This document discusses pigments and their separation using chromatography techniques. It outlines different pigments like chlorophyll, carotenoids, and xanthophyll and shows their structures. It then explains the principles of paper chromatography and high performance liquid chromatography (HPLC) for separating pigments, detailing steps like filtration, column packing, elution, and detection using a diode array detector to produce a chromatogram.

2. Pigments
Lycopene
Chlorophyll-a
Chlorophyll Carotenoid Xanthophyll
Canthaxanthin

3. Chlorophyll
Mg2+
Phytol Tail
Pheophytin
Pheophorbide
Mg2+
Chlorophyllide
Mg2+
Mg2+
Chlorophyll degradation pathway
H
H H
H

4. Chlorophyll (Chl-a) Absorption Spectrum
Carotenoid (Lycopene) Absorption Spectrum

6. Loading of the Pigment extract
Solvent run
Paper Chromatogram
Retention Partition
(Separation)
Elution Identification Quantitation
Pigment Separation Principle (Paper Chromatography)

7. Filtration Sonication Loading Auto sampler
Column
HPLC (High Performance Liquid Chromatography)
?What happens in the column

8. Flow Cell
Column packing
More polar analyte
Less polar analyte
TS
Inlet
Pore spaces Interstitial
area
(mobile
phase)
Support particle Nonpolar
Bonded
phase
Octyl silane (C8)
DAD (Diode Array Detector)
Chromatogram
Reversed phase Binary Gradient Elution at 60°C by C8 Column