22
Anal. Chem. 1983, 55, 22-29
supported in part by grants from the Faculty Research Committee of Miami University, the Research Corporation, and the donors of the Petroleum Research Fund, administered by the American Chemical Society. Infrared spectra were taken on a Perkin-Elmer Model 680 spectrometer funded by the National Science Foundation through Grant TFI-8022902.
Donation of the 9533 liquid chromatograph by IBM to the Chemistry Department is appreciated. R. W. Siergiej gratefully acknowledges support provided by a Dissertation Fellowship from Miami University. This work was presented at the 183rd American Chemical Society Meeting, Las Vegas, NV, March 30, 1982.
Liquid Chromatography/Proton Nuclear Magnetic Resonance Spectrometry Average Composition Analysis of Fuels James F. Haw,’
T. E. Glass, and H. C.
Dorn”
Department of Chemistty, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 2406 1
The use of a NMR spectrometer as a continuous flow llquld chromatographlc detector (LC/‘H NMR) generates a proton spectrum of each hydrocarbon class present In the sample. A detailed set of equations Is presented which permtfs LCPH NMR Integration data from petroleum fuels to be Interpreted as an average compostflon for each chromatographlc fractlon. Ouantltles calculated for each aromatic fraction Include: the number average molecular weight, average degree of substltutlon on aromatlc rings, the absolute number of moles of each structural type of carbon, an average structure (devoid of stereoisomer Information), the total number of moles of carbon In each chromatographlc fraction, and numerous other propertles of Interest In fuel characterization. The method Is demonstrated for artHlcal fuels of known composltlon, for two experlmental aviation fuels, and for a fuel blending stock sample which had been fully characterlzed at an Independent laboratory by gas chromatography and GC/MS. The LC/‘H NMR average composltlon method Is shown to be very accurate for the monocycllc aromatic (substtfuted benzenes and tetrallns) and dlcycllc aromatic (substituted naphthalenesand acenaphthenes) fractions of petroleum fuels. Average molecular weights for these fractions can be routinely determined at an accuracy of 1 4 daltons. The other quantltles are also determined at a hlgh degree of accuracy. The appllcablllty of the LC/’H NMR method to the aliphatic fractlon of fuel samples Is restrlcted by dlfflculties In accounting for quaternary carbons and cycloalkanes.
Often in fuel analysis it is unnecessary (even undesirable) to identify and quantify every component. In these cases, certain average compositional data (vide infra) may be related to desirable or undesirable properties of the sample. A familiar average compositional datum is aromaticity which is easily measured via 13C NMR. Identification and quantitation of every compound also permit aromaticity to be calculated. This also provides a great deal of additional information, often obtained a t great expense. This paper delineates systematic methodology for determining average compositional data for the aromatic fractions of low boiling fuels. Inherent in the determination of average compositional data are the following questions. How much information can be extracted reliably from a spectrum with as little a priori knowledge as possible? Present address: Department of Chemistry, Colorado State University, Ft. Collins, CO 80523. 0003-2700/83/0355-0022$0 1.50/0
TOwhat extent can the method be made independent of crude correlations derived from a limited set of test samples? Can a method be developed that is reliant on only one measuring device rather than on a collection of different instruments? In considering which instrumental method is most appropriate for petroleum fuel analysis, the following considerations apply. The resulting information must unambiguously correlate with structure. In other words, a peak in a certain position must indicate a specific structural unit and no other. Furthermore, the intensity (or area) of each peak must be directly proportional to the relative population of that structural unit in the sample. The proportionality should be linear to avoid the need for calibration curves. In addition, the proportionality should be independent of the remainder of the structure. For this reason infrared absorption data are unsuitable. All of the above requirements are readily met by proton NMR spectrometry. With careful attention to experimental parameters, 13C NMR is also a suitable candidate. Other techniques (e.g., mass spectrometry) have greater sensitivity for trace constituents, but since average composition is desired here, the sensitivity of modern NMR spectrometers is entirely adequate. The scheme presented here is based totally on the use of ‘H NMR analysis of several easily obtained liquid chromatographic fractions. The option of using one piece of 13C NMR data (aromaticity of the bulk sample) to derive additional information is also discussed. NMR has been of interest to fuel chemists ever since early high-resolution instruments became available. Since this time, several “average structure” schemes using NMR in association with other techniques have been developed. For the most part, these techniques have been proposed for total aromatic cuts (obtained by open column liquid chromatography on silica gel). These methods will not be reviewed in detail since this has been done by Clutter et al. (I). Each method does have limitations which deserve mentioning. In 1958, Williams (2) reported a method for determining an average structure for fuel cuts. Quantitative ‘H NMR, elemental analysis, molecular weight data, other mass spectral data, and semiempirical correlations based on a poorly defined quantity (branchiness index) were used as input data for calculations that gave the average numbers of each carbon type in a hypothetical average molecule. Williams was severely limited by existing instrumentation, but his work was very impressive for its time. Proceeding in a similar vein, Brown and Ladner (3, 4 ) were able to interpretate IH NMR spectra of soluble coal-derived samples in terms of an average structure framework. Knight (5) employed 13CNMR, IH NMR, average molecular weight 0 1982 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 55, NO. 1, JANUARY 1983
data (from mass spectrometry or WO),arid elemental analysis to arrive at an average sitaucture. This method has some nice features since the carbon backbone is examined directly. But like many of these methods, it requires a rather large and diverse quantity of data to be acquired. Clutter et al. (1) developed a method based solely on lH WMR spectrometry. In order to calculate average parameters for monocyclic and dicyclic aromatics, certain assumptions are made which are of questionable validity. For example, monocyclic and dicyclic aromatic ring protons are determined from one spectrum, with a dividing line at 7.05 ppm. Our fuel samples rarely show any hint of a valley at or near this dividing line, even at 200 MHz. They further assumed that the average number of alkyl substituents on benzene rings was identical with the average number of substituents on naphthalene rings. This assumption is necessary to solve their equations. The fraction of monocyclic aromatics is then calculated by an iterative process based on these assumptions This method may give good results (or a t least show trends) for certain sets of samples. But for experimental fueb (which may be doped with additives to alter performance) the calculated values may be in considerable error. The akwe methods suffer because they were developed for the anallysis of fractions containing the total aromatic content of the fuel. The need to somehow estimate the relative proportion of compounds of different classes (e.g., substituted benzenes and substituted naphthalenes) complicates the calculativie process. Modern normal-phwe liquid chromatography is readily able to separate low boiling fuels into distinct hydrocarbon classes. Aliphatics, monocyclilc aromatics, dicyclic aromatics, and tricyclic aromatics readily may be collected as separate fractions with the total separation requiring between 5 and 30 min (depending on choice of column, solvent, and flow rate). A suitable solvent is 1,1,2-trichlorotrifluoroethanewhich is less expensive than most chromatographic solvents and yields no proton signals. Chloroform-d may be used as a polar additive at little additional cost. This permits fractions collected from the chromatographic column to be submitted directly for lH NMR analysis without the manipulation difficulties and potential sample loss problems associated with LC solvent removal and subsequent uptake in a deuterated solvent. The high sample load capacity of semipreparative HPLC columns and the high sensitivity of modern spectrometers permit the total analysis to be done with a single injection of 100 pL or less. Much smaller injections can be made at the cost of lower signal to noise or increased spectral acquistion time.
EXPERIMENTAL SECTION A series of seven model mixtures, designed to resemble typical fuel samples, were prepared from reagent grade chemicals. One of these (a standard model mixture used frequently in this lab, designated “model C”) was prepared by mixing 13.29 g of n-butylbenzene, 18.47 g of n-pentane, 10.16 g of rn-xylene, 13.07 g of tetralin, 95.70 g of n-nonane, 56.20 g of hexadecane, 200.40 g of isooctane, 43.65 g of n-hexane, 85.67 g of dodecane, and 12.80 g of naphthalene. The remaining six model mixtures were prepared by adding known quantities of an additional compound to model C. Two National Aeronautic and Space Administration experimental fuel samples wlere supplied by the Air Force Aero Propulsion Laboratory (Wright-Patterson Air Force Base, OH) for analysis. The two NASA fuels were designated NASA-Lewis 3s and NASA-Lewis 3B. In addition, one Naval Research Laboratory (NRL) sample (81-3,a blending stock of alkylbenzenes) is reported. All samples were subjected to on-line L,C/lH NMR analysis without pretreatment. A Whatman Magnum9 silica gel-PAC column (500 mm X 9 mm i.d.) was used for all neparations. The packing in this column is silica gel derivatized t o introduce amino and cyano functionalities to the surface. Retention of aromatic hydrocarbons on this column is generally superior to silica gel columns. A special
23
activation sequence was used to remove polar compounds (e.g., methanol) from the column. These polar compounds slowly bleed off with nonpolar solvents and create large background signals in the proton spectra. The following sequence completely removes this background: 50 mL of 10% acetonitrile-d3(99%-d,Aldrich) in chloroform-d (99.8%-d, Aldrich) was followed by 60 mL of chloroform-d. The chromatographic solvent, 97.5% l,l,l-trichlorotrifluoroethane(Miller-StephansonChemical Co.) and 2.5% chloroform-d, was then pumped through the column. Column equilibration was generally achieved after 60 mL. A second source of background is particular to semipreparativeLC columns. Large internal diameter columns do not completely flush the alkane fraction due to partially stagnant regions near the inlet, outlet, and wall. In terms of LC/lH NMR this effect can result in a very small amount of aliphatic material being present in the spectra of the aromatic fractions. For fuels of low aromaticity, the signal is intense enough to make the measurement of CH2and CH3‘H integrals for alkyl aromatics erroneously high. A correction for our column was determined by injecting a very low aromaticity fuel and measuring the relative aliphatic signal intensities in each file. This tailing problem is apparently not present in analytical scale columns which have smaller internal diameters. The chromatographic solvent contained 0.05% (v/v) hexamethyldisiloxane (HMDS, Merck) as a chemical shift and quantitiation reference. The solvent was not degassed since dissolved oxygen reduces proton spin-lattice relaxation time values to several seconds. A Waters M-45 pump equipped with a needle valve to create a lo00 psi back-pressure was used. The M-45 pump requires back-pressure to activate its pulse dampener. A Valco injector equipped with a 100-pL sample loop was used. A 1-mL rinse of solvent followed by 1 mL of sample was used to ensure that the sample loop was thoroughly flushed. All samples were injected neat. A guard column was used as a matter of course. A Jeol FX-200 nuclear magnetic resonance spectrometer equipped with an Oxford 4.7-T superconducting solenoid magnet (54 mm bore) was used to obtain ‘H spectra at 199.50 MHz. A floppy disk system was used for data storage and each diskette had sufficient storage for 58 (1024 point) LC/IH NMR spectra. A flow cell designed for quantitative work was used for all analyses. This cell is described in detail in ref 10. Further details on flow probe design may be found in ref 8. The average composition equations were incorporated in a BASIC program which was run on a HewletbPackard HP-85. A copy of this program is available upon request. Aromaticity was measured from 13C spectra obtained at 50 MHz. Tris(acetylacetonato)chromium(III), C r ( a ~ a c )was ~ , added to each sample to reduce 13C spin-lattice relaxation times for aromaticity measurements. Gated decoupling was used for NOE suppression. Long pulse delays were also used to further ensure quantitative 13C spectra. All 13C spectra were run under conventional (spinning) conditions in 10-mm sample tubes.
RESULTS AND DISCUSSION Our laboratory is currently interested in several applications of NMR of flowing systems, principally the use of NMR as an on-line, continuous flow HPLC detector (6-9). The equations in our average composition method are applicable to both fraction collection (off-line) and directly coupled systems (on-line LC/lH NMR). Most laboratories will prefer to use the off-line approach, at least until a commercial flow probe becomes available. Whichever approach is used, the effort spent in the preliminary separation step is a small price for the resulting more explict and reliable calculations (vide infra). For the purposes of this paper, the average composition of a low boiling fuel sample is defined in the following manner. For each hydrocarbon class, the absolute moles of each distinct carbon type (with associated hydrogens) in a specified aliquot is determined. An example of a distinct carbon type is an unsubstituted aromatic ring carbon (which has one associated hydrogen). Substituted aromatic ring carbons (having no associated hydrogen) clearly constitute a separate type of carbon. Having determined the number of moles of each carbon type in a fuel aliquot, it may be possible to normalize
24
ANALYTICAL CHEMISTRY, VOL. 55, NO. 1, JANUARY 1983
MONOCYCLIC AROMATIC FRACTION
c&z’oL
%n
DICYCLIC AROMATIC FRACTION
P P M 9
8
7
6
5
4
3
2
1
Flgure 3. A 200-MHz LC/lH NMR spectrum of the monocyclic aromatic fraction of NASA-Lewis 3B.
Flgure 1. Representative carbon types for the monocyclic and dicyclic aromatic fractions.
P P M 9
PPM
2.5
2.0
1.5
1.0
0.5
d
Flgure 2. A 200-MHz LCI’H NMR spectrum of the allphatic fraction of NASA-Lewis 38. Integration regions are symbolized.
these data to a hypothetical average molecular structure. Although the average structures have no real significance themselves (since they are hypothetical constructs of the normalized compositional data), quantities derived from the average structure (e.g., average molecular weight) can be valuable. Table I defines some of the major average compositional quantities discussed in this paper. Symbols based on the letter C refer to carbon types. Compounds with three aromatic rings are neglected in this present treatment because of their uniform low levels in the fuels we have examined to date. However, extension of this method to include tricyclic aromatics is certainly feasible. Many monocyclic aromatic carbon types have counterparts in the dicyclic aromatic fraction. Redundant definitions, symbols, and equations will be minimized by using the right superscript x (x = m or d) which designates monocyclic aromatics or dicyclic aromatics. An asterisk (*) in the left superscript position of symbols based on C indicate that a normalized carbon type is indicated. A subscript (e.g., aCH,) is used to specify the carbon type. The various carbon types are illustrated for several molecular structures presented in Figure 1. Figures 2-4 show the on-line LC/lH NMR spectra for the alkane, monocyclic, and dicyclic aromatic fractions of a jet fuel. In addition, these figures indicate the various ‘H NMR integration regions for the various hydrogen types (e.g., Hm,, etc.). We have previously shown that these fractions are clearly separated with base line resolution between each fraction, confirmed via a refractive index detector on-line between the HPLC column and the NMR flow probe. The various chemical shift regions which are integrated to produce input parameters are indicated. Superscripts a, m, and d
8
7
6
5
4
3
2
1
Flgure 4. A 200-MHz LC/’H NMR spectrum of the dicyclic aromatic fraction of NASA-Lewis 38. The broad signal at -3.5 ppm is background from the probe which has since been eliminated.
(alkane, monocyclic aromatic, and dicyclic aromatic) indicate that separate but analogous terms exist for the three fractions. In a separate paper (9),we have demonstrated quantitation via on-line LC/lH NMR. A result from that study (which is entirely valid for both on-line and off-line fraction collection) relates the integrated area of the reference peak to the known number of moles of equivalent reference protons in each fraction (eq 1). In this equation, n, is the number of
K ( P ) = n,M,V“/H,”
x = a, m, or d
(1)
equivalent protons in the reference; for Me4Si n, = 12 and for HMDS n, = 18. M, is the molar concentration of reference. In our laboratory, the reference (usually hexamethyldisiloxane (HMDS) because of its low volatility relative to Me,Si) is added directly to the chromatographic solvent. We most commonly use a HMDS concentration of 0.500 g/L. H”,refers to the integrated area of the reference peak in the appropriate fraction: aliphatic, monocyclic aromatic, or dicyclic aromatic. The K ( vb) terms are designated as reference response factors. In the following discussion, monocyclic aromatics include alkylbenzenes plus tetralins and indans. Dicyclic aromatics are taken to be alkylnaphthalenes and acenaphthene derivatives which are analogous to indans in the monocyclic fraction. The inclusion of biphenyl derivatives in the dicyclic fraction is discussed later. Having shown how measurements of hydrogen types may be made via LC/IH NMR data and the reference response factor formalism, it is now possible to develop a series of equations relating hydrogen type composition to carbon type composition. In ref 7, we presented equations for the fraction of substituted sites (FSS”)and average degree of substitution (ADS”). These equations are not repeated here. In the following discussion, the equations for the monocyclic and dicyclic fractions are presented together. The superscript x is
ANALYTICAL CHEMISTRY, VOL. 55, NO. 1, JANUARY 1983
25
Table I. Definitions of Representative Symbolsa 'H NMR integrated area €or the reference peak (HMDS) of the aliphatic monocyclic aromatic or dicyclic wr aromatic fractions (x = a, m, or d (aliphatic, monocyclic aromatic, or dicyclic fraction)) ' H NMR integrated area for methine groups (R,C-H) in the alkane fraction HaCH IH NMII. integrated area for methylene groups (R-CH,-R) in the alkane fraction 'H NMR integrated area for methyl groups (R-CH,) in the alkane fraction CH3 'H NMR integrated area for aromatic hydrogens in the monocyclic aromatic or dicyclic aromatic fracHXAr tion ( x = m or d) 'H NMR integrated area for aliphatic ring hydrogen a t o aromatic rings (e.g., a-methylene hydrogen in HXoning tetralrn) in the monocyclic or dicyclic aromatic fractions (x = m or d ) IH NMli integrated area for aliphatic methylene hydrogen a to aromatic rings (e.g., methylene group in HXaCH, ethylbenzene) for the monocyclic or dicyclic aromatic fractions (x m or d) 'H NMli integrated area for aliphatic methyl hydrogen a to aromatic rings (e.g., methyl groups for HXaCH3 toluene and/or 0-methylnaphthalene) for the monocyclic or dicyclic aromatic fractions (x = m or d) ' H NMII. integrated area for aliphatic hydrogen groups not adjacent to aromatic rings (Le., greater than HX>a a ) for the monocyclic or dicyclic aromatic fractions (x = m or d) moles of unsubstituted aromatic carbon for the monocyclic or dicyclic aromatic fractions (x = m or d) C Xun moles o f substituted aromatic carbon for the monocyclic or dicyclic aromatic fractions (x m or d) CXmb moles of' methyl carbon a to aromatic rings (x = m or d) 'o~CH, moles of' methylene carbon 01 to aromatic rings (x = m or d) CXaCH, moles of aliphatic ring carbon a to monocyclic aromatic rings (e.g., a-methylene carbons in tetralin) Cm&g moles of aliphatic methylene carbon not bonded to aromatic rings (i.e., greater than a ) C"CH,>a moles of terminal chain methyl carbon not bonded to aromatic rings (Le,, greater than a ) CH, >a moles of bridgehead carbon for the dicyclic aromatic fraction (i.e., * C d B ~= 2.0) CdBH the total carbon in a given fraction (Le., alkane, monocyclic, or dicyclic fraction) C"tota1 ADSX the average degree of aromatic substitution for the monocyclic or dicyclic fractions MWX the number average molecular weight for the monocyclic or dicyclic fraction the carbon aromaticity for the total sample (Le., the total aromatic carbon (CA,(t,,tal)) divided by total fa carbon (Ctotal)) the partial carbon aromaticity contribution for the monocyclic aromatic or dicyclic aromatic fractions fa" (i.e., f a = f a m + f a d ) F" the fraction of total carbon in each chromatographic fraction (i.e., Fatotalt Fmtotal+ Fdtotal= 1)
;p*
If the cara See Figure 1 for further clarification of symbols and Figures 2-4 for defined 'H NMR integration regions. bon types indicated below have a superscript asterisk (e.g., *Cmun), this denotes the number of each carbon type in the hypothetical average molecule. used to indicate that separate, but analogous, terms exist for the two fractions (i.e., x .- m or d). For some quantities (e.g., average molecular weight) this is not possible and separate equations are presented. Two common problems with previous average composition methods have been relating proton integrals to carbon content and normalizing the data to an average molecule. In the LC/lH NMR method, the number of aromatic ring carbons is known for both of thle aromatic cuts. The existence of an aromatic ring proton imlplies the existence of an unsubstituted aromatic ring carbon. Likewise, the existence of three aCHB protons implies the existence of an a carbon and a substituted aromatic ring carbon. These absolute mole values are obtained via the product of the reference response factor with the appropriate proton integration value weighted for proton/ carbon ratio (e.g., for an aCHz group, 2 mol of protons in the aCHz region implies the existence of 1 mol of a carbons). Unsubstituted aromatic ring carbons are given by eq 2.
Substituted aromatic ring carbons are given by eq 3.
For the monocyclic fraction, normalization is obtained via the following equation. The number of carbons of each type in the "average structure" of the monocyclic aromatic fraction is obtained by dividing the absolute number of moles of each carbon type by the appropriate normalization constant and reference response factor. This is illustrated by eq 6 for *e,. The asterisk denotes the number of each carbon type (*Cx,) in the hypothetical average molecule instead of the absolute number of moles of carbon (Pun) in the injected sample.
For the dicyclic fraction (here assumed to be naphthalenes and acenaphthenes), the normalization constant is given by the following equation.
(7) The normalized dicyclic fraction carbon types are obtained by analogy to eq 6. Naphthalene molecules also contain two bridgehead carbons.
1 C d = ~-[Cdun 4 ~ + CdsubI (3) At 200 MHz and above, the various a proton types are sufficiently spectrally resolved that quantities such as C d H 3 may be separately defined (Table I and eq 4). In a similar manner, equations for CxaCH2, C'snring, and CXnCH can be easily written except with denominators of 2, 2, and I., respectively.
*CdBH
=
CdBH/K(Vd)Nd
=2
(8) (9)
The region of the proton spectra of the monocyclic and dicyclic fractions upfield of 2 ppm contains all alkyl chain protons a t positions P, y, 6, and higher with respect to the aromatic ring. Division of this region into distinct subregions (e.g., PCH,) is impractical for most fuel samples. The region H",, is instead integrated as a whole and equations for *C'CH*>~ and *CXCH2>* have been derived.
26
ANALYTICAL CHEMISTRY, VOL. 55, NO. 1, JANUARY 1983
These equations assume that no branching exists beyond the a position. Experience in our laboratory indicates that this is a reasonable assumption for aviation fuels. Alkyl substitution on aromatics tends to take the form of n-alkyl and tetralin. Similarly, we frequently ignore the acHregion. Even if branching is present beyond the a position, the accuracy of the average properties calculated below (e.g., average molecular weight) will be unaffected. The absolute number of moles of alkyl carbon in positions greater than a (e.g., CXCH3>a and CXCH2>a) can be calculated directly from *CXCH3>,and *CXCH2>,, respectively.
C ” C H ~ > *~ C ” C H , > P K ( ~ )
(12)
C’CH~>~ = * C ” C H ~ > Pp) K(
(13)
The total number of moles of aromatic carbon in the monocyclic aromatic fraction and the total number of moles of carbon (alkyl plus aromatic) in this fraction are calculated via eq 14 and 15, respectively. CmAr(total) Cmtotal
=
CmAr(total)
-k
=
c”un
CmaCH3
+ Cmsub
(14)
+ CmaCH2 + CmaCH + Cmaring (15)
The average molecular weight of the compounds in this fraction may also be calculated.
m”= 13*Cm,n + 12*Cmsub+ 15*C”,cH3 + 14*CmaCH2
+ 13*CmacH+ 14*Cmaring+14*CmCH2>a + 15*CmCH3>a (16)
This follows in a straightforward manner. Since the number of carbons of each type in the ”average molecule” is determined, the weight in daltons of every carbon (and associated hydrogens) can be summed. The result is the number average molecular weight rather than the less useful weight average (1). Total absolute moles of dicyclic aromatic ring carbon and total absolute moles of all carbon in the dicyclic aromatic chromatographic fraction can now be calculated. CdAr(total)
Cdbtal = CdAr(total)
=
Cdun
+ Cdsub + CdBH
(17)
+ CdaCH3 + CdaCHz + CdaCH + Cdaring (18)
The average molecular weight of this fraction now follows as before.
md= 13*Cdun+ 12*Cdsub+ 14*Cd,CH2
12*CdBH + 1 5 * C d , ~ + ~3 + 13*Cd,CH + 14*Cdaring + 14*Cdc~2>a+ 15*CdCH3>s
(19)
For fuel samples, one of the most commonly measured average compositional properties is the carbon aromaticity (fa). It is defined as the numerical ratio of aromatic ring carbons to total carbons in the sample. It is conveniently (and most directly) measured by 13C NMR. The conditions necessary for obtaining quantitative 13C spectra are now well understood (10, 11). If the aromaticity is known for the sample, the partial aromaticities for the monocyclic and dicyclic fractions may be calculated assuming the absence of tricyclic aromatics. The partial aromaticities are defined such that their sum is equal to the total aromaticity. fa
= fa* + f,d
(20)
The fraction of total carbon in each chromatographic peak (relative to the entire sample) may now be calculated. The values for the monocyclic and dicyclic fractions are obtained explicitly. The value for the aliphatic fraction is obtained by difference and will be in error if there are appreciable amounts of latter eluting materials (e.g., phenanthrenes) or noneluted polars (e.g., phenols). ptotal
(
c = (fa9
cxtotal)
(22)
CXAr(total)
And for the aliphatic peak
In the above discussion, it has been assumed that the dicyclic fraction is composed exclusively of alkylnaphthalenes and acenaphthene. Coal-derived fuel samples can contain some biphenyl derivatives, usually as minor constituents of the dicyclic fraction. Compounds of these types have elution characteristics similar to alkylnaphthalenes (8). Acenaphthenes will not introduce error. The aCHz proton signal of acenaphthene has a unique chemical shift so an explicit treatment analogous to that for tetralins (via Cd&) is possible. Biphenyls can introduce error, but only for normalized compositional data. For naphthalene, eight protons imply the existence of ten carbons. For biphenyl, ten protons imply the existence of twelve carbons. If a dicyclic fraction is 100% biphenyl, eq 2 will give the correct value for Cd,. Equation 8 will give a value for CdBHthat will be 25% too high. The error in Cdk total will only be 4%. The fraction of substituted sites for dimethylbiphenyl (0.2) will be correctly calculated but the calculated ADSd will be 20% low. The normalization equation for dicyclics (eq 7 ) should have a denominator of ten for biphenyls rather than eight which was derived for naphthalenes. Computations of normalized average composition (e.g., ADSd and data via the above equations are typically -20% low for pure biphenyl fractions. Biphenyls are typically a = 0.811). The average compositional data for the dicyclic (naphthalene) fractions of these fuels are very similar.
(mm
(mm
The total moles of carbon in each fraction (Catotal,Cmtotal, and Cd,d) are presented for each sample. Also presented are: aromaticities (fa), partial aromaticities (fam, fad), total moles of aromatic ring carbon (C"Ar(tot& CdAr(htd))r and fraction of total carbon in each LC peak (Fatotal,Ptotal, Fdtotal).These data also illustrate the stark contrast between 3 s and 3B. Sample 35 has a higher level of both monocyclic and dicyclic aromatics (P,d = 0.573, FdtOtal = 0.217). In contrast, sample 3B has the lowest levels of both monocyclic and dicyclic aromatics ( P t o t d= 0.192, Fd = 0.136). Sample 3 s contains a large quantity of relatively simple monocyclic aromatics (e.g., xylenes), and sample 3B contains a small quantity of more complex monocyclic aromatic compounds (e.g., tetralins, polysubstitution, longer chain alkyl groups). I t is particularly interesting that the fraction of total carbon in the naphthalene fraction differs significantly in these two samples. It should also be noted that sample 35 was also subjected to GC/MS analysis. Biphenyls and acenaphthenes were not detected in these samples a t levels above 5% (vide supra). Substituted phenanthrenes were not included in the analytical scheme for the fuels discussed above; however, they are occasionally present in our samples at detectable levels. The same LC/'H NMR average compositional formalism is applicable given adequate signal to noise. In principle, mixtures of many types could be characterized by a formalism analogous to the one developed here for fuel samples. Although the results of only two actual fuels are reported in this paper, it should be mentioned that we have analyzed -75 other fuels by this approach.
CONCLUSIONS The LC/lH NMR average composition formalism demonstrates that a considerable amount of information can be extracted from IH NMR spectra of mixtures. A key requirement is that some information must be known about the class of compounds composing each mixture. In this formalism, hydrocarbon classes separated by normal phase liquid chromatography constitute the mixtures. The knowledge of compound class (e.g., monocyclic aromatic hydrocarbons) permits the spectral data to be interpreted explicitly as average compositions for each class. The LC/lH NMR method allows carbon framework data to be obtained indirectly via the proton spectra and elution volumes. Direct observation of the carbon framework (via I3C NMR) should also remain a fruitful area of fuel characterization research. Spectral editing pulse sequences (13-15), with or without prior chromatographic fraction collection, are potential avenues of research. In the LC/'H NMR average composition treatment of the alkane fraction, a critical problem is the determination of quaternary carbons. Spectral
Table V. Average Structural Parameters for NASA Fuels 3s and 3B Monocyclic Aromatic Fraction sample 3s 3B
*CmUn *Cmglb 3.85 3.19
sample 3s 3B
*cdUn
*CmaCH,
*CrnaCH
0.320 0.777
0.008 0.034
1.71 1.57
*Crnaring *CmCH,>a
0.106 0.423
0.043 1.82
* C m c ~ , > @ADSm 0.328 0.811
2.15 2.81
Dicyclic Aromatic Fraction *cdsub
*c~BH
2.02 1.73
2.00 2.00
5.98 6.27 alkanes
3s 3B
*CmaCH,
2.15 2.81
*cdaCH,
* c ~ ~ C H , *C'CH,>@
1.90 1.61
0.117 0.117
0.001 0.079
MW" 113.3 153.9
*cdCH,>@
ADS^
MWd
0.117 0.117
2.02 1.73
158.1 155.2
Absolute Number of Moles and Fractional Aromaticity Data alkyl aromatics naphthalenes
Catotal
Fatotal
C"Ar(total)
0.00262 0.004 83
0.210 0.672
0.00314 0.00063
C"total
0.00446 0.001 20
fa" 0.403 0.101
Fmtotal
Cd.4r(total)
0.573 0.00139 0,192 0.000714 -
Cdtotal
0.00169 0.00085
fad
0.179 0.114
Fdtotal
fa
0.217 0.582 0.136 0.215
29
Anal. Chem. 1983, 5 5 , 29-32
editing sequences could1 permit direct measurement of this quantity.
LITERATURE CITED Clutter, D. R.; Petrakls, L.; Stenger, R. L., Jr.; Jensen, R. K. Anal. Chem. 1972, 44, 1395-1405. Williams, R. B. ASTM Spec. Tech. Pub/. 1958, STP 2 2 4 , 168-194. Brown, J. K.; Ladner, W. R.; Sheppard, N. Fuel 1959, 3 9 , 79-86. Brown, J. K.; Ladner, VV. R. Fuel 1959, 3 9 , 87-96. Knight, S. A. Chem. Imd. 1987, 1920-1923. Haw, J. F.; Glass, T. E.: Hausler, D. W.; Motell, E.;Dorn, H. C. Anal. Chem. 1960, 5 2 , 1135--1140. Haw, J. F.; Glass, T. E.; Dorn, H. C. Anal. Chem. 1981, 5 3 , 2327-2332. Haw, J. F.; Glass, T. E.; Dorn, H. C. Anal. Chem. 1981, 5 3 , 2332-2336. Haw, J. F.; Glass, T. E.; Dorn, H. C. J . Magfl. Reson. 1982, 4 9 , 22-3 1. Dorn, H. C.; Wooton, D. L. Anal. Chem. 1976, 4 8 , 2146-2148.
(1 1) Gray, G. A. Anal. Chem. 1975, 4 7 , 545A-564A. (12) Hardy, Dennis; Hazlett, R. N., Naval Research Laboratory, private communication. 1981, (13) Burum, D. P.; Ernst, R. R. J . Magn. Reson. 1860, 3 9 , 163-168. (14) Bendall, M. R.; Doddrell, D. M.; Pegg, D. T. J . Am. Chem. Soc. 1981, 103, 4603-4605. (15) Cookson, D. J.; Smith, B. E.;White, N. J . Chem. Soc., Chem. Commun. 1981, 12-13.
RECEIVED for review February 6,1982. Resubmitted July 26, 1982. Accepted September 21,1982. This work was supported by the Naval Research Laboratory (Washington, DC) and the United States Air Force Aero Propulsion Laboratory (Wright-Patterson Air Force Base, OH). J. F. Haw holds a Virginia Mining and Minerals Resources and Research Institute Fellowship.
Determination of Vasodilators and Their Metabolites in Plasma by Liquid Chromatography with a Nitrosyl-Specific Detector Wlng C. Yu" and E. Ulku Goff Thermo Electron Corporation, Analytical Instruments, 10 1 First Avenue, Waltham, Massachusetts 02254
A speclflc and sensltivcs method for the determination of the nitrate esters of glycerol, Isosorblde, and pentaerythrltol has been developed. The Instrumentation involves high-performance liquid chromatography (HPLC) Interfaced to a nltro/ nitrosyl-speciflc detector (TEA analyzer). The lower limit of detection of the methodl is 0.1 ng for each of glycerol trlnltrate (GTN) and pentaerythrltol tetranltrate (PETN) and 0.2 ng for lsosorbide dlnltrate (ISDN). At the 5 ng level, the relatlve standard deviations are f4.1%, f2.2%, and f7.3% for GTN, PETN, and ISDN, respectively. The isocratlc condltlons developed by uslng HPLCITEA provide a useful tool for the routlne analysis of plasma or blood samples In bloavallability studies.
Organic nitrate esters such as glycerol trinitrate (GTN), isosorbide dinitrate (ISDN), and pentaerythritol tetranitrate (PETN) have been widely used as vasodilators in the treatment of angina pectoris. Despite many publications, the pharmacokinetics of these drugs and their metabolites in humans are not well eiatablished, posing questions as to the efficacy and efficiency of the formulated drug. The problem is due, in part, to the limitation of available analytical instrumentation for detecting low levels of the drugs and their metabolites in circulating blood. A survey of analytical techniques that have been used in the determination of these compounds include spectrophotometry ( I ) ,polarography (Z), carbon-14 radioactivity labeling ( 3 - 4 ,thin-layer Chromatography (6,7), gas chromatography (8-IZ),liquid chromatography (I3-I6), and digital plethysmography ( I 7). Of these techniques, gas chromatography coupled with electron capture detection is most commonly used. Electron capture, though sensitive, suffers from lack of reproducibility, detector contamination, and excessive retention times for the separation of some metabolites. Also, special techniques m w t be employed to maintain linearity of the detector response.
Recent advances in high-performance liquid chromatography (HPLC) offer another approach in the determination of these thermally unstable compounds. The use of the UV detector in conjunction with normal- or reversed-phase HPLC is a logical choice. However, in a matrix as complex as blood or plasma where the levels of therapeutic drug and metabolites are present at the low parts per billion range, the UV detector cannot properly fulfill the need because of its relative lack of specificity and limited sensitivity. For enhanced sensitivity and selectivity, specific detectors have to be used. Lafleur (18) et al. recently used a nitrosyl-specific detector, the TEA analyzer, in the identification of explosives a t trace levels by interfacing it with high-performance liquid chromatography (HPLC/TEA). Spanggord (19) et al. also reported on the application of HPLC/TEA in the determination of nitroglycerin and its metabolites in blood. Both methods utilized solvent programming techniques. In this report we describe an analytical method for the selective and sensitive determination of glycerol trinitrate, isosorbide dinitrate, pentaerythritol tetranitrate, and their metabolites, in plasma by HPLC/TEA using isocratic conditions.
EXPERIMENTAL SECTION Equipment. The high-performanceliquid chromatograph was constructed by combining a solvent pump (Altex, Model 110) with an injector (Waters Associates, Model U6K). The columns used were 10 Mm Ultrasil NH2,25 cm long by 4.6 mm i.d. (Altex),and 10 wm pBondapak CN, 30 cm long by 3.9 mm i.d. (Waters Associates). The detector was a TEA Model 502 analyzer (Thermo Electron). Data aquisition was achieved with System I computing integrator (Spectra-Physics). Chemicals. The HPLC solvents, methylene chloride, chloroform, methanol, ethyl acetate, and isooctane, were distilled in glass (Burdick and Jackson). Sep-PAK C18cartridges (Waters Associates) and 0.5-fim Millex-SR filters (Millipore) were used. HPLC Procedure. Isocratic conditions were employed for the analysis of the vasodilators and their metabolites. The Ultrasil
0003-2'700/83/0355-0029$01.50/00 1982 American Chemical Society
