Energy & Fuels 1993,7, 194-199
194
Identification of Metalloporphyrins by Third-Derivative UV/Vis Diode Array Spectroscopy David H. Freeman* and Delphine Castres Saint Martin Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742
Christopher J. Boreham Division of Continental Geology, Bureau of Mineral Resources, BMR, GPO 378, Canberra, Australia Received December 21, 1992
Qualitative analysis of organic compounds is expedited by third-derivative processing of diode with k0.02 nm precision, or 1/100 of the present diode array UV/vis spectra which can locate ,A, interval. The technique was applied to over 50 metalloporphyrins, primarily geological residues of ancient chlorophyll, and the results suggest broader applicability. Among metalloporphyrins, the Soret band wavelength, A(Soret),varies with the type of functional group structure on the periphery of the 20-carbon porphyrin chromophore. Distinctive A(Soret)ranges are defined by exocyclicstructure (butano, propano, ethanol, or lack thereof (etio). Among etioporphyrins A(Soret) varies with the number of alkyl substituents. Etio- and ethanoporphyrin isomers with the same alkyl groups are least differentiated. Fifty or more spectral classes can be specified by including A(a) and A@), or higher than that when other data, Le., molecular mass or chromatographic data, are added. Moreover, such results can be obtained 1000-foldfaster than conventional NMFt and MS structure determinations. The new technique allows one to differentiate quickly among congeners, homologues, and isomers, to use wavelength data to guide chemical isolations of known components from mixtures, and to expedite the isolation of unknowns or new structures when the technique is adjoined to HPLC separations.
Introduction This paper presents a spectroscopic innovation designed to facilitate the qualitative analysis of chlorophyll residues known as porphyrins, Le., petroporphyrins or geoporphyrins. Scientific curiosity in porphyrins was stimulated by Treibs who noted their likely origin from algae, bacteria, or heme.' These complex suites of molecular fossils are widely found in sedimentary deposits and petroleum. The residues consist of complex suites of metalloporphyrins and free base porphyrins whose separation and identification are often tedious. While the present work focuses on porphyrin compounds, broader applications are implicit in the results of the study. The isolation, identification, and interpretation of individual porphyrin structures is of growing interest to the field of organic geochemistry. Some porphyrins, but not all,can be related to specific ancestral or extant types of photosynthetic organisms.2 Alterations can and do occur. The relative abundance5 of various porphyrin structures, for example, the exocycle/etioporphyrin, i.e., DPEP/ETIO, ratio changes with depth or temperatureS3y4 The distribution of porphyrin structures may help correlate a migrated petroleum with its generative source rocks, indicate specific depositional environments, and/or tell about its thermal history.3-7 (1)Treibs, A. Angew. Chem. 1936,42, 682-6. (2)Ocampo, R.; Bauder, C.; Callot, H. J.; Albrecht, P. Geochim. Cosmochim. Acta 1992,56, 745-761. (3)Dydik, B.M.; Alturki, Y. I. A.; Pillinger, C. T.;Eglinton, G. Nature 1975,256,563-5. (4)Barwise, A. J. G.; Roberts, I. Org. Geochem. 1984,6,167-176.
As the interpretational platform expands, so does the need for an efficient analytical system. Mixtures of alkylporphyrins (and their carboxyl derivatives) can be routinely extracted and separated into porphyrin subgroups, but the isolation and structure determination of pure components presently involve major investments of time. Let us examine this analytical process more closely. A preliminary view of porphyrin mixtures is provided by HPLC or by mass spectrometry, MS.8 The assignment of molecular structure is provided by MS and NMR techniques after extensive isolation has been carried out. Presently, the isolation and NMR are the slow steps. This approach becomes even slower if independent synthesis is required for a structural confirmation. Although a year or more may be required for each analysis,Q-12NMR and MS results are structurally definitive, or nearly so,13 The complexity of metalloporphyrin HPLC patterns ranges (5)Sundararaman,P.; Boreham, C. J. Geochim. Cosmochim. Acta 1991, 55,389-395. (6)Moldowan, J.; Sundararaman, P.; Schoell, M. Org. Geochem. 1986, 10, 915-926. (7)Shi, J.;Mackenzie, A. S.; Alexander, R.; Eglinton, G.; Gower, A. P.; Wolff, A. P.; Maxwell, J. R. Chem. Geol. 1982,35, 1-31. (8) Sundaraman, P. Anal. Chem. 1985,57,2204-6. (9)Quirke, J. M. E.; Eglinton, G.; Maxwell, J. R. J . Am. Chem. SOC. 1979,101, 7693-7. (10)Chicarelli, M. I.; Kaur, S.; Maxwell, J. R. ACS Symposium Series; Filby, R. H., Branthaver, J. F., Eds.; American Chemical Society: Washington, DC, 1987;Vol. 344,p p 4C-67. (11)Ocampo, R.; Callot, H. J.; Albrecht, P. Ibid. pp 68-73, (12)Verne-Mismer, J.; Ocampo, R.; Bauder,R.; Callot, H. J.; Albrecht, P.Energy Fuels 1990,4 , 639-643. (13)Verne-Mismer, J.; Ocampo, R.; Callot, H. J.; Albrecht, P. Tetrahedron Lett. 1988,29, 371-4.
0887-0624/93/2507-0194$04.00/00 1993 American Chemical Society
Identification of Metalloporphyrins from the relatively uncomplicated RPLC pattern of Messel Shale extract2J4to highly complex patterns in crude oil and asphalt ~ a m p l e s . ~ JThe ~ J ~true complexity of geological samples is difficult to estimate in terms of the overall number of isomers, homologues, and congeners in any sample type. About 504 framework structures have been identified so far,10J4J7but the total number is probably much higher. Approximately 240 were cited in recent work on Boscan oil.’* There is a need for rapid qualitative determination of organic chemicals, in general, and especially for metalloporphyrins. The feasibility of applying high precision UV/vis spectroscopy to this purpose is the subject of this article. The absorbance maxima of metalloporphyrins include an intense Soret band located near 400 nm along with weaker ,f3 and a bands, respectively, at approximately 515 and 550 nm for nickel porphyrin, Nip, and 530 and 572 nm for vanadyl porphyrin, V0P.19920Small but significant shifts in absorption wavelength, related to variations in framework structure, were reported among metalloporphyrin comple~es.’~ It followsthat UV/vis measurements of reference structures can help identify unknowns. The correlation of structure and wavelength is enhanced by instrumental accuracy and, especially, by high wavelength precision. In the present study, it will be demonstrated how Soret band wavelengths alone provide an empirical basis for sorting porphyrins into major structure subgroups. Then, we will report how wavelengths of all three absorption bands, combined with other readily accessible measurements, can provide a rapid qualitative specification of porphyrin structure. Among free base porphyrins the Soret band is followed by several absorption band^.^^,^^ The possible correlation of UV/vis wavelength with free base chromophore behavior was not studied in this work. Wavelength Precision in Diode Array Spectrometry. Improved wavelengthprecision is logicalto approach via odd order derivative spectroscopy.21 Raw absorbance data (and, more generally, even order absorbance derivatives) have zero slope at their absorbance maxima, while odd order derivatives show a steep zero crossing at the wavelength of the absorbance maximum. Digitized absorbance measurements from diode array spectrophotometers are particularly convenient for derivative data processing. While first-derivative spectra can be used for wavelength determination, the higher odd order derivatives provide a high-pass filter which discriminates against broad band interferences that degrade the accuracy of spectrophotometric measurement. Higher spectroscopic derivatives contain amplified noise that needs to be suppressed by data smoothing. The third derivative, as a compromise between chemical and random noise, was selected for the present study. The mathematical requirements for a third-derivative (14)Boreham, C.J.; Fookes, J. R. C. J . Chromatogr. 1989,467,195208. (15)Sundararaman, P. Anal. Chem. 1985,57,2204-2206. (16)Aizenshtat, Z.;Sundararaman, P. Geochim. Cosmochim. Acta 1989, 53,3185-3188. (17)Callot, H. J.;Ocampo, R.; Albrecht, P. Energy Fuels 1990,4,6339. (18)Ping’an, P.; Eglinton, G. Energy Fuels 1992,6,215-225. (19)Gouterman, M. The Porphyrins; Dolphin, D., Ed.; Academic Press: New York, 1978;Vol. 111, pp 1-165. (20)Falk, J. E. Porphyrins and Metalloporphyrim; Elsevier: New York, 1964. (21)O’Haver, T. C. Clin. Chem. 1979,25,1548-1553.
Energy & Fuels, Vol. 7, No. 2, 1993 195 algorithm are as follows. For derivative order, n,the use of n 1 boxcar smooths was found to be optimal.22 A third-derivative algorithm with an odd number of terms aligns the wavelength scale of the derivative to that of the diodes. Further, the bandwidth of the smoothing/derivative algorithm should not be too different from that of the absorption band.23 Derivative algorithms are easily generated in forms that are suitable for spreadsheet computation. Third-derivative algorithms are defined by their initial Pascal coefficients (1, -3,3, -1). After three three-point boxcar smooths, this becomes (1,0, 0, -3,0,0,3,0,0, -1) which contains an even number of terms. A final four-point smooth yields a 13-term algorithm (1, 1, 1, -2, -3, -3, 0, 3 , 3 , 2, -1, -1, -1). When measured against a porphyrin bandwidth of ca. 30 nm, this algorithm spans 13 diodes, or 24 nm for the diode array spectrophotometer (2 nm interval) used in this work.
+
Experimental Section Measurements were made with a diode array spectrophotometer, Hewlett Packard HP8452a (2 nm diode spacing) after 60min lamp warm-up and 1-s sample acquisition times. Porphyrin spectra were taken in accordance with our own solvent assurance recommendation^,^^ namely limited exposure to light and use of freshly distilled dichloromethane or that stabilized by 50 ppm each of cyclohexene and triethylamine. Quartz or glass cuvettes, 1 cm square cross section, were used. Background scans were updated preceding each measured sample and were checked for porphyrin staining of the cuvette. Spectral data was transferred to a magnetic floppy disk in the controller, an IBM PCAT. The spectrum derivative was computed automatically with a Macintosh computer using macrodriven Wingz software where the algorithms were user selected. The Wingz script was written by Prof. T. C. O’Haver. Output included tabulated absorbance maxima (0th derivative), interpolated third-derivative wavelengths a t zero crossings, and an interpolated even order algorithm for quantitative analysis.20 The wavelength measurements were calibrated against a holmium oxide standard. A narrow third-derivative algorithm (1, -2,0,2, -1) obtained by two-point signal averaging was used to conform to the narrow absorbance bands of this standard. The standard was also measured by two other instruments, Cary Model 14 and Guilford Response spectrophotometers. The measured and publishedz5values agreed to within 0.2 nm. The 13-term algorithm was tested extensively for its preciseness for measuring wavelength, Amax, a t the zero crossing. Comparison was made to a nine-term algorithm obtained after three twopoint boxcar smooths and one three-point smooth. Both algorithms span less than the nickel or vanadyl porphyrin bandwidth of 28-36 nm. The 13-term algorithm gave unexpectedly good precision of A0.03 nm, or 1/60 of the instrument’s 2-nm diode interval. The third derivative provided by the HP8452a software was equally exact (agreement within 0.010.03 nm in limited tests). The nine-term algorithm showed 0.06 nm standard error and 0.5 nm discrepancies and was not used further. Wavelength artifacts were avoided by sticking closely to a solvent assurance protocol.z4 The use of a single solvent avoids solvent dependent wavelength effects.z6 T o reduce errors the concentration range was bounded by absorbance values (above baseline, or above background) between 0.03 < A < 1.5. These raw absorbance maxima, unimproved by (22)O’Haver, T.C.;Begley, T. Anal. Chem. 1981,53, 1876-1878. (23)Freeman, D. H.; OHaver, T. C. Energy Fuels 1990,4,688+94. (24)Freeman, D. H.; Castres Saint Martin, D. Energy Fuels 1992,6, 532-534. (25)Edisbury, J.R.PracticalHintson AbsorptionSpectroscopy; Hilger and Watts: London, 1966. (26)Freeman, D. H.; Swahn, I. Energy Fuels 1990,4,699-704.
Freeman et al.
196 Energy & Fuels, Vol. 7, No. 2, 1993 Table I. NiP SDectral Datae abs wavelength (nm) c, Soret @band aband
porph rin
codl
20.00000000 20.10121021 20.10121221 20.11121121 20.11121221 20.12101221 20.12121021 20.12121201 20.12121212 20.12121221 20.22222222h
20 28 30 30 31 30 30 30 32 32 36
average Soret range 22.101210’21 22.111210’21 22.111211”21 22.120210’21 22.121110’21 22.121210’21 22.121211’21 22.121211”21 22.1212101”O 22.1212101”l
30 31 32 31 31 32 33 33 30 31
average Soret range 23.1112101’1 23.1212101’1
31 32
average 24.1012100’0 24.1012100’1 24.1112100‘1 24.1212100’0 24.1212100’1
29 30 31 31 32
average Soret range 24.0~0~111221 BZ 32 25.121211’0’1 34
385.4 389.27 389.74 391.55 390.15 389.69 389.69 389.65 390.45 390.68 391.68 389.6 385.4-391.6 392.81 393.09 394.35 393.17 393.46 393.33 393.83 394.54 394.08 394.18 393.7 392.8-394.6 395.67 395.87 395.8 398.47 398.35 397.80 398.91 398.71 398.4 397.8-399.0 398.28 394.13
abs ratios S/a
alB
11
7.7 6.1 6.4 5.1 6.9 6.6 6.5
1.2 2.2 2.4 2.3 2.7 2.7 2.3 2.7
6.2
2.8
6.4
2.5
503.9 512.80 513.73 515.48 514.82 513.66 513.65 513.53 514.90 514.66 515.50 513.1
537.1 548.95 549.83 551.74 551.37 549.81 549.71 549.69 551.38 551.24 551.66 549.1
512.47 513.56 514.78 512.84 513.63 513.53 513.88 514.70 513.27 513.39 513.6
549.52 552.36 552.98 551.07 552.16 552.06 552.05 552.77 551.51 551.57 551.8
6.9 8.1 8.6 8.6 8.5 7.9 9.7 8.8 9.1 8.9 8.5
1.7 1.8 1.8 1.8 1.9 1.8 2.1 1.9 1.9 1.8 1.9
516.36 516.42 516.4 518.60 518.76 518.59 519.80 519.34 519.0
551.65 551.81 551.7 552.65 552.83 553.33 554.43 553.99 553.4
10.7 7.8 9 11.4 10 9.7 11.4 10.7
2.0 1.7 2 1.3 1.2 1.6 1.5 1.5 1.4
526.16 513.96
568.41 552.63
11
4.4 8.3
1.8 1.8
a The wavelength data are considered accurate to within 0.1 nm. bReference compound is assumed not to occur naturally and is excluded from wavelength averages.
structures
Table 11. VOP Spectral Data cn Soret Pband a band
20.00000000“ 20.10121221 20.11121221 20.12121212 20.12121221 20.22222222“ 22.121210’21 22.1212101”l 23.1212101’1 24.1212100’0 24.0’0’111221 Bz 24.0’0’111221 Bz
20 30 31 32 32 36 32 31 32 31 32 32
399.4 405.94 406.42 406.62 406.68 407.3 410.53 411.00 411.53 412.58 413.97
523.8 531.88 532.56 532.80 532.63 533.2 533.30 533.53 535.36 536.75 544.72
559.4 569.62 570.55 570.70 570.48 570.9 573.03 572.21 572.65 573.59 578.74 591.33
S/a
alp
23 9.5 7.8
0.8 1.7 1.5
8.9
1.7
13 16 20 14 8.2
1.2 1.2 1.0 0.9 2.4
See Table I. interpolative or signal averaging algorithms, were used to calculate the ASIA, and A,IAB ratios in Tables I and 11. Satisfactory wavelength data were often obtained with less than 1pg of sample. In serial dilutions from 10 to 0.1 pg/mL, a pooled standard deviation of A0.02 nm was calculated from 40 independent measurementsz6of vanadyl porphyrins with etio-I and octaethyl structures. On that basis the third-derivative algorithm should be recognized as an interpolative data processing technique capable of wavelength precision within 1/100of the diode spacing of 2 nm. This is a marked improvement over Fourier-domain interpolation whose precision was to within 1/10 of the pixel spacing in a comparable photodiode array study.2I In previous work it was found that nickel porphyrin spectra became difficult to reproduce at the lower concentrations. A solvent assurance protocolz4allowed manipulating of Soret, a~
(27) Lepla, K. C.;Horlick, G. Appl. Spectrosc. 1990, 8, 1259-1269.
and @-bandintensities so they would fall within the prescribed absorbance limits. Among numerous sets of dilution data, the pooled standard deviation for the nickel porphyrins was *0.03 nm. In a more stringent test of reproducibility, 26 independent porphyrin isolates (8pairs, 2 trios, and 1quartet) showed a pooled standard deviation of 0.13 nm. The larger deviationsmay reflect small amounts of impurities whose contributions would have been veiled in the dilution tests. Assuming randomly propagated deviations, a pair of independently isolated porphyrins having the same structure are expected to agree within 4 2 X 0.13 nm or f0.2 nm, or with 99% confidence to within 10.5 nm. Most of the experimental work (HPLC, MS, NMR, etc.) for isolation and identification of porphyrins measured in this work has been described e1~ewhere.l~Four synthetic C30 nickel etioporphyrin isomers were provided in addition by H. J. Callot. Purified metal complexes of porphine and octaethylporphyrin were obtained through MIDCENTURY, P.O. Box 217, Posen, IL 60469. An extract of Messel Shale was fractionated by RPLC in MeOH carrier. The two most dominant components were isolated after rechromatography on a bonded aminez8column (ZORBAX, 4.6 x 250 mm) with dichloromethane and hexane carrier.
Nomenclature Petroporphyrin nomenclature has veered from Fisher and revised Fisher29names toward ideograms,1°J2J4too many to be easily memorized, and which are not easily encoded in ASCII. The followingmnemonic structure code was developed to facilitate database tabulation of the alkylporphyrin structures. First, a two-digit prefix defines the tetrapyrrole core, beginning with 20 as the number of carbon atoms in an etio core. Then, for exocyclic (i.e., cycloalkano, CAP) porphyrins: 22 means ethano (commonly DPEP), 23 propano, 24 butano, and 25 refers to di-DPEP structure. 26 and a suffix stands for benzu-DPEP (bz) or tetrabenzo-DPEP (tb) structures. Examples are given in Figure 1. The next eight numerals designate alkyl substituents. Numerals 0, 1,2, etc., respectively, indicate -H, -Me,-Et, etc. The substituents occupy beta positions 2,3,7,8,12, 13,17, and 18. The substituent code starts at position 2. To illustrate, 2(1) uses the (1)to indicates a methyl group at the 2-position. Naming continues clockwise around the tetrapyrrole ring. Hence, the sequence for etioporphyrin (III), which is 2,7,12,18-tetramethy1-3,8,13,17tetraethylporphyrin, becomes “20.” followed by 2(1), 3(2), 7(1), 8(2), 12(1), 13(2), 17(2),and 18(1). Dropping the 2, 3, 7, ... gives the etio (111) structure code, 20.12121221. Note: the sum of the digits in the code name is equal to the number of carbon atoms. The preceding instructions allow one to envision the structure mentally and with minimal effort from the code name. To minimize the number of synonyms and to help connote geologically significant porphyrin-chlorophyll relationships, the code naming is augmented by simple orientation rules. Methyl groups, when present, are aligned at positions 2,7, 12, and/or 18 and ethyl groups at 3,8,13, and/or 17. Exocyclicsubstituents occur between pyrrole and methine carbons, such as 13,15 or 15,17 cycloalkano porphyrins, or CAPS. These are named clockwise using primes and double primes instead of superscript numerals.29 Thus, 22.121211’21 indicates a (28) Freeman, D. H.; Angeles, R. M. 193rd ACS National Meeting, Denver, CO, April 5-10, 1987; Abstr. PETR 41. (29) Bonnett, R. In The Porphyrins; Dolphin, D., Ed.; Academic: New York, 1978; Vol. I, pp 1-13.
Identification of Metalloporphyrins
22.121211’21
20.12121221 (Etio Ill)
22.121210’21 (DPEP)
22.121 21 01’1
23.1112101’1
26.1200’1021 bz
24.12121 00’1
25.12121 1’0’1 Top halt tenlalive atsignmenl.
Figure 1. Alkylporphyrin framework structures, with 10-digit
codes that are structure specific and data base compatible. 13’-methyl; the chl-c type structure, 22.1212101”1, has a 15”-methyl. Some porphyrins have exocyclic structure connecting adjacent @-carbonson the pyrrole ring. In that case, the cycloalkyl substitution on A, B, C, or D rings (i.e., benzo or tetrahydrobenzo) is marked by primes on adjacent numbers to designate the pendant C4Hz or C4H4 moiety, and a suffix to indicate degree of saturation, Le., bz for benzo. To set up a structure orientation prior to naming, we used ChemDraw software to rotate, flip, and thereby align the structure for naming. An advantage of the mnemonic code is it allows the user to envision porphyrin structure with very little practice-analogous to ortho, meta, and para with aromatic structures. It also facilitated Email communications between collaborating workers on different continents, and for keeping track of porphyrins whose nomenclature varies among different authors and publications. Results
Over 50 metalloporphyrins were measured. After data from duplicate structures were averaged for wavelengths and absorbance ratios, the spectral properties of 28 natural nickel porphyrins plus several reference compounds were measured and are presented in Table I. A smaller set of vanadyl porphyrins are reported in Table 11. The Soret band wavelengths of vanadyl porphyrins include the common geological descendent of chl a,
Energy & Fuels, Vol. 7, No. 2, 1993 197 22.121210’21, along with averaged results for three independent isolations of that widely observed compound. Excellent spectroscopic agreement among these different isolates was observed. Wavelength data for vanadyl structures are also reported for their corresponding nickel structures in Table I. The results in Tables I and I1 show Soret band wavelengths of metalloporphyrins fall into separate and distinctive ranges for etio, ethano, propano, and butano structure groups. The wavelength gap between these groups, according to results obtained so far, significantly exceeds measurement uncertainty. It follows that metalloporphyrin as unknowns can be divided, at least tentatively, into their respective structure groups based on wavelength. In other words, Soret band wavelength provides a qualitative index of structure type. This is significant because, by comparison, molecular ion mass spectrometry can show a mass difference of 2 amu between etio and exocyclic structures, while the Soret band wavelength divides the exocyclic group into wavelength subgroups corresponding to ethano, propano, and butano. As an example, if we stick to the specified conditions of measurement, there is no way for an isolated nickel butanoporphyrin to be confused with a nickel propano type. Absorbancevalues could not be determined as extinction coefficients, although this may be possible in future work. The Soret/a and CY/@ absorbance ratios in Table I are only marginally structure sensitive. The measured CY/@ ratios for etio and ethano structures agree with published values.30 The nickel propano and butano groups contain too few measured structures to search for trends within these groups. Among the butanos, two C31 structures show distinctly different wavelengths while the C29 and C30 structures have nearly identical wavelengths but different masses. We are concerned next with several questions: To what be expected to extent can a precise tabulation of A,, differentiate organic congeners and isomers? What are the limitations, and what new applications avenues are possible to enhance the study of mixtures, including the application of HPLC-derivative UV/vis to help isolate new compounds? The nickel ethano set is large enough to permit a preliminary estimate of the extent to which precise measurements of all three wavelengths (Soret, @ and CY) will facilitate qualitative analysis. The structural specificity of this UV/vis approach is indicated in Figure 2 by absent correlation in a coplot of wavelength and skeletal mass expressed as the number of carbons. These data were plotted relative to the common chlorophyll a descendent using AA = A(samp1e) - A(22.121210’21), and correspondingly, AC, = C, - 32. Among the Ni porphyrins within the 392.8-394.6-nm Soret band wavelength range as measured so far, based on absorption wavelengths and mass to the nearest number of carbon atoms, each structure is distinct including C34 di-DPEP. Proceeding one step further, it is possible to estimate the maximum number, C, of nickel ethano structures that can be classified using only UV/Vis wavelength and molecular mass. Here, C is a qualitative capacity which (30) Baker, E.W.; Corwin,A. H.; Klesper, E.; Wei, P. E. J. Org. Chem. 1968,33,3144-3148.
Freeman et al.
198 Energy & Fuels, Vol. 7, No. 2, 1993
2c
number of hydrogen, or nor, substituents. This nor effect is conspicuous with the porphine structure, 20.00000000. It is the etio end member with only hydrogens as pyrrole substituents and it has the lowest Soret band wavelength.
1C
Limitations
0’ b I
2210121021
* ......
22 11 121021
..
O[
r
yu
-t
6 N
---e
22111211’2:
--it--
2212021021
I-
!
*.. ..
1c
22 1211 1 w21
--*- 22121211’21
P2
--*--
-
2c
-f
2212121:’2‘ 221212101’0 221212101’1 2512121101
-3 c
Soret
p
a
cn
t Absorbance 4 Bands
Figure 2. Wavelength data, AX = X - X(ref) and mass data, AC = C, - 32, calculated relative to the common 22.121210’21 reference. The 11 compounds (including the C32 reference) demonstrate an absent correlation that is fundamental to wavelength-derived qualitative analysis. is analogous to peak capacity in ~hromatography.~~ Consider the ethanoporphyrins where the range of Soret band wavelength is 1.6 nm, and the a and @ band ranges are both 2.7 nm. Figure 2 shows no correlation between Soret wavelength, a-band wavelength, and mass. This is the basis for the qualitative strength in the present approach. There is some redundancy, however, in that half of the ethano structures show nearly constant differences between their a and @ Wavelengths. Two porphyrin structures can be assumed different if their wavelengths differ by about 20.3 nm. On that basis, the Soret region provides C(S) = 5 (i.e., 1.5 nm/0.3 nm), while the a-bands provide C(a) = 9 (2.7 nm/0.3 nm). Using Soret and a wavelengths alone, the qualitative capacity within the nickel ethano range is estimated: C(overal1) = C(S)C(a)= 45. This is somewhat conservative because it omits the @-bandinformation. In several instances we have isolated a nickel porphyrin which exceeded a 0.3-nm difference from its reference wavelength, but in each case the discrepancy was removed by more intensive separation. Note use of wavelength to guide a separation. Additional information, such as molecular ion mass or better yet, high-performance liquid chromatography, or HPLC, can easily expand the qualitative capacity. Consider the effect of mass. The carbon number, N , of the present ethano group ranges from 30 to 33 for which C(N) = 4. The qualitative capacity of UV/vis-ms within the ethano group is the product C(S)C(a)C(N)whose value is 180,or larger still if one includes C(@).A lower qualitative capacity might apply to the less common butano and propano bands, based on their possibly being more closely spaced on the wavelength axis. Some of the compounds are amenable to wavelength classification of another kind. Observe among the etioporphyrins that the Soret band wavelength varies with the (31) Giddings, J. C. Anal. Chem. 1967, 39, 1027-8.
Dissimilar structures can fall into the same wavelength group. To illustrate, the Soret band of the nickel di-DPEP structure (25.121211’0’1) lies inside the ethano wavelength region. In this instance, UV/vis alone fails to distinguish between ethano and di-DPEP frameworks. Other cases like this may may be found. To resolve ethano from diDPEP, these structures have unequal saturation and differ in mass by 2 amu. Now we will examine spectral differentiation among isomers. Among the C30 nickel etioporphyrins, there are four which have practically the same absorbance wavelengths. This quartet also has a fixed alkyl group distribution of one hydrogen, four methyl and two ethyl groups. (Count the O’s, 1’9, and 2’s in the mnemonic name after the decimal point.) Distinctively separate from this quartet is another C30 isomer, 20.11121121, which has zero hydrogen count and therefore experiences no %or effect” as described earlier. This suggests that structural isomers having constant alkyl substituent distributions may be least differentiated by their UV/vis absorption spectra. Similar results are seen with the C31 ethanoporphyrins. Three isomers with similar wavelengths have the same alkyl substituents of five methyl and two ethyl groups. The trio is distinctly different, as the reader can easily find, from another C31 ethanoporphyrin, 22.120210’21. Similarly, the two C31 nickel butanoporphyrin isomers have distinctly different wavelengths and alkyl distributions. As spectral similarity can now be expected for compounds having the same alkyl groups, we prepared a Ni ethanoporphyrin, 22.122110’21, which is not known to occur naturally, by nickelating the corresponding free base provided by Timothy Lash. As expected, all three absorbance wavelengths were within 0.2 nm of those of its similarly substituted isomer, the common ethanoporphyrin, 22.121210’21. Can similar isomers be distinguished another way? It is well-known that HPLC, especially reverse phase, is practically invincible when it comes to isomers separation, As a specific and this applies to the nickel ~0rphyrins.l~ test, we found with C18 RPLC in methanol carrier that the preceding isomers of nickel porphyrins, 22.121210’21 and 22.122110’21, exhibited conspicuous retention differences. While it is not new for HPLC to be augmented by scanning UV/vis spectroscopy for identifying chemical compounds, the present approach leads to the idea that wavelength and retention data are synergistic in terms of qualitative analysis and they can be obtained together quickly, precisely and even automatically. Applications One application of UV/vis wavelength qualitation is when the compounds are already chromatographically pure. This leads to an example of in which HPLC and UV/vis wavelength can work together to help separate or classify mixtures. The two most dominant components of a Messel Shale extract were isolated by RPLC from chromatograms that
Identification of Metalloporphyrins
Energy & Fuels, Vol. 7,No. 2, 1993 199
Table 111. Wavelength (nm) Comparison Tests on Nickel Porphyrins Isolated from Messel Shalea s a m d e code
22.121210’21 reference Messel Shale 24.1212100’1 reference Messel Shale
XS
XR
X”
HPLC ref
393.33 393.49
513.33 513.48
552.06 552.13
SK 7,this work
398.71 398.77
519.34 519.51
553.99 554.05
2 2
SK 11, this work
Average discrepancy among the six bands compared above is
0.11nm. T h e calculated discrepancy is 0.14,or d2(0.1),where 0.1 is the accuracy claimed in Tables I and 11.
were congruent with those of other ~ o r k e r s . ~HPLC J~ components SK7 and S K l l were obtained as practically pure isolates. Based on previous identifications,2J4specifically based on retention order, these fractions correspond to 22.121210’21 and 24.1212100’1. The relevant data are given in Table 111. Initial but partial confirmation is provided in that the Soret band of SK7 is an ethano wavelength type, and S K l l is a member of the butano wavelength group. The wavelength agreement between the measured Messel isolates and the reference values in Table I is within expected error limits. Independently, we have isolated the common C32 DPEP, 22.121210’21, from immature geological samples. Its chromatographic retention is useful as a reference mark as it is often a major comonent in a sample, and it has become a straightforward procedure to monitor its purification via wavelength convergence to the data in Tables I and 11. It is well-knownthat the spectral absorbance of amixture of solutes is often additive in terms of concentration and spectral extinction. The spectra of nickel or vanadyl porphyrin suites, representing numerous porphyrins present in geologic samples, often closely resemble the spectra of a single porphyrin. A suite of porphyrins tends to show slightly broadened peaks and is by appearance in the form of an “average” porphyrin and not as a lumpy spectrum of imperfectly coalesced peaks. It is proposed that the average absorbance wavelength of a mixture of solutes with similar absorbance wavelengths may be additive with respect to the concentration and specific wavelength of each. From Tables I and 11,a suite of etiorich porphyrins, as would be extracted from a rock or a petroleum sample, should have a lower average Soret band wavelength than that of an exocycle-rich mixture. We have observed the expected wavelength shift in response to thermal stress, as discussed at the beginning of this article, and it will be described more fully in a future publication. In a somewhat different context, the alp absorbance ratio has been reported to vary with thermal stress33 in a way that is not consistent with expectations based on Table I.
Conclusions The present study has shown that highly precise wavelength data can readily be obtained from diode array (32)Sundararaman, P. Biological Markers in Sediments and Petroleum; Moldowan, J . M., Albrecht, P., Philp, R. P., Eds.;Prentice Hall: Old Tappan, NJ, 1992,pp. 313-319. (33)Baker,E. W.;Palmer, S.E.; Huang, W. Y. InitialRep. DSDP,U.S. Govt. Printing Off.,Washington, DC,1977;Vol. 41,pp 825-37.
spectrophotometers. The present tests of reproducibility suggest combined potential accuracy and precision of 0.03 nm, which is less than 1/60 of the 2-nm-wavelength separation between diodes. The study of a number of congeners among nickel porphyrins shows subtle differences between homologues and isomers are correlated with correspondingly varied wavelengths. This can be used to assist compound separation and identification. Wavelength determinations from diode array instruments are made quickly, requiring a few minutes of instrument time, or less if done on-line. Wavelengths that differ from known structures can indicate mixtures, or chemicals outside the data base. Porphyrin mixtures as they occur in nature are complex in a chromatographic sense, and many new porphyrin structures remain to be identified. Isolation of such chemicals from mixtures must be accompanied by convergence of absorbance wavelength to that of the pure chemical. Thus, the present technique offers a wavelength criterion for chemical purity. Derivative UV/vis spectroscopy and suitable HPLC retention data, aided by MS, provide powerful tools for tracking and scouting chemical compounds. A combination of precise spectral and retention data are more significant in a qualitative sense than either kind of data alone. Moreover, definitive structure determination without NMR, is feasible when compounds are specified by a proper combination of spectral and chromatographic techniques. The gain in analysis speed needs to be emphasized. Porphyrin separations have usually been monitored by retention position alone, aided in part by spectral ratios (14)while the type of exocyclicstructure (ethano, propano, etc.) had to be deduced from NMR (NOE) experiments, as they cannot be inferred from molecular ion mass determination. UV/vis scanning and derivative computation require only a few minutes, while NMR (NOE) structure assignment often requires weeks. The UV/vis approach allows porphyrins to be indexed qualitatively approximately 1000 times more quickly than NMR. Further, UV/vis properties of strong light absorbing chromophores requires less than 0.1 pg while porphyrin sample mass requirements for NMR are 1000-foldhigher. We conclude that derivative wavelength data, coupled with chromatographic data, are consistent with new qualitative opportunities, such as hyphenated HPLCdiode array spectroscopy with postrun, or even real time, estimation of component structure. The isolation of pure chemicals, until now a limiting step in porphyrin analysis, can be expedited by wavelength-guided enrichment.
Acknowledgment. We gratefully acknowledge assistance from Peter Hambright, Henri J. Callot, and Timothy Lash who provided several porphyrins for this study. T. C. O’Haver provided data handling in the form of a WingZ macro program. S. Kondragunta provided sample data on the Messel Shale. This work was jointly supported by Amoco Production Co., Marathon Oil Co., and Unocal Corp. C.J.B. publishes with the permission of the Director, Bureau of Mineral Resources, Geology and Geophysics, Canberra, Australia.
