mixture of digitalis glycosides can be achieved in 12 min. The separation is sufficiently good for quantitative work (19). Impurities originating from the chloroform used in the extraction and traces of reagent are eluted before the derivatives. The first peak corresponds to t o (non-sorbed component, dodecylbenzene). The unidentified peak is an impurity of the desacetyl lanatoside C standard. The lowering of the polarity of the aglycones and glycosides due to the derivatization step results in a better chromatographic behavior. The eluents used can be of lower polarity and viscosity; peak broadening due to strong adsorbentadsorbate interactions is reduced and the improved selectivity permits an isocratic separation of very complex mixtures.
CONCLUSIONS Esterification of the digitalis glycosides with 4-NBC1 can be carried out simply and quantitatively. With a sufficient excess of reagent, completely substituted derivatives are obtained. The liquid-chromatographicproperties of the glycosides are considerably improved. The strong absorption and the favorable ,A, (260 nm) of the 4-nitrobenzoates permits a very sensitive and simple detection both in HPLC and TLC. For all these reasons, this technique is superior to previously existing methods for trace analysis of digitalis glycosides. The method should be widely applicable t o the derivatization of non-aromatic OH groups which are not sterically hindered. I t seems particularly promising for the wide field of carbohydrate analysis where a more rapid and sensitive method could mean a real breakthrough in analytical knowhow. Further work on the application of this technique to the
separation and quantitation of glycosides and sugars is in progress.
ACKNOWLEDGMENT The authors thank H. R. Loosli for taking the NMR spectra.
LITERATURE CITED (1) J. W. Myrick, J. Pharm. Sci., 58, 1018 (1969). (2) T. Higuchi and E. Brochmann-Hanssen, "Pharmaceutical Analysis", Interscience Publishers, New York, London, 1961, p 56. (3) D. Wells, B. Katzung, and F. H. Meyers, J. Pharm. Pharmacoi., 13, 389 (1961). (4) L. F. Cullen, D.L. Packman, and G. J. Papariello, J. Pharm. Sci., 59, 697 (1970). (5) A. 2 . Britten and E. Njau, Anal. Chim. Acta, 76, 409 (1975). (6) D.P. Page, FDA By Lines, 1, 1 (July 1974). (7) S. Mardh, Clin. Chim. Acta, 44, 165 (1973). (8) G. H. Burnett and R. L. Conklin, J. Lab. Clin. Med., 78, 779 (1971). (9) R. E. Chambers, Ciin. Chim. Acta, 57, 191 (1974). (10) G. S.Ahluwahia and Z . J. Kuczala, Clin. Chem.. 21, 270 (1975). (1 1) R. C. Boguslaski and C. L. Schwartz, Anal. Chem., 47, 1583 (1975). (12) E. Watson and S. K. Kalman, J. Chromatogr., 56, 209 (1971). (13) E. Watson, P. Tramell, and S. K. Kalman, J. Chromatogr., 69, 157 (1972). (14) F. J. Evans, J. Chromatogr., 88, 411 (1974). (15) W. Lindner and R. W. Frei, J. Chromatogr., 117, 81 (1976). (16) S.Siggia, "Instrumental Methods of Organic Functional Group Analysis", Wiley-lnterscience, New York, 1972, pp 1-74. (17) F. A. Fitzpatrick and S. Siggia, Anal. Chem., 45, 2310 (1973). (18) R. M. Cassidy, D. S. Legay, and R. W. Frei, Anal. Chem., 46, 340 (1974). (19) F. Nachtmann, H. Spitzy, and R. W. Frei, J. Chromatogr., 122, 293 (1976).
RECEIVEDfor review January 15, 1976. Accepted June 14, 1976. F. Nachtmann thanks Sandoz Ltd. for a grant in support of his thesis work.
Exclusion Chromatography with Multiple Detectors for Following Compositional Changes of Petroleum Residuals during Desulfurization E. W. Albaugh" and R. C. Query Gulf Research & Development Company, Pittsburgh, Pa. 15230
The changes in size, molecular weight, sulfur distribution, and aromaticity during catalytic desulfurization have been shown. Also, a method for continuously monitoring the sulfur content of chromatographic effluents has been demonstrated.
Recently, the feasibility of rapidly following the changes in molecular weight distribution and aromaticity as a function of molecular weight by utilizing exclusion chromatography (GPC) with multiple detectors was reported for petroleum residuals ( I ) . In that work, samples of identical weight (mass) were separated with a set of exclusion columns and the effluent was monitored with a differential refractometer (RID), an ultraviolet absorption detector (UAD), and a flame ionization detector (FID). By comparing the chromatograms from the RID and UAD of samples taken a t different stages of desulfurization, estimates of the changes in aromaticity could be made. A comparison of the changes in the chromatograms from the FID gave an estimate of the changes in mass distribution as a function of molecular weight.
One of the problems with the previous work was the inability of the FID to produce equal response for the various classes of petroleum hydrocarbons. For saturates, the response was comparatively high; while with resins and higher molecular weight species, the response was low. In the current work, this problem has been minimized by incorporating the improved Pye LCM2 FID for following changes in weight distribution. Also included in this current work are preliminary data on instrumentation for obtaining, on an analytical wale, the sulfur distribution as a function of molecular weight.
EXPERIMENTAL The liquid chromatograph was fabricated at GR&DC and was of conventional design. It consisted of a solvent reservoir, degasser, pumping system, sample injection valve, three Waters 4-ft X 3/s-in. lo4linear Styragel columns, an oven, and detectors. Benzene was used as solvent and pumped at a flow rate of 1.7 ml/min. The experimental details of the system have been reported previously ( I , 2). The Pye System I1 flame ionization detector used previously ( I ) was replaced by the Pye LCM2. This instrument converts the sample to carbon dioxide and then to methane which is determined with a
ANALYTICAL CHEMISTRY, VOL. 48, NO. 11, SEPTEMBER 1976
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COMBUSTION FURNACE
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Figure 4. FID mass distribution 60
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Figure 2. Weight distribution based on preparative scale GPC separations
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flame ionization detector. The instrument was evaluated with oil, resin, and asphaltene fractions from residuals ( 3 ) .The maximum deviation in response was approximately 10%. However, residual samples before and after desulfurization gave essentially the same response. Under the evaporating conditions necessary for removal of the benzene chromatographic solvent, the response for normal hydrocarbons decreased below C14 and Clz was not detected. To determine the sulfur distribution continuously on an analytical scale, the flame ionization detector of a Pye System I1 detector was replaced with a Dohrmann microcoulometer. To accomplish this, a quartz combustion tube was utilized with a heated transfer line that connected through quartz ball joints to the Dohrmann furnace (Figure 1).A vacuum pump was attached to the exit line of the Dohrmann titration cell to maintain normal gas flows. Under vacuum, the solution in the iodine reference side arm was drawn into the main titration cell body. To overcome this problem, a hollow tube was placed from the reference cell to the main cell body. Oxygen was used in the combustion tube of the Pye instrument. To obtain sufficient sensitivity, it was necessary to spray the sample on the transport wire ( 4 ) . Furnace temperatures were optimized by the manufacturers' recommended procedures ( 5 , 6 ) . Preparative scale separations were made, fractions isolated, weighed, and analyzed for sulfur; and molecular weights were determined as previously described (2). Sulfur contents were determined in accordance with ASTM D1552.
RESULTS AND DISCUSSION In the desulfurization of petroleum residuals, three parameters t h a t can be important are the changes i n weight, sulfur, and aromaticity distributions as a function of size and molecular weight. 1580
MOLECULAR
II I
WEIGHT
Figure 5. Sulfur weight distribution from preparative GPC separation
Changes in Size and Molecular Weight Distribution. T h e amount of cleavage in desulfurization is dependent upon t h e conditions used. Thus, in evaluating catalysts, process conditions, and changes in sample composition, t h e change in size and molecular weight distributions can be useful. Most residuals cannot be totally analyzed by gas chromatography for carbon number distribution because of their low volatility, while gravities and molecular weights will give only averaging information about the total sample. GPC is not limited by volatility and produces a size separation for residuals (2). In order t o convert t h e size distribution shown by GPC into molecular weight, it is necessary t o use a calibration technique. Figure 2 shows the preparative scale GPC separation on t h e original residue (4.1%sulfur) and the desulfurized product (0.6% sulfur). These chromatograms show t h a t t h e size distributions of both samples are monomodal and symmetrical and t h a t desulfurization has narrowed t h e size distribution. Shown in Figure 3 are the calibration curves (molecular weight vs. elution volume) obtained by determining the molecular
ANALYTICAL CHEMISTRY, VOL. 48, NO. 11, SEPTEMBER 1976
I EVI
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Figure 6. Sulfur distribution on analytical scale as a function of molecular weight
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Flgure 7. UAD chromatograms (condensed aromatics) as function of molecular weight weight of selected fractions from the preparative separation. The calibration curves show a different size molecular weight relationship beyond a molecular weight of approximately 800. After desulfurization, the compounds above 800 molecular weight have a greater molecular volume for a given molecular weight. Apparently, these larger molecules unfold and swell as sulfur bridge systems are destroyed and ring systems are hydrogenated. The GPC chromatograms obtained on an analytical scale using the Pye LCM2 are plotted in Figure 4 using calibration curves based on the preparative scale fractions. These chromatograms show the original charge has a molecular weight range from 12 000 to 250 with a peak molecular weight of 450. After desulfurization, the molecular weight range extends from 5000 to 230 with a peak molecular weight of 470. Changes in Sulfur Distribution. Figure 5 shows the sulfur distribution obtained from the preparative separation before and after desulfurization. From these data, it can be seen that desulfurization removes sulfur across the entire molecular weight range. Chromatograms obtained with the Dohrmann microcoulometer are shown in Figure 6. These rapid analytical chromatograms show the same distribution and changes in sulfur content with desulfurization as the preparative scale data, i.e., the sulfur is distributed throughout the entire molecular weight range and the distributions are monomodal. T h e changes in sulfur distribution follow a similar trend to that of the condensed aromatics (Figure 7). Changes in Aromaticity. The changes in aromaticity can be judged from the chromatograms derived from the UAD and RID ( I ) . In Figure 7 are shown the chromatograms from the UAD a t 313 nm and 365 nm for the original residue and the desulfurized product. Many condensed aromatic systems absorb a t these wavelengths. T h e ultraviolet spectra of these samples show a nearly uniform decrease in absorbance at the different wavelengths (Figure 8). Spectra from GPC fractions
I
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Figure 8. Ultraviolet absorption spectra of samples from desulfurization, 0.2 mg/ml each .-
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Figure 9. RID chromatograms before and after desulfurization as a function of molecular weight of different weights from the same sample are also similar. Thus, the chromatograms in Figure 7 approximate the distributions of the condensed system as a function of molecular weight. In the original sample, i t is seen that the condensed systems are distributed throughout the same and that the molecular weight range covered (38 000 to 250) is larger than that indicated by the mass detector (12 000 to 250). This is primarily due to the greater sensitivity of the UAD. T h e amount of material in the indicated 12 000 to 38 000 range is very small. The 38 000 molecular weight is an extrapolated value; however, it does represent compounds of much higher molecular weight range than 12 000. As the sulfur content is reduced, the molecular weight range of the condensed aromatics decreases in about the same manner as the total sample. Also, condensed aromatic compounds are found throughout the entire molecular weight range after desulfurization, and their aromaticity decreases with increased desulfurization.
ANALYTICAL CHEMISTRY, VOL. 48, NO. 11, SEPTEMBER 1976
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Another look a t the change in aromaticity is obtained from the refractive index chromatograms (Figure 9). In the original sample, the large positive area (above baseline) indicates a large amount of condensed aromatics with a molecular weight range from about 20 000 to 250. This is within experimental agreement of the UAD data. T h e negative alkyl area in the positive profile indicates the presence of saturates plus possibly substituted noncondensed aromatics. Their molecular weight range is 610 to 320 with the peak a t 425. As the sulfur content is reduced to 0.6%, the amount of condensed aromatics decreases and the molecular weight range is decreased to approximately 9000 to 225. The amount of saturates plus alkyl-substituted monoaromatics increases and their molecular weight range extends from 275 to 900 with a peak molecular weight of 400. The information provided here supplements that determined on a class basis, Le., percentage of asphaltenes, resins, aromatics, and saturates. One of this technique’s main limitations is the qualitative nature of the information. Not all condensed aromatic systems absorb a t 313 nm and 365 nm, while some noncondensed heterocyclic compounds do. With refractive index, some noncondensed aromatics do have a refractive index greater than benzene. However, the data to date on isolated petroleum fractions indicate that these detectors (UAD and RID) respond to the condensed aromatic systems regardless of whether they are in the asphaltenes, resin, or aromatic class; and the changes they depict are those
of the whole sample. Because of the different absorptivities and refractive indices of the various compounds, absolute values cannot be placed on the aromaticities. However, comparison of one chromatogram with another clearly shows the changes in condensed aromaticity. T h e same problem exists for the alkyl-substituted monoaromatics and aliphatic compounds as shown by the RID. Because of the variation in refractive index with structure, absolute values have not been calculated. However, with a series of crude oils, a semiquantitative correlation was found between saturate content and the corresponding area used here. When the saturates were removed from these samples, the negative peak disappeared indicating a very low alkyl-substituted monoaromatic contribution.
LITERATURE CITED W. Albaugh and P. C. Taiarico, J. Chromatogr.,74, 233 (1972). (2) E. W. Albaugh, P. C. Talarico, €3. E. Davis, and R. W. Wirkkala,Prepr. Pop. Nat. Meet. Div. Petrol. Chem.,Am. Chem. Soc., 15, No. 2 A225 (1970). (3) D. M. Jewell, E. W. Albaugh, B. E. Davis, and R. G. Ruberto, lnd. Eng. Chem., Fundam., 13, 278 (1974). (4) J. H. vanDijk, J. Chromatogr. Sci., I O , 31 (1972). (5) Technical Manual, Pye LCMP Liquid ChromatographPublication No. 299-365, Pye Unicam, Cambridge, England. (6) Operating Instructions for Dohrmann Micro.Coulometric Titrating System, Model C200 Dohrmann,Mountainview, Calif. 94040. (1) E.
RECEIVEDfor review May 21, 1975. Accepted May 21, 1976.
Molecular Sieve Adsorption Method for Determination of nParaffins in C12-C32 Petroleum Distillates V. R. Sista and G. C. Srivastava”’ Indian Institute of Petroleum, Dehadrun, India
Based on the principle of preferential adsorption of n-parafflnic hydrocarbons on molecular sieves, a method was developed for direct estlmation of n-paraffins In petroleum distillates (boiling range, 200-470 “ C ) .The effect of temperature on the desorption of n-paraffins was studied. The method is accurate to f l wt %.
The demand for n-paraffinic hydrocarbons (np) is increasing with time. For the production of single cell protein and detergents, n p present in the mi‘ddle distillates are preferred. It has been felt that the presence of hydrocarbons other than n p may deteriorate the quality of the final product especially in the case of single cell protein production. Thus, need arises to check the purity of the feed. Besides, knowledge of n p content in kerosene and gas oil fractions is essential for process development studies. Based on the principle of adsorption of n p on molecular sieves, methods developed (I, 2) have been summarized by Chen and Lucki and these authors have given two methods for the determination of np content in gas oil fractions ( 3 ) .As has been reported by them, the methods are suitable only when there is a significant quantity of cyclic and branched Present address, IIP Proteins Project, Gujarat Refinery, Baroda, India. 1582
hydrocarbons. When these methods were applied to determine the percentage purity of n p in the normal paraffinic feed, prepared by any of the processes, inaccurate results were obtained. All the methods reported deal with the estimation of unadsorbed hydrocarbons (cyclic and branched) which, if present in low quantities, would give erroneous results. In case larger samples are used, a fairly high quantity of molecular sieves (40 g/g of np) would be required. The method described here is the direct estimation of n p using a lesser quantity of molecular sieves as well as a test sample and can be applied to determine the percentage purity of the n p sample as well as for quantitative determination of n p content in kerosene and gas oil fractions.
EXPERIMENTAL Materials. Molecular Sieve type 5A, lhs-inch pellets (Linde Co.); naphtha (boiling range: 80-100 “ C ) ; acetone (LR Grade, BDH); n hexadecane (BDH); n-dodecane (Fluka); n-docosane (Fluka); ndotriacontane (Fluka); dewaxed gas oil fractions (boiling range: 200-370 “C and 300-460 O C ) ; np prepared from gas oil fractions of Ankleshwar crude oil by the urea adduction method. Preparation of Dewaxed Gas Oil. An Ankleshwar gas oil fraction was subjected to successive urea adduction till no more adduct was formed. This dewaxed gas oil was used as a sample representing cyclic and branched hydrocarbons. Experimental Setup. The U-shaped sorption tube (herein called “tube”) employed was similar to the one used by Nelson et al. ( 4 ) .A suitable furnace having capacity up to 350 “ C was used. The disturbance caused in the system due to pressure variation was overcome
ANALYTICAL CHEMISTRY, VOL. 48, NO. 11, SEPTEMBER 1976
