral data . The 8-receptor cluster covers (1) DTLET; (2) DTLET ; (3) DADLE ; (4) a 2 -Leu 5 -enkephalin ; and (5) DPDPE . The it-receptor cluster includes (6) DAGO ; (7) Met 5 -enkephalin ; and (8) f-endorphin . The alternate 8 receptor cluster is composed of (9) Leu 5 -enkephalin ; and (10) Leu 5 -enkephalin amide . The K-cluster of th e dynorphins contains (11) B(1—13) ; (12) A(1—13) ; (/3) A(1—9) ; (14) A(1—11) ; and (15) A(1—13) amide . No receptor preference was reported for PLO 17 or ICI 174 864 .

exchange proved to be a straight forward quality control procedure for these dru g and other potential biotech drug systems 131] . Although there is no direct correlatio n between the spectroscopic differences and the thermodynamic stability difference s between enantiomeric metal complexes, there is still a good precedent in these result s to consider further investigations of chiral ligand exchange separations of metaboli c products, such as glucuronides, by HPLC and CE . In the continuation of [31] a quantitative structure—activity relationship (QSAR ) model was derived for the neuropeptides [331 that correlated receptor sites for know n drug substances with the CD spectral properties of the mixed chiral ligand complexes . From a principal component analysis treatment of the CD spectral data it was determine d that all spectra could be fitted using only five components . A two-dimensional cluste r analysis using only the first two principal components spatially separated drugs by thei r affinities for 8, ®, and K brain receptors (Fig . 31 .4) . Of more direct interest in the present context is the degree of specificity achieved i n the analysis of the aminoglycoside antibiotics (Fig . 31 .5) . The curves in Fig . 31 .5 are actually plotted as the differences between the spectrum for the reagent and the spectr a for the mixed Cu(II)—L-tartrate—antibiotic complexes . The drugs can be assayed in dru g formulations after extraction with little evidence of interference problems . While these analyses were done on bulk systems, derivatization with a chiral meta l complex is adaptable to chromatographic methods in a post-column reactor, if th e metal chosen is labile to ligand substitution . Chiral discrimination among the potentia l



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150 100 50

ellipticity(mdeg)

(a)

API

(b )

__du/4

¼

0 -5 0 (f)

-100 -150 400

wavelength(nm) ' ' 500 600

700

Fig . 31 .5 . CD spectra for : streptomycin (a) ; neomycin (b) ; amikacin (c) ; gentamycin (d) ; and kanamycin (e) , plotted as the difference from the spectrum for the parent complex (I) .

analyses is a consequence of the subtleties of the interactions between the competin g chiral ligands with the metal and with each other .

31 .5 .3. LC system s Excellent summaries of the many problems associated with the separation of carbohydrates by HPLC, their measurement using chiroptical detectors, and of LC-chiroptica l detection in the determination of enantiomeric purity, were given by Armstrong an d coworkers in two relatively recent review articles [34,351 . In the first of these, 3 4 different anomers were separated on cyclodextrin columns using a range of solven t mixtures . Monomeric sugars that do not undergo conversion between anomeric forms , such as aldoses substituted in the C-1 position, are the least of the problems . Like al l diastereoisomers, anomers have different retention times and the signs of the chiropti c properties are opposites . In the interconversion process, when it occurs, the initia l observed rotation for one of the anomers would be seen to diminish with time (mutarotation) until equilibrium is established . The equilibrium position is rarely a 50 : 5 0 mixture so there is a residual chiroptic signal . For samples in bulk, the signal correlates well with solution concentration . Analyse s that rely on separations are more difficult to perform and especially so when mutarotation half lives correspond closely with chromatographic resolution times . Dependin g upon the extent of the correspondence, separations may be complete, partially complete , or totally incomplete . With prior information on the kinetics of the mutarotation, colum n parameters such as flow rate, solvent composition, and temperature can be managed t o maximize the separation . It turns out that this is one instance where lowering the colum n temperature leads to greater separation efficiency . Although not specifically written for carbohydrates, the second article [35] cover s some of the more critical issues that must be considered in HPLC separations wher e commercial chiroptical detectors are used, either alone or in tandem with conventiona l detectors . The specific application described in the article is the determination o f References pp . 1148—1149



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enantiomeric ratios . The subject is relevant because some sugars occur naturally a s a mixture of the D- and L-forms, and the development of methods with which t o distinguish between the enantiomers is of analytical importance . So, the opinions expressed by the authors are pertinent to the role of chiroptic detectors in the chira l chromatographic separation and determination of carbohydrates . The first opinion was that the sensitivities of polarimetric and CD detectors with non-laser illumination are very limited, so relatively large samples have to be injected , requiring great care to avoid overloading the column, most especially chiral columns . The introduction of off-line commercial, dedicated, CD detectors for LC has take n care of a great part of this deficiency . Next, the linear range of the CD detector i s very narrow compared to absorbance . When absorbance and CD detectors are used i n series for enantiomeric ratio or purity determinations [18–21], the limiting detector i s CD . Whenever the mole fraction is greater than 0 .9 or less than 0 .1, as one hope s it would be in a test for enantiomeric purity, the absolute and relative errors becom e so large that the method was not recommended . Considerable improvements, however , have since been made and are discussed later. The authors also strongly recommende d that chiroptic devices, in general, should not be used as stand-alone detectors in LC fo r several reasons, e .g . peak areas for enantiomers do not always add up to give the tota l concentration ; tail-front aberrations in the chromatogram that are obvious in absorbance detection are absent in chiroptical detection, etc . What Armstrong et al . have done is to focus attention on the less obvious constraint s that will be there in LC detection even when the technology has advanced to th e point that the sensitivity of chiroptical detectors is competitive with other methods . The general consensus is that dual detectors are still the best solution for chiral analyte s 136-411 . Concepts that have evolved as answers to these experimental limitations that ar e capable of improving upon the accuracies of enantiomeric purity determinations ar e the g-factor [39–41] and principal component analysis (PCA) treatments 1401 of elute d band intensities as a function of time . These are especially useful in cases where bands are asymmetrical 1351 . The g-factor is defined as the ratio of the CD intensity to th e absorbance intensity, AA/A . One attribute of this factor is that for an enantiomericall y pure material the g-ratio does not change with concentration . A change in the g-facto r during the elution of a band is a clear indication of an enantiomeric impurity . Idea l traces documenting the total resolution of bands for two pure enantiomers in a racemi c mixture would consist of two horizontal lines with g-values of equal and opposite signs . Over the time interval between the eluting bands, the traces are separated by signal s that are excessively noisy, since calculated g-factors in these ranges where no CD-activ e species is being eluted, correspond to zero divided by zero . With PCA the number o f components that are co-eluted can be derived by reduction of a matrix of intensity vs . concentration vs . time data for a series of solutions with prepared concentrations [40] . In most reviews of LC detectors, chiroptic devices are regularly dismissed with onl y a cursory reference . The reiterated criticism is always the lack of sensitivity, a constrain t that is aggravated even more by the need to miniaturize sample volumes . For a lot of samples, and especially biological and clinical samples, a very small quantity is al l that is ever available, so the criticism is a valid one . At semi-micro levels, excellent



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sensitivity is achieved using a Xe-lamp, if the `cells' have a narrow bore and extra lon g pathlengths (25—50 mm) . Micro-cell designs were developed by Boehme [36] and b y Kuo and Yeung [37] . In the latter design, the device was used to identify and measure a series of urinary sugars . One answer to poor sensitivity is to convert to laser illumination , which has been described and critically evaluated for polarimetric applications [18] . Laser-induced fluorescence-CD was also developed for microbore chromatography [42 ] and subsequently for capillary electrophoresis [16] . Another laser-based option is the thermal lens detector [43] . Its use, however, is limited to the visible range and, as such , it is not applicable to the determination of underivatized carbohydrates . Lloyd et al . [15] in their development of a diode-laser LC instrument with polarimetric detection for a sample cell on the order of 8described the separation of the components of a glucose syrup solution under both isocratic and gradient elutio n conditions, and preliminary investigations in the analysis of visibly opaque sugar syrups , e .g . molasses . They point out an extra experimental concern when using gradient elutio n with polarimetric detection which is defocusing of the laser beam caused by the rapi d changes in the RI of the eluent . Ways to minimize the solvent effect were discusse d by Reitsma and Yeung [44] . Laser-based polarimetric detection linked with ion-pai r chromatography was used for the determination of the four analogs of gentamycin [45 ] and the three analogs of erythromycin [46] . The degree of selectivity is very impressiv e since the analogs differ structurally by only one substituent group . Mass detection limit s were reported to be 35 and 12 ng, respectively . Both antibiotics were also determine d in spiked milk samples after a simple work-up . Derivatization of the analyses was not a prerequisite . Following the conclusions and recommendations of Armstrong and Jin 134], couple d detectors, e .g . polarimetry or CD detection in tandem with RI or absorbance, respectively, are the most prevalent options . Aldoses, as a general rule, are determined b y HPLC using absorbance detection around 300 nm . But adding polarimetry does increas e the selectivity in the analysis of mixtures . For instance, gel permeation chromatograph y and reversed phase HPLC separations with RI and polarimetric detectors in series, wer e used very effectively in the characterization of amylodextrins, including cyclodextrins , and starch . For starch a method to determine the molecular weight distribution is also de scribed [47] . In another example, RI and polarimetry detection were used in series for th e analysis of HPLC separated methylated oligo- and polysaccharides, e .g . scleroglucan , amylose, and waxy-corn starch [48] . In the separation of the depolymerized hydrolyzate , fragments from dimer through tetramer are identified by the values of their specific rotations compared to that for an injected internal standard . Because each peak elutes a s a doublet, it is possible to determine the a/P-anomeric ratio . O-Anomers in general have lower rotations which enables their easy distinction from the a-forms . The authors clai m to have developed a sub-milligram method for determining the chirality of sugars .

31 .5 .4 . LC systems with induced chiralit y Structural and conformational information for carbohydrates in solution are deduce d from CD spectra measured in the UV and vacuum-UV ranges [7] . Data collection is an References pp. 1148—1149



Chapter 3 1

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extremely difficult experiment and interferences are so great that the range is unsuitabl e for chemical analysis . Para-benzoate, anthroate, and other derivatizing agents that absorb in the near UV have also been used for structural and conformational assignments i n the saccharides . Exciton coupling theories are used to interpret the spectra in terms o f linkage isomerisms and the local stereochemistry between neighboring groups [7,8] . These and similar derivatization reagents might just as easily be used for chemica l analysis . If derivatizations are done pre-column, the separation efficiencies might be affected significantly . Prospects are better for on-line post-column derivatizatio n reactors, see above . Derivatization of molecules for CD detection also occurs intramolecularly. For in stance, the union of chirality in the carbohydrate moiety of glycosides, with unsaturatio n in the base in such compounds as nucleosides and nucleotides, saponins and flavones , etc ., forms a basis from which the application of chiroptical detection methods wil l become very significant . The emergence of glycotechnology and the development o f methods for the manufacture of new drug substances, such as glycoproteins, glycolipids , proteoglycans, etc ., is another area which will have a major impact on pharmaceutic s in the very near future . In conjunction with these advances, analytical methods wil l be needed that are able to measure the optical (enantiomeric) purity of the new drug s both as standards and in formulations, and to discriminate them from their structurall y related analogs in quality-control applications . The laser-based polarimetric detection o f the erythromycins noted above 1461 is an instance in point, and with it the special valu e of a chiroptic detector is established .

31 .6 . CONCLUSIO N As chiroptic detector technology develops and selectivities and sensitivities ar e increased, multiple wavelength and even full spectral methods will become practica l realities . Add to this the power of multivariate regression analysis methods, and th e whole area of chemical analyses of chiral compounds by chromatographic methods wil l change significantly. The obvious success of the direct chemical analyses included i n the present discussion appears to signify that only partial separations will be sufficien t for analytical chromatography .

31 .7 . REFERENCE S 1. 2. 3. 4. 5. 6. 7.

A . Cotton, Compt . Rend ., 120 (1895) 98 T.M . Lowry, Optical Rotatory Power, Longmans Green, London, 193 5 T.M . Lowry, Optical Rotatory Power, Longmans Green, London, 1935, p . 193—19 8 C . Djerrasi, Optical Rotatory Dispersion and Circular Dichroism in Organic Chemistry, McGraw-Hill , New York, 1960 G . Schnatzke (Ed .), Optical Rotatory Dispersion and Circular Dichroism in Organic Chemistry , Heyden, London, 1967 B . Jirgensons, Optical Activity of Proteins and Other Macromolecules, Springer-Verlag, Berlin, 197 3 W.C . Johnson Jr ., Advances in Carbohydrate Chemistry, Vol . 45, Academic Press, New York, 1987, p. 73



Chiroptical Detectors for HPLC of Carbohydrates 8. 9. 10. 11.

12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48.

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