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Gas chromatographic determination of phytol
ANALYTICAL
BIOCHEMISTRY
Gas
17, 521-525 (1966)
Chromatographic
ROBERT
K. ELLSWORTH
Division
Determination AND
HAROL’D
of Phytol’ J. PERKINS
of Science and Mathematics, State University Plattsburgh, New York 12101
Received May
College,
31, 1966
The pathway of chlorophyll biosynthesis may be studied by administering, to green plants, l”C-labeled precursors, extracting and purifying the pigments, and finding and comparing the molar specific activities of the chlorophylls and their various degradation products (1). In certain experiments with sodium acetate-l-l% as a precursor (2-4), we have attempted to measure the incorporation of l”C into the phytol (C,,H,,OH) moiety of chlorophyll a by subtracting the molar specific activities of methyl pheophorbide a or of pheophorbide ‘a from the molar specific activity of the corresponding pheophytin a. Although it is reasonable to expect phytol to be derived from acetate via mevalonate in view of its isoprenoid structure, the results of these calculations indicated little or no label in the phytol. The puzzling nature of these results made it desirable to determine the specific activity of the phytol directly. Because a suitably accurate and sensitive method for the quantitative determination of microgram amounts of phytol was not available, the procedure described in this paper was developed. MATERIALS
AND METHODS
The phytol was estimated by gas-liquid partition chromatography on a 10 ft X l/4 in. stainless-steel column packed with 20% silicone fluid (SF-96) on an inert support of 60/80 mesh regular firebrick. The chromatograph used was a Wilkens Aerograph (Wilkens Instrument and Research, Inc., Walnut Creek, Calif.), model A-90-P, equipped with a thermal conductivity detector. The Brown Recorder (MinneapolisHoneywell Regulator Co., Brown Instrument Division, Philadelphia, Pa.) was equipped with a disc integrator (Disc Instruments, Inc., Santa
Anita, Calif.). Helium was used as the carrier gas. Injections into the chromatograph were made with a Hamilton (Hamilton Co., Whittier, Calif.) 10 ,~l syringe (701N-w/g). ‘A preliminary 48th Canadian
report Chemical
of the work described in this paper was presented at the Conference held in Montreal, Canada, in ,Jlme 1965. 521
522
ELLSWORTH
AND
PERKINS
Authentic phytol was obtained from Nut.ritional Biochemical Corp. (Cleveland, Ohio), Mann Research Laboratories (New York 6, N. Y.), and K & K Laboratories (Plainview, N. Y.). Preliminary experiments showed that, when phytol was allowed to remain on the column for a relatively long time (i.e., 5 min), two peaks were observed. Presumably these peaks represent incomplete resolution 8
7
6
5 II (3 ii = Y 4 w a
4
?
2
I
RETENTION
TIME
IN
MIN
FIG.
ml/min.
1. Typical Column
chromatogram temperature
for 25O”C,
pure phytol at a carrier gas flow injector temperature 280°C.
rate
of 75
of the four phytadienes (cis-trans isomers) recently found by Blumer and Thomas (5) to result from the pyrolysis of phytol on the chromatographic column at 200°C. Collection of these peaks, and infrared analysis of them using a Perkin-Elmer 221G spectrophotometer, supported this presumption. To determine all of the phytadienes simultaneously (i.e., to group all the phytadienes under one peak), we found that retention times shorter than 3 min were necessary. Of the factors influencing retention time, the flow rate of the carrier gas was the most significant.
DETERMINATION
523
OF PHYTOL
Analyses performed at column temperatures of 250” and carrier gas flow rates of 37, 65, and 75 ml/min showed that, the phytadienes were grouped under one symmetrical peak at a carrier gas flow rate of 75 ml/min. Injector and detector temperatures of 280” and 32O”C, respectively, were used in obtaining these chromatograms. A typical chromatogram for a carrier gas flow rate of 7;i ml/min is shown in Figure 1. To st.udy the relationship between integrator counts and amount of phytol, weighed amounts of phytol were volumetrically diluted with TABLE Integrator
Counts
per
Microgram of Solrltion
1 I’hytol Injected
(pure
Concentration
Integrator counts /a PhYtol
m pbtol ,iimzG
21.7 87.1 174.1 217.7 290.2 453.4 870.8
versus
phytol) Mean : S.D.:
7.61 7.64 7.25 7.85 7.55 7.96 4.98 7.64 kO.22
reagent-grade petroleum ether (boiling range 35-60°C) and 1 ~1 injections of each concentration were made. Typical results are presented in Table 1. Each entry is the mean of five injections. RESULTS
AND
COMMEKTS
Except for pure phytol, the data in Table 1 show that the number of integrator counts per microgram phytol is constant regardless of the concentration of the injected solution. The discrepancy in the case of pure phytol is attributed to the difficulty experienced in making quantitative injections of this viscous compound into the chromatograph. In computing the mean shown in Table 1 the result for pure phytol has therefore been excluded. The value of 7.64 +- 0.22 integrator counts per microgram phytol was sufficiently accurate and sensitive for our purposes. In attempts to reproduce, on different, clays, the data of Table 1, aftcl the chromatograph had been turned off and then programed as nearly as possible to the original conditions, it was found that the absolute value of the integrator counts per microgram phytol was quite variable from day to day (on one occasion, a value of 13.6 + 0.3 integrator counts
524
ELLSWORTH
AND
PERKINS
per microgram phytol was obtained). In all cases, however, the standard deviation was satisfactorily low, and many experiments of this nature have established that a linear relationship between integrator counts per microgram of phytol and the weight of phytol injected can be readily obtained on any one day. Because of the difficulty in programming the gas chromatograph to precisely the same operating conditions from day to day, it is essential, however, to include one standard solution in a series of injections of phytol solutions of unknown concentrations. The consistently low standard deviation suggests that it is not necessary to inject more than one standard solution per series of unknowns provided that the standard solution used has a phytol concentration below 500 dd Gas chromatographic analysis of three different authentic phytols gave a mean value of 7.64 2 0.09 integrator counts per microgram phytol. That the standard deviation is low, suggests that the commerical phytol samples were of a similar degree of purity. Infrared spectroscopic examination of these preparations verified this conclusion. Evidence indicating that the pyrolysis of the phytol was complete and that the recovery of the phytadienes was quantitative when the injector temperature was 280°C and the column temperature was 250” was established by collecting the reaction mixture (i.e., phytadienes), weighing the mixed phytadienes, and reinjecting a known concentration of phytadienes into the chromatograph. The number of integrator counts/pg phytadienes, collected from the column, was compared with the number of integrator counts/pg phytadienes obtained by pyrolysis at the injector port of phytol solutions of known concentration. That the assumption (implicit in the latter calculation) that 100% conversion of the phytol occurred at the instant of injection onto the column was valid was verified by comparing qualitatively, and quantitatively, the chromatograms obtained when pure phytol was injected with those obtained when the collected phytadienes were injected. These data are shown in Table 2. Further evidence of the quantitative pyrolysis of pytol was established by injecting 5 ~1 of pure phytol (4.35 mg) into the chromatograph. During a continuous run of 8 hr, no additional peaks were observed on the chromatograph. Clearly, the method for the quantitative determination of phytol described in this paper cannot be used for the analysis of phytol present as one component of a mixture inasmuch as the intentionally poor resolution of the gas chromatography would probably result in the cochromatography of other components of the mixture with the phytadiene peak. Thus this method requires that the sample be pure phytol, a restriction that is not serious when the method is used as
DETERMINATION
Comparison those
525
OF PHTTOL
TABLE 2 of Integrator Counts/pg Phytadienes Collected from Columns from Phytadienes Obtained by Pyrolysis of Phytol Solutions
Solution
Solution ooncn., pig/All
Phytadiene concn., /.lg/&d
Phytol in petroleum ether Phytol in petroleum ether Phytadienes in petroleum ether
81.5 40.8 31.7
76.5 38.3 31.7 Mean: S.D.:
with
Integrator counts/pg phytadienes
15.1 15.0 14.6 14.9 ZkO.2
intended for the determination of phytol obtained from the hydrolysis of chlorophylls or pheophytins. Such preparations, when made from pigments of a high degree of purity, are not contaminated with other compounds. The practical limit of sensitivity of this method for determining phytol is about 10 pg, and the maximum error at these phytol levels is 3%. SUMMARY
An accurate and sensitive procedure has been developed for determining the diterpene phytol, C,,H,,OH. Vapor-phase chromatography on a column consisting of 20% silicone fluid (SF-96) on an inert support of SO/SO mesh firebrick resulted in complete pyrolysis of the phytol to a mixture of presumed phytadienes when the injector temperature was 280°C and the column temperature was 250”. If the retention time is kept below 3 min, all of the pyrolysis, products nppctar under one symmetrical peak and yuant#itative estimation of tlie amount of phytol present in an unkown sample can be made on samples containing as little as 10 pg of phytol. The maximum error at these phytol levels is 35%. ACKNOWLEDGMENTS The authors gratefully acknowledge the skilled technical assistance of Barry Newman with the experiments described in this paper and critical reviews of the manuscript by Drs. V. Morley and D. Shearer of the Analytical Chemistry Ftrsearch Service, Canada Department of Agriculture, Ottawa, Canada. This research was supported in part by the United States Atomic Energy Conimission under contract number AT(30-l)-3559. REFERENCES 1. PERKINS, 2. PERKINS, 3. ROBERTS, 4. PERKINS, 5. BLUMER,
H. J., AND ROBERTS, D. W. A., Biochinl. Riophys. Actn 58, H. J., AND ROBERTS, D. W. 8., Carl. J. Bat. 41, 221 (1963). D. W. A., AND PERKINS, H. J., Biochirn. Biophys. Actu 58, H. J., AND ELLSWORTH, R. Ii., unpublished results, 1964. M., AND THOMAS, D. W., S&ace 147, 1148 (1965).
486
(1962).
499
(1962)
BIOCHEMISTRY
Gas
17, 521-525 (1966)
Chromatographic
ROBERT
K. ELLSWORTH
Division
Determination AND
HAROL’D
of Phytol’ J. PERKINS
of Science and Mathematics, State University Plattsburgh, New York 12101
Received May
College,
31, 1966
The pathway of chlorophyll biosynthesis may be studied by administering, to green plants, l”C-labeled precursors, extracting and purifying the pigments, and finding and comparing the molar specific activities of the chlorophylls and their various degradation products (1). In certain experiments with sodium acetate-l-l% as a precursor (2-4), we have attempted to measure the incorporation of l”C into the phytol (C,,H,,OH) moiety of chlorophyll a by subtracting the molar specific activities of methyl pheophorbide a or of pheophorbide ‘a from the molar specific activity of the corresponding pheophytin a. Although it is reasonable to expect phytol to be derived from acetate via mevalonate in view of its isoprenoid structure, the results of these calculations indicated little or no label in the phytol. The puzzling nature of these results made it desirable to determine the specific activity of the phytol directly. Because a suitably accurate and sensitive method for the quantitative determination of microgram amounts of phytol was not available, the procedure described in this paper was developed. MATERIALS
AND METHODS
The phytol was estimated by gas-liquid partition chromatography on a 10 ft X l/4 in. stainless-steel column packed with 20% silicone fluid (SF-96) on an inert support of 60/80 mesh regular firebrick. The chromatograph used was a Wilkens Aerograph (Wilkens Instrument and Research, Inc., Walnut Creek, Calif.), model A-90-P, equipped with a thermal conductivity detector. The Brown Recorder (MinneapolisHoneywell Regulator Co., Brown Instrument Division, Philadelphia, Pa.) was equipped with a disc integrator (Disc Instruments, Inc., Santa
Anita, Calif.). Helium was used as the carrier gas. Injections into the chromatograph were made with a Hamilton (Hamilton Co., Whittier, Calif.) 10 ,~l syringe (701N-w/g). ‘A preliminary 48th Canadian
report Chemical
of the work described in this paper was presented at the Conference held in Montreal, Canada, in ,Jlme 1965. 521
522
ELLSWORTH
AND
PERKINS
Authentic phytol was obtained from Nut.ritional Biochemical Corp. (Cleveland, Ohio), Mann Research Laboratories (New York 6, N. Y.), and K & K Laboratories (Plainview, N. Y.). Preliminary experiments showed that, when phytol was allowed to remain on the column for a relatively long time (i.e., 5 min), two peaks were observed. Presumably these peaks represent incomplete resolution 8
7
6
5 II (3 ii = Y 4 w a
4
?
2
I
RETENTION
TIME
IN
MIN
FIG.
ml/min.
1. Typical Column
chromatogram temperature
for 25O”C,
pure phytol at a carrier gas flow injector temperature 280°C.
rate
of 75
of the four phytadienes (cis-trans isomers) recently found by Blumer and Thomas (5) to result from the pyrolysis of phytol on the chromatographic column at 200°C. Collection of these peaks, and infrared analysis of them using a Perkin-Elmer 221G spectrophotometer, supported this presumption. To determine all of the phytadienes simultaneously (i.e., to group all the phytadienes under one peak), we found that retention times shorter than 3 min were necessary. Of the factors influencing retention time, the flow rate of the carrier gas was the most significant.
DETERMINATION
523
OF PHYTOL
Analyses performed at column temperatures of 250” and carrier gas flow rates of 37, 65, and 75 ml/min showed that, the phytadienes were grouped under one symmetrical peak at a carrier gas flow rate of 75 ml/min. Injector and detector temperatures of 280” and 32O”C, respectively, were used in obtaining these chromatograms. A typical chromatogram for a carrier gas flow rate of 7;i ml/min is shown in Figure 1. To st.udy the relationship between integrator counts and amount of phytol, weighed amounts of phytol were volumetrically diluted with TABLE Integrator
Counts
per
Microgram of Solrltion
1 I’hytol Injected
(pure
Concentration
Integrator counts /a PhYtol
m pbtol ,iimzG
21.7 87.1 174.1 217.7 290.2 453.4 870.8
versus
phytol) Mean : S.D.:
7.61 7.64 7.25 7.85 7.55 7.96 4.98 7.64 kO.22
reagent-grade petroleum ether (boiling range 35-60°C) and 1 ~1 injections of each concentration were made. Typical results are presented in Table 1. Each entry is the mean of five injections. RESULTS
AND
COMMEKTS
Except for pure phytol, the data in Table 1 show that the number of integrator counts per microgram phytol is constant regardless of the concentration of the injected solution. The discrepancy in the case of pure phytol is attributed to the difficulty experienced in making quantitative injections of this viscous compound into the chromatograph. In computing the mean shown in Table 1 the result for pure phytol has therefore been excluded. The value of 7.64 +- 0.22 integrator counts per microgram phytol was sufficiently accurate and sensitive for our purposes. In attempts to reproduce, on different, clays, the data of Table 1, aftcl the chromatograph had been turned off and then programed as nearly as possible to the original conditions, it was found that the absolute value of the integrator counts per microgram phytol was quite variable from day to day (on one occasion, a value of 13.6 + 0.3 integrator counts
524
ELLSWORTH
AND
PERKINS
per microgram phytol was obtained). In all cases, however, the standard deviation was satisfactorily low, and many experiments of this nature have established that a linear relationship between integrator counts per microgram of phytol and the weight of phytol injected can be readily obtained on any one day. Because of the difficulty in programming the gas chromatograph to precisely the same operating conditions from day to day, it is essential, however, to include one standard solution in a series of injections of phytol solutions of unknown concentrations. The consistently low standard deviation suggests that it is not necessary to inject more than one standard solution per series of unknowns provided that the standard solution used has a phytol concentration below 500 dd Gas chromatographic analysis of three different authentic phytols gave a mean value of 7.64 2 0.09 integrator counts per microgram phytol. That the standard deviation is low, suggests that the commerical phytol samples were of a similar degree of purity. Infrared spectroscopic examination of these preparations verified this conclusion. Evidence indicating that the pyrolysis of the phytol was complete and that the recovery of the phytadienes was quantitative when the injector temperature was 280°C and the column temperature was 250” was established by collecting the reaction mixture (i.e., phytadienes), weighing the mixed phytadienes, and reinjecting a known concentration of phytadienes into the chromatograph. The number of integrator counts/pg phytadienes, collected from the column, was compared with the number of integrator counts/pg phytadienes obtained by pyrolysis at the injector port of phytol solutions of known concentration. That the assumption (implicit in the latter calculation) that 100% conversion of the phytol occurred at the instant of injection onto the column was valid was verified by comparing qualitatively, and quantitatively, the chromatograms obtained when pure phytol was injected with those obtained when the collected phytadienes were injected. These data are shown in Table 2. Further evidence of the quantitative pyrolysis of pytol was established by injecting 5 ~1 of pure phytol (4.35 mg) into the chromatograph. During a continuous run of 8 hr, no additional peaks were observed on the chromatograph. Clearly, the method for the quantitative determination of phytol described in this paper cannot be used for the analysis of phytol present as one component of a mixture inasmuch as the intentionally poor resolution of the gas chromatography would probably result in the cochromatography of other components of the mixture with the phytadiene peak. Thus this method requires that the sample be pure phytol, a restriction that is not serious when the method is used as
DETERMINATION
Comparison those
525
OF PHTTOL
TABLE 2 of Integrator Counts/pg Phytadienes Collected from Columns from Phytadienes Obtained by Pyrolysis of Phytol Solutions
Solution
Solution ooncn., pig/All
Phytadiene concn., /.lg/&d
Phytol in petroleum ether Phytol in petroleum ether Phytadienes in petroleum ether
81.5 40.8 31.7
76.5 38.3 31.7 Mean: S.D.:
with
Integrator counts/pg phytadienes
15.1 15.0 14.6 14.9 ZkO.2
intended for the determination of phytol obtained from the hydrolysis of chlorophylls or pheophytins. Such preparations, when made from pigments of a high degree of purity, are not contaminated with other compounds. The practical limit of sensitivity of this method for determining phytol is about 10 pg, and the maximum error at these phytol levels is 3%. SUMMARY
An accurate and sensitive procedure has been developed for determining the diterpene phytol, C,,H,,OH. Vapor-phase chromatography on a column consisting of 20% silicone fluid (SF-96) on an inert support of SO/SO mesh firebrick resulted in complete pyrolysis of the phytol to a mixture of presumed phytadienes when the injector temperature was 280°C and the column temperature was 250”. If the retention time is kept below 3 min, all of the pyrolysis, products nppctar under one symmetrical peak and yuant#itative estimation of tlie amount of phytol present in an unkown sample can be made on samples containing as little as 10 pg of phytol. The maximum error at these phytol levels is 35%. ACKNOWLEDGMENTS The authors gratefully acknowledge the skilled technical assistance of Barry Newman with the experiments described in this paper and critical reviews of the manuscript by Drs. V. Morley and D. Shearer of the Analytical Chemistry Ftrsearch Service, Canada Department of Agriculture, Ottawa, Canada. This research was supported in part by the United States Atomic Energy Conimission under contract number AT(30-l)-3559. REFERENCES 1. PERKINS, 2. PERKINS, 3. ROBERTS, 4. PERKINS, 5. BLUMER,
H. J., AND ROBERTS, D. W. A., Biochinl. Riophys. Actn 58, H. J., AND ROBERTS, D. W. 8., Carl. J. Bat. 41, 221 (1963). D. W. A., AND PERKINS, H. J., Biochirn. Biophys. Actu 58, H. J., AND ELLSWORTH, R. Ii., unpublished results, 1964. M., AND THOMAS, D. W., S&ace 147, 1148 (1965).
486
(1962).
499
(1962)