Optical Multichannel Analyzer for Characterization of Fluorescent Liquid Chromatographic Petroleum Fractions J. I?.Jadamec and W. A. Saner” U.S. Coast Guard Research and Development Center, Avery Point, Groton, Connecticut 06340
Y. Talmi Princeton Applied Research Corporation, P.0.Box 2565, Princeton, New Jersey 08540
The contlnuous, real tlme, acquisltion of fluorescence emklon spectra of eluting petroleum oil fractions, separated by high pressure liquld chromatography, was accomplished using an Optlcal MultichannelAnalyzer (OMA) N-type detector coupled to a spectrofluorometer. A complete readout (frame scan) of the detector target Is achieved every 32 ms. The spectral region simultaneously monitored by the detector (“spectral window”) may be varled dependlng on the spectral characteristlcs of the elutlng fraction. The spectral response of the Optical Multlchannel Analyzer detector covers the entlre 190-900 nm region. However, In thls study the spectral wlndow covered the 290-400 nm range only.
The widespread use of fluorescence spectrometric techniques for the identification of polynuclear aromatics derived from petroleum oils has been summarized by Pop1 ( I ) . McKay and Latham (2) and Lloyd (3, 4 ) used fluorescence spectrometry (emission and excitation) to characterize handcollected fractions of petroleum oils separated by gel-permeation and straight phase liquid-chromatography. All collected chromatographic bands were analyzed by conventional fluorescence spectrometric techniques. The use of rapid scanning spectrometers (RSS) equipped with flow cells in place of stop-flow solution drive mechanisms are potentially very useful not only as detectors for liquid chromatography, but primarily as spectral scanners of eluting peaks during an actual chromatographic analysis. Applications of the RSS for scanning absorption in the ultraviolet and visible regions have been primarily limited to recording spectral changes resulting from biochemical and electrochemical reactions or chemical reaction kinetics. Wightman’s work (5) provides a noteworthy example of a computercontrolled RSS application in anion protonation studies. Only recently has the RSS (silicon vidicon) been applied as an absorption detector for ion-exchange chromatography (6). This paper reports the preliminary evaluation of a silicon-intensified target (SIT), electronic imager (7), as a multichannel spectrofluorometer detector for liquid chromatography. The SIT, controlled by the Optical Multichannel Analyzer (OMA), was specifically tested for its capability to simultaneously monitor the fluorescence emission spectra of petroleum derived polyaromatics during their elution from a liquid chromatographic column. Petroleum samples were studied in an attempt to augment fingerprinting techniques for oil spill identification. The optical-fiber faceplate of the SIT was coated with a very efficient UV-to-visible scintillator (8)that extended the spectral response of the detector far into the vacuum UV region. The development of an efficient UV scintillator was necessary since many aromatic compounds, present in petroleum oils, have major fluorescence emissions 1316
ANALYTICAL CHEMISTRY, VOL. 49, NO. 9, AUGUST 1977
in the 280-400 nm spectral range.
EXPERIMENTAL Liquid Chromatograph. Waters Associates Model 660 Solvent Programmer,two Model 6000A pumps, and a Model U6K injector were interfaced with a Waters Associates 440 Absorbance Detector, equipped with dual channel UV detection. Column effluent was first monitored at 254 nm (both channels, but at different sensitivities) prior to passage through a 10-pL flow cell mounted in a Farrand Spectrofluorometer. All chromatographic separations were performed on a 0.64 X 30 cm column packed with Waters HBondapack Cp,. Teflon tubing (0.012-inch i.d. X 4-inch) connected the spectrofluorometric flow cell to the end of the stainless steel tubing (0.009-inch i.d.). Two Perkin-Elmer Model 56 recorders were used, one on each detector channel. The column was maintained isothermally by means of a column heater block connected to a Haake Water Bath, Model FE. Samples were injected using side-port Auto Sampler Syringes (Precision Sampling Corp.) Spectrofluorometer-OMA. A Princeton Applied Research Corporation (PARC) Optical Multichannel Analyzer (Model 1205A) controlling a SIT detector (PARC Model 1205D) was interfaced with a Farrand Optical Mark I Spectrofluorometer. The standard 28000 grooves per inch grating in the emission monochromator was replaced with a 14000 grooves per inch grating in order to double the simultaneous spectral coverage of the detector, to 110 nm. The emission monochromator was converted into a polychromator via the use of a folding mirror that diverts the diffracted light from the exit slit toward the SIT faceplate (vertically mounted exactly at the focal plane of that polychromator). The emission spectra are detected on the target of the SIT (12.5 mm long and 5 mm wide, which is electronically divided into 500 detection channels) and can be stored (for 32 ms) much like a photographic emulsion, but in a ready-to-process digitized form. The OMA console allows the use of the SIT in either the real time or the accumulation mode. Signal averaging is performed by accumulatingmany scans (32 ms each) in memory. Fluctuations due to random noise, average towards zero. Weak signals accumulate in each scan and are thus pulled from the background (multiplex advantage). A Tektronix 604 CRT display was used as a real-time output device. Fluorescence spectra were either photographed from the CRT screen or recorded (from the memory) on a strip chart. Reagents. De-ionized,glass distilled water and spectroquality methanol (MCBMX 475) were used as the mobile phase in the liquid chromatograph. The solvents were not degassed, but were filtered using 0.22-pm pore diameter Millipore filters (GSWPO 4700) for water and 0.22-pm pore diameter Fluoropore filters (FHOPO 4700) for methanol. The aromatic standards were obtained from Duke Standards Co., Palo Alto, Calif.; solutions of the standards were prepared in spectroquality methanol (MCBMX 475). Glacial acetic acid (reagent grade) was used to acidify methanol to prepare the oil extracting solution (0.4% acid in methanol). Sample Preparation. Petroleum oils were extracted with acidified methanol after drying, according to a procedure previously described (9). The extracts were not concentrated prior to introduction to the column; injection volumes were varied (from
E M B A N D W I D T H 5 nm
EM BANDWIDTH 2 nm
EM BliNDWlOTH 0 5 "rn
>
5 5 b W
E
4 K
r-
I,
E 6
PERKINELMER
EXBANDWIDTH 10 nm
: 4
Fluorescence emission spectra of 320 ppb naphthalene in methanol: 1-cm cuvette, Ex X 270 nm, Em 1 290-400 nm, 100 (A - B) OMA accumulations
Figure 4.
400
EX AND EM BANDWIDTHS
K
A
m
a
1 E r n CUVETTE
m
&
s
a
40
a0
nm
Figure 2.
A 350 nm
Ka
nm
Fluorescence emission spectra of Ovalene (plastic std) Ex
r,"":
300
n1
Comparison of fluorescence spectra from a standard cuvette and a flow cell of 2 ppm naphthalene in methanol: 100 (A - B) OMA accumulations, Em bandwidth 0.5 nm Figure 5.
PERKIN ELMER E M BANDWIDTH 2 nrn E X AND EM BANDWIDTHS
'
'
,bo
"m
Figure 3. Ex X 300
10"lFLOWCELL
Fluorescence emission spectra of p-terphenyl (plastic std) nm
2.5 to 75 yL) in order to counter the effects of weathering (loss of the polyaromatic fraction by oxidation and solution, i.e. natural environmental weathering processes). Separation Procedure. Samples were chromatographed using a gradient from 50/50 methanol in water to 100% methanol (linear increase) in 50 min, at a flow rate of 1 mL/min (-700 psig). Column temperature was held constant at 60 "C. The chromatograph was allowed to pump 100% methanol for 20 min after completion of the gradient to ensure that all oil components were eluted. The column was regenerated by using a reverse gradient (100% to 50% methanol in water) for 3 min and equilibrated by pumping the initial gradient composition for an additional 23 min before the next injection.
R E S U L T S AND DISCUSSION S p e c t r a l Resolution of t h e OMA a n d Comparison of Flow Cells to S t a n d a r d Cuvettes. Figure 1shows a typical OMA response (400-680 nm) to a mercury pen-ray light source. The OMA was coupled to a JACO 0.3-m Ebert mounting polychrometer, PARC Model 1208 with a 150 grooves per mm grating blazed at 500 nm. With a simultaneous spectral coverage of 280 nm, the system has sufficient spectral resolution to clearly distinguish the 577-579 nm Hg doublet (Each OMA channel, approximately, spans 0.56 nm). The OMA was allowed to accumulate signal for 1s (32 scans) before the spectrum was recorded.
Fluorescence emission spectra of naphthalene at different concentrations in the flow cell: 100 (A - B) OMA accumulations
Flgure 6.
Figures 2 and 3 compare emission spectra of plastic fluorescence standards (Starna Ltd., London) that were detected by the OMA and a Perkin-Elmer MPF-SA Spectrofluorometer (photomultiplier tube detector). Both OMA spectra represent an integrated signal of 10 consecutive scans (0.32 s). The spectra were recorded in the (OMA) A - B mode where the digitized background accumulated in memory B is subtracted from the digitized raw spectra accumulated in memory A. The emission spectrum of naphthalene (320 ppb) in methanol in a standard 1-cm cuvette at varying emission bandwidths is shown in Figure 4. An upper time limit of 3 s (100 scans) was imposed on the OMA signal accumulation time. Since liquid chromatographic bands with minimal resolution allowed only 15-20 s between peak maxima, 3 s was nominally set as an upper time limit for scanning so that fluorescence emission spectra could be obtained sufficiently fast to preclude the need to stop-flow. Loss of spectral resolution is evidenced at the 5-nm emission bandwidth setting. Figure 5 compares the 3-s accumulated spectra of a 2-ppm naphthalene solution in a 10-rL flow-cell and in a standard 1-cm cuvette. The reversal of the two major peak ratios in the flow-cell spectrum is a result of optical distortion, while the decrease in signal-to-noise is a result of decreased flow-cell optical efficiency (increased light dispersion) and also lower fluorescence intensity due to a decrease in pathlength. ANALYTICAL CHEMISTRY, VOL. 49, NO. 9, AUGUST
1977
1317
recording the CRT display, was only 1/15 s (and unsynchronized with the OMA scan) each spectrum shown in Figure 9 actually comprises two separate (and consecutive) real time spectra superimposed on one another. Because the spectral response changed with time (elution), this double-frame superimposition meant a slight deterioration in image fidelity. [A much more powerful and accurate data acquisition, of time dependent spectral information, can be accomplished by interfacing the OMA to a digital magnetic tape formatter (such as Kennedy Model 9232). Such a system is capable of a real-time recording of about 20000 OMA spectra (500 channels every 32 ms) within 10-12 min.]
MINUTES
Figure 7. Liquid chromatogram of aromatic standards
Liquid Chromatographic Separation of a Petroleum Extract and OMA Response to Eluting Fractions. Figure 10 is the chromatogram of the methanol extract of a #2 fuel
Figure 6 illustrates the effect of emission bandwidth on signal-to-noise ratio and spectral resolution. It is clear that a very small loss of resolution, if any, occurred as a result of widening the bandwidth from 0.5 to 2 nm. Nevertheless, to avoid any potential slit limitation on spectral resolution, the narrowest possible slit (0.5 nm) was used.
oil supplied in an interlaboratory collaborative study of oil spill identification methods. The fluorescence emission spectra of the numbered peaks are shown in Figure 11. Reproducibility of the fluorescence spectra is shown by duplicate spectra taken from a second chromatographic separation of the sample. All spectra were obtained by accumulation of 92 consecutive scans. The spectra were recorded on a Farrand Model 100 stripchart recorder using the fastest OMA plot time (16 8). Intermediate fractions were avoided in order to increase purity of outside fractions with marginal chromatographic resolution. Although only a few compounds as yet have been positively identified by means of their OMA spectra, a substantial number of p-terphenyls are suspected of being present (fractions 14, 19, and 20), on the basis of spectral similarities to a p-terphenyl standard and published data. In fact, the identification of eluting compounds from petroleum oils was not our primary concern. Rather, any peak identifications were incidental to comparisons of corresponding OMA spectra of liquid chromatographicfractions from oil spill and source samples. Although it was realized that fluorescence does not always distinguish between members of homologous series, or parent aromatics from alkylated derivatives, it was felt that chromatographic retention time differences should spacially separate any nondiscriminatory fluorescencespectra sufficiently to eliminate confusion. Similarly, when chromatographic retention time differences were negligible for two compounds, it was felt that comparisons of their fluorescence spectra would allow their differentiation. An example of this latter case is discussed below. Fractions 17 and 18 show very dissimilar, yet reproducible spectra for compounds with very minimal chromatographic
Aromatic Standards-Chromatographic Separation, OMA Identification. The gradient-elution chromatogram of a mixture of five aromatic hydrocarbon standards is shown in Figure 7. Each numbered peak represents 440 ng of standard introduced onto the column. The band eluting between peaks 3 and 4 is due to contamination of the distilled water. This unintended extraction (trace enrichment) occurred primarily during column equilibration of 50% methanol in water, but also during the actual chromatographic separation over the gradient range from 50 to 80% methanol in water. For methanol concentrations greater than 80%, the impurities from water were not retained on the column. Since the contamination did not fluoresce, it caused no interference with the OMA response, and no attempt was made to remove it. Figure 8 shows the five fluorescence emission spectra (ex. X 270 nm, excitation bandwidth 10 nm, emission bandwidth 2 nm) corresponding to the five peaks in Figure 7. Each spectrum represents an OMA accumulation of 92 scans. The spectra were recorded by photographing the CRT display. Spectral response time dependency is shown in Figure 9, where consecutive fluorescence spectra of fluorene were recorded every 5 s, during its elution from the column into a 10-pL flow-cell (flow rate of 1 mL/min). Since the OMA was operating in the real time mode (30 scans/s) and the shutter speed of the scope camera, used for 1
325
2
nrn
435
3
325
nrn
435
Figure 8. CRT screen photographs of spectra (92 OMA scans) of aromatic standards taken during elution (Ex bandwidth 10 nm, Em bandwidth 2 nm, Ex A 270 nm) 1318
ANALYTICAL CHEMISTRY, VOL. 49, NO. 9, AUGUST 1977
Table I. Retention Time Reproducibility of Major Peaks from an Oil-Methanol Extract Peak a
b 1
3 4 C
9 13 19 21 28 29 32
Run 89
Run 81
Run 90
Run 75
Run 61
Run 86
X
Var.
Std dev
15.4 17.3 19.4 21.0 22.1 23.4 27.0 30.1 33.8 36.6 39.4 39.8 42.0
15.6 18.0 20.4 22.0 23.0 25.3 28.4 30.9 34.4 36.9 39.6 40.0 42.1
15.8 17.7 19.8 21.3 22.4 23.7 27.1 30.0 33.6 36.2 39.1 39.4 41.8
15.8 17.8 19.8 21.4 22.4 23.9 27.2 29.8 33.4 36.2 39.0 39.4 41.6
16.2
15.4 17.4 19.6 21.1 22.2 23.4
15.7 17.72 19.9 21.47 22.55 24.03 27.5 30.2 33.86 36.52 39.3 39.66 41.88
0.077 0.085 0.143 0.159 0.166 0.459 0.28 0.175 0.126 0.076 0.048 0.054 0.030
0.277 0.291 0.379 0.399 0.407 0.677 0.529 0.418 0.356 0.279 0.219 0.233 0.172
18.1
20.4 22.0 23.2 24.5 27.8
-
34.1 36.7 39.4 39.7 41.9
~
~~
Table 11. Retention Time Reproducibility of Major Peaks from a Five-Component Aromatic Standard Mixture Run No. Peak 1 Peak 2
Peak 3 Peak 4
Peak 5
69 51 70 53 54 87 74 73
17.40 18.20 17.80 18.00 17.80 18.24 17.20 17.58
27.30 27.70 27.60 27.60 27.48 27.46 26.90 26.96
35.60 36.50 36.20 36.30 35.90 35.80 35.20 35.28
Mean
17.78 20.00 27.38 32.39 35.85 0.120 0.087 0.079 0.089 0.195 0.346 0.294 0.280 0.298 0.442
Var.
Std dev
19.60 20.30 20.20 20.20 20.10 20.30 19.60 19.68
32.28 32.60 32.80 32.70 32.40 32.40 31.90 32.00
410
300
Multiple exposure photograph of the CRT screen, at indicated elution time intervals, of the fluorene peak (real time monitoring). Each exposure represents two separate OMA scans and displays Figure 9.
Ill i
0
20
40
MINUTES
Figure 10. Liquid
chromatogram of #2 fuel oil (sample 1)
separation. Fractions 15 and 16 and 19 and 20 are also examples of distinct and reproducible spectra for compounds with minimal chromatographic separation. Fraction 6, taken on a peak maximum, shows a fluorescence spectrum different from fractions 6a and 7 . Fraction 6a, taken between fractions 6 and 7, was sampled in order to observe a transition spectrum. Although no discernible slope changes are evident from the UV absorption chromatogram, the OMA spectra indicate the presence of other compounds “buried” in the downslope of fraction 6. The capability of the OMA to distinguish a compound present in the unresolved envelope is evident in fraction 8.
The OMA spectrum taken in a chromatographic “valley” has the spectral characteristics of acenaphthene (10). Since the triggering of the OMA (start of accumulation)was done manually (based on recorder pen slope changes on the UV absorption detector), duplication of the various data acquisition points for the OMA was not absolutely reproducible (especially for rapid slope changes and minimally distinguishable absorption variations). This timing problem is clearly evidenced in the duplication of the spectrum corresponding to fraction 8. The fact that some spectral characteristics of fraction 7 are evident in that duplicate (8) suggests that it was acquired too early. Fractions 22 through 25 comprised a single, large compound peak on the UV chromatogram which was indicated by multiple slope changes. Intermediate fractions 22a and 23a were intentionally recorded from the OMA at non-optimal times in order to observe spectral composites of more than one compound and transition spectra midway between spectra of much higher purity. Spectrum 22a, taken during a slope change, shows spectral characteristics similar to fraction 23; fluorescing bands located at the same wavelengths are indicated by letter. Spectrum 23a was taken just prior to spectrum 24; overall spectral characteristics between 23a and 24 are very similar, although 23a shows less resolution than 24. (This situation is similar to the decreased resolution in the duplication of fraction 8.) Retention Time Reproducibility. Different extracts of the #2 fuel oil whose chromatogram is shown in Figure 10 were chromatographed by two different individuals over a 16-day time frame. Retention time data for major peaks indicated in Figure 10 are summarized in Table I. Table I1 lists retention time data for the five aromatic standards (Figure 7). While retention time variability decreases for aromatic standards eluting from 17 to 27 min, variations increase for standards eluting from 22 to 36 min. On the other hand, variability in retention time for oil components increases over ANALYTICAL CHEMISTRY, VOL. 49, NO. 9, AUGUST 1977
1319
.-
E
-'TI. 5--
c
---\
1320
ANALYTICAL CHEMISTRY, VOL. 49, NO. 9, AUGUST 1977
C v)
.-50 E u-
075r
I
\9 2 90
A
400
290
6
400
Figure 14. CRT screen photographs of spectra (92 OMA scans) of fluorene (A) and 1-methyl fluorene (B) taken during elution
0151 10
20
30
40
50
MEAN ELUTION TIME (MINUTES)
Figure 12. Retention time reproducibility for aromatic standards (A) and oil components (B)
3
20
40
MINUTES
Figure 13. Liquid chromatogram of #2 fuel oil (sample 2) the elution range of 16 to 24 min and decreases after 25 min. Figure 12 plots standard deviations vs. mean retention time for the five aromatic standards and for indicated oil peaks. Naphthalene and fluorene (peaks 1 and 9 from the oil; peaks 2 and 3 from the standards, respectively) are common to both mixtures. Both compounds were identified in the oil sample on the basis of their spectral (fluorescence) similarity to the standard compounds as well as by comparison of their spectra to that of published data (10). Although the mean retention times of these two compounds in both mixtures is comparable (within 0.1 min), the retention time variances were substantially higher with the complex oil mixture. It is primarily this enhanced retention time variability that renders the identification of chromatographic bands, on the basis of retention time values alone, somewhat tenuous. This fact is illustrated in the following example. Figure 13 shows the chromatogram of a second #2 fuel oil supplied in an interlaboratory oil spill testing program. The labeled component in Figure 13 is eluted within 0.2 min of peak #9 (fluorene) in Figure 10. Since the standard deviation of the retention time for fluorene in fuel oil samples was 0.53 min, previously calculated, it is obvious that based on retention time values alone, the unknown labeled component in Figure 13 could be identified falsely as fluorene. Figure 14 shows CRT screen photographs of fluorene (A) and of the unknown spectrum (B), labeled peak A in Figure 13, as very similar but not identical to that of fluorene. The
unknown spectrum, did however match very well with the published spectrum of 1-methyl fluorene (11). The presence of this fluorene homologue, intrinsic to only one of these #2 fuel oils, is indicative of two distinct oils, even though this may be the only fraction out of 30 or more which is, in fact, different.
FUTURE OIJTLOOK Although this report has clearly demonstrated the practical use of the OMA as a molecular fluorescence multichannel detector for liquid chromatography,it is in our opinion merely a scratching of the surface. When interfaced to a computer, the system could generate, very rapidly, essential information concerning both the excitation and emission spectra of each eluted component. Retaining (in the computer’s memory) the ratio values of various peaks in the emission spectrum of a pure compound will later allow the user to determine the degree of purity of that compound as it is being eluted from the column. Consequently it will be possible to determine the optimal time for data acquisition, i.e., minimal overlap with other eluting compounds. A computer program could be written that will allow automatic (delayed) triggering of the OMA, whenever a new spectral feature, i.e., an absorption transition point, is monitored by the “in-tandem’’single channel UV-absorption detector. Finally, a pattern recognition program could be written, which will provide a positive identification of a particular oil spill by comparing its digitized spectral and temporal (retention time values) characteristics to those of known (and stored) oil samples.
ACKNOWLEDGMENT We acknowledge the contribution of S. Buchanan of the Coast Guard Research and Development Center who carried out chromatographic separations. We also credit E. Callet, S. Cravitt, W. Sombathy, and H. Madlin of Farrand Optical Company and lastly D. Baker of Princeton Applied Research Corporation for the design and formulation of the OMA/ spectrofluorometer system.
LITERATURE CITED ( 1 ) M. Popl, M. Stejskal, and J. Mostecky, Anal. Chem., 48, 1581 (1974). (2) J. F. McKay and D. R. Latham, Anal. Chem., 44, 2132 (1972). (3) J. B. F. Lloyd, J . Forensic Scl. SOC.,11, 235 (1971). (4) J. B. F. Lloyd, Anakst (London), 100, 529 (1975). (5) R. M. Wightman, R. L. Scott, C. N. Reilley, R. W. Murray, and J. N. Burnett, Anal. Chem., 48, 1492 (1974). (6) A. McDowell and H. Pardue, Anal. Chem., 48, 1815 (1976). (7) Y. Talmi, Anal. Chem., 47, 697A (1975).
(8) Y. Talmi and D. C. Baker, Internal publication, Princeton Applied Research Gorp., 1976. (9) W.A. Saner, G. E. Fltzgerald, and J. P. Welsh, Anal. Chem., 48, 1747 (1976). (10) I. B. Berlrnan, “Handbook of Fluoremnce Spectra of Aromatic Molecules”, Academic Press, New York, 1971, p 345. (11) Ref. IO, p 202.
RECEIVED for review March 21,1977. Accepted May 23,1977. This paper was presented in part a t the 1977 Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Cleveland, Ohio, March 1977. ANALYTICAL CHEMISTRY, VOL. 49, NO. 9. AUGUST 1977
1321
