An improved method for the rapid and easy separation of leaf pigments and their derivatives by thin-layer chromatography

Iriyama, Keiji; Yoshiura, Masahiko; Shiraki, Masaru; Yano, Shinji; Saito, Shiro

doi:10.1016/0003-2697(80)90527-8

Cited by 9 publications

(6 citation statements)

References 5 publications

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“…One of the most common degradation products is the loss of the magnesium ion from chlorophyll resulting in the formation of the colored pheophytin at an R f higher than the original chlorophyll. Pheophytin a and b are observed at R f values consistent with literature reports (7) in addition to the corresponding chlorophyll a and b when dried or frozen spinach was used to prepare extract (Figure 1). These degradation products are also found in commercially available purified chlorophyll a and b samples.…”

Section: Resultssupporting

confidence: 88%

“…Since the chromatographic separation will be affected by even small quantities of water, a bulk separation of the extract using a separatory funnel (or comparable microscale techniques) followed by drying of the organic layer with an appropriate drying agent is often required (2,3,6,(11)(12)(13)(14). A further complication arises in this separation owing to the well-documented limited stability of the chlorophyll pigments (7)(8)(9)(10). This can be confusing to the student since the sample can produce both the expected pigments and colored degradation products with similar R f values.…”

Section: Resultsmentioning

confidence: 99%

“…Also many of the chromatographic solvent systems reported for this separation do not adequately resolve chlorophyll a and chlorophyll b from each other or from other pigments present in the sample. A further complication exists in that the pigments to be isolated are susceptible to degradation (7)(8)(9)(10)(11) resulting in the observation of materials that are not representative of the pigments in the plant. We present significant improvements in both the sample preparation and the mobile phase for the extraction and separation of plant pigments.…”

mentioning

confidence: 99%

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An Improved Method for the Extraction and Thin-Layer Chromatography of Chlorophyll a and b from Spinach

2004

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A direct method of extracting plant pigments from spinach leaves into a dry organic solvent is presented. A powder is obtained by grinding a mixture of raw spinach, drying agent, and sand (1:1:2) that can be extracted directly into acetone in 10 minutes to provide a sample suitable for chromatographic analysis. This method avoids liquid–liquid extractions and subsequent drying as is found in previously reported methods. Removal of the magnesium ion from chlorophyll pigments was observed by treating the acetone extract with a strong ion-exchange resin for three minutes. The loss of magnesium ion is often observed as a degradation product of chlorophyll samples. An improved solvent system consisting of petroleum ether, cyclohexane, ethyl acetate, acetone, and methanol in 60%, 16%, 10%, 10%, and 4% quantities, respectively, is also reported. TLC separation using this extraction method and solvent system allows for the clear separation of chlorophyll a and b from all other extracted pigments.

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Section: Resultssupporting

confidence: 88%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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An Improved Method for the Extraction and Thin-Layer Chromatography of Chlorophyll a and b from Spinach

2004

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“…Thin-layer chromatography of the initial acetone pigment extract, using various supports and specific eluents, were tried (Choudhury and Bajaj, 1979;Iriyama et al, 1980;Loevenschuss and Wakelyn, 1973), as well as different chromatographic techniques (Jones et al, 1972;Scholz et al, 1981;Sievers and Hynninen, 1977). Pigment separation was achieved, although its quantification still remained very difficult because of lipid presence.…”

Section: Methodsmentioning

confidence: 99%

Chlorophyll and carotenoid presence in olive fruit (Olea europaea)

Mínguez‐Mosquera¹,

Garrido‐Fernández²

1989

J. Agric. Food Chem.

109

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“…Other methods such as thin-layer chromatography (32,34,50), paper chromatography (18), and normal-phase HPLC (48) have also been employed. However, reversed-phase HPLC seems superior to these methods with respect to stability of the pigments during analysis, resolution capacity, and analysis time.…”

Section: Discussionmentioning

confidence: 99%

Pigment and acyl lipid composition of photosystem I and II vesicles and of photosynthetic mutants in barley

Henry

Mikkelsen

Møller

1983

Carlsberg Res. Commun.

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The pigment and acyl lipid composition ofphotochemically active stroma lamellae and inside-out photosystem II vesicles have been investigated. Mechanical disruption followed by phase partitioning in an aqueous two phase system resulted in a clean separation of the photosystems. A qualitative and quantitative determination of the pigment content was achieved using reversed-phase high performance liquid chromatography. A single chromatographic separation provided adequate resolution of neoxanthin, violaxanthin, lutein, chlorophyll b, chlorophyll a, and 13-carotene and was completed in 22 min. For each molecule of P700, the stroma lamellae contained 166 molecules of chlorophyll a, 14 molecules of chlorophyll b, 0 molecules of neoxanthin, 3 molecules of violaxanthin, 7 molecules of lutein, and 13 molecules of 13-carotene. For each molecule of P680 the photosystem II vesicles contained 250 molecules of chlorophyll a, 100 molecules of chlorophyll b, 7 molecules of neoxanthin, 7 molecules of violaxanthin, 22 molecules of lutein, and 4 molecules of l~-carotene. The pigment content of three photosynthetic mutants chlorina-[2, viridis-zb63 and viridis-zd69 was determined. On a chlorophyll basis, these mutants generally showed a reduced carotenoid content, except that chlorina-f2 had an increased level ofl3-earotene and viridis-zd69 an unaltered level of lutein.The acyl lipid composition of stroma lamellae, photosystem II vesicles and lamellar systems was also determined. Although the photochemical activities of the stroma lamellae and photosystem II vesicles were enriched compared to the parent lamellar system, more than 50% of the acyl lipids in the subchloroplastic membranes were degraded or severely modified resulting in a drastically reduced content of monogalactosyldiglyceride and digalactosyldiglyceride in the stroma lamellae and photosystem II vesicles. Due to the lipolytic degradation the level of saturation in the gatactolipids was higher in stroma lamellae and photosystem II vesicles compared with the lamellar system. Stroma lamellae do not contain the light-harvesting chlorophyll a/b-protein, but were enriched in phosphatidylglycerol and trans-3-hexadecenoic acid.Abbreviations: Chl = chlorophyll; ChI-P = chlorophyll-protein; DG = digalactosyldiglyceride; GLC = gas-liquid chromatography; HPLC = high-performance liquid chromatography; MG = monogalactosyldiglyceride; PC = phosphatidylcholine; PG = phosphatidylglycerol; PI = phosphatidylinositol; SL = sulfoquinovosyldiglyceride; TLC = thin layer chromatography.

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An improved method for the rapid and easy separation of leaf pigments and their derivatives by thin-layer chromatography

Cited by 9 publications

References 5 publications

An Improved Method for the Extraction and Thin-Layer Chromatography of Chlorophyll a and b from Spinach

An Improved Method for the Extraction and Thin-Layer Chromatography of Chlorophyll a and b from Spinach

Chlorophyll and carotenoid presence in olive fruit (Olea europaea)

Pigment and acyl lipid composition of photosystem I and II vesicles and of photosynthetic mutants in barley

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