amount of sample delivered by the valve and it also was well within +2%. The limit of detection of water can be improved by using a valve which delivers a larger sample. AS indicated in Figure 1, carbon dioxide is also determinable. Carbon dioxide may be present per se in nitrogen tetroxide or may be formed in the analytical procedure by oxidation of dissolved or suspended organic matter. Since ethylene dichloride is freely soluble in nitrogen tetroxide, it was used as a calibrant for the carbon dioxide peak; samples of nitrogen tetroxide containing dissolved ethylene dichloride gave proportionally increased carbon dioxide and water peaks (stoichiometric). Perhaps ethylene dichloride may be useful as a calibrant for both carbon and hydrogen because it offers a weight advantage over the direct introduction of carbon dioxide or water. The halogen content of the ethylene dichloride does not appear in the gas effluent from the hot copper; apparently, it forms copper(1) chloride and remains in the column or condenses in the line leading to the chromatograph. The shape of the nitrogen peak is symmetrical as long as there is sufficient copper in the column packing t o reduce nitrogen tetroxide; when the copper is spent, nitric oxide will be formed and the nitrogen peak in the chromatogram appears unsymmetrical because nitric oxide has a longer retention time than nitrogen and is not cleanly resolved. The
column packing can be regenerated by repeated injections of small portions of methanol while the column furnace is maintained at 400 "C to 500 "C. During regeneration, the water peak is monitored; when it reaches a low, constant value, regeneration is complete. Alternatively, the combustion tube can be repacked with fresh copper. A calibration plot for water was also obtained with methanol-water mixtures that had been analyzed for their water content by the Karl Fischer procedure. The calibration plot obtained in this way (by-passing the furnace) was in all ways identical with the one obtained from mixtures of water and nitrogen tetroxide. A methanol-water mixture may be used for a periodic check of the calibration of the chromatographic system, once it is established that the operating conditions of a given copper-copper oxide column are such that the water content of a calibrated mixture of nitrogen tetroxide as indicated by the gas chromatograph record is the same as that obtained from a methanol-water mixture. RECEIVED for review June 30, 1967. Accepted October 31, 1968. This work was performed for the Jet Propulsion Laboratory, California Institute of Technology, sponsored by the National Aeronautics and Space Administration under Contract NAS7-100.
Analysis of Tea Flavanols by Gas Chromatography of Their Tr imethylsilyl Derivatives Albert R. Pierce, Harold N. Graham, Seymour Glassner, Howard Madlin,' and Jorge G. Gonzalez Thomas J. Lipton, h e . , 800 Sylvan Ave., Englewood CIifs, N.J. 07632 An accurate quantitative gas chromatographic method for the determination of tea flavanols as their trimethylsilyl ethers is described. Two sets of isothermal conditions are used to separate the derivatives on an 8-foot glass column packed with 3% OV-1 supported on 60-80 mesh Gas Chrom Q. The method is convenient and accuracy and reproducibility are good. Pure TMS ether calibration standards were not commercially available nor could they be successfully prepared. The need for pure standards was overcome through the unique use of a gas density balance detector to determine directly the exact yield of the derivatization reaction. THEORGANOLEPTIC properties of green tea (consumed mostly in Japan and China) are partially determined by the quantities and proportions of the various flavanols present: catechin (CAT), epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECG), and epigallocatechin gallate (EGCG) ( I ) . These flavanols are enzymatically oxidized during the fermentation of green leaf to black tea to produce the normal tea of commerce for this country and most of the tea drinking world. This black tea is manufactured by a process that crushes the green tea leaf which breaks the vacuoles and thereby causes the flavanols to come into contact with the tea polyphenolase enzyme. The resulting oxidations bring into
Present address, Foster D. Snell, Inc., 29 West 15th Street, New York, N. Y. (1) C . R. Harler, "Tea Manufacturer," Oxford University Press, London, 1963,2-3. 298
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existence the characteristic black tea properties which are partially dependent on the original flavanol content (2, 3). The enzymic reactions are terminated by heat treatment when the process is considered to be complete. Leaf to be manufactured as green tea is made enzymatically inactive before maceration, thereby preventing most flavanol oxidation. Paper and column chromatographic methods for the separation and quantitative analysis of these chemicals have been reported (4-13). The methods are time consuming and require very carefully controlled conditions in order to obtain reproducible results. The flavanols cannot be directly de(2) E. A. H. Roberts, J . Sci. Food Agr., 9, 381 (1958). (3) G. W. Sanderson, Tea Quart., 36, 172 (1965). (4) A. E. Brandfield, M. Penny, and W. B. Wright, J . Chem. SOC., 1947, 32. (.5 .) R. A. Cartwright - and E. A. H. Roberts, Chem. Znd. (London), 1954, 1389. 16) ~,K. M. Dzhemukhadze and G. A. Shalneva, Biokhimiya, 20, 336 (1955). (7) A. L. Kursanov and M. N. Zaprometov, ibid., 14,466 (1949); Chem. Abstr.. 44. 9786 (1950). (8) E. A. H. Roberts and D. J. Woods, Biochem. J., 53, 332 (1953). (9) M. S . Shipalov, M. A. Bokuchava, and G. A. Soboleva, Biokhimiya, 23, 390 (1958); Chem. Absfr.,52, 17359e (1958). (10) M. N. Zaprometov, Fiziol. Rust., 5, 46 (1958); Chem. Abstr., 53, 131f(1959). (11) M. Nakagawa and H. Torii, Agr. Biol. Chem. (Tokyo), 28, (3), 160 (1964). (12) M. Nakagawa and H. Torii, ibid., 28, (8), 497 (1964). (13) L. Vuataz, H. Brandenberger, and R. H. Egli, J . Chromatogr., 2, 173(1959).
termined by GLC because of their low vapor pressures. The gas chromatographic analysis of trimethylsilyl derivatives of naturally occurring chemicals with low vapor pressures, such as carbohydrates, steroids, amino acids, and polyphenols has been examined by many researchers (14-18). A. A. Bondarovich et al. (14) reported fair resolution of an artificial mixture of the TMS derivatives of tea polyphenols by GLC. Waiss, Lundin, and Stern (18) studied the TMS ethers of some flavanoid compounds, including catechin, by NMR. The majority of the reported work on polyphenols has been based on the silylation procedure of Langer, Connell, and Wender (16), namely the reaction with hexamethyldisilazane (HMDS) and trimethylchlorosilane (TMCS). Klebe, Finkbeiner, and White (15) reported that bis-trimethylsilylacetamide (BSA) is a superior silylating agent. Our work corroborated Klebe's findings and, consequently, BSA was incorporated into a procedure for the determination of tea flavanols. This paper describes the extraction, derivatization, and gas chromatographic techniques involved in the analysis. A unique method of obviating the need for calibration standards by utilizing a gas density balance detector is also described. EXPERIMEhTAL
Apparatus. The gas chromatograph was a Barber Colman series 5000 Dual Column Chromatograph with dual hydrogen flame ionization detectors used in the dual column, dual detector mode. The recorder was a Moseley, Model 1701. Two 8-foot X 4-mm U-shaped glass columns were packed with 17 grams 3 % OV-1 on 60-80 mesh Gas Chrom Q, (packing prepared by Applied Science Laboratories, Inc.). The glass columns were washed with methanol and dried with a stream of filtered air. Column material was packed with vibration and tapping. The packed columns were conditioned overnight at 300 "C. A Gow-Mac Gas Density Balance Detector (GDB) Model No. 373 4-W-2 was installed in a Barber Colman Model 5002 detector bath and this module was utilized with a 2-foot X 0.25-inch stainless steel column packed with 3P;: OV-1 on 60-80 mesh Gas Chrom Q in a Barber Colman Model 5076 column oven. The GDB was powered by a Gow Mac 40-05-C power supply. Reagents. Karl Fischer grade pyridine was obtained from Kodak. Triphenylbenzene and coronene were obtained from the Aldrich Chemical Co. and exhibited single peaks by GLC. Bis-trimethylsilylacetamide (BSA) silylating reagent was purchased from the Aldrich Chemical Co. Catechin and epicatechin are commercially available and were purchased from the Mann Research Laboratories, Inc. Epigallocatechin, epicatechin gallate, and epigallocatechin gallate were isolated from tea extracts in our tea research laboratory. Procedure. PREPARATION OF DERIVATIVES. TMS derivatives were prepared by the method of Klebe, Finkbeiner, and White (15). A 5-10 mg portion of the sample was dissolved in 2 mi of pyridine in a 5-ml flask; 2 ml of BSA were added, the flask was stoppered tightly and the contents were allowed to react for 15 min at room temperature. The reaction mixture could then be injected directly into the gas chromatograph. (14) H. A . Bondarovich, A. S. Giammarino, J. A. Renner, F. W. Shephard, A. J. Shingler, and M. A. Gianturco, J. Agr. Food Chem., 15, 36 (1967). (15) J. F. Klebe, H. Finkbeiner, and D. M. White, J. Amer. Chem. Soc.. 88. 3390 (1966). (16) S.' H.'Langer, S.'Connell, and I. Wender, J. Org. Chem., 23, 50 (1958). (17) C. C.' Sweeley, R. Bentley, M. Makita, and W. W. Wells, J . Amer. Chem. SOC.,85,2497 (1963). (18) A. C. Waiss, R. E. Lundin, and D. J. Stern, Tetrahedron Lerr., 1964, 513.
CHROMATOGRAPHIC CONDITIONS.A complete analysis of the flavanols requires two sets of isothermal conditions. The TMS derivatives of CAT, EC, and EGC were separated at a column temperature of 250 "C. The detector block and injection port were both at 340 "C. Flow rates were as follows: Helium, 200 cc/min (inlet pressure 40 psi); hydrogen, 70 cc/min; and air, 400 cc/min. A second set of conditions was required for the separation of the TMS derivatives of ECG and EGCG. For this separation the temperatures of the column bath, detector block, and injection port were 300, 340, and 340 "C, respectively. Helium flow rate in the measuring column was 187 cc/min. Hydrogen, air, and helium flow rates for the compensating column were the same as those described for the lower temperature operation. Injected sample aliquots were approximately 2 pl in volume, adjusted appropriately to produce on scale peaks at an amplifier attenuation of 3 x 10-l2 ampere with a 5-mV recorder. The GDB detector was used only to determine the efficiency of the derivatization reactions. The temperatures of the column bath, detector block, and injection port were 260, 260, and 340 "C, respectively. Nitrogen flow in the measuring column was 37 cc/min and in the reference side of the detector 150 cc/min. The injection of 20 p1 sample aliquots produced full scale response with no amplifier attenuation on the 1-mV recorder. CALIBRATION AND ANALYSIS.Calibration curves were plotted from prepared mixtures of the flavanols and internal standards. The flame ionization detector was used for all determinations. Triphenylbenzene was used as the internal standard for the analysis of CAT, EC, and EGC at 250 "C. Coronene was used as an internal standard for the analysis of ECG and EGCG at 300 "C. Two appropriate sets of mixtures were prepared in which the concentration of each component was varied within the range expected in tea. At least three different mixtures for each temperature level were prepared and injections made in triplicate. Peak areas were calculated from the peak heights and peak widths at half height. The calibration curves were prepared by plotting the area ratio (flavanol/int. standard) cs. weight ratio (flavanol/int. standard) for each flavanol. The plots were based on the weight of the pure flavanols and not on the TMS derivatives. Fresh or fresh frozen tea leaf was mixed with granulated dry ice (the dry ice volume being 3-4 times that of the tea) and passed once through a Micro-Pulverizer. This produced a very fine powder of frozen tea leaf and dry ice. The dry ice was quickly removed (in 10 minutes) with a stream of nitrogen and a 12-gram sample (approximately 22z tea solids) was weighed into a 125-ml glass-stoppered Erlenmeyer flask. The sample was extracted with 50 ml of pyridine for 20 minutes on a mechanical shaker, and then poured through a Whatman No. 90 filter paper into a 100-ml round bottom flask. Approximately 50 ml of pyridine were used to wash the filter and residue. The filtrate was then taken to dryness under vacuum on a Rotovapor evaporator in a water bath at 40 "C. All solvent and water must be removed; this takes about 15 minutes. The residue was then taken up in exactly 50 ml of pyridine and 2 Drierite chips were added. A 2-ml aliquot of this liquid was placed in a 5-ml glass stoppered flask, 2 ml of BSA were added, and the reaction was allowed to proceed for 15 minutes at room temperature. To analyze dried tea, 3 grams of leaf were weighed into a 125-ml Erlenmeyer flask and extracted with 50 ml of pyridine and 5 ml of water at room temperature for 15 minutes on a mechanical shaker. The water is added to effect extraction of the flavanols from the dried tea. After extraction, sample preparation is the same as for fresh frozen tea. Triphenylbenzene and coronene were added quantitatively to the derivatized sample. The weights of internal standards were approximately 1.5 mg in each case. Four microliters of the sample were then injected into the gas chromatograph. The exact amount of sample injected was not important, provided the peaks did not vary drastically in size from those obtained VOL. 41,NO. 2, FEBRUARY 1969
299
EGC
65.m YIHUTES
L
5
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Figure 2. Gas chromatogram with the Gas Density Balance Detector showing quantitative derivatization of flavanols 5
10
15
20
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Figure 1. Gas chromatograms of TMS derivatives of flavanols found in tea ( A ) at 250 "C ( B ) at300 "C 1. Epicatechin 4. Epicatechin gallate 2. Catechin 5. Epigallocatechin gallate 3. Epigallocatechin
in developing calibration data--i.e., 1/q--3/4 of full scale at 3 X 10-12 A. The 300 'C run was usually made first. Duplicate analyses were made on each sample. Results were reported as per cent pure flavanol in a moisture free sample. RESULTS AND DISCUSSION
I t has been found that the TMS derivative of the flavanols can be determined by GLC on low loaded columns. While hexamethyldisilazane (HMDS) and trimethylchlorosilane (TMCS) could be used for derivatization, BSA was adopted because of the advantages of increased reactivity and absence of interfering precipitates. N,O-Bis-trimethylsilyl-trifluoroacetamide (BSTFA) was also tested and produced results similar to that of BSA for this application. Klebe, Finkbeiner, and White (15) suggested that even though both silyl groups of the BSA can be exchanged in cases where equilibrium position favors transfer, as with alcohols, carboxylic acids, and amines, it is more convenient to use a full mole of BSA per exchangeable proton. It is not necessary to remove the monosilylacetamide for gas chromatographic analysis. The phenolic hydroxyl groups are rapidly silylated at room temperature. No acids or bases are involved and no difficult-to-remove by-products are formed. A column packed with 3% SE-30 on 60-80 mesh Gas Chrom Q gave only a fair separation of the flavanol-TMS ethers. Catechin and epicatechin TMS ethers could not be separated satisfactorily for quantitative analysis. A column of OV-1 on 60-80 mesh Gas Chrom Q, however, afforded good separation of the flavanol-TMS ethers (Figure 1). OV-1 is a silicone rubber compound with a higher molecular weight
3z
300
ANALYTICAL CHEMISTRY
Prepared sample 34.7% EC EGC 65.3% and greater polarity than SE-30. Irreversible absorption occurred on a column of OV-17 on Gas Chrom Q. Glass columns were arbitrarily selected. An experiment with a stainless steel column showed no apparent detrimental effects. Quantitative programmed temperature gas chromatography (PTGC) was studied extensively in order to speed up the analysis time. Reproducible, linear calibration data were obtained for a single component, catechin, when chromatographed with an internal standard, However, when known mixtures of the flavanols were chromatographed against an internal standard, the data was neither repeatable nor linear, although great care was taken to control all instrumental and procedural parameters. PTGC was therefore restricted to use as a semiquantitative screening technique. Proof of Derivatization Efficiency. Calibration curves are usually plotted with data obtained from prepared mixtures of pure standards. The TMS derivatives of the flavanols are not commercially available, nor can they be readily isolated from derivatization reaction mixtures. The efficiency of derivatization of pure flavanols was determined so that reaction mixtures could be used for calibration. Quantitative silylation of the flavanols was substantiated in several ways. GLC studies to determine the optimum reaction time for silylation showed that after 15 minutes no additional derivative was formed, nor did application of low heat produce any further silylation. Only in the case of catechin was it possible to isolate the pure TMS derivative for use as a calibration standard. A calibration curve was established using this pure catechin-TMS with triphenylbenzene as an internal standard. Samples of catechin were then derivatized and the response relative to triphenylbenzene compared to the calibration data. The per cent silylation was 95.5% with a standard deviation of 1.71. The infrared spectrum of the catechin derivative showed three bands associated with the trimethylsilyl group at 8.0, 11.9, and 13.2 microns which are not present in the spectrum
of pure catechin. The absence of absorption due to hydroxyl groups was also noted. The completeness of the derivatization reaction was confirmed through gas chromatography using a gas density balance detector (Gow-Mac Instrument Co., Madison, N. J.) The G D B detector (19, 20) has not received wide acceptance in the instrumental laboratory even though its invention (21) almost coincided with that of gas chromatography. It has, however, the unique characteristic of yielding quantitative data without calibration, a valuable asset for determining the extent of a reaction when the products cannot be isolated. The use of the detector could become increasingly significant in the field of derivative gas chromatography where the commercial availability of standards generally lags behind the requirements of the latest technology. Conditions were established using a Gow-Mac Model 373-4 W2 Gas Density Balance Detector and a 2-foot 3% OV-1 column for the separation of two mixtures, one containing triphenylbenzene and catechin and the other containing epicatechin and epigallocatechin. EGC was selected because experimental derivatization indicated that it required the longest reaction time for complete derivatization, probably due to the sterically hindered hydroxyl group at the 3 position. The flavanols were derivatized with BSA. Twenty microliters of the reaction mixtures were injected onto the column and the concentration, in weight per cent, was calculated (Figure 2). The concentration is determined by multiplying each peak area by a corresponding area factor in order to obtain a corrected peak area:
where :
M , = the molecular weight of the component M g = the molecular weight of the carrier gas Each corrected peak area, when divided by the sum of all the corrected areas and multiplied by 100, gives the weight per cent of the component. In Table I, concentrations determined with the GDB detector are compared to the theoretical concentrations calculated from the starting weights, assuming TMS derivatization of all the hydroxyl groups. The experimental results agree closely with the theoretical, indicating that derivatization is essentially quantitative. The application of the GDB detector to this type of problem was successful in spite of the unusually high operating temperature for this type of detector. Comparable work with this detector on high molecular weight compounds has not been reported. The sensitivity of the GDB is inversely proportional to the temperature and directly proportional to the difference between the molecular weights of the carrier gas and each component. The great difference between the molecular weights of the flavanol derivatives (Mol Wt EGCTMS = 946) and nitrogen was sufficient to offset the effect of the detector cell temperature (260 "C) which is high for sensitive GDB operation. The high detector temperature does have one advantage in that the detector is more stable at high temperatures. Optimal flow rate adjustment also contributed to the suc(19) C. L. Guillemin and Mlle. F. Auricourt, J. Gas Chromatogr., 1, (lo), 24 (1963). (20) C. L.. Guillernin and Mlle. F. Auricourt, ibid., 2, 156 (1964).
I
5
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EGCG 13.0%
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15
20
Figure 3. Gas chromatograms of typical analysis of fresh green tea leaf
Table I. Results of GDB Analysis of Known Mixtures Calculated Mixture Theoretical % GDB) F 1. Triphenylbenzene 28.7 21.5 EGC-TMS 71.3 12.5 2. EC-TMS 53.2 55.2 EGC-TMS 46.8 44.8 3. EC-TMS 34.7 35.5 EGC-TMS 65.3 64.5 4. Triphenylbenzene 36.8 38.0 63.2 62.0 EGC-TMS Average of two injections. 0
Table 11. Results of Recovery Experiments EC theoretical, % Sample EC added, mg rng EC found, rng Recovery 20.4 100.2 20.33 A 10.16 20.7 96.3 B 12.15 21.54 21.1 95.0 C 12.02 22.19 20.4 102.8 D 9.63 19.80 22.6 96.3 E 13.33 23.50 Average 98.1 Standard deviation, 3.27 ECG ECG theoretical, ECG % Sample added, mg mg found, mg Recovery 56.5 99.1 A 25.46 57.09 43.25 98.8 B 12.15 43.78 40.0 97.5 41.03 C 9.40 52.56 50.7 96.6 D 20.93 61.56 62.5 101.6 E 29.93 Average 98.6 Standard deviation, 1.91
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Sample Reference No. description 1. Experimental tea= (this paper) (13) 2. Ceylon tea leaf (12) 3. Japanese tea leaf. 4. Japanese green tea (this paper) (11) 5. Japanese green tea a Represents average of several sets of data.
Table 111. Comparison of Methods Per cent EGCG ECG EGC 2.96 3.64 10.04 2.75 2.72 10.55 3.3 4.3 10.1 7.3 2.1 3.5 6.1 2.0 2.3
EC
CAT
1.20 0.63 1.6
0.24 0.35 ... 0.2
1.o
0.7
...
Total 18.90 16.90 19.2 14.1 11.1
cessful detection. The greater the difference between the measuring and reference gas flow rates, the greater will be the sensitivity of the GDB detector (21-23). Evaluation of Method. The extraction procedure was evaluated by performing a series of recovery experiments. Samples of fired green tea, previously analyzed for flavanols, were fortified with known amounts of epicatechin and epicatechin gallate. An average recovery of 98.3% was obtained as shown in Table 11.
The flavanol content of several fresh samples of experimentally grown tea was determined by this method. The results reported in Table I11 indicate that the method compares favorably with those reported in the literature. As expected, different flavanol concentrations are observed for green tea and fresh leaf samples. The first three samples were fresh leaf and should be compared separately from samples 4 and 5 which are fired green tea. The chromatograms of a typical two stage analysis of fresh green tea are shown in Figure 3.
(21) A. J. P. Martin and A. T. James, Biochem. J., 63, 138 (1963). (22) A. G. Nerheim, ANAL.CHEM., 35, 1640 (1963). (23) J. T. Walsh and D. M. Rosie, J . Gas Chromatogr., 5, 232 (1967).
RECEIVED for review August 22, 1968. Accepted November 6, 1968. Paper presented to the Division of Analytical Chemistry 153rd Meeting, ACS, Miami Beach, Fla., April 1967.
General Considerations Concerning Atmospheric Aerosol Monitoring with the Hydrogen Flame Ionization Detector R. Wayne Ohline,' Edwin ThallY2and Ping Hwat O e y Department of Chemistry, New Mexico Institute of Mining and Technology, Socorro, N. M. 87801 The primary nonorganic constituent of the atmosphere giving a response with the hydrogen flame ionization detector is sodium. Consequently, it appears that continuous, nonattended operation of the detector for organic aerosol monitoring can be achieved if ion pulses arising from sodium-containing particles can be subtracted from the total charge delivered in the pulse mode. To understand the response characteristics for the detector to sodium salts, the rate of ion formation has been studied, and first-order rate constants for the ionization of sodium in five small hydrogen diffusion flames have been determined. Rate data obta-ined from these studies indicate that the detector can detect particles of sodium chloride 0.1 p in diameter. THEPOTENTIALITIES of the hydrogen flame ionization method for the detection and sizing of aerosols have been realized for some time (1-3). However, an instrument which will monitor an actual polluted atmosphere has not yet been perfected. Our work indicates that sodium-containing aerosols are responsible for most of the charge delivered to the detector in
(1) R. W. Ohline, ANAL.CHEM., 37, 93 (1965). (2) H. Frostling and P. H. Lindren, J . Gas Chromatogr., 4, 243 (1966). (3) W. L. Crider and A. A. Strong, Rec. Sei. Instrum., 38, 1772 (1967). 302
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pulse form. Hence, if charge pulses arising from sodiumcontaining aerosols can be subtracted from the total charge delivered in pulse form, a detector useful for atmospheric organic aerosol monitoring may result. Our approach has been based largely on the work of Lawton and Weinberg (4,who demonstrated that useful rates of ionization data can be obtained in flames by measuring the charge yield under saturation conditions (i.e., where the potential on the collecting electrodes is not so small that ions recombine before collection, or so large that ion multiplication occurs). Their approach is clarified by examination of a current-potential curve for the flame ionization phenomena (Figure 1). In region a, recombination and escape of ions from the collecting region are competing with the ion-collecting ability of the field. In region b (the saturation region), increase in potential produces no charge yield increase. Hence, all ions are being collected and, therefore, one must conclude that a rate process is being observed. Lawton and Weinberg demonstrated that plots of the logAuthor to whom inquiries should be directed. Present address, Institute of Polymer Science, The University of Akron, Akron, Ohio. (4) J. Lawton and F. J. Weinberg, Proc. Roy. Soc., Ser. A , 277,
468 (1964).
