LIQUID CHROMATOGRAPHY/INFRARED SPECTROSCOPY 1...

LIQUID CHROMATOGRAPHY/INFRARED SPECTROSCOPY 1

LiquidChromatography/InfraredSpectroscopy

Govert W. SomsenUniversity of Groningen, Groningen,The Netherlands

Tom VisserNational Institute of Public Health andEnvironmental Protection, Bilthoven,The Netherlands

1 Introduction 1

2 Coupling Modes and Operating Principles 22.1 Flow-cell Approach 22.2 Solvent-elimination Approach 3

3 Flow-cell Techniques 43.1 Cell Types and Infrared Detection

Modes 43.2 Use in Liquid Chromatography 63.3 Use in Flow-injection Analysis 7

4 Solvent-elimination Techniques 84.1 Deposition Substrates and Infrared

Detection Modes 84.2 Early Interfaces 94.3 Spray-type Interfaces 10

5 State of the Art 15

6 Perspective and Future Developments 17

Abbreviations and Acronyms 18

Related Articles 19

References 19

Analytical techniques that combine liquid chromatography(LC) and infrared (IR) spectroscopy have been developedprimarily to permit specific detection and/or identificationof sample constituents. LC is an important and extensivelyused method for the separation of mixtures into theirindividual components. IR spectroscopy is very useful forthe characterization of functional groups and has strongcompound-identification capabilities which are especiallysuited for the differentiation of structural isomers. Over thepast years the coupling of LC and IR spectroscopy (LC/IR)has been accomplished by two different approaches. Thefirst and simplest approach is to use a flow cell throughwhich the effluent from the LC column is passed while

the IR spectra are continuously measured. The merits offlow-cell IR detection include ease of operation, real-timedetection and low maintenance, but its main disadvantagelies in the significant IR-absorption of the solventscommonly used in LC. These absorptions seriously limitboth the detection sensitivity and the obtainable spectralinformation. The second approach involves eliminationof the LC solvent prior to IR detection. In this approachan interface is used to evaporate the eluent and depositthe separated compounds onto a substrate suitable for IRdetection. The primary advantages of solvent-eliminationLC/IR are the possibility to obtain full spectra ofthe analytes and the considerably enhanced sensitivitywhen compared to flow-cell detection. Unfortunately,common LC solvents, and particularly aqueous eluents,are not easily removed and therefore the evaporationinterfaces are often rather complex. This article reviews thedevelopments, practical aspects, applications and currentstatus of LC/IR, covering both coupling approaches. Itfollows that despite the unfavorable detection limits, flow-cell LC/IR can be useful for the specific and quantitativedetection of major components of mixtures. However,solvent-elimination-based IR-detection should be usedwhen small amounts of sample constituents have to becharacterized with a high level of confidence. In general,the practical use of IR detection in LC is still limited,but the advent of various (commercial) flow-cell andinterface designs shows that LC/IR is more and morebeing recognized as a feasible and rewarding technique.

1 INTRODUCTION

IR spectroscopy has a high potential for the elucidationof molecular structures. The IR spectrum of a poly-atomic molecule is based on molecular vibrations, eachspecifically dependent on atomic masses, bond strengthsand intra- and intermolecular interactions. As a conse-quence, the entire IR spectrum of an organic compoundprovides a unique fingerprint, which can be readilydistinguished from the IR-absorption patterns of othercompounds including isomers. In other words, when ref-erence spectra are available, most compounds can beunambiguously identified on the basis of their IR spectra.Moreover, characteristic absorption bands can be usedfor compound-specific detection.

LC is a powerful and versatile separation technique,which can handle a wide range of sample types andcompound classes. Because of the widespread use of LCand the (growing) need for analytical procedures thatprovide confirmation and/or identification of sample con-stituents, much effort has been – and still is – devoted tothe coupling of LC with spectrometric techniques such

Encyclopedia of Analytical ChemistryEdited by Robert A. Meyers. John Wiley & Sons Ltd, Chichester. ISBN 0471 97670 9

2 INFRARED SPECTROSCOPY

as mass spectrometry (MS), IR and nuclear magneticresonance (NMR) spectroscopy. Today, with modernFourier transform infrared (FTIR) instrumentation rou-tinely available, spectra can be recorded from nanogram,or even picogram, amounts of pure substance so that IRdetection, in principle, is suited for molecular recognitionat analyte levels frequently met in LC. Unfortunately,because of the (spectral) characteristics of the mobilephase, the coupling of LC and IR spectroscopy (LC/IR)is not straightforward and often requires the constructionof special flow cells or the development of rather complexinterfaces. Therefore, compared with other LC detectionmodes such as ultraviolet/visible (UV/VIS) absorptionspectroscopy or MS, the use of IR detection in LC is stillrather limited. Nevertheless, progress in interfacing tech-niques during the last decade has brought LC/IR to a stageof analytical utility which suggests that LC/IR may wellbecome a commonly available and applied technique.

2 COUPLING MODES AND OPERATINGPRINCIPLES

In the first LC/IR systems.1,2/ flow cells were used in afashion analogous to LC with on-line UV/VIS absorptiondetection. In order to circumvent interfacing difficultiesrelated to the IR absorptions of the mobile phase, in1979 Kuehl and Griffiths.3/ developed the first solvent-elimination based LC/IR set-up in which the eluentis evaporated prior to IR detection. Since then twoapproaches can be discerned in LC/IR, namely, the flow-cell approach and the solvent-elimination approach. Inthe contemporary practice of LC/IR both approachesare applied, although the detection limits and spectralinformation obtained with either approach may differconsiderably. The principles, applications, merits andlimitations of flow-cell and solvent-elimination LC/IRhave been reviewed in a number of books and papers..4 – 8/

2.1 Flow-cell Approach

The simplest way to couple LC and IR is to let the columneffluent pass directly through a flow cell suited for IRmeasurements. The IR absorption of the LC effluent iscontinuously monitored and spectral data are collectedon-the-fly and stored throughout the chromatographicrun. During or after the run, the spectra and/or IRchromatograms are computed and absorption due to themobile phase is subtracted. In a flow-cell design, bandbroadening caused by detection is easily minimized.

Unfortunately, the absolute sensitivity of IR spec-troscopy is relatively poor compared to spectrometrictechniques like MS, UV/VIS and fluorescence spec-troscopy. Moreover, solvents suited for LC generally

have many absorption bands in the IR region, whichleads to serious limitations of flow cell LC/IR interfacing.Firstly, absorption bands of analytes may be obscured byeluent absorptions. In other words, in flow-cell LC/IR, thespectral information that can be obtained is limited anddepends on the window provided by the mobile phaseused. Moreover, ill-considered subtraction of solventabsorption bands may lead to the erroneous conclu-sion that there is no absorption of the analyte in thecorresponding spectral regions. Secondly, gradient elu-tion can hardly be applied as the changing compositionof the mobile phase frustrates proper subtraction of thebackground. To some extent, the second derivative of aspectrum can be used to compensate for this problem,but accurate spectral correction is virtually impossible.Thirdly, the pathlength of the flow cell has to be limitedto ensure a certain spectral window and sufficient energyreaching the IR detector. For organic solvents commonlyused in LC/IR, the effective pathlength rarely exceeds1 mm which, bearing in mind Beer’s law, seriously reducesthe analyte detectability. For aqueous eluents, the largesttolerable pathlength is even shorter. As can be seen fromthe spectrum of water in Figure 1, most of the mid-IRregion between 4000 and 1000 cm�1 is opaque, even at avery short pathlength of 10 µm. It implies that a practicalcombination of reversed-phase liquid chromatography(RPLC) and IR via a transmission flow cell is restricted tospecific applications. Finally, the use of signal averagingto improve the signal-to-noise ratio (S/N) of the spectra islimited owing to the short time that is available for analy-sis under dynamic conditions. Occasionally, the principleof stop-flow can be used to enhance the S/N without toomuch loss in chromatographic performance.

The choice for a certain mobile phase and flow-cellproperties is primarily determined by chromatographicconsiderations and the desired spectral information. Inprinciple, the spectral window of the solvent can bevery small for quantitative analysis as the measurement

20

10

Tran

smitt

ance

(%

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0

4000 3000

Wavenumbers (cm−1)2000 1000

Figure 1 IR transmission spectrum of water recorded using aflow cell with silver bromide windows and an optical pathlengthof 10 µm.


of a single wavenumber, e.g. the band maximum, issufficient. Contrary, qualitative information desires IRtransparency over a much wider spectral region in orderto determine the presence or absence of functionalgroups, or to identify a compound by its IR fingerprintregion (1300–600 cm�1). Obviously, the spectral windowof the mobile phase is inversely proportional to thecell thickness and thus, an excess of sensitivity can betraded for more spectral information and vice versa.The experimental parameters in LC/IR are, therefore, acompromise between chromatographic and spectroscopicconsiderations giving maximum cell thickness and solventtransparency..5 – 8/

In order to minimize the problems associated witheluent absorption, the choice of the solvent in flow-cellLC/IR is generally restricted to chlorinated alkanes anddeuterated solvents. These solvents leave relatively widewindows in the spectrum, although even these eluentsinevitably obscure parts of the spectral functional groupregion (4000–1300 cm�1) and the fingerprint region. Theuse of a small percentage of a more polar modifier inthe eluent, as is quite common in normal-phase liquidchromatography (NPLC), may already prohibit effectivedetection. Owing to the short optical pathlength, theabsolute limit of detection in on-line LC/IR often is inthe order of a few micrograms, which implies that analyteconcentrations of 1–10 g L�1 have to be injected to obtainidentifiable spectra.

Tran et al..9/ proposed a completely different flow-cell LC/IR technique with, potentially, higher sensitivity.Different from the conventional way of measuring theIR absorption over a certain wavenumber region, thistechnique, called IR thermal lens spectrometry, is basedon the measurement of the temperature rise that isproduced in an illuminated sample by non-radiativerelaxation of the energy absorbed by molecules upon IRlaser excitation. Detection is carried out either directly,i.e. at a specific wavelength where only the analyte ofinterest absorbs, or indirectly, i.e. at a wavelength whereonly the mobile phase absorbs. Obviously, no spectralinformation is obtained with this quantitative technique,and the wavenumber region that can be covered withIR-tunable lasers is limited.

In conclusion, flow-cell IR detection is not a widelyapplied technique in LC as result of the interference ofabsorption bands of the mobile phase and the limitedsensitivity of IR spectroscopy. Applications largely focuson dynamic systems where low detection limits arenot crucial and solvents appropriate for IR detectioncan be selected more or less freely, that is, withouthaving unacceptable detrimental effects on the analyticalperformance. Examples of such techniques are gelpermeation chromatography (GPC), also referred to assize-exclusion chromatography (SEC), and flow-injection

analysis (FIA). An important feature of the flow-cellLC/IR technique is the ability to monitor the elutionof compounds with specific structural characteristics, i.e.functional groups. Besides, apart from regions of solventopacity, spectra can be obtained instantaneously fromany point in a chromatographic peak.

2.2 Solvent-elimination Approach

The compatibility and time-domain difficulties connectedto flow-cell IR detection can be circumvented by couplingLC and IR spectrometry via a substrate suitable forIR detection. In this indirect approach, the eluentis eliminated and the chromatographically separatedcompounds are immobilized on the substrate prior tothe collection of IR spectral data. The immobilization ofthe chromatogram is accomplished by using an interfacewhich evaporates the eluent and continuously deposits thecolumn effluent onto the moving substrate. In this way,interference-free IR spectra of the deposited compoundscan be recorded independently from the LC conditionsand the sensitivity of the FTIR spectrometer can be fullyexploited. A schematic of a solvent-elimination LC/IRset-up is depicted in Figure 2.

Besides the possibility to acquire full spectra, there aresome additional advantages of the solvent-eliminationapproach with respect to flow-cell IR detection. By carefulcontrol of the interface performance and the speed of thesubstrate, concentrated deposits may be obtained whichwill enhance analyte detectability. Spectral analysis canbe performed without any time constraints since thechromatogram is stored on the substrate. This impliesthat signal averaging can be used and analyte spots can beanalyzed repeatedly. For instance, after rapid screeningof the deposited trace applying low spectral resolutionand only one scan per spectrum, high-resolution spectrawith a high S/N can be recorded for a few interesting partsof the chromatogram. Moreover, spectrometric analysisof the stored chromatogram in principle can be carriedout at any convenient time or place, which may be helpfulwhen suitable IR facilities are limited and spectrometershave to be shared.

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LC

IF

Sub

TS

Figure 2 Schematic representation of a set-up for sol-vent-elimination LC/IR; LC, liquid chromatograph; IF, inter-face; Sub, deposition substrate; T, transfer; S, spectrometer.


Evidently, the solvent-elimination approach to LC/IR is(technically) more complicated than on-line IR detection.It requires an interface which should adequately effectthe evaporation of the eluent and, at the same time,maintain the chromatographic resolution during thedeposition process. In this respect, the LC flow rate,the composition of the eluent, the nature of the analytesand the substrate material are important factors. Forexample, small volumetric flows of a volatile solvent maybe readily evaporated, while rapid elimination of aqueouseluents will require an interface with enhanced solvent-elimination power. Elimination of the eluent may alsobe hampered by the presence of non-volatile additivessuch as buffer salts. Next to eluent evaporation, ideallythe interface also should provide compound depositioninto narrow spots in order to minimize band broadeningand achieve optimum IR sensitivity. However, completeeluent evaporation and compact analyte deposition maywell be irreconcilable goals. Therefore, in solvent-elimination LC/IR reduced flow rates, non-bufferedeluents and/or eluents with a low (or even zero) waterpercentage are frequently used.

The compounds analyzed by solvent-eliminationLC/IR, of course, should be considerably less volatilethan the eluent to accomplish their deposition. Since LCis used for non-volatiles in particular, this condition isgenerally met. The quality of the used substrate shouldnot be affected by either the eluent or the deposited com-pounds. The substrate also must be compatible with theselected IR mode without introducing additional inter-ferences. Furthermore, the physico-chemical propertiesof the substrate may influence the efficiency of ana-lyte immobilization. For instance, residual eluent easilyspreads over a substrate with a hard and smooth surface,while it may be effectively sorbed by a powder.

During the last decades, the combination of LCand IR via solvent elimination has been pursued byseveral research groups, which designed quite a numberof different interface concepts with varying success.The common goal of all these LC/IR systems is tosensitively acquire IR spectra of mixture constituentsthat are free from spectral interferences and can beused for identification purposes. Through the years, the

IR detection limits obtained with solvent-eliminationLC/IR have improved gradually from the microgram tothe low- or sub-nanogram range. Despite the progress,today there is no single ‘‘perfect’’ solvent-eliminationinterface available: every described system has its specificlimitations with respect to, for example, flow rate,composition of the eluent and/or achievable sensitivity.The research activities in the field, which until nowhave been dominated by the problem of simultaneouseluent-evaporation and compound-deposition, are stillon-going. Nevertheless, during the last years, several newcommercial interfaces based on the solvent-eliminationapproach have been introduced..10,11/

3 FLOW-CELL TECHNIQUES

3.1 Cell Types and Infrared Detection Modes

A variety of flow cells, differing in optical material,pathlength and cell volume, is available for LC/IRdetection purposes. The cells, including correspondingbeam condensing optics, generally fit in the standardoptical bench of the IR spectrometer. The distancebetween the flow cell, i.e. the IR spectrometer, andthe LC system is kept as short as possible in orderto minimize deterioration of the chromatography. Asnoted in section 2.1, the selection of a certain cell typeand, particularly, the optical pathlength, depends on therequired information.

The choice for a specific window material is mainlydetermined by the properties of the LC eluent and thespectral region that has to be monitored. A fully IR-transparent material such as potassium bromide (KBr),for instance, cannot be used with RPLC. Instead, moreexpensive water-insoluble materials such as calciumfluoride and zinc selenide (ZnSe) have to be chosen.The optical and physical properties of some commonlyused IR-window materials are presented in Table 1. Inall cases, in order to obtain an identifiable IR spectruma minimum amount of analyte has to be present in thedetection cell during the time of measurement. Accordingto Beer’s law, the minimum identifiable concentration

Table 1 Optical materials for use in IR flow cells

Material Spectral window (cm�1) Solubility in water Sensitive to

Silver chloride 4000–350 slightly soluble complexing agentsCalcium fluoride 4000–1100 insoluble ammonium salts; acidsQuartz 4000–2400 insoluble hydrofluoric acid; hot sulfuric acidPotassium bromide 4000–400 very soluble water; methanolZinc selenide 4000–450 insoluble acids; strong alkalisKRS-5 4000–250 slightly soluble complexing agentsPolyethylene 630–30 insoluble organic solvents


decreases when the pathlength of the cell is increased.However, extending the pathlength also results in anincrease of the eluent absorbance, thus limiting thespectral window. Commonly, the volume of the flowcell has to be minimized to such an extent that only asmall part (1% or less) of an LC-peak volume will fill thecell. Obviously, this places a significant limitation on theobtainable sensitivity. Through the use of microbore LCcolumns, the volume of the flow cells and LC peaks canbe made more compatible and, for that reason, micro-LC/IR is often preferred. Besides a reduced solventconsumption, microbore LC may also offer higher peakconcentrations in the cell. At the same time, however, itshould be noted that the sample capacity (both in massand volume) of micro-LC columns is rather limited..12/

Two types of commercially available flow cells canbe distinguished for LC/IR: transmission cells andattenuated total reflection (ATR) cells. The principleof a transmission flow cell is depicted in Figure 3. Thebasic part of the cell consists of an IR-transparent cavityor two IR-transparent windows separated by a metalspacer. The optical set is mounted into a metal bodybetween flexible rings to prevent breakage. The LCeffluent enters and exits the cell via capillary tubesconnected to an assembly of universal fittings and theIR beam passes perpendicularly through the LC flow.Additional equipment can be purchased for operationat elevated temperature and pressure. Zero-dead-volume(ZDV) flow cells for IR detection have been developedto minimize the volumes needed to connect the cell tothe column, which is very important when microbore LCis used. In this type of cell the LC effluent is directly led

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Figure 3 Cavity flow cell for IR transmission detection.

into the cavity of the optical element (Figure 4). Flowcells, micro-flow cells and ZDV-cells can be purchasedin many variations, rigid and demountable, differingin window material, pathlength (0.001–0.5 mm) andinternal volume (0.1–10 µL). Micro-cells are generallyused in combination with a beam condenser to obtain asufficiently high energy throughput and thus enhance theS/N of the recorded signal and/or spectra.

The second type of flow cell is based on the phe-nomenon of ATR and is called cylindrical internalreflectance cell or CIRCLE cell..13/ The principle ofthe CIRCLE cell is depicted schematically in Figure 5.The cell consists of a cylindrically shaped IR-transparentrod crystal with cone shaped ends that is incorporated ina boat-type cell body made of stainless steel (SS) or glass.The effluent of the LC column flows around the opticalcrystal while the interrogating IR beam enters the crystalat one end, reflects off the internal surfaces of the crystaland then exits at the other end. The cell body fits into asmall optical bench with special input and output optics.Several crystal materials can be used but ZnSe is com-monly preferred because of its high IR transparency, highrefractive properties and insolubility in water. CIRCLE

cells can be equipped with heating or cooling jackets

O-ring

LC-fitting

IR-transparentwindow

LC-tubing

Cell body

Figure 4 Schematic representation of a ZDV flow cell for IRtransmission detection.

IR-beam Detector

LC-flow (in)

LC-flow (out)

Rod crystal

Figure 5 Schematic representation of an IR flow cell forcylindrical internal reflectance detection (CIRCLE cell).


too. The cell design tends to involve a relatively largesample volume and efforts have been made to reduce theinternal volume of CIRCLE cells to 1–25 µL in orderto allow their efficient application in LC. The effectivepathlength of a CIRCLE cell is defined by the numberof reflections in the optical element and, therefore, sensi-tivity can be enhanced by using longer optical elements.These, however, also imply an increased cell volume and,thus, extra broadening of the LC peaks. CIRCLE cellscannot be used for quantification in a straightforwardmanner since the penetration depth of the IR radiation inthe LC eluent is limited (typical 1–5 µm) and wavelengthdependent. Special algorithms are used to compensatefor this.

3.2 Use in Liquid Chromatography

As outlined in the previous sections, flow-cell IR detectionis not the principal choice in LC. Yet, it is a valuablealternative and additional method to obtain specificquantitative and structural information on analytes. Inview of the relatively poor sensitivity, IR detection isrestricted to applications in which low detection limits arenot required. In GPC, for example, column capacities andsample concentrations are usually high. Gel permeationchromatography/infrared (GPC/IR) is well suited forthe characterization and quantification of compositionalchanges throughout a (bio)polymer mass distribution.Besides, the structural differences between the polymercomponents (usually homologues) often are small whichimplies that one or two spectral windows will suffice forspecific detection.

Applications of on-line LC/IR, including automaticsubtraction of the solvent background, had already beendescribed in the mid-1970s..1,2/ Injected amounts at sub-microgram level were found to be feasible for detectionin both NPLC and RPLC separations. In subsequentstudies, Taylor et al..13,14/ demonstrated that microboreLC (column i.d., less than 1 mm) offers improvedsensitivity compared to conventional LC (column i.d.,4.6 mm) because of the higher sample concentrationin the detector cell. Furthermore, use of halogenatedhydrocarbons as eluent was shown to offer better spectralspecificity owing to the higher IR-transparency of thesesolvents. A reasonable number of applications of flowcell LC/IR in a variety of disciplines has been developedsince. Saunders and Taylor,.15/ for example, applied on-line GPC/IR with tetrahydrofuran (THF) as mobile phasefor the determination of the nitrogen content of cellulosenitrates. A cylindrical micro flow cell with a pathlengthof 1 mm and an internal volume of 4 µL was employedin combination with a beam condenser. The extinctionof the O�N�O asymmetric stretching band was usedfor the quantification of primary and secondary carbon

nitration. An improved resolution and quantificationwas achieved by spectral derivatization. On-line GPC/IRis also a viable analysis method in the polymer field.One method employs high-temperature GPC with flow-cell IR detection to characterize the molecular weightdistribution of high-density polyethylenes..16/ Tri- anddichlorobenzene were used as mobile phase as thesesolvents do not exhibit interfering absorption in the CH2-stretching band region (3000–2700 cm�1). Furthermore,a quartz flow cell with a relative long pathlength of1 cm could be used in order to enhance the sensitivity.The method compares favorably with gradient elutionfractionation combined with 13C-NMR spectroscopiccharacterization.

The strong IR absorption of water, methanol andacetonitrile, limits the application of on-line reversed-phase liquid chromatography/infrared (RPLC/IR) tothe analysis of samples with relatively high analyteconcentrations, such as wines, beverages and cellulosesolutions. Various examples of this type of analysis havebeen described in the literature. Recent applications arethe identification and quantification of sucrose, glucoseand fructose in soft drinks,.17/ and the specific analysis ofmonosaccharides, alcohols and organic acids in wine..18/

In both studies, separations were achieved with ion-exchange columns using an aqueous eluent. IR detectionwas performed with a 25-µm pathlength flow cell andminimal identifiable concentrations of typically 1 g L�1

were obtained.When a high sensitivity or selectivity is required, con-

ventional RPLC solvents cannot be used with IR flowcells. Therefore, alternative methods have been devel-oped to circumvent the IR-opacity problems imposedby the aqueous mobile phase, while maintaining theadvantages of on-line detection. One approach is theextraction of the analytes into a more IR-transparent sol-vent. Another approach is the use of deuterated solventswhich virtually have the same chromatographic proper-ties but do not absorb in the spectral region of interest. Anexample of the first method is the dynamic extraction ofthe analytes from the aqueous effluent into chloroform orcarbon-tetrachloride..19/ The on-line liquid–liquid extrac-tion (LLE) is carried out in an extraction coil followedby continuous separation of the aqueous and organicphases by a hydrophobic membrane. Subsequently, theorganic phase, carrying the extracted analytes, is mon-itored in a common IR flow cell. A basically differentpost-column extraction method has been developed byMesserschmidt..20/ In this method, the RPLC effluentis diluted with water, and the analytes of interest aretrapped on several small solid-phase extraction (SPE)columns. These columns are dried with a flush of nitro-gen, and sequentially eluted with a small volume oftetrachloromethane into an IR flow cell allowing the


individual spectra of the analytes to be recorded. Anadditional advantage of this method is the improvedminimal identifiable concentration as a result of ana-lyte concentration on the SPE column. This techniquehas been successfully applied by DiNunzio.21/ for theseparation and identification of active compounds anddegradation products in pharmaceutical samples.

In on-line LC/IR, deuterated solvents can be attractivesubstitutes for conventional (hydrogenated) solvents. Theabsolute absorbance of deuterated solvents is usuallysmaller and, more importantly, their IR absorption bandsare shifted to different spectral regions. Additionally,deuterated solvents and their hydrogenated counterpartsshow very similar elution properties. With respect toon-line extraction procedures, the use of deuteratedeluents has advantages in terms of simplicity, speedand maintenance of the chromatographic resolution. Amajor drawback is their high price. A detailed study onthe utility of deuterated eluents in micro-column RPLC,NPLC and GPC with flow-cell IR detection was carriedout by Fujimoto et al..22/ It follows that the problemof interfering solvent absorption bands can be (partially)solved by a deliberate choice of a deuterated eluent. Chenand Kou,.23/ for instance, used deuterated methanol andwater instead of the non-deuterated solvents to overcomethe strong interfering absorptions that hinder the effectivedetection and quantification of lipid fractions by on-lineLC/IR. Remsen and Freeman analyzed proteins using on-line gel permeation chromatography/Fourier transforminfrared (GPC/FTIR) with a deuterium oxide eluentto remove water and other detection-interfering low-molecular-weight compounds and to achieve a rapidhydrogen exchange..24/ Amounts of 50 µg per proteincould be detected in the amide-I region between 1600and 1300 cm�1, and important information on the proteinconformation could be obtained.

3.3 Use in Flow-injection Analysis

FIA is a well-established analytical technique, basedon the automated injection of a series of samples ina continuous carrier stream. In FIA various detectorsare used, mainly for quantitative purposes. Amongstthese detection modes, flow-cell IR is not a frequentchoice because of the interfering solvent absorptionsand the relative poor sensitivity compared to UV/VISand fluorescence detection..25/ In several cases, however,on-line flow-injection analysis/infrared (FIA/IR) is anappropriate alternative for the rapid determination andquantification of a specific analyte in a simple mixture. InFIA, there is no need for an eluting solvent and, therefore,in contrast with LC, a carrier solvent suitable for IRdetection can be selected quite easily. In this respect,FIA/IR has a wider application range than LC/IR. Still,

the flow cells used in FIA/IR are not different from theones used in LC/IR and the resemblance in detectionapproach is evident. Commonly, FIA/IR is applied forsingle-component analysis but since IR spectra comprisea range of absorption frequencies, multi-componentanalysis can in principle be carried out as well. Obviously,the IR absorption bands of the carrier should notspectrally interfere with the marker band of each analyte.

Guzman et al..26/ compared the characteristics oftransmission and CIRCLE cell detection in FIA/IRfor multicomponent analysis of ternary solvent mixtures.With tetrachloromethane as carrier, the transmission cellprovided better sensitivity in the continuous-flow mode.The shorter effective pathlength of the CIRCLE cell wascompensated for by applying the stopped-flow techniquewhich allowed data averaging and, thus, enhancement ofthe S/N of the ATR spectra. Analyte concentrations of1–2% (v/v) could still be measured.

The principles and applications of flow-cell FIA/IRhave been studied extensively by the group of de laGuardia..27 – 33/ The effects of flow rate, injection volume,cell volume and optical pathlength on the performanceof the FIA/IR analysis of o-xylene in solutions of hexane,were investigated..29/ Further optimization was achievedin the development of a method for the automateddetermination of benzene in gasoline..30/ In a furtherstudy, incorporation of an analyte-enrichment step wasproposed to improve the detection limits of on-lineFIA/IR..31,32/ Preconcentration was carried out by trap-ping the analytes on an SPE cartridge. After drying of thecartridge with nitrogen gas, the analytes were eluted withdichloromethane and detected by IR transmission spec-troscopy using a micro flow cell. For aqueous solutionsof the pesticide carbaryl and its major metabolite 1-naphthol, detection limits of 0.36 mg L�1 and 1.6 mg L�1,respectively, were obtained after preconcentration of100-mL samples. Quantification was achieved by spe-cific detection of the bands at 1744 cm�1 (carbaryl CDO)and 1276 cm�1 (1-naphthol C�O) (Figure 6). FIA/IR isalso a useful alternative for the quantitative analysis ofmineral oil and grease..33/ Analogous to the Interna-tional Organization for Standardization (ISO) procedurefor the quantitative analysis of mineral oil in water andsoil, this method is based on measurement of the aro-matic, olefinic and aliphatic C�H stretching bands inthe 3200–2700 cm�1 region. The samples were extractedwith tetrachloromethane by LLE or microwave-assistedextraction. Next, the extracts were analyzed by FIA/IRat a sampling frequency of 60 h�1 using a quartz flowcell with a 10-mm pathlength. The detection limit was1 mg L�1 when 300 µL of the extract was injected.

Miller et al..34/ used FIA/IR for the simultaneous detec-tion of succinylcholine chloride and bethanechol chloride,


0.80

0.60

0.40

0.20

0.00

1850 1650 1450 1250

Abs

orba

nce

Wavenumber (cm−1)

A B

Figure 6 Absorbance spectra of carbaryl ( ) and 1-naphthol(- - -) dissolved in CHCl3 showing peaks at (A) 1741 cm�1

and (B) 1276 cm�1. (Reproduced by permission of ElsevierScience from Y. Dagbouche, S. Garrigues, M. de la Guardia,Anal. Chim. Acta, 314, 203–212, 1995.)

two pharmaceutically important compounds. The com-pounds were specifically monitored at 1075 cm�1 and953 cm�1, respectively, using a CIRCLE cell. The detec-tion limit was about 0.02% (w/v) at a throughput of 60samples per hour. FIA/IR was also applied to the quantifi-cation of acetylsalicylic acid, caffeine and paracetamol inpharmaceutical formulations..27,28/ Using a chlorinatedsolvent the extinction of a characteristic absorptionband could be measured for each of the analytes inthe 1800–1500 cm�1 region. Commercially available flowcells with an optical pathlength of 0.117 mm (5 µL) or0.17 mm (7.2 µL) were used. Two FIA/IR methods forautomated analysis in clinical and process chemistry havebeen developed by Kellner et al..35,36/ The first methodaimed for the determination of glucose and urea after on-line enzymatic digestion to gluconic acid and ammoniumcarbonate, respectively. A 25-µm pathlength transmis-sion cell was used to monitor the aqueous flow from theenzyme detector. The performance of the system wassatisfactory for relative clean samples such as standardsolutions, fruit juices and soft drinks. However, for rou-tine application, for example in blood analysis, furtherimprovement of the reproducibility is still required. Thesecond FIA/IR method was used to determine the amy-loglucosidase activity during starch hydrolysis processes.The IR flow cell (pathlength, 49 µm) was constructed oftwo different window materials in order to compromisebetween high transparency (ZnSe) and low reflectiv-ity (calcium fluoride). Because of the high viscosity ofthe starch solutions, a stopped-flow method was usedto obtain maximum reproducibility and sensitivity. Thechanges in the IR-spectra of the process mixture appearedto be directly correlated with the enzyme activity.

4 SOLVENT-ELIMINATION TECHNIQUES

4.1 Deposition Substrates and InfraredDetection Modes

The solvent-elimination approach in LC/IR involves theuse of an eluent-evaporation interface that deposits theLC-separated compounds onto an IR-compatible sub-strate. With most described set-ups, after immobilizationof one or more chromatograms, the substrate is trans-ferred to the IR spectrometer where spectra from thedeposited spots are recorded. The deposited traces onthe substrate may be moved (stepwise) through the inter-rogating IR beam so that, when scanning is complete,continuous IR chromatograms can be reconstructed bycomputer software. In some designs, IR detection is exe-cuted during the immobilization process, within 5–20 safter deposition, allowing spectra to be obtained in realtime. Obviously, such a design requires a dedicated detec-tor set-up. Dependent on the type of substrate used (seebelow) and/or size of the deposited spots, often specialoptics, such as a (diffuse) reflectance unit, a beam conden-sor or an IR microscope, are used to scan the depositedcompounds.

Basically three types of substrates and correspondingIR modes are used in solvent-elimination LC/IR: pow-der substrates for diffuse reflectance Fourier transforminfrared (DRIFT) detection; metallic mirrors for reflec-tion/absorption (R/A) spectrometry; and IR-transparentwindows for transmission measurements. In early solvent-elimination LC/IR designs DRIFT detection of analyteson potassium chloride (KCl) powder was used, but todayother, more convenient, detection modes are preferred.DRIFT as such is one of the most sensitive IR modesand sub-microgram identification limits can be achievedwhen the residual solvent is evaporated quickly fromthe powder. If the eluent is not highly volatile, it candraw analyte away from the KCl powder surface intothe substrate. This will limit the sensitivity, because theeffective penetration depth of a DRIFT measurement isnot more than 100 µm. To overcome this problem, diffusetransmittance spectrometry has been applied instead ofDRIFT, using a layer of KCl powder on an IR-transparentsubstrate..37/ The main limitations of DRIFT detection inLC/IR, show up during application. Reproducibility ishard to control since factors such as sample homogene-ity, sample load and compactness of the powder layersignificantly influence the DRIFT analysis. Reorientationof the DRIFT matrix as a result of sample depositionmay lead to a poor background compensation. Carefulfilling of cups or trays with the powder substrate is verytime-consuming and has to be repeated for every analysis.Finally, common substrates such as KCl powder cannotbe used in combination with aqueous eluents. In view ofthe overriding importance of RPLC, this is a very serious


restriction. Some authors have used diamond powder as awater-resistant DRIFT substrate, but it is expensive (andthus not disposable) and not easy to clean.

Front-surface aluminum mirrors, which are suitablefor R/A detection, are compatible with aqueous elu-ents and are relatively easy to handle. Compounddeposition on this type of substrate requires efficientsolvent-elimination interfaces because residual solventwill easily spread over the hard and smooth reflectivesurface. The band intensities in the R/A spectroscopy arelargely governed by a double-pass transmittance mecha-nism, so that spectral data analogous to transmission dataare obtained. Some useful results of solvent-eliminationLC/IR using mirrors have been reported, although severalauthors.38 – 40/ have reported evidence of band asymmetryand spectral distortions. Aspects such as specular anddiffuse reflection from the analyte, thickness and micro-crystallinity of the spot, and optical characteristics of thesubstrate affect the shape and intensity of R/A spectraobtained from analytes on aluminum mirrors. The useof an IR-transparent germanium disc with a reflectivebacking has been proposed in order to reduce spectraldistortions. This type of disc is used in the commer-cially available LC-Transform LC/IR interfaces..10/ Thecleaning of aluminum mirrors in between analyzes is quitedelicate: the thin metal layer is fragile and can be damagedeasily by rubbing.

Most favorable results in solvent-elimination LC/IRare obtained with IR-transparent deposition substratesthat allow straightforward transmission measurements.So far mainly KBr and ZnSe windows have beenapplied in experimental LC/IR set-ups. These substrateshave a hard and smooth surface and, therefore, eluentelimination has to be fast to achieve proper depositions.ZnSe is a water-resistant, inert material, while KBrusually cannot be used in combination with RPLC.Deposited compounds on ZnSe can be removed simply,with water or alcohol, so that one window can beused repeatedly. With ZnSe, good-quality IR spectrawith symmetrical band profiles can be recorded fromdeposited spots. When the size of the sample spots issmall and microscopic optics are used for measurement,the sensitivity of ZnSe transmission measurements ishigher than the sensitivity of DRIFT measurements..40/

ZnSe windows also cause fewer spectral artifacts thanmirror substrates for R/A detection..39/ Many LC/IRstudies demonstrated that spectra obtained using ZnSe,closely resemble conventional KBr-disk transmissionspectra. Consequently, existing spectral libraries andsearch programs can be used for identification purposes,which is very important for the acceptance of IR as avaluable detection technique.

The quality and appearance of spectra obtained withsolvent-elimination LC/IR will be influenced by the

morphology of the deposited analytes. The morphologywill depend primarily on parameters such as eluentcomposition, evaporation rate, temperature and nature ofthe substrate and the analytes. During solvent eliminationsome compounds will form nice crystals while otherswill deposit as an amorphous layer. Also, some analyteswill deposit as a smooth film, whereas others may formirregular clusters. When the spot thickness exceeds acertain level, the effect of light scattering may becomeapparent. A compound may also exhibit polymorphism sothat mutually (slightly) different spectra can be obtainedfor the same compound. In general, IR detection ofdeposited compounds on IR-transparent substrates doesnot pose serious problems. However, analyte morphologyshould always be taken into consideration during spectralinterpretation.

In solvent-elimination LC/IR the identification limitsusually improve when the width of the analyte spotsis decreased. A prerequisite for this gain is the use ofthe appropriate detector and sampling optics. Optimumsolvent-elimination interfaces can produce analyte spotswith a width as small as 100–300 µm. For these deposits,the focus of a conventional beam condensor is too largeand the use of an IR microscope is indicated. Frequently,the sensitivity enhancement is rationalized by consideringthe relatively increased spot thickness only (Beer’s law!),but this approach is too simple. From more completeS/N considerations it follows that the good sensitivity ofIR microscopic detection essentially results from the lownoise level of the IR detectors in IR microscopes..4/ Inother words, to achieve the most sensitive IR detection inLC, the width of the analyte deposits should have the samedimensions as the microscope detector area (typically,100–200 µm). Of course, as with any IR experiment,the S/N ratio also can be improved by increasing themeasurement time (signal averaging). As outlined insection 2.2, this advantage can be exploited to its fullextent in solvent-elimination LC/IR, although at the costof an increased time of analysis.

4.2 Early Interfaces

The solvent-elimination systems that were developed inan early stage, generally used KCl-powder substrates forDRIFT detection or flat KBr windows for transmissiondetection. The first working interface for the coupling ofNPLC and IR was designed by Kuehl and Griffiths..3,41/

The organic eluent was led through a heated concen-trator tube and dropped into a series of cups filledwith KCl powder suited for DRIFT analysis. A carouselrotated the cups into the IR spectrometer where iden-tifiable spectra could be recorded for sub-microgramamounts of analyte. In order to allow for RPLC sepa-rations, the aqueous effluent was first on-line extracted


with dichloromethane which, after continuous phase sep-aration, was directed through the concentrator to the KClcups..42/ For extracted compounds good-quality spectrawere obtained. The carousel–DRIFT method was alsoadopted for narrow-bore NPLC (use of 1-mm i.d. column)by reducing the size of the KCl cups and by omitting theconcentrator tube..43/ Using a similar set-up, Kalasinskyet al..44,45/ coupled both narrow-bore NPLC and RPLCwith DRIFT. The KCl-powder substrate was held eitherin a ‘train’ of compartments or in a continuous trough.Aqueous eluents could be used by on-line conversion ofthe water into methanol and acetone via a post-columnreaction with 2,20-dimethoxypropane (DMP). The identi-fication limits of these systems were 1–3 µg, typically.

The early DRIFT-based systems for the first timedemonstrated that solvent-elimination LC/IR can provide(much) better sensitivity and spectral quality thanflow-cell based LC/IR. However, the systems weremechanically complex and tedious to work with, andDRIFT detection appeared to be strongly affected bysmall disturbances of the KCl-powder surface and by thepresence of (residual) water.

Jinno et al..46 – 47/ proposed the use of micro-LCcolumns (i.d., 0.3 mm) in solvent-elimination LC/IR inorder to alleviate the problem of the evaporation of largeeluent volumes. The effluent (5 µL min�1) from eithera GPC or an NPLC was led directly to a moving KBrplate which was covered by a stream of heated nitrogen.Subsequently, the plate with the deposited track wasscanned by IR transmission spectroscopy using a 3ðbeam condensor. The potential of the approach (termed‘‘buffer-memory’’ technique) was illustrated by theanalysis of a mixture of dithiocarbamate metal complexesby three spectroscopic techniques..47/ In a modified set-up the linearly moving KBr plate was replaced by arotating KBr disk which, after the chromatographic runwas finished, was transferred to a special rotation modulein the IR spectrometer..48/ In order to permit the use ofRPLC, a SS wire net (WN) was used instead of an KBrwindow..49/ IR transmission measurements were possiblebecause after deposition and drying the analytes werepartly suspended in the metal meshes.

The ‘‘buffer-memory’’ technique demonstrated theusefulness of the storage of a continuous chromatogramon a flat substrate. Besides, it was considerably simplerthan the DRIFT methods. However, at least severalmicrograms of analyte were needed for a positive IRidentification. These amounts often exceeded the samplecapacity of the micro-columns and required unrealisticallyhigh concentrations to be injected.

4.3 Spray-type Interfaces

When using the ‘‘buffer-memory’’ technique for com-pound deposition on flat substrates, it is not possible

to eliminate organic or aqueous eluents at flow rateshigher than about 5 µL min�1 without spreading the com-pounds over a large area of the substrate surface. Toachieve a more viable coupling of LC and IR, the use ofinterfaces with enhanced evaporation power is essential.The solvent-elimination interfaces developed in the lastdecade all use some kind of spraying to induce rapideluent evaporation. Heat, gas, electric potential and/orultrasonic vibrations are used to break up the LC elu-ent stream into small droplets. Some designs incorporateexisting (commercial) equipment, while others have beenbuilt from scratch.

In the thermospray (TSP) interface, originally devel-oped for liquid chromatography/mass spectrometry(LC/MS), a directly heated tube evaporates part ofthe column effluent to an expanding vapor causingnebulization of the remaining effluent. As a result, amist of desolvating droplets emerges from the tube.In the TSP-based LC/IR systems, nebulization is per-formed at atmospheric pressure and the spray is directedtowards a deposition substrate. Eluent flow rates of upto 1 mL min�1 can be handled. Griffiths and Conroy.50/

reported preliminary results on the use of TSP for LC/IR,but the first really working interface was introduced byJansen..51/ With a home-made TSP, the LC effluent wassprayed on a SS IR-reflective tape which moved throughan optical accessory in the IR spectrometer (Figure 7).Most of the eluent was eliminated directly by the TSPand residual solvent was evaporated off the tape byheating. The immobilized chromatogram was monitoredcontinuously and full IR spectra were recorded. Sev-eral simple polymer samples (20–80 µg injected) wereanalyzed by GPC/IR using an organic eluent at a flowrate of 0.5–1 mL min�1, but some low-molecular-weightmonomers were too volatile to be deposited. The charac-terization of two Irganox-type polymer additives (100 µgeach), which were separated by RPLC using an eluent

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Figure 7 Schematic of TSP–moving belt interface for LC/IR;1, moving SS tape; 2, TSP; 3, heater; 4, IR reflectance cellmounted in the IR spectrometer.


(0.5 mL min�1) with 30 vol% water, was also shown.Robertson et al..52/ further optimized the TSP–movingbelt interface by studying the effect of the TSP tempera-ture and distance to the substrate. The system was usedfor the analysis of amino acids, saccharides, carboxylicacids, antioxidants and polymers,.53,54/ and analyte iden-tification could be achieved down to concentrations of50 µg mL�1 or about 2.5 µg injected.

The main advantage of the TSP-based systems isthat relatively high flow rates (0.5–1 mL min�1) ofboth organic and aqueous eluents can be handled andconventional-size LC can thus be used. Furthermore,spectral data are acquired during the run, which givesthe IR detector an essentially on-line character. On theother hand, the high temperature of the TSP may induceanalyte losses by evaporation or thermal degradation andthe analyte spots on the moving tape are still quite largewhich results in a moderate IR sensitivity.

The particle beam (PB) interface was modified forLC/IR by de Haseth et al..55,56/ and Wood..57/ In thisinterface the LC eluent is nebulized by helium anddirected into a desolvation chamber where most ofthe liquid is vaporized. The mixture of gas, vaporand condensed analyte molecules (i.e. particles) isaccelerated into the momentum separator where theanalytes travel straight through the skimmer cone,while the gas and vapour are pumped away. ForIR detection an IR-transparent substrate is placedin the particle-beam path to collect the analytes of

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Helium supply

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Rotarypump

Rotarypump

Ballvalve

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0.5-in. o.d. glass tube

0.75-in. o.d. glass tube

0.5-in. glass O-ring fitting

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KBr plate located inslot cut into glass

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Figure 8 Schematic of PB interface for LC/IR.

interest (Figure 8) and after deposition, the substrate istransferred to the IR spectrometer for analysis. Untilnow stationary substrates have been used in liquidchromatography/particle beam/infrared (LC/PB/IR), thatis, no complete chromatograms, only fractions, wereanalyzed. The particle beam/infrared (PB/IR) interfacecan effect the elimination of aqueous eluents at flowrates of up to 0.3 mL min�1 as was demonstrated by theanalysis of erythrosin B and p-nitroaniline (50 µg each)by RPLC/IR..56/ Since the PB interface has strong eluent-elimination capacities, it was believed that interferencecaused by buffers might be small..58/ The interfaceindeed appeared to be able to process a 0.3 mL min�1

flow of buffered eluent, but the buffer salts were nevercompletely eliminated. Best results were obtained witheluents buffered with ammonium acetate, although bufferbands were clearly present in the IR spectra recordedfrom microgram amounts of analyte. When phthalateor phosphate buffers were used, the analyte spectrawere completely dominated by absorption bands of thebuffer salts. Spectral subtraction procedures could beused to recover spectra from 130-µg depositions but wereunsuccessful at the 13-µg level. PB/IR has been used as atool for the determination of protein structures..59 – 61/

For b-lactoglobulin and lysozyme it was shown thattheir structural integrity is maintained during the PBdesolvation process and the subsequent deposition on thesubstrate. In addition, lysozyme appeared to retain itsbiological activity. The sample loads in these experimentsgenerally were quite high (5–500 µg).

The PB interface can effectively remove both organicand aqueous solvents. However, relevant applications inLC/IR would still require the construction of a devicethat allows the continuous deposition of a complete chro-matogram on a moving substrate. The PB/IR analysis ofcompounds at the nanogram level has been indicated,.55/

but the reported sample quantities mainly are in the (high)microgram range. The modest analyte detectability nodoubt is related to the fact that the efficiency of analytetransfer in the PB interface probably is 5–10% only.

The potential of electrospray (ESP) nebulization formicro-LC/IR was studied by Raynor and co-workers..62/

A high electrical potential is used to form a sprayof charged droplets at the end of a capillary filledwith flowing liquid. As a result of charge density, theinitial droplets break up into smaller droplets whichfacilitate solvent evaporation. Use of low flow rates(typically 1–20 µL min�1) is indicated in order to obtaina stable ESP. The ESP is formed under atmosphericconditions and a sheath flow of nitrogen gas is applied toenhance eluent evaporation (Figure 9). The ESP interfacewas used to deposit the effluent from a micro-RPLCcolumn onto a ZnSe plate..62/ After LC separation,20-ng amounts of caffeine and barbital could be analyzed


25-µm capillaryHigh-voltagecable (+3 KV)

(Evacuatedchamber)

(Vacuumpump)

Gas

Taperedstainless steelelectrospray tip

ZnSe plateStage

Earthed cable(a)

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High-voltagecable (+3 KV)

25-µm fused silicacapillary tubing(0.25-mm o.d.)

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Solvent stream formsTaylor cone at tip

SolventN2

Focused stream ofelectrostatically chargeddroplets

Earthed disc(b)

Figure 9 Schematic of ESP interface for (a) micro-LC/IR(b) with the ESP tip in detail. (Reproduced by permission ofHuthig Publishing from M.W. Raynor, K.D. Bartle, B.W. Cook,J. High Resolut. Chromatogr., 15, 361–366, 1992.)

successfully using an IR microscope. Identification of 2 ngcaffeine appeared to be possible, although subtractionof the interfering bands from a siliceous impurity wasrequired. Stable ESP conditions were achieved withhexane, dichloromethane, acetonitrile, methanol andseveral aqueous solvents, but problems were reported forpure water. Until now there have been no further studieson electrospray/infrared (ESP/IR) detection in LC.

During pneumatic nebulization a high-speed gas flowis used to disrupt the liquid surface and to form small,fast-moving droplets. Organic solvents can be rapidlyevaporated by pneumatic nebulization, while removalof aqueous solvents is possible when the nebulizer gas

Nitrogen gas

HPLC effluent

Tee

Reflective surface

Gearbox

Drive shaft(a)

Effluent deposit

Sample track

(b)

Figure 10 Schematic of a narrow-bore NPLC and IR systemwith pneumatic nebulization; (a) side view during deposition;(b) top view of collection mirror. (Reproduced by permissionfrom J.J. Gagel, K. Biemann, Anal. Chem., 58, 2184–2189, 1986, American Chemical Society.)

is heated. Pneumatic nebulizers have been used in sev-eral solvent-elimination LC/IR designs, among which arethe most successful so far. One of the first nebulizer-based LC/IR methods involved the continuous sprayingof the effluent from a narrow-bore NPLC column ona rotating IR-reflective disk (Figure 10)..63/ The efflu-ent was mixed with nitrogen gas to form a fine sprayand the immobilized chromatogram was analyzed byrotating the disk through a 3ð -condensed IR beamwhile recording R/A spectra. The system was tested withpolycyclic aromatic compounds (200–800 ng each) whichwere separated using hexane–dichloromethane as elu-ent (30 µL min�1). Good-intensity spectra were obtained,although some spectral deviations were observed. Thispneumatic nebulizer design was improved to accomplishelimination of aqueous solvents..38/ A heated nitrogenflow served as an evaporation-enhancing and spray-focusing sheath gas. Eluents containing up to 55% watercould be handled at 30 µL min�1 and a number of isomericnaphthalenediols (500 ng each) were separated and iden-tified. Again the recorded R/A spectra showed anomalies.These spectral problems could be partially solved by usingan IR-transparent germanium disk with a rear surfaceof aluminum as substrate..64/ This pneumatic nebulizer


LC/IR design is commercialized by Lab Connections..10/

The instrument consists of a sample-collection moduleand an optics module for R/A analysis. So far the com-mercial interface has been applied mainly in the field ofGPC/IR..65,66/ It was also used for the identification oftriclosan, an antibacterial agent, in toothpaste..67/

A simple but effective concentric flow nebulizer (CFN)for the coupling of narrow-bore LC and IR spectrometrywas constructed by Lange et al..68/ The interface consistsof two concentric fused-silica capillaries. The LC columneffluent is led through the inner capillary and heatedhelium gas through the outer capillary (Figure 11a). Thehot gas facilitates the evaporation of the solvent and thefocusing of the spray emerging from the inner tube. Toenhance the elimination of aqueous eluents, the CFN andthe ZnSe substrate were placed in a vacuum chamber(Figure 11b). IR microscopy was used for optimumdetection. The CFN can handle eluents with up to 100%water at a flow-rate of 50 µL min�1 and identifiable spectraof analytes can be recorded down to the low-nanogramrange. The CFN was also installed in an evacuatedcompartment which included the IR-microscopic opticsand a motor to translate the ZnSe window..69/ Withthis system, an RPLC effluent (50 µL min�1) couldbe continuously deposited on the moving substrate.After immobilization of the chromatogram, spectraldata could be collected without the need to transportthe substrate from the Chromatograph to the IRspectrometer. To further improve the on-line character ofthe system, a modified CFN was installed on the opticalbench of a Tracer (Biorad, Dusseldorf, Germany)gas chromatography–IR interface which allows spectralacquisition in real time..70/ So far, only some preliminaryresults with this on-line LC/IR system have beenreported. Unfortunately, proper analyte spectra cannotbe obtained with the CFN/IR system when using non-volatile buffers, because of strong co-deposition ofbuffer salts..71/ However, if sufficient vacuum pumpcapacity is applied, a 1 mM ammonium acetate buffer canbe completely eliminated, although higher ammoniumacetate concentrations cause interference.

Somsen et al..39/ proposed a spray-jet interface for thecoupling of narrow-bore RPLC and IR spectrometry.In this interface a heated nitrogen flow provides pneu-matic nebulization of the column effluent (20 µL min�1)which is led through a SS needle that protrudes througha spray nozzle (Figure 12). With ZnSe as substrate andIR microscopy for detection, identification limits in the10–20-ng range were achieved for quinones and poly-cyclic hydrocarbons. The narrow-bore RPLC/IR systemwas used for the impurity profiling of a steroids,.72/

for the isomer-specific characterization of chlorinatedpyrenes,.73/ and for the identification of additives inpolymer samples..74/ Furthermore, the suitability of the

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Figure 11 CFN for (a) narrow-bore LC/IR and (b) interfacechamber; A, electric connections; B, LC effluent inlet; C, heliumgas inlet; D, heating wire; E, concentric fused-silica tubes; F,stage; G, magnet for stage rotation; H, vertical positioner.(Reproduced by permission from A.J. Lange, P.R. Griffiths,D.J.J. Fraser, Anal. Chem., 63, 782–787, 1991, AmericanChemical Society.)

interface for GPC/IR was demonstrated by analyzingpolystyrene oligomers..74/ When RPLC is applied, thespray-jet LC/IR system is limited with regard to the LCflow rate, the water percentage of the eluent and the han-dling of buffered eluents. In order to take away these lim-itations, a post-column LLE module (phase segmentor,extraction coil and phase separator), was inserted on-lineand the organic phase, carrying the extracted analytes,was sent to the evaporation interface..75/ The resultingliquid chromatography/liquid–liquid extraction/infrared(LC/LLE/IR) system can handle eluents with highwater percentages (20–100 vol%) at flow rates up to0.2 mL min�1 and provides identification of compoundsat the sub-milligram per liter level. Since the salts arenot extracted, non-volatile buffers can be used without


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causing interference. The detectability of the analyteswas further improved by incorporation of on-line SPEfor analyte enrichment (Figure 13). With such a system,triazine herbicides, including several isomers, could beidentified at the low-microgram per liter level in riverwater (Figure 14)..76/

In an alternative approach to improve the compatibilityof the spray-jet interface with RPLC, the eluent flow ratewas reduced to 2 µL min�1..77/ To obtain a useful spray,a make-up liquid (20 µL min�1 of methanol) was addedto the micro-LC effluent. As a consequence, the perfor-mance of the interface became independent of the watercontent of the eluent, so that gradient elution was possible.A micro-precolumn for on-line trace enrichment wasapplied to improve detection limits. With a 40-µL samplevolume, good-quality IR chromatograms and analytespectra were recorded at the low-milligram per liter level.

In an ultrasonic nebulizer a spray is formed by deposit-ing the LC effluent on a transducer that is vibrating atultrasonic frequencies. For LC/IR purposes, the sprayis directed towards a substrate by a carrier gas. Castleset al..78/ used ultrasonic nebulization for the depositionof compounds separated by narrow-bore RPLC on adiamond-powder substrate suitable for DRIFT detection.Spectra of satisfactory quality were obtained for injec-tions of 3 µg of analyte. In some instances, the completeand direct evaporation of the eluent by the ultrasonicnebulizer was not achieved because the vibrating sur-face was not uniformly effective and occasionally largedroplets were formed which wetted the diamond powder.Dekmezian and Morioka developed an interface forhigh-temperature GPC/IR which involved an ultrasonicnebulizer..79/ The nebulizer was placed in a vacuumchamber and sprayed the column effluent on a set

P4

P3

P2

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ZnSe

TrT

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Pr

Waste

Recycle

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V1 V2

S

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Figure 13 Set-up for LC/IR with on-line SPE and post-column LLE; P1, pump for sample loading and SPE-column rinsing; P2and P3, eluent pumps; P4, organic phase pump; S, solvent selection valve; V1-V3, six-port injection valves; Pr, SPE column; Anal,analytical column; T, T-piece; EC, extraction coil; PS, phase separator; R, restrictor; UV, UV absorbance detector; I, spray-jetinterface; H, gas heater; ZnSe, ZnSe deposition substrate; TrT, translation table; FTIR, FTIR microscope.


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Figure 14 SPE and LC/LLE/IR chromatograms of river Meuse water samples spiked with five triazines: (a) 20 mL (30 µg L�1),(b) 50 mL (6 µg L�1) and (c) 100 mL (2 µg L�1). IR spectra of peaks 1, 3 and 5. Chromatogram representation, (a) Gram–Schmidtor (b and c) spectral window (1650–1500 cm�1). Peaks: 1, simazine; 2, atrazine; 3, sebutylazine; 4, propazine and 5, terbutylazine.

of heated KBr discs, which were subsequently ana-lyzed by IR transmission spectrometry. The system wasapplied to the determination of compositional changesof ethylene–propylene rubbers. An interface comprisingan ultrasonic nebulizer in a vacuum chamber is used byLab Connections in their LC-Transform 300 Series..10/

This commercial device sprays the chromatographic efflu-ent on a rotating germanium collection disk suited forR/A analysis (see above). The system was used for the

quantitative analysis of copolymers by GPC/IR.80/ andfor steroid analysis by RPLC/IR..81/

5 STATE OF THE ART

In the contemporary practice of LC/IR both flow-cell and solvent-elimination approaches are applied.


Since the flow-cell procedure cannot get around thedetection limitations imposed by the LC eluent, it hasdeveloped into a special-purpose method with restrictedapplicability. The IR absorptions of any solvent invariablytake up parts of the mid-IR spectral region and, therefore,the main power of IR spectroscopy, i.e. the reliableidentification of compounds, cannot be fully exploitedin flow-cell IR detection. Nevertheless, making use of thespectral windows of the eluent, flow-cell IR spectroscopycan serve as a moderately sensitive, compound-specificdetection technique. Occasionally, it is used as a universal,fast and low-cost method to obtain quantitative andstructural information on major constituents of samples.Various types of flow cells are commercially available, andthe experimental set-up and practice of flow-cell LC/IR isrelatively simple and well-suited for routine applications.

When the objective of IR detection in LC is theunambiguous identification of (low-level) constituents ofmixtures, coupling via solvent elimination should be theapproach of choice. Solvent elimination procedures offerthe possibility

ž to record spectra over the entire mid-IR regionwithout interference from the eluent;

ž to perform signal averaging in order to improve theS/N of the spectra; and

ž to contain a relatively large part of the chromato-graphic peak within the IR beam.

As a result, the solvent-elimination approach provides aset-up which features increased sensitivity and enhancedspectral quality, two important conditions for effectiveanalyte identification. The most recent commercial

LC/IR systems which are presently available,.10,11/ aresolvent-elimination devices. Interestingly, also in gaschromatography and supercritical-fluid chromatographyanalyte-deposition-based IR detection has proven to bemore sensitive and versatile than flow-cell-based tech-niques. On the other hand, one should realize that thevibrational information obtained after solvent elimina-tion is different from the vibrational information obtainedwith flow-cell detection (condensed-phase spectra againstsolution-phase spectra). Recently, this difference wasused to determine subtle molecular features of drugmetabolites which were analyzed by both flow-cell andsolvent-elimination LC/IR..82/

Today, the vast majority of LC separations is carriedout by means of RPLC and, not surprisingly, research inthe field of LC/IR has concentrated on the developmentof interfaces that are suitable for the elimination of aque-ous eluents. Table 2 summarizes the characteristics of thevarious solvent-elimination reversed-phase liquid chro-matography/Fourier transform infrared (RPLC/FTIR)systems which have been developed during the last years.The systems based on TSP, PB and ultrasonic nebuliza-tion can handle relatively high flows of aqueous eluentsand allow the use of conventional-size LC, which evi-dently is an advantage. However, these systems oftenexhibit moderate, or even unfavorable identification lim-its and, therefore, their analytical applicability is limited.Until now, the most favorable results have been obtainedwith pneumatic nebulizers which essentially represent thestate of the art in solvent-elimination LC/IR.

The pneumatic interfaces combine rapid solventelimination with a relatively narrow spray. This implies

Table 2 Characteristics of solvent-elimination RPLC/IR systemsa

Type of interfacing IR mode Substrate Eluent flow rateb Limit of identificationb Reference(µL min�1)

mass (ng) conc (mg L�1)

Concentrator after LLE DRIFT KCl powder 800 100 10 42Deposition after DMP DRIFT KCl powder 50 1000 1000 44Buffer memory trans SSWN 4 1000 10 000 49TSP R/A SS tape 500 10 000 – 51

R/A SS tape 1000 1000 25 52PB trans KBr window 300 1000 200 56, 58ESP trans-micr ZnSe 4 1 10 62Pneumatic nebulizer R/A Al mirror 30 30 30 38

trans-micr ZnSe 50 1 17 69trans-micr ZnSe 20 5 3 39, 73

. . .after LLE trans-micr ZnSe 200 30 0.2 75. . .after SPEC LLE trans-micr ZnSe 200 50 0.001 76

. . .after SPECmake-up trans-micr ZnSe 2 20 0.02 77Ultrasonic nebulizer DRIFT Diamond powder 40 1000 1000 78

R/A Ge disk 500 100 20 81

a DMP, on-line reaction with DMP; make-up, addition of excess methanol; trans, transmission; trans-micr, transmission with IR microscope;– , concentration and injection volume not stated.

b Typical values.


that analytes can be deposited, for example on ZnSe,in a narrow track of spots and IR microscopy can beapplied effectively to achieve mass sensitivities in thelow-nanogram range. Bearing in mind that often onlypart of the injected amount of analyte is actually ana-lyzed, this means that the mass detectability of theseLC/IR systems approaches a level close to the minimumthat can be identified by IR spectroscopy. The systemsbased on pneumatic nebulization are limited with regardto flow rate (2–50 µL min�1) and water percentage of theeluent. The tolerable water content of the eluent dependson the flow rate. Flow rates of 2–5 µL min�1 of even purewater can be eliminated, but for 20–50 µL min�1 flows ofaqueous eluents, enhancement of the solvent evaporationefficiency is required, for example by mixing the effluentwith nitrogen gas.38/ or by placing both the nebulizer andthe deposition substrate inside a vacuum chamber..68/ Thetedious evaporation of water can also be circumventedby applying on-line LLE of the aqueous effluent with anorganic solvent. Such a system allows much higher flowrates (0.2 mL min�1) and percentages of water (up to 100vol%)..75,76/ Of course, the required LLE module adds tothe complexity of the system and the analytes must have asufficiently high extraction efficiency. Solvent-eliminationRPLC/IR with gradient elution poses the problem of theefficient evaporation of an eluent with a changing watercontent. One solution involves the increase of the temper-ature of the nebulization gas during the gradient run,.38/

another the addition of excess methanol to a micro-LC effluent in order to mask the changes in its watercontent..77/

The injection volumes that can be handled with micro-and narrow-bore LC columns, are at most 1–2 µL.The identification limits in terms of concentration unitsof the pneumatic nebulizer-based systems thereforeare in the low-milligram per liter range, which issufficient for a number of analytical applications..72 – 74/

By using the LLE-pneumatic nebulizer combination, thedetectability can be improved to sub-milligram per literlevels because larger LC columns – and, thus, increasedinjection volumes – can be used. With an adequate trace-enrichment procedure such as on-line SPE, the LC/IRdetection limits can be further improved down to thelow-microgram per liter level..76/ The use of bufferedeluents is generally avoided in solvent-elimination LC/IR,since buffer salts may seriously affect the deposition anddetection of the analytes. With pneumatic nebulizers evenvolatile buffer salts are rarely completely eliminated. Infact, until now the LLE-pneumatic nebulizer combinationis the only described LC/IR system which allows the use ofnon-volatile buffer salts without introducing interfacingdisturbances and/or spectral interference..75,76/

Effective solvent elimination by the pneumatic nebu-lizers allows the use of deposition substrates with a hard

and smooth surface such as ZnSe windows. With thesesubstrates interference- and distortion-free transmissionspectra are obtained which can be readily comparedwith conventional KBr-disk IR spectra. This implies thatlibraries of condensed-phase reference spectra can beused for spectral recognition and identification. Com-pounds of various nature such as quinones, steroids, drugs,polymer additives and herbicides have been analyzed suc-cessfully by pneumatic-nebulization-based LC/IR. Theinterfaces can handle most types of analytes, although toovolatile compounds will be evaporated by the nebulizergas and therefore will not be deposited on the sub-strate. Thermal degradation of analytes is commonly notobserved during pneumatic nebulization, despite the factthat the nebulizer gas is heated to rather high tempera-tures (70–180 °C). Probably, due to the rapid evaporationof the solvent, the spray droplets are cooled considerably.

6 PERSPECTIVE AND FUTUREDEVELOPMENTS

In the last fifteen years, LC/IR has emerged as apotentially powerful tool for the specific detectionof major components (flow-cell approach) or for theidentification of (minor) constituents of complex mixtures(solvent-elimination approach). With respect to commonLC detection techniques such as UV/VIS absorptiondetection, the sensitivity of flow-cell IR detection is ratherpoor and its merits therefore mainly lie in the abilityto quantitatively monitor absorptions that are specificfor the analyte or for a certain functional group. Sincethe limitations of flow-cell LC/IR are inherent in thetechnique, no vast improvements can be anticipated inthe future. Some gain in performance may be achieved byoptimization of the flow-cell design and use of advancedFTIR spectrometers, but these improvements will bemodest and not essentially change the applicability offlow-cell IR detection. Employment of chemometricaltechniques, however, may be useful particularly when thesignal or spectrum is the result of the absorbance of twoor more substances.

The usefulness of solvent-elimination LC/IR to providestructural information and/or identification of unknownshas been demonstrated convincingly since 1990. Unfor-tunately, the development of coupling techniques pro-ceeded, and still proceeds, quite slowly and until now mostinterfaces have been used only by their designers. Nev-ertheless, the difficulty of solvent-elimination LC/IR, i.e.simultaneous eluent evaporation and analyte deposition,seems to be a technical rather than a fundamental prob-lem. In other words, the development of an overalleffective and routinely applicable interface probably is


a matter of time, effort and technological innovations.Of course, solvent-elimination LC/IR has to competewith other identification techniques of which today on-line LC/MS undoubtedly is one of the most important.Quite a number of LC/MS interfaces have been devel-oped and commercialized, but still each interface has itsspecific limitations. Furthermore, MS techniques cannotdiscriminate between isomers. Hence, even with adequateLC/MS techniques available, there is a need for alter-native and complementary detection techniques whichindependently confirm MS-based identifications and dif-ferentiate between structurally highly similar compounds.A recent study demonstrated that LC/IR can make aviable contribution to identification analysis in a researchsetting that includes MS and NMR..82/ Prerequisite forthe successful implementation of IR detection was a goodunderstanding of the relative strengths and weaknessesof each technique, and the integration of analysis in thetotal research program.

In order to enhance the acceptance of solvent-elimination LC/IR, several items of interest shouldbe considered. The practicality of the technique forreal-life samples should be demonstrated more exten-sively. The applications described so far.72 – 74,76/ indicatethat LC/IR can indeed be used for the characteri-zation and unambiguous identification of target andunknown compounds. LC/IR is particularly useful for thedistinction of isomeric compounds.77 – 79/ which cannotbe distinguished by LC/MS. Another item of atten-tion is the development and use of appropriate on-line sample-treatment procedures to improve analytedetectability. Despite the low-nanogram identificationlimits, the detectability in concentration units of eventhe best LC/IR systems will not be sufficient to meetcurrent demands in bio- and environmental analysis. On-line SPE can improve the concentration detectabilityby one or two orders of magnitude. Obviously, suchan improvement is unlikely to be obtained by opti-mization of interfacing and/or IR detection only. Thefirst studies indicating this advantage in both flow-celland solvent-elimination IR-detection have already beenreported..31,32,76,77/

Concerning the viability of solvent-elimination LC/IR,the availability and use of commercial interfaces alsois essential. The LC-Transform interface (Lab Con-nections) has been available now for several years,but unfortunately few applications have been reported.Because this solvent-elimination system uses a mirrorsubstrate and standard IR equipment, both the IR sen-sitivity and spectral quality are limited. In a more viableapproach an IR-transparent substrate should be usedtogether with microscopic IR detection. Such a configu-ration is used by the Infrared Chromatograph interface(Bourne Scientific). In this commercial and automated

design the LC column effluent is deposited on a movingZnSe window which instantaneously passes through thefocused beam of the IR spectrometer allowing spectra andIR chromatograms to be recorded in real time. The place-ment of the chromatograms on the substrate is controlledby computer which also keeps a record of the posi-tion of deposited compounds. The IR Chromatograph

seems promising but, as it has been introduced onlyrecently, it is still too early to assess its merits. The han-dling of the obtained spectral data also is a matter ofconcern in solvent-elimination LC/IR. The identificationof analytes on the basis of their IR spectra often is adifficult operation. Therefore, the automated retrievalof spectra in reference collections, and the computeri-zation of spectral interpretation are important. Severalsuch procedures have already been introduced in thevibrational-spectroscopic field and high priority shouldbe given to their implementation in the separation field.

Finally, it should be noted that the solvent-eliminationapproach in LC is not restricted to IR detection butcan, in principle, be applied to any spectrometrictechnique which requires the compounds of interestto be present as deposits..83/ An example of such ananalyte-deposition-based detection technique is matrix-assisted laser desorption/ionization (MALDI) MS. Froma technical viewpoint the collector systems developedfor the coupling of LC or capillary electrophoresiswith MALDI/MS show a strong similarity with solvent-elimination LC/IR systems.

ABBREVIATIONS AND ACRONYMS

ATR Attenuated Total ReflectionCFN Concentric Flow NebulizerDRIFT Diffuse Reflectance Fourier

Transform InfraredDMP DimethoxypropaneESP ElectrosprayESP/IR Electrospray/InfraredFIA Flow-injection AnalysisFIA/IR Flow-injection Analysis/InfraredFTIR Fourier Transform InfraredGPC Gel Permeation ChromatographyGPC/FTIR Gel Permeation Chromatography/

Fourier Transform InfraredGPC/IR Gel Permeation Chromatography/

InfraredIR InfraredISO International Organization for

StandardizationLC Liquid ChromatographyLC/IR Liquid Chromatography/Infrared

Spectroscopy


LC/LLE/IR Liquid Chromatography/Liquid–liquidExtraction/Infrared

LC/MS Liquid Chromatography/MassSpectrometry

LC/PB/IR Liquid Chromatography/ParticleBeam/Infrared

LLE Liquid–liquid ExtractionMS Mass SpectrometryMALDI Matrix-assisted Laser Desorption/

IonizationNPLC Normal-phase Liquid ChromatographyNMR Nuclear Magnetic ResonancePB Particle BeamPB/IR Particle Beam/InfraredR/A Reflection/AbsorptionRPLC Reversed-phase Liquid

ChromatographyRPLC/FTIR Reversed-phase Liquid

Chromatography/FourierTransform Infrared

RPLC/IR Reversed-phase LiquidChromatography/Infrared

S/N Signal-to-noise ratioSEC Size-exclusion ChromatographySPE Solid-phase ExtractionSS Stainless steelTHF TetrahydrofuranTSP ThermosprayUV/VIS Ultraviolet/VisibleWN Wire NetZDV Zero-dead-volume

RELATED ARTICLES

Biomedical SpectroscopyInfrared Spectroscopic Multicomponent Assay of Bio-fluids

Environment: Water and WasteInfrared Spectroscopy in Environmental Analysis

Infrared SpectroscopyGas Chromatography/Infrared Spectroscopy

Mass SpectrometryLiquid Chromatography/Mass Spectrometry

Nuclear Magnetic Resonance and Electron SpinResonance SpectroscopyHigh Performance Liquid Chromatography/NuclearMagnetic Resonance

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