Anal Bioanal Chem (2002) 373 : 508515 DOI 10.

1007/s00216-002-1249-3

O R I G I N A L PA P E R

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)

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

Abstract The reversed-phase chromatography of a number of polymer additives has been undertaken with on-line characterisation via a combination of diode array UV, 1H NMR, IR spectroscopy and mass spectrometry. This combination of spectrometers enabled the on-flow collection of full UV, 1H NMR, IR and mass spectra for a range of common polymer additives in amounts ranging from ca. 230 to 900 g on-column. The practical difficulties associated with multiple hyphenation and potential future developments are discussed. Keywords HPLC Polymer additives Multiple hyphenation Spectroscopic characterisation

IR, UV, NMR and mass spectra [3, 4,5] have been reported. In a recent series of experiments, we have investigated the potential of such an HPLC-IR-UV(DAD)NMR-MS system in combination with D2O at high temperatures for the separation and analysis of ecdysteroids in plant extracts [6] and model pharmaceuticals [7] to obtain on-line spectra. Here, we describe further studies to evaluate this system, using a number of common polymer additives as models, with a more conventional reversedphase chromatographic system. These results are compared with those from earlier studies on the separation and spectroscopic characterisation of polymer additives with normal phase eluents and off-line FT-IR following on-line collection using various commercial (LC-Transform) interfaces [2,3].

Introduction
The use of techniques such as HPLC-MS and HPLC-NMR is now routine in many laboratories for the analysis of mixtures. This is possible as a result of the development of robust chromatographic interfaces that have evolved to the extent that relatively few compromises need to be made by either separation system or spectrometer. The successful development of this type of hyphenation has led to the construction of more sophisticated systems combining more than one spectrometer. Such multiple hyphenation (hypernation) is perhaps most widely developed for HPLC-NMR-MS (reviewed in [1]). Here, the complementary nature of the NMR and MS data provide a powerful and efficient method for the characterisation and identification of chromatographic peaks. However, even more elaborate concatenations of instruments are possible and systems capable of providing NMR, IR, MS [2] and

Experimental section
Reagents The model polymer additives (structures in Fig. 1) used in this investigation were Irganox 245 (triethyleneglycol bis-3(3-tertiarybutyl-4-hydroxy-5-methylphenylpropionate), BHA (butylated hydroxyanisole, 2-tertiary-butyl-4-methoxyphenol), BHT (butylated hydroxytoluene, 2,6-di-tertiary-butyl-4-methylphenol), Bisphenol A and Topanol CA (1,1,3-tris-(2-methyl-4-hydroxy-5-tertiarybutylphenyl)butane) (purchased from Sigma, Poole UK, Fluka, Gillingham, UK and Aldrich, Gillingham, UK). Samples were dissolved, at concentrations of 4699 g L1 (see text) in (CD3CN) (99 atom%, Aldrich, UK). For this investigation, a test mixture comprising Bisphenol A, BHA, Irganox 245, BHT and Topanol CA was prepared and analysed as described below. In addition, the various model analytes were chromatographed individually. The HPLC system consisted of a Constametric 3200 HPLC pump (Laboratory Data Control, Stone, Staffs UK) which delivered CD3CN-D2O (80:20 v/v) at 1.0 mL min1 to a 1004.6 mm Hypersil H5ODS (5 m 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 tubing, to a Bio-Rad FT-IR model FTS3000 Excaliber spectrometer (Cambridge, MA USA). The spectrometer was fitted with a Spectra Tech (Stamford, CT USA) Micro Circle Cell ATR (attenuated total reflectance) high-pressure stainless steel flow cell of 25 L volume. The flow cell was fitted with a zinc selenide ATR crystal. The spectrometer was purged with dry nitrogen to minimise any

D. Louden A. Handley LGC, 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, UK e-mail: Ian.Wilson@astrazeneca.com

509 was carried out in the pseudo-2D-mode at 500.13 MHz using a flow-through probe of 3 mm-i.d. with a cell volume of 60 L. Typically, 16 scans/FID per increment were acquired into 8 K data points each with a spectral width of 8278 Hz. Spectra were acquired using the NOESYPRESAT pulse sequence (Bruker Spectrospin, UK) to suppress residual water and acetonitrile resonances. 90-Pulses were used with an acquisition time of 0.5 s, a relaxation 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 constructed for these studies is shown schematically in Fig. 2. The present system showed a number of differences from that used in our previous investigations on hypernation for the characterisation of polymer additives. Thus, in the earlier study [3] a triple quadrupole mass spectrometer was employed rather than the single quadrupole instrument used here, whilst the flow probe of the NMR spectrometer had a volume of 120 rather than 60 L. 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 designed for on-line deposition of chromatographic peaks on a rotating germanium disk for subsequent off-line spectroscopy. In contrast, with the current system, all of the spectra (including IR) were acquired on-flow. Another difference in layout for this system was that the post column splitter, used to send the minor portion of the flow to the MS, was placed immediately after the HPLC column rather than after the UV detector, as used previously. In the current system, the bulk of the eluent from the splitter was directed first to the FT-IR, from which it then flowed to the UV and NMR spectrometers. Finally, the variable wavelength UV detector that had been employed to monitor the chromatographic separation was replaced with a UV-VIS DAD spectrophotometer, capable of providing spectra over the range 1881000 nm. An important limitation of the prototype system described here was the lack of an integrated data capture system and, as indicated in Fig. 2, spectra were acquired by each individual spectrometer. This lack of integration inevitably meant that some of the advantages obtained from acquiring all of the spectral data simultaneously were negated because of the need for several operators to control the various instruments, and for the manual collation of the spectra post-run.

Fig. 1 Structures of the model compounds water vapour interference of the collected spectra. Any residual signal from water vapour in the collected data was subtracted using a water vapour reference spectrum. Spectra were acquired with the kinetics software, enabling Gram Schmidt reconstruction of the on-flow data. 57 Scans per spectrum (10 s acquisition time) were collected using a sensitive MCT (mercury cadmium telluride) liquid nitrogen-cooled detector. The spectra were acquired at 8 cm1 spectral resolution. The sample data were ratioed against those for a background spectrum of the flowing solvent through the cell prior to injection of the sample solution, thus automatically subtracting out the solvent spectrum from the sample spectra. The remainder of the flow (ca. 5%) was directed to a Micromass Platform single quadrupole mass spectrometer (Micromass UK, Wythenshawe, UK) via 4.5 M of 75 m-i.d fused silica capillary tubing (this length of tubing generated sufficient back pressure to direct the bulk of the flow to the FTIR). Mass spectra were measured over a range of 115650 Daltons. Prior to introduction of the sample into the ion source of the mass spectrometer, the eluent was mixed with a make up flow of 90:10 (v/v) methanol/water. This was delivered using a second Constametric 3200 pump and was introduced via a t-piece at 0.5 mL min1. Positive ion spectra were recorded with a cone voltage of 25 V, a scan time of 0.9 s and an inter 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 array detector (Bruker, Coventry, UK) via 30 cm of 0.005-i.d. PEEK tubing. UV spectra were collected over the wavelength range 1881000 nm. From the UV detector the solvent stream was directed to the NMR via 280 cm of 0.010-i.d. PEEK tubing. All of the instrumentation described above was located outside the 5 Gauss line of the stray magnetic field generated by the 500 MHz NMR spectrometer. NMR spectra were acquired using a Bruker DRX-500 NMR spectrometer. On-flow 1H NMR detection

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

510 Fig. 2 A schematic representation of the HPLC-IR-UVNMR-MS system used in these investigations

of NMR spectroscopy, of allowing the observation of exchangeable protons on e.g. phenolic hydroxyls. The volatility of the solvent was also valuable in simplifying the conditions required for evaporation when the eluent was collected for off-line FT-IR. However, unmodified chloroform was unsuitable when electrospray MS was included in the system. This was due to the dryness of the solvent that prevented ionisation due to the lack of a suitable proton donor. Useful MS data were only obtained when the chloroform was modified with deutero-methanol (CD3OD) and ammonium acetate (10 g L1 in D2O) [3]. Whilst this expedient enabled mass spectra to be obtained, it also resulted in the appearance in the NMR spectra of additional, unwanted, resonances for these additives, and the loss of signals for exchangeable protons. In the current series of studies where solvent volatility was not a consideration, reversedphase chromatography, on conventional C-18 bonded packing materials was used, to more easily obtain MS data. The relatively non-polar nature of these polymer additives required the use of high proportions of acetonitrile in the mobile phase to ensure elution in a reasonable time. The resulting large signal for the protons present in the acetonitrile methyl resonance meant that it was better to use deuterated (CD3CN) acetonitrile rather than the protonated form of the solvent. Suitable chromatography of the test compounds was obtained with D2O-CD3CN 20:80(v/v). The chromatography of a test mixture consisting of Bisphenol A (371 g), BHA (228 g), Irganox 254 (332 g), BHT (992 g) and Topanol CA (338 g) is shown in Fig. 3. Baseline resolution was achieved for Irganox 254, BHT and Topanol CA, but the earliest eluting components, Bisphenol A and BHA, chromatographed as a single peak. However, as discussed below Bisphenol A eluted in the leading edge of the combined peak and BHA in the tailing edge, enabling spectra to be obtained for each component. In addition to their chromatography as a mixture, each compound was also chromatographed individually in order to obtain a reference library spectrum.

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

Infrared spectroscopy The quantities of compound present in the chromatographic peaks from the test mixture generally enabled the acquisition of good quality IR spectra on-flow. Thus, examination of the IR spectra from the leading and tailing edges of the combined peak of Bisphenol A and BHA revealed clear diagnostic spectra for the individual components (Fig. 4a,b). Similarly, diagnostic spectra were obtained for both Irganox 245 and BHT and representative spectra for these compounds are illustrated in Fig. 4c,d). The only difficulty encountered in obtaining IR spectra from the test mixture under these conditions was seen with Topanol CA. For this compound the signals for the analyte coincided with those of the solvent, and spectra

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could not be acquired. Where spectra were obtained, the spectral matches against standards were very high (99.12% for Bisphenol A, 91.58% for BHA, 97.36% for Irganox 245 and 97.23% for BHT). Ultraviolet spectroscopy As seen with IR spectroscopy, the quantities of polymer additives used here provided no particular challenge with respect to sensitivity for the UV/VIS-DAD spectrometer. The resulting 2D-UV chromatogram for the five-compound test mixture is shown in Fig. 5. The first eluting peak is composed of a mixture of Bisphenol A and BHA and although the peak appears symmetrical (Fig. 3), spectroscopic examination showed the components to be partially resolved, with Bisphenol A eluting in the leading edge of the peak and BHA in the tailing edge. Evidence for the non-homogeneous nature of this peak can be seen in the distorted shape of the contour plot for the 260 300 nm region as shown in Fig. 3. The individual UV spectra obtained from this two-component peak (inset into Fig. 5) reflect this in the different UV maxima of the two analytes. Thus Bisphenol A gave a secondary UV maximum absorption at 275 nm, whilst that for BHA was at 285 nm. These values are comparable to those obtained for the individual standards chromatographed under the same conditions. Similarly, the UV spectra for the remaining compounds in the test mixture were comparable to those of the individual standards. Representative UV spectra obtained for each component in the mixture are shown in Fig. 5.

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

Fig. 5ae The 2D-UV chromatogram (254 nm) for the mixture shown in Fig. 2. The inset UV spectra are for Bisphenol A, BHA, Irganox 245, BHT and Topanol CA as indicated

512 Fig. 6ae The 2D-1H NMR chromatogram for the mixture shown in Fig. 2 with spectra for a Bisphenol A, b BHA, c Irganox 245, d BHT and e Topanol CA extracted from the 2D-profile

NMR spectroscopy In initial experiments an attempt was made to use CH3CN rather than CD3CN as the organic modifier. However, whilst it was possible to obtain spectra using CH3CN, obtaining adequate suppression of the signals due to the solvent protons caused difficulties in on-flow measurements. This problem was circumvented by the use of fully deuterated CD3CN. In this way, diagnostic 1H NMR spectra were obtained for all of the compounds in the test mixture. In the case of Bisphenol A and BHA which chromatographed as a single peak, it was still possible to obtain the appropriate spectra from the leading and tailing edges of the peak, as was the case with IR and UV spectroscopy. The pseudo-2D-NMR data obtained for the test mixture are shown in Fig. 6 together with the 1D-spectrum of the individual components extracted from the data (Fig. 6ae). The spectra were of good quality and showed all 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 on its structure. Thus, the spectrum comprised two sets of triplets 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 carboxy-function). The latter were significantly attenuated as a result of the suppression of the residual water signal (3.58 ppm). A single line at 3.44 ppm represented the central ethylene glycol moiety. The aromatic protons resonated at 6.73 and 6.8 ppm, whilst the tert-butyl group and methyl group gave signals at 1.27 and 2.09 ppm, respectively. Suppression of the residual acetonitrile signal (1.9 ppm), resulted in the slight distortion of the singlet at 2.09 ppm. The quantities of each of the compounds in the

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

Mass spectrometry As with the UV spectroscopy described above, the quantities of material present in the peaks were more than adequate to obtain spectra for the five additives in the model mixture. The total ion current (TIC) obtained is shown in Fig. 7a. Deuterated radical cations were obtained for Bisphenol A, BHA and BHT (mass chromatograms are shown in Fig. 7c,e,f). In the case of Topanol CA, a molecular ion was not observed as the compound fragmented in the ion source to give an ion at m/z 192 (Fig. 7b). Irganox 254 was observed as a polydeuterated molecular ion at m/z 592 (mass chromatogram shown in Fig. 7d). The much greater degree of deuteration of Irganox 245 than would have been expected on the basis of the exchangeable protons on the structure represents an unusual finding and the mass spectrum obtained shows an unusual isotope pattern (Fig. 7d). Given that the NMR data obtained concomitantly showed no evidence for facile deuterium exchange, these data suggested that deuteration occurred in the ion source of the MS rather than in the HPLC mobile phase. Further, off-line, investigation of this phenomenon suggested that this polydeuteration involved the replacement of the protons adjacent to the carbonyl group (unpublished observations). One feature that is also apparent from some of the mass chromatograms shown in Fig. 7 (and particularly in Fig. 7a,b) is that there are periodic disturbances in the baseline. These oscillations would seem to have a period of ca. 1 min and it is noteworthy that the peak for Topanol

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Fig. 7af Mass spectra obtained for the mixture shown in Fig. 2 with 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)

CA appears to be split into two in phase with this cycle. These periodic oscillations are also well illustrated in the two-dimensional mass chromatogram in Fig. 8a, where there is the appearance of regular bands of masses at ca 1 min intervals (most apparent following the elution of Irganox 245, at ca. 3.0 min). However, it is also noteworthy that the various peaks for the analytes are also irregular over shorter timescales. An example of this is seen in Fig. 8b where the trace for m/z 484, one of the higher mass ions associated with Irganox 245 (see Fig. 8c), can be seen to be formed from three distinct peaks. None of these phenomena were observed in the UV and NMR chromatograms. It seems likely that these observations are an artefact, linked in some way to the use of two separate pumps to deliver the eluent from the HPLC column and the make up flow to the mass spectrometer. The measured piston cycle time is ca. 10 and 20 s for this type of pump at flow rates of 1 and 0.5 mL min1 respectively. An irregular flow of solvent into the MS might result in compounds becoming deposited in the probe (e.g. as a result of the probe temperature changing) and then being evaporated as conditions in the cycle changed. It is also possible that changing solvent composition through this hypothetical cycle could have had a similar effect. It may be relevant to note that these oscillations and effects on peak shape were not observed in studies where more polar, water soluble, compounds were analysed with pure D2O as the chromatographic eluent and methanol/water (90:10) as the make up flow [7,8]. Clearly the elimination of this unwanted artefact would be desirable in any future work. Irrespective of the reason for this artefact, it might be prudent to employ eluents and make up flow solvents that are matched in terms of solvent composition as a matter of course in future designs.

Fig. 8 a 2D-Mass chromatogram for the mixture shown in Fig. 2. b Mass chromatogram for m/z 484. c Mass spectrum for Irganox 245 taken at RT 3.01 min

514 Fig. 9ad Spectra for the unknown, identified on the basis of a NMR, b UV, c IR and d MS data as BHT

Using this system, spectroscopic data were also acquired on a suspected polymer additive which was thereby conclusively identified as BHT. The combined spectroscopic data for this material are shown in Fig. 9. As we have noted in previous hypernation studies, the best sensitivity that can be achieved with any particular system is that attainable by the least sensitive spectrometer(s). However, the quantities of material used in the test mixture here were not designed to explore the limits of detection of this layout, but to compare the utility of onflow, on-line spectroscopy compared to a mixed on-line off-line method. In that context, the minimisation of the solvent interferences in the 1H NMR spectrum of BHT resulting from the use of the CD3CN-D2O mobile phase, compared to the highly modified chloroform system needed previously, is noteworthy. In addition, being able to obtain IR spectra on-line clearly has the potential for the more rapid analysis of mixtures. With respect to IR spectroscopy however, the off-line methodology provided good quality spectra with no interferences from the mobile phase and the potential for very high sensitivity. However, in considering the potential of hypernation in relation to the results described for this prototype system, it should be borne in mind that the instrumentation used in this study was relatively modest by modern standards and certainly was not state-ofthe-art. Whilst we made no particular investigation of sensitivity in this study, our previous experiments [6,7] with this layout suggest that for NMR and IR (generally the least sensitive instruments in the array of spectrometers assembled here) at this flow rate, ca. 100 g of each compound would be need to obtain an identifiable spec-

trum. There is no doubt that sensitivity could be improved and good results in the low g- and even ng-range have been achieved in miniaturised flow NMR systems that have been linked to a variety of separations [8, 9, 10,11]. Furthermore, the current layout was designed to further test the concept of hypernation and there is no doubt that the system was not fully optimised with respect to limiting peak broadening and flow rates etc. Rather than demonstrating the full potential that could be achieved with a state-of-the-art hypernated system, the studies reported here provide a pointer to what could be done.

Conclusions
The value of HPLC-NMR-MS is now accepted, and such combinations are commercially available. However, it is still not clear whether the benefits of hypernated systems of the type described here outweigh the costs of assembling such an array of instruments. However, the results illustrated using this prototype hypernated system, together with our previous studies [3, 4, 5, 6, 7,8], do demonstrate that the concatenation of chromatography to a wide range of spectroscopic detectors does not present overwhelming difficulties. With further optimisation to minimise band broadening, and using state-of-the-art instrumentation, we have no doubt that diagnostic UV, IR, 1H NMR and MS spectra could be obtained on-flow with modest quantities of material. Should such a system be considered to represent a worthwhile objective, progress would also be required to produce fully integrated data acquisition systems in order for such hypernated systems to achieve their full potential.

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