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Semiquantitative paper chromatographic separation of chlorophylls a and b
ANALYTICAL
BIOCHEMISTRY
101,
Semiquantitative
103-106
(1980)
Paper Chromatographic of Chlorophylls a and 6 S. BURKE
Department
AND
S.
ARONOFF
of Chemistry, Simon Fraser British Columbia, V5A 1%. Received
Separation
University, Canada
Burnaby,
June 25, 1979
[14C]Chlorophyll (chl) a has been utilized to demonstrate the contamination of chl b by (probably) oxidation products of chl a in thin-layer or paper chromatography. By circular chromatography of both chlorophylls as their pheophytins, the contamination of chl a (as pheophytin a) in chi b (as pheophytin b) may be reduced to 0.15-0.35.
While numerous methodologies exist for quantitative determination of chlorophylls (chls)’ a and b in solution, e.g., by absorption spectrophotometry or flourimetry, physical isolation is required for the determination of individual specific activities of radiochlorophylls. Previous investigators concerned with this problem showed that repeated paper chromatography of the chlorophylls did not provide analytically pure compounds (1). Other investigators have claimed radiochemical purity in paper chromatographed compounds on the basis of constancy of specific activity of the chlorophylls and their derivatives (2). If, however, both chlorophylls are radioactive initially, the degree of intercontamination is difficult to assess, especially in the presence of overlapping oxidation products. Our study of the problem involved the use of radiochemically pure [14C]chl a* and ’ Abbreviations used: chl(s), chlorophyll(s); tic, thinlayer chromatography; Solvent 1, hexanes: diethyl ether: n-propanol (70:30:0.5): Solvent 2, hexanes: diisopropyl ether:n-propanol (70:30:0.5); Solvent 3. petroleum ether (30/60):acetone:i-propanol(90:7.5:0.45). * Although initially radiochemically pure by our criteria (see Materials and Methods), chl a during storage in sealed glass vials evacuated to ~10~’ Torr had degraded at times by as much as 20% prior to use, as shown by the subsequent chromatography. 103
nonradioactive
chl 6. It concentrated
on
(a) development of an optimal separation system using circular chromatography; (b) investigation of possible sources of cross contamination, i.e., contamination of the chlorophylls with each other; and (c) employment of derivatization (pheophytinization) and chromatographic procedures to overcome cross-contamination problems. MATERIALS
AND METHODS
Chlorophylls a and b. Chlorophylls a and b were obtained from dandelion leaves in the immediate vicinity. The chlorophylls were prepared by standard procedures (4). The occasional use of Siliclad (Clay Adams, Inc.) coating (1% followed by extensive water washing) appeared to decrease capillary transport along the wall of the chromatographic column. Pigments were dried for 24-28 h on a high-vacuum line at 510-l Torr and stored in sealed glass ampoules. [‘4ClChlorophyl/ a. The blue-green alga, Anacystis nidufans, obtained from the American Type Culture Collection, was maintained on their No. 616 medium. It produces only chl a ; chl b is not detectable even in trace quantities. 0003-2697/80/010103-04$02.00/O Copyright All rights
0 1980 by Academic Press, Inc. of reproduction in any form reserved.
104
BURKE
AND
A culture of A. nidulans was fed 14COZ, generated from Ba14C0, (New England Nuclear, 5 mCi/mmol), until >99.5% uptake occurred. The culture was then permitted to grow an additional 24-30 h and the [14C]chl a was isolated as above. Under these conditions the [14C]chl a comprised 13-15% of the 80% acetone extract. The several preparations used had a specific activity range of 20,000-40,000 cpm/mg. Radiochemically pure [14C]chl a, as used in these studies, is defined as chl a: (a) synthesized by an organism genetically devoid of chl b, such as the blue-green alga; (b) chromatographed at least twice on sucrose columns; (c) devoid of all other pigments, as determined by visual analysis of cellulose tic (3) of the final product from (b), and (d) having a blue/red absorbancy ratio of absorption bands, supporting evidence for the absence of carotenoids (2). Spectrophotometry. Spectra were determined using a Cary 17; analysis employed the extinction coefficients of Hoffman and Werner (4). Radioactivity measurements. Radioactivity was measured by the counting of planchets using a proportional end-window multiplanchet counter (Nuclear Chicago, Model No. 4342). The efficiency for 14C was
ARONOFF
12-14%. Single counts were made for 100 min, resulting in a background of 10.4 2 0.97 cpm at the 99% confidence level. Circular chromatography. During sample application O2 was controlled, in part, by flooding the atmosphere of the loading region with NP, resulting in a measured 0, content of less than 2%. Circular chromatography was performed on Whatman No. 3 (40-cm-diameter) paper disks by means of the device shown in Fig. 1. In this apparatus the rate of addition of developer was controlled by the size of the wick, the pressure of which on the chromatography paper was adjustable. Developers and solvents. Three different developers were tested: Solvent 1, (5) hexanes:diethyl ether:n-propanol (70:30:0.5); Solvent 2, hexanes:diisopropyl ether:n-propanol (70:30:0.5); and Solvent 3, (3) petroleum ether (30/60):acetone:i-propanol (90: 7.5:0.45). Hexanes and petroleum ether were reagent grade; acetone was spectral grade. The alkyl ethers were freed of peroxides immediately prior to use (6). Pheophytinization. The chlorophylls were converted to their pheophytins by the addition of 1% oxalic acid (w/v) in acetone in the dark at room temperature for 20 min. Under these conditions, chl a is completely pheophytinized and chl b almost completely. By the use of 1% acetic acid under similar
I
ENLARGED VIEW TEFLON WAFER
‘GLASS
SUPPORT
OF
TANK
FIG. 1. Radial chromatography apparatus. The rate of development is regulated by the crosssectional area of the cotton or wool wicks and by the contact area of the wick with the chromatogram. The chromatogram is readily visible through the translucent Teflon sheets.
SEMIQUANTITATIVE
CHLOROPHYLL
circumstances but with an abbreviated time, e.g., 5 min, it is possible to convert chl Q to its pheophytin with virtually no effect on chl6. While the acetic acid procedure resulted in the gross separation of chl 6 from pheophytin a, the method was clouded by the overlap or near-overlap of degradation products of chl a with chl h to the extent of 2-3% of total chl a activity. In the oxalic acid procedure, the resulting pheophytins were diluted with an equal volume of 1% aqueous CaCl,, transferred to petroleum ether, washed three times with water, dried with anhydrous Na,SO,, further dried on a high-vacuum line (room temperature) for 2 days, and stored in vclcuo (10-l Torr). RESULTS
AND DISCUSSION
Circular chromatography of radioactive chl a and nonradioactive crude chlorophyll extracts (that is, extracted pigments not purified by column chromatography) were compared using the three different solvent systems. The optimal loading capacity for this paper was 1 mg of chlorophyll. Development proceeded until the pheophytin u bands had almost reached the paper’s edge, at which time gross and visibly clear zones separated chls CI and b; furthermore, chls a and h were separated clearly from their parent compounds, as were the carotenoids. Activity in the chls b + b’ bands relative to the chl a were: Solvent 1, 2.02.5%; Solvent 2,2.0-2.5%/o; Solvent 3, -3%. Chromatography of only chl a with Solvent 1 resulted in appreciable tailing of the bands, attributed to peroxide formation in the diethyl ether. Solvent 2, containing diisopropyl ether and known to form peroxides more rapidly than ethyl ether, showed enhanced tailing, while tailing with Solvent 3 was appreciably reduced compared to that with Solvent 1. Thus, Solvent 3 was considered the optimal developing solvent of these three. A further parameter in this choice was the lower volatility of the polar
DETERMINATION
105
component of the eluant (i.e., the propanol vis-&vis the ether). A concomitant concern, perhaps more pronounced in the chromatography of the purified pigment than in the crude pigment extract, is that of oxidation of the chlorophylls, especially chl LI, and the near-coincidence of the chromatographic affinity of one of the chl a oxidation products with that of chl b (4). These oxidation products, when present, are prominent components of the radioactivity found in the chl b band and are absent or in much lower quantity in the crude pigment extracts containing natural antioxidants. Had this radioactivity been due strictly to tailing of the chl a band, then a loss of chl a by pheophytinization would have decreased activity in the chl b region. When neat chl a is pheophytinized, the radioactivity in the chl b region actually increases, suggesting that tailing is not the only and possibly not the major reason for contamination (see Fig. 2). The use of Teflon sheeting above and below the chromatography paper was conceived as an attempt to minimize oxidation during development. While this technique provided only marginal success, a synergistic effect encouraged its use, namely, that development time was diminished appreciably (from 8-12 to 3-4 h). It was not possible to store small quantities of the purified chlorophylls for several weeks or longer without change. Pigments were stored in vucuo following 48 h of roomtemperature drying at 510-l Torr. As seen in Fig. 2b, our methodology appears inadequate to prevent some degree of degradation, e.g., allomerization to purpurins. Thus, chl a dried as above yielded 3-4% of its activity in such compounds following 1 to 2 weeks of storage. A further 12% was found as pheophytin a and another 2.53.0% remained at the origin. The same sample, exposed to air an additional 12 min, resulted in increased activity at the origin without appreciable increase in pheophytin a or in the ailomerized region (Fig. 2a).
106
BURKE AND ARONOFF
0 Ocm -
28
! 56
184
1------1
7890 Ocm16 50 PomOn Of plgmenl bands
11.2
140
cage 16.8 o'kve'
I
134150168
FIG. 2. Sectoral analysis of radial chromatograms. Sectors were cut with arbitrary annuli of dimensions shown in the abscissa. The percentage of total activity found in each sector is given above each sector. The position of chl b is indicated as lying within the broken lines. Both chl regions include the primed (a’ and b’) compounds-which are separated clearly on the chromatogram. (b) Distribution of activity following chromatography of [‘%]chl a using Solvent 3. (a) Same as (b) but applied pigment allowed to stand in air 12 min prior to chromatography. (c) Same as (b) but chl a converted to its pheophytin prior to chromatography.
Differential pheophytinization of chls a and b is performed readily, the rate constant for the former being approximately an order of magnitude larger (7). While visual chromatography, following treatment of chls a and b with 1% acetic acid in acetone, appeared to result in only two pigmentswere sepachl b and pheophytin u-which rated widely on the chromatogram, the use of [‘4C]chl a showed that such treatment also resulted in an increase of allomerization products. Consequently, it was decided to separate both pigments as pheophytins. In the following experiment, 1% oxalic acid in acetone was utilized for pheophytinization, this acid being much stronger than acetic and its action correspondingly more
rapid. As shown in Fig. 2c, under our conditions 85% of the neat chl a treated with 1% oxalic acid in acetone appeared as pheophytin a, 4.5% as residual chl a, and 5.5% as allomerized or other degraded pigment not found at the origin, with 5.3% degraded chlorophyll remaining at the origin. Under these same conditions, when using [14C]chl a added to the nonradioactive crude chlorophyll extract, it is possible to separate pheophytin b from pheophytin a, with the former containing between 0.15 and 0.35% of the activity originating from chl a. A typical protocol therefore involved two steps: (a) crude pigments from a leaf extract were mixed with [14C]chl a and chromatographed with Solvent 3; (b) the chl b band from this chromatogram was mixed with an approximately equal amount of nonradioactive chl a, and the mixture was pheophytinized and circularchromatographed with Solvent 3. Pheophytin b, so obtained, had a radiochemical purity of 99.65-99.85%. Stated conversely, the contamination due to chl a was 0.35-0.15%. ACKNOWLEDGMENTS We wish to thank our head machinist, Mr. Frank Wick, and our head glassblower, Mr. Peter Hatch, for their skills in fabrication of the radial chromatography apparatus. This research was supported, in part, by a grant from the National Research Council of Canada.
REFERENCES 1. Shlyk, A. A., Rotfarb, R. M., and ‘Lyachnovich, Ya. P. (1975) Bull. Znsr. Biol., 115-120 (in Russian). 2. Chan, A. S. K., Ellsworth, R. K., Perkins, H. J., and Snow, S. E. (1970)J. Chromarogr. 47,395399. 3. Bacon, M. F. (1965)J. Chromatogr. 17,322-326. 4. Svec, W. A. (1978) in The Porphyrins (Dolphin, D., ed.), Vol. C, Academic Press, New York. 5. Ikemori, M., and Arasaki, S. (1977) Bull. Japan. Sot.
Phycol.
25, 58-66.
6. Vogel, A. I. (1956) in Practical Organic Chemistry, 3rd ed., Longmans, Green, London. 7. Joslyn, M. A., and Mackinney, G. (1938) J. Amer. Chem. Sot. 60, 132-136.
BIOCHEMISTRY
101,
Semiquantitative
103-106
(1980)
Paper Chromatographic of Chlorophylls a and 6 S. BURKE
Department
AND
S.
ARONOFF
of Chemistry, Simon Fraser British Columbia, V5A 1%. Received
Separation
University, Canada
Burnaby,
June 25, 1979
[14C]Chlorophyll (chl) a has been utilized to demonstrate the contamination of chl b by (probably) oxidation products of chl a in thin-layer or paper chromatography. By circular chromatography of both chlorophylls as their pheophytins, the contamination of chl a (as pheophytin a) in chi b (as pheophytin b) may be reduced to 0.15-0.35.
While numerous methodologies exist for quantitative determination of chlorophylls (chls)’ a and b in solution, e.g., by absorption spectrophotometry or flourimetry, physical isolation is required for the determination of individual specific activities of radiochlorophylls. Previous investigators concerned with this problem showed that repeated paper chromatography of the chlorophylls did not provide analytically pure compounds (1). Other investigators have claimed radiochemical purity in paper chromatographed compounds on the basis of constancy of specific activity of the chlorophylls and their derivatives (2). If, however, both chlorophylls are radioactive initially, the degree of intercontamination is difficult to assess, especially in the presence of overlapping oxidation products. Our study of the problem involved the use of radiochemically pure [14C]chl a* and ’ Abbreviations used: chl(s), chlorophyll(s); tic, thinlayer chromatography; Solvent 1, hexanes: diethyl ether: n-propanol (70:30:0.5): Solvent 2, hexanes: diisopropyl ether:n-propanol (70:30:0.5); Solvent 3. petroleum ether (30/60):acetone:i-propanol(90:7.5:0.45). * Although initially radiochemically pure by our criteria (see Materials and Methods), chl a during storage in sealed glass vials evacuated to ~10~’ Torr had degraded at times by as much as 20% prior to use, as shown by the subsequent chromatography. 103
nonradioactive
chl 6. It concentrated
on
(a) development of an optimal separation system using circular chromatography; (b) investigation of possible sources of cross contamination, i.e., contamination of the chlorophylls with each other; and (c) employment of derivatization (pheophytinization) and chromatographic procedures to overcome cross-contamination problems. MATERIALS
AND METHODS
Chlorophylls a and b. Chlorophylls a and b were obtained from dandelion leaves in the immediate vicinity. The chlorophylls were prepared by standard procedures (4). The occasional use of Siliclad (Clay Adams, Inc.) coating (1% followed by extensive water washing) appeared to decrease capillary transport along the wall of the chromatographic column. Pigments were dried for 24-28 h on a high-vacuum line at 510-l Torr and stored in sealed glass ampoules. [‘4ClChlorophyl/ a. The blue-green alga, Anacystis nidufans, obtained from the American Type Culture Collection, was maintained on their No. 616 medium. It produces only chl a ; chl b is not detectable even in trace quantities. 0003-2697/80/010103-04$02.00/O Copyright All rights
0 1980 by Academic Press, Inc. of reproduction in any form reserved.
104
BURKE
AND
A culture of A. nidulans was fed 14COZ, generated from Ba14C0, (New England Nuclear, 5 mCi/mmol), until >99.5% uptake occurred. The culture was then permitted to grow an additional 24-30 h and the [14C]chl a was isolated as above. Under these conditions the [14C]chl a comprised 13-15% of the 80% acetone extract. The several preparations used had a specific activity range of 20,000-40,000 cpm/mg. Radiochemically pure [14C]chl a, as used in these studies, is defined as chl a: (a) synthesized by an organism genetically devoid of chl b, such as the blue-green alga; (b) chromatographed at least twice on sucrose columns; (c) devoid of all other pigments, as determined by visual analysis of cellulose tic (3) of the final product from (b), and (d) having a blue/red absorbancy ratio of absorption bands, supporting evidence for the absence of carotenoids (2). Spectrophotometry. Spectra were determined using a Cary 17; analysis employed the extinction coefficients of Hoffman and Werner (4). Radioactivity measurements. Radioactivity was measured by the counting of planchets using a proportional end-window multiplanchet counter (Nuclear Chicago, Model No. 4342). The efficiency for 14C was
ARONOFF
12-14%. Single counts were made for 100 min, resulting in a background of 10.4 2 0.97 cpm at the 99% confidence level. Circular chromatography. During sample application O2 was controlled, in part, by flooding the atmosphere of the loading region with NP, resulting in a measured 0, content of less than 2%. Circular chromatography was performed on Whatman No. 3 (40-cm-diameter) paper disks by means of the device shown in Fig. 1. In this apparatus the rate of addition of developer was controlled by the size of the wick, the pressure of which on the chromatography paper was adjustable. Developers and solvents. Three different developers were tested: Solvent 1, (5) hexanes:diethyl ether:n-propanol (70:30:0.5); Solvent 2, hexanes:diisopropyl ether:n-propanol (70:30:0.5); and Solvent 3, (3) petroleum ether (30/60):acetone:i-propanol (90: 7.5:0.45). Hexanes and petroleum ether were reagent grade; acetone was spectral grade. The alkyl ethers were freed of peroxides immediately prior to use (6). Pheophytinization. The chlorophylls were converted to their pheophytins by the addition of 1% oxalic acid (w/v) in acetone in the dark at room temperature for 20 min. Under these conditions, chl a is completely pheophytinized and chl b almost completely. By the use of 1% acetic acid under similar
I
ENLARGED VIEW TEFLON WAFER
‘GLASS
SUPPORT
OF
TANK
FIG. 1. Radial chromatography apparatus. The rate of development is regulated by the crosssectional area of the cotton or wool wicks and by the contact area of the wick with the chromatogram. The chromatogram is readily visible through the translucent Teflon sheets.
SEMIQUANTITATIVE
CHLOROPHYLL
circumstances but with an abbreviated time, e.g., 5 min, it is possible to convert chl Q to its pheophytin with virtually no effect on chl6. While the acetic acid procedure resulted in the gross separation of chl 6 from pheophytin a, the method was clouded by the overlap or near-overlap of degradation products of chl a with chl h to the extent of 2-3% of total chl a activity. In the oxalic acid procedure, the resulting pheophytins were diluted with an equal volume of 1% aqueous CaCl,, transferred to petroleum ether, washed three times with water, dried with anhydrous Na,SO,, further dried on a high-vacuum line (room temperature) for 2 days, and stored in vclcuo (10-l Torr). RESULTS
AND DISCUSSION
Circular chromatography of radioactive chl a and nonradioactive crude chlorophyll extracts (that is, extracted pigments not purified by column chromatography) were compared using the three different solvent systems. The optimal loading capacity for this paper was 1 mg of chlorophyll. Development proceeded until the pheophytin u bands had almost reached the paper’s edge, at which time gross and visibly clear zones separated chls CI and b; furthermore, chls a and h were separated clearly from their parent compounds, as were the carotenoids. Activity in the chls b + b’ bands relative to the chl a were: Solvent 1, 2.02.5%; Solvent 2,2.0-2.5%/o; Solvent 3, -3%. Chromatography of only chl a with Solvent 1 resulted in appreciable tailing of the bands, attributed to peroxide formation in the diethyl ether. Solvent 2, containing diisopropyl ether and known to form peroxides more rapidly than ethyl ether, showed enhanced tailing, while tailing with Solvent 3 was appreciably reduced compared to that with Solvent 1. Thus, Solvent 3 was considered the optimal developing solvent of these three. A further parameter in this choice was the lower volatility of the polar
DETERMINATION
105
component of the eluant (i.e., the propanol vis-&vis the ether). A concomitant concern, perhaps more pronounced in the chromatography of the purified pigment than in the crude pigment extract, is that of oxidation of the chlorophylls, especially chl LI, and the near-coincidence of the chromatographic affinity of one of the chl a oxidation products with that of chl b (4). These oxidation products, when present, are prominent components of the radioactivity found in the chl b band and are absent or in much lower quantity in the crude pigment extracts containing natural antioxidants. Had this radioactivity been due strictly to tailing of the chl a band, then a loss of chl a by pheophytinization would have decreased activity in the chl b region. When neat chl a is pheophytinized, the radioactivity in the chl b region actually increases, suggesting that tailing is not the only and possibly not the major reason for contamination (see Fig. 2). The use of Teflon sheeting above and below the chromatography paper was conceived as an attempt to minimize oxidation during development. While this technique provided only marginal success, a synergistic effect encouraged its use, namely, that development time was diminished appreciably (from 8-12 to 3-4 h). It was not possible to store small quantities of the purified chlorophylls for several weeks or longer without change. Pigments were stored in vucuo following 48 h of roomtemperature drying at 510-l Torr. As seen in Fig. 2b, our methodology appears inadequate to prevent some degree of degradation, e.g., allomerization to purpurins. Thus, chl a dried as above yielded 3-4% of its activity in such compounds following 1 to 2 weeks of storage. A further 12% was found as pheophytin a and another 2.53.0% remained at the origin. The same sample, exposed to air an additional 12 min, resulted in increased activity at the origin without appreciable increase in pheophytin a or in the ailomerized region (Fig. 2a).
106
BURKE AND ARONOFF
0 Ocm -
28
! 56
184
1------1
7890 Ocm16 50 PomOn Of plgmenl bands
11.2
140
cage 16.8 o'kve'
I
134150168
FIG. 2. Sectoral analysis of radial chromatograms. Sectors were cut with arbitrary annuli of dimensions shown in the abscissa. The percentage of total activity found in each sector is given above each sector. The position of chl b is indicated as lying within the broken lines. Both chl regions include the primed (a’ and b’) compounds-which are separated clearly on the chromatogram. (b) Distribution of activity following chromatography of [‘%]chl a using Solvent 3. (a) Same as (b) but applied pigment allowed to stand in air 12 min prior to chromatography. (c) Same as (b) but chl a converted to its pheophytin prior to chromatography.
Differential pheophytinization of chls a and b is performed readily, the rate constant for the former being approximately an order of magnitude larger (7). While visual chromatography, following treatment of chls a and b with 1% acetic acid in acetone, appeared to result in only two pigmentswere sepachl b and pheophytin u-which rated widely on the chromatogram, the use of [‘4C]chl a showed that such treatment also resulted in an increase of allomerization products. Consequently, it was decided to separate both pigments as pheophytins. In the following experiment, 1% oxalic acid in acetone was utilized for pheophytinization, this acid being much stronger than acetic and its action correspondingly more
rapid. As shown in Fig. 2c, under our conditions 85% of the neat chl a treated with 1% oxalic acid in acetone appeared as pheophytin a, 4.5% as residual chl a, and 5.5% as allomerized or other degraded pigment not found at the origin, with 5.3% degraded chlorophyll remaining at the origin. Under these same conditions, when using [14C]chl a added to the nonradioactive crude chlorophyll extract, it is possible to separate pheophytin b from pheophytin a, with the former containing between 0.15 and 0.35% of the activity originating from chl a. A typical protocol therefore involved two steps: (a) crude pigments from a leaf extract were mixed with [14C]chl a and chromatographed with Solvent 3; (b) the chl b band from this chromatogram was mixed with an approximately equal amount of nonradioactive chl a, and the mixture was pheophytinized and circularchromatographed with Solvent 3. Pheophytin b, so obtained, had a radiochemical purity of 99.65-99.85%. Stated conversely, the contamination due to chl a was 0.35-0.15%. ACKNOWLEDGMENTS We wish to thank our head machinist, Mr. Frank Wick, and our head glassblower, Mr. Peter Hatch, for their skills in fabrication of the radial chromatography apparatus. This research was supported, in part, by a grant from the National Research Council of Canada.
REFERENCES 1. Shlyk, A. A., Rotfarb, R. M., and ‘Lyachnovich, Ya. P. (1975) Bull. Znsr. Biol., 115-120 (in Russian). 2. Chan, A. S. K., Ellsworth, R. K., Perkins, H. J., and Snow, S. E. (1970)J. Chromarogr. 47,395399. 3. Bacon, M. F. (1965)J. Chromatogr. 17,322-326. 4. Svec, W. A. (1978) in The Porphyrins (Dolphin, D., ed.), Vol. C, Academic Press, New York. 5. Ikemori, M., and Arasaki, S. (1977) Bull. Japan. Sot.
Phycol.
25, 58-66.
6. Vogel, A. I. (1956) in Practical Organic Chemistry, 3rd ed., Longmans, Green, London. 7. Joslyn, M. A., and Mackinney, G. (1938) J. Amer. Chem. Sot. 60, 132-136.