JPS60142988A - Separation of chlorophyll and pheophytin - Google Patents

Abstract

PURPOSE:Chlorophylls are subjected to high-speed liquid chromatography using tightly sealed silica gel packed under high pressure as the solid phase to achieve rapid, simple and low-cost separation of high-purity chlorophylls of a, b, a' and b' derivatives. CONSTITUTION:Chlorophylls are subjected to partition high-speed liquid chromatography using silica gel which has been packed under high pressure and tightly sealed as the solid phase and a 3:97 isopropyl-n-hexane mixture as an eluent to effect separation into high-purity chlorophyll a, chlorophyll b, chloropyll a' and chlorophyll b'. Then, individual products are dissolved in ethyl ether, respectively, stirred vigorously together with HCl to separate the lower phase, then solvent is distilled off to give pheophytin a, b, a' and b'.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for separating chlorophylls and pheophytins. More specifically, the present invention relates to a method for separating chlorophylls and pheophytins, which is characterized by separating chlorophylls and pheophytins by high-performance liquid chromatography using high-pressure packed and sealed silica gel as a stationary phase.

It is known that chlorophylls and pheophytins, which have Mg removed from their chemical structure, exist as pigments in the green organs of higher plants.

Conventionally, methods for separating and purifying these pigments from green plants include dioxane treatment, sucrose column chromatography, ion exchange resin column chromatography, and alternating current distribution using a combination of various solvents. The law is known.

These dyes undergo slight molecular modification by air, heat, and solvents, resulting in the production of structurally similar derivatives.

Therefore, among the chlorophylls separated and purified by conventional methods, chlorophyll a (hereinafter referred to as
Ch7. a), chlorophyll b (hereinafter referred to as chz,
b), these isomers, chlorophyll a'
(hereinafter referred to as Ch, 4.a'), rioo fill b'
(hereinafter referred to as Cht, b'), pyrochlorophyll a, pyrochlorophyll b, pheophytin a (
(hereinafter referred to as pheo, a), pheophytin b (hereinafter referred to as PbO2, b), pheophytin a/ (hereinafter referred to as Pheo, a'), pheophytin b' (hereinafter referred to as Pheo, b'), pyrophytin It is known that pheophytin a, pyropheophytin b1, pheophobide a1, pheophobide b, and various other derivatives exist.

We have established a rapid and effective method for isolating each of these derivatives, not only to ensure reliable detection of chlorophyll and its modified products, but also to conduct reliable in vitro characterization of the extremely unstable chlorophyll. It has been essential to prepare samples that are of essential importance.

Conventionally used methods for preparing reliable research samples include heptadose column chromatography and ion exchange resin column chromatography. These methods can be applied on a laboratory scale (several milligrams) and can be applied to Cb4 in chlorophyll. This is an example in which only a, chz, and b are roughly separated, and the reality is that various types of the aforementioned rust conductor compounds are mixed in each separated sample. This is due to the drawback of conventional column chromatography, which has a low separation ability, and also because it requires a long time to operate, making it extremely unstable, meaning that rolophyll is pointlessly exposed to the air or solvent atmosphere. This is due to the production of various lucidic conductors that are modified products, and it is currently extremely difficult to obtain pure samples.

It is known that the use of highly reactive silica gel to separate chlorophyll causes molecular denaturation, making the results completely unreliable (Brauma
n, Grime). The present inventor also attempted column chromatography using the conventional natural dripping method using silica gel as a packing material, but as a result, the operation required a long time, meaninglessly increasing the contact between chlorophyll and silica gel, resulting in the decomposition of chlorophyll. This resulted in the following.

The present inventor has developed highly pure chlorophyll derivatives, Cb4. a, Cht, b, chz, a
', cbz, b' were successfully obtained. Therefore, the present invention provides a method for separating chlorophylls and isolating various highly pure chlorophyll derivatives by high performance liquid chromatography using silica gel packed and sealed under high pressure as a stationary phase. According to this method, pheophytins are purified as Pheo, a 1 Pheo, b,
It can also be isolated as Pheo, a', pheo, b'.

The present inventor has successfully removed chlorophyll hydrolysis products (chlorophyll, phephobide, and other water-soluble pigments) that are insoluble in nonpolar solvents mainly used in normal-phase silica gel columns. 5 hours aimed at
By using ephaaex LH-20 column chromatography as a pretreatment method for injection into a silica gel column, we can produce high-purity chlorophyll and its derivatives on a medium scale (tens of milligrams, or grams depending on the size of the column). succeeded in. In other words, some of the chlorophyll mixture separated and purified by conventional methods is hydrolyzed, which has no solubility at all in the developing solvent, which is mainly a nonpolar solvent used in silica gel normal phase columns. product or other water-soluble dyes are mixed in. When such dyes are injected into a silica gel column, they are deposited on the silica gel and do not easily elute. Not only does it take a long time to clean the column, but it also accelerates column deterioration, significantly shortening its lifespan, and creating a huge economic burden. Be strong. Alternatively, the reality is that washing the column frequently with a polar solvent significantly reduces the separation ability of the column, resulting in a very high cost, so the inventors of the present invention, when using a normal phase silica gel column, 5 pigments unsuitable for its use
We have developed a separation method using a 5 ephadex column and a silica gel column, in which the residue is separated and removed by ephadex LH-20 column chromatography and then injected into silica gel HPLC. Part 5ephad
Compared to using a sucrose column instead of an ex column, it is easier to fill the column uniformly, wash it, and regenerate it.
The 5ephadex column is so simple that it cannot be compared with a sucrose column. The conventional drawback of using silica gel to separate chlorophyll, which causes deterioration, has been solved by dramatically shortening the operation time using the method of the present invention, which uses a high-pressure packed and sealed column and applies the HPTJC method. .

In addition to the above-mentioned separation method, the present inventor further developed an analytical HP method using silica gel as a stationary phase as a chlorophyll analysis method.
An LC analysis method was also developed. Conventionally, id? was used to determine the purity of chlorophyll. The I7-layer chromatography (TLC) method has been recommended for many years; a',
Even if ch, z, b, , cht, and b' can be separated, the four types of pheophytins cannot be effectively separated. Furthermore, because conventional methods generally have long operating times and dyes are exposed to air for long periods of time, these techniques are susceptible to experimental error. Although it is still a micro analysis in recent years,
Silica gel was used as a stationary phase for analysis of chlorophyll] (P
The TJC method and the HPLC method using a reversed stationary phase in which silica gel is modified with a C8 or C18 alkyl group have been reported, but in order to simultaneously separate chlorophylls and pheophytins using these analytical methods, solvent programming is required. (gradient), which currently requires a long time for analysis. The method developed by the present inventor is characterized by using a high-pressure packed and sealed silica gel column as the stationary phase, and is an isocratic method that does not use gradientene as a separation solvent. This is a revolutionary analysis method that allows eight structurally similar chlorophyll derivatives to be separated extremely quickly, and the analysis can be completed in just a few minutes.

EXAMPLES The present invention will be described below with reference to Examples.

Example (a) Removal of chlorophyll hydrolysis products and water-soluble dyes using 5ephadex LH-20 column Column size: 200 myn x 26 mm (cocked glass column) Packing material: 5ephadex LH-20 developing solution W:
n-Hexane Chloroform-3 Chlorophyll 1 extracted and purified from freeze-dried chlorella powder by a known method
．． 00117 is dissolved in a developing solvent and developed after injection into a column. In order of elution 7x, (-phytin, Ch, 4.a
, Cht, b, hydrolysis product, water-soluble pigments, so Ch,, 5. a and Ch, 1. ．． 1] Separate only the portion. The hydrolyzate and water-soluble dye degrade very slowly, so their separation is clear.

The resulting ChL,a, Cht,b mixture is obtained as a solid by immediately removing the solvent in a rotary evaporator.

(b) Ch by preparative high performance liquid chromatography,
4. a, Ch4. a', chz, b, cht, b'
Separation column: stainless steel (250mm x 20m
m) Filler Silica gel (particle size 5 microns) Developing solvent: Isopropyl alcohol: n-hexane = 3:97 Flow rate = 10 g/min Wavelength: 430 nm 307 ng of the solid obtained in (a) was added to 3. tul
Dissolve in developing solvent and inject. Developed isocratically in the same solvent, and in the order of elution Cht, a', Cht,
Since they are a, chz, b', cht, and b, a solid is obtained by separating each of them and quickly removing the solvent using a rotary evaporator. By repeating this operation,
Obtain the required amount of each chlorophyll derivative. Further, 20 m7 of each chlorophyll substance was taken and the same operation was repeated three times to obtain highly pure ChL, a
, Ch., 4. a', ChL, b.

ChL,b' is obtained.

(c) Pheo, a, Pheo, a', Pheo
, b , Pheo, b' separation (respective pure Ch obtained in bl, 4.a, Cht, a', cbz
, b, chz, b' each 20 mg in ethyl ether 5
After dissolving the solution in a liquid solution and transferring it to a separating funnel, add 10 mg of 4N-HCL and shake vigorously. After separation, discard the lower layer and wash with distilled water until the washing solution becomes neutral.

Next, the ether layer is dehydrated with anhydrous Na2'S'04, and then the solvent is quickly removed using a rotary evaporator to obtain a solid. Through this operation, each isomer is partially produced, and at the same time, a portion of the hydrolyzate is also produced. (a) above;
By carrying out the operation (b), they can be removed (1) and a pure product can be obtained.

(d) Elemental analysis The main analysis was carried out by Analytische LabOratOr.
This was done at the request of Germany (West Germany). Samples were handled in the dark in an inert gas atmosphere and dried for 3 hours at 80°C 1Q and lmmHg immediately before analysis. Ph
The calculated analytical values for eo, b and Pheo, b' differ by 0.5 to 0.7% depending on the element, but the analytical values for other components are within the nominal measurement error (specification ±0. 2
%), which agrees with the calculated value for anhydrous dye.

(El Analytical column size by analytical HPLC, 150 mm x 4.6 M Luzo size = 20 μm.Developing solvent: Isozoalcohol: n-hexane-1
, 5: 98.5 Flow rate: 1 mg/min Wavelength: 430 nm Other conditions were the same as preparative HPLC.

(f') Ultraviolet-visible wavelength measurement A spectrophotometer UV1DEC-650 manufactured by JASCO Corporation (microcomputer control, double monochrome type, wavelength accuracy 0.3nffL absorbance minimum reading value 0.0001) was used. The spectral properties of each derivative were measured in ethyl ether, acetone, and benzene.

(gl CD scroll measurement ChL, a and Cht, a', Chz, bi and chz, b
', Pheo, a and Pheo, a', Pheo, b and p
Since heo and b' cannot be distinguished by spectroscopic properties, they were measured by circular dichroism (CD). This analysis was conducted at the Faculty of Engineering, University of Tokyo. Measurement was done using JASCO Go20.
A model automatic spectropolarimeter was used. Benzene was selected as the solvent, and the wavelength ranged from 700 nm to the limit wavelength of benzene2.
It was measured by eating a set meal at a speed of IQ nm 7 minutes at 70 nm+j. The sample was placed in a fused silica cell with a dense column and an optical path length of 1 cm, and the concentration was set to 5-7×10M.

Total elemental analysis (see Table 1) and analytical HPLC (see FIG. 3) were performed on each of the eight pigments isolated by the method of the present invention to evaluate purity.

For Pheo, b and Pheo, b', the analytical value and calculated value differ by 0.5 to 0.7 depending on the element, but for other pigments, the elemental analysis value is based on the nominal measurement error (as per the specifications). It agrees with the calculated value for anhydrous dye within ±0.2%). Therefore, the drying conditions used here (80°C, 0
, IHHg, 3 hours), it can be seen that crystal water was almost completely removed.

Previously, 5train et al.
00'c, 0,001 mm Hg, 1 hour) is recommended (see Reference 19 below), Fong and KO.
eB'ter is Cht, even if a is dried most strictly, it is 1
Although it was stated that one molecule of water is attached to each molecule (see Document 20 below), the results obtained by the method of the present invention are as follows.
These claims are denied.

Still, some researchers have assumed that chL at the time of weighing is monohydrate when calculating the molar extinction coefficient (ε) (see Reference 2.21.22 below); Considering the results obtained, if these reporters had properly dried the samples before weighing,
The calculated value of 2E is larger than the true value.

Since the purity of isomers (epimers) cannot be guaranteed by elemental analysis alone, the results of analytical HPLC for the eight purified dyes are shown in FIG. 3.

From this figure, Cht, a 1Ch, 4. a', chz, b
, ch4. b', Pheo, a, Pheo, a
', Pheo, b 5 99.9%, 99.5%, 99.5%, 99.4chi, 9 for Pheo, b', respectively
It can be seen that purity of 5%, 91%, 99.5%, and 85% was achieved. In fact, the only impurities are the respective epimers. This was done by recording an HPLC chart at a detection wavelength of 254 nm to confirm that no impurities that absorb ultraviolet light were present.

The purity of Pheo species is cht! The relatively low value compared to 1111 is mainly due to the fact that the efflux times of the epimers are close to each other. In fact, by repeating the HPLC purification operation, even higher purity Pheos could be obtained. Another reason for the low purity of Pheos is that Hynninen and S.
j, as suggested by the results obtained by CV e r B for Pheo, a' (see Reference 2 below), ch
This may be because the interconversion between Pheo molecules is faster than that of Pheo species.

The purity of ChL,a obtained by the method of the invention is extremely high (99.9%). Compare this value with the purity of the best cht,a sample currently available on the market (two lots from one manufacturer are named sample 1 and sample 2, and one purchased from another manufacturer is named sample 3). did. All commercially available products are special grade reagents, and immediately after opening the sealed ampoule, analytical HPLC using the method of the present invention can be performed.
I put it on. Sample 1 has a purity of 98.9% and contains Cht,a' (Q, 9%) and Pheo,a (0
, 2%). Cht,a purity of sample 2 is 97.90%, which is slightly lower than that of sample 2, and ChL,a' (1,
57%), chz, b' (0,28 section), PhθO,b
' (0,14%), carotene (0,07%) and P
heo,a (0.04%). Sample 3 contained Cb, /=, a' (2%) as the main impurity, and two other unidentified compounds as trace components, so the purity was evaluated as less than 98%. . When calculating these IQ centages, 430 n
The molar extinction coefficients of all components in m were taken into account.

In order to clarify the separation ability of the analytical HPLC method according to the present invention, isolated chzs and pheos were subjected to HPLC under the same conditions. The results are shown in Figure 4. Although the peaks of ChL, a' and pheo, a coincidentally overlap here, it can be seen that eight structurally similar compounds can be separated extremely quickly. With only four ChLs, separation can be completed within 5 minutes depending on increasing the flow rate or shortening the column.

The UV-visible absorption characteristics of the a'-type and b'-type dyes matched those of the a-type and b-type, respectively. Therefore, for the four dyes in three organic solvents, the peak wavelength (λmax, ±Q, 3nn+), the molar extinction coefficient of the red peak [ε□ax (Red)), and the blue band/
The red band absorption ratio CA (Blue)/A (Red)') is summarized and shown in Table 2.

Considering the importance of the εmax value in the spectral properties of chlorophyll pigments, the obtained εmax (
The absolute value of εmax (Red) and the previously reported value are compared below. ・The actual measured value of εmax described in the literature (unit: 1.0'
M-"on-1) includes the following.

■chz, a [6,61(24
), 8.63 (19), 8.68 (21), 8.76 (
25), 8.85 (26), 9.02 (27), 9.1
2(28)), in acetone [5,1(24), 7.8
8(25): ) in benzene [5,47(24) )
;■Cht,b C5,10(2
6), 5, 15 (28), 5.21 (24), 5.
61 (]9), 5.63 (25, 27), 5.78 (
21)], in acetone [4,2(24)];■P
heo, a : :), :c chi # 1-7 jlz middle [5,40(26) ) ; ■ Pheo, b ::) x
Chill x -7-/l/medium [: 3.63(26)]
. The value reported by Trurnitt and Co. (see reference 24 below) is clearly too small, but this is probably due to the presence of impurities. 5aVer et al.
The ax value (see reference 21 below) is given by Cht-H as the molecular formula.
Since it was calculated assuming 2O, it would have to be multiplied by 0.98 if the sample had been properly dried. As you can see from the above brief summary,
As a result of the method of the present invention, the emax values obtained are close to the largest among those described in the literature and are among the most frequently cited for Ch, /, , a and Chz, b K in diethyl ether. 5trai in 1963
It is closer to the value of Sm1th and ]3eniteZ' reported in 1955 (see Reference 27 below) than the value of Sm1th and ]3eniteZ' reported in 1955 (see Reference 27 below). Vernon (Reference 3 below)
1) is 8.27xlO' as εmax in acetone by adopting the values of Sm1th and Benltez.
(Cht,a) and 4.86X10'(cbz,
b), which is again close to the value obtained by the inventor.

Based on this measurement result, Cb4 in acetone and diethyl ether. A mixture of a, chz, b and Phe
A formula for quantifying each component from a mixture of o, a and Pheo, b by absorbance measurement was derived (Table 3).

The quantitative formula for this pole reported up to 1966 was 5vec
(see Document 7 below), but there are some differences compared to the results obtained by the present inventor. For example Cht,a in acetone.

A mixture of and chz, seven
The previous value of k4 is 9.78.0.99.21.
40.4. ．． It is 65.

Circular dichroism (CI) spectra The CD spectra and absorption struts of eight types of dyes in benzene are shown in FIGS. 5(A) to 5(D). Subjected to this measurement,
pH, eo, a and Pb, eO of Aft dissolved in benzene
The epimer purity of the sample 9b is h 9(j T
o or more and about 0.93%, and the purity of the other six types of dyes is the same as in FIG. 3. As can be easily seen from Fig. 5, between a pair of epimers, the absorption thread is
However, in contrast, the CD spectrum differs significantly from L<. As far as the present inventor knows 1), (j) of Cbt monomers
There are 3 papers that discuss this. These are the results of Cbt, a in ether and 0.5 ethanol-CC and Pheo, a in ether (see Reference 32 below)
, ChL in ether and methanol, a, Chmu a
', cb4. b, chz, b', results (see reference 33 below), and Cht, a, chz, a in THF
,',Pheo,aSPheo,a,' (see Document 2 below). Below, we will mainly focus on the purity of the sample and compare the current CD data with previously reported data. For the sake of simplicity, the comparison will be limited to the three silk electronic transitions that appear in the mouth region, namely QX, Qy, and 5oret absorption bands.

The name of these electronic transitions is weiss (Reference 34 below)
), and the axis of transition moment Cx, V is 1.
) are shown in FIG.

m Qy transition region The so-called “red absorption peak” of ChI-derivatives is due to Qy
Corresponding to the (0+' 0 ) transition, thezatilite in the vicinity of Ama belongs to the Qy (1,0) band (see References 34 and 35 below). For all four dyes that investigated prime ('), a strong negative CD was observed at Q, y (0,0).
A clear negative (CD) satilite appears along with the large Qy(1,0) peak. In contrast, dyes without primes have a total (CD) peak in these absorption peaks.
(chL,b and Pheo,a) or weakly negative CDL (Cb4.a and Pheo,a).
b). The weak optical activity observed in the latter two dyes is due to
This is probably due to the coexistence of prime-dominant dyes (epimers) as impurities. In previous reports (References 32 and 33 below), a strong negative CD peak was obtained even with a derivative without a prime, but our results are completely contrary to this. According to estimates, if we assume that the optical activity of the molecule is not significantly affected by the type of solvent, then the Cht used in the study cited in 2.
The samples of a, chz, and b have significant (at least 2
It turns out that it contains the prime-dominant pigment of 0).

KohVi diethyl ether, (later cited document 32.33),
Ethanol (Reference 32 below), methanol (Reference 3 below)
This is thought to be due to the rapid interconversion between epimers in a nuclear solvent such as 3) (see Reference 3 below). Based on these results, we cannot help but have some doubts about the validity of the detailed discussion on the electronic state of Chl- derivatives (References 32, 34, and 35 mentioned below), which was based in part on previously reported CD data. I don't get it.

(21QX transition region A series of QX transitions appear in the "valley" of the absorbing waves (Reference 34.35). cht, a, Pheo
, a, Pheo, and b have Q and X(0,0) absorption peaks at 580.539.560 nm, respectively, and all are accompanied by clear QX (], , O) satilites on the short wavelength side. For pigments that do not have a prime, Pheo, a is Q
There is no optical activity in this electronic domain, except for a very weak negative peak at the x(o,o) wavelength. In contrast, the primed dye shows a fairly strong CD, but its sign (positive) is opposite to that for the Qy transition described above. A similar behavior is observed in diethyl ether for cht, a
' (Reference 33 below) and Pheo, a' in THF
It is reported in (Reference 2 below).

ChL, b′ (and cht
The optical activity of ,b) is exceptionally weak, but this may be related to the fact that the absorption spectrum is extremely blurred.

(3) According to the results of molecular orbital calculations in the 5-oret band region (Reference 34.35, mentioned below), the 5-Oret absorption peak is almost degenerate and consists of two electronic transitions BX (0
, 0), and By(0, O). All four primed dyes exhibit a single strong positive CD in the 5Oret absorption peak. This is a CD from BX and B7
This suggests that the contributions to On the other hand, dye CD7 without prime. The existence of two electronic transitions appears to be reflected in the extinction. In other words, maximum and minimum energy occur at wavelengths separated by 70 to JQOmev, and the midpoint wavelength of these approximately coincides with the 5 Oret absorption peak wavelength.

When passing through the 5oret absorption peak from the long wavelength side to the short wavelength side, the CD of only Cbz, a changes from minimum to maximum, but for the other three species (chz, b, PheO, a, P
heO,b) changes from maximum to minimum. The CD slayers that have been reported so far for dyes without primes (
In Documents 2.32.33) mentioned below, such maximum-minimum changes are not seen at all, and in both cases, the CD maximum wavelength and the absorption peak wavelength match well. Given that the CD intensity of primed compounds is significantly higher [7], as mentioned in section 2, if the CBT Xanzol used in previous studies contained more than 20% of primed compounds as impurities. If so, such a result is understandable. Chta,b before preparing diethyl ether solution
Even if Xansol is pure, the purity of the epimer after dissolving it in such a nuclear solvent (see Reference 3 below) cannot be guaranteed.

References including those referred to in the above description are listed below.

(]l Proc, Natl A, Cad, Sci
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7227-7231° (21z, Naturforsch, 1981,
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[Brief explanation of the drawing]

No. 111: Chemical structural formulas of chlorophyll a, chlorophyll b, chlorophyll a /, chlorophyll b',
FIG. 2 is a diagram showing analysis of various derivatives of pheophytins. Patent Applicant Nankata Rakuhin Kogyo Co., Ltd.

Claims (1)

[Claims] Separation of chlorophylls characterized by separating chlorophylls by high performance liquid chromatography using silica gel packed and sealed at high pressure as a stationary phase to obtain chlorophyll a, chlorophyll b, chlorophyll a' and chlorophyll b' with high purity. Method.