Standard Operating Procedure GARNet 7
Analysis of Arabidopsis thaliana for Flavonoids.
Author: Jennie Lewis
Last Modified: October 2002
Standard Operating Procedure
Analysing Arabidopsis thaliana plants for Flavonoids.
Materials:
Equipment:
1.5ml eppendorf tubes
Methanol
Polished Water
Acetic Acid
Acetonitrile
Naringenin
Injection vials and caps
Tapered low volume inserts
Gilson pipettes and tips
Balance
Whirlimix
Centrifuge
HPLC with Diode Array Detector
Column: Lichrosorb RP C18 10μ 250x4.6mm
Guard cartridge: Lichrosorb RP C18 10μ
Procedure:
Step 1: Plant Extraction
1.1
Label three 1.5ml eppendorf tubes with the plant material to be
analysed and Replicate 1, 2 or 3.
1.2
Make up 1mg/ml solution of Naringenin in water.
1.3
Weigh out 100mg fresh, frozen plant material or 10mg freeze dried
plant material, prepared for analysis following SOP GARNet 4, into
each labelled eppendorf tube.
1.4
To each tube add 300μl 80:20 Methanol:Water.
1.5
To each tube add 100μl of Internal Standard Naringenin (1mg/ml in
water).
1.6
Mix the plant material and the solvent by holding the tubes in a bench
top whirlimix for 1 minute.
1.7
Leave the tubes to stand for 10 minutes on the bench.
1.8
Repeat step 1.6.
1.9
Centrifuge the tubes at 18000g for 5 minutes.
1.10
Label injection vials with the appropriate plant material and replicate
number and into each place a tapered low volume glass insert.
1.11
Transfer 50μl of the supernatant, post centrifugation, to the
appropriately labelled injection vial and cap the vial.
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Standard Operating Procedure GARNet 7
Analysis of Arabidopsis thaliana for Flavonoids.
Author: Jennie Lewis
Last Modified: October 2002
Step 2: HPLC Analysis
2.1
Make up Mobile Phases A: 0.01% Acetic Acid and B: Acetonitrile
2.2
Set up the HPLC with a Lichrosorb RP C18, 10μ, 250x4.6mm column
with in line guard cartridge
2.3
Programme the HPLC with the following gradient and flow rate of
1ml/min.
Time (minutes)
0
5
20
45
55
60
% Mobile Phase A
100
93
93
78
0
100
% Mobile Phase B
0
7
7
22
100
0
2.4
Set the diode array detector to scan at 190-550nm and to selectively
monitor at 254nm.
2.4
Equilibrate the column in 100% Mobile Phase A until a flat base line is
maintained.
2.5
Inject 25μl of each prepared sample in to the HPLC system.
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