Table 11. Comparison of Theoretical a n d Experimental Elution Times for a Linear Density Program= Retention t i m e , hr
Bead diameter,
2339 3117
A
Theory
Experiment
7.87 15.02
7.2 15.1
a The experimental results and conditions are reported in Figure 8; the theoretical results are obtained from Equations 34, 39, and Figure 4 .
The solutions to Equation 39, shown in Figure 4, along with Equation 34, can be used for the prediction of the programmed density retention parameters of the experiment reported in Figure 8. Table I1 shows the results of this comparison. Once again agreement is very good, with the average deviation being less than 5%. This reinforces the conclusion that the experimental phenomena are much as described by theory. General Conclusions. The excellent agreement of theoretical and experimental retention parameters in SFFF shows that the technique can be manipulated and opti-
mized in accordance with theoretical guidelines. One general guideline of major importance is that high field strengths are necessary to improve the speed of separation and to extend its range to smaller particles and macromolecules ( I ) . This conclusion is not altered by virtue of programming; the general function of a program is simply to provide a range of conditions in proper sequence for fractionating a diverse sample (9). The prototype experimental system employed here is unable to use high rotational speeds because of the vulnerability of the special seal used to transport solvent into and out of the centrifugal basket. Field strengths did not exceed 500 g's in the present study. An improvement in the seal design would provide a superior system. This and other design improvements should make it possible to reduce the large elution times reported here. More importantly, the method would then be applicable to complex mixtures including various proteins, nucleic acids, and cell particles of smaller dimensions than those studied here. RECEIVEDfor review March 21, 1974. Accepted July 12, 1974. This investigation was supported by Public Health Service Research Grant GM 10851-17 from the National Institutes of Health.
Simultaneous Esterification of Carboxyl and Hydroxyl Groups with Alcohols and Heptafluorobutyric Anhydride for Analysis by Gas Chromatography John B. Brooks, Cynthia C. Alley, and John A. Liddle Center for Disease Control, Public Health Service, US.Department of Health, Education, and Welfare, Atlanta, Ga. 30333
A reagent mixture consisting of heptafluorobutyric anhydride (HFBA), pyridine (py), and ethanol was used to esterIfy carboxyl groups wlth the alcohol, and to derivatize hydroxyl and amine groups with the anhydride. HFBA catalyzed esterification of carboxyl groups and py catalyzed derivatization of carboxyl, hydroxyl, and amine groups. Ethanol acted both as an esterifying agent and as a stabilizing agent for ethyl ether. Esterification of carboxyl groups and derivalization of hydroxyl and amine groups were reproducibly performed with or without heating the sample. Heating the sample briefly produced a moderate increase in derivatization, but an increased amount of HFBA and more anhydrous conditions were required. A simple drying procedure for aqueous extracts for which MgS04 is used is described. The procedures were demonstrated to be useful for studying body fluids with gas chromatography. Diesterification of hydroxy acids was confirmed by mass spectrometry.
The carboxyl and hydroxyl groups of acids and alcohols are frequently esterified for analysis by gas-liquid chromatography (GLC). Carboxyl groups are usually esterified by heating the acid with an alcohol in the presence of a catalyst such as boron trifluoride or sulfuric acid, and the hydroxyl groups of both hydroxy acids and alcohols are esterified for GLC analysis with an anhydride such as heptafluorobutyric anhydride (HFBA). Diesterification of hydroxy 1930
acids for GLC analysis is frequently performed in two steps. Esterification of the carboxylic group with an alcohol is followed by esterification of the hydroxyl group with HFBA ( I , 2 ) . In addition to derivatizing hydroxyl groups, HFBA is frequently used to derivatize amines for GLC analysis (3,4 ) . During the course of our GLC studies with HFBA derivatives of both the p H 2 diethyl ether extraction of hydroxy acids and alcohols, and the p H 10 chloroform extractions for amines which contained residual ether from the pH 2 extraction, we observed extraneous GLC peaks in the chromatograms. Extraneous peaks were peaks that were not present when ethanol was added as a stabilizer or could not be detected when other solvents were used. When ethyl ether containing ethanol was used as a stabilizing agent and small quantities of ethanol were included along with the HFBA and pyridine (py), the extraneous peaks were not observed. In later work involving analysis of derivatized extracts containing hydroxy acids we discovered by GLC-mass spectrometry that not only were the hydroxyl groups esterified with HFBA in 3 minutes without heating the sample, but, in addition, the carboxyl groups were es(1)J. B. Brooks and C. C. Alley, Anal. Cbern., 46, 145 (1974). (2)J. E. Brooks, D. S. Kellogg. L. Thacker, and E. M. Turner, Can. J. MicrobioL, 18, 157 (1972). (3)J. E . Brooks, C. C. Alley, J. W. Weaver, V. E. Green, and A. M. Harkness, Anal. Cbem., 45, 2083 (1973). (4)J. B. Brooks, W. B. Cherry, L. Thacker. and C. C. Alley, J. lnf. Dis., 126, 143 (1972).
ANALYTICAL CHEMISTRY, VOL. 46, NO. 13, NOVEMBER 1974
terified with ethanol. The purpose of this study was to further study and improve the diesterification and amine derivatization procedures and to test their reproducibility.
EXPERIMENTAL Reagents. The ethyl ether used in the study was Baker's analyzed reagent grade containing ethanol as a stabilizer. Chloroform was of pesticide quality (Matheson, Coleman, and Bell). Pyridine was Mallinckrodt spectrophotometric grade, and HFBA was reagent grade (Pierce Chemical Co.). A blank was run on all solvents by extracting 2 ml of distilled water with 20 ml of the solvent and then treating the extract in the same manner as the derivative preparation described below. Standards. Standard solutions of hydroxyhippuric, homovanillic, 3,4-dihydroxyphenylacetic,and benzoic acids were prepared by diluting 300 pmoles of each to 10 ml with diethyl ether. One-tenthml aliquots were used to prepare derivatives. Standards of salicylic, 0 - hydroxyphenylacetic, p - hydroxyphenylacetic, and p - hydroxyphenylpyruvic acids were prepared by diluting 300 @molesof each acid to 10 ml with distilled water. One-tenth-ml aliquots were diluted to 2 ml with distilled water and then extracted a t pH 2 with 20 ml of diethyl ether. Individual lactic acid st.andards were prepared by diluting 3.6 pmoles to 10 ml with ether. A 0.1-ml aliquot was used to prepare derivatives. A standard mixture was prepared that contained 10.8 X lo-; mole of lactic acid, 7.8 X mole of salicylic acid, 3 X 10-6 mole of p - hydroxyphenylacetic acid, 9.7 X mole of benzyl alcohol, 1.9 X mole of hexylamine, 5.0 X mole of 6-phenylethylamine, and 9.4 X loe6 mole each of putrescine and cadaverine per 2 ml of distilled water. Other alcohol and amine standard mixtures were prepared as described (3, 4 ) . The HFBA-ethyl esters, and HFBA alcohol and amine derivatives were prepared as described below. Procedures f o r Preparation of Esters a n d Amine Derivatives. Two-ml samples of either svnovial fluid or aqueous standard mixtures containing hydroxy acids, alcohols, and amines were acidified to ahout pH 2 with 0.2 ml of 50% (v/v) H2S04. The acidified samples were extracted by vigorously shaking with 20 ml of diethyl ether to obtain the hydroxy acids and alcohols. The samples were then made basic (about p H 10) with 8N NaOH and re-extracted with 20 ml of chloroform. Both the ether and chloroform extracts were placed in a 50-ml beaker and evaporated with a gentle stream of clean dry air to about 1 ml. Next the samples were transferred to 12- X 75-mm test tubes with a disposable Pasteur pipet. Care was taken to discard any visible layer of moisture (bottom layer in the ether extract and top layer in the chloroform extract) present in the pipet. Next, the samples were dried with MgS04 and treated as follows. First, about 100 mg of MgS04 was added to the concentrate (the MgS04 was about 0.1 the volume of the concentrate). The contents of the test tube were shaken thoroughly, briefly centrifuged, and the solvent layer was decanted into another clean dry 12- X 75-mm test tube. One ml of solvent was added to the sedimented MgS04; the sample was shaken and centrifuged, and the solvent layer was decanted and combined with the previously decanted layer. The MgSO4 was discarded. At this point, the samples were further concentrated by air to about 0.1 ml. Next, 0.2 ml of chloroform was added to the concentrated ether extracts. The test tube was shaken to mix the contents, and the samples were again evaporated by air to about 0.1 ml. During the evaporation procedure, most of the ether was removed leaving the concentrate in the chloroform layer. The samples were again drawn up into a disposable pipet. Any visible aqueous layer (top) was discarded. The chloroform layer (bottom) was placed into a clean dry 12- X 75-mm test tube. Non-extracted standard mixtures that were dissolved in ether were evaporated to almost dryness and 0.1 ml of chloroform was added. Next, both the extracts and standard solutions were treated with ca. 0.01 ml of a solution consisting of one part of ethanol and four parts of chloroform. After the contents of the tube were mixed, they were treated with ca. 0.01 mi of py which had been previously diluted with three parts chloroform to one part py. Once again the sample reagent was mixed by shaking, and ca. 0.03 ml of HFBA was added. Then the tube was shaken again, and the chloroform was evaporated to about one-half a drop. The tube was stoppered with a cork, taped, and heated in a boiling water bath for 4 minutes. After the sample was heated, it was cooled under tap water and then diluted with ca. 0.14 ml of chloroform. Next the sample was treated with ca. 0.09 ml of a O.1N HCI. The test tube was shaken thoroughly to extract into the aqueous layer substances that interfere with subsequent GLC analvsis. and the contents of the tube were again drawn up into a
disposable pipet. The chloroform layer was deposited back into the test tube and the aqueous (top) layer discarded. The washing procedure was repeated with 0.1N NaOH. The sample was permitted to stand for 30 minutes. The chloroform layer was deposited into a clean dry test tube and the aqueous layer was discarded. It is important to keep the volume of the chloroform layer a little above 0.1 ml during the washing procedures to avoid sample loss. Next, with a gentle stream of dry air, the chloroform in the test tube was evaporated until it was almost but not, completely dry. Either 0.1 ml of ether or acetone was added as a final solvent for GLC anaiysis. Apparatus. A Perkin-Elmer gas chromatograph Model 900 equipped with a 63Ni 10-mCi detector, a Beckman four-way and three-way switching valve, and a 25.4-cm potentiometric recorder were used. The instrument was operated with two coiled glass columns (0.3-cm i.d. by 7.3-m length). One column (nonpolar) was packed with Chromosorb w 80/lOO mesh (AW-DMCS H.P.) that was coated with 3% OV-1 (Applied Science Laboratories). The second column (polar) was packed with TA33 Tabsorb (Regis Chemical Co.). The switching valves permitted a comparative analysis of the effluent from either column on a single detector or the flow from both columns to be vented. The instrument was operated isothermally for 4 minutes a t 70 "C, then programmed for a linear increase of 4 "C per minute to 225 "C; then it was held isothermally a t 225 "C for 32 minutes. The temperature of the injector was 225"C, the manifold temperature was 250 "C, and the detector temperature was 275 "C. The electrometer was attenuated so that full scale was obtained at 8 X A. The detector polarization was set a t 10. Oxygen-free nitrogen (Matheson) was used as the carrier gas a t a flow rate of 50 ml/minute. The carrier gas line was modified between the manifold and detector to permit a flush gas to be used. A flush gas improves the base line and overload characteristic of the detector, and permits carrier gas to flow through the detector when the columns are vented. The flush gas was regulated through the detector so that the combined flow of nitrogen from the column and flushing system was 67 ml/minute. The recorder was operated with an input signal of 1 mV and a chart speed of 76.2 cm/hour. New columns were conditioned for 12 hours a t 245 "C, and longer if necessary, to obtain a stable base line. The columns were also conditioned each morning before use by heating them a t 245 "C for 30 minutes. Mass Spectra. A LKB Model 9000 gas chromatograph-mass spectrometer was used. The resolution of the instrument was about 1000 mle; the temperature of the ion source was 290 "C; and the electron energy was 70 eV. Acceleration voltage was 3.5 ; the scan ( m l e ) limits were from 0 to 500; mle scan speed used was 6 (0 to 500 m / e in 16 seconds); and the UV oscillograph chart speed was 5 cm per second. Effluent from the gas chromatogr:ph was monitored by a total ion current detector. The instrument was equipped with a coiled glass column (0.3-cm i.d. by 3.6-m length) which was packed with Chromosorb W 80/100 mesh (AW-DMCS H.P.) coated with 3% OV-1. The gas chromatograph was operated isothermally for 5 minutes a t 70 "C; then it was programmed for a linear increase of 5 OC/min to 225 O C . Helium was used as the carrier gas with a flow rate of 36 ml per minute. The recorder was operated with an input signal of 2 mV (full scale) and a chart speed of 76.2 cmhour.
RESULTS AND DISCUSSION The GLC data taken from a retention time comparison of known standards of lactic and salicylic acids on polar and nonpolar columns indicated that the peaks shown (Figure 1, curve B ) were HFBA-ethyl esters of lactic and salicylic acids, respectively. Additionally, analysis by both ECGLC and mass spectrometry of the alcohols, acids, hydroxy acids, and amines listed under Standards above, revealed that primary and secondary alcohols and amines had formed HFBA derivatives; the acids, ethyl ester derivatives; and the hydroxy acids, HFBA-ethyl ester derivatives. The general reaction for diesterification is shown in Scheme I. Additionally, when propanol or butanol were substituted for ethanol, the carboxyl group was esterified with the substituted alcohol. Gas chromatograms of HFBA and HFBAethyl esters of a standard mixture of lactic acid, salicylic acid, p -hydroxyphenylacetic acid, and benzyl alcohol are shown in Figure 1, curve C. Mass spectra of the HFBA-
ANALYTICAL CHEMISTRY, VOL. 46, NO. 13, NOVEMBER 1974
1931
0
il
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ethyl esters of lactic, salicylic, and p -hydroxyphenylacetic acids are shown in Figure 2. The mass spectrum of the HFBA-ethyl ester of lactic acid (Figure 2, top graph) presents evidence that diesterification occurred. The molecular ion a t mfe 314 is present. The strongest peak in the spectrum (not used as the base peak due to its low mass) occurs at m f e 29 and indicates the presence of CzH5. Strong evidence of esterification with HFBA is presented in the fragments a t mfe 69,100, 119, 169, and 197. The fragment a t mle 169 was the second most intense peak in the spectrum, and this peak was used as the base peak in the preparation of the graph. In the graph of the spectrum of the HFBA-ethyl ester of salicylic acid (Figure 2, middle graph), there is again strong evidence of diesterification. The molecular ion is present at m f e 362. There is an intense fragment a t m f e 29 indicative of C2H5, and the base peak at m f e 317 which may have been formed by the loss of CzH50 again indicates esterification with ethanol. Evidence for esterification with HFBA is shown in fragments at m f e 69, 169, and in the total mass shown by the molecular ion. The mass spectrum of the HFBA-ethyl ester of p - hydroxyphenylacetic acid (Figure 2, bottom graph) is quite different from that of the HFBAethyl ester of salicylic acid. As in the case of the HFBAethyl ester fragmentation pattern of lactic acid, the fragment a t m f e 29 (not considered the base peak) is the largest peak in the spectrum and indicates the presence of C2H5. The m f e 303 fragment (considered the base peak) possibly indicates the loss of CsH502. The substituted benzene ring is indicated in the fragments a t mfe 90, 106, and 163. Unlike the fragmentation pattern of the HFBA-ethyl ester'of salicylic acid, there is a strong fragment a t mle 78. Esterification of the hydroxyl group with HFBA is confirmed by the fragments a t m f e 69,100,119, and 169. The best combination of reagents for the maximum amount (based on peak rise) of diesterification with a clean background was studied by varying the concentration of HFBA, py, and ethanol. Additionally, a comparison was made between derivatization with and without heating the sample. Based on maximum derivatization and a clean background, the best observed combination of active reagents, when the reaction mixture wasn't heated, was ca. 0.02 ml per derivative of HFBA, ca. 0.01 ml of a mixture of one part pyridine to three parts chloroform, and ca. 0.01 ml of a mixture of one part of ethanol to four parts of chloroform. Derivatization of acids, hydroxy acids, alcohols, and amines was reproducibly obtained in 3 minutes without heating the sample. Evaporation of the chloroform and subsequent heating of the reaction mixture with ca. 0.03 ml of HFBA and one drop each of py and ethanol diluted 1:4 in chloroform produced a moderate increase in derivatization of all the functional groups mentioned above. However, when trace amounts of water were present, as is often 1932
10
20
40
30
50
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TiME
- - - - - - - - - - - - - - 225"C Figure 1. EC-GLC chromatograms of extracts treated with HFBA-
py-ethanol Curve A is a pH 10 chloroform extraction of synovial fluid taken from a patient with juvenile rheumatoid arthritis. Curve B is the pH 2 ethyl ether extract of the same synovial fluid as described for Curve A, and Curve C is a pH 2 ethyl ether extraction of a standard mixture of hydroxy acids and an alcohol
the case with concentrated sample extracts, little or no derivation of the hydroxyl and amine groups was obtained. Drying the concentrated extract with small amounts of MgS04 before adding the reagents and increasing the amount of HFBA from 0.02 to 0.03 ml prevented derivatization loss while heating. Loss of sample during the drying step with MgS04 was determined by comparing anhydrous samples treated and not treated with MgS04. There was an overall net increase of about 20% in the size of the chromatographic peaks when derivatization was done by drying the concentrated sample with MgS04 and heating to form the derivatives. Trace amounts of water did not prevent reproducible derivatization with HFBA when the sample was not heated. Loss of sample was considerable when MgS04 was used in larger amounts to dry the ether extract before evaporation or when the MgS04 was not washed with solvent and the supernatant combined with the supernatant from the initial drying step. In addition, increasing the amount of HFBA from four to six drops in the sample that was not heated did not increase the amount of derivatives formed, and it produced a huge tailing peak that was not eliminated by the washing procedure. Both ether and acetone were satisfactory final solvents for the derivatives, but ether was slightly superior to acetone because peak concentration was increased. The major advantages of the techniques described above for preparing derivatives for GLC analysis are as follows: (i) The specificity of the reaction of carboxylic acids with alcohols to form esters and with HFBA to esterify hydroxyl groups can be advantageously used in a single simple esterification procedure which occurs rapidly with or without heating the reaction mixture. (ii) Both the diesterified hydroxy acids and esterified alcohols are highly electron cap-
* ANALYTICAL CHEMISTRY, VOL. 46, NO. 13, NOVEMBER 1974
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ANALYTICAL CHEMISTRY, VOL. 46, NO. 13, NOVEMBER 1974
1933
turing and pive full scale response at low nanomole and picomole concentrations. (iii) Ethanol can be used both as an esterification agent and as a stabilizing agent to prevent formation of extraneous peaks when HFBA is used in the presence of trace amounts of ethyl ether. (iv) The HFRA reagent mixture will react under the same condition with amines to produce derivatives that are highly electron capturing. The capacity of HFBA-py to catalyze esterification of carboxyl groups was further tested by substituting propanol, butanol, and fi-trichloroethanol for ethanol in the reagent mixture. Both GLC and mass spectral analysis revealed that the carboxyl groups of the acids were esterified with the substituted alcohol: however, only etharlol showed stabilizing properties to HFBA in the presence of ethyl ether. In another study in which we used /3-trichloroethanol and substituted borontrifluoride (BF?) for HFRA-py as a catalyst, we found that, as judged by peak she, HFRA-py catalyzed the formation of more ester than did BF?. When two alcoho!s, ethanol and butanol, were included in the reaction mixture along with lactic acid, two peaks were obtained. One peak corresponded in retention time on both polav and nonpolar columns to the HFBA-ethvl ester of lactic acid and the second peak to the HFBA-botvl ester of lactic acid. When the alcohol standard mixture ( 3 ) was cornbjwd with the acid standard mixture ( 3 ) and derivatized with HFBA-py-ethanol, the alcohols were esterified with HFBA and the acids with ethanol. There was no detectable interference between the dilute alcohol in the Sample and esterification of the carboxyl groups with ethanol. Thus, the data. indicate that the alcohol must be present in
sufficient concentration before it can compete for esterification of the carboxyl group in the presence of HFBA-py. However, there is enough ethanol present in ethyl ether that has been stabilized with ethanol to compete with larger amounts of other alcohols for esterification of the carboxyl group in the presence of HFBA-py. The procedure described above reproducibly produced derivatives that can be effectively used with EC-GLC to selectively detect minute quantities of hydroxy acids, alcohols, or amines in small amounts (2 ml) of synovial fluid (Figure 1, curves A and B ) . The chromatogram was obtained by analysis of 2 ml of synovial fluid taken from a patient with juvenile rheumatoid arthritis. The patient was receiving salicylate treatment and this acid was easily detected (Figure l, curve B ) . Identification of the compound was confirmed by GLC mass spectrometry as the HFBAethyl ester derivative of salicylic acid. Basic extractable HFBA reactive compounds were detected in the pH 10 extraction (Figure 1, curve A ) , but these have not been identified. The above procedure has been applied to the study of a limited number of synovial fluid samples taken from people with arthritis, and several EC-GLC profiles have been obtained that differ according to the form of arthritis involved
RECEIVEDfor review April 22, 1974. Accepted July 22, 1974. TJse of trade names is for identification only and does not constitute endorsement by the Public Health Service or by the U.S. Department of Health, Education, and Welfare.
Alternating Current Linear Sweep and Cyclic Voltammetry at a Dropping Mercury Electrode with Phase-Selective Fundamental and Second Harmonic Detection H. Blutstein and A. M. Bond Department of Inorganic Chemistry, University of Melbourne, Parkville, Victoria, 3052, Australia
The analytical application of phase-selective ac linear sweep voltammetry (fundamental and second harmonic) with scan rate synchronized to a dropping mercury electrode is considered. Commercially available instrumentation was adapted to provide the technique, and the theoretical response was obtained over a wide range of operating conditions for both the faradaic and charging current components. The techniques combine the advantages of fast scan rates (up to 200 mV/sec used in this work), extremely high reproducibility (better than 1 % at the 10-6Mlevel), and linear calibration curves over a wide concentration range. With the second harmonic method, flat base lines were obtained despite the growth of the mercury drop during the scan duration. and this would appear to be the preferred technique. At the high frequencies necessitated by the condition AEwt >> vf, slight non-ideality leads to sloping base lines in the fundamental mode. Comparison with the dc method shows considerable advantage of ac techniques with respect to resolution. Cyclic ac voltammograms can also be obtained at the dropping mercury electrode with the same instrumentation. 1934
Limitations associated with the analytical use of conventional dc polarography include the relatively poor sensitivity result,ing from the high contribution of the charging current at low concentrations, difficulties in measuring wave heights, which frequently show non-ideal exponential shape current-potential curves, and the length of time required to record a po1arogra.m. Many of the recent advances in polarographic methodology have been aimed a t minimizing these limitations and making polarography more competitive with other commonly used instrumental analytical techniques such as molecular spectroscopy, chromatography, and atomic absorption spectrometry. The use of phase-selective fundamental ac polarography (I ), second order ac techniques (1 ), pulse and current sampled dc polarography (2) successfully discriminates against the charging current. These methods frequently increase the sensitivity by two or more orders of magnitude over conventional dc polarography. (1) D. E. Smith in "Electroanalytical Chemistry," A. J. Bard, Ed., Marcel Dekker, New York, N.Y.. 1966, Vol. 1, Chap. 1. (2) H. Schmidt and M. yon Stackelberg, "Modern Polarographic Methods," Academic Press, New York, N.Y., 1963.
ANALYTICAL CHEMISTRY, VOL. 46, NO. 13, NOVEMBER 1974
