Gas Chromatog ra phy of Trimethylsilyl Derivatives of Compounds Related to Chloramphenicol PAUL D. SHAW Department o f Plant Pathology, University o f Illinois, Urbana, 111.
b A method is described for the preparation and gas chromatography of the trimethylsilyl derivatives of some compounds related to chloramphenicol. Several of these compounds are eluted in approximately the order of their molecular weights. Changes in substituents, in certain cases, however, cause variations in retention time which cannot be explained on a molecular weight basis. The apparent molar response of the hydrogen flame detector to the phenylserinol derivatives is increased by the presence of an acyl group on the side chain but is not affected by changes in structure of the side chain.
D
studies on the biosynthesis of chloramphenicol by Streptomyces aemzuelae and of other naturally occurring nitro compounds, it became necessary to separate chloramphenicol from structurally related compounds and to determine it quantitatively in the presence of these related materials. Paper chromatographic methods have been described for the separation of various aromatic nitro compounds (2, 8), and if individually present, chloramphenicol and certain related compounds can be URINO
Table 1. Derivatives of threo-lPhenyl-2-amino-lt3-propanediol
H NHR’
6Hk
Com-
R
pound
1 H
2 3
4
5 6
7
8 9 10 11
12 13
14 15 16 17 18
KO; KO, KOp
Molecular
R’
weights
H H H CHaCO OHCCO CHgFCO (CHs)zCHCO CHsClCO CHFzCO CHC1,CO CFaCO CHClzCO CH2BrCO CHCliCO
167 182 212 254 268
cc1,co
CHBrClCO CHBrzCO CBr,CO
All are of the D-threo- configuration except compounds 1, 10, 11, and 13, which are DL. The antibiotic, chloramphenicol, is compound 12. The identities of these compounds were confirmed by comparison of their melting points with the values reported in the literature (7). Chromatograms were run on an F & M Model 609 gas chromatograph with a hydrogen flame detector and using a 6-foot coiled column with an internal diameter of 3/1,3 inch. The c o l u m was packed with 1.5 % SE30 silicone on 100- to 120-mesh Gas Chrom P (Applied Science Laboratories). The support had been pretreated with dimethyldichlorosilane before use (6). Methods. The trimethylsilyl derivatives were prepared by the method of Bentley et al. (1). In a typical experiment, about 10 mg. each of compounds 3, 4, and 12 were dissolved in 2.0 ml. of pyridine. Aliquots (0.01 t o EXPERIMENTAL 0.5 ml.) were transferred to 1-ml. volumetric flasks and treated with 0.05 Materials. Trimethylchlorosilane ml. each of trimethylchlorosilane and and hexamethyldisilazane were obhexamethyldisilazane. After standing tained from Applied Science Laborafor 10 minutes a t room temperature, tories, State College, Pa. Pyridine the mixtures were diluted to 1.0 ml. was dried over potassium hydroxide with pyridine and the tubes stored in pellets before use. All compounds used a desiccator until used. Pyridine was in these studies except N-acetyl-pchosen as solvent because of the high nitroplienylserinol were gifts of Parke, solubility of the phenylserinol derivaDavis and Co., Ann Arbor, Mich. This tives and the short time required for compound was prepared by the method formation of the trimethylsilyl derivaof Rebstock (7). The structures of tives. these compounds are shown in Table I. For the chromatography of the They are derivatives of the parent compound, l-phenyl-Zamino-l,3-pro- synthetic mixture shown in Figure 1, trimethylsilyl derivatives were prepared panediol (compound l ) , which has been from solutions containing about 4 mg. given the trivial name, “phenylserinol.” determbed quantitatively by biological assay. However, these techniques do not lend themselves readily to the quantitative estimation of a mixture of such compounds usually present in an S. venezuelae growth medium or a cellfree extract. Recently, several reports have described the sepamtion of phenols (@, carbohydrates (1, S), and sterols (6, 10, 12) by vapor phase chromatography as trimethylsilyl ethers. Since the conversion of a hydroxyl group to its trimethylsilyl ether has the effect of increasing the volatility and decreasing the polarity of the compound bearing the hydroxyl group, such derivatives might be useful in the vapor phase Chromatography of the relatively nonvolatile chloramphenicol.
4,lO
I
272
Figure 1 . Gas chromatography of phenylserinol derivatives
290
Range 100 Attenuation 3 2
282 289
308 308 323 333 356 358 368 412 49 1
0
5
15
10
TIME
1580
0
ANALYTICAL CHEMISTRY
20
IN M I N U T E S
25
,#MOLES
(~10')
Figure 2. Variations of peak area with sample size of phenylserinol derivatives
0 p-Nitrophenylsarinol 0
Chloramphenicol N-Acetyl-p-nitrophenylserinol
per ml. of each component. Volume of the injected sample was 4 pl. The flash heater was maintain:d a t 312' C. and the detector a t 280' C. The gas flow rates (ml. per minutie) a t the start of each chromatogram were: helium, 93; hydrogen, 45; oxygen, 333. The column temperature was programmed from 110' C. a t 6.4" C. per minute for 12 minutes, then a t '2.3' C. per minute to 260' C. Each (component of the mixture was first separately chromatographed under these same conditions. When two materials appeared to have the same retention time, they were also chromatographed together to confirm their identification. For the quantitative estimation of each component of a mixture of compounds 3, 4, and 12, the flash heater waij held a t 275' C. and the detector a t 245' C., and the initial gas flow ratec, (ml. per minute) hydrogen, 44; were : helium, 85 oxygen, 297. The column temperature was programmed from 140' to 220" C. a t a rate of 6.4' C. per minute, and then the column was allowed to operate isothermally for 5 minutes. Peak areas were measured by triangulation. The areas given in Figurc? 2 were computed on the basis of zero attenuation, and the molar response ie the area in square millimeters divided 3y the micromoles of compound injected, RESULTS AND DISCUSSION
Seventeen derivatives of phenylserinol were separable into 1.3 peaks, as shown in Figure 1. The numbers identifying each peak refer to the compounds in Table I. I n four cases, individual members of a pair conld not be separated -3 and 11, 4 and lC, 8 and 13, and 16 and 17. Nine of the phenylserinol derivatives are eluted in approximately the order of their molecular weights (Figure 3). Deviations from linearity, especially in the first few compounds eluted, are a result of changing the rate of tempersture programming ,after 12 minutes. Of the remaining eight compounds examined, only thoiie having fluorine in the acyl group formed any sort of
pattern; these three (compounds 6, 9, and 11) are eluted in the reverse of the expected order. The remaining five compounds are randomly distributed; the monobromoacetyl derivative has a retention time shorter than expected on the basis of its molecular weight, whereas the bromochloroacetyl and glyoxalyl derivatives have longer retention times. Attempts to detect the trimethylsilyl derivative of the tribromoacetyl derivative were unsuccessful. In this case steric hindrance by the bulky bromine atoms may have prevented the formation of the trimethylsilyl derivative. The effects on retention time of substitution in the position para- to the serinol side chain are variable. The substitution of an amino group (compound 2) or a nitro group (compound 3) for the para-hydrogen atom of phenylserinol (compound I) causes no change other than that attributable to an increase in molecular weight. The exchange of the nitro group of chloramphenicol (compound 12) for a methoxyl group (compound 10) causes a decrease in retention time greater than that due to a decreased molecular weight. The substitution of a methanesulfonyl group (compound 14) increases retention time. A series of closely related compounds would be expected to be eluted from the relatively nonselective SE-30 column in order of decreasing volatility. It was reported by Luukkainen et al. (6) that in the case of sterol trimethylsilyl ethers, the elution pattern followed approximately the order of molecular weights. However, variations in the molecular weights of the trimethylsilyl phenylserinol derivatives did not always lead to predictable changes in relative retention times. These results suggest that the volatility of these compounds is not necessarily directly related to their molecular weights. Variability is particularly apparent in the derivatives having fluorine in their acyl groups. The retention times of these compounds decrease progressively as fluorine is substituted for hydrogen in the acetyl group of N-acetyl-pnitrophenylserinol. The decreased retention time of the trifluoroacetyl derivative in relation to p-nitrophenylserinol and N-acetyl-p-nitrophenylserino1 is not surprising in view of the greater volatility of trifluoroacetic acid and its derivatives. For example, cholestanyl trifluoroacetate is eluted from an SE-30 column before cholestanol and cholestanyl acetate (11). Similar experiments with sterol mono- and difluoroacetates are not available for comparison, however. Figure 2 shows t h a t gas chromatography can serve as a quantitative method for the determination of phenylserinol derivatives. These compounds shown were chosen because they
I60 180 200 220 2 4 0 260 283 300 320 340 MOLECULAR WEIGHT
360 380 600 120
Figure 3. Relationship of retention time to molecular weight of phenylserin01 derivatives
often occur together in our enzymatic reaction mixtures. They were chromatographed as mixtures. For these compounds there is a linear relationship between concentration and peak area. However, the slope of the line for the unacetylated compound is about one half that of the acetyl derivative and chloramphenicol. Some of the other available derivatives were also examined, and as can be seen in Table 11, most of these compounds give the same response as the acetyl derivative within experimental error. Unfortunately. some of the derivatives contained impurities and were not available in sufficient quantities for purification, so their molar responses could not be determined. The low response of compounds 3, 8, and 15 probably cannot be due to the presence of impurities, since they give single peaks on the gas chromatograph, and their melting points agree closely with those reported in the literature ( 7 ) . It was observed that phenylserinol (compound 1) and p-aminophenylserinol (compound 2), both of which have free amino groups, also appear to give a lower molar response than the acyl derivatives. Unfortunately, these two compounds contained volatile impurities, so an exact comparison could not be made. However, if it is assumed
Table 11. Response of Hydrogen Flame Detector to Derivatives of fhreo-lPhenyl-2-amino- 1,3-propanedioI
Corn-
pound
Relative molar
response 0.46 1.00 0.93
1.06
Relative Corn- molar pound response 10 11
0.96 1.05
12 14
1.12 0.96 0.66 0.98
15 0.71 17 9 1.02 Relative molar remonae = molar response of compound 0.94
molar response of compound 4
VOL. 35, NO. 11, OCTOBER 1963
1581
that the molar response of the impurities is no greater than that of the phenylserinol derivatives and if they have approximately the same molecular weights, the relative molar response for phenylserinol is 0.57 and for p-aminophenylserinol 0.50. Since both of these compounds have free amino groups potentially able t o form silyl amines (analogous to hexamethyldisilazane), the mole fraction of silicon may be important in determining the response of the detector t o these compounds. An alternative and perhaps more likely explanation is that the amino groups of the phenylserinol derivatives are not silylated, and the trimethylsilyl ethers may be partially lost by adsorption onto the column or injection port. &o it is possible that because of a charge on some of the amino groups, the silyla-
tion reaction was incomplete and these salts were nonvolatile. Sweeley has reported that charged groups will not react with silanes ( 9 ) . ACKNO WLEDGMENl
The gifts of the compounds used in these studies by Harry M. Crooks, Jr., Parke, Davis and Co., and the helpful discussions with C. C. Sweeley on certain phases of the work are gratefully acknowledged. LITERATURE CITED
(1) Bentley, R., Sweeley, C. C., Makita,
M., Wells, W. W., Biochem. Biophys. Research Commun. 11,14 (1963). ( 2 ) Gottlieb, D., Robbins, P. W., Carter, H. E., J . Bacterid. 72, 153 (1956). (3) Hedgley, E. V., Overend, W. G , , Chem. Ind. (London) 1960, p. 378.
(4) Horning, E. C., Moscatelli, E. 8., Sweely, C. C., Ibid., 1959, p. 751. (5) Langer, S. H., Pantsges, P., Wender, I., Ibid., 1958, p. 1664. (6) Luukkainen, T., VandenHeuvel, W.
J. A., Haahti, E. 0. A., Horning, E. C., Biochem. Biophys. Acta 52,599 (1961). ( 7 ) Rebstock, M. C., J . Am. Chem SOC. 72,4800 (1950). ( 8 ) Smith, G. N., Worrel, C. S., Arch. Biochem. 28,1(1950). (9) Sweeley, C. C., personal communi-
cation.
(10) VandenHeuvel,
W. J. A., Creech, B. G., Horning, E. C., Anal. Biochem.
4,191 (1962). (11) VandenHeuvel,
W. J. A , , Haahti, E. 0. A., Horning, E. C., J . A m . Chem.
SOC.83,1513 (1961).
(12).Wells, W. W., Makita, hl., Anal. Bzochem. 4,204 (1962).
RECEIVEDfor review February 11, 19G3. Accepted July 1 1 , 1963. Work supported by a grant from the National Institute of -4llergy and Infectious Diseases (E-4258).
Tet racya noquinodimet hanide as a Color Reagent for Inorganic Thin Layer Chromatography LEONARD F. DRUDING Deparfmenf of Chemistry, Rufgers, The State Universify, Newark 2, N. J.
b The separation of the alkali, alkaline earth, and some mono- and divalent post-transition metal ions by thin layer chromatography using silica gel adsorbent is reported. Two solvent systems, 5% glacial acetic acid in ethanol, and 5% 2N HCI in f butanol were used, with the former providing excellent resolution of the alkali metals while the latter is more effective for the remaining ions. The R, values for all ions in each solvent are reported. The use of tetracyanoquinodimethanide (TCNQ-) as a color reagent for these ions was investigated and found particularly sensitive for the detection of monovalent ions.
W
MANY paper chromatographic separations of inorganic ions have been reported over the past years, comparatively few have been reported using the thin layer technique (6-8). This nen- method of separation shares the advantage of applicability to microamounts and offers greater speed and convenience than paper chromatographic methods. Separation and identification of the alkali and alkaline earth metal ions was of immediate interest. Seiler and Rothweiler (6) used 1% glacial acetic acid in absolute ethanol to separate LiT, Na+, Ki+, and Mg+2 on a plate coated with silica gel. With their system, they were able to achieve a maximum Rr of about 0.4 for Li+, with lesser values for
1582
HILE
ANALYTICAL CHEMISTRY
the other ions. A solvent system that would produce a greater degree of separation was desired; this study was extended to include all of the alkali and alkaline earth metal ions. The second part of the research was devoted to the investigation of a more sensitive means for detecting and identifying the various ions of these two families. In the past, violuric acid was used for both paper chromatography, and in Seiler's work. This process can be time consuming, since the chromatograms must be heated for a t least 30 minutes at 120" C. to develop the color. Recent work by Acker and Hertler (1) and Melby et al. (4)on cyanocarbons has shown that tetracyanoquinodimethan (TCKQ) can easily be reduced to an anion radical (TCNQ-), and that this anion forms insoluble salts with many mteal cations, including the alkali metal ions (4). Both the anion, TCNQ-, and the salts are intensely colored. From these intense colors, it appears that TCNQ- may be a more sensitive reagent for the identification of the alkaline earth ions. Also investigated were some mono- and divalent post-transition metal ions whose chemical behavior with TCKQ- suggested inclusion. As tetracyanoquinodimethan itself appears to be a stronger a-acid than tetracyanoethylene (TCXE) (0, this suggests the use of TCiYQ as a color reagent in place of T C N E for the detection of aromatics (9).
EXPERIMENTAL
Preparation of Silica Gel. One drawback of all commercial adsorbents, including those without added binder, is the presence of large quantities of cations,. especially those of sodium, magnesium, calcium, and iron. It is necessary to remove these by washing the silica gel with acid and distilled mater (8). Two hundred and fifty grams of silica gel, (Kieselgel without binder, Arthur H. Thomas Co. or Silica Gel H, Brinkmann Instrument Co.) were stirred with 500 ml. of 1:l (v./v.) HC1-mater for 2 hours, then filtered and washed with 200 ml. of acid, 700 ml. of distilled water, and finally 200 ml. of benzene. The deionized silica gel was dried at 110" C. This silica gel \vas applied directly, instead of rice starch being added as a binder. The resulting plates were quite fragile, and extreme care had to be exercised in spraying color reagent on the plates. Preparation of Plates. Glass plates, 200 x 200 mm., were coated with the deionized silica gel by spraying on a slurry of the adsorbent (2). Silica gel, 12 grams (enough for three plates), was mixed with 40 ml. of water. This was poured into a n atomizer attached to the laboratory air line. One slow pass with the spray produced a plate thickness of about 250 to 300 microns. The plates were activated by drying at 110" C. for a t least 1 hour. Development of Chromatogram. Each of the metal ions was originally present as either the nitrate or chloride salts. Two microliters of a O.1M
