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LABORATORY PROCEDURES FOR PLANT CELL LINES

REFERENCE NO.: PC/1998/3/1.6


TITLE: DESCRIPTION OF METHODS FOR STANDARD HPLC ANALYSIS


INTRODUCTION

The following conditions and methods are used for the standard HPLC analysis of methanolic extracts.

PROCEDURE

Equipment: The following equipment is used: solvent delivery system: 2 pumps contraMetric 3000, LDC Analytical, 1990; detector: SM4000, LDC Analytical, 1990; autosampler: Marathon, Spark Holland, 1990; Dynamic + Static HPLC Mixing Chamber, Spark Holland, 1990; pump control and data analysis system: Thermochrom Vers. IV, LDC Analytical, 1990.)

Solvents used: Solvent A: Water + 0.01 % phosphoric acid

Solvent B: Methanol + 0.01 % phosphoric acid

Column
for routine standard: 4 x 100 mm, Nucleosil C18, 7 mm
for analytical standard: 4 x 250 mm, Nucleosil C18, 7 mm

Flow rate: 1 ml/min

Detection: wavelength: 280 nm, range: 0.01

Gradients:

Method 1

0 min; 100% solvent A; 0% solvent B; 1ml/min
1 min; 100% solvent A; 0% solvent B; Injection
31 min; 0% solvent A; 100%solventB;
36 min; 0% solvent A; 100%solventB;
31 min; 100% solvent A; 0%solventB;
44 min; 100% solvent A; 0% solvent B;

Method 6 (Prewash)

0 min; 0% solvent A; 100% solvent B; 0ml/min
1 min; 0% solvent A; 100% solvent B; 1ml/min
15 min; 100% solvent A; 0% solvent B;

Method 7 (Postwash)

0 min; 100% solvent A; 0% solvent B; 1ml/min
10 min; 0% solvent A; 100%solventB;
14 min; 0% solvent A; 100% solvent B; 1ml/min
15 min; 0% solvent A; 100 %solvent B; 0 ml/min

 


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