960
Anal. Chem. 1984, 56,906-971
first case and octadecanal in the second. Identities of the two aldehydes were confined by matching retention times with authentic samples measured on two different stationary phases (SE-30 and Carbowax-20M). In addition, the 2,4-DNP derivatives of authentic hexadecanal and octadecanal perfectly matched the retention times of peaks 1 and 2 in HPLC. While the above examples are representative of biologically interesting applications, this preconcentration and chromatographic procedure can be used in a variety of chemical studies. Depending upon the information required and/or a previous knowledge of sample composition, the isolation procedure could be simplified through elimination of certain steps. In addition, the wide linear range and the possibility of further chemical studies from collected fractions make this method quite useful for a variety of analytical problems. Registry No. Octadecanal, 638-66-4;hexadecanal, 629-80-1; 2-heptanone, 110-43-0; nonanal, 124-19-6; 2-pentadecanone, 2345-28-0; l-carvone, 6485-40-1; 2-octanone, 111-13-7.
(4) Kuwata, K.; Ueborl, M.; Yamasaki, Y. J. Chromarogr. Scl. 1979, 17, 264-268. (5) Fung, K.; Grosjean, D. Anal. Chem. 1081, 5 3 , 188-171. (6) Llebezeit, G. HRC CC, J . High Res. Chromafogr. Chromatogr. Commun. 1980, 5(4), 215-216. (7) Sellm, S. J. Chromatogr. 1977, 136, 271-277. (8) Rhodes, G. R.; Miller, M.; McConnell, M. L.; Novotny, M. Clin. Chem. (Winston-Salem, N . C . ) 1981. 27 (4), 580-585. (9) Raymer, J.; Wiesler, D.;Novotny, M.; Asa, C.; Seal. U. S.; Mech. L. D. Experienfia , in press. (IO) Scoggins, M. W. Anal. Chem. 1973, 45, 2204-2207. (11) Keeney, M. Anal. Chem. 1957, 2 9 , 1489-1491. (12) Novotny, M.; Lee, M. L.; Bartle, K. D. Chromatographia 1074, 7 (7), 333-338. (13) Rhodes, G.; Holland, M. L.; Wiesler, D.; Novotny, M.; Moore, S. A,; Peterson, R. G.; Felton, D. Experlentla lg82, 3 8 , 75-77. (14) Scott, R. P. W.; Kucera, P. J . Chromarogr. 1979, 185, 27-41. (15) Yang, F. J. HRC CC, J . High Res. Chromatogr. Chromatogr. Commun 1980, 3 , 589-590. (16) Novotny, M.; Alasandro, M.; Konishl, M. Anal. Chem. 1983, 55, 2375-2377. (17) Schaumberg, H. H.; Spencer, P. S. J . Neuropathol. Exp. Neurol. 1977, 36, 276-299. (18) Spencer, P. S.; Sabri, M. I.; Schaumberg, H. H.; Moore, C. L. Ann. Neurol. 1978, 5 , 501-507.
.
LITERATURE CITED (1) Raymer, J.; Novotny, M. I n "Trace Analysis"; Lawrence, J. F., Ed.; Academic Press: New York, In press. (2) Lipari, F.; Swarin. S. J. J . Chromatogr. 1982, 247, 297-306. (3) Mansflekl, C. T.; Hodge, E. T.; Hege, R. B., Jr.; Hamlin, W. C. J. Chromafogr. Sci. 1977, 15, 301-302.
RECEIVED for review November 14, 1983. Accepted January 26,1984. This work was supported by Grant No. GM 24349 from the Institute of General Medical Sciences,Department of Health and Human Services, US. Public Health Service.
Determination of Basic Nitrogen Compounds in Coal-Derived Products by Microbore Liquid Chromatography with Fourier Transform Infrared Spectrometric Detection Patricia G . Amateis and Larry T. Taylor* Department of Chemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061-0699 Hlgh-performance llquld chromatographycoupled wlth Fourier transform infrared spectrometry was employed for the analysls of bask nltrogen compounds such as anilines, pyrldlnes, and qulnollnes. A mlcrobore amlno (NH,) bonded phase column and a 7030 CDCI,:CCI4 0.02 % trlethylamlne mobile phase were used to achleve a separation of model compounds wlth detectlon afforded by flow-cell FTIR. The resultlng I R spectra of such compounds were studled as were resolutlon and column overloading consideratlone wlth mlcrobore columns. An Identical chromatographlc system was applied to the analysls of hlghly polar coal-derlved products. I n addRlon to some of the nltrogen compounds expected, such as tetrahydroqulnollne and qulnollne, carbonyl species includlng y-butyrolactone were found to be present.
+
In ow laboratory we have investigated via high-performance liquid chromatography coupled with Fourier transform infrared spectrometry nonpolar substances ( I ) , neutral nitrogen compounds (2), and phenolic material (3) in coal-derived liquids. Emphasis is now being placed on the analysis of basic nitrogen compounds such as azaarenes and amines in similar samples. These basic compounds are environmentally important and many have been shown to possess mutagenic activity ( 4 , 5 ) . Furthermore, the presence of these compounds in coal-derived products is important regarding the extent of the liquefaction process and the quality and stability of the resulting synfuels (6). Previous chromatographic determinations of amines and azaarenes were performed in various ways. Work with HPLC
resulted in separations via both the reversed-phase and normal-phase modes (7-9). Various mobile phase systems and columns were employed in these studies to separate azaarene compounds containing one to several rings. Cation exchange chromatography was found to separate primary aromatic amines from azaarenes. An ion-pairing chromatographic study (10) suggested that heterocyclic nitrogen compounds were retained as a group to a greater extent than amines. These trends are related to the basicity of the compounds. Standard ultraviolet detectors were used in all of the work cited above. Thin-layer chromatography (11) and GC/MS were also employed to separate and identify basic nitrogen compounds. Ho et al. (4) and Schiller (12) analyzed synfuel products for alkyl-substituted pyridines and multiring azaarenes by GC/MS. Tomkins and Ho (13) performed GC/MS on synfuel samples after derivatization with trifluoroacetic anhydride. Derivatization is often necessary because many nitrogen compounds are not resolved by GC and free amines cannot be distinguished from their methylated azaarene isomers because their parent ions are identical. It has also been suggested that electron impact mass spectra of nitrogen heterocycles give little structural information of relevance in identifying specific isomers (14, 15). We have chosen HPLC in the normal-phase mode for the identification of azaarenes and aromatic amines in coal-derived products. Our mode of detection is on-line Fourier transform infrared spectrometry. This analytical method offers advantages over many GC/MS methods in that nonvolatile material may be handled, derivatization need not be performed prior to analysis, and isomer identification is possible. The disadvantages due to sensitivity and mobile phase in-
0003-2700/84/0358-0966$01.50/0@ 1984 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 56, NO. 6, MAY 1984
frared transparency have been partly addressed by employing microbore columns and deuterated and/or halogenated chromatographic solvents. The use of low flow rates (pL/min) with microbore columns results in low solvent consumption which has advantages with respect to cost and disposal. Microbore columns also provide an increase in eluent concentration over analytical scale columns for a similar amount of injected material. EXPERIMENTAL SECTION Materials. Chloroform and carbon tetrachloride were obtained from Burdick and Jackson Laboratories, Inc. (Muskegon, MI). Deuterated chloroform (99.6% deuterated) and triethylamine were obtained from Aldrich Chemical Co. (Milwaukee, WI). Hexane and methanol were purchased from Fisher Scientific (Fair Lawn, NJ). All solventa were dried and maintained over molecular sieves before use. Model compounds were also purchased from Aldrich Chemical Co. Equipment. Separations were performed on two 50 X 1mm id. coupled microbore columns packed with 10-km amino ("2) bonded silica by CM Laboratories (Nutley,NJ). A microanalytical chromatography system (MACS) from EM Science (Gibbstown, NJ) was employed which consisted of a MACS 100 pump capable of operating at low flow rates (pL/min), a Rheodyne Model 7413 injector with 0.5, 1.0-, and 5.O-pL internal loops, and a MACS 700 variable-wavelength ultraviolet detector with a 0.5-pL 1-mm path length flow cell. A Nicolet 6000C FTIR equipped with mercury-cadmiumtelluride detectors (Models 7010A and 7010B) was used for IR detection of chromatographicseparations. The flow cell consisted of a modified Nicolet cell holder with a 0.2-mm KBr flow cell. The overall volume of this cell was estimated to be 3.2 pL. The standard Nicolet FTIR software package was used to collect 4 cm-' resolution spectra with a time resolution between spectra of approximately9.5 s (e.g., each spectrum is a ratio of 12 sample scans to 24 background scans at a mirror velocty of 0.59 cm/s). Sample Preparation. Model mixtures of amines and azaarenes were prepared by dissolving the compounds in the mobile phase. The mixture consisted of approximately 4 pg of the earlier eluting components and 6-7 pg of later eluting components for FTIR detection. With UV detection the total mass of model compounds injected was 1 pg. The coal-derived sample employed in this study was received from the Kerr-McGee Corp. (Cresent, OK); liquefaction of Wyodak No. 3 coal was carried out by them with 1,2,3,4-tetrahydroquinolineas the process solvent. After the process had been completed,gases were vented and the reaction vessel was emptied. The reaction vessel was then washed with tetrahydrofuran to remove material (residue and process solvent) remaining in the vessel. Material which was solubilized in THF was designated THF solubles and was chosen for analysis via pHPLC/FTIR. Thii sample was subjected to a preliminary preparative separation on a "Bond Elut" NH, column (2.8 mL capacity) from Analytichem International (Harbor City, CA) in order to provide simpler fractions for analysis. Four fradions were generated by successive elution with one volume of hexane and two volumes each of carbon tetrachloride, chloroform,and methanol. pHPLC/FTIR results for fraction 2 (eluted with CC1,) will be discussed in this paper. One microgram of this fraction was injected when UV detection was utilized whereas up to 50 pg of the sample was injected when employing FTIR detection. RESULTS AND DISCUSSION Separation of Basic Nitrogen Model Mixture. Thirteen heterocyclic nitrogen compounds and aromatic amines (Table I) were investigated in our laboratory by pHPLC/FTIR as representatives of basic polar compounds that could be present in coal-derived materials. Although reversed-phase systems have been routinely used for separation of basic nitrogen compounds, use of FTIR detection with ow current flow cell precludes the employment of aqueous and alcoholic mobile phases due to intense absorbance of these solvents over a wide range in the IR region. We have chosen, therefore, to employ a normal-phase mode.
967
Table I. Heterocvclic Nitrogen Compounds and Aromatic Amines-
v3 0
1.
N-Methylanil i n e
2.
7,8-Benzoquinol i n e
3.
1,2,3 ,4-Tetrahydroqui no1 ine
o-Toluidine @ 3
5.
2-Benzyl p y r i dine
pKab
24.75
4.05
24.96
4.21
25.17
5.03
27.04
4.44
27.82
5.13
27.07
5.83
ti
"2 4.
m
R.T.~
mcti %2
6.
Quinaldine
7.
4-Azafluorene
28.91
8.
9.
5.6-Benzoquinol i n e
29.64
4.90
29.95
5.11
\
10.
2,3-Cyclohexenopyridine
11.
Aniline
12.
3-picoline
0' 0
13. P y r i d i n e
@"
@
30.68
30.99
4.63
31.72
5.68
32.03
5.25
aRetention time, minutes bAqueour values
The solvent chosen as mobile phase must have good chromatographic properties and large regions of IR transparency. Several different normal-phase systems were investigated. Most systems met with little success. Cyano, polar aminocyano, and silica columns had excessive retention times, irreversibly adsorbed the nitrogen compounds, and caused peak tailing with mobile phases of chloroform or binary mixtures of chloroform and acetonitrile. However, a bonded phase amino (NH,) column was found to elute the compounds with 70:30 CDC13:CC14+ 0.02% triethylamine (TEA), a solvent system compatible with our IR detector. The single-beam FTIR spectrum of this mobile phase is presented in Figure 1. The bold line in the spectrum represents regions of IR transparency. The break in this line is a region of total IR absorbance (less than 5% transmittance) for which compensation cannot be made during an pHPLC/FTIR run. Therefore, information is lost in this region in the file spectra that are obtained during a flow cell experiment. A wide range of transparency is achieved with this mobile phase. Carbon tetrachloride is known to be a very good IR solvent, and by use of deuterated chloroform the valuable carbon-hydrogen stretching region becomes transparent. A small amount of TEA was also needed in the mobile phase in order to prevent tailing of the basic nitrogen compounds on the NH, column. TEA, no doubt, deactivates unreacted silanol groups on the column packing material. At only 0.02%, TEA presents no interference in the IR region. The nitrogen models are listed in Table I with their retention times calculated from single injections and their corresponding aqueous pK, values. Several mechanisms have been proposed for retention on NH, columns. Hammers et al. (16) suggested that the nitrogen atom in amines and
968
ANALYTICAL CHEMISTRY, VOL. 56, NO. 6, MAY 1984 7 0 : 3 0 CDCL3:CCLk
. 3.C2%
TEO
/\
a
B
Flgure 1. Single beam FTIR spectrum of the mobile phase 70:30 CDCI,:CCI, -I- 0.02% triethylamine, 0.2 mm path length cell.
heterocyclic compounds forms a hydrogen bond with unreacted silanol groups on the packing material. The NH2 moiety bonded on the column material was also suggested to form a donor-acceptor complex with an electrophilic part of the sample molecule (e.g., an aromatic ring). Other workers (8,9)found that retention was dependent on steric availability of the electron pair of the basic aza nitrogen. Our retention data indicate base strength and steric hindrance both influence retention on the NH2 column under our chromatographic conditions. There appears to be a general trend in our data which suggests that stronger bases have longer retention times (Table I). This observation supports the idea of hydrogen bonding with free silanol groups. However, when the nitrogen functionality is sterically hindered, hydrogen bonds cannot as readily form, and such compounds are seen to elute earlier than would be expected on the basis of their pKa values alone. For example, on comparison of quinoline (pK, = 4.90) with quinaldine (pKa = 5.83) and pyridine (pKa = 5.25) with 3picoline (pK, = 5.68) the more basic compound of each pair actually elutes prior to the less basic material because of the steric effect of the nearby methyl group on the ring nitrogen in the more basic compound. The mixture of 13 compounds was chromatographed as discussed above employing both UV (254 nm) and FTIR detection. Both chromatograms are presented in Figure 2. Figure 2B is a Gram-Schmidt (17) reconstructed chromatogram. The two chromatograms differ in appearance because UV monitors electronic excitation and IR monitors vibrational excitation modes. Seven peaks are reasonably resolved with UV detection whereas eight peaks are observed in the GramSchmidt reconstruction. The numbers above the peaks in Figure 2B correspond to file numbers for the individual spectra. Examinationof individual IR spectra obtained during the pHPLC/FTIR run allows peak assignment of all 13 model compounds. Because IR scans are taken continuously throughout the LC run, the front, middle, and back portions of a peak may be examined to see how the peak composition changes with time. This sometimes allows “spectroscopic” resolution of compounds for which chromatographic resolution is poor. Figure 3 illustrates such a case. File spectrum no. 166 (Figure 2B) corresponds to a peak maximum and is shown to be quinoline (Figure 3). The peak under consideration (Figure 2B) has a slight shoulder indicating the presence of another component. File spectrum no. 173 (Figure 3) identifies this compound to be 5,6-benzoquinoline. The arrow in Figure 2B indicates the point of elution of this compound. Representative “on-the-fly”spectra of 1,2,3,4-tetrahydroquinoline (file no. 123), quinaldine (file no. 147), aniline (file no. 184), and 4-azafluorene (file no. 155) are shown in Figure 4. These particular spectra were taken at the maximum of each peak and are typical of the solution-phase IR spectra of these nitrogen compounds. Azaarenes have very weak IR bands and lack a unique vibrational mode such as the carbonyl mode in ketones or the -OH mode in phenols. The charac-
FILE NV(BERS
Flgure 2. Separation of basic nitrogen compounds: (A) UV detection (254 nm) numbers over peaks correspond to numbers in Table I,total of 1 p g of models injected; (B) FTIR detection, numbers over peaks correspond to file numbers of individual spectra, peaks are numbered to correspond with the numbers in Table I.
DFN 166 QUINOLINE
ff
11 7,s-Benroquinoline
I1
Figure 3. File spectra of quinoline and 5,6-benzoquinohe (obtained during the pHPLC/FTIR run) and 7,8-benzoquinoline (an off-line static spectrum).
teristic bands for azaarenes appear in the 1600-1450 cm-I region and are combinations of ring C=C and C=N stretches. In addition, bands of variable intensity appear in the regions 1300-1180 cm-l and 1100-1000 cm-’ which are caused by ring CH deformations. In spite of these weak absorbances,IR can easily distinguish isomers as shown in Figure 3 (7,8-benzoquinoline vs. 5,6-benzoquinoline). The spectra of 1,2,3,4-tetrahydroquinolineand aniline (Figure 4) do not show the expected NH stretch at 3440-3460 cm-l. Instead, an N-D band is present in the region 2600-2400 cm-l. Exchange of hydrogen for deuterium has apparently taken place during the separation of the materials on the column. Spectra of “-containing compounds dissolved in
ANALYTICAL CHEMISTRY, VOL. 56, NO. 6, MAY 1984
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WAVENUMBERS
WAVENUMBERS
File sDectra obtained during- the ALHPLCIFTIRrun: 1,2,3,4-tetrahydroquinoIine(file 123),quinaldine (file 147),4-azafluorene (file 155), aniline (file 184j. Flgure 4.
the mobile phase but not in contact with the column material surprisingly show only the NH stretch. Amount Injected vs. Column Efficiency. A relatively large amount of material was injected onto the microbore column during the pHPLC/FTIR analysis of the model mixture and the coal-derived sample. Therefore, overloading of the column and loss in resolution were major concerns. Comparison of Figure 2A and Figure 2B suggests that a loss in resolution is not exhibited. Peak widths of 15 pL and 12 p L were calculated for the first peak in the UV and FTIR &omatograms, respectively. A study was undertaken to learn how column efficiency deteriorated with increasing sample mass injected. Increasing amounts (0.25-50 pup) of 2,6-dimethylquinoline were injected onto 50-cm amino microbore column. The flow rate of 50 hL/min chosen for this study was convenient in terms of time but not optimal for this column. Detection was by UV at various wavelengths. The limiting sample mass for injection is typically set at the point where there is a loss in column efficiency of 10% of the total theoretical plates. This point occurs for this column at approximately 7 fig injected. Above 7 pg injected, a significant loss in resolution, however, does not seem to be occurring upon visual inspection of the chromatographic trace. Analysis of the Coal-Derived Sample. Conditions for the separation of the coal-derived sample were identical with those employed in the model mixture analysis. A small-scale preliminary preparative separation was performed prior to rHPLC/FTIR analysis in order to remove the relatively large amount of 1,2,3,4-tetrahydroquinoline present (THQ) in the THF solubles sample. The preliminary fraction which eluted with hexane was indeed found by UV detection to contain almost exclusively THQ. Preliminary fraction 2 was eluted with carbon tetrachloride and was found to contain only a small amount of THQ. Numerous other components, which are discussed herein, have been detected via pHPLC/FTIR.
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Separation of coal-derived sample (fraction 2 of THF solubles): (A) UV detection (254 nm); (B) FTIR detection, numbers over peaks correspond to file numbers of individual file spectra. Figure 5.
Fractions 3 and 4 which are expected to be chemically different from fraction 2 have yet to be investigated. Figure 5 presents the pHPLC separation of fraction 2 of the THF solubles with UV and FTIR detection. As noted earlier, a relatively large amount ( 50 p g ) of sample had to be injected to obtain IR spectra with a reasonable signal/noise ratio for the low intensity bands of basic nitrogen-containing molecules. The total amount injected exceeds the amount at which column overloading is anticipated. Many components, however, are included in the 50 pg that was injected; therefore, each component was considerably less than 50 pg. The chromatograms in Figure 5 also do not show a striking loss in resolution with FTIR detection vs. UV detection. FurN
970
ANALYTICAL CHEMISTRY, VOL. 56, NO. 6, MAY 1984
DFN 250
N
E
I-BUTVROLACTONE
&
1800 1670 1590 WAVENUMBERS
190
Figure 8. Carbonyl stretching region (1800-1600 cm-I) for selected spectra obtained during the pHPLC/FTIR run of the coal-derived sample, taken from files 190-255.
thermore, any loss in chromatographicresolution will be more than compensated for by the additional information and spectroscopic resolution provided by FTIR detection. Individual file spectra were examined in order to gain information about the eluting material. Two major components are 1,2,3,4-tetrahydroquinoline(file 203) and quinoline (file 225). The N-H stretch at 3434 cm-l and the N-D stretch at -2600 cm-l and a side-by-sidecomparison with the neat THQ spectrum confirm the assignment. Similar comparisons support the quinoline assignment. No other file spectrum had an N-H (N-D) band suggesting the absence of primary and secondary amine functionality. A survey of all the files indicated the presence of several different carbonyl species. Figure 6 shows the carbonyl stretching region (1800-1600cm-l) for selected spectra taken throughout the entire run. The band at 1600 cm-l which is present in every file is due to an aromatic C=C ring stretch. Early in the run (file 190), before elution of THQ, bands at 1677 and 1690 cm-l are observed. After THQ elutes an unique band at 1660 cm-l is seen (file 217) prior to elution of quinoline. FinalIy, compounds elute around files 244-250 with a characteristic FTIR absorbance at 1774 cm-'. The 1774-cm-'band is higher in frequency than is seen in many types of carbonyl compounds and is therefore more easily assigned. Carbonylic materials expected to exhibit absorption in this region would include anhydrides, lactones, acid peroxides,and vinyl and phenyl esters (18). Anhydrides and acid peroxides have a second band around 1800 cm-I. Both vinyl and phenyl esters have a band near 1210 cm-' which our file spectrum does not exhibit. A five-membered y-lactone has a band at 1770 cm-l(I9). The presence of such a species is likely since THF was used to isolate the original sample. It is known that in the presence of coal-derived material T H F adducts readily to the coal material and can be converted to y-butyrolactone under rather mild conditions. A static IR spectrum of y-butyrolactone is compared with file 250 in Figure 7. The similarity is striking. Obviously, some of the extra IR bands in file 250 are due to another compound(s) eluting in the vicinity of the lactone. Additional support for this assignment comes from the observation that y-butyrolactone exhibits, under the same chromatographic conditions, a chromatographic retention time that is consistent
WRVENUMBERS
Comparison of file 250 (from pHPLC/FTIR run of coalderived sample) with y-butyrolactone (statlc spectrum). Flgure 7.
2-HYDROXYPYRIDINE
-
'1000
3560
3120
2680 22'10 WAVENUMBERS
1800
1360
920
Figure 8. Static I R spectrum of 2-hydroxypyridineand file spectra 190 217 from the pHPLC/FTIR run of the coal-derived sample.
and
with the material giving rise to file 250. File 190 exhibits bands at 1690 and 1677 cm-l whereas file 217 exhibits only a single band at 1661 cm-l (Figure 8). Ketones, aldehydes, and amides, for example, have C=O stretches in these regions. However, there is no indication of a N-H (or N-D) stretch for a primary or secondary amide nor the characteristic C-H doublet near 2820 cm-l and 2720 cm-l for an aldehyde. A tertiary amide exhibts an amide I band near 1650 cm-'. This would correspond to the band in
971
Anal. Chem. 1984, 56, 971-978
file 217 but not file 190. A type of ketone is possible. Aryl ketones give higher energy bands like those in file 190 whereas an a,@-unsaturatedketone exhibits lower energy bands as in file 217. An hydroxypyridine or similar compound could be responsible for the IR absorbance in this region (19). Many of these types of compounds exist exclusively in the keto form (20). Single injections of 2-hydroxypyridine and 4-hydroxypyridine show that these compounds do elute with retention times similar to those of compounds in the sample. Figure 8 shows the IR spectrum of 2-hydroxypyridine for comparison. There is a large amount of saturation in the compounds as evidenced by the relatively intense CH stretching modes below 3000 cm-l. This is perhaps an indication of alkyl side chains on an aromatic ring. Further work must be done to determine exactly the compounds giving rise to for the carbonyl bands seen in the LC/IR run. In conclusion, we have shown that basic nitrogen compounds (azaarenes and amines) can be chromatographed on a system amenable to IR detection. FTIR detection provides a wealth of information not available from typical LC detectors. This mode of detection can extend the resolution afforded by the column by allowing multiple components in one peak to be distinguished. Various coal-derived samples and polyfunctional model system will be analyzed during future work in our laboratory. Methods for further concentrating the basic nitrogen componentswill also be investigated.
108-99-6; pyridine, 110-86-1; y-butyrolactone, 616-45-5; 2hydroxypyridine, 142-08-5.
Registry No. N-Methylaniline, 100-61-8;7,8-benzoquinoline, 230-27-3; 1,2,3,4tetrahydrcquinohe,635-46-1; o-toluidine,95-53-4;
RECEIVEDfor review November 23,1983. Accepted February 1, 1984. The financial assistance provided by the Electric Power Research Institute, the Department of Energy Grant DE-FG22-81PC40799, and the Commonwealth of Virginia is gratefully appreciated.
2-benzylpyridine, 101-82-6;quinaldine, 91-63-4; 4-azafluorene, 244-99-5; quinoline, 91-22-5; 5,6-benzoquinoline, 85-02-9; 2,3cyclohexenopyridine, 10500-57-9; aniline, 62-53-3; 3-picoline,
LITERATURE CITED (1) Johnson, C. C.; Taylor, L. T. Anal. Chem. 1983, 55, 436-441. (2) Brown, R. S.;Taylor, L. T. Anal. Chem. 1983, 55, 1492-1497. (3) Amateis, P. 0.; Taylor, L. T. Chromatographla, in press. (4) Ho, C.-h.; Clark, 6. R.; Guerin, M. R.; Ma, C. Y.; Rao, T. K. Prepr. Pap.-Am. Chem. SOC., Div. Fuel Chem. 1979, 24 (l),281-291. (5) Haugen, D. A.; Peak, M. J.; Suhrbler, K. M.; Stamoudis, V. C. Anal. Chem. 1982, 54, 32-37. (6) Ford, C. D.; Holmes, S. A,; Thompson, L. F.; Latham, D. R. Anal. Chem. 1881, 53, 831-836. (7) Stubby, C.; Steli, J. R. P.; Mathleson, D. W. J. Chromatogr. 1879, 777,313-322. (6) Dong, M.; Locke, D. C. J . Chromatogr. Scl. 1977, 75, 32-35. (9) Colin, H.; Schmitter, J. M.; Guiochon, G. Anal. Chem. 1981, 53, 625-631. (10) Holy, N. L.; Lln, T. Yi J. Llq. Chromatogr. 1979, 2, 687-695. (11) Narang, A. S.;Choudhury, D. R.; Richards, A. J . Chromatogr. Scl. 1982, 20. 235-237. (12) Schiller, J. E. Anal. Chem. 1977, 49, 2292-2294. (13) Tomkins, 8. A.; Ho, C.-h. Anal. Chem. 1982, 54, 91-96. (14) Burchiil, P.; Herod, A. A.; Pritchard, E. Fuel 1983, 62, 20-29. (15) Schmitter, J. M.; Colln, H.;Excoffier, J. L.; Arplno, P.; Guiochon, 0. Anal. Chem. 1982, 54, 769-772. (16) Hammers, W. E.; Spanler, M. C.; DeLigny, C. C. J. Chromatogr. 1979, 174,291-305. (17) deHaseth, J. A.; Isenhour, T. L. Anal. Chem. 1977, 49, 1977-1961. (18) Socrates. G. "Infrared Characterlstic Group Frequencies"; Wiiey-Interscience: Chichester, New York, 1980;pp 58-59, 93. (19) Conley, R. T. "Infrared Spectroscopy"; Allyn and Bacon : Boston, MA, 1972;p 152. (20) March, J. "Advanced Organic Chemistry"; McGraw-Hili: New York, 1977;p 74.
Dual-Wavelength Absorbance Ratio 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
I n principle, the ratlo of the absorbances measured at two dlflerent wavelengths Is characteristlc for a glven solute and provldes the means to recognize unknown components In succeslve chromatograms, a sltuatlon encountered In lattice dedgn optknlzatlon of llquld chromatographic separations. I n practlce, the appllcabllHy of the absorbance ratio Is restricted by chrornatographlc and Instrumental Ilmltatlons, such as unresolved peaks, base Ilne drWl and offset, flnlte sampllng rate, and peak talllng. Model calculatlons and practlcal examples are used to formulate guidellnes for the use of dual wavelength detection for solute recognltlon. I t Is shown that the Influence of the mobile phase composltlon upon the UV spectra Is probably the most severe restrlctlon In practlcai appllcations.
In recent years there is an increasing interest in developing efficient and automatic optimization schemes for high-performance liquid chromatography (HPLC). The schemes proposed can be distinguished in two categories. One type uses the sequential simplex procedue ( I , 2), which requires
no knowledge about the solutes or their expected retention behavior, but is usually slow (10-40 runs) and offers no guarantee that the true optimum has been obtained. The other type is based on a lattice design, whereby the optimum is predicted from a response surface calculated from a limited number of initial runs (3). Recently, we have introduced a mixed procedure, where the predictions based on a limited lattice design are refined in successive chromatograms of the sample (4). Although the lattice design approach is more rapid (five to eight runs), it requires that the sample solutes are recognized in successive chromatograms. With the common detectors (RID, UV) such a recognition can only be based on peak areas (5), which creates problems in the case of overlapping peaks or response variation between chromatograms run with different solvents. Specific detectors (electrochemical, fluorometric) could detect some solutes but will often overlook others. The powerful mass spectrometer is expensive and difficult to interface (6). The potential of using UV absorbance data at more than one wavelength for solute identification was first recognized in a theoretical analysis by Ostojic (7). Its practical appli-
0003-2700/84/0356-097 1$01.50/0 0 1984 American Chemlcal Society
