(model 111) or is the result of solvent participation in the transfer of the proton from the sulfur atom to the nitrogen atom of the DTC acid (model IV). Solvent participation is likely to occur in the case of DTC acids with small alkyl substituents (e.g., dimethyl- or diethyl-DTC acid) as the studies with mixed solvents indicated ( 3 ) that for these DTC acids the solvent is able to approach closely the N-C bond (See Scheme 1).
f
\
I
H
(W Scheme 1
The present authors propose that the solvent isotope effect observed in the decomposition of DTC acids containing small alkyl substituents is mainly due to the difference in position of either the deuteron or the proton in the hydrogen bond. Indeed, in agreement with zero point energy considerations, the probability of finding the deuteron close to the nitrogen atom is larger than the equivalent probability for a proton (6). As a result, the fractional charge on the nitrogen atom (6) R. E. Rundle, J . Phys., 25,487 (1964).
(model IV) will be larger when a deuteron rather than a proton occupies the “hydrogen bond.” We have shown previously how an increase of fractional charges in the DTC acid molecule enhances the decomposition rate (3). However, in the case of DTC acids containing large alkyl groups (e.g., diisopropyl-DTC acid), the solvent is less able to approach the N-C bond as is reflected in the great instability of these acids (3). The lower decomposition rate of diisopropyl-DTC acid in D?O suggests that the rate-determining step in the decomposition of that compound is the transfer of proton from the sulfur atom to the nitrogen atom (model 111). Indeed, the transfer of a deuteron from one base to another occurs at a slower rate than that of a proton. The opposite solvent isotope effects seen in Table I thus lead to the conclusion that, depending on the availability of a solvent molecule near the N-C bond, the rate-determining step in the decomposition of DTC acids is either the proton (or deuteron) transfer from the sulfur to the nitrogen atom or the decomposition of the intermediate IV. The solvent isotope effect in the decomposition of dibutyl-DTC acid (Table I) clearly illustrates the competition between these two possible rate-determining steps. If the above arguments are a true interpretation of the experimental results, the solvent isotope effect may be regarded as confirming the coilclusion reached by studies in mixed solvents (3); namely, that the rates of decomposition of DTC acids are primarily governed by the ease of approach of the solvent to the N-C bond. Solvent isotope effects have the peculiarity to throw light on the influence of the proton transfer on the overall reaction rate. ACKNOWLEDGMENT
The authors are grateful to Dr. P. M. Laughton and Dr. C. H. Langford, Carleton University, for helpful discussions. RECEIVED for review March 22, 1971. Accepted June 7, 1971. This paper constituted a part of the MSc. thesis of K. I. Aspila. The authors are grateful to the National Research Council of Canada for research grants.
Gas Chromatographic Determination of PeniciIIins Charles Hishta, David L. Mays, and Michael Garofalol Bristol Laboratories, Dicision‘of’Bristol-Myers Company , Syracuse, N . Y.
NUMEROUS CHEMICAL METHODS are available for the quantitative determination of penicillins; the identity of the penicillin must usually be determined by a different procedure, such as infrared spectrophotometry or thin-layer chromatography. Official microbiological methods for determining penicillins suffer from high variability and time-consuming procedures. A gas chromatographic procedure for the indirect identification of penicillins was reported by Kawai and Hashiba ( I ) . Organic acids produced by alkaline cleavage were Present address, Xerox Corporation, Rochester, N. Y . (1) S. Kawai and 1530
S. Hashiba, Burwcki Kugakir, 13. 1223 (1964).
converted to methyl esters and separated on a 3 . 5 % SE-30 column. Wolfe (2) and coworkers chromatographed the methyl ester of L-phenethicillin. A single peak without evidence of column decomposition was obtained from two columns coupled in series, 1% SE-30 on glass beads (70 cm) followed by 5 % NGS on Chromosorb (40 cm). The chromatography of methyl esters of several 6-amino penicillanic acid derivatives was reported by Evrard (3) in 1964. Some of these derivatives were separated on a 0.4% SE-52 column (2) S. Wolfe, Queens University, Kingston, Ontario, personal communication, 1964. (3) E. Evrard, M. Claesen, and H. Vanderhaeghe, Nature, 201, 1124 (1964).
ANALYTICAL CHEMISTRY, VOL. 43, NO. 11, SEPTEMBER 1971
. . -CH-COO'
//C - N 0
Penicillin G
Penicillin V
0
R=
G C H , - C - I1
Rg
G O - C H 2 - C -II
Phenethicillin
R.
Methicillin
R=
0
Figure 2. Composite chromatograms indicating the separation of penicillin G and penicillin V isothermally at 245 "C 5-a-Cholestane, (2) Penicillin G, (3) Penicillin
(1) V
Oxocillln
R=
Cloxacillin
R=
Dicloxoci IIln
Figure 1. Structure of penicillins
without evidence of decomposition. Martin ( 4 ) pioneered the detection of trace quantities of penicillins G and V by gas-liquid chromatography of the methyl esters. Other antibiotics, including Neomycin, Paromomycin, and Kanamycin, have recently been gas chromatographed ( 5 , 6). This paper reports the separation and quantitative determination of several penicillins by gas chromatography. EXPERIMENTAL Apparatus. A Varian Aerograph Model 2100 gas chromatograph equipped with flame ionization detector was used with gas flow rates of 165 to 215 ml/min for helium, 85 ml/min for hydrogen, and 260 ml/min for air. Column oven temperatues of 245 and 275 "C were used; injector and detector temperatures were maintained at 275 "C. Column. A 4-mm i.d. X 660-mm glass U-tube column was packed with 2 % OV-17 (Applied Science Laboratories, State College, Pa.) on 80-100 mesh Supelcoport (Supelco, Inc., Bellefonte, Pa.). The column packing was prepared by the standard slurry-filtration method. Before it was packed, the empty glass column was thoroughly rinsed with methanol and acetone, dried, conditioned 30 min with a 5 % (w/v) HMDS (Ohio Valley Specialty Company, Marietta, Ohio) solution in toluene to silylate reactive sites, and again rinsed with methanol and acetone, and dried. The packed column
F i g u r e 3. C o m p o s i t e chromatograms indicating the separation of D-phenethicillin and L-phenethicillin isothermally at 245 "C (1) 5-a-Cholestane, (2) DPhenethicillin, (3) L-Phenethicillin
(4) J. Martin, R. Robinson, and R. Bezjian, "The Determination of Sub-Microgram Amounts of Penicillin by Gas Chroma-
tography,'' presented at the 17th Annual Pittsburgh Conference on Applied Spectroscopy, Pittsburgh, Pa., Feb. 1966. ( 5 ) K . Tsuji and J. H. Robertson, ANAL.CHEM. 41. 1332 (1969). (6) Ibid.,42, 1661 (1970). ANALYTICAL CHEMISTRY, VOL. 43, NO. 1 1 , SEPTEMBER 1971
1531
Figure 4. Chromatogram of methicillin isothermally a t 275 "C
3
5-~Cholestand-one, (2) Methicillin
(1)
1
Table I. Calibration Data for 245 "C and 165 ml/min Carrier Flow Rate Relative standard Relative deviation Relative response of response Compound retention time factor factoi, %a 1.00(2.3min) 1.0 .. . 5-a-Cholestane 2.5 1.5 Penicillin G 1.65 2.4 1 .o D-Phenethicillin 1.60 1.71 2.4 1.3 L-Phenethicillin 2.05 2.5 1.7 Penicillin V a Relative standard deviations were calculated from the response factors obtained before rounding off.
had a theoretical plate height of 0.6 mm for silylated Penicillin V. Buffer Solution. A saturated aqueous solution of ammonium sulfate was adjusted to pH 2.2 with concentrated sulfuric acid. Internal Standard-Silylating Reagent. A 50 v/v solution of HMDS in pyridine containing 0.375 mg/ml of 5-a-cholestane or 5-a-cholestan-3-one (Mann Research Laboratories, Inc., New York, N. Y.) was prepared. Reference Standards. Penicillin reference standards (Figure 1) (Bristol Laboratories Control Division house standards) were dissolved in water at a concentration of 20 mg/ml. To 2.00 ml of the standard solution, 8.00 ml of chloroform and 2.0 ml of pH 2.2 buffer were added. The mixture was immediately shaken vigorously for 1 min and centrifuged. A 2.00-ml aliquot of the organic phase was transferred to an 8 . 2 4 serum vial for silylation. Silylation Procedure. To each vial was added 2.00 ml of internal standard-silylating reagent. The vials were sealed, mixed, and allowed to stand at room temperature with oc1532
2
3 4 5 Time ( m i d
6
7
8
Figure 5. A single chromatogram showing the separation of oxacillin, cloxacillin, and dicloxacillin isothermally at 275 "C (1) 5-a-Cholestan-3-one,(2) Oxacillin, (3)
Cloxacillin, (4) Dicloxacillin ~
~
~~
_
_
_
~
Table 11. Calibration Data at 275' and 215 ml/min Carrier Flow Rate Relative standard
Relative
Relative deviation response of response factor factor, %a
Compound retention time 5-a-Cholestan3-one 1 00 (2 0 min) 1 0 Methicillin 1 51 2 6 Oxacillin 1 58 2 5 Cloxacillin 2 16 2 1 Dicloxacillin 2 83 3 3 Relative standard deviations were calculated from the factors obtained before rounding off
2 2 2 2
4 5 1 3
response
casional shaking. Silylation was essentially complete within 10 minutes for penicillin G, penicillin V, D- and L-phenethicillin, and methicillin. Oxacillin, cloxacillin, and dicloxacillin required up to 60 minutes for complete silylation. Two microliters were injected into the chromatograph. RESULTS AND DISCUSSION
The merit of the gas chromatographic determination of penicillins is the capability to separate, identify, and quantitate penicillins in a single procedure. Unfortunately, two pairs in the set of penicillins tested have similar retention times
ANALYTICAL CHEMISTRY, VOL. 43, NO. 11, SEPTEMBER 1971
under the experimental conditions. Typical chromatograms indicating the separation of several penicillins are shown in Figures 2 to 5. Relative retention times, as well as response factors and their relative precision, are shown in Tables I and 11. Precision was determined for each compound by calculating the relative standard deviation of response factors obtained from six separate preparations of reference standard. Response factors were calculated from peak areas of internal standards and reference standards, measured by the peak-height times half-width method. An LBK 9000 gas chromatograph-mass spectrometer was used to confirm the identity of the silyl derivatives of each penicillin. Chromatographic peaks were shown to be the trimethylsilyl esters of the intact penicillins by observation of their molecular ions.
CONCLUSIONS
Good precision of response factors of reference standards indicates the direct applicability of the procedure to penicillin prime bulk material. Combined with suitable sample preparation the method could be extended to include bulk blends, tablets, syrups, and other commercial preparations. ACKNOWLEDGMENT
The supply of purified D- and L-phenethicillin from the Bristol Laboratories Research Division is acknowledged. R. D. Brown of the Control Division performed the mass spectrometric analyses.
RECEIVED for review April 22, 1971. Accepted June 7, 1971.
CORRESPONDENCE
I
The Internal Reflection Probe SIR: Double-pass internal reflection plates for Internal Reflection Spectrometry (IRS) (1) permit a light beam to enter and exit through one and the same end of the plate. These plates can then be used as “Internal Reflection Probes” for recording spectra of liquids and powders and for reaction monitoring since the free end can be immersed into the sample material (Figure la), thus eliminating the need for special cells (2). The double-pass geometry was conceived to overcome the difficulty of building vacuum chambers, dewars, and ovens having two optical windows, such as used in IRS surface studies with single-pass plates. The double-pass geometry requires only one optical window. This geometry also simplifies the design of the transfer optics for their use in spectrometers and, furthermore, permits use of reflection plates of any length without altering the optics. Studies were made for NASA on the potential use of such a probe for dipping into the surface of the moon to record spectra of moon dust. The advantage that IRS offers is that particulate matter, regardless of size, does not scatter light in its interaction with the evanescent wave (3), and, therefore, spectra of powders can be recorded without elaborate sample preparation. The prisms (or equivalent mirrors) in Figure l a rotate the slit image from the usual vertical orientation to a horizontal orientation, and the mirrors direct the light beam at the required angle so that light will travel via multiple reflection, vertically along the length of the reflection plate. A more sophisticated structure, the vertical double-pass plate, was developed ( 4 ) to minimize the number of optical components required to achieve the same result. The VDP plate, shown in (1) N.J. Harrick, “Internal Reflection Spectroscopy,” Interscience, Division of J. Wiley & Sons, New York, N. Y., 1967. (2) N. J. Harrick, ANAL.CHEM., 36, 188 (1964). Flexible optical fibers may also be used for this purpose as demonstrated by W. N. Hansen, ibid.,35,765 (1963). (3) N.J. Harrick and N. H. Riederman, Spectrochim.Acta, 21,2135 (1965). (4) N.J. Harrick, Appf. Opt., 5, l(1966).
r.;
a
f
1
.c
: i :,e
b C
Figure 1. Internal reflection probes a . Doublepass plate; b. Vertical double-pass plate; c. Doublepass, double-sampling plates. The free end can be dipped into liquids, powders, etc. for recordingspectra
ANALYTICAL CHEMISTRY, VOL. 43, NO. 11, SEPTEMBER 1971
e
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