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Anal. Chem. 1985, 57,962-968
Multiwavelength Absorbance Detection for Solute Recognition in Liquid Chromatography Anton C. J. H. Drouen,* Hugo A. H. Billiet, and Leo De Galan
Laboratorium voor Analytische Scheikunde, Technische Hogeschool Delft, Jaffalaan 9, 2628 BX Delft, The Netherlands
4
Exlstlng software has been expanded to extract full Informatlon from UV spectra collected wlth a photodlode array detector in liquid chromatography. A four-step operator-interactlve strategy Is developed to establish the elution order of an unknown sample, a situation encountered In lattlce deslgn optlmlzaUon of LC separatlons. Flrst, a maxbnum absorbance plot Is used to dlvlde the chromatogram In base line separated peak groups. Second, the minimum number of solutes in each group Is established from elutlon proflies at selected wavelengths. Third, representatlve spectra for each solute are stored at selected elution times. Flnaily, the spectra from different chromatograms are matched. Examples demonstrate that composite proflles of severely overlapping solutes can be unraveled by judlclous human Interpretatlon.
The identification of solutes eluting from a liquid chromatograph forms a major problem. The retention times and peak areas provided by one-dimensional detectors (RI or single-wavelengthUV) are adequate only in the simple situation that a well-separated solute must be matched against a limited number of references. Neither condition is likely to be met in practice. The statistical analysis of Davis and Giddings (1)shows that peak purity is lost rapidly even when the number of solutes in the sample is well below the peak capacity of the chromatogram. For the true identification of an unknown solute, featurerich molecular spectra are probably indispensable. Unfortunately, the coupling of infrared or mass spectrometry with liquid chromatographyhas not yet been realized satisfactorily. In this study we address the challenge of recognizing unknown solutes in successive chromatograms, run under different conditions. Such a situation is encountered in lattice design techniques for the optimization of LC separations. The schemes developed by Kirkland et al. (2) and by ourselves (3-5) are based on a few chromatograms run a t selected mobile phase compositions. The response surface of the optimization criteria can then be calculated over the entire parameter space, provided that the retention time of each solute in each chromatogram is known. The commonly used strategy of injecting all solutes separately is not only slow but obviously not feasible for unknown samples. In that case we need a tool to match the corresponding solutes in different chromatograms. In a previous paper (6) we analyzed the potential of the two-wavelengthabsorbance ratio for this purpose. Although peak overlap could be detected down to a peak separation of only O.b, solute recognition generally required a separation exceeding 1.1%. Even then recognition was hampered by instrumental limitations and by the influence of the mobile phase composition on the absorbance ratio. This approach gains power when more wavelengths and, hence, more ratio values are collected. We then soon reach the stage where it becomes profitable to record the entire absorption spectrum. This is especially true since the mechanically scanning (7) and vidicon based (8) spectrophotometers have been superseded by the fast scanning linear 0003-2700/85/0357-0962$0 15010
photodiode array detectors, that record an entire spectrum in less then 10 ms with a repetition rate of 10 Hz. As has been shown by Horlick (9) the quality of the spectra is sufficient for modest identification purposes. Similarly, Wegner (10) identified cosmetic dyes by comparing their UV spectra with those of 20 pure standards. On the other hand, for the distinction of rather similar pharmaceutical compounds, Fell (11)had to increase the revealing power of the UV spectra by taking the second derivative. In all these examples the solute peaks were almost completely separated in the liquid chromatogram. Almost by definition, such an ideal situation will not be encountered in HPLC optimization schemes that intend to improve an initially poor separation. It then becomes mandatory to fist determine the number of solutes in a composite elution profile and, next, to extract spectra that can be used to match correspondingsolutes in successive chromatograms. The first step has received some attention in the literature. A major problem is to condense the massive amount of data collected during the chromatographic run into a manageable and easily interpretable format. Three-dimensional plots of all data in time-absorbancewavelength space are difficult to interpret and obscure many details. The isoabsorbance contours in the wavelength-time domain introduced by Fell (12) reveal more details but take a long time to plot and some experience to read. The peak purity may be tested more easily from chromatograms recorded at several wavelengths (13) or from a few spectra taken at selected positions in the compoeite peak profile (14). In the present study some of these techniques will be evaluated for the purpose of recognizing and matching solutes in different chromatograms. The approach developed relies on human interpretation of the data. Mathematical techniques such as factor analysis and deconvolution (15)will not be considered. In a previous study (16)we have shown that principal component analysis can be used to extract suitable spectra from a composite profile of three moderately overlapping peaks. In the present study we shall be interested in more difficult situations. EXPERIMENTAL SECTION The chromatograms were obtained with equipment consisting of a Hewlett-Packard chromatograph equipped with a 100 X 4.6 mm HP column packed with 5-pm RP-18 (Waldbronn, Germany). The detector was the Hewlett-Packard Model 1040Afast scanning LDA detector connected to a "-85 desktop computer, equipped with inputloutput, plotterlprinter, mass storage and advanced programming ROMs, 16 kbyte additional memory, and a HP-IB IEEE-488 interface. A HP7470A graphics plotter and a HP82910M dual 5l/., in. flexible disk drive were connected to the HP-85 via the HP-IB bus interface. The software for handling the 1040A, the storing of data, and plotting was the standard software version I (EVALU I) supplied by Hewlett-Packard. Additional software was developed in BASIC on the HP-85 to present the maximal-absorbancechromatogram,normalized spectra plot, and selected multiwavelength chromatogram as discussed below. RESULTS AND DISCUSSION The standard software, EVALU 1, supplied with the Hewlett-Packard 1040A detector allows a continuous storage of 0 1985 American Chemical Soclety
ANALYTICAL CHEMISTRY, VOL. 57, NO. 6, MAY 1985 963
spectra recorded with a given time interval up to 1o/s. Unfortunately, the resulting (long) data file is rather inaccessible with the supplied software (EVALU I) and only permits the retrieval of complete spectra at a fairly slow speed. Alternatively, predetermined cross sections of the UV data can be obtained in two other options. In one option a spectrum is stored only at an inflection point in the chromatogram, resulting in three spectra for each single peak (upslope, apex, and downslope). The other option allows a continuous recording of the chromatogram for up to eight preselected wavelengths. In practice, this software package was found to be very useful in assessing the purity of chromatographicpeaks either from the simultaneous record of eight wavelengths or from the spectra collected at the upslope and downslope of a peak. On the other hand, the preselected positions of spectrum recording did not allow matching of corresponding solutes in successive chromatograms unless they were reasonably separated. An illustrative example is offered by the analysis of diphenylamines considered in our previous paper on twowavelength absorbance ratio recording (6). Chromatograms run with methanol (MeOH) or tetrahydrofuran (THF) as organic modifier showed only four peaks that could not be matched, even when it was clear that one peak in tetrahydrofuran was impure. However, when a shoulder peak in the chromatogram run with acetonitrile (ACN) revealed the presence of a fifth solute, they were readily matched in all three chromatograms. We recall, that even this simple example could not be solved with the two-wavelength absorbance ratio (6). This and other examples demonstrate that for more demanding problems a greater freedom to choose the wavelengths and the spectrum positions is desirable. Therefore, the software was modified to enlarge the scope of the photodiode array detector and to gain access to all data collected during a record of full spectra with constant time interval throughout the chromatogram. To reduce the postchromatographic processing time and also to releave storage capacity of the background memory, no spectra are stored during the base line, except for a few used to correct for background (mobile phase) absorption. Spectra are also rejected when the absorbance falls below a threshold value (for the HP1040A this threshold is h5 mAU). The data are stored in a two-dimensional array according to wavelength and time, and can be subjected to the following manipulations. Similar to EVALU 1 a complete spectrum can be retrieved at any time slot. Also, a chromatogram can be drawn for any wavelength, rather than at eight preselected wavelengths. In addition, chromatograms can be drawn for the total absorbance or the maximal absorbance a t each time slot. On the basis of this software a four-step procedure was developed for solute recognition in successive, complex chromatograms. In the first step a chromatogram is divided into a series of base-line separated peak profiles. Since each profile may contain more then one solute or visibly discernible peak, we shall refer to this as a peak group. In the second step the number of solutes in each peak group is determined. In the third step a representative spectrum is seleded for each solute. Ideally, these spectra should resemble those of the pure solutes, but in the case of severe overlap this is not always possible. In the fourth and final step the spectra selected in successive chromatograms are matched to locate the position of corresponding solutes. The procedure will now be elucidated with the optimization of the separation of polynuclear aromatic hydrocarbons (PAH). The sample contains an unknown number out of 16 reference components distributed by the NBS (reference material 1647). Again the three initial chromatogramsare run
0. 00
tima. min
I* 00
Figure 1. The total absorbance (Atot),maximal absorbance (Amax), and single wavelength (330nm) elution profiles of a mixture of polynuclear aromatlc hydrocarbons in 65 % ACN.
in ACN, MeOH, and T H F and the data are collected as specified above. If desired, a single wavelength chromatogram can be traced with a pen recorder to check the operation of the chromatograph,but this is not essential for the procedure. The first task is to select the peak groups in the three chromatograms with a minimal effort without the risk of overlooking a component. Any single-wavelength plot is clearly inadequate, whereas a plot of all wavelengths is unpractical. The pseudoisometric three-dimensional representations of wavelength-time absorbances (so-called spectrochromatograms (17)or 3D plots (18)) are difficult to interpret, Low-absorption bands may be hidden behind highabsorption peaks and can be revealed only by rotating the 3D plot. In our experience the isoabsorbance contour plots proposed by Fell (12) are more useful, but they take a long time (3-6 h) to construct and the absorbance contour values must be selected with care to convey the full information. In analogy to the total ion current monitored in gas chromatography/mass spectrometry, the total absorbance can be recorded for the LC/UV combination (12). Here the total absorbance is simply the sum of all absorbances measured at a certain time slot. It offers the advantage of a rapid general survey of spectral information, but it entails the risk of overlooking solutes with a very narrow absorption band that has a low total absorption. Therefore, we prefer to also record the maximal absorbance chromatogram, in which each point refers to the highest absorbance value in the spectrum stored a t that moment. Solutes with narrow but high absorption bands are easily visible. Together with the total absorbance chromatogram all peaks will be detected. Figure 1 shows the total absorbance, the maximal absorbance, and a single wavelength chromatogram of the PAH mixture eluted in ACN. In all representations the highest value has been normalized to a full scale deflection. For the particular wavelength chosen (330 nm) two peak groups (1 and 2) are missed. In this particular example the total absorbance plot is slightly more revealing than the maximum absorbance plot. We have also observed examples where the reverse is true. In general, however, the relative heights of the chromatographic peaks vary considerably in both representations. It is, therefore, recommended to plot the chromatogram also with an expanded scale factor (about 10-fold the threshold value for the maximal absorbance plot, e.g., 50-100 mAU full scale for the HP1040A). This is done for the maximal absorbance plot of the PAH mixture in all three binary eluents in Figure 2. The numbers entered into the figure refer to the elution order of the corresponding solute to be clarified below. From Figure 2 we observe in ACN 6 peak groups (indicated by Roman numerals) with 10 clearly revealed peaks and 2 shoulders indicating at least 12 solutes. In MeOH we see again 6 peak groups, but only 10 distinct peaks. In THF we observe only 3 peak groups with 5 peaks, indicating a severe overlap in this eluent. Obviously, complete separation is achieved in none of the three eluents.
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IV
V
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Figure 2. Maximal absorbance elution profiles of 13 PAH’s recorded in 65% ACN, 80% MeOH, and 40% THF (thick lines scaled to 1 AU, thin Nnes scale expanded to 70 rnAU). Roman numerals indlcate base
line separated peak groups. The determination of the number of solutes in a particular peak group is elucidated in Figure 3 for some representative examples from the ACN chromatogram. We start by plotting a series of spectra collected during the elution of the peak group and normalized to the maximum absorbance in each spectrum. If all spectra coincide as in frame A1 of Figure 3, we conclude that the peak group (I in Figure 2) consists of a single solute. Of course, a similar picture results when two (or more) solutes coelute with exactly the same retention time or when they possess exactly the same UV spectrum. Such a situation cannot be revealed and can only be detected in the consecutive analysis when the solute recognition procedure fails to come to a satisfactory conclusion. Any variation in the sequence of absorption spectra as shown in the other A frames of Figure 3 indicates an impure peak group containing more than one solute differing in both retention time and UV spectrum. To determine the number of solutes, we select in each frame the wavelengths where the absorbance shows a large variation. For those wavelengths, indicated by numbeirs in frames A of Figure 3, elution profiles are plotted in frames B. Again, the profiies are normalized to a common maximum value. A pure peak will now show only one maximum (frame Bl),whereas an impure peak group gives rise to more than one maximum. The number of isolated maxima in the multiwavelength elution profiles or the number of “maxima” appearing in one single wavelength elution profile gives an indication of the number of solutes present in the peak group. Care must be taken when maxima are close to each other, because small sh3h can be caused by the influence of a nearby eluting solute. The two maxima in frame B2 point to a t least two solutes in
Peak group IV, whereas the presence of at least three solutes in peak group V is evident from frame B3. In a similar way the third and sixth peak groups in the ACN chromatogram (Figure 2) are readily concluded to contain two and one solutes, respectively. The second peak group analyzed in frames A4 and B4 of Figure 3 poses more problems. The presence of three solutes is readily apparent from frame B4. That the small shift in the first elution profile, observed between the wavelength 230 and 250-262 nm, actually indicates the presence of a fourth component is perhaps less obvious. The total number of solutes in the sample is thereby raised from an initial estimate of 12 to at least 13. The procedure for the chromatogram run in MeOH is similar to the one in ACN and will not be discussed further. Again 13 solutes are found, lending more trust in the number found for the chromatogram run in ACN. In the present example the greatest challenge resides in the second and third peak group of the chromatogram in\THF. This is elucidated in Figure 4. The first peak group in the THF run is pure as demonstrated by frames A1 and B1 of Figure 4. The second peak group contains at least four solutes: three indicated by the shift in the maxima of the main peak and one at the shoulder on the front of the peak (frame B2, Figure 4). The last peak group in THF contains certainly three solutes as is clear from the three maxima in frame B3 of Figure 4. However, the small shift in the maximum of the central elution profie indicates the presence of yet another solute in this peak group. This leads us to a confirmed number of only nine solutes in THF. On the basis of the normalized spectra and multiwavelength elution profiles, we can put a reasonable figure to the number of solutes present in the sample. In the chromatograms run with ACN and MeOH we have found 13 solutes distributed over six peak groups. In the chromatogram run with THF we have located only nine solutes. Therefore, the three peak groups in THF must contain four additional solutes. Obviously, the 13 solutes must still be matched between the three chromatograms. The next step, therefore, is the extraction of a representative spectrum for each solute. This is most simple for a pure peak arising from a single solute. Any spectrum sampled during passage of the peak is suitable, but the one taken at the maximum of the elution profile shows minimal noise and least interference from residual background. Examples are shown in frames C1 of Figures 3 and 4. The similarity of these two spectra indicates that the first eluting solute in ACN and THF is probably the same component. When a peak group consists of two solutes, the most representative spectra are found at the front and the tail end of the elution profile. In locating the positions where the spectra are extracted, a compromise must be sought between acceptable signal to noise ratio and spectral purity. The normalized elution profiles in frame B of Figure 3 can be used to locate the optimum positions. If, as in frame B2, the two solutes are reasonably separated, fairly pure and noise-free spectra can be readily obtained. They are plotted in frame c2.
If, however, the two solutes overlap t o a large extent, the result is less satisfactory. The two spectra recorded at 2.3 and 2.5 min in frame C4 of Figure 3 show very little difference despite the fact that they have been taken at the upslope and downslope positions of the elution profile. In that case the purity of the spectra can be enhanced by subtracting two spectra taken at either side of the elution maximum. This is illustrated in Figure 5. Figure 5A shows a part of the elution profile of frame B4 in Figure 3 retrieved from the data file for two wavelengths. The trace for a wavelength of 250 nm indicated the earlier
ANALYTICAL CHEMISTRY, VOL. 57, NO. 6, MAY 1985 065
A1
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E34
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Flgure 3. Analysls of peak group I, IV, V, and I1 in ACN (Flgure 2). Frames A represent spectra taken during elution of peak group. Frames 6 present elution profiles at wavelengths taken from frames A. Frames C present representatbe solute spectra at elution times taken from frame 6. Frames D present representative solute spectra taken from peak group 111 and VI.
eluting solute, the other trace for 230 nm indicates a second, later eluting solute. We now take first a full spectrum at 2.35 min, where the absorbance difference between the two wavelengths is largest. A second spectrum is taken at 2.5 min, where the absorbance at 230 nm is the same. Due to the severe overlap the two spectra are rather similar as shown in Figure 5B. However, their difference spectrum (2.35-2.5), also presented in Figure 5B, is representative for the earlier eluting solute. In a similar way, the substraction of two spectra taken a t 2.5 and 2.3 min yields a spectrum representative for the later eluting solute (Figure 5C). When in a complex peak group more than two solutes overlap, the multiwavelength elution profiies can again be used to locate the optimum positions to extract the appropriate spectra. In frame B3 of Figure 3 it is seen that the three
Table I. Peak Assignment Resulting from Matching the Spectra Collected in Figure 3 and Figure 4 peak group
65% ACN
1 2
1 2-3-4,5 6, 7 8-9 10-11,12 13
3
4 5 6
50% MeOH
1 2-3-4,5 6, 7 8-9
40% THF 1 2?-4-3-5-6-7 8, 9-12-10-13,
11
10-11,12
13
solutes in this particular peak group elute with retention times of 7.64,7.99, and 8.52 min. Spectra retrieved a t 7.99 and at 8.52 min will be representative for the two later solutes and little disturbed by the previous eluting solute. However, a
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m1
AI
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Figure 4. Analysis of peak group I, 11, and 111 in THF (Figure 2). Frames A represent spectra taken during elution of peak group. Frames B present elution proflles at wavelengths taken from frames A. Frames C present representative solute spectra at elution times taken from frame 8.
spectrum retrieved at 7.64 min would be significantly disturbed by the later eluting second solute. Therefore, a representative spectrum for the first solute is extracted at 7.54 min. If necessary or desired, the spectrum subtraction technique explained in Figure 5 can be used to enhance the characterisitic features in the spectrum. Again, the greatest challenge is the extraction of relevant spectra for the 12 solutes contained in the composite peaks of the THF chromatogram. The first peak group in T H F is pure, as indicated by the frames A1 and B1 in Figure 4. The relevant spectrum is selected at the top and plotted in frame C1. For the second peak group the spectra recorded during the elution of the shoulder on the front of the profile indicate one solute. The spectrum at 5.22 rnin is taken ae representative and reproduced in frame C2 of Figure 4. Spectra plotted during the elution of the main peak of this peak group reveal only a small and gradual change of the recorded spectra. This indicates a severe overlap in contrast with less overlapping solutes in which case the recorded spectra show a stepwise transition. In these situations no spectra can be selected that resemble the spectra of the pure solutes. However, characteristic absorption bands may still be observed and these can be used in the final step of matching the spectra to reveal the
elution order in the successive chromatograms. As an example frame C2 in Figure 4 shows one spectrum with some characteristic absorption bands of the middle peak of the second peak group (6.00 min). In the third peak group (frame B3 in Figure 4), the two extreme elution peaks seem to be pure (no change in the recorded spectra) and representative spectra are selected at 7.83 and 9.41 min. The spectra collected across the central elution profile in frame B3 show only small differences again indicating a severe overlap of solutes. Spectra at 8.10 and 9.01 min are selected for characteristic absorption bands and plotted in frame C3 of Figure 4. We now come to the final step of matching the selected spectra of the three recorded chromatograms to reveal the elution order. This matching is rather straightforward for the spectra selected from the chromatogramsin ACN and MeOH and results in the same elution order (compare the numbers in Figure 2). Again the challenge is the elution order in THF and this will be discussed in some detail. In doing so, we shall refer to the solutes by their elution order in ACN (Figure 2). Representative spectra are collected in Figure 3. For solutes 2 and 3 the difference spectra in Figure 5B,C are used. These solutes must now be identified in the chromatogram run in
ANALYTICAL CHEMISTRY, VOL. 57, NO. 6, MAY 1985
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Figure 5. (A) Partial elution profile of peak group I1 recorded In 65% ACN for 230 (-) and 250 (-- -) nm. (B) Spectra taken at 2.35 and
2.5 min together with the difference spectrum (2.35 - 2.5) representative for the first eluting solute. (C) Spectra taken at 2.5 and 2.3 min together with the difference spectrum (2.5 2.3) representative for the later eluting solute.
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T H F by matching the spectra with those in Figure 4. We first take the almost pure spectra of solutes 1,4,8, and 11. The spectrum of solute 1 (frame C1, Figure 3) matches that of the first eluting solute in T H F (frame C1, Figure 4). The spectrum of solute 4 (frame C4, Figure 3) is recognized in the THF spectrum taken at 5.22 min. Similarly, solutes 8 and 11 (frames C2 and C3 in Figure 3) are easily matched with the T H F spectra taken at 7.83 and 9.41 min (frame C3 in Figure 4). For the other nine solutes a direct match is not possible and the spectra must be searched for characteristic absorption bands. The high absorption band at 230 nm of solute 3 (Figure 5C) is also seen in the T H F spectrum at 5.46 min, whereas characteristic absorption bands of solutes 5 (254),6 (288 and 358 nm), and 7 (240,260,272,308,318,and 332 nm) are all found in the composite T H F spectrum at 6.00 min in frame C2 of Figure 4. We conclude,therefore, that the second peak group in the chromatogram in THF contains not only solutes 3 and 4 but also solutes 5, 6, and 7. In the third peak group of the THF chromatogram solutes 8 and 11 have already been identified above. Characteristic bands of solutes 12 and 13 (344,362, and 382 nm) are also seen in the spectra recorded for this peak group (frame C3 in Figure 4). Probably solute 12 preceeds solute 13 also in THF, because the absorption at 330 nm (indicative for solute 12) increases in going from 8.10 to 9.01 min in THF. These time slots also reveal characteristic bands of solute 9 (276 and 288 nm) and solute 10 (254 nm). Consequently, solutes 8,9,12,10,13, and 11 are found in that order in the third peak group of the THF chromatogram. The only unidentified component is solute 2, for which the difference spectrum in Figure 5B shows characteristic absorption bands at 260,288, and 298 nm. Unfortunately,solutes
967
6 and 7 also absorb at 286 and 260 nm, respectively. By analogy with the elution order in ACN and MeOH, solute 2 is, therefore, arbitrarily located in the second peak group in THF. The complete results of the solute matching are presented in Table I. During the continuation of the optimization procedure there was no indication that one of the elution orders was incorrect. Unfortunately, the optimum ternary composition of 4.8% THF, 57.2% ACN, and 38% H 2 0 yielded only a small improvement of the separation, compared to the separation already achieved with the binary mobile phase of 65% ACN. After the entire procedure had been completed, the elution order was checked by separate injection of each solute in the sample. These injections gave the same result as that shown in Table I. This example shows that with some ingenuity of the operator the elution order in successive chromatograms can be established,even when there is a severe overlap of many components. Obviously, the components must show some characteristic absorption bands by which they can be identified. It may be clear that collecting all measured spectra during the elution of a chromatogram in computer memory, permits the selection of appropriate wavelengths to reveal overlapping solutes. The normalized spectra plot (frames A in Figures 3 and 4) can be recalled directly from memory. The consecutively constructed normalized wavelength plots (frames B in Figures 3 and 4) give a strong indication of the number of solutes eluting in a peak group. Another advantage is offered when selecting the most pure spectra because, naturally, more spectra are available to single out this spectrum. The same applies for the correction of interfering solutes. The limitations of the present procedure are basically the same as those discussed for the previous sample, where solute matching was done with the standard software EVALU 1. It is highly desirable if not mandatory that an almost pure spectrum for each solute can be extracted from at least one of the initial chromatograms. However the extended software offers more potential to extract such almost pure spectra. When two or more solutes retain a severe overlap in all initial chromatograms,the procedure can lead to wrong conclusions. ACKNOWLEDGMENT
The work described in this paper has been supported by HewletbPackard, Waldronn, West Germany,through the loan and disposition of equipment. This support is gratefully acknowledged. LITERATURE CITED (1) Davls, J. M.; Glddlngs, J. C. Anal. Chem. 1983,55, 418. (2) Glajch, J. L.; Klrkland, J. J.; Squlre, K. M.; Mlnor, J. M. J . Chromatogr. 1980, 199, 57. (3) Schoenmakers, P. J.; Drouen, A. C. J. H.; Billiet, H. A. H.; De Galan, L. Chromatographia 1982, 15, 688. (4) Drouen, A. C. J. H.; Billiet, H. A. H.; De Galan, L. Chromatographia 1982,16,4a. (5) Haddad, P. R.; Drouen, A. C. J. H.; Billlet, H. A. H.; De Galan, L. J . Chromatogr. 1983,282, 71. (6) Drouen, A. C. J. H.; Bllliet, H. A. H.; De Galan, L. Anal. Chem. 1984, 56, 971. (7) Denton, M. S.; DeAngells, T. P.; Yacynych, A. M.;Helneman, W. R.; Gilbert, T. W. Anal. Chem. 1976,48, 20. (8) Pardue, H. L.; McDowell. A. Anal. Chem. i978, 4 8 , 1815. (9) Horlick, G.; Ccdding, E. G. Anal. Chem. 1973,45, 1490. (10) Wegener, J. W. M.; Grunbaur, H.J. M.; Fordham, R. J.; Karcher, W. J . Llq. Chromatogr. 1984, 7 , 809. (11) Fell, A. F.; Scott, H. P.; Gill, R.; Moffat, A. C. Chromatographia 1982, 16, 69. (12) Fell, A. F.; Clarck, B. J.; Scott, H. P. J . Chromatogr. 1984,286, 261. (13) Byllna, A.; Sybilska, D.; Grabowsky, Z . R.; Koszewskl, J. J . Chromatogr. 1973,8 3 , 357. (14) George, A. S.;Maute, A. Chromatographla 1982, 15, 419. (15) Lawton, W. H.; Sylvestre, E. A. Technometrlcs 1971, 13, 617.
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(16) Kateman, G.;Essers, R.; Vandeglnste, E. G. M.; Drouen, A. C. J. H.; Biiiiet, H. A. H.;De Galan, L.; Frank Jnz, J.; Duine, J. A. “The possibllites of Multiwavelength Detection In Blochemicai Separations Using LiquM Chromatography”; Analytical Methods and Problems in Biotechnology, 17-19 April 1984. Leeuwenhorst congress Center, Noordwijkerhout, The Netherlands. (17) Overzet, F.; Ghilsen, R. T.; Drenth, E. F. H.; De Zeeuw, R. A. J . Chro-
matogr. 1982, 240, 190. (18) Hewlett-Packard, Publication No. 12-5953-0095, 8/83 printed in the Federal Republic of Germany.
RECEIVED for review November 26, 1984. Accepted January 30, 1985.
High-Performance Liquid Chromatography-Controlled Potential Coulometry as an Absolute Method for Determining the Purity of Reference Standards Gary W.Schieffer Analytical Chemistry Department, Norwich Eaton Pharmaceuticals, Inc.,’ Norwich, New York 13815
A coulometric detector made of crushed reticulated vltreous carbon was used to determine the purity of reference standards by counting the number of coulombs generated as each compound eluted from a hlgh-performance llquld chromatographic column. The purity of acetamlnophen, L-dopa, ascorbic acid, hydroquinone, and 2,5-dlhydroxybenzoic acid could be determined with a relatlve accuracy and precision of 0.5 % or better by using reversed-phase columns wtth flow rates of less than 3 mL/mln and injecting the optimum range of 100-400 nmol of sample. The coulometrlc yield was monitored with a downstream amperometrlc detector.
Absolute methods, i.e., methods that do not require a pure form of the analyte as a standard, are desired whenever possible for assessing the purity of organic compounds to be used as reference standards for other methods. Controlled potential coulometry is a highly accurate and precise absolute method that uses the faraday, 96 487 C/equiv, as the standard. However, when performed in the classical batchwise manner with a mercury pool electrode for reductions or a platinum gauze electrode for oxidations, the technique suffers from poor selectivity. Any impurity or degradation product that is electroactive at or below the set potential will contribute to the number of coulombs counted for the analyte, possibly resulting in a false assumption of purity. For this reason, increasing the resolution of controlled potential coulometry by interfacing it with high-performance liquid chromatography (HPLC) would be desirable. Johnson and Larochelle (1) interfaced low-pressure liquid chromatography with a porous platinum electrode that operated with 100% conversion efficiency. An ion-exchange column was used to separate metal ions, which were then determined coulometrically. Strohl and Curran (2) used a reticulated vitreous carbon (RVC) electrode with a dead volume of 7.8 mL to perform controlled potential coulometry of organic compounds in flowing solutions. Curran and Tougas ( 3 ) designed an RVC electrode with a symmetrical counter electrode to serve as a coulometric detector for HPLC. Although the dead volume was 150 pL, the mass-sensitive nature of the detector yielded an effective dead volume of 7.8 pL. Despite conversion efficiency approaching 100% for flow rates less than 1 mL/min, the study focused on trace analysis with ‘A Procter & Gamble Company.
standards. In addition, the 150 p L actual dead volume precluded development of an efficient dual electrode system (4, 5). Other electrochemical detectors (6-8) and a commercial dual electrode system (5) have been designed to yield a coulometric response; however, studies with these systems have also centered on trace analysis with standards. This paper explores the feasibility of interfacing a coulometric detector with HPLC for compound purity determination without standards. EXPERIMENTAL SECTION Reagent Purity. Ascorbic acid (Hoffman-LaRoche, Nutley, N( ), L-dopa (Ajinomoto Co., Inc., Tokyo), and acetaminophen (k.allinckrodt, Inc., St. Louis, MO) were all reference standards ceT*tifiedat 100% by a variety of conventional spectroscopic, chromatographic, and titrimetric techniques including iodine titration for ascorbic acid, perchloric acid titration for L-dopa, and tetrabutylammonium hydroxide titration and p-(2,4-dinitrophen0xy)acetanilide gravimetric assay for acetaminophen. The purity of hydroquinone (EastmanKodak Co., Rochester,NY), 2,5-dihydroxybenzoicacid (Eastman Kodak Co.) and folic acid (Sigma Chemical Co., St. Louis, MO) was checked by an HPLC procedure in which the peak eluting from a reversed-phaseHPLC column was quantitatively collected and diluted to a known volume. The UV absorbance of this solution was then compared with the absorbance of a peak similarly collected but with the column removed and replaced by empty tubing. The purity of hydroquinone and 2,5-dihydroxybenzoicacid was estimated to be 100% by this procedure, while folic acid purity was estimated to be 93.9%. The samples were dried at 110 “C for 1 h prior to use, if necessary. Electrochemical Cells. The crushed RVC coulometric cell construction was similar to that shown in Figure 2 of ref 4 except that the body was constructed of Teflon instead of Plexiglas and the 2.2 cm by 2.8 mm i.d. cation exchange membrane tube was replaced by a 2.4 cm by 4.4 mm i.d. Vycor glass tube (Corning Glass Works, Corning, N Y ) sealed with O-rings at either end. A Coulochem Model 5100A dual electrode coulometric detector (Environmental Science Associates, Bedford, MA) was also used for some initial studies. A thin-layer cell of conventional design consisting of a 3.0-mm diameter glassy carbon disk and a 0.125-mm thick Teflon spacer was constructed in the laboratory and placed downstream from the coulometric cell. All potentials reported for the cells constructedin the laboratory are given with respect to a silver-silver chloride reference electrode (SSCE) which was 0.1 M in potassium chloride. The potentials for the Coulochem Model 5100A are given with respect to the proprietary internal pseudoreferenceelectrode. All coulometric determinations were made at an applied potential (normally
0003-2700/85/0357-0968$0 1.50/0 0 1985 American Chemical Society
