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Electron capture-gas chromatography for sensitive assay of abscisic acid
530
Electron
SHORT
Capture-Gas Assay
COMMUNICATIONS
Chromatography of Abscisic
for
Sensitive
Acid
Abscisic acid (ABA), an endogenous plant growth inhibitor, has been reported to occur in a number of species at a concentration of about 100 parts per billion on a fresh weight basis (1). Recently the gas chromatography of abscisic acid was reported (24). These investigations have employed flame ionization detectors which can detect a minimum of 1V2 to 10-l pg of abscisic acid. A more sensitive detector such as the electron capture detector, which has a limit of detection of 1V6 to 10e5 pg, would be of benefit for measurement of ABA in gas chromatography. However, electron capture detectors are sensitive only to certain types of compounds. According to the molecular characteristics described by Lovelock (5) with regard to the ability of substances to capture electrons, the structure of abscisic acid suggests that it may possess electroncapturing capabilities. Therefore, an investigation was undertaken to determine the feasibility of using an electron capture detector in conjunction with gas chromatography to detect small amounts of ABA. We have found that the methyl ester of ABA has electron-capturing properties of a very high order. Instrumentation. The gas chromatograph used was a Barber-Colman, model 5000, containing a glass U-column 6’ X 3 mm i.d. packed with 3% DC 200 on Analabs SD 60-70 mesh, or 2% SE 30 on Gas-Chrom Q 6s 80 mesh. Columns were routinely treated with Silyl-8 column conditioner (Pierce Chemical Co.). The column temperature was 200” with injection and detector temperatures of 210” and 225”, respectively. An argon ionization detector was operated in the electron capture mode. The ionization source was a 20 mCi strontium-90 foil. Argon carrier gas was used at a flow rate of 120 ml/min. Electrometer attenuation was 300. Esterification. Abscisic acid was esterified with diazomethane following the micro method of Schlenk and Gellerman (6). The esterification apparatus was made with Teflon tubing and polyethylene stoppers. Solvents used were redistilled and stored in bottles equipped with Teflon cap liners. Results. Using the DC 200 column we were able to quantitatively detect 100 picograms (lVLo gm) of the synthetic (RS) - (+) cis-transand trans,trans- isomers of ABA. Response with this column was approximately linear to 15 nanograms. By employing 2% silicone gum
SHORT
COMMUNICATIONS
531
rubber (SE 30) as the liquid phase, which gives a shorter retention time of 1.8 min, 10 picograms (10-l* gm) of ABA could be quantitatively detected. The limit of detection with this column was 1 picogram with linearity to 1 nanogram. Separation of the isomers was complete on both columns; resolution factors were greater than 2. When six 100 nanogram samples of ABA were prepared and chromatographed, the mean peak height of a 1 ng sample injected in 1 ,pl of ethyl acetate was 22.3 cm +1.40 cm at the 95% confidence level based on a standard deviation of 0.546. That the method is applicable to ABA in plant extracts as well as to synthetic ABA is demonstrated by the analysis of apple juice. Two liters of fresh apple juice was reduced in volume to 500 ml in a flash evaporator using a water bath temperature of 60”. The ABA was extracted from the juice at pH 3 with methylene chloride. The methylene chloride was taken to dryness in a Aash evaporator, and the ABA cont.ained in the residue applied to the top of a silica gel partition column using the acetone/heptane transfer method previously described (7). The 1.5 x 9 cm silica gel column was made from 8.0 gm of Mallinckrodt’s silicic acid, and hydrated with 5.0 ml of 0.5 formic acid as described by Powell (7). The column was eluted stepwise with 25 ml batches of 0.5, 1, 3, 5, and 10% n-butyl alcohol in n-hexane solvents, all solvents being saturated with 0.5 M formic acid. The 3, 5, and 10% fractions were collected and bulked (ABA is found mostly in the 5% fraction), and taken to dryness in a flash evaporator. One-thirtieth of the residue containing the ABA was methylated with diazomethane. After methylation the ether was evaporated in a stream of nitrogen, and the residue dissolved in 1 ml of ethyl acetate. One microliter of the ethyl acetate solution, representing 0.07 ml of the apple juice, was injected into the gas chromatograph. A chromatogram of ABA in apple juice is shown in Figure 1. The ABA peaks, when compared with known amounts of ABA, indicate the presence of approximately 150 pg/liter of cis,trans-ABA, and an estimated 1/3 this quantity of the trans,trans-isomer. This quantity is somewhat greater than that reported by others (3, 8). Other large peaks are solvent contaminants which have since been eliminated. That these chromatogram peaks represented methyl a#bscisate was shown by comparing gas chromatographic retention Cmes, and micro infrared (9) and fluorescence spectra of methyl abscisate collected from the gas chromatograph, using as reference materials cis,trans-ABA from Shell Development Co., and a ci.s,trans/trans,transmixture supplied by Reynolds Tobacco Company. For the fluorescence analysis, the method of A. H. Hatch (personal communication) was used. The collected Me-ABA was heated in 50%
532
SHORT COMMUNICATIONS Me. cis-ABA
9
16.5
12 Time
(mini
FIG. 1. GLC of methylated apple juice extract. Me. c&ABA = methyl cis,tran-s-abscisic acid. Me. tram-ABA = methyl ester of trans-trans-abscisic
ester of acid.
aqueous H&30, for 2 min at 120”, then cooled under running water. The fluorescence spectrum of this solution was determined with an AmincoBowman spectrophotofluorometer. The excitation h,,, was at 445 IQL and the fluorescence X,,,,, at 495 rnp, which agreed with that obtained with authentic ABA. We have also applied the method successfully to vegetative apple material, which presents a more formidable cleanup problem. Our results indicate that electron capture-gas chromatography can be used for the detection and analysis of ABA in plant extracts. ACKNOWLEDGMENT Synthetic abscisic acid from Shell Development Company, was a gift through the courtesy of Dr. J. van Overbeek.
Modesto,
California,
REFERENCES 1. MILBOEIROW,B. V., Phta 76,93 (1907). 2. DAVIS, L. A., HEINZ, D. E., AND A~DICOTT, F. T., Plant Physiol.
43, 1389 (1968).
SHORT
533
COMMUNICATIONS
3. GASKIN, P., AND MACMILLAN, J., Phytochemistry 7, 1699 (1968). 4. LENIN, J. R., BOWEN, M. R., AND SAUNDERS, P. F., Nature 220, 86 (1968). 5. LOVEMCK, J. E., Anal. Chem. 33, 162 (1961). 6. SCZILENK, H., AND GELLERMAN, J. L., Anal. Chem. 32, 1412 (1960). 7. POWELL, L. E., Plant PhysioE. 39,836 (1964). 8. PIENIAZEK, J., AND RUDNICKI, R., BUZZ. Acctd. Polon. Sci. 15, 251 (1967). 9. POWELL, L. E., Plant Physiol. 42, 1460 (1967). SCHUYLER LOYD
Department
E.
D.
SEELEY
POWELL
of Pomology
Cornell University Ithaca, New York I@50 Received December 16, 1969
Effect
of Solution Polyacrylamide
Components Gel
on Large-Pore Formation
Since Davis (1) and Antoine (2) first described the use of riboflavinsensitized photopolymerization of acrylamide/N,N’-methylenebisacrylamide (BE) mixtures to form large-pore stacking gels for electrophoresis discs, several authors (3-5) have used this photopolymerization technique to form gels which are useful for various specific separations and applications. Although photopolymerized gels have been used extensively in separations, little is known concerning the effect of the added components, i.e., accelerators, buffers, viscosity improvers, leading and trailing ions, and proteins, ‘on the rate and course of photopolymerization. In addition, Oster (6, 7) has shown that both oxygen and hydrogen donors effect riboflavin-sensitized photopolymerizations of aqueous acrylamide, and we recently reported (8) that both proteins and amino acids have a dramatic effect on riboflavin-sensitized photopolymerizations. Since workers are often puzzled by their inability to photopolymerize reproducibly and consistently low concentrations of acrylamide-BIS solutions to gels, we have proceeded to determine the effect of typical solution additives on formation of polyacrylamide gels. Additives, alone and in combination, are found to have a marked effect on the photopolymerization rate and gelation time of polyacrylamide-BIS gels. The sources of materials (1, 8) and photopolymerization techniques (1, 8, 9) have been described. The dye-fade times and the times of firm gel formation were determined for each run. Aliquots (50 ~1) were withdrawn
Electron
SHORT
Capture-Gas Assay
COMMUNICATIONS
Chromatography of Abscisic
for
Sensitive
Acid
Abscisic acid (ABA), an endogenous plant growth inhibitor, has been reported to occur in a number of species at a concentration of about 100 parts per billion on a fresh weight basis (1). Recently the gas chromatography of abscisic acid was reported (24). These investigations have employed flame ionization detectors which can detect a minimum of 1V2 to 10-l pg of abscisic acid. A more sensitive detector such as the electron capture detector, which has a limit of detection of 1V6 to 10e5 pg, would be of benefit for measurement of ABA in gas chromatography. However, electron capture detectors are sensitive only to certain types of compounds. According to the molecular characteristics described by Lovelock (5) with regard to the ability of substances to capture electrons, the structure of abscisic acid suggests that it may possess electroncapturing capabilities. Therefore, an investigation was undertaken to determine the feasibility of using an electron capture detector in conjunction with gas chromatography to detect small amounts of ABA. We have found that the methyl ester of ABA has electron-capturing properties of a very high order. Instrumentation. The gas chromatograph used was a Barber-Colman, model 5000, containing a glass U-column 6’ X 3 mm i.d. packed with 3% DC 200 on Analabs SD 60-70 mesh, or 2% SE 30 on Gas-Chrom Q 6s 80 mesh. Columns were routinely treated with Silyl-8 column conditioner (Pierce Chemical Co.). The column temperature was 200” with injection and detector temperatures of 210” and 225”, respectively. An argon ionization detector was operated in the electron capture mode. The ionization source was a 20 mCi strontium-90 foil. Argon carrier gas was used at a flow rate of 120 ml/min. Electrometer attenuation was 300. Esterification. Abscisic acid was esterified with diazomethane following the micro method of Schlenk and Gellerman (6). The esterification apparatus was made with Teflon tubing and polyethylene stoppers. Solvents used were redistilled and stored in bottles equipped with Teflon cap liners. Results. Using the DC 200 column we were able to quantitatively detect 100 picograms (lVLo gm) of the synthetic (RS) - (+) cis-transand trans,trans- isomers of ABA. Response with this column was approximately linear to 15 nanograms. By employing 2% silicone gum
SHORT
COMMUNICATIONS
531
rubber (SE 30) as the liquid phase, which gives a shorter retention time of 1.8 min, 10 picograms (10-l* gm) of ABA could be quantitatively detected. The limit of detection with this column was 1 picogram with linearity to 1 nanogram. Separation of the isomers was complete on both columns; resolution factors were greater than 2. When six 100 nanogram samples of ABA were prepared and chromatographed, the mean peak height of a 1 ng sample injected in 1 ,pl of ethyl acetate was 22.3 cm +1.40 cm at the 95% confidence level based on a standard deviation of 0.546. That the method is applicable to ABA in plant extracts as well as to synthetic ABA is demonstrated by the analysis of apple juice. Two liters of fresh apple juice was reduced in volume to 500 ml in a flash evaporator using a water bath temperature of 60”. The ABA was extracted from the juice at pH 3 with methylene chloride. The methylene chloride was taken to dryness in a Aash evaporator, and the ABA cont.ained in the residue applied to the top of a silica gel partition column using the acetone/heptane transfer method previously described (7). The 1.5 x 9 cm silica gel column was made from 8.0 gm of Mallinckrodt’s silicic acid, and hydrated with 5.0 ml of 0.5 formic acid as described by Powell (7). The column was eluted stepwise with 25 ml batches of 0.5, 1, 3, 5, and 10% n-butyl alcohol in n-hexane solvents, all solvents being saturated with 0.5 M formic acid. The 3, 5, and 10% fractions were collected and bulked (ABA is found mostly in the 5% fraction), and taken to dryness in a flash evaporator. One-thirtieth of the residue containing the ABA was methylated with diazomethane. After methylation the ether was evaporated in a stream of nitrogen, and the residue dissolved in 1 ml of ethyl acetate. One microliter of the ethyl acetate solution, representing 0.07 ml of the apple juice, was injected into the gas chromatograph. A chromatogram of ABA in apple juice is shown in Figure 1. The ABA peaks, when compared with known amounts of ABA, indicate the presence of approximately 150 pg/liter of cis,trans-ABA, and an estimated 1/3 this quantity of the trans,trans-isomer. This quantity is somewhat greater than that reported by others (3, 8). Other large peaks are solvent contaminants which have since been eliminated. That these chromatogram peaks represented methyl a#bscisate was shown by comparing gas chromatographic retention Cmes, and micro infrared (9) and fluorescence spectra of methyl abscisate collected from the gas chromatograph, using as reference materials cis,trans-ABA from Shell Development Co., and a ci.s,trans/trans,transmixture supplied by Reynolds Tobacco Company. For the fluorescence analysis, the method of A. H. Hatch (personal communication) was used. The collected Me-ABA was heated in 50%
532
SHORT COMMUNICATIONS Me. cis-ABA
9
16.5
12 Time
(mini
FIG. 1. GLC of methylated apple juice extract. Me. c&ABA = methyl cis,tran-s-abscisic acid. Me. tram-ABA = methyl ester of trans-trans-abscisic
ester of acid.
aqueous H&30, for 2 min at 120”, then cooled under running water. The fluorescence spectrum of this solution was determined with an AmincoBowman spectrophotofluorometer. The excitation h,,, was at 445 IQL and the fluorescence X,,,,, at 495 rnp, which agreed with that obtained with authentic ABA. We have also applied the method successfully to vegetative apple material, which presents a more formidable cleanup problem. Our results indicate that electron capture-gas chromatography can be used for the detection and analysis of ABA in plant extracts. ACKNOWLEDGMENT Synthetic abscisic acid from Shell Development Company, was a gift through the courtesy of Dr. J. van Overbeek.
Modesto,
California,
REFERENCES 1. MILBOEIROW,B. V., Phta 76,93 (1907). 2. DAVIS, L. A., HEINZ, D. E., AND A~DICOTT, F. T., Plant Physiol.
43, 1389 (1968).
SHORT
533
COMMUNICATIONS
3. GASKIN, P., AND MACMILLAN, J., Phytochemistry 7, 1699 (1968). 4. LENIN, J. R., BOWEN, M. R., AND SAUNDERS, P. F., Nature 220, 86 (1968). 5. LOVEMCK, J. E., Anal. Chem. 33, 162 (1961). 6. SCZILENK, H., AND GELLERMAN, J. L., Anal. Chem. 32, 1412 (1960). 7. POWELL, L. E., Plant PhysioE. 39,836 (1964). 8. PIENIAZEK, J., AND RUDNICKI, R., BUZZ. Acctd. Polon. Sci. 15, 251 (1967). 9. POWELL, L. E., Plant Physiol. 42, 1460 (1967). SCHUYLER LOYD
Department
E.
D.
SEELEY
POWELL
of Pomology
Cornell University Ithaca, New York I@50 Received December 16, 1969
Effect
of Solution Polyacrylamide
Components Gel
on Large-Pore Formation
Since Davis (1) and Antoine (2) first described the use of riboflavinsensitized photopolymerization of acrylamide/N,N’-methylenebisacrylamide (BE) mixtures to form large-pore stacking gels for electrophoresis discs, several authors (3-5) have used this photopolymerization technique to form gels which are useful for various specific separations and applications. Although photopolymerized gels have been used extensively in separations, little is known concerning the effect of the added components, i.e., accelerators, buffers, viscosity improvers, leading and trailing ions, and proteins, ‘on the rate and course of photopolymerization. In addition, Oster (6, 7) has shown that both oxygen and hydrogen donors effect riboflavin-sensitized photopolymerizations of aqueous acrylamide, and we recently reported (8) that both proteins and amino acids have a dramatic effect on riboflavin-sensitized photopolymerizations. Since workers are often puzzled by their inability to photopolymerize reproducibly and consistently low concentrations of acrylamide-BIS solutions to gels, we have proceeded to determine the effect of typical solution additives on formation of polyacrylamide gels. Additives, alone and in combination, are found to have a marked effect on the photopolymerization rate and gelation time of polyacrylamide-BIS gels. The sources of materials (1, 8) and photopolymerization techniques (1, 8, 9) have been described. The dye-fade times and the times of firm gel formation were determined for each run. Aliquots (50 ~1) were withdrawn