increased numbers of moments and/or histograms yielded similar percentages. Increasing the number of variables to 20 improved the efficiencies by only a few tenths of a per cent. One interesting outcome was found in calculating the correlation coefficients for the variables. For moments with the same parity or x 2 n - l ) the correlation coefficients were large (greater than 0.95) indicating that little new information can be added by including higher moments. Although the above mentioned 10 variable contraction has been successfully used in transformation studies (7), a further improvement was desired. Examination of statistical parameters indicated there was insufficient peak position information in the contraction. To obtain more position information, two sets of partial averages and moments were included. The ranges were determined by the sample means. The “low” set had the range:
(x”
1 l X l X
(4)
and the upper $,et:
(7) C. F. Bender and B. R. Kowalski, ibid., 45, 590 (1973).
Moments with n = 2, 3, and 4 were used for the three ranges leading to twelve variables including the means. Three more variables were included in the final 15 variable contraction ( F ) , the total number of peaks, H o , ~ ~and - ~ ,the , m / e value of the largest peak. As in all cases the data were autoscaled ( 2 ) . The classification efficiency of this case is also given in Table I. Clearly little information has been lost in the 9fold contraction. CONCLUSIONS
Moments have been used to contract the representation of low resolution mass spectra to fifteen variables with only a slight degradation (average less than 1 %) in classification efficiency when compared to a calculation using all the data and R < 3. Such a contraction gives rise to a considerable savings in computational effort. This new representation also allows further detailed pattern recognition studies with small manageable data sets. RECEIVED for review July 12, 1972. Accepted October 24, 1972. This work was performed under the auspices of the U. S. Atomic Energy Commission. H. D. Shepherd is a Military Research Associate (USN).
Rapid Methylation of Micro Amounts of Nonvolatile Acids Monte J. Levitt Departments qf Pathology and Obstetrics and Gynecology, Unicersitj, of Pittsburgh School of Medicine, Pittsburgh, Pa. 15213
A DIAZOMETHANE GENERATING SYSTEM is described which permits microgram or smaller amounts of complex organic acids to be quantitatively esterified without detectable side-product formation. Multiple samples can be processed in sequence at two-minute intervals. The methyl esters produced are suitable for further analysis by electroncapture gas chromatography. The apparatus is constructed from components present in most laboratories, and may be dismantled easily for storage. EXPERIMENTAL
Description of the Esterification Apparatus. An esterification technique ( I ) utilized with milligram amounts of acids has been modified to permit nanogram amounts of acids to be treated with minimal handling in a manner compatible with subsequent analysis by electron-capture gas chromatograph y . The system consists basically of a generator and a split stream of inert gas (Figure 1). The generator is a 20- X 150-millimeter side-arm test tube with a 2-hole rubber stopper. Through one hole of the stopper is inserted a dropping-tube consisting of a piece of glass laboratory tubing with a stopcock. An inert gas is delivered through an inlet tube constructed from a length of laboratory glassware which is bent at 90” and inserted through the other hole in the stopper to extend to the bottom of the generator. Diazomethane formed in the generator is delivered to the samples through a glass capillary pipet, bent at 90°, and connected to the side-arm of the generator by means of a 2-centimeter length of Tygon tubing. [For permanent installation, shrinkable polyethylene or Teflon (Du Pont) tubing could be used.] (1) H. Schlenk and J. L. Gellerman, ANAL.CHEM., 32, 1412 (1960). 618
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ANALYTICAL CHEMISTRY, VOL. 45, NO. 3, MARCH 1973
The two pieces of glass are butted against each other to avoid leaching any contaminants from the plastic tubing. Nitrogen or other inert gas is passed through a molecular sieve filter and then through copper tubing which has been cleaned of manufacturing oils by heating or washing with solvents. The gas stream is split by a T-connector, part going to the generator, and part to a glass capillary pipet for evaporating the samples after esterification. A micro-valve in the gas line aids in regulating the flow of gas through the generator. Removable metal-to-metal and metal-to-glass connections are conveniently made with Swagelok or Cajon fittings (Crawford Fitting Company, Cleveland, Ohio). All glass components are fire-polished to avoid initiating a diazomethane explosive reaction ( 2 ) . Operation of the Esterification Apparatus. The generator with attached delivery tube is clamped in a fume hood behind an explosion shield, then 2 milliliters each of ethyl ether a n d 2-(2-ethoxyethoxy)ethanol (Carbitol, Aldrich Chemical Company, Milwaukee, Wis.) are added. Approximately 25 milligrams of N-methyl-N-nitroso-p-toluenesulfonamide (Diazald, Aldrich Chemical Company) are added and rinsed to the bottom of the generator with 3 milliliters of ethyl ether. The rubber stopper is then inserted in the generator and the inlet tube is connected to the gas line. Inert gas is passed through the system for approximately 30 seconds, during which time 1 milliliter of 60z aqueous potassium hydroxide is added to the dropping tube. Then the gas flow is momentarily interrupted and the stopcock is opened to allow the KOH to drain into the generator. After the stopcock is closed, gas flow is resumed at the rate of 1-3 bubbles per second. Ethereal diazomethane is swept out of the gen(2) Th. J. de Boer and H. J. Backer, “Organic Syntheses,” N. Rabjohn. Ed.. Coll. Vol. 4, John Wiley and Sons, New York, N.Y., 1963, p 250.
FILTERED
- GLASS DROPPING
COPPER TUBING
TUBE
RUBBER STOPPER
Figure 1. Apparatus for esterifying micro amounts of nonvolatileacids
ON
I'
-9"GLASS PIPET
-9"GLASS
PIPET
erator by the gas stream and is introduced into the samples by means of the delivery tube. Each acid to be esterified is dissolved in 1 milliliter of ethyl ether and 0.1 milliliter of methanol. The alcohol aids in solubilizing the acid and also accelerates the esterification reaction ( I ) . The sample tube is held so that the tip of the delivery tube reaches to the bottom of the sample solution. The amount of ethereal diazomethane delivered to the sample solution is dependent upon the rate of gas flow and the length of time of exposure to the gas stream. Usually, 1 minute is sufficient for a pale yellow color to appear in the sample solution, indicating an excess of diazomethane. The sample is then set aside for approximately 1 minute to permit esterification to be completed. The sample solution is next evaporated to dryness under the stream of inert gas delivered at the other end of the system. Submicrogram amounts of samples are processed conveniently in 3- to 5-milliliter conical tubes so that the esters are deposited as dry residues in the tips and are ready for further treatment. Multiple samples can be esterified at 2-minute intervals, which leaves sufficient time to rinse the outside of the delivery tube with solvent between samples. One charge of diazomethane precursor is sufficient to esterify at least ten samples. After all the samples are esterified, ethereal diazomethane is vented into the fume hood. When the production of bubbles in the generator has ceased, the rubber stopper is removed and ethyl ether containing 10% acetic acid is cautiously added dropwise to discharge any traces of diazomethane and to neutralize the base. After the contents are flushed down the drain, the generator can be stored with the delivery tube inside it. RESULTS AND DISCUSSION
To determine the suitability of this system for the esterification of small amounts of unsaturated and hydroxylated acids, reactions were performed with 100-microgram amounts of bile acids (Supelco, Inc., Bellefonte, Pa.). The esters were examined by flame-ionization gas chromatography at 250 "C on a 6-meter glass column packed with 3 % OV-17. Retention times were measured relative to cholesterol added to the acids as an internal standard. The retention times of synthesized methyl esters of lithocholic acid, deoxycholic acid, chenodeoxycholic acid, and cholic acid were identical to the retention times of known samples of the esters (Supelco, Inc.). No other chromatographic peaks were detected. Samples of lithocholic acid ranging from 10 to 1000 micrograms were esterified and analyzed in the same manner to
Table I. Gas Chromatographic Behavior of Methyl Esters Synthesized from Different Amounts of Lithocholic Acid Ratio of methyl esters to cholesterol Micrograms 'Retention Peak of acid time area 1000 500 100 50 10
1.49 1.49 1.49 1.49 1.49
1.31 1.26 1.36 1.31 1.27
determine the yield of ester. As shown in Table I, the ratio of the peak area for the ester compared to the peak area for cholesterol included as an internal standard was constant over the range of lithocholic acid concentration studied. Completeness of esterification was also attested to by the finding that re-treatment with diazomethane did not alter the arem of the chromatographic peaks obtained. The esterification procedure is equally satisfactory when nanogram quantities of complex acids are treated and examined by electron-capture gas chromatography. Samples of 100 nanograms of prostaglandins F1, and F1, were esterified as described, then converted to volatile, electron-capturing derivatives (3) by reaction with heptdfluorobutyric anhydride (Columbkd Organic Chemicals, Columbia, S. C.). The tri-heptafluorobutyrate methyl esters were examined by gas chromatography at 200 "C on a 3-meter glass column packed with 3x OV-1. Less than one nanogram of each derivative could be detected easily, and a blank treated the same way contained essentially no electron-capturing material. More than one peak was observed for each derivative, but mass spectral analyses showed that the extra peaks were not the result of the esterification process ( 3 , 4 ) . The observed quantitative esterification in 2 minutes a t room temperature is not due solely to the small amounts of acid used, since similar rates of reaction have been observed when milligram amounts of acids were treated with ethereal (3) . , M. J. Levitt, J. B. Josimovich, and K. D. Broskin, Prostaglandins, 1,121 (1972). (4) M. J. Levitt and J. B. Josimovich, Abstracts, 62nd Annual Meeting of the American Society of Biological Chemists, San Francisco, Calif., June 1971, No. 166. \
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ANALYTICAL CHEMISTRY, VOL. 45, NO. 3, MARCH 1973
619
diazomethane in the presence of methanol ( I , 5, 6). The brief reaction period and low temperature employed may account for the fact that the reaction tubes were free of oil or polymer observed previously (7) after esterification with diazomethane. Since diazomethane is capable of entering into many reactions (8), it is desirable to minimize the reaction time in order to reduce the likelihood of obtaining unexpected reaction products-especially with complex biological acids (9). The use of freshly-generated diazomethane also reduces the risk of side-product formation (10, 11). Es-
terification procedures which employ distilled diazomethane are more complex, have added safety hazards, and are subject to contamination from co-distilled chemicals (12). ACKNOWLEDGMENT
Mrs. Kathy D. Broskin is thanked for laboratory assistance, and John B. Josimovich for helpful discussions. Prostaglandins were kindly provided by John E. Pike of The Upjohn Company.
(5) J. Churacek, M. Drahokoupilova, P. Matousek, and K. Komarek, Chromatographia, 2, 493 (1969). (6) G. G. McKeown and S. I. Read, ANAL.CHEM., 37,1780(1965). (7) W. R. Morrison, T. D. V. Lawrie, and J. Blades, Chem. b7d., Lo/zdori, 1961, 1534. (8) H. B. Hopps, Aidrichimica Acta, 3,9 (1970). (9) P. G.Simmonds, R. C. Pettitt, and A . Zlatkis, ANAL.CHEM., 39,163 (1967). (10) M. L. Vorbeck, L. R. Mattick, F. A. Lee, and C. S . Pederson, ibid., 33,1512 (1961). (11) R. Roper and T. S. Ma, Microchem. J., 1,245 (1957).
RECEIVED for review July 13, 1972. Accepted October 16, 1972. This work was performed in the Department of Obstetrics and Gynecology of The University of Pittsburgh Medical Center and was supported in part by National Institutes of Health Grants HD 00227 and 05791. Mass spectral analyses were performed at the Pittsburgh Facility for Biomedical Research, supported by National Institutes of Health Grant 00273. (12) D.Kubik and V. I. Stenberg, Chem. hid., London, 1965,248.
Design and Evaluation of a Low Cost Recording Spectropolarimeter Stephen J. Simon' and Karl H. PearsonZ Department of Chemistry, Cleceland State Unioersity, Cleueland, Ohio 44115 THERECENT PUBLICATION of Reinbold and Pearson ( I ) describes the modifications done to a Perkin-Elmer Model 141 polarimeter to obtain continuous optical rotatory dispersion data over the entire spectral region of 650-240 nanometers. These modifications consisted of removing the light source of the Model 141 polarimeter and replacing this with a Bausch and Lomb 250-mm double grating monochromator, Catalog No. 33-86-66, and a Bausch and Lomb 150-watt high inAn tensity xenon light source, Catalog No. 33-86-20-01. adjustable optical bench was easily fabricated to support and allow optical alignment of the new continuous light source and monochromator with the polarimeter. To the above modified spectropolarimeter, a modified Bausch and Lomb synchronous wavelength drive, Catalog No. 33-86-67, was attached directly to the wavelength drive mechanism of the monochromator. This wavelength drive provides synchronous wavelength scanning speeds of 5,25, and 125 nm/min forward and slewing speeds of 500 nm/min in both forward and reverse wavelength directions. A Coleman Model 165 multi-speed, multi-millivolt recorder was added which, when connected to the optional Perkin-Elmer transmitting potentiometer, Catalog No. 141-4000, of the Model 141 polarimeter, provided the observed optical rotation in analog form. Figure 1 is a photograph of the completed modified recording spectropolarimeter described above, showing the attachment of the B & L monochromator, the wavelength drive and the xenon light source to the Perkin-Elmer Model 141 polarimeter which is connected via the transmitting potentiometer to Present address, Department of Chemistry, Wayne State University, Detroit, Mich. 48202. Author to whom all correspondence should be addressed. ( 1 ) P. E. Reinbold and K . H. Pearson, ANAL.CHEM., 43,293(1971). 620
ANALYTICAL CHEMISTRY, VOL. 45, NO. 3, MARCH 1973
Table I. Wavelength Drive Scan Speed us. Recorder Speed Wavelength drive speed Recorder speed, mm/min nml min 5.0 10 20 60 120 240 0.50 0.25 0.083 0.04 0.02 1.Oa 5 0.21 0.10 1.25 0.42 2.5 5.0 25 1.04 0.52 12.5 6.25 2.08 125 25.0 a Chart values give number of nanometers/millimeter of the strip chart. Table 11. Transmitting Potentiometer Setting us. Millivolt Setting of the Recorder Setting of
transmitting potentiometer 4
Millivolt setting on recorder
2 5 1 .OO" O.4OOa 0.200" 4.00" 1.60" 3 0.800" 20.0" 8.00" 2 4.00" Chart values give range of strip chart in degrees rotation. 1
10 2.00" 8 .OO" 40.0" of optical
the Coleman Model 165 recorder. Table I shows the available speeds of the recorder and wavelength drive and the resulting calibration of the strip chart in nanometers/millimeter. The Coleman Model 165 recorder provides four millivolt settings to give three useful decade ranges in degrees of rotation depending on the setting of the transmitting potentiometer of the polarimeter (Table 11). Potentiometer settings
