handbook of vibrational spectroscopy || thin layer chromatography and vibrational spectroscopy

Thin Layer Chromatography and VibrationalSpectroscopy

John M. ChalmersUniversity of Nottingham, UK

1 INTRODUCTION

Thin layer chromatography (TLC; planar chromatography)and high-performance thin layer chromatography (HPTLC)are widely used separation techniques for nonvolatile org-anic molecules. They have distinct advantages in simplic-ity, low cost, speed and relative ease of use, and highthroughput.1–11

It is not the purpose of this article to discuss in detailthe practices and merits of TLC nor the attributes andprinciples of the vibrational spectroscopy approaches usedto characterize TLC fractions. The latter are all coveredcomprehensively elsewhere within this Handbook, and theformer are covered by other references.1–10 Rather, we con-centrate on the synergy between the selectivity of TLC andspecificity of vibrational spectroscopy techniques, and par-ticularly on approaches for combining these two disciplinesto enhance analytical science investigations, their limita-tions and applications.

In TLC, the sample is carried by the mobile liquid phase(the solvent) through a stationary adsorbent, a finely dividedsolid, coated onto a support such as a glass, aluminum orplastic plate. Typically, these supports have dimensions of10 ð 20 cm or 20 ð 30 cm. Among the common adsorbentsare silica gel and alumina (both inorganic) and celluloseand polyamide (both organic). Stationary phases in whichthese supports are bonded with nonpolar materials are usedin reversed-phase TLC. The partitioning of solutes betweenthe mobile and stationary phases depends primarily on therelative strengths of their adsorption to the solid-phasesurface and their relative solubility in the mobile phase.

Samples in solution are spotted (deposited) towards oneedge of a TLC plate; this edge is then immersed in a sol-vent; capillary action then draws a solvent front upwardsthrough the adsorbent (Figure 1). Sample components movewith the solvent and migrate to different distances andare separated in a linear direction, according to their dif-ferential partitioning characteristics. Separated components(fractions) on the TLC plate may be visibly seen as col-ored spots or highlighted under a UV lamp, or throughchemical visualisation by reagent reaction.1–3,5 Spots aredifferentiated by Rf (retardation factor) values, which is theratio of the distance travelled by the solute to that travelledby the solvent front. If a reference compound has beenincluded with the sample, Rrel is the ratio of the distancetravelled by the solute to that travelled by the referencecompound. Identifications of commonly encountered sub-stances may be based simply on their Rf value under a setof standard experimental conditions; or a compound may beidentified by comparing its Rf value with those of standardsspotted alongside the sample on the same plate. However,neither of these proves identity unequivocally, and alter-native methods of structural fingerprinting are required,of which vibrational spectroscopy techniques are clearlycandidates.

2 TLC STATIONARY PHASES

Important TLC adsorbent properties are polarity, acid–basenature, adsorption character, chemical and thermal stabilityand colour.1–11 The particle size and layer thickness ofthe adsorbent are typically in the ranges 2–50 µm andabout 100–250 µm, respectively, for routine application.In HPTLC, the layers are prepared from particles of a

Handbook of Vibrational Spectroscopy, Online © 2006 John Wiley & Sons, Ltd.This article is © 2006 John Wiley & Sons, Ltd.This article was published in the Handbook of Vibrational Spectroscopy in 2006 by John Wiley & Sons, Ltd.DOI: 10.1002/9780470027325.s2907

1684 Hyphenated Techniques

TLC plate

Chromatographic tank

Solvent

Cover

“Start” − spotted sample

TLC SpotsSolvent front

Figure 1. Diagram of solute separation by TLC.

narrow size distribution and mean diameter between 5 and15 µm. Phosphor, or another fluorescent indicator, may beincorporated in the adsorbent. A sample (fraction) spot willquench this fluorescence so that it is detectable from thebackground when the plate is interrogated under a UV lamp.A small quantity of binder is also mixed with the adsorbentto improve adhesion to the plate (support). Common bindersare gypsum and starch.

To vibrational spectroscopists trying to identify low con-centrations of separated fractions as adsorbates, many ofthe stationary-phase properties are anathemas! The phasesand binders are strongly infrared absorbing, and manyexhibit strong Raman features; fluorescence, either intrinsicto the substrate or arising from added fluorescing agents,is clearly a major bugbear for Raman studies. The parti-cle size covers the range of mid-infrared wavelengths; thiscauses either significant scatter problems for transmissionmeasurement approaches, or can give rise to considerableintrusive specular reflection components in direct diffusereflection measurements (Section 3.2). Moreover, the spec-trum of a fraction will resemble that of a surface-bondedspecies and rarely be identical to that of a solid or solutionphase standard held in a library. Nevertheless, many publi-cations report feasibility studies and applications of in situidentification of TLC spots, by both infrared and Ramantechniques.

3 TLC COUPLED WITH MID-INFRAREDSPECTROSCOPY

Although many investigations have been concerned within situ measurements, for the reasons given above, manyinfrared developments have also targeted analyte identifi-cation after its removal from the TLC plate. For historicalreasons, we discuss the latter first in the next section,because these were the early approaches prior to the eraof modern Fourier transform infrared (FT-IR) and Ramansystems and FT-IR sampling procedures.

3.1 Spot removal and identification bymid-infrared spectroscopy, pre-FT-IR

One of the earliest (1965) reported sampling techniquesdescribed an approach for obtaining infrared spectrafrom 50–100 µg of compounds in solution in carbontetrachloride.12 The adsorbent containing the compoundwas scraped from the chromatogram and packed into adisposable Pasteur-type pipette. The compound was elutedthrough some glass wool to the tapered end of the pipettewith a few drops of a suitable eluent. The end of thecapillary was then cut off, the elution solvent evaporatedfrom the capillary, and the residue (analyte) redissolvedin a few microlitres of the infrared solvent (CCl4) andcentrifuged into a cavity type microcell for measurementof its infrared spectrum. Tricks of the trade involved:12

“rolling and tilting the capillary with the hands until thedroplet of solvent was near the cut end; watching the solventevaporate, then placing the cut end of the capillary in thecavity-cell filling hole; centrifuging briefly to transfer thesolution, and then plugging the cell with a drop of mercury”.An interesting procedure to try to get passed by today’ssafety auditors!

Procedures13–18 based on dispersing the separated frac-tion in KBr disc preparations were those most commonlyinvestigated from the late 1960s until the advent of mod-ern FT-IR spectrometers. Rice13 employed a direct transfertechnique. The TLC adsorbent around an outlined teardrop-shaped spot was removed from the plate and the glasssupport surrounding it cleaned carefully with a solvent.Powdered KBr was formed into an approximately 0.2 cmwide by 0.6 cm long line such that one end of the line wasin physical contact with the point of the teardrop. A solventwas then added dropwise at the blunt end of the teardrop,until the sample had eluted to the far end of the KBr line.About 6 mg of the far end of this KBr line was then formedinto a KBr micropellet and its IR spectrum recorded. Inanother approach, de Klein14 again removed the TLC adsor-bent support from around the spot and cleaned the glasssupport. A semicircular wall of finely divided KBr powderwas built around the tip of the spot but at a distance ofabout 1–2 mm away. Solvent was then added to the spotso as to concentrate the compound (solute) at the tip of thespot; solvent spillage and spread in the gap between the spotand the KBr were regulated so that the KBr sucked up thesolvent and, along with it, the compound. A 1.5-mm KBrdisc was then formed for infrared analysis. Recovery andcontamination were significant factors that were dependenton the TLC adsorbent.

These spot-removal practices required high patience andgood manipulative skills. Ingeniousness became routinelypracticable with the development and commercialization


Thin Layer Chromatography and Vibrational Spectroscopy 1685

Vial

Cap

Solution

Clip support

Wick-Stick™25 mm

Thickness ∼2 mm

8 mm

Figure 2. Diagram of a Wick-Stick.

of the Wick-Stick,15 a porous triangle of pressed KBr,of dimensions 2.5 cm high, 0.8 cm wide and 0.2 mm thick(Figure 2). Garner and Packer15 reported in 1968 a pro-cedure whereby a Wick-Stick was stood on adsorbentscraped from a TLC plate contained in a glass vial. Solventadded to the vial base climbed to the tip of the Wick-Stick, preferentially evaporating at its apex and depositingany nonvolatile solute at its tip. Then, 1–2 mm of theWick-Stick is subsequently broken off and prepared asa KBr micropellet for an infrared transmission examina-tion. At this time, satisfactory spectra for qualitative anal-ysis were obtained from about 10–50 µg of a sample ina 1.5 mm micro-KBR disc and beam-condensing optics.Wick-Sticks are still employed today for a variety ofapplications, including TLC spot identifications, and havebeen utilized in diffuse reflection procedures, see below.The Wick-Stick disc approach has also been used suc-cessfully to analyze TLC-separated low-solubility productswith a high-boiling solvent, such as 1-bromonaphthalene,heated to 170 °C.16

3.2 In situ measurement of TLC spots bymid-infrared spectroscopy

In situ infrared studies on TLC plates have includedtransmission,19–21 diffuse reflection,22–33 and photoacous-tic34,35 spectroscopy. In the mid-1970s, Griffiths andcolleagues19–21 used thin layers (about 100 µm thickness)of silica gel and alumina adsorbent on AgCl plates to facil-itate measurement of chromatographically sorbed materialsby transmission infrared spectroscopy. In a preliminarystudy,19 although low quantities (a few micrograms) ofcompounds could be detected, adsorbent scatter and absorp-tion severely limited the spectral window available (approx-imately 2200–1250 cm�1) for interrogation and was notviable as a reliable fingerprinting method. Improvementswere made to this feasibility study by minimizing scatterby post-treating the alumina TLC plate with an infrared

mulling oil, notably Fluorolube, a perfluorohydrocarbonoil, with a refractive index that closely matched that ofthe adsorbent.20 The samples in this study were chlorinatedpesticides. The IR detection limits were of the order of10 µg cm�2 for these relatively weakly absorbing samples.Differences between KBr disc spectra and spectra of com-pounds adsorbed onto alumina and silica gel were noted thatrelated to sorption mechanisms of the adsorbent. (In thisstudy,20 in situ Raman measurements were also attempted,but achieved much lower detection sensitivity than the FT-IR measurement.) Although detection levels were improvedto about 10 ng (methylene blue) by using programmedmultiple development,21 the desire for using routine com-mercial plates focused interest on diffuse reflection as apotentially more user friendly and convenient approach.

One of the first reported in situ diffuse reflection mea-surements was made from a commercially available alumi-num-foil-backed silica gel TLC plate. This facilitated easypunching out of the spots with a 6 mm diameter file punch,so that the sample could be located readily at the focus ofthe diffuse reflection measuring device.22 In this work anda follow-up publication,23 although microgram and submi-crogram levels of an adsorbate could be detected, both thespectral contrast and content were poor. The high attenua-tion of the infrared beam by the silica substrate precludedobservations over much of the spectral range. Also, thenature of the silica surface in the region of the spot wasaltered both by the presence of the adsorbate and from thesolvent travel, such that it was difficult to select an appro-priate background against which to ratio the single-beamsample spectrum. Frequency shifts and intensity changesresulted in noisy uneven baselines.

Reporting on in situ studies on nine different small size(2.5 ð 10 or 2.5 ð 7.5 cm) TLC plates, mounted for exam-ination in a commercial diffuse reflection accessory, Zuberet al.24 stressed the important requirement of obtaining arepresentative TLC blank, which matched the adsorbentat the sample spot as closely as possible. They accom-plished this by developing simultaneously a second plate,and recording a reference spectrum from this plate at anequivalent Rf value to that of the spot on the sample plate.This spectrum was then subtracted appropriately from thatof the analyte spot. Good quality spectra from pharmaceuti-cal substances were recorded from concentrations of about10 µg per spot. These spectra were significantly influencedby the adsorption characteristics of the TLC substrate, andwere different from the spot after its removal from the plateand analyzed in transmission as a KBr disc.

Not only do different substrates give rise to differingspectra, but also the problem may be exacerbated by othercomponents of the TLC layers. In a study using three silicagels, Bauer and Kovar25 showed that an interaction of acids



and bases with components (e.g. binders), of TLC layerscan lead to greatly altered spectra. They recommended theuse of a water-resistant silica gel for the analysis of sub-stances with acidic properties and/or acidic mobile phases,but standard layers with neutral binders when basic mobilephase components are employed. Thus, as pointed out forexample by Zuber et al.24 and others,26,27 unless a library ofTLC-infrared standards is available, one is generally limitedto generic classification rather than specific identification.The problem of the limitations of in situ identification ver-sus spot removal techniques is illustrated in Figures 3 and4.36 In Figure 3, much of the X–H stretching and fingerprintregions are heavily obscured by the cellulose TLC substrate,and subtraction techniques offer little reward in isolatinga useful spectrum characteristic of the adsorbate. (Brim-mer and Griffiths37 have highlighted some of the spec-tral differences that can appear in diffuse reflection spec-tra on strongly infrared absorbing substrates, and warnedagainst confusing features appearing as a consequence ofanomalous dispersion or reststrahlen bands with chemicalchanges in the analyte compound). Yet (in this case36) elu-tion of the spot from the plate to the tip of a Wick-Stick,which is then rubbed onto silicon carbide paper and exam-ined in diffuse reflection mode, yields a Kubelka–Munkspectrum over the range 4000–500 cm�1, characteristic ofthe polymer antioxidant (Figure 4). As seen in Figure 3, the

diffuse reflection spectrum recorded directly from a cellu-lose TLC plate essentially precludes observation of adsor-bate infrared absorption bands in the regions 3500–3200,1450–950 and below 600 cm�1. Nevertheless, an advantageof using cellulose in preference to other chromatographicthin layers is its relatively lower infrared absorption char-acteristics. This attribute was used in a qualitative andquantitative assessment of drug (narcotics) analysis overthe range 2–10 µg.28

If a generic library of TLC/FT-IR spectra is availablethen in situ spot identification becomes a more realisticmeans of identifying substances within a specific class.Also, automated interrogation of the TLC separation canrepresent a more efficient process. Glauniger et al.27 devel-oped a computer-controlled x–y stage for use with a diffusereflection unit and reported a feasibility study on the sep-aration of three benzodiazepines. During measurement, thex–y stage supporting the developed TLC plate is translateddiscontinuously at a certain rate beneath the diffuse reflec-tion optics in the direction the plate was developed, whileinterferograms are recorded and stored continuously. Typ-ically, a step movement of 200 µm with an interferogramregistration time of 3 s enables a co-addition of 25 singlescans at 8 cm�1 resolution per point of the chromatogram.In real time, simultaneous with display of recorded spec-tra, a Gram–Schmidt infrared chromatogram is generated

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Figure 3. Diffuse reflection spectra recorded from commercial cellulose TLC plate (Al foil backed) before (a) and after (b) spottingwith 20 µg of Irganox 1076. The phenolic –OH and carbonyl stretching bands of the Irganox 1076 are visible; however, the remainderof the spectrum is obscured. (Reproduced from reference 36 by kind permission of the Society of Applied Spectroscopy.)



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Figure 4. Diffuse reflection spectrum generated following removal of the TLC spot (of Figure 3b) and concentration of the Irganox1076 to the tip of a Wick-Stick by elution with solvent. (Reproduced from reference 36 by kind permission of the Society of AppliedSpectroscopy.)

and or functional group chromatograms reconstructed anddisplayed. Stationary measurements may, of course, alsobe employed. In these, the effect of anomalous dispersionwas minimized by aligning the diffuse reflection unit suchthat the intensity of the ca 1250 cm�1 reststrahlen bandof silica was low by ensuring its reflection maximum waslocated between 1255 and 1250 cm�1. This criterion (forthis case) was derived from an investigation comparingspectra recorded at alignments of maximum throughputwith those measured by progressive lowering of the sam-ple surface in a diffuse reflection unit, in which it wasnoted that as its intensity reduced its maximum movedtowards lower wavenumbers. Alignment for optimum spec-tral contrast of the sample with minimal interference fromTLC substrate effects can be difficult, because this positiondoes not necessarily coincide with a measured maximumthroughput in a FT-IR spectrometer. Specular (and dif-fuse specular) reflection contributions often dominate themeasured signal intensity! Stahlmann and Kovar29 reportdeveloping their own library of “TLC silica gel infraredspectra” of over 400 substances of forensic and pharma-ceutical interest. They searched against this library in astudy of impurities produced in chlordiazepoxide present intablet formulations subjected to stress tests (50 °C and 75%humidity). Again, an automated diffuse reflection assemblywas used, although two-dimensional TLC was employedto achieve higher analyte concentration spots, and lowerthe limit of detection, when impurities were present belowthe 0.3% level. Unequivocal identification of the impuritiesdemoxepam, nordazepam and aminochlorobenzophenoneare reported. The possibilities and limitations of assays fromTLC/FT-IR measurements have been reviewed and consid-ered by Frey et al.30

Complex mixture analysis may sometimes benefit fromdual separations followed by spectroscopic fingerprinting ofthe isolated fractions. High performance liquid chromatog-raphy (HPLC) has been coupled with TLC/FT-IR.26,31 TheHPLC effluent is transferred via a capillary tube onto thesurface of the TLC plate, which is mounted on a trans-lation table, driven in a linear direction. After the soluteshave been trapped on the TLC plate, and remaining HPLCsolvent driven off, the plate is then developed by elution ina direction perpendicular to the deposition direction.

By comparison with diffuse reflection, there have beenfew reported investigations on characterising TLC spots insitu by FT-IR photoacoustic spectroscopy (PAS). In 1982Lloyd et al.34 reported photoacoustic (PA) detection by bothmicrophonic and piezoelectric transducer (PZT) techniques.In much the same way as for some diffuse reflection inves-tigations, discs were excised from the TLC plate and thenplaced in the sample cup of a PAS cell. For the PZT detec-tion a transducer disc was attached with an epoxy cement tothe back of the TLC plate. In both approaches the signal-to-noise ratio was poor. Detection limits were some 70 timesworse than diffuse reflection measurements, even with atwo-fold increase in spectrum accumulation time.35 In theearly PAS study,34 a major source of interference was thedesorption of bound water from the TLC plate into thePA cell, requiring that TLC plates be left in an oven forseveral days to remove loosely bound water prior to theirFT-IR/PAS examination. Much improved mid-infrared PAspectra of adsorbates were reported 3 years later by White.35

In these, 70 scans at 8 cm�1 resolution were signal averagedfrom samples deposited from solution directly onto 0.32 cmdiameter silica gel wafers punched out from silica gel TLCplate. The prepared samples were purged in the PA cell



for 30 s with helium to remove CO2 and H2O interference.As with the previous PA measurements, a saturated PAresponse was observed in the region of the intense Si–O–Siabsorption region around 1000–1200 cm�1, and no spectralinformation relating to the analyte could be revealed inthis region. And, although good quality spectra of ¾50 µgof caffeine, acetylsalicylic acid and 4-hydroxy-acetanilinedeposited on silica gel were recorded, as with diffuse reflec-tion studies of adsorbates, these bore little resemblance toabsorbance spectra generated from a transmission measure-ment of the sample in a KBr disc. The author concluded,“due to interactions between adsorbed species and silicagel substrate, comparisons of TLC derived spectra withtransmittance spectra may not yield meaningful results. Adifference spectrum library of silica gel adsorbed substanceswould be more useful for reference purposes.”35

These problems with obscuring of key spectral regionsby TLC adsorbents and analytes yielding adsorbed speciesspectra rather than conventional transmission-like spectraled to the continued development and evaluation of alter-native strategies. These included: chromatogram transfer ofdeposits onto a substrate more amenable to infrared stud-ies, evaluating more “infrared-friendly” materials as TLCsubstrates, and the continued experimentation with FT-IRexaminations on solvent removal techniques of individualspots. These are discussed in the next sections.

3.3 Sample (chromatogram) transfer techniquesfor TLC/FT-IR

This strategy for overcoming the problems associatedwith in situ measurements off conventional TLC platesinvolves essentially orthogonal elution of the chromatogramdeveloped on a conventional TLC substrate onto an infraredtransparent substrate. In the method developed by Shaferet al.,38 a developed section of an aluminum-backed TLCplate was turned sideways and attached to an aluminumstrip in which a row of 1.2 mm wide ð 4.0 mm deep cupshad been drilled. Each cup was packed with a finelyground infrared-transmitting glass composed of germanium,antimony and selenium. In contact with the glass, at the baseof each hole was a bundle of glass fibers, which were alsopressed into intimate contact with the stationary phase of theTLC plate (Figure 5). This was then developed by solventelution in a direction perpendicular to the first development,so that TLC spots are transferred from the TLC plate ontothe ground glass in the cups. After evaporating off thesolvent, the spectrum of each eluate (spot) is measuredby diffuse reflection. (The Ge–Sb–Se glass was preferredover powdered KCl because it is nonhygroscopic.) Closecomparisons were achieved between a spectrum recordedof a spot by this method and its spectrum recorded by

Chromatoplate

Metal block Glass fibers

Diffuse reflectance powder

Diffuse reflectance medium

Wicks to contact chromatoplate

(a)

(b)

Figure 5. Diagrams of an apparatus for transferring (eluting) aTLC chromatogram from a developed TLC plate onto a mediummore suited to infrared diffuse reflection measurement. (Figuresreproduced from, (a) reference 38 with kind permission fromAnalytical Chemistry, and (b) reference 72 with kind permissionof Spectroscopy magazine, Advanstar Communications, Inc.)

a conventional transmission technique, see example inFigure 6. (This procedure was developed into a commercialaccessory, Section 6.) An extensive study of coal extractsusing this approach has been reported.39

To minimize the risk of contamination associated withzone removal from a TLC plate, workers at the ShimadzuCorporation40–42 made use of a new commercially avail-able TLC plate, the Empore TLC sheet, in developinga TLC/FT-IR accessory. This had the advantage that theadsorbent is self-supporting and does not require a backingsuch as glass or aluminum. Consequently, zones developedon this sheet may be eluted directly into a thin dried, groundKBR overlayer coated onto the developed plate (Figure 7).The assembly is then scanned automatically underneath thefocus of a diffuse reflection unit for FT-IR measurements.Figure 8 compares the ultraviolet chromatogram beforetransfer with a Gram–Schmidt reconstructed infrared chro-matogram after transfer. This shows that there is little losssuffered of chromatographic resolution on transfer. In theirpublication40 the authors show results from a separatedstandard mixture, which contained 15 µg each of anhydrouscaffeine, phenacetin and noscapine. Figure 9 compares aspectrum of anhydrous caffeine recorded in situ on the TLCplate with that recorded in diffuse reflection from the spotafter transfer to the KBr overlayer. Also shown in Figure 9is a diffuse reflection spectrum recorded directly from stan-dard anhydrous caffeine. The problems of in situ and theadvantages of adsorbate transfer measurements are clearlyevident. The approach is adaptable to thin layer (or paper)chromatographic media which do not have an impermeablesupport such as glass or aluminum.



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Figure 6. (a) Diffuse reflection spectrum recorded from 1,4-dihydroxyanthraquinone on sample transfer accessory after elutionfrom 40 µg spotted onto a silica gel TLC plate. (b) Referencespectrum obtained from chloroform solution in a sealed cell.(Reproduced from reference 38 by kind permission of AnalyticalChemistry.)

3.4 Alternative stationary phases formid-infrared FT-IR/TLC studies

With spot removal or transfer methods there is alwaysan increased risk of contamination, and irreproducibilityof results. To allow for improved in situ analyses, zirco-nium oxide, ZrO2, has been investigated as a chromato-graphic material for thin-layer separation.43 The reagentwas obtained as crystalline, monoclinic zirconia micro-spheres, approximately 5–10 µm in diameter with a surfacearea of 25 m2 g�1. A water slurry was prepared and spreadover a microscope slide, which was then dried. Diffusereflection spectra were then recorded from the developedplate. Zirconia has relatively much lower absorption overthe range 4000–1100 cm�1 than either silica or alumina,and so greater coverage of the mid-infrared region is avail-able for observing adsorbate bands. Also, it was observedthat greater influences concerning structural features could

TLC sheet

TLC sheet

Sheet holder

Sheet holder

Fixing screw

Transfer solvent

KBr powder layer

Sheet holder

Sheet holder

Base plate

(a)

(b)

Figure 7. TLC fraction zone transfer technique; (a) sheet holdersfor the TLC sheets: (b) cross-section of the accessory to showthe transfer process. (Reproduced from reference 41 by kindpermission of Analytical Sciences.)

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Figure 8. Comparison of chromatograms generated from TLCseparation of a mixture of anhydrous caffeine (A), phenacetin (B),and narcotine (C). The upper trace is the ultraviolet chromatogrambefore transfer, whereas the lower trace shows the infrared recon-structed chromatogram after zone transfer using the apparatusshown in Figure 7. (Reproduced from reference 41 by kind per-mission of Analytical Sciences.)

be drawn from spectra of a spot on zirconia than couldbe with the same material on either silica or alumina.Although zirconia exhibited a greater affinity for retainingacidic groups than silica, no sacrifice of chromatographicresolution was observed.43,44

The potential for using zirconia to circumvent the strongbackground stationary phase interferences from more con-ventional TLC substrates led to the development of a newmicrochannel TLC technique.44 Zirconia-packed grooves



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Figure 9. FT-IR diffuse reflection spectra of anhydrous caffeine:(a) in situ recorded from fraction separated on an Empore TLCsheet; (b) from the TLC fraction after its transfer from the TLCsheet to KBr powder; and (c) from a standard reference sample.(Reproduced from reference 40 by kind permission of the Societyof Applied Spectroscopy.)

with dimensions 400 µm ð 200 µm ð 5 cm in a brass plateserved as the chromatoplate, from which, when a chro-matogram had been developed, in situ diffuse reflectionspectra were recorded. Figure 10 compares transmission

spectra of four materials with their diffuse reflection spectrarecorded from the compounds after their separation ona microchannel zirconia TLC plate. Within this set, thespectra of the fractions are clearly distinct and can bealigned with their transmission spectrum counterpart.

Other heavy metal oxides such as titania, TiO2, offersimilar possibilities for this type of approach and have beenevaluated but not (to our knowledge) openly reported.

3.5 Spot removal and identification bymid-infrared FT-IR spectroscopy

As indicated in Section 3.1, eluting of spots post devel-opment of a TLC plate to the tip of a Wick-Stick orequivalent is still much practised today. The concentratedeluate may then be examined either in transmission as aprepared micro-KBr disc or in a diffuse reflection measure-ment. A similar alternative approach proposed the use ofalkali-halide pellets made by compressing 0.7 g of driedKCl powder in a 13 mm die.45 These were placed over anexcised TLC spot sited at the bottom of a flat-bottomedglass tube, and solvent used to elute the analyte fromthe TLC plate into the KCl pellet, in a manner analo-gous to a Wick-Stick preparation procedure. The pelletwas then placed in the cup of a diffuse reflection unitfor infrared examination. In a study of additives extractedfrom a polypropylene and subsequently separated on asilica gel TLC plate, it was observed that the intensityof the diffuse reflectance spectrum on KCl of one ofthe additives (Topanol OC) decreased with time, disap-pearing completely after about 10 min. This highlights alimitation of spot removal and transfer techniques – frac-tions are bound to TLC plates, but in this case the addi-tive, having a high vapor pressure at room temperature,volatilized from the KCl! Readily identifiable spectra ofthe polymer additives after extraction were achieved atthe 40 µg level. Iwaoka et al.46 reported higher sensitiv-ities (for a vitamin E derivative) through use of both“Gothic roof shaped” pyramids and microtriangles of KBrfor TLC spot identifications. These were attached to theside of a developed TLC plate, and orthogonal elutionused to transfer the separated fractions to them. They werethen formed into semimicro KBr pellets for transmissionmeasurements.

Chen and Smart47 adopted a very different strategy byinvestigating FT-IR characterization of the sample afterthermal desorption from the developed TLC plate. Aseparated TLC spot is scraped off from the plate andloaded into the sample pan of a thermogravimetric analyzer(TGA)/FT-IR system. A detection limit of 0.8 µg wasreported for methyl benzoate on silica gel. Clearly, ifthis procedure were routinely practicable, it would have



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a significant advantage in identification through librarymatching, since gas phase spectra are generated, and thereis absence of interferences from a solid substrate or matrix.However, the method is only appropriate to thermally stablesubstances that can be desorbed from thermally stable TLCstationary phases.

4 TLC COUPLED WITHNEAR-INFRARED SPECTROSCOPY

Although absorption bands of compounds are relativelyweak in the near-infrared region, consisting of essentiallyovertone or combination modes involving X–H fundamen-tals in the mid-infrared range, direct measurement of spotson TLC plates has potential in that adsorbents such as sil-ica gel have no strong absorptions in this region. A FT-IRspectrometer fitted with an InSb detector and coupled to amicroscope has been used in the range 7200–4000 cm�1 tointerrogate a silica gel plate thin-layer separation of threephospholipids.48 The phospholipids were chosen becausethey have essentially no absorption bands in the UV or vis-ible regions, and would require derivatization for properdetection. Spectra were recorded directly from the platein a diffuse transmission-like mode. Figure 11 shows anear-infrared chromatogram constructed from the integratedabsorbance signal between 4600 and 4000 cm�1. Good cal-ibration (densitometry) curves were generated for the threephospholipids using both absorbance and second-derivativedata; the linearity improved with the latter approach, anda detection limit of 1 µg was estimated. For 10 depositionsand developments of a 5 µg spot, a within-plate variation of

8

PCPE

PA

Scanning distance (mm)16 240

1

2

3

Inte

grat

ed a

bsor

banc

e

4

5

6

Figure 11. Near-IR chromatogram of separated phospholipidson an HPTLC plate. The amount of each phospholipid was10 µg. The plate was scanned at 1.6 mm min�1. The inte-grated absorbance signals between 4600 and 4000 cm�1 wereacquired and smoothed twice. (PC D phosphatidylcholine, PE Dphosphatidylethanolamine, PA D phosphatidic acid). (Reproducedfrom reference 48 by kind permission of the Society of AppliedSpectroscopy.)

about 2.5% and an interplate variation of about 3.5% wereassayed. This was reported as being better than previouslyreported charring methods.

Fong and Hieftje49 constructed a near-IR filter photome-ter designed to examine in a transmission mode smallportions of TLC plates. In this feasibility study a cooled PbSdetector (2 mm ð 2 mm element) was placed about 4 mmbeneath the plate to maximize collection of the diffuselytransmitted radiation. Although the near-infrared densito-meter set-up was suboptimal, detection limits of less than1 µg of selected sugar samples were demonstrated. The pho-tometer comprised 18 near-infrared bandpass filters. Thetransmitted intensity of one was used for normalization,whereas the signal from the remaining 17 were utilized ina PLS calibration.

The use of near-infrared spectroscopy as a detectiontechnique for TLC has been the subject of two feasibil-ity articles by Ciurczak and colleagues, one on quantita-tive measurements50 and one on qualitative applications.51

Mustillo and Ciurczak have also reviewed the developmentand role of near-infrared detection in TLC.52 In the densit-ometry work, a fibre optic bundle attachment was used toreduce the illuminated area and thereby enhance sensitivity.Although no advantage was gained in the use of derivativespectroscopy for the quantitative measurements, second-derivative spectra were used in the qualitative application.

5 TLC COUPLED WITH RAMANSPECTROSCOPY

Raman studies have focused almost exclusively on in situmeasurements. An advantage of Raman over infrared is thatin general a wider wavenumber range may be investigatedin a measurement, as there is not the cut-off near 400 cm�1

experienced with mid-infrared measurements. (Also, silicagel and water are weak Raman scatterers.) Success dependson factors such as sample scattering strength, concentra-tion, stability, fluorescence, and polarity and backgroundscattering/fluorescence from the TLC substrate. Two majorproblems for Raman investigations are spot spreading andfluorescence. There is a mismatch between a typical TLCspot size and the diameter of the excitation laser beam atthe sample point focus. As a consequence only a smallamount of an eluted spot is sampled. Many of the reportedfeasibility studies deal with spotted compounds (i.e. directdeposition), where the spot diameter may typically be afew millimeters, rather than chromatographically developedplates, where spread may be very much larger! In onestudy, low-concentration TLC spots were enriched up to100-fold for a micro-Raman study.53 Postchromatography,



opposing solvent elution was effected either side of a TLCspot, reducing its area typically to 0.05–0.3 mm2.

To circumvent or minimize fluorescence effects, Fouriertransform (FT)-Raman has been employed. Anotherstrategy, and one for which there are collectively manymore publications, makes use of resonance Ramanand/or surface enhancement approaches (both surfaceenhanced Raman spectroscopy (SERS) and surfaceenhanced resonance Raman spectroscopy (SERRS)) withvisible lasers and dispersive spectrometers to increase theassociated Raman intensities to well above that of thefluorescence interference.

5.1 In situ identification of TLC fractions byFT-Raman spectroscopy

Key advantages of using near-infrared laser excited FT-Raman spectrometers for in situ Raman analysis of TLCplates are avoidance of both fluorescence and laser-inducedsample damage.54 Figure 12 compares the FT-Raman spec-tra of a silica gel, a cellulose and a polyamide-11 TLCplates. An alumina plate, however, gave intense backgroundfluorescence. By contrast to infrared, it is evident from itsweaker background that the silica-gel plate is the most suit-able substrate. In a practical demonstration Everall et al.54

eluted a mixture of 200 µg each of three polymer addi-tives, erucamide, Irganox 1010 and Topanol OC. The spotswere visualized by iodine staining. Although the mixturewas deposited into a spot a few millimeters in diame-ter, after elution the erucamide spot was about 25 mm2 inarea, the Irganox 1010 occupied about 75 mm2, whereas

1998.5 1598.5

(c)

(b)

(a)

1198.5

Raman shift /cm−1

Ram

an in

tens

ity

798.5 398.5

Figure 12. Comparison of the FT-Raman spectra of differentTLC plates: (a) silica gel, (b) cellulose, and (c) polyamide 11.(Reproduced from reference 54 by kind permission of the Societyof Applied Spectroscopy.)

1802.5

(b)

(a)

1402.5 1002.5

Raman shift /cm−1

Ram

an in

tens

ity

202.5602.5

Figure 13. FT-Raman spectra of TLC fractions eluted from amixture of erucamide, Topanol OC, and Irganox 1010. Onlyerucamide (a) and Irganox 1010 (b) gave recognizable spectra atthe loadings and spot sizes. (Reproduced from reference 54 bykind permission of the Society of Applied Spectroscopy.)

the Topanol OC was spread over more then 200 mm2. Thespots were interrogated using a back-scattering Raman con-figuration, with a laser-beam focus diameter at the sampleof about 1 mm. The spectra of Figure 13 were recordedwith a laser (Nd : YAG) power of 2.5 W. Under these con-ditions, the Topanol OC was not detectable, presumablybecause of the extent of sample spread. (In the discussionof Section 3.5, Topanol OC was noted to exhibit a highvapor pressure at room temperature. It may just be that thisproperty led also to some evaporation of the material, if itbecame less tightly adsorbed on the TLC substrate becauseof warming by the excitation laser beam!) None of the spotsgave acceptable Raman spectra using 514.5 nm excitationfrom an argon-ion laser and dispersive spectrometer sys-tem, owing to strong background fluorescence; they were afaint yellow colour, possibly from residual iodine from thestaining process. The backscattering geometry employedafforded a 4-µm diameter laser beam spot at the sample.As with infrared studies, when compared to conventionalsolid state spectra some differing relative band intensi-ties, band broadening and shifting was apparent, due toadsorbate–adsorbent interaction. From direct depositions,an identification limit of 10 µg per square millimeter wasestimated.

In another study, Petty and Cahoon,55 analyzing elutedpharmaceutical compounds highlighted the necessity forsubtracting from a spectrum recorded from a TLC spotthat of a background spectrum recorded from a bare areaof the TLC plate after chromatography. Deresolved spec-tra derived from higher concentration spots were then



800

0

2500

5000

(c)

(b)

(a)

1100 1400

Raman shift /cm−1

1700

Ram

an in

tens

ity (

arbi

trar

y un

its)

Figure 14. Micro-FT-SERS spectra recorded from TLC spotsfrom 5 µl solutions of crocetin at differing concentrations spottedonto the silica gel TLC plate: (spectrum a) 10�3 M, (b) 10�5 M,and (c) 10�6 M. (Reproduced from reference 57 by kind permis-sion of the Spectrochimica Acta.)

subjected to library searching using different search algo-rithms against a library of 400 FT-Raman spectra at16 cm�1 resolution. They ascertained that the residual base-line effects were more significant than noise in affecting thesearch results.

For weak scatterers the detection limits by FT-Ramanspectroscopy may be in the high-microgram range.54,56

Rau56 reported on the feasibility of in situ measurementsof spots from silica gel TLC plates by both FT-Raman andFT-SERS (see Section 5.3). The potential for rapidly finger-printing pharmaceutical products at the 40 µg or less levelwas demonstrated with FT-Raman. Caudin et al.57 havereported measurements using FT-Raman microspectroscopyon a strong scatterer, the model biological molecule all-trans-crocetin. With a spot size of 7 mm and a laser-beamwaist of 8 µm, they calculated the mass detection limitunder illumination (analyzed mass) for the microprobe FT-SERS was 0.02 fg (Figure 14 spectrum b). However, forFT-Raman they calculated a mass detection limit of ca0.02 pg (although the latter exhibited for the strongest bandat 1540 cm�1, a signal-to-noise ratio of only about 2 : 1to 3 : 1!).

5.2 TLC resonance Raman spectroscopy

Nanogram levels of metalloporphyrins spotted onto TLCplates have been characterised on dried silica gel TLCplates by resonance Raman spectroscopy.58 Sensitivity wasagain very dependent on spot dispersion. An unfocusedlaser beam was used in a backscattering geometry to min-imize photodecomposition, and the plate skewed at anangle of 3–10° perpendicular to the laser beam, to min-imize collection of reflected light and diminish backgroundscatter.

5.3 TLC/SERS

Tran in 198459 reported on the in situ identification ofpaper chromatogram spots by surface enhanced Ramanscattering. The dried, developed paper chromatogram wassprayed to wetness with a silver colloidal hydrosol. Ramanspectra were then recorded from the wet chromatogram.Submicrogram quantities of three separated dyes weredetected and identified using 4 mW of an unfocused514.5 nm laser line. The SERS spectra of the dyes wereof good signal-to-noise ratio and relatively free from back-ground scatter. The SERS intensity was found to be depen-dent on chromatography paper type, and related to thecompetition between paper–dye and dye–silver interac-tions. No Raman scattering was observed from the spotsprior to spraying with the silver colloidal hydrosol.

After drying fluorescent indicator free HPTLC plates,Koglin60 sprayed to wetness the TLC plates with silvercolloidal solutions by a spray atomizer. Low laser power(10 mW) 514.5 nm radiation at the spot yielded character-istic SERS spectra from analytes (benzoic acid, purine,nitropyrene) as 2–4 mm diameter (20–30 ng) activatedspots deposited onto silica gel plates. A micro-SERS/TLCspectrum was also reported, suggesting picogram, or less,quantities were detectable. This approach has been usedto analyze developed chromatograms from the separa-tion of nucleic acid derivatives.61 Significant differenceswere observed between TLC/SERS spectra compared withnormal Raman spectra of molecules in aqueous solu-tion. Relative intensities changed, some band shifts greaterthan 10 cm�1 were noted,60,61 and some band broadeningoccurred. The position of the ring-breathing mode in theregion 600–750 cm�1 was highlighted as being a diagnosticof the methylation sites for guanine and adenine derivatives.

The influence of the supporting matrix (TLC adsorbentin this case) and the protocol of hydrosol preparation canhave profound effects on the both the intensity and stabil-ity of SERS signals.62 Soper and Kuwana62 constructed aremote-sensing Raman spectrometer to study these effects.Optical fibers were used both to carry the excitation light(514.5 nm) to the TLC plate and to collect the Raman-scattered radiation. Sodium borohydride and sodium citratesols were prepared by the methods of Creighton et al.63

and Lee and Miesel,64 respectively. The model compoundstudied was the dye pararosaniline, which was depositedmixed with the Ag colloidal hydrosol onto conventionalTLC plates. In a preliminary experiment, the most intenseRaman bands for pararosaniline were observed with thecitrate sol preparation with the highest concentration Agconcentration (0.18 g L�1). This sol exhibits large electronicabsorption at the wavelength of the excitation radiation, anda partial degree of aggregation. This sol was then used in



a study to maximize the SERS signals from various typesof TLC plates. Of those examined, enhanced SERS inten-sities were observed with an aluminum oxide TLC plate,possibly attributed to a pH effect. Adsorption of the dyewould be facilitated in this case by the alkalinity of the alu-minum oxide, in contrast to silica, which would inhibit theadsorption of the dye onto the sol. Stability of the SERS sig-nal was observed to be greater for the (hydrophilic nature)aluminum oxide TLC plate compared with a (hydropho-bic nature) reversed-phase TLC plate. For the aluminumoxide plate, SERS signals increased immediately (for about1 min) after deposition of the Ag–dye complex, and thenfell sharply after about 5 min, followed essentially by aslow decay. The rate of these losses, of evaporation andphotodecomposition, respectively, were greater for higherlaser powers. The maximum SERS intensity measured inthis study yielded a detection limit of about 180 fmol (33 pg)of dye deposited onto the TLC plate.

Silver colloidal SERS activated nano-HPTLC plateshave been combined with micro-Raman investigationsof molecules of biological, environmental and organicinterest,65,66 (Figure 15). An implication again in thesestudies was the influence on TLC/SERS intensities of thepolarity and hydrophilic nature of the silver-activated TLCplates.65

Compared with the potential for rapidly fingerprintingpharmaceutical products at the 40 µg or less level withFT-Raman (Section 5.1), Rau56 also demonstrated submi-crogram identifications for some dyes by FT-SERS. In

Wavenumber / cm−1

400.00 600.00 800.00 1000.00 1200.00

364.

00

405.

60 840.

00

1125

.6

1232

.0

716.

00

995.

20

Inte

nsity

Figure 15. SERS/HPTLC microprobe analysis of dibenzofuran:10 mW, 632 nm laser spot focus of 1 µm; 50 pg of dibenzofuran onMerck HPTLC silica gel 60 plate, activated with Ag (Creightonsolution). (Reproduced from reference 65 by kind permission ofthe Journal of Planar Chromatography.)

the FT-Raman microspectroscopy comparison by Caudinet al.,57 the calculated mass detection limit for the SERSmeasurement was 0.02 fg (Figure 14 spectrum b).

Three possibilities for SERS activation of TLC platesare:67 post-activation of the developed chromatogram by,for example, spraying the plate with Ag colloidal solutions(e.g. reference65), simultaneous activation of the plate withspotting of the sample by, for example, dissolving thesample in Ag colloid,62,68 or the pre-activation of TLCplates.67 Matejka et al.67 used the latter approach in anear-infrared excitation FT-SERS study of heterocyclic andaromatic species spotted onto TLC plates. Silver particleswere generated on TLC plates by first dipping them intoa saturated solution of silver oxalate. The plate was thenplaced into an oven to decompose (pyrolyze) the adsorbedoxalate. Commercial plates coated with an F254 dye werefound to be unsuitable for near-infrared FT-SERS, becausethey produced highly fluorescent backgrounds, even if theexcitation wavelength was in the near-infrared. In the casestudied here of 2,20-biypyridene adsorption, a much greaterintensity and higher signal-to-noise ratio spectrum wasrecorded from a large-pore silica-gel sorbent than withplates coated with an aluminum oxide neutral sorbent.As with other investigations, spectra again resembledchemisorbed species.

Finally, the Hadamard transform technique has also beenemployed for the imaging of SERS/TLC spots.68,69

5.4 TLC/SERRS

In the previous section, it was mentioned that Soper andKuwana62 from their investigations reported that the mostintense SERS signals occurred when an electronic absorp-tion of the hydrosol coincided with or overlapped with thatof the excitation wavelength of the laser radiation. This sit-uation has the potential to produce the SERRS effect. In afollow-up publication, Soper et al.70 investigated the opti-mization of parameters for using their set-up for couplingLC to TLC/SERRS for the LC analyte detection.

6 COMMERCIAL SYSTEMS

Several commercial TLC/vibrational spectroscopy inter-faces have been developed, although not all remain mar-keted. These include an FT-IR zone transfer system40–42

developed by the Shimadzu Corporation, Japan, that wasoutlined in Section 3.3. Another transfer system,71–73 re-fined from the development work of Shafer et al.,38 becamethe CHROMALECT marketed by Analect Instruments,Irvine, CA, USA (Figure 16). The cup holder, which



BA

C

A

C

B

C

D

FE

A

B

(a)

(c)

(b)

Figure 16. Diagrams of the CHROMALECT TLC/FT-IR interface. (a) Optitrain body (A) containing a row of sample cups (B)to hold separated components; the TLC plate (C) is attached to the underside prior to sample transfer. (b) End view, the samplecomponents move up the TLC plate (A) and through the Optitrain’s wicks (B) to the surface of the infrared transparent powder (C).(c) Transfer assembly: the Optitrain (A), the solvent injection syringe (B), the air knife (C), airflow over the sample cups (D), solventoverflow relief (E), and connections to water bath circulation (F). (Reproduced from reference 73 by kind permission of John Wiley& Sons, Inc.)

Figure 17. Bruker TLC Raman stage. (Reproduced from refer-ence 74 by kind permission of Bruker Analytik GmbH.)

is stepper-motor driven so that after transfer individualcups may be translated into the sampling position ofthe diffuse reflection sampler, is called an Optitrain.71–73

An accessory based upon that described by Glauningeret al.27 has been developed by Bruker Analytik GmbH,for both FT-IR diffuse reflection and FT-Raman74,75

applications. The infrared accessory has been used in arecent chemometrics FT-IR approach for rapid screeningfor metabolite overproduction.76 Figure 17 shows a pictureof the Bruker GmbH TLC Raman stage.74

7 APPLICATIONS

Although the majority of papers referenced in the discus-sions above, particularly those concerned with in situ mea-surements, are mostly concerned with feasibility studies andmany are from deposited spots rather than chromatographi-cally developed TLC plates, some contain specific illustra-tions of applications. Some other applications may be foundin the following: TLC/infrared analysis of water, wastewater and sludge;77 TLC/FT-IR of drugs;78–80 TLC/FT-IRof sodium edetate78 and edetic acid in water.81



8 CLOSING SUMMARY

The list of references is evidence of the keen interestin developing methods to couple the selectivity of TLCto the specificity of vibrational spectroscopy techniques.Many of the references cover investigations undertaken inrecent years, indeed in 1999 Stahlmann82 published a Ten-Year Report on HPTLC/FT-IR Online Coupling. A widerange and variability of applications are discussed in thisreview,82 covering the fields of pharmaceutical, biological,forensic and environmental analysis.

There is no doubt that FT-IR and enhanced Raman tech-niques have more than adequate sensitivity for many TLCspot identifications discussed in this article. However, theirdetection sensitivity generally is much less than that ofother more conventional detection methods, such as ultra-violet–visible, and HPTLC approaches may have detectionsensitivities in the low-nanogram range.11 Spot removalapproaches enable conventional spectra to be recorded read-ily, although the process is less efficient. However, in situspectra on TLC spectra will always resemble more thatof a chemisorbed species than commonly held referencestandards in commercial libraries of condensed or solutionphase compounds. A main problem for in situ Raman lieswith minimizing spread or concentrating the adsorbate toa size where Raman is able to sample the majority ratherthan a small portion of it. For in situ FT-IR examinations,the inherent problems of adsorbent absorption strength mustbe lived with. Alternative strategies may emerge that mayminimize these in some circumstances. Notwithstanding,investigations into improved coupling are likely to continue,and applications will grow as the synergy gains recognitionamongst industrial analysts.

ACKNOWLEDGMENTS

I would like to thank the help given in compiling thisarticle by Neil Everall (ICI plc, UK), Paul Turner (BrukerUK Ltd.), Richard Jackson (Bruker, Inc., USA) and PeterGriffiths (University of Idaho, USA).

ABBREVIATIONS AND ACRONYMS

HPTLC High-performance Thin Layer ChromatographyPZT Piezoelectric TransducerTGA Thermogravimetric Analyzer

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