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Gradient Composition

The document outlines the procedure for conducting a gradient composition test in HPLC, including preparation of mobile phases and flushing of channels. It provides a detailed gradient table with specific time intervals, flow rates, and pressure settings, along with instructions for running the system and calculating concentrations. The final chromatogram must be checked for gradient steps, ensuring actual concentrations do not deviate more than ± 2 from set values.

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820 views2 pages

Gradient Composition

The document outlines the procedure for conducting a gradient composition test in HPLC, including preparation of mobile phases and flushing of channels. It provides a detailed gradient table with specific time intervals, flow rates, and pressure settings, along with instructions for running the system and calculating concentrations. The final chromatogram must be checked for gradient steps, ensuring actual concentrations do not deviate more than ± 2 from set values.

Uploaded by

sanjeetsandhu1992
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as DOCX, PDF, TXT or read online on Scribd

6.1.1.

1 Gradient Composition:
[Link].1 Connect the union between injector and Detector. Before starting the Gradient composition test,
flush the all channels for 15 minutes initially with hot water (100%), Methanol (100%) then IPA
(100 %) followed by Methanol (100%) and water (100%).
[Link].2 Prepare Mobile phase: (A and C): Milli -Q Water, (B and D): 0.4% acetone aqueous solution.
[Link].3 Perform flow rate calibration before performing Gradient composition and purge all the HPLC
channels individually with flow 5ml/ min for 5 minutes with 100% composition.
[Link].4 Fill the gradient table parameter as mention below:

TIME (in Flow


A % B % C% D% Pressure
minutes) ml/min

0 50 0 50 0 2.0 400 bar

1 50 0 50 0 2.0 400 bar

1.01 40 10 40 10 2.0 400 bar

4 40 10 40 10 2.0 400 bar

4.01 30 20 30 20 2.0 400 bar

7 30 20 30 20 2.0 400 bar

7.01 20 30 20 30 2.0 400 bar

10 20 30 20 30 2.0 400 bar

10.01 10 40 10 40 2.0 400 bar

13 10 40 10 40 2.0 400 bar

13.01 0 50 0 50 2.0 400 bar

16 0 50 0 50 2.0 400 bar

21 50 0 50 0 2.0 400 bar

26 50 0 50 0 2.0 400 bar


[Link].5 Run the System for stabilization before testing.
[Link].6 Run the time programmer as per the above table by setting wavelength 265 nm and column oven
temperature at 40°C.
[Link].7 To run sequence, keep HPLC vial with water at -1 and add 0.1 µl in acquiring method parameter.
[Link].8 Calculate the concentration for each conc. Label 20%,40%, 60 %,80% and 100%.
Template No. Page No. 1 of 2
[Link].9 The actual concentration should not deviate by more than ± 2 of set value.
[Link].10 height at set % conc.
Calculation: Actual conc. = ----------------------------------------------------------- × 100
height at 100%conc.
[Link].11 The chromatogram obtained shall be checked for gradient step.

Template No. Page No. 2 of 2

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