TESTING METHOD Coding ENLASA

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QUANTIFICATION OF Reference

GIBERELIC ACID Date of issue: 30/07/2019

Page: 1 de 2

 High performance liquid chromatography.

1. Team
2. HPLC chromatograph
3. UV variable wave detector
4. Recorder
5. Column M-Bendapak C- (Water A550510 P / N 2734)
6. Polycarbonate membranes (45 microns)

 Reagents
1. Methanol distilled in glass or equivalent
2. 5% solution of KH2PO4 Analytical Grade
3. 8.5% w / w phosphoric acid solution
4. IRS Giberelic Acid Standard

 Mobile phase preparation

1. Mix 70% v / v of KH2PO4 and 25% v / v of CH3OH
2. Filter
3. Degassing and let it reach room temperature.

 Chromatography conditions:
1. Volume, injection 20 microliter
2. Flow = 2ml / min
3. UV detector at 204 nm
4. Room temperature
5. Rank 0.4 AUPS in the recorder
 TESTING METHOD Coding ENLASA
Revisión: 0

QUANTIFICATION OF Reference

GIBERELIC ACID Date of issue: 30/07/2019

Page: 2 de 2

 Preparation of the standard:

Weigh 50 mg carefully. IRS gibberellic acid in a 100 ml volumetric bottle: Dilute to volume
with unfiltered mobile phase and mix.

 Test Preparation:

Weigh 50 mg carefully. Sample gibberellic acid in a 100 ml volumetric bottle: Dilute to

volume with unfiltered mobile phase and mix.

 System adequacy test

Chromatograph duplicate injections of the standard preparation and detect a peak with
retention time between 7 and 9 minutes. The peak response for duplicate injections should
not vary by more than 2% if this accuracy is not obtained, continue injecting the standard
preparation until this 2% concordance is obtained for successive injections

 Process

Inject exactly the same volume of each test preparation and chromatograph for
approximately 20 min. Measure the response peak for gibberellin (G3) (Retention time = 7-
9 min) obtained with the standard preparation obtained from the system adequacy test for
the calculation.

 Calculations:
𝑅𝑒𝑠𝑝𝑜𝑛𝑠𝑒 𝑜𝑓 𝐺𝐴3 𝑖𝑛 𝑠𝑎𝑚𝑝𝑙𝑒 ∗ 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝑚𝑔 ∗ 𝑝𝑢𝑟𝑖𝑡𝑦 𝑜𝑓 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑
𝑅𝑒𝑠𝑝𝑜𝑛𝑠𝑒 𝑜𝑓 𝐺𝐴3 𝑖𝑛 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 ∗ 𝑚𝑔 𝑡𝑒𝑠𝑡