Anal. Chern. 1984, 56,251-256
25 1
Preparation of Chlorophylls and Pheophytins by Isocratic Liquid Chromatography Tadashi Watanabe,* Akinori Hongu, and Kenichi Honda Department of Synthetic Chemistry, Faculty of Engineering, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan
Masataka Nakazato, Mitsuo Konno, and Sadao Saitoh Research and Development Division, Nampo Pharmaceutical Co., Ltd., 1-6, Nihonbashi-honcho, Chuo-ku, Tokyo 103, Japan
Isocratlc high-performance liquid chromatography (HPLC) wlth silica gel as a stationary phase provides a powerful means for rapld preparative Isolation (on a 20-50 mg level) of chlorophylls ( a , a’, b , and b’) and pheophytlns ( a , a’, b , and b’). The purity and Identity of the Isolated pigments have been conflrrned by complete elemental analyses and analytlcal HPLC; the purity levels were 99.9, 99.5, 99.5, 99.4, 95, 91, 99.5, and 85% for chlorophyll a , a’, b , and b’ and pheophytln a , a’, b , and b’, respectively, wlth the sole lmpurities being almost totally the corresponding epimers. UVvisible spectrometric data (In dlethyl ether, acetone, and benzene) and CD spectra (In benzene) of the purified plgments are presented.
Green organelles of higher plants contain, among others, chlorophyll (Chl) a and b, and also Mg-free pheophytin (Pheo) a (1). These play a central role in the primary stage of photosynthesis, the details of which still remain to be clarified. It is well-known that heat or solvent treatments of these pigments cause slight modifications of the molecules giving, in total, eight structurally similar derivatives: Chla, a’, b, and b’, and the corresponding Pheos, as illustrated in Figure 1. The a and a’form (or b and b’form) are called C10-epimers to each other. Although their distinction is practically impossible by absorption spectrometry they behave quite differently with respect to optical activity (2) and intermolecular aggregation behaviors (3). Thus, not only for unambiguous detection of plant pigments and their alteration products (4) but also for reliable in vitro characterization ( 5 ) of these important, occasionally interconvertible and rather degradable pigments, it is essential to establish a rapid and effective procedure for their isolation. Until the mid-19709, preparative scale isolation and microanalyses of these compounds have relied largely upon column chromatography and thin-layer chromatography (TLC), respectively (6, 7). In general, these procedures are not effective in separating the four Pheos. Further, due to a long operation time and occasional exposure of the pigments to air, these methods are liable to lead to experimental artifacts. In recent years the HPLC technique has been applied to the separation of plant pigments (4,8-18), though mostly on a microanalytical scale (4,10,11,13-18). In many cases the reversed-phase mode, where Cg- or CIs-alkyl coated silica gel serves as a stationary phase, has been employed (4,8, 9,12, 13,15-18). In such a system, solvent programming is usually required for simultaneous separation of Chls and Pheos (4, 9,13,16). Moreover it is probable that colorless long-chain aliphatic compounds, eluted together with the pigments,
contaminate the latter to necessitate further purification in preparative scale isolation. Iriyama and co-workers (10, 14) and Stransky (11) used uncoated silica gel as a stationary phase and obtained good microanalytical results. Even a simultaneous separation of Chls and Pheos appears possible by an isocratic procedure (10, 14). In view of this, we attempted in the present work to apply the silica-based HPLC to preparative isolation of the eight Chl derivatives. Despite an objection raised by Braumann and Grimme (16) that the use of “reactive” silica gel would lead to erroneous results, we were able to prepare very pure pigments, as revealed by elemental analyses, analytical HPLC, and spectroscopic measurements. This is the first example where the rapidity and simplicity of the HPLC technique have been successfully combined for medium-scale preparation of Chl derivatives. The purity of Chla thus prepared was much higher than that of the best Chla samples commercially available. On this basis the UV-visible spectrometric parameters for the purified pigments have been reinvestigated in three organic solvents.
EXPERIMENTAL SECTION Extraction and Partial Purification of Chla + b Mixture. The procedure described by Iriyama et al. (14) was employed with some modification. For pigment extraction, 100 g of lyophilized and powdered Chlorella was blended with 250 mL of methanol for 2 h and filtered, and the residue washed with an additional 250 mL of methanol. Fifty milliliters of dioxane was added to the filtrate + wash solution and then distilled water (50 mL) was added dropwise until the formation of a dark green precipitate. The suspension was allowed to stand for 1 h in a refrigerator, the mixture was filtered, and the solid material was washed with a methanol/dioxane/water mixture (1O:l:l by volume) until the wash solution was colorless. The solid was drained by squeezing it between filter papers and redissolved in acetone (200 mL) plus dioxane (20 mL). Forty milliliters of distilled water was dropwise added under stirring to give a dark green precipitate. The solid, obtained by filtering this suspension, was washed with an acetone/dioxane/water mixture (5:l:l by volume) and drained between filter papers; this was followed by drying in a vacuum desiccator at room temperature. Then the solid was put in nhexane, crushed into fine particles with a spatula, and washed until the supernatant solution lost the yellow color. The solid was separated by filtration and redissolved in diethyl ether (200 mL) plus hexane (100 mL), and the solution was transferred to a 500-mL separatory funnel. The solution was washed four to five times with 80% methanol and then three times with distilled water. After treatment with anhydrous Na2S04,the diethyl ether + hexane layer was evaporated on a warm water bath under a stream of nitrogen. A final drying of the residue in a vacuum desiccator gave 1.1g of a crude mixture consisting predominantly of Chla + b. It took about 8 h to complete this partial purification. Partial Epimerization. The crude Chla + b mixture (1.1g) thus obtained was redissolved in 200 mL of chloroform. After the solution was allowed to stand for 2 h in a refrigerator, the
0003-2700/84/0356-0251$01.50/0 0 1984 American Chemical Society
252
ANALYTICAL CHEMISTRY, VOL. 56, NO. 2, FEBRUARY 1984 R‘
F] C02CH3
b’
CHO CHO
C02CH3 H C02CH3
Structure and part of carbon numbering of the four Chls. Replacement of Mg2+ by two protons gives the corresponding Pheos. Figure 1.
solvent was evaporated on a warm water bath under nitrogen atmosphere. This treatment promoted partial epimerization (Chla ---* a’, Chlb --+ b ? without noticeable side reactions. Preparative HPLC Separation of Chla, a’, b , and b’. The HPLC apparatus consisted of a Model SP-800-9 high-pressure pump (Nihon Seimitsu Kikai), a Model UVILOG-7 variable wavelength detector (Oyo Bunko Kiki) set at 430 nm, and a Model 7125 sample injector (Rheodyne, USA) equipped with a 5mL loop. A stainless steel column (250 mm X 20 mm i.d.) was packed, by the laboratory of Senshu Scientific, Ltd., at a packing pressure of 250 kg/cm2, with a 5 - ~ m silica gel (Nucleosil 50-5, Macherey-Nagel, FRG). About 3 mL of 2-propanol/hexane (3:97, v/v) containing 20 to 50 mg of the mixture of Chla, a’, b, and b’, prepared by the above procedure, was injected and eluted isocratically with the same solvent. Each pigment fraction was collected, the solvent evaporated immediately in a rotary evaporator, and the solid stored at 0 “C. This procedure was repeated several times until each solid Chl weighed 40-50 mg. Finally, the sample was subjected three times to the same HPLC to obtain extra pure Chla, a’, b, and b‘. Pheophytinization. Twenty milligrams of purified Chla was dissolved in 20 mL of diethyl ether. A stream of N2, containing gaseous HCl by passing through a concentrated HCl solution, was bubbled into the ether solution at a rate of 100 mL/min. After 1min the solution was transferred to a 100-mL separatory funnel, and washed with distilled water until the aqueous phase was neutral. The ether layer was treated with anhydrous Na2S04and evaporated in a rotary evaporator to obtain Pheoa in a solid state. The same procedure was applied to Chla’, b, and b’to give respective pheophytins. HPLC Purification of Pheoa, a’, b , and b’. Since the pheophytinization treatment promotes partial epimerization as well as formation of trace amounts of side products (as verified by analytical HPLC), it was necessary to further purify each Pheo pigment. To this end, 20 mg of each Pheo was subjected at least twice to the preparative HPLC described above, to obtain purified Pheoa, a’, b, and b’. Elemental Analysis. The complete elemental analyses of the eight purified derivatives have been conducted at the Analytische Laboratorien (Engelskichen, FRG). The samples had been handled in inert atmosphere under darkness and dried at 80 “C for 3 h at 0.1 mmHg just before analysis. Analytical HPLC. Analytical HPLC was carried out for routine examination of the purity of Chls and Pheos. Here the column size was 150 mm X 4.6 mm i.d., the loop size was 20 pL, the eluent was 2-propanol/hexane with the content of the former ranging from 1 to 2% (v/v), and the flow rate was usually 1 mL/min. Other conditions were the same as in the preparative HPLC mentioned above.
,
40
,
30
I
20 Time / min
1
I
10
0
Flgure 2. HPLC separation of a Chl mixture (20 mg) after the partial epimerization treatment. Pressure was 38 kg/cm2. See text for other details of operation.
UV-Visible Spectra Measurements. A microcomputeraided, double monochromator type spectrophotometer Model UVIDEC-650 (JASCO), with a wavelength resolution of 0.3 nm and a minimum absorbance reading of IO-*, was used for the spectrometric characterization of the purified compounds in ethyl ether, acetone, and benzene. The sample concentration was around 5 X lo4 M. CD Spectra Measurements. An automatic spectropolarimeter Model 5-20 (JASCO) was used for circular dichroism (CD) measurements of the eight pigments. Benzene was chosen as the solvent, in view of the sufficiently slow interconversion between epimeric species in this medium (3). This limited the shorter wavelength end to 270 nm, up to which the spectra were recorded from 700 nm at a scan rate of 10 nm/min. The sample was contained in a stoppered 1-cm fused silica cell at a concentration of (5-7) X lo4 M. Solvents. The solvents used for analytical HPLC were reagent grade. In measurements of UV-visible absorption spectra and CD spectra, spectrograde solvents (Merck) were used without further purification. In all other operations, analytical grade solvents were used after distillation. RESULTS AND DISCUSSION Efficiency of the Preparative HPLC. Figure 2 shows a typical HPLC chart for a mixture (20 mg) of the four Chls after the partial epimerization treatment. At this stage, a tentative assignment of each peak was made by observing a change in the peak height through a shift of the detection wavelength and also by the coincidence of retention times of Chla’ and b’ with those of Chla’ and b’ prepared according to Hynninen et al. (3). This assignment is to be substantiated further by elemental analysis and UV-visible spectrometry. Note that the peak separation is excellent, and the elution time of 40 min, or the eluent volume of 400 mL, is sufficient for complete separation of the four pigments. A 5-mL sample containing 50 mg of the Chl mixture showed a similar separation behavior, but with a slightly enhanced overlap between Chla and b’bands. The Chl mixture before the partial epimerization treatment showed a to ‘/,-fold lower content in the primed derivatives than that in Figure 2. Purity of the Isolated Pigments. The purity of each of the eight derivatives thus isolated was estimated by complete elemental analyses (Table I) and analytical HPLC (Figure 3). Except for Pheob and b’which exhibit a 0.547% deviation from theory for some elements, the analytical data are consistent with the anhydrous form of pigment within the nominal accuracy of measurement (&0.2%,as specified by the analyst). Hence the drying condition employed here (80 “C, 0.1 mmHg, and 3 h) appears sufficient for nearly complete removal of water of crystallization, though Strain et al. (19) recommended a more extensive drying (100 “C, 0.001 mmHg, and 1h). The
ANALYTICAL CHEMISTRY, VOL. 56, NO. 2, FEBRUARY 1984
253
Table I. Analytical Data for the Eight Chl Derivativesa elemental analyses, % found (calcd) compd (formula)
a
mol wt
C
H
N
893.503 893.503 907.486 907.486 871.213 871.213 885.197 885.197
73.86 (73.93 74.03 (73.93 72.77 (72.80 72.82 (72.80 75.87 (75.83 75.86 (75.83 74.18 (74.63 75.30 (74.63
8.01 (8.12) 8.21 (8.12) 7.80 (7.77) 7.87 (7.77) 8.41 (8.56) 8.49 (8.56) 8.36 (8.20) 8.59 (8.20)
6.13 (6.27) 6.15 (6.27) 6.22 (6.17) 6.11 (6.17) 6.40 (6.43) 6.38 (6.43) 5.92 (6.33) 5.83 (6.33)
0 9.11 (8.95) 8.83 (8.95) 10.42 (10.58) 10.39 (10.58) 9.32 (9.18) 9.26 (9.18) 11.54 (10.84) 10.28 (10.84)
Mg
2.88 2.78 2.79 2.81
(2.72) (2.72) (2.68) (2.68)
The samples were dried at 80 "C for 3 h at 0.1 mmHg just before analysis. Pheophytins
H
5 min
Chlorophylls
572
Flgure 3. Analytical HPLC charts for purified Chls and Pheos. 2Propanol content (v/v) in the eluent was 2 % for Chls and 1.5% for Pheos. See text for other details of operation.
present finding argues against the statement of Fong and Koester (20) that even the driest Chla contains an equimolar amount of water. In the calculationsof molar absorptivity (E),several workers (2,21,22) assumed that Chls were in a monohydrated state at the time of weighing. Our results indicate that this assumption would have led to higher values o f t if the specimen had been properly dried before weighing. The influence of the drying condition on water removal was examined by analyzing the four Chls for C, H, and N after drying them under a less rigorous condition (60 OC, 1mmHg, and 3 h). Least-squares treatments of the analytical data gave x = 0.98,0.09,0.36, and 0.47 for Chla, a', b, and b', respectively, when each molecule was expressed as (anhydrous formula).xH20. Thus, we can conclude that the Chl derivatives, isolated by the present HPLC procedure and dried at 80 "C and 0.1 mmHg for 3 h, are substantially pure in the sense of atomic composition. Elemental analysis alone does not assure the isomeric (epimeric) purity. Figure 3 shows the results of analytical HPLC for the eight purified compounds. The purity levels of 99.9, 99.5, 99.5, 99.4, 95, 91, 99.5, and 85% have been attained with Chla, a', b, and b'and Pheoa, a', b, and b', respectively. It is seen that practically the sole impurities are the corresponding epimers, with the exception of Chlb'and Pheoa'which contain trace amounts (0.2%or below) of Pheob and Chla, respectively. According to Scholtz and Ballschmiter (231, contact of Chla with methanol, as in our pretreatment,
leads to the formation of allomerized products. The latter are eluted between Chla and b (16). We suppose that a very small peak seen at 30 min in Figure 2, showing a wavelength dependence similar to that of Chla, might correspond to such alteration products. However, these compounds can be easily removed by successive, repeated preparative HPLC purifications with solvents other than methanol, and hence do not appear in the chromatograms of Figure 3. The absence of UV-absorbing contaminants has been verified by recording the HPLC charts also at 254 nm. The relatively low levels of purity observed with Pheos come mainly from the proximity in elution peaks of the two epimeric species. In effect, we were able to obtain Pheos with higher purity by further repeating the HPLC purification procedure. Another factor may be an easier interconversion between epimeric forms of Pheos in comparison with Chls, as suggested by the results of Hynninen and Sievers (2) on Pheoa'. The remarkably high purity (99.9 %) of Chla thus prepared was compared with the purities of commercially available Chla samples (samples 1 and 2 being two lots purchased from a manufacturer, and sample 3 from another manufacturer). The latter are all "guaranteed reagents" and were subjected to the present analytical HPLC just on opening the sealed ampules. Sample 1was found to be 98.9% pure, containing Chla'(0.9%) and Pheoa (0.2%) as impurities. Sample 2 gave a somewhat poorer result; the Chla purity was 97.90%, with Chla'(1.57%), Chlb'(0.28%), Pheob' (0.14%), a carotene (0.07%), and Pheoa (0.04%) being the impurities. The purity of sample 3 was estimated to be lower than 98%, since it contained Chla'(2%) as a main impurity and minor amounts of two unidentifiable compounds. The molar absorptivities at 430 nm of all the components have been taken into account in the calculation of these percentages. It is important to note that the four Chla samples (Chla isolated by the present procedure, and samples 1-3) gave essentially only one spot in thin-layer chromatography (TLC) on silica or sucrose. This demonstrates that TLC, which has long been recommended as a final means of purity check of photosynthetic pigments (7), is not sufficiently effective to separate 1% or lower levels of homologues from the main body of Chla. For the purpose of demonstrating the separation performance of the present analytical HPLC procedure, mixtures of the isolated Chls and/or Pheos were subjected to the HPLC under identical conditions. Fhe results are illustrated in Figure 4. It is seen that the eight, structurally similar compounds can be determined very rapidly though there occurs an accidental overlap between Chla'and Pheoa. As far as only the four Chls are concerned, their separation can be completed within 5 min by using a higher flow rate or with a shorter column. UV-Visible Spectrometry. The UV-visible absorption characteristics of the primed derivatives were found to be identical, within experimental error, with those of the nonprimed ones. Table I1 summarizes the peak wavelengths,A( f0.3 nm), the molar absorptivities at the red peaks [€-(red)],
254
ANALYTICAL CHEMISTRY, VOL. 56, NO. 2, FEBRUARY 1984
Table 11. Spectrometric Parameters for Chla and b , and solvent diethyl ether hmaX(red)/nm hmax (blue)/nm emax(red)x 10-4/M-1cm-’, A( blue)/A(red) acetone
hm,(red)/nm h ( blue)/ nm emax(red)x 10-4/M-1cm-’ a A (blue)/A(red)
benzene
a
I
Xma,(red )/nm h m, (blue)/nm emax(red)x 10-4/M-1cm-l A(blue)/A (red) Average of four or more independent measurements, Pheophytins
Mixture
,
1 1
I/
Chlorophylls
Pheoo and b at Room Temperature (ca. 20 “C) Chla Chlb Pheoa 660.3 641.9 667.9 428.4 451.9 408.4 8.98 i. 0.07 5.67 i. 0.02 5.26 t 0.10 1.285 2.813 2.036
Pheob 654.6 432.7 3.48 i 0.02 4.934
661.6 429.8 8.13 t 0.05 1.230
665.9 409.2 4.60 t 0.03 2.251
653.3 433.8 2.93 t 0.02 5.230
671.6 665.4 646.2 414.8 432.5 457.9 5.31 i 0.06 7.97 -f 0.05 5.62 t 0.05 2.039 1.272 2.698 using ca. 1 0 mg of sample in each.
656.7 439.5 3.23 t 0.02 4.718
l
644.6 455.8 4.76 i 0.03 2.867
Table 111. Equations for the Determination of Chlorophylls and Pheophytins in Two Solvents, [a] = h,A, - k,Ab [b] = k 3 A b - k,A, mixture Chla t b Pheoa + b Chla t b Pheoa t b
Time /min
Figure 4. Analytical HPLC charts for a mixture of four Chls, a mixture of four Pheos, and a mixture of the eight compounds. 2-Propanol content (v/v) in the eluent was 1.4%.
and the blue/red absorbance ratios [A(blue)/A(red)] for the four pigments in three organic solvents. The absolute values of cmax(red)obtained in the present work are compared with those reported previously, in view of their particular importance for spectrometric characterization of the pigments. The literature values of emax (in IO4 M-l cm-l), arising from original determinations, are as follows: Chla in diethyl ether [6.61 (24),8.63 (19),8.68 (21),8.76 (25), 8.85 (26),9.02 (271, and 9.12 (28)],in acetone [5.1 (24) and 7.88 (25)],and in benzene [5.47 (24)]; Chlb in diethyl ether [5.10 (26),5.15 (28),5.21 (24),5.61 (19),5.63 (25,27),and 5.78 (21)]and in acetone [4.2 (24)]; Pheoa in diethyl ether [5.40 (26)]; and Pheob in diethyl ether [3.63 (2611. The values of Trurnitt and Colmano (24)are evidently too small, probably due to the presence of impurities. The emax vaues reported by Sauer e t al. (21) were based on a formula Chl-H20 and hence should be reduced by a factor of 0.98 if their samples had been properly dried (see above). Seely and Jensen (29) obtained 8.51 X lo4 as €,,(red) of Chla in diethyl ether and 7.82 X lo4in benzene, on the assumption that emax for Chla be equal to that for ethyl chlorophyllide a in acetone, 7.66 X lo4 (30). This brief review shows that our emax values are among the highest ones seen in the literature and, with respect to Chla and b in diethyl ether, are closer to the values of Smith and Benitez (27) reported in 1955 than to the most frequently cited values of Strain et al. (19) in 1963. Vernon (31) used the values of Smith and Benitez to obtain 8.27 X lo4 (Chla) and 4.86 x lo4 (Chlb) as the eman values in acetone, which are again close to ours. Table I11 shows the spectroscopic equations, derived from the present measurements, for determining Chla and b or Pheoa and b, in their binary mixtures in acetone and diethyl ether. Svec (7) summarizes such equations reported by 1966, which are somewhat different from the present results; for instance, the previous values of k l , k2, k S ,and k4 for Chla + b in acetone are 9.78, 0.99, 21.40, and 4.65, respectively.
solvent acetone acetone diethyl ether diethyl ether
ki
k,
k3
11.35 21.21 10.02 17.38
1.90 5.87 0.54 2.95
19.69 33.84 16.12 26.70
k,
3.73 13.11 2.08 7.41
a [a] = concentration of a species (mg/L), [b] = concentration of b species (mg/L), A, = absorbance (path length 1 cm) at the red peak of a species, Ab = absorbance (path length 1cm) at the red peak of b species.
CD Spectra. The CD and absorption spectra of the eight derivatives in benzene are illustrated in Figure 5. The epimeric purities, before dissolution in benzene, of Pheoa and Pheob samples submitted to these measurements were >99% and ca. 93%, respectively, while the other six compounds were of the same purity as those in Figure 3. It is immediately noted in Figure 5 that, for a given pair of epimers, the CD spectra are considerably different, although the absorption spectra are practically identical with each other. T o our knowledge, only three reports are available in the literature concerning CD of monomeric Chl species studied here: Chla in diethyl ether and 0.5% ethanol-CC1, and Pheoa in diethyl ether (32),Chls a, a’, b, and b’in diethyl ether and methanol (33),and Chls a and a’and Pheos a and a’in T H F (2). In what follows we compare the present CD data with the previously published results, mainly in the context of sample purity. For simplicity, the comparison is limited to the three sets of electronic transitions in the visible range, namely, the Qy, Q,, and Soret absorption bands. Here the notations are taken from Weiss (34),and the axes of transition moments are depicted in Figure 1. (1) The Qr Transition Region. The so-called “red absorption peak” of Chl derivatives corresponds to the Qy(O,O) transition, and the nearest satellite is assigned to the Qy(l,O) band (34,35). For each of the four primed species, an intense negative CD is associated with Qy(O,O) and a well-defined negative satellite with Qy(l,O). On the other hand, the ellipticity of the nonprimed species is absent (Chlb and Pheoa) or weakly negative (Chla and Pheob) at these transitions. It is most probable that the latter weak ellipticities result mainly from the coexistence of primed species as impurities. These results are in sharp contrast with the literature (32,33),where significantly negative CD peaks are reported for nonprimed derivatives. Visual inspection suggests that, unless the optical activity of the molecules depends crucially on the solvent, Chla and b samples used in those works contained a fair amount (at least 20%) of primed species, due to rapid epimeric in-
ANALYTICAL CHEMISTRY, VOL. 56, NO. 2, FEBRUARY 1984 1
I
I
*15u
c
,
I
A Chl-g 8 g’
+20E‘
I
-
*20
-
*15t
I
I
255
I
B Chl-b 8
434
\ I I 700
w 300
Wavelength / n m
400
500
600
700
600
700
Wavelength / nm
-‘ 1
h
11
*15
*10
11CD
b’
/I
w 300
400
500
Wavelength / nm
Flgure 5. UV-visible circular dichroism (CD) and absorption (Abs) spectra for (A) Chla and a’, (B) Chlb and b’, (C) Pheoa and a’, and (D) Pheob and b’ in benzene. [e] and e denote the molar ellipticity and molar absorptivity, respectively.
terconversion (3)in such nucleophilic solvents as diethyl ether (32,33),ethanol (32),and methanol (33). These findings could in turn cast some doubt on the validity of detailed discussions concerning the electronic states of Chl derivatives (32,34,35) based partly on the previous CD data. (2) The QxTransition Region. A series of Q, transitions occur in the “valley” of the absorption spectrum (34,35). The Q,(O,O)absorption peaks appear a t 580,539, and 560 nm for Chla, Pheoa, and Pheob, respectively, with well-resolved Q,( 1,0)satellites to the shorter wavelengths. The nonprimed derivatives exhibit almost no optical activity in this region, except for Pheoa which gives a very weak negative peak at Q,(O,O). In contrast, the primed derivatives show fairly intense CD, but here the sign is opposite to that for Qytransitions described above. Similar observations have been reported for Chla’in diethyl ether (33) and Pheoa’in THF (2). The optical activity of Chlb’as well as Chlb is exceptionally weak in the Q, transition region; this may be related to the very diffuse feature of their optical absorption spectrum. (3) The Soret Band Region. According to the results of molecular orbital calculations (34,35),the Soret absorption peak consists of two nearly degenerate electronic transitions, B,(O,O) and B,(O,O). All of the four primed derivatives give single, strongly positive CD spectra at this absorption peak, suggesting that the two transitions contribute to CD spectra
in a similar manner. In contrast, the CD spectra of nonprimed species apparently reflect the existence of the two transitions: they show a maximum and a minimum at wavelengths separated by 70-100 meV in energy, with the center wavelength roughly coinciding with the Soret absorption maximum. Through the absorption peak from longer to shorter wavelength, Chla alone gives a minimum-to-maximum change in CD spectra, while the other three nonprimed derivatives give maximum-to-minimum changes. None of the previous works on CD spectra of the nonprimed species ( 2 , 3 2 , 3 3 )was able to demonstrate such a maximum-minimum crossover in the Soret absorption band; in all cases a close agreement was noted between the CD maximum wavelength and the absorption peak wavelength. In view of the extremely high CD intensity of the primed species, this is not surprising if t h e Chl samples used in those works contained, as estimated above, >20% primed derivatives as impurities. Even the “chromatographic purity” of Chla or b sample before preparation of a diethyl ether solution (33)would not assure their epimeric purity after dissolution in such a nucleophilic solvent ( 3 ) .
CONCLUSIONS It is evident that any degradation of Chl derivatives, induced by adsorption to a rather reactive adsorbent (silica gel) during the HPLC procedure, can be minimized by use of a
258
Anal. Chem. 1984, 56,256-262
short operation time. The results of UV-visible spectrometry on purified pigments reveal lack of purity control in many of the published works. In particular, we have shown that not only the sample purification but also the proper choice of a solvent (benzene), causing little epimeric interconversion (3), is essential for obtaining reliable CD data.
ACKNOWLEDGMENT The authors are grateful to K. Iriyama, The Jikei University School of Medicine, for valuable comments on Chl separation. Registry No. Chla, 479-61-8;Chla', 22309-13-3;Cub, 519-62-0; Chlb ', 22309-14-4; Pheoa, 603-17-8; Pheoa', 75598-38-8; Pheob, 3147-18-0; Pheob', 75498-61-2; benzene, 71-43-2; diethyl ether, 60-29-7; acetone, 67-64-1. LITERATURE CITED (1) Kllmov, V. V.; Dolan, Ed.; Shaw, E. R.; Ke, 8. Proc. Nati. Acad. Sci. U.S.A. 1980, 77, 7227-7231. (2) Hynnlnen, P. H.; Slevers, G. Z. Nafurforsch., 6: Anorg. Chem., Org. Chem. 1981, 366, 1000-1009. (3) Hynninen, P. H.; Wasielewski, M. R.; Katz, J. J. Acta Chem. Scand., Ser. 6 1979, 633, 837-6413. (4) Schwartz, S. J.; Woo, S. L.; von Elbe, J. H. J . Agric. Food Chem. 1981.29, 533-535. (5) Seely, G. R. I n "Primary Processes of Photosynthesis"; Barber, J., Ed.; Elsevier: Amsterdam, 1977; pp 1-53. (6) Strain, H. H.; Svec, W. A. I n "The Chiorophylls"; Vernon, L. P., Seely, G. R., Eds.; Academic Press: New York, 1966; pp 21-66. (7) Svec. W. A. I n "The Porphyrins"; Dolphin, D., Ed.: Academic Press: New York, 1978; Vol. V, pp 341-399. (8) Eskins, K.; Scholfield, C. R.; Dutton, H. J. J. Chromatogr. 1977, 735, 217-220. (9) Shoaf, W. T. J . Chromatogr. 1978, 752, 247-249. (10) Irlyama, K.; Yoshlura, M.; Shiraki, M. J . Chromatogr. 1978, 754, 302-305. (11) Stransky, H. 2.Nafurforsch., C: 6iosci. 1978, 33C, 836-840.
(12) Schoch, S. 2.Naturforsch., C: 6iosci. 1978, 33C, 712-714. (13) Braumann, T.; Grimme, L. H. J. Chromatogr. 1979, 770, 264-268. (14) Iriyama, K.; Shikraki, M.; Yoshlura, M. J. Li9. Chromatogr. 1979, 2 , 255-276. (15) Falkowski, P. G.; Sucher, J. J . Chromatogr. 1981, 273, 349-351. (16) Braumann, T.; Grimme, L. H. Biochim. Biophys. Acta 1981. 637, 8-17. (17) Shioi, Y.; Fukae, R.; Sasa, T. 6iochim. Biophys. Acta 1963, 722, 72-79. (18) Shioi, Y.; Sasa, T. Biochim. Blophys. Acta 198S, 756, 127-131. (19) Strain, H. H.; Thomas, M. R.; Katz, J. J. Biochim. Biophys. Acta 1963, 75, 306-311. (20) Fong, F. K.; Koester, V. J. 6iochim. Biophys. Acta 1976, 423, 52-64. (21) Sauer, K.;Llndsay Smlth, J. R.; Schultz, A. J. J. Am. Chem. SOC. 1966, 88, 2681-2688. (22) Hynnlnen, P. H. Z. Nafurforsch., 6: Anorg. Chem., Org. Chem. 1981, 368, 1010-1016. (23) Scholtz, B.; Ballschmiter, K. J . Chromatogr. 1981, 208, 148-155. (24) Trurnltt, H. J.; Colmano, G. 6iochim. 6lophys. Act8 1959, 37, 434-447. (25) Jeffrey, S.W.; Humphrey, G. F. Blochem. Physioi. Pf/anz. 1975, 767, 191-194. (26) Goedheer, J. C. I n "The Chlorophylls"; Vernon, L. P., Seely, G. R., Eds.; Academic Press: New York, 1966; pp 147-184. (27) Smith, J. H. C.; Benitez, A. I n "Modern Methods of Plant Analysis"; Peach, K., Tracey, M., Eds.; Springer: Heldelberg, 1955; Vol. 4, pp 142-1 96. (28) Zscheile, F. P.; Comar, C. L. Bot. Gaz. 1941, 702, 463. (29) Seely, G. R.; Jensen, R. G. Spectrochim. Acta 1985, 21, 1835-1845. (30) Holt, A. S.; Jacobs, E. E. Am. J . Bot. 1954, 4 7 , 710. (31) Vernon, L. P. Anal. Chem. 1960, 32, 1144-1150. (32) Houssier, C.; S a w , K. J . Am. Chem. SOC. 1970, 02,779-791. (33) Prokhorenko, I.R.; Lobachev, V. M.;Kutyurin, V. M. Zh. Obshch. Khim. 1976 46, 2147-2151. (34) Weiss, C. I n "The Porphyrins": Dolphin, D., Ed.; Academic Press: New York, 1978; Vol. 111, pp 211-223. (35) Petke, J. D.; Maggiora, G. M.; Shipman, L.; Christoffersen, R. E. Photochem. Phofobioi. 1979, 30, 203-223.
RECEIVED for
review August 3, 1983. Accepted October 3,
1983.
Characterization of Reversed-Phase Liquid Chromatography Columns with Retention Indexes of Standards Based on an Alkyl Aryl Ketone Scale Roger M. Smith Department of Chemistry, University of Technology, Loughborough, Leicestershire LE11 3TU, United Kingdom
The advantages of using a retention Index scale based on the alkyl aryl ketones for reporting retentlons in HPLC are dlscussed and a method is presented to characterize the varlations in the retentlon propertles of reversed-phase liquid chromatography column-eluent combinatlons by using the retentlon Indexes of a set of reference compounds, toluene, nitrobenzene, p-cresol, and 2-phenylethanoi. These compounds were selected, by using multlvarlant analysls, to glve the optimum discrlminatlon between eluents and columns. The method Is compared with alternative methods using capacity factors and selectivity ratios and enables the specific column lnteractlons to be studied.
The rapid spread of high-performance liquid chromatography has largely been due to the availability of stable alkyl-bonded reversed-phase column materials. These materials are prepared by a number of manufacturers (l), but each may use different bonding reactions for the alkyl groups, different
shapes and porosities of silica, and different reactions to cap unreacted silanol groups (2). As a consequence nominally identical columns (e.g., ODs-silicas) from different sources may give markedly different retentions (3-5). Even with the same method, there are batch to batch variations, which together with unannounced changes in manufacturing methods have given rise to concern about column reproducibility (6). These differences between columns also make interlaboratory comparisons difficult and thus limit the use of HPLC techniques in Official and Standards methods as well as hindering studies of the mechanism of retention. A range of techniques have been used to compare different bonded phases, including pyrolysis-GLC (7), but most methods are based on the reversed-phase separation of a test mixture. The capacity factors (iz? or selectivities (a)are then used for manufacturers' quality control (8-10) or to compare different materials (lI,12), although as discussed later many of these tests are inappropriate and may be misleading. More fundamental studies have used nitrobenzene in hexane to test for unreacted silanols (13) or determined the physical pa-
0 1984 American Chemical Society 0003-2700/84/0356-0256$01.50/0
