1. It was known that a various pheophorbides derived chlorophyll were active for anti hepatitis virus.

Design an experiment using various chromatographic methods, determining the pheophorbides. Answer: Pheophorbide in sediment samples is important compounds as indicators of early diagenetic process. However, there is no remarkable difference in absorption and/ or fluorescence spectral properties of chlorophyll or its degradation products. The excitation and emission maximal wavelengths of these compounds in acetone were as follows: pheophorbide (410 nm/ 670 nm), respectively. High performance liquid chromatography has been used for separation and identification of chlorophylls in sediment. A sensitive fluorescence high performance liquid chromatography is advantageous for the determination of chlorophylls. Experimental Apparatus The high performance liquid chromatograph was a Hitachi model 655 equipped with a Rheodyn 7125 syringe-loading sample injector (20 mm3 loop) and a Zorban ODS column (250 mmX4.6 mm I.D.). The fluorescence detector was Hitachi model F-1000 spectrofluorometer with a 12 mm3 flow cell. The excitation light source was a 150 W xenon arc lamp and the detector was a R-928F photomultiplier. Materials and Reagents Chlorophyll a was isolated from confrey (Symphytum officinalle L.) with methanol and acetone using dioxane method, and was purified by the use of cellulose column chromatography. Pheophorbide a was prepared from chlorophyll a by addition of 1 mol dm-3 hydrochloric acid. Pheophorbide a was prepared from chlorophyll a according to the Hynninen method. All chemicals were of reagent grade. Chromatographic conditions Separation of chlorophyll and chlorophyll derivatives was performed at 25C by using acetonitrileacetone (45 : 55 v/ v) as the mobile phase at a flow rate of 0.6 cm3 min-1. The effluent from the column was monitored with a spectrofluorometer by using an excitation wavelength of 410 nm and an emission wavelength of 670 nm. Result Determination of chlorophylls by HPLC A typical chromatogram obtained by the mixed standard solutions of chlorophyll a, pheophytin a, pheophorbide a, and methylpheophorbide a is shown in Fig. 1. For a reversed phase type column, Zorbax ODS, a variety of solvent conditions were examined to obtain the complete separation of chlorophyll and chlorophyll derivatives. The most suitable capacity factors (k') were obtained by using acetonitrile-acetone (45 : 55 v/ v) for chlorophylls. The k' values at 25 C were 0.26

for pheophorbide a, 0.33 for methylpheophorbide a, 1.78 for chlorophyll a, and 4.23 for pheophytin a, respectively. The determination of chlorophyll a and pheophytin a was carried out by means of the peak height method. And the relative standard deviation inherent in the peak height method for chlorophyll a and pheophytin a was less than 1.8% (for 9 determinations). However, the peak for methylpheophorbide a was greatly influenced by the tailing peak of pheophorbide a. This indicates that coexistence of even a small amount of pheophorbide a causes a large error in the determination of methylpheophorbide a. In order to eliminate the error from pheophorbide a, the narrow base line method was used for the quantitative analysis of methylpheophorbide a.

Determination of methylpheophorbide a by the narrow baseline method The narrow baseline method for the spectrophotometric determination was first proposed by Commins. In these studies, the baseline was set by connection of two points each about 5 nm apart from the peak on the spectrum. This baseline was called a narrow baseline (NBL) and the height to the peak from the NBL was taken as analytical data. In this paper, the unit on abscissa (wavelength/ nm) is replaced by "retention time/ min". The peak height of methylpheophorbide a increased with increasing peak height of pheophorbide a, as shown in Fig. 2. The observed value of methylpheophorbide a was 30% higher than the theoretical value, when the peak height of pheophorbide a was equal to that of methylpheophorbide a. Therefore, the narrow baseline method was applied for the determination of methylpheophorbide a by HPLC. In order to set a narrow baseline, we determined the two peaks (a and b) each 5 min apart from the peak of methylpheophorbide a on

the chromatogram, as shown in Fig. 3. The analytical signal (I) was given in the next equation; I=1o-(Ia+Ib)/2, where Io, Ia and Ib were peak height of the maximum peak, point a and point b, respectively. The relative standard deviation inherent in this method for a sample containing 8.1X10.8 mol dm-3 was less than 2.4% (for 6 determinations).

Determination of chlorophylls in core samples

Core samples collect from the Inland Sea of Japan were analyzed by this method. Two grams of the wet sample were transferred to a centrifuge tube containing 20 cm3 of acetone and shaken for 5 min. The suspension was centrifuged at 2000 r.p.m. for 10 min. The total extracts that were obtained from 5 separate runs were collected through a separatory funnel. The yellow supernatant solution thus obtained (acetone extract, ca. 100 cm3) was mixed with 10 cm3 of petroleum ether and then was added to an equial volume (approximately) of saturated sodium chloride solution. In these procedures, chlorophylls were extracted to the petroleum ether layer. This layer was evaporated in vacuo and was made up to 10 cm3 with acetone or mobile phase {acetonitrile : acetone=45 : (v/ v)}. Five mm3 of the resulting solution was injected in a sample loop of the HPLC with a microsyringe. The results are summarized in Table 1. These results confirm that HPLC combined with the narrow baseline method will contribute, as a convenient technique, to the determination of chlorophyll and its degradation products.

1. Yoshitake, Yoshiaki et al. Determination of Pheophorbide alpha andMethylpheophorbide alpha by Fluorescence High Performance Liquid Chromatography. Analytical Science. 1986, Vol.2