Plant extracts gradient elution

The problem of the separation of samples containing components of widely different polarities is difficult because of general elution. This can be solved by use of gradient elution. As has been observed, in TLC separation of plant extracts, gradient elution markedly improves the separation of spots owing to stronger displacement effects... 

Fig. 2.6.1. RP-LC-ESI-MS analysis of flocculation sludge from a Barcelona drinking water treatment plant. Column Cig LiChrolute 250 X 4.6 mm, 5 pm, gradient elution with ACN-water. Upper trace total ion current (TIC), lower traces extracted ion chromatograms for NPEOs, raE0 = 1-5. Inset ESI mass spectrum of NPEO oligomeric mixture.

The active components of the herbaceaous perennial plant Hypericum perforatum are antiinflammatory, antidepressive and healing agents, therefore, their analysis is of considerable importance for health care. Samples were prepared by extracting the dried flowering tops by hot methanol. RP-HPLC separations were performed in an ODS column (250 X 4.6 mm i.d. particle size 5 pm) thermostated at 30°C. The steps of gradient elution are listed in Table 2.49. 

This study evaluated the impurity profile of untreated water from a textile plant in Portugal [35]. The organic material was concentrated by extraction from 11 of water into dichloromethane and HPLC-NMR and HPLC-MS experiments were carried out using a reverse-phase separation with an acetonitrile/ D2O gradient elution with H NMR spectroscopic observation at 600 MHz. For the HPLC-NMR studies, the samples were further fractionated into two pools according to their HPLC retention times. The HPLC-NMR studies were carried out in the stop-flow mode and the combination of NMR and MS results yielded the identification or tentative identification of 14 compounds, comprising mainly surfactants, anthraquinone dyes and nonylphenol-related molecules. 

An example for the separation for flavonoids with HP-RPC is the screening method employed for the systematic identification of glycosylated flavonoids and other phenolic compounds in plant food materials by Lin et al20 These authors used an analytical 4.6 mm x 250 mm 5 pm C18 silica column at 25 °C with linear gradient elution (eluent A (0.1% FA in water and eluent B 0.1% FA in ACN) at 1.0 ml min-1. DAD was performed at 270, 310, 350, and 520 nm to monitor the UV/VIS absorption. The LC system was directly coupled to an ESI mass spectrometer without flow splitting and the mass spectra acquired in the positive and negative ionization mode. The same analytical scheme (aqueous MeOH extraction, reversed-phase liquid chromatographic separation, and diode array and mass spectrometric detection) can be applied to a wide variety of samples and standards and therefore allows the cross-comparison of newly detected compounds in samples with standards and plant materials previously identified in the published literature. 

As a consequence of the toxicity related to the presence of aristolochic acid in plant preparations, several health institutions, such as the US Food and Drug Administration, Therapeutic Goods Administration have recently published safety information to prevent further cases of intoxication (information available at web address http //www.cfsan.fda. gov/ dms/ds-bot.html) [471], So detecting aristolochic acids in plant species that could be used in herbal remedies, and also in herbal preparations of uncertain composition, has attracted great priority in recent years to help prevent future adverse reactions. Aristolochic acids present in medicinal plants or herbs are analyzed by soxhlet extraction followed by TLC in the Chinese pharmacopoeia [412]. Another report used multiple ultrasonic extraction followed by HPLC analysis [472]. Ong s laboratory reported a method using a home made pressurized liquid extraction (PLE) system in dynamic mode to extract aristolochic acid in medicinal plants, followed by gradient elution HPLC [473]. Several scientific communities described various analytical methods for... 

NPLC is most suitable for separation of nonionic and moderately polar compounds, especially for lipophilic samples that are too strongly retained by RPLC. Lipids differing in the number and position of double bonds, tocopherol, carotenoids, fat-soluble vitamins, and steroids in pharmaceuticals can be successfully separated by NPLC on silica gel or alumina columns. Mixed lipid classes in the extracts of animal or plant tissues can be analyzed on silica columns or on columns with bonded polyvinyl alcohol using complex solvent gradients. Gradient-elution... 

Gradient development. Gradient developments have been used to achieve separation of complex mixtures such as plant extracts, dyes, etc. However, their application to lipid separation is not very common. Golkiewicz (1996) discusses in detail stationary phase gradients and mobile phase gradients, the theory behind solvent selection, automated techniques and applications of gradient elution in TLC. 

HPLC-PDA detection offers an alternative for the cost-effective separation of taxanes and taxines in complex plant extracts. The spectrum index plots in Fig. 2 depict the chromatographic analysis of the taxine-purified extract on a Cig column with gradient elution. The UV spectra of the detected peaks are illustrated in the upper part of the chromatogram. A clearer prospect of the corresponding... 

In a very nice separation by Zhou et al. [1034], 10 aiylamines (aniline, benzidine, o-anisidine, quinoline, o-toluidine, 4,4-methylenedianiline, 3,3 -dimethoxy- and 3,3 -dimethylbenzidine, 4-aminobiphenyl, W,Ar-dimethylaniline) conunonly found in a dye process plant were extracted fiom water and baseline resolved and exhibited excellent peak shapes on a base deactivated C g column (X = 254 nm). A 30/70 (hold 8min) -> 90/10 (at 16min hold 4min) acetonitrile/water gradient eluted all compounds within 15min. A linear range of O.l-lOOmg/L was reported. 

An isocratic separation using a 35/25/40 acetonitrile/methanol/water (0.1% H3PO4) mobile phase eluted all components in 10min but generated poorer resolution for three pair of analytes (as compared with the gradient). Linear working ranges of 0.2-50 mg/L of plant extract were reported. 

Matysik and Benes (1991) determined the anthocyanin content in the petals of red poppy during flower development by six-stage polyzonal-stepwise gradient gel TLC-densitometiy of the separated zones at 465 nm. Matysik (1992) determined anthocyanins in plant extracts by stepwise gradient elution with various combinations of the mobile-phase ethyl acetate-isopropanol-water-acetic acid on silica gel 60 plates. Densitograms were recorded at 465 or 580 nm. 

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