Energy & Fuels 1990, 4 , 688-694
688
1.20 (t, 3 H, &-Me [n-Pr,Et]),-1.83, (br s, 2 H, NH); UV-vis A, (relative absorbances) 418 (1.00), 522 (0.0861, 5 $4 (0.1051, 608 (0.063), 666 (0.383). MS analyzed as components of the natural mixture: samole
calc mass calc M
formula
C3eH42N405 (Et,Et] [n-Pr,Et] C3,H,,N405 [i-Bu,Et] CaHgN405
610.3155 624.3312 638.3468
+1
611.3234 625.3391 639.3547
found 611.3230 625.3376 369.3544
Zinc(I1) 9-Deoxo-2-ethy1-3-methyl-Bmph-e/~ (26). Zinc(I1) Bniph-e-triol (420.8 mg) was dissolved in 1,2-dichloroethane(70 mL). Zn12 (893.3 mg; 1.5 equiv) and then NaCNBH3 (878.2 mg; 7.5 equiv) were added. The reaction was allowed to proceed for 2 h at room temperature. It was then diluted with CH2C12,washed three times with water, dried over anhydrous Na2S04, and evaporated to dryness. The residue was chromatographed on a Brockmann Grade I11 neutral alumina column eluting with 25% cyclohexane/CHzC12. The main band was collected and evaporated to give 231 mg of product (66% yield based on molecular weight of the [Et,Et]homologue): 'H NMR, ppm 9.66,9.53(each s, 2 X 1 H, a-meso-H and /3-meso-H),4.70 (m, 2 H, 10-CHz),4.65 (4, 1 H, 8-H), 4.23 (m, 1 H, 7-H), 3.95 (s, 3 H, &-Me),4.05-3.84 (m, containing 2a-CH2,4a-CH2,5a-CH2and 9-CH,), 2.52,2.20 (m,
7-CH2CHz),1.90 (t,3 H, 5b-Me), 1.76, 1.72 (two overlapping q, 6 H, 2b-Me and 4b-Me [Et,Et]), 1.49 (d, 3 H, %Me), 1.24 (t, 3 (relative absorbances) 406 (1.00), H, 4c-Me [n-Pr,Et]);UV-vis A, 512 (0.063), 578 (0.057),620 (0.210). MS analyzed as components of the natural mixture: samDle
[Et,Et]
formula
calc mass
calc M+1
found
C36H42N402Zn626.2599 627.2678 626.2634
(627.2730) [n-Pr,Et] C3,HUN4OZZn 640.2756 641.2835 641.2855 [i-Bu,Et] C~HMN,OzZn 654.2912 655.2991 655.2987
Free Base 9-Deoxo-Bmph-e(19). The zinc(I1)complex was demetalated by shaking with 10% aqueous HC1 for 5 min. It was then washed with water, NaHC03, and twice more with water, dried over anhydrous Na2S04,and evaporated to dryness. The NMR spectrum, UV-vis spectrum, and TLC of this compound were all identical with those of the compound obtained from the deoxygenation of Bmph-c. See compound 19 above.
Acknowledgment. This research was supported by a grant from the National Science Foundation (CHE-8619034).
Derivative Spectrophotometry of Petroporphyrins David H. Freeman* and Thomas C. O'Haver Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742
Received June 11, 1990. Revised Manuscript Received August 20, 1990
Derivative spectroscopy is found to be ideally suited to porphyrin geochemical analysis through numerical differentiation of digitized data provided by diode array spectrophotometry. Choices are made among several derivative and data-averaging algorithms in order to establish proper conformity to porphyrin peak width and to suppress the non-porphyrin background, random noise, and spectral distortion while enhancing the graphic resolution. Illustrative applications of the algorithms are reported. Micropowdered shale (516 pm) was extracted ultrasonically and gave porphyrin assays with *870 relative standard deviation (RSD). Similarly, dilution tests gave concentration ratios with fl70reproducibility. Derivative extinction coefficients, needed for Beer's law in derivative form, were obtained for Ni2+and Vrv02+porphyrin (etio-I) standards in dichloromethane and ethyl acetate with *2% RSD. The resulting gains in analytical precision and speed lead directly toward more reliable study of porphyrin biomarkers.
The petroporphyrins are geological pigments with distinctive rubylike colors. Their analytical determination by spectrophotometry is prone to certain interferences, and its improvement will be considered here. Definitive qualitative analysis for porphyrin pigments is based on X-ray' and NMR2 methods for structure determination. Traces of numerous exocyclic and etioporphyrin-type structures occur in geological extracts as homologous groups3 although an unusual pure metalloporphyrin in crystalline form, a b e l ~ o n i t e is , ~ also known. (1) Ekstrom, A.; Fookes, C. J. R.; Hambley, T.; Loeh, H. J.; Miller, S. A.; Taylor, J. C. Nature 1983, 306, 173-174. (2) Sanders, J. K. M.; Waterton, J. C.; Denniss, I. S. J. Chem. SOC., Perkins Trans. 2 1978, 1150-1157. (3) Thomas, D. W.; Blumer, M. Ceochim. Cosmochim. Acta 1964,28, 1147-1 154.
0887-0624/90/2504-0688$02.50/0
T h e utility of petroporphyrins for exploring the geological record was linked by Philp to the development of improved analytical technique^.^ Of increasing interest is the finding of distinctive petroporphyrin-precursor relationships ascribed to bacterial,6 algal,7-10and heme"J2 (4) Storm, C.,B.; Krane, J.; Skjetne,T.;Talnaes, N.; Branthaver,J. F.; Baker, E. J. Science 1984, 223, 1075-1076. (5) Philp, R. P. Mass Spectrosc. Reu. 1985, 4 , 1-54. (6) Ocampo, R.; Callot, H. J.; Albrecht, P. J . Chem. SOC.Chem. Commun. 1985, 200-201. (7) Ocampo, R.; Callot, H. J.; Albrecht, P.; Kintzinger, J. P. Tetrahedron Lett. 1984,25, 2589-2592. ( 8 ) Verne-Mismer, J.; Ocampo, R.; Callot, H. J.; Albrecht, P. Tetrahedron Lett. 1988,29, 371-374. (9) Verne-Mismer, J.; Ocampo, R.; Callot, H. J.; Albrecht, P. Ibid. 1990, 31, 1751-1754 (Chl b). (10) Chicarelli, M. L.; Maxwell, J. R. Ibid. 1984, 25, 4701-4704.
0 1990 American Chemical Society
Derivative Spectrophotometry of Petroporphyrins inputs. These qualitative perspectives are the fruit of extensive experimental efforts requiring more than a year of study per geological facies. The partial resolution provided by HPLC was sufficient to show that petroporphyrins are altered in response to their burial depth.13 An underlying feature of previous isolation techniques has been the need to modify the procedure for each different ~amp1e.l~ Clearly, in order to develop the practical utility of the petroporphyrins as biomarkers, improvements are needed, not the least of which is the development of faster standard methods as well as reference materials and standards. The analytical goal in the present work is the rapid and accurate measurement of petroporphyrins as isolated groups or mixtures, including the expectation that such improvements should be applicable to isolated porphyrin compounds or standards. The standard error in any analysis is the starting point for method validation.15 This quantity in petroporphyrin analysis is rarely reported, and this may reflect uncertainty in defining either the precision or the accuracy of the measurement. Moreover, petroporphyrin assay is presently inexact because the required analytical properties of petroporphyrins in geological mixtures have not yet been determined. The partial resolution provided by petroporphyrin HPLC"j needs to be improved, but even so, accurate HPLC analysis also requires extinction data for isolated compounds. Fortunately, such data are now starting to emerge.17J8 For the analysis of porphyrin mixtures, an average molar extinction coefficient would provide an approximate basis for improved accuracy. A molar extinction coefficient of 20 000 was citedlg for a mixture of vanadyl petroporphyrins. This value is low enough to suggest predominant exocyclic ring structure,18,mgiven that vanadyl porphyrins with simpler alkyl substituents have higher va1ues.17J8*21,22 There is also a critical need to adopt reliable solvent conditions for spectrophotometry, especially in view of our recent finding that halogenated solvents may be linked to spectrophotometric irrepr~ducibilityl~ that may be explained by porphyrin chemical degradation.23 Another serious problem is the non-porphyrin background that tends to dominate impure petroporphyrin spectra%,%as illustrated in Figure la. Signal amplification alone (Figure l a , inset) boosts both the background and (11)Bonnett, R.; Burke, P. J.; Czechowski, F.; Reszka, A. Org. Geochem. 1984,6,177. (12)Bonnett, R.; Burke, P. J.; Czechowski, F.; Reszka, A. Fuel 1987, 66,515-520. (13)Mackenzie. A. S.:Quirke. J. M. E.: Maxwell. J. R. Aduances in Organic Geochemistry 197'9;Douglas, A. G., Maxwell, J. R., Eds.; Pergamon: Oxford, 1980;pp 239-248. (14)Quirke, J. M. E. Metal Complexes in Fossil Fuels; Filby, R. H., Branthaver, J. F.. Eds.: American Chemical Societv: Washington. DC. 1987: No. 344. DD 308-331. (15)Keith,'f.'H.; Crummett, W.; Deegan, J., Jr.; Libby, R. A.; Taylor, J. K.; Wentler, G. Anal. Chem. 1983,55,2210-2218. (16)Aizenshtat. Z.; Sundararaman, P. Geochim. Cosmochim. Acta 1989,53,3185-3188. (17)Popp, B. P.; Hayes, J. H.; Boreham, C. J. Energy Fuels, submitted for publication. (18) Freeman, D. H.; Swahn, I. D.; Hambright, P. Energy Fuels, submitted for publication. (19)Hodgson, G. W.; Peake, E.; Baker, B. L.; K.A. Clark Volume, Athabasca Oil Sands: Carriav. M. A.. Ed.: Research Council of Alberta, Information Series; Researih Council of Alberta: Edmonton, Alberta, 1963;NO.45,pp 75-100. (20)Hodgson, G. W.; Baker, B. L. Chem. Geol. 1967,2,187-198. (21)Buchler, J. W.; Eikelman, G.; Puppe, L.; Rohbak, K.; Schneehage, H. H.; Weck, W. Liebigs Ann. Chem. 1971,745, 135-151. (22)Edwards, L.; Dolphin, D. H. J.Mol. Spectrosc. 1970,35,90-109. (23)Freeman, D. H.; Castres St. Martin, D. Unpublished data. (24)Nuzzi, M.; Casalini, A. Rio. Combust. 1974,28,49-58. (25)van Eggelpoel, A. Adu. Org. Geochem. 1964,24,227-242.
Energy & Fuels, Vol. 4, No. 6,1990 689
'1
a
t
10.6
cn
n U
350
550
450
650
Wavelength (nm)
f *) N
2nd
5
Derlvatlve
0 N
2
0.3
-
0.28
'
'
'
'
500
Wavelength =o.o1o;
-0.020 u
500
.
,
'
I
550
'
.
-
'
.
,
-0.010
(nm) '
0
'
.
'
'
0
Wavelength
. t1 0.010
0
5
600
550
-
I
600
(nm)
121 111112
F i g u r e 1. Spectrum of chromatographically deasphalted Cero Negro crude oil in (a) shown with inset absorbance scale expansion by lox. T h e derivative spectrum for this sample is shown with expanded wavelength scale with a near-optimal algorithm in (b) and, for comparison, with suboptimal algorithms in (c).
the sought (analyte) spectrum, and so it provides no selectivity. For measuring real samples or extracts, as opposed to solutions of isolated pure compounds, proper background definition," correction, or suppression, as discussed here, are necessary aspects of reliable petroporphyrin assay. Petroporphyrin spectrophotometry can be improved mathematically, instrumentally, or by selective chemical enrichment. The latter is not without risk in view of known decomposition on active ads or bent^.'^^^^ Highly precise diode array spectrophotometry has the advantage that the derivative approach is particularly convenient to apply to digitized absorbance data. This mathematical (26)Millson, M. F.;Montgomery, D. S.; Brown, S. R. Geochim. Cosmochim. Acta 1966,30, 207-221.
690 Energy & Fuels, Vol. 4, No. 6, 1990
a
: n
Freeman and O’Hauer
0.010
0.030 NIP
0.005 0.000 ~
-
-
-
0.025 -
~
9.005
0
.
0
2
0
0.015 0.010
-0.015
Pn
0.005
9.020
0.000
9.025
-0.005
01
9.030 -9.010 500
550
600
Wavelength
(nm)
b P
380-400-nm region. It is more effective to work in the 500-600-nm range where the non-porphyrin background has a less threatening magnitude and a more gentle curvature. In this region the NiP and VOP a-bands and the less intense but confirmative @-bandsare available and are recommended for quantitative analysis. Their derivative spectra are shown in Figure 2a. Derivative spectroscopy includes data smoothing which improves the signal-to-noise ratio. While differentiation suppresses the broad band non-porphyrin background, too much smoothing causes distortion or flattening of the analyte spectral absorbance bands.n An optimal approach can provide better precision, increased sensitivity, and, therefore, an efficient pathway toward improved accuracy. In derivative spectroscopy, Beer’s law is obeyed for well-behaved solute-solvent combinations in the following general form:
P
P 0.2
2
1
n *)
N
a
Absorbance
500
550
Wavelength
-0.020 0.025 600
(nm)
Figure 2. Derivative spectra shown for individual NiP and VOP etio-I standards in (a). The mixed standards are shown in (b) where the more highly resolved derivative spectrum can be compared.
approach suppresses the broad band non-porphyrin background while selectively amplifying the narrower band analyte spectrum. The numerical derivative calculation normally incorporates signal averaging to improve the preci~ion.~’-~~ Reliable analytical methods must also assure the recovery of the measured anal@, or else they should provide a measure of its loss. A different type of problem is created by the use of analytical methods that are not designed to be robust; then there is a need to tailor such methods to individual sample^.'^*^^ By contrast, a robust analysis is designed to provide the broadest possible applicability of method to sample. This problem has been at least partly solved with an efficient separation of whole fractions or groups of nickel porphyrin (Nip), vanadyl porphyrin (VOP), and mixed metalloporphyrin acids.31 For analytical purposes, given the potential for uncertain recovery of porphyrins during the early preparative steps, the derivative technique can help to pinpoint and help reduce losses of accuracy. The practical advantages of derivative spectroscopy are well established for clinical analysis,28 and they are applicable to the present problem. Differentiation of a digitized spectrum of a crude porphyrin extract discriminates against the non-porphyrin background and it is suppressed as a result. Even so, a sufficiently intense impurity background can easily obscure the Soret band in the (27) O’Haver, T. C.; Begley, T. Anal. Chem. 1981, 53, 1876-1878. (28) O’Haver, T. C. Clin.Chem. 1979,25, 1548-1553. (29) Enke, C. G.; Nieman, T. A. Ibid. 1976,48, 705-712A. (30) Baker, Earl W.; Palmer, S. E. In Geochemistry of Porphyrins. The Porphyrins, Structure and Synthesis,Part A ; Dolphin, D., Ed.; Academic Press: New York, 1978; pp 485-551. (31) Freeman, D. H.; Angeles, R. M.; Keller, S. Prep.-Am. Chem. SOC.,Diu. Pet. Chem. 1988,33, 231-238.
The derivative of absorbance with respect to wavelength is proportional to the derivative extinction coefficient, the molar concentration, C, of absorber (solute or analyte), and to the path length, 2,through which the light must pass. Equation 1 can be expressed with a simpler notation, i.e., A” = c”C2, which we will use later on. In the present discussion, numerical differentiation will be referred to digitized absorbance values Ai for diodes at corresponding wavelength values Xi. It is convenient to refer to the wavelength and absorbance values Xo and A , a t an absorbance maximum. Consider several adjacent data pairs (A,, A-J, (&, A,), (A,, A , ) where each pair is separated by fixed increment, A = Xi+, - Xi. If we consider the absorbance series A-,, A,, A,, the first numerical derivative is simply a table of first differences, [A, - A-,I, [ A , - A,], etc. Each difference [Ai+, - Ai] is referred to a wavelength halfway between Xi+, and hi. Similarly, the second derivative is a table of second differences. By subtracting the previous two bracketed terms we obtain the second derivative, [ A , - 2Ao + A_,]. This second derivative is now referred to the center wavelength, A,, noting that Beer’s law measurements are taken at A., The second-derivative absorbance is thus obtained from a measured maximum absorbance, A,, by subtracting the average of two absorbances A , and A-l, each separated by the wavelength increment, A, or some multiple of it, nA. This is mathematically identical with the Allen correction32-33which is used to improve accuracy in clinical spectrophotometric analysis. In general, A2A/AX2= A_, - 2A0
+ A,,
(2)
where the separation increment is nA. The second derivative, A”, has a negative value due to its downward concavity. However, to keep second-derivative absorbance maxima pointing upward, we will follow the graphic convention of plotting and calculation based on -A /’ a t Am=. The derivative technique enhances the fine structure of spectral curves at the cost of a loss in signal-to-noise ratio. The latter is readily compensated by use of sliding average data moo thing.^^ It is helpful to note that when the number of smooths equals the derivative order, n , the Pascal coefficients in eq 2 with values of 1, -2, and 1 are restored with each pair separated by an equal number, n, of zeros. This is illustrated by the entry for A223(Xo)in Table 1. Optimal data smoothing is reached when the number of sliding averages is equal to one more than the derivative order; i.e., three smooths for a second-derivative
Derivative Spectrophotometry of Petroporphyrins
Energy & Fuels, Vol. 4 , No. 6, 1990 691
Table I. Development of Second-Derivative Absorbance, Sliding Average Value at a Reference Wavelength, X, wavelength L4 A_, h2 L1 X, A, X2 A, XI absorbance A_, A_, A_, A_, A, Al A2 A, A4 first smooth A 201(A-J A201 A201 (A-1) A213(X,) second smooth A213( A-1) A213(Xo) A213( A-1) A223( Xo) third smooth A223(X-1) A223(X,3) A 223 ( LI) A223( A,)
1 -2 1 1 -2 1 1 -2 1 -1 0 -1
1 1
1 1
11/31
1 -1 0 -1 1 1 -1 0 -1 1 1 -1 0 -1 1 0 0 -2 0 0
11/31 11/31 11/31 11/91
0 1 1
0 - 2 0 0 0 -2 1 0 0 1 -2 -2
0 0 -2 -2
1 0 0 1
1 1 1 0 1
11/91 11/91 1 11/91 1 11/27)
The table begins with the unsmoothed second derivative:
A201(&) = A-1 - 2Ao + A+ which is combined with A201 values on both sides of Xo to give the average value, A213. After a second sliding average one obtains A223(X0) = (1,0,0,-2,0,0,1)(1/9). This
twice-smoothed three-point sliding average has its averaging coefficient 11/91 in brackets. The noise in A “ diminishes with increased smoothing in the order A201 (unsmoothed) > A213 > A223 > A233 (near optimum).
spectrum.27 The Pascal coefficients in eq 2 can be highlighted by a shorthand notation; i.e., A” = (1,-2,1). T o keep a more explicit nomenclature as simple as possible, the symbol ANSP will be used to denote, respectively, the absorbance A after the Nth derivative, the number of smooths, S, or sliding averages, and P, the number of points in the sliding average. In sliding average smoothing, the average value of several consecutive absorbance values replaces the central value. The order of differentiation and smoothing are unimportant. Table I illustrates a spreadsheet development of a second derivative (N = 2) algorithm after two smooths ( S = 2) of three points (P = 3), which gives A223 = (l,O,O,2,0,0,1){1/9},where the 11/91 results from the double averaging. Similarly, an optimal three-point smoothing algorithm is approached by the S = N + l algorithm,n A233 = (1,1,1,-2,-2,-2,1,1,1)~1/27~. Because A233 contains nine data points, it should be 3lI2-fold more precise than the three-point algorithm A223. As suggested by Table I, increased smoothing broadens the scope of a derivative algorithm, and eventually, this causes distortion. The derivative algorithm should therefore conform to the bandwidth of the absorbance peak. It is intuitively obvious that the span of the derivative algorithm should not greatly exceed the peak bandwidth under which analyte signal is acquired and beyond which non-porphyrin interference may be introduced. For our particular spectrophotometer the wavelength separation between adjacent diodes is A = 2 nm and, using Table I, the A201 algorithm is across 3 diodes with a span of 4 nm, while the A235 algorithm is across 15 diodes for 28 nm. Since the baseline width of a typical petroporphyrin peak is ca. 30 nm, the latter algorithm is near the intuitive limit. (For the fourth derivative with A”” = -1,-4,6,-4,1, A453 is across 15 diodes while A455 spans 25 diodes.) Algorithm width must be considered in terms of two extremes, excessive noise due to insufficient smoothing and excessive peak distortion due to too much smoothing. These are illustrated in Figure IC. We will not provide a mathematical argument as to which algorithm is exactly optimal. However, for second-derivative spectroscopy the A233 algorithm must be nearly so as it provides low magnitudes for residual background curvature, algorithm bandwidth relative to the absorbance bandwidth, short-
term noise, spectral distortion, and random analytical errors. T o be complete, the scope of this inquiry must include the higher order derivative algorithms. To illustrate, if a background spectrum can be represented by a simple power series with up to third-order terms, the fourth derivative will suppress that background because the derivative of X3 is 0. In comparing graphic applications of second- and fourth derivative spectra with equal smoothing, A ”” provides a lower absorbance peak bandwidth as well as more highly pronounced satellite bands. One can foresee certain instances where the fourth derivative may be preferred because of its greater background suppression. The experiments reported here consist of efforts to test the merits of derivative spectroscopy on sample extracts of shale and petroleum following published separation procedures31 with some modifications as discussed next.
Experimental Section A Hewlett-Packard HP9852a diode array spectrophotometer controlled by an IBM PCAT with HP software was used in this work. Data was transferred as needed to a spreadsheet program where various derivative algorithms were tested. All solvents were reagent grade or the equivalent, but this by no means assures accuracy. As discussed elsewhere,IaB porphyrin quantitation in the sub-ppm range is reliable in dichloromethane containing cyclohexene stabilizer (Fisher, No. D134) and much less so with cyclohexane preservative (sic) (Baker Chemical Co., No. 9315-3). Further, Ni” and VIVOporphyrins dissolved in chlorinated solvent without curtailed exposure to light are prone to photodegradati~n.~~ Sedimentary rock samples were pulverized in an organic solvent slurry using a Mixor Mill (Spex Industries, Inc., Metuchen, NJ). Sample preparation was consistently monitored by visible light microscopy until the particle size was below 16 pm.34- The powdered sample was extracted with dichloromethane in a 23mm-i.d. vial immersed in a 125-W ultrasonic bath at 35 OC. An 8-mm metal ball was placed in a covered extraction vial while the vial was moved over a 12-mm distance at 2 cycles/s. The ball remained practically stationary while motion of the vial kept the powdered rock in suspension which is necessary for efficient extraction. Emphasis was placed on precise technique, including the ultrasonic extraction of small particles in suspension which is comparable to other extractive methods which have been reviewed.% We have not proved that any method provides complete extraction of the porphyrin content of a rock sample. The concentration of metalloporphyrins from geoorganic extracts is subject to major asphaltic interference that shows up in the form of chromatographic fouling. However, as found by F e r g u ~ o nmacroporous ,~~ silica gel (preferably 250 A, 30 pm; Amicon, Danvers, MA, Product No. 84240) permits a rapid and efficient separation of metalloporphyrins from the so-called asphaltene component^.^^^^^ Deasphaltation of shale extract dissolved in dichloromethane involves simple chromatographic filtration through macroporous silica. If this is not done, the extract may precipitate in alkane solvent. The second step separation31 of deasphalted extract into NiP, VOP, and a more polar (carboxyl) metalloporphyrinfractions has proved to be straightforward with many samples that we have examined, including all shale extracts and all but one crude oil (Wilmington). Special care was taken to incorporate good spectrophotometric practices which provide *2% reprod~cibi1ity.l~ Quality assurance included frequent testing for sorption of porphyrin on the glass spectrophotometric cuvettes. The use of quartz cuvettes was found to be satisfactory for NiP and VOP extracts dissolved in dichloromethane. To avoid contamination, routine periodic baking of washed glassware was carried out overnight at 450 “C. (32)Allen, W.M. J . Clin. Endocrinol. 1950,10, 71-83. (33)Jensen, S.B.;Oliver,R. W. A. Clin. Chim. Acta 1973,44,443-448. (34)Ferguson, W.S.Bull. Am. Assoc. Pet. Geol. 1962,46,1613-1620. (35)Monin, J. C.;Pelet, R.; Fevrier, A. Inst. Fr. Pet., Reo. 1978, 33, 223-240. (36)Lewan, M. D.Geochim. Cosmochim. Acta 1983,47,1471-1479.
Free man and 0 ’Haver
692 Energy & Fuels, Vol. 4, No. 6,1990 0
.
0
8
1
7
0
.
0
2
S
a N
a
a
N
E
f
n
a
0.03 350
450 Wavelength
-
h
550
650
(nm)
0.16 I
1‘0.01
=oi E
m
0.14-
In 0
E a u E m
c
N
a
0.120.10-
M
0.08of MurDhv Co. Core a i 6571 11
0.06 350
!
i
-0.05 650 I
450 550 Wavelength (nm)
Figure 3. Reflectance spectrum of unpyrolyzed Woodford shale shown below in (a), with its derivative spectrum shown above. In (b) is shown a different shale sample in which neither the reffectance spectrum nor its derivative shows evidence for metalloporphyrins.
Results Derivative spectroscopy provides a more selective utilization of spectroscopic data for purposes of graphic display and for quantitative analysis. The graphic aspect is illustrated in Figure l b , which presents a derivative spectrum of a deasphalted fraction of Cero Negro crude oil as shown in Figure la. The derivative graph provides a more detailed view of traces of the NiP and VOP present in this mixture. For comparison, derivative spectra of NiP and VOP standards are shown separately in Figure 2a and then as a standard mixture in Figure 2b. The derivative spectrum in Figure 2b is less confusing graphically than the coplot of the usual absorbance spectrum. The suitability of the A N S P algorithms is considered next. The A233 algorithm allowed a precise comparison of VOP isolated from Woodford shale: before and after hydrous pyrolysis, as well as VOP isolated from the generated oil. The results (a-band), taken from a study in progress,39were wide-ranging from a low of 0.4ppm NiP in untreated rock to a high of 790 ppm VOP in the oil generated by hydrous pyrolysis. In some instances, however, low-level NiP &bands showed A ”values of the wrong sign. This indicated the presence of higher order An (n > 2) terms in the background and justified the use of a fourth-derivative algorithm. The derivative technique may be used to advantage in other applications. One of our activities is to search for (37) Holden, P. Energy Fuels, submitted for publication. (38)Constantinides, G.;Arich, G.Research on Metal Complexes in Petroleum Residues. Sixth World Petroleum Congress: Frankfurt, Germany, 1963; Verein zur Forderung des 6. Welt-Erdol Kongresses: Hamburg, West Germany, 1963; Paper V-11, pp 65-77. (39)Freeman, D. H.; Lewan, M. D. To be published.
unusually rich sources of geoporphyrins. Reflectance spectroscopy3’ was tested for its potential to serve as a rapid analytical screen. To illustrate the findings, the reflectance spectrum of a compressed powdered drill core rich with vanadyl porphyrin is shown in Figure 3a and indicates the presence of petroporphyrins based on the confirmatory presence of the a- and @-bands. The A,, values are red shifted, which is a ligand field shift or “solvent” effect.38 The reflectance technique was used to identify metalloporphyrin-rich shale members, shown in Figure 3a, flanked by end members, illustrated in Figure 3b, that did not appear to contain detectable metalloporphyrins. Since nondetection is associated with sample noise, that will be considered next. The non-porphyrin background noise was found to vary among the samples studied. Accordingly, it is possible or even likely that trace porphyrin analysis will eventually indicate a false positive, Le., a spurious detection of a substance that is actually below the detection limit. The American Chemicial Society has recommended for qualitative or quantitative analysis that the magnitude of the analyte signal should be, respectively, at least 3 or 10 times the standard deviation obtained with comparable analyte-free ~amp1es.l~ The way this rule is applied, and the definition of a suitable sample blank, needs to be considered with care. While the porphyrin content of extracted matter, or bitumen, is measured after extraction, reflectance spectroscopy may respond to porphyrin signal from porphyrins that ordinarily might be nonextractable. As demonstrated in the cited hydrous pyrolysis study, the a-band derivative can be allowed only if it exceeds the weaker @-bandwith the correct mathematical sign. A stringent requirement for quantitative analysis would be for the derivative a- and @-bandabsorbances to have a ratio that falls within certain prescribed limits. Such criteria, as well as personal judgement, can be used to help avoid false negatives or false positives when assessing the petroporphyrins in weak spectra such as that illustrated in Figure 3b. Quantitative Spectroscopy. Quantitative analysis of petroporphyrins was explored by applying different derivative algorithms to spectra of the same VOP group extract after varied dilution. The absorbance spectra of diluted shale extract are referred in Table I1 to the spectrum of a VOP (etio-I) standard.18 Derivative calculations of concentration and of mass balance are based on the following. Given that A” = &” (Beer’s law), and keeping path length 2 constant, then the measured sample concentration is C ( x ) = A ” ( x ) C ( s ) / A ” ( s )where , (x) refers to the unknown in the sample and (s) refers to that in the standard. Several A N S P algorithms are compared in Table 11. The reader is invited to examine the different algorithms used in rows 22-27. Included is the second-derivative of the algorithm, Ahp2d = (-4,-4,-1,4,10,4,-1,-4,-4)(1/400) present diode array spectrophotometer. The related concentrations C ( x ) were calculated for rows 30-35. The results in Table I1 show that estimated concentrations C(x) of diluted VOP petroporphyrins vary to some extent with the algorithm, even though each algorithm is applied identically to the unknown ( x ) and to the standard (s). Algorithms with five point sliding averages (P = 5) gave higher results compared to those with a three-point average. The fourth derivative, A433, gives a lower result. These differences are more than 3 times the standard deviation with which a single sample can be measured repetitively,18 and it should be feasible to determine experimentally which algorithm is most exact. A possible
Derivative Spectrophotometry of Petroporphyrins
Table 11. Dilution Study of Woodford Shale VOP Extract (HP879) B C D E
A 1 2 3 4 5 6 7
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52
Energy & Fuels, Vol. 4, No. 6,1990 693
Absorbance for Sample Volume (V) V=5mL V=10mL V=50mL 1.4387 0.7762 0.1767 0.8625 0.1933 1.6057 1.8249 0.9768 0.2153 1.0781 0.2349 2.0068 2.1582 1.1704 0.2528 1.2457 0.2678 2.2756 1.3100 0.2807 2.3883 2.4147 1.3220 0.2828 2.2858 1.2424 0.2677 1.9982 1.0863 0.2369 0.9071 0.2017 1.6545 1.3675 0.7564 0.1714 1.1457 0.6371 0.1469 0.5715 0.1338 1.0156 0.1256 0.9149 0.5242
x 556 558 560 562 564 566 568 570 572 574 576 578 580 582 584 A223 A225 A233 A235 A433 Ahp2d
0.1130 0.0744 0.1006 0.0607 0.0191 0.0246
ANSP Values 0.0629 0.0412 0.0557 0.0332 0.0108 0.0136
C223 C225 C233 (2235 c433 Chp2d
for V = V(C223) V(C225) V(C233) V(C235) V(C433) V(Chp2d)
Calculated Total VOP (pg) 5 mL 10 mL 116.1 129.4 124.6 138.1 117.3 129.9 130.1 142.3 101.1 114.2 117.4 130.3
9.96 pg/mL VOP Etio Standard 0.0934 0.1193 0.1617 0.2151 0.2894 0.3767 0.4663 0.5180 0.5042 0.4228 0.3108 0.2090 0.1315 0.0865 0.0542
0.0123 0.0081 0.0110 0.0066 0.0021 0.0027
Calculated Concentrations (pg/mL) 23.23 12.94 2.54 24.92 13.81 2.73 23.46 12.99 2.56 26.02 14.23 2.81 20.23 11.42 2.25 23.48 13.03 2.57
F
0.0484 0.0297 0.0427 0.0232 0.0094 0.0104 Concn Ratio: C (10)/ C(50) 5.10 5.06 5.07 5.07 5.07 5.08
50 mL 126.9 136.3 128.1 140.4 112.6 128.3
A223 = (-Dl3 + 2D10 - D7)/9, A223 = (-Dl5 + 2D10 - D5)/25 A233 = (2SUM(DlO:D12) - SUM(D7:DS) - SUM(D13D15))/27 A235 = (2SUM(D8:D12) - SUM(D3:D7) - SUM(D13:D17))/125 A453 = (SUM(D3:D5) - 4(SUM(D6:D8)) + G(SUM(D9:DlI)) - 4(SUM(D12:D14)) + SUM(D15:D17))/243 Ahp2d = (10D10 + 4(D9 + D11 - D6 - D7 - D13 - D14) - D8 - D12)/400 (2223 = 9.96D19/H19
source of bias may be that standard is a single compound while the sample consists of many porphyrins with different molecular structures and different spectral properties” (with a definable average extinction coefficient). A contributing problem, up to an estimated several percent of error, may arise for the following reason: diode array instrumentation does not quite provide A,, exactly because the true A, seldom coincides with one of the diodes. Concentration ratios of petroporphyrins in a fixed amount of sample extract, diluted from 10 to 50 mL are indicated by the C(l0 mL)/C(50 mL) ratios. The several derivative algorithms applied to these bitumen dilution data show that concentration ratios are obtained within f l % statistical variation. A determination of mass balance was obtained during the dilution tests. The VOP mass, product of concentration of VOP and sample volume, using the different algorithms, is estimated in Table I1 as 130 pg after dilution a t 10- and at 50-mL volumes. The results show good agreement for the respective algorithms at 10 and 50 mL. The results at 5 mL volume are significantly worse. This
is because substantial departures from Beer’s law occur above ca. 20 pg/mL NiP and VOP concentrations.ls The results from the A233 and Ahp2d algorithms, in the absence of more exact standards, appear to be appropriate for petroporphyrin analysis, except when the fourth derivative is mandated by background. Until more appropriate standards are incorporated (by averaging) into spectrophotometric analysis of petroporphyrin mixtures, such analysis can be referred only approximately to any single compound as a standard. A digitized portion of the visible spectrum of VOP (etio) reference solution is given in the far right column in Table 11. The maximum in the zero- and second-derivative absorbances occur at 570 nm (within 1nm of the true Am=). The corresponding A233 and Ahp2d derivative values are 0.0427 and 0.0104, respectively, using the expressions on lines 47 and 51. To illustrate the calculation of concentration using C = A”/t”Z (eq 1) with a cell for which Z = 1 cm, the concentration of the VOP analyte can be calculated from (I/&’) values given in Table I11 for VOP etio-I standards
Freeman and O'Hauer
694 Energy & Fuels, Vol. 4, No. 6, 1990 Table 111. Derivative Reciprocal Extinction Coefficients (I/d'lua/(mL*cm)l) for Nip-E and VOP-E" compd VOP-E VOP-E Nip-E Nip-E
solvent MC EtOAc MC EtOAc
n 4 3 5 5
(1/;:'233) 232 f 6 182 f 3 258 f 3 201 f 4
(l/c%p2d) 955 f 25 750 f 13 1051 f 15 826 f 16
A,,
570.7 f 0.1 568.8 f 0.0 551.4 f 0.0 550.3 f 0.1
" E = etio-1 structure, MC = dichloromethane, and EtOAc means ethyl acetate. Results calculated from measurements reported in ref 18. N.B. f refers to the standard deviation of n determinations. Table IV. Representative Tests of Analytical Precision in Petroporphyrin Assays of Homogenized Shale Samples Extraction with Varied Solvent for 90 min at 60 OC extraction solvent VOP. u e l e ED:MeOH 9:l" 26.9, 27.1 ED:MeOH 2:l 25.9, 24.5 ED 23.8, 24.3 av (n = 6) 24.6 SD (n = 6) 1.9 0.08 RSD ( n = 6) With Varied Extraction Times in ED:MeOH (19:l) a t 65 "C time, min VOP, pg/g VOP (COOH), rg/g 20 24.2 1.84 20 22.3 1.54 80 23.1 1.15 80 27.2 1.4 160 26.7 1.62 av 24.4 1.5 SD (n = 5) 1.95 0.22 RSD (n = 5 ) 0.08 0.15 ED stands for dichloroethane.
in dichloromethane. To follow this, refer in Table I1 (cell C27) to the value Ahp2d = 0.0136, which refers to a VOP extract in dichloromethane solvent. The determined VOP concentration is c = A"(l/t")(l/Z) = 0.0136 (955 pg/ (mL.cm))(l/l cm) = 12.99 pg/mL, which should be rounded to 13.0 pg/mL. Note that this agrees with the value 13.03 (cell C35), which should also be rounded to 13.0 pg/mL based upon a single VOP-E reference solution (column F). The average value given in Table I11 is expected to be more accurate. In assessing the ruggedness of any assay of geological samples, it is helpful to determine the precision of replicate determinations of different portions of the same sample. A precision of f l 6 % RSD (relative standard deviation) was reported earlier using conventional spectrophotometry.31 A series of subsamples from unpyrolyzed Woodford (HPO) shale were extracted and measured. Tests of varied extraction times and different solvents are illustrated in Table IV. On this basis, analytical reproducibility appears to be feasible within AO.1 RSD or A1070 relative error. Pure methanol, or ethyl acetate which is worse, is not
1 2 3 4 4 5 5 6 6
Table V. Assays of Individual Shale Grains" shale samdeb WD4 (HPO) WD9 (HP9) grain no. mass, mg VOP, pg/g mass, mg VOP, pg/g 72.3 123.2 30.1 94.7 148.8 30.4 95.6 28.1 22.3 116.0 53.0 80.9 94.7 72.3c 83.6 63.3 75.3e 94.7 62.1d 88.7 107.6 57.1d 122.6 56.1e 60.ge 102.6
av value, pg/g SD, rg/g RSD (n = 6)
52 (n = 6) 22 (n = 6) 0.4 (n = 6)
44 (n = 4) 17 (n = 4) 0.4 (n = 4)
OExtracted for 20 min in dichloromethane a t 35 O C . bHP9 means with hydrous pyrolysis; HPO means without. Repeated assay of grain extract. d*e Assayed subsamples of homogenized grain.
suitable alone for porphyrin extraction. Additional experiments wel'e carried out to explore the effect of sample size. Particle-to-particle variability in petroporphyrin content is indicated in Table V. If this variability were truly random among the 0.1-g samples, then the interparticle variations should obey the n1/2rule and a 4-fold reduction of sample-to-sample variability would be predicted for a 1.6-g sample. This was verified approximately by finding RSD values below 0.1 for VOPrich rock samples of 1 g. It is concluded that derivative spectroscopy facilitates the precise analysis of petroporphyrins, but only in the context of careful preparative techniques, stability in solvent, avoidance of cuvette contamination, reduced exposure to light, etc. The use of automated data acquisition and data handling helps to eliminate human error. The present implementation of derivative spectroscopy takes full advantage of a highly accurate diode array instrument. For the most part, the derivative technique provides a major improvement in terms of interference suppression with improved precision, sensitivity, speed, and reliability. The results indicate a markedly improved approach to the precise measurement of porphyrin mixtures that should be equally applicable to isolated porphyrins. As the extinction properties of individual porphyrin coefficients are now becoming available," the next step is to improve the accuracy.
Acknowledgment. This work was supported in part by a gift from the Amoco Production Co. We are grateful to Irvine D. Swahn, Charles E. Walsh, and Peter N. Holden, who provided measurements we have discussed. Jerry L. Clayton (USGS) and Michael D. Lewan (Amoco Production Co.) provided shale and oil samples used in this work. Registry No. NIP, 14055-19-7; VOP, 129849-31-6; dichloromethane, 75-09-2; ethyl acetate, 141-78-6.
