Abstract The reversed-phase chromatography of a num-ber of polymer additives has been undertaken with on-linecharacterisation via a combination of diode array UV, 1H NMR, IR spectroscopy and mass spectrometry. Thiscombination of spectrometers enabled the on-flow collec-tion of full UV, 1H NMR, IR and mass spectra for a rangeof common polymer additives in amounts ranging fromca. 230 to 900 µg on-column. The practical difficulties as-sociated with multiple hyphenation and potential futuredevelopments are discussed.

Keywords HPLC · Polymer additives · Multiple hyphenation · Spectroscopic characterisation

Introduction

The use of techniques such as HPLC-MS and HPLC-NMRis now routine in many laboratories for the analysis ofmixtures. This is possible as a result of the developmentof robust chromatographic interfaces that have evolved tothe extent that relatively few compromises need to be madeby either separation system or spectrometer. The success-ful development of this type of hyphenation has led to theconstruction of more sophisticated systems combiningmore than one spectrometer. Such multiple hyphenation(“hypernation”) is perhaps most widely developed forHPLC-NMR-MS (reviewed in [1]). Here, the comple-mentary nature of the NMR and MS data provide a pow-erful and efficient method for the characterisation andidentification of chromatographic peaks. However, evenmore elaborate concatenations of instruments are possibleand systems capable of providing NMR, IR, MS [2] and

IR, UV, NMR and mass spectra [3, 4,5] have been re-ported. In a recent series of experiments, we have investi-gated the potential of such an HPLC-IR-UV(DAD)-NMR-MS system in combination with D2O at high tem-peratures for the separation and analysis of ecdysteroidsin plant extracts [6] and model pharmaceuticals [7] to ob-tain on-line spectra. Here, we describe further studies toevaluate this system, using a number of common polymeradditives as models, with a more conventional reversed-phase chromatographic system. These results are com-pared with those from earlier studies on the separation andspectroscopic characterisation of polymer additives withnormal phase eluents and off-line FT-IR following on-linecollection using various commercial (“LC-Transform”)interfaces [2,3].

Experimental section

Reagents

The model polymer additives (structures in Fig.1) used in this in-vestigation were Irganox 245 (triethyleneglycol bis-3(3-tertiary-butyl-4-hydroxy-5-methylphenylpropionate), BHA (butylated hy-droxyanisole, 2-tertiary-butyl-4-methoxyphenol), BHT (butylatedhydroxytoluene, 2,6-di-tertiary-butyl-4-methylphenol), BisphenolA and Topanol CA (1,1,3-tris-(2-methyl-4-hydroxy-5-tertiary-butylphenyl)butane) (purchased from Sigma, Poole UK, Fluka,Gillingham, UK and Aldrich, Gillingham, UK). Samples were dis-solved, at concentrations of 46–99 µg µL–1 (see text) in (CD3CN)(99 atom%, Aldrich, UK). For this investigation, a test mixturecomprising Bisphenol A, BHA, Irganox 245, BHT and TopanolCA was prepared and analysed as described below. In addition, thevarious model analytes were chromatographed individually.

The HPLC system consisted of a Constametric 3200 HPLCpump (Laboratory Data Control, Stone, Staffs UK) which deliv-ered CD3CN-D2O (80:20 v/v) at 1.0 mL min–1 to a 100×4.6 mmHypersil H5ODS (5 µm particle size), HPLC column (Hichrom,Reading, UK, Batch No. 4421, SER. NO. H5ODS-5886).

From the column, the eluent entered a splitter from which ca.95% of the flow was directed, via 110 cm of 0.020”-i.d. PEEK tub-ing, to a Bio-Rad FT-IR model FTS3000 Excaliber spectrometer(Cambridge, MA USA). The spectrometer was fitted with a Spec-tra Tech (Stamford, CT USA) Micro Circle Cell ATR (attenuatedtotal reflectance) high-pressure stainless steel flow cell of 25 µLvolume. The flow cell was fitted with a zinc selenide ATR crystal.The spectrometer was purged with dry nitrogen to minimise any

Dave Louden · Alan Handley · Eva Lenz · Ian Sinclair ·Steve Taylor · Ian D. Wilson

Reversed-phase HPLC of polymer additives with multiple on-line spectroscopic analysis (UV, IR, 1H NMR and MS)

Anal Bioanal Chem (2002) 373 :508–515DOI 10.1007/s00216-002-1249-3

Received: 29 October 2001 / Revised: 28 December 2001 / Accepted: 21 January 2002 / Published online: 7 March 2002

ORIGINAL PAPER

D. Louden · A. HandleyLGC, The Heath, Runcorn, Cheshire, WA7 4QD, UK

E. Lenz · I. Sinclair · S. Taylor · I.D. Wilson (✉ )AstraZeneca Pharmaceuticals, Mereside, Alderley Park, Macclesfield, Cheshire SK10 4TG, UKe-mail: Ian.Wilson@astrazeneca.com

© Springer-Verlag 2002

water vapour interference of the collected spectra. Any residualsignal from water vapour in the collected data was subtracted us-ing a water vapour reference spectrum. Spectra were acquired withthe kinetics software, enabling Gram Schmidt reconstruction of theon-flow data. 57 Scans per spectrum (10 s acquisition time) werecollected using a sensitive MCT (mercury cadmium telluride) liq-uid nitrogen-cooled detector. The spectra were acquired at 8 cm–1

spectral resolution. The sample data were ratioed against those fora background spectrum of the flowing solvent through the cellprior to injection of the sample solution, thus automatically sub-tracting out the solvent spectrum from the sample spectra.

The remainder of the flow (ca. 5%) was directed to a Micro-mass Platform single quadrupole mass spectrometer (MicromassUK, Wythenshawe, UK) via 4.5 M of 75 µm-i.d fused silica capil-lary tubing (this length of tubing generated sufficient back pressureto direct the bulk of the flow to the FTIR). Mass spectra were mea-sured over a range of 115–650 Daltons. Prior to introduction of thesample into the ion source of the mass spectrometer, the eluent wasmixed with a make up flow of 90:10 (v/v) methanol/water. Thiswas delivered using a second Constametric 3200 pump and was in-troduced via a t-piece at 0.5 mL min–1. Positive ion spectra wererecorded with a cone voltage of 25 V, a scan time of 0.9 s and aninter scan delay of 0.1 s. The multiplier was set to 400 V.

From the FT-IR the eluent flowed to a Bruker UV-diode arraydetector (Bruker, Coventry, UK) via 30 cm of 0.005”-i.d. PEEKtubing. UV spectra were collected over the wavelength range188–1000 nm. From the UV detector the solvent stream was di-rected to the NMR via 280 cm of 0.010”-i.d. PEEK tubing.

All of the instrumentation described above was located outsidethe 5 Gauss line of the stray magnetic field generated by the 500 MHz NMR spectrometer. NMR spectra were acquired using aBruker DRX-500 NMR spectrometer. On-flow 1H NMR detection

was carried out in the pseudo-2D-mode at 500.13 MHz using aflow-through probe of 3 mm-i.d. with a cell volume of 60 µL. Typ-ically, 16 scans/FID per increment were acquired into 8 K datapoints each with a spectral width of 8278 Hz. Spectra were ac-quired using the NOESYPRESAT pulse sequence (Bruker Spec-trospin, UK) to suppress residual water and acetonitrile reso-nances. 90°-Pulses were used with an acquisition time of 0.5 s, arelaxation delay of 0.7 s and a mixing time of 100 ms.

Results and discussion

Instrumental layout

The layout of the HPLC-IR-UV-NMR-MS system con-structed for these studies is shown schematically in Fig.2.The present system showed a number of differences fromthat used in our previous investigations on hypernation forthe characterisation of polymer additives. Thus, in the ear-lier study [3] a triple quadrupole mass spectrometer wasemployed rather than the single quadrupole instrumentused here, whilst the flow probe of the NMR spectrometerhad a volume of 120 rather than 60 µL. In the earlier work,IR spectra were obtained by directing either the whole [2]or a portion (ca. 50% [3]) of the eluent to an interface de-signed for on-line deposition of chromatographic peakson a rotating germanium disk for subsequent off-linespectroscopy. In contrast, with the current system, all ofthe spectra (including IR) were acquired on-flow. Anotherdifference in layout for this system was that the post col-umn splitter, used to send the minor portion of the flow tothe MS, was placed immediately after the HPLC columnrather than after the UV detector, as used previously. Inthe current system, the bulk of the eluent from the splitterwas directed first to the FT-IR, from which it then flowedto the UV and NMR spectrometers. Finally, the variablewavelength UV detector that had been employed to mon-itor the chromatographic separation was replaced with aUV-VIS DAD spectrophotometer, capable of providingspectra over the range 188–1000 nm.

An important limitation of the prototype system de-scribed here was the lack of an integrated data capturesystem and, as indicated in Fig.2, spectra were acquiredby each individual spectrometer. This lack of integrationinevitably meant that some of the advantages obtainedfrom acquiring all of the spectral data simultaneouslywere negated because of the need for several operators tocontrol the various instruments, and for the manual colla-tion of the spectra post-run.

Chromatography

In previous studies the separation of polymer additiveswas achieved on polymer-based size exclusion columns.When analysis was performed in the context of HPLC-NMR-IR, deuterated chloroform (CDCl3) alone was foundto be a suitable eluent [2]. This solvent allowed goodNMR and IR spectra to be obtained with minimal inter-ference with the additional benefit, from the point of view

509

Fig.1 Structures of the model compounds

510

of NMR spectroscopy, of allowing the observation of ex-changeable protons on e.g. phenolic hydroxyls. The volatil-ity of the solvent was also valuable in simplifying the con-ditions required for evaporation when the eluent was col-lected for off-line FT-IR. However, unmodified chloroformwas unsuitable when electrospray MS was included in thesystem. This was due to the dryness of the solvent that pre-vented ionisation due to the lack of a suitable proton donor.Useful MS data were only obtained when the chloroformwas modified with deutero-methanol (CD3OD) and ammo-nium acetate (10 g L–1 in D2O) [3]. Whilst this expedientenabled mass spectra to be obtained, it also resulted in theappearance in the NMR spectra of additional, unwanted,resonances for these additives, and the loss of signals forexchangeable protons. In the current series of studieswhere solvent volatility was not a consideration, reversed-phase chromatography, on conventional C-18 bondedpacking materials was used, to more easily obtain MS data.The relatively non-polar nature of these polymer additivesrequired the use of high proportions of acetonitrile in themobile phase to ensure elution in a reasonable time. The re-sulting large signal for the protons present in the acetoni-trile methyl resonance meant that it was better to usedeuterated (CD3CN) acetonitrile rather than the protonatedform of the solvent. Suitable chromatography of the testcompounds was obtained with D2O-CD3CN 20:80(v/v).The chromatography of a test mixture consisting of Bis-phenol A (371 µg), BHA (228 µg), Irganox 254 (332 µg),BHT (992 µg) and Topanol CA (338 µg) is shown in Fig. 3.Baseline resolution was achieved for Irganox 254, BHTand Topanol CA, but the earliest eluting components,Bisphenol A and BHA, chromatographed as a single peak.However, as discussed below Bisphenol A eluted in theleading edge of the combined peak and BHA in the tailingedge, enabling spectra to be obtained for each component.In addition to their chromatography as a mixture, eachcompound was also chromatographed individually in orderto obtain a reference library spectrum.

Infrared spectroscopy

The quantities of compound present in the chromato-graphic peaks from the test mixture generally enabled theacquisition of good quality IR spectra on-flow. Thus, ex-amination of the IR spectra from the leading and tailingedges of the combined peak of Bisphenol A and BHA re-vealed clear diagnostic spectra for the individual compo-nents (Fig.4a,b). Similarly, diagnostic spectra were ob-tained for both Irganox 245 and BHT and representativespectra for these compounds are illustrated in Fig. 4c,d).The only difficulty encountered in obtaining IR spectrafrom the test mixture under these conditions was seenwith Topanol CA. For this compound the signals for theanalyte coincided with those of the solvent, and spectra

Fig.2 A schematic represen-tation of the HPLC-IR-UV-NMR-MS system used in theseinvestigations

Fig.3a–e HPLC trace 254 nm for a Bisphenol A (371 µg), b BHA(228 µg), c Irganox 245 (332 µg), d BHT (992 µg) and e TopanolCA (338 µg)

could not be acquired. Where spectra were obtained, thespectral matches against standards were very high(99.12% for Bisphenol A, 91.58% for BHA, 97.36% forIrganox 245 and 97.23% for BHT).

Ultraviolet spectroscopy

As seen with IR spectroscopy, the quantities of polymeradditives used here provided no particular challenge withrespect to sensitivity for the UV/VIS-DAD spectrometer.The resulting 2D-UV chromatogram for the five-com-pound test mixture is shown in Fig.5. The first elutingpeak is composed of a mixture of Bisphenol A and BHAand although the peak appears symmetrical (Fig.3), spec-troscopic examination showed the components to be par-tially resolved, with Bisphenol A eluting in the leadingedge of the peak and BHA in the tailing edge. Evidencefor the non-homogeneous nature of this peak can be seenin the distorted shape of the contour plot for the 260–300 nm region as shown in Fig.3. The individual UVspectra obtained from this two-component peak (inset intoFig.5) reflect this in the different UV maxima of the twoanalytes. Thus Bisphenol A gave a secondary UV maxi-mum absorption at 275 nm, whilst that for BHA was at285 nm. These values are comparable to those obtainedfor the individual standards chromatographed under thesame conditions. Similarly, the UV spectra for the re-maining compounds in the test mixture were comparableto those of the individual standards. Representative UVspectra obtained for each component in the mixture areshown in Fig.5.

511

Fig.4a–d IR spectra for a Bisphenol A, b BHA, c Irganox 245and d BHT obtained for the mixture shown in Fig.2

Fig.5a–e The 2D-UV chro-matogram (254 nm) for themixture shown in Fig.2. Theinset UV spectra are for Bis-phenol A, BHA, Irganox 245,BHT and Topanol CA as indi-cated

NMR spectroscopy

In initial experiments an attempt was made to use CH3CNrather than CD3CN as the organic modifier. However,whilst it was possible to obtain spectra using CH3CN, ob-taining adequate suppression of the signals due to the sol-vent protons caused difficulties in on-flow measurements.This problem was circumvented by the use of fullydeuterated CD3CN. In this way, diagnostic 1H NMR spec-tra were obtained for all of the compounds in the test mix-ture. In the case of Bisphenol A and BHA which chro-matographed as a single peak, it was still possible to ob-tain the appropriate spectra from the leading and tailingedges of the peak, as was the case with IR and UV spec-troscopy. The pseudo-2D-NMR data obtained for the testmixture are shown in Fig.6 together with the 1D-spec-trum of the individual components extracted from the data(Fig.6a–e). The spectra were of good quality and showedall of the expected signals for the analytes. For example,in the case of Irganox 245, the 1H NMR spectrum (Fig.6c)showed all the signals that would be anticipated based onits structure. Thus, the spectrum comprised two sets oftriplets at 2.49 and 2.69 ppm (from the propionate group),two sets of ‘doublets-of-doublets’ at 3.55 and 4.08 ppm(from the ethylene-glycol moiety neighbouring the car-boxy-function). The latter were significantly attenuated asa result of the suppression of the residual water signal(3.58 ppm). A single line at 3.44 ppm represented the cen-tral ethylene glycol moiety. The aromatic protons res-onated at 6.73 and 6.8 ppm, whilst the tert-butyl groupand methyl group gave signals at 1.27 and 2.09 ppm, re-spectively. Suppression of the residual acetonitrile signal(1.9 ppm), resulted in the slight distortion of the singlet at2.09 ppm. The quantities of each of the compounds in the

test mixture were quite adequate to obtain on-flow 1H NMRspectra with good signal-to-noise ratios.

Mass spectrometry

As with the UV spectroscopy described above, the quan-tities of material present in the peaks were more than ade-quate to obtain spectra for the five additives in the modelmixture. The total ion current (TIC) obtained is shown inFig.7a. Deuterated radical cations were obtained forBisphenol A, BHA and BHT (mass chromatograms areshown in Fig.7c,e,f). In the case of Topanol CA, a molec-ular ion was not observed as the compound fragmented inthe ion source to give an ion at m/z 192 (Fig.7b). Irganox254 was observed as a polydeuterated molecular ion atm/z 592 (mass chromatogram shown in Fig.7d). Themuch greater degree of deuteration of Irganox 245 thanwould have been expected on the basis of the exchange-able protons on the structure represents an unusual findingand the mass spectrum obtained shows an unusual isotopepattern (Fig.7d). Given that the NMR data obtained con-comitantly showed no evidence for facile deuterium ex-change, these data suggested that deuteration occurred inthe ion source of the MS rather than in the HPLC mobilephase. Further, off-line, investigation of this phenomenonsuggested that this polydeuteration involved the replace-ment of the protons adjacent to the carbonyl group (un-published observations).

One feature that is also apparent from some of themass chromatograms shown in Fig.7 (and particularly inFig.7a,b) is that there are periodic disturbances in thebaseline. These oscillations would seem to have a periodof ca. 1 min and it is noteworthy that the peak for Topanol

512

Fig.6a–e The 2D-1H NMRchromatogram for the mixtureshown in Fig.2 with spectrafor a Bisphenol A, b BHA,c Irganox 245, d BHT and

e Topanol CA extracted fromthe 2D-profile

CA appears to be split into two in phase with this cycle.These periodic oscillations are also well illustrated in thetwo-dimensional mass chromatogram in Fig.8a, wherethere is the appearance of regular “bands” of masses at ca1 min intervals (most apparent following the elution ofIrganox 245, at ca. 3.0 min). However, it is also notewor-thy that the various peaks for the analytes are also irregu-lar over shorter timescales. An example of this is seen inFig.8b where the trace for m/z 484, one of the higher massions associated with Irganox 245 (see Fig.8c), can be seento be formed from three distinct peaks. None of these phe-nomena were observed in the UV and NMR chromato-grams. It seems likely that these observations are an arte-fact, linked in some way to the use of two separate pumpsto deliver the eluent from the HPLC column and the makeup flow to the mass spectrometer. The measured pistoncycle time is ca. 10 and 20 s for this type of pump at flowrates of 1 and 0.5 mL min–1 respectively. An irregularflow of solvent into the MS might result in compoundsbecoming deposited in the probe (e.g. as a result of theprobe temperature changing) and then being evaporatedas conditions in the cycle changed. It is also possible thatchanging solvent composition through this hypotheticalcycle could have had a similar effect. It may be relevant tonote that these oscillations and effects on peak shape werenot observed in studies where more polar, water soluble,compounds were analysed with pure D2O as the chro-matographic eluent and methanol/water (90:10) as themake up flow [7,8]. Clearly the elimination of this un-wanted artefact would be desirable in any future work. Ir-respective of the reason for this artefact, it might be pru-dent to employ eluents and make up flow solvents that arematched in terms of solvent composition as a matter ofcourse in future designs.

513

Fig.7a–f Mass spectra obtained for the mixture shown in Fig.2with a TIC, b m/z 192 (Topanol CA), c m/z 221 (BHT), d m/z 592(Irganox 245), e m/z 181 (BHA) and f m/z 136 Bisphenol A)

Fig.8 a 2D-Mass chromato-gram for the mixture shown inFig.2. b Mass chromatogramfor m/z 484. c Mass spectrumfor Irganox 245 taken at RT3.01 min

Using this system, spectroscopic data were also ac-quired on a suspected polymer additive which was therebyconclusively identified as BHT. The combined spectro-scopic data for this material are shown in Fig.9.

As we have noted in previous hypernation studies, thebest sensitivity that can be achieved with any particularsystem is that attainable by the least sensitive spectrome-ter(s). However, the quantities of material used in the testmixture here were not designed to explore the limits ofdetection of this layout, but to compare the utility of on-flow, on-line spectroscopy compared to a mixed on-lineoff-line method.

In that context, the minimisation of the solvent inter-ferences in the 1H NMR spectrum of BHT resulting fromthe use of the CD3CN-D2O mobile phase, compared to thehighly modified chloroform system needed previously, isnoteworthy.

In addition, being able to obtain IR spectra on-lineclearly has the potential for the more rapid analysis ofmixtures. With respect to IR spectroscopy however, theoff-line methodology provided good quality spectra withno interferences from the mobile phase and the potentialfor very high sensitivity. However, in considering the po-tential of hypernation in relation to the results describedfor this prototype system, it should be borne in mind thatthe instrumentation used in this study was relatively mod-est by modern standards and certainly was not “state-of-the-art”. Whilst we made no particular investigation ofsensitivity in this study, our previous experiments [6,7]with this layout suggest that for NMR and IR (generallythe least sensitive instruments in the array of spectrome-ters assembled here) at this flow rate, ca. 100 µg of eachcompound would be need to obtain an identifiable spec-

trum. There is no doubt that sensitivity could be improvedand good results in the low µg- and even ng-range havebeen achieved in miniaturised flow NMR systems thathave been linked to a variety of separations [8, 9, 10,11].Furthermore, the current layout was designed to furthertest the concept of hypernation and there is no doubt thatthe system was not fully optimised with respect to limit-ing peak broadening and flow rates etc. Rather thandemonstrating the full potential that could be achievedwith a state-of-the-art hypernated system, the studies re-ported here provide a pointer to what could be done.

Conclusions

The value of HPLC-NMR-MS is now accepted, and suchcombinations are commercially available. However, it isstill not clear whether the benefits of hypernated systemsof the type described here outweigh the costs of assem-bling such an array of instruments. However, the resultsillustrated using this prototype hypernated system, to-gether with our previous studies [3, 4, 5, 6, 7,8], dodemonstrate that the concatenation of chromatography toa wide range of spectroscopic detectors does not presentoverwhelming difficulties. With further optimisation tominimise band broadening, and using state-of-the-art in-strumentation, we have no doubt that diagnostic UV, IR,1H NMR and MS spectra could be obtained on-flow withmodest quantities of material. Should such a system beconsidered to represent a worthwhile objective, progresswould also be required to produce fully integrated data ac-quisition systems in order for such hypernated systems toachieve their full potential.

514

Fig.9a–d Spectra for the “un-known”, identified on the basisof a NMR, b UV, c IR and d MS data as BHT

515

References

1.Wilson ID (2000), J Chromatogr A 829:3152.Ludlow M, Louden D, Handley A, Taylor S, Wilson ID (1999)

Anal Comm 36:853.Ludlow M, Louden D, Handley A, Taylor S, Wright B, Wilson

ID (1999) J Chromatogr A 857:894.Louden D, Handley A, Taylor S, Lenz E, Miller S, Wilson ID,

Sage A (2000) Anal Chem 72:39225.Louden D, Handley A, Taylor S, Lenz E, Miller S, Wilson ID,

Sage A, Lafont R (2001) J Chromatogr A 910:2376.Louden D, Handley A, Lafont R, Lenz E, Taylor S, Sinclair I,

Wilson ID (2002) Anal Chem 74:288

7.Louden D, Handley A, Lenz E, Taylor S, Sinclair I, Wilson ID(2001) The Analyst 126:1625

8.Wu N, Peck TL, Webb AG, Magin RL, Sweedler JV (1994) J Am Chem Soc 116:7929

9.Behnke B, Schlotterbeck G, Tallarek U, Strohschein U, TsengLH, Keller T, Albert K, Bayer E (1996) Anal Chem 68:1110

10.Wu N, Peck TL, Webb AG, Magin RL, Sweedler JV (1994)Anal Chem 66:3849

11.Schewitz J, Gfrorer P, Pusecker K, Tseng LH, Albert K, BayerE, Wilson ID, Bailey N, Scarfe GB, Nicholson JK, Lindon JC(1998) The Analyst 123:2835