Ionization techniques chlorophylls

FAB and LSIMS are matrix-mediated desorption techniques that use energetic particle bombardment to simultaneously ionize samples like carotenoids and transfer them to the gas phase for mass spectrometric analysis. Molecular ions and/or protonated molecules are usually abundant and fragmentation is minimal. Tandem mass spectrometry with collision-induced dissociation (CID) may be used to produce abundant structurally significant fragment ions from molecular ion precursors (formed using FAB or any suitable ionization technique) for additional characterization and identification of chlorophylls and their derivatives. Continuous-flow FAB/LSIMS may be interfaced to an HPLC system for high-throughput flow-injection analysis or on-line LC/MS. 

Among natural products in this category, vitamin B12 and chlorophylls in particular have been extensively examined using the newer ionization techniques. Again the emphasis has been on model studies on known structures though some of these have relevance to aspects of their biosynthesis or the mechanism of their biological activities. 

Due to the high mass, low volatility, and thermal instability of chlorophylls and derivatives, molecular weight determination by electron impact (El) MS is not recommended. Desorption-ionization MS techniques such as chemical ionization, secondary ion MS, fast-atom bombardment (FAB), field, plasma- and matrix-assisted laser desorption have been very effective for molecular ion detection in the characterization of tetrapyrroles. These techniques do not require sample vaporization prior to ionization and they are effective tools for allomerization studies. 

The most recent progress in MS analysis of chlorophylls has been obtained with the development of atmospheric ionization methods such as atmospheric pressure chemical ionization (APCl) and electrospray ionization (ESI). These techniques have demonstrated much more sensitivity than thermospray ionization, detecting chloro-... 

Development of fast, accurate, and reproducible high-performance liquid chromatography (HPLC) methods has offset the use of traditional open-column and TLC methods in modern chlorophyll separation and analysis. A number of normal and reversed-phase methods have been developed for analysis of chlorophyll derivatives in food samples (unit F4.4), with octadecyl-bonded stationary phase (C]8) techniques predominating in the literature (Schwartz and Lorenzo, 1990). Inclusion of buffer salts such as ammonium acetate in the mobile phase is often useful, as this provides a proton equilibrium suitable for ionizable chlorophyllides and pheophorbides (Almela et al., 2000). 

In widespread use since 1982 (Barber et al., 1982), FAB and LSIMS are matrix-mediated techniques. The most effective matrix for static FAB/LSIMS analysis of chlorophylls and their derivatives is 3-nitrobenzyl alcohol (van Bree-men et al., 1991a), whereas glycerol provides adequate sensitivity and a more robust system during continuous-flow FAB/LSIMS (van Breemen et al., 1991b). Ionization and desorption of the chlorophyll analyte occur together during the bombardment of the matrix by fast atoms (or ions) to produce molecular ions, M+-, and protonated molecules, [M+H]+. 

Zissis, K.D., Dunkerley, S., and Brereton, R.G. 1999. Chemometric techniques for exploring complex chromatograms Application of diode array detection high performance liquid chromatography electrospray ionization mass spectrometry to chlorophyll a allomers. Analyst 124 971-979. 

It was not possible to use MS for the characterization and identification of chlorophylls and its derivatives until the development of desorption methods (desorption ionization) appropriate for nonvolatile and thermolabile compounds. The mass spectmm of chlorophylls has been obtained using laser desorption [92-94], field desorption [95], plasma desorption [96,97], fast atom bombardment (FAB) [28, 98-101], in-beam electron ionization [102], and electrospray ionization (ESI) [103]. A combination of the techniques of desorption and tandem mass spectroscopy (MS/MS) has also been used for the charactmzation of chlorophylls and derivatives [ 104—107]. The latest research in this field coupled HPLC with MS, using as ionization source FAB [108-110] or atmosphoic pressure chemical ionization (APCI) [111-115]. 

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