Integrity of Chlorophyllous Pigments in Silica Normal-Phase HPLC

Abstract

KeywordsChlorophyll a, chlorophyll a', fast atom chromatography bombardment mass spectrometry, high-performance liquidWe have been utilizing silica normal-phase HPLC for the preparation and analysis of chlorophylls (Chls)1, and have detected two molecules of Chl a' (C132 epimer of Chl a) in close association with P700, the primary donor of photosystem (PS) I of higher plants and cyanobacteria.2-4 However, this claim has occasionally met serious skepticism based on previous warnings against the use of siliceous adsorbents for the chromatography of chlorophylls.5,6The questions raised so far concern the identity of each peak, denaturation and recovery in the column, and the integrity of the pigments during extraction.Though we have repeatedly addressed these questions"2, we now feel that it is appropriate to assure the assignments and to compare the performance of our normal-phase HPLC with that of reversed-phase HPLC, which is generally considered to be sufficiently inert with respect to the elution behaviors of chlorophyllous pigments. Experimental InstrumentationFour HPLC systemsl''-9, of which the operational conditions are summarized in Table 1, were employed in the present work.1H-NMR spectra were recorded on a 270 MHz NMR spectrometer (JEOL GSX-270) for acetone-d6 solutions with tetramethylsilane as an internal standard. The pulse angle was 45° (4.3 µs), the acquisition time 5.5 s, and 64 transients were accumulated. The measurements were carried out at 25° C. Resonance peaks were assigned by comparisons with those reported by Hynninen and Lotjonen.10 Positive-ion FAB-MS measurements were performed with a JEOL JMS-700 MStation. Pigments (25 nmol) were dissolved in chloroform (10 µl), and 1 sl of the solution was submitted to analysis using 3-nitrobenzyl alcohol as the matrix.ll-13 Ions were produced by

HPLC determination of photosynthetic pigments during greening of etiolated barley leaves

“…The Chl a ′/Chl a molar ratio was obtained directly from the peak areas because their fluorescence quantum yields are the same (Watanabe et al, unpublished result). The Chl b /Chl a molar ratio was deduced from calibration curves as described previously [7, 8]. The amounts of loaded Chls a and b were always within the linear region of calibration curves (data not shown).…”

Separation and Determination of Minor Photosynthetic Pigments by Reversed-phase HPLC with Minimal Alteration of Chlorophylls

“…Peak 7 was assigned to Chl a′ from the absorption spectrum and retention time comparison with the standard sample. 21 In our previous work, reversed-phase HPLC analysis of Chl a′ purified by normal-phase HPLC, containing less than 0.1% of Chl a as the sole impurity, showed 3.9% of Chl a′ allomers. In contrast, the same sample on the normal-phase medium showed practically no allomers.…”

“…Standard samples of Chl a, Chl b and Pheo a were prepared as described previously. 11,21 A standard sample of PhQ was purchased from Sigma and used without further purification. The purity of the standard samples determined by reversedphase HPLC was as follows: Chl a, 98.6% (impurities were Chl a allomers and Chl a′); Chl b, 98.7% (impurities were Chl b allomers and Chl b′); Pheo a, 99.3% (Pheo a′ was the sole impurity); PhQ, 95.7%.…”

“…In contrast, the same sample on the normal-phase medium showed practically no allomers. 21 This indicates that Chl a was oxidized during reversed-phase HPLC, probably due to the polar nature of the eluent. Under the HPLC conditions developed here, Chl a purified by normal-phase HPLC showed less than 0.1% of Chl a′ and Chl a allomers; this confirms the inertness of the HPLC conditions to Chl a (Fig.…”

“…Chl a was purified by preparative normal-phase HPLC. 21 Detection was done with a fluorescence detector (λex = 425 nm, λem =660 nm).…”