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C18 column after run Mobile phase contain Edetate, TBAHS

Discussions about methods, troubleshooting and best practices across both pharmaceutical and biopharmaceutical analysis.

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Hi all experts,
I am Van, from VietNam.
Currently I am having a problem with C18 column. In running progress retention time and theoretical plate of the peak decrease gradually (first time retention of peak is 14.4 min, second time is 13.0 min and third time is 11.2 min) and the next analysis after first time analysis some impurities co-eluted. May be the column have losing performance.
I tried many ways to wash this column from water/MeOH/Acetonitrile to acetic acid 1%/MeOH/Acetonitrile. But they are not effective.
So that, now we only run this column with this mobile phase only 2 times. Please help me give the flushing method for wash and restore this column after run this mobile phase.

I used it for analysis Risedronate impurities with mobile phase (I referred from "Risedronate tablets" of BP2020) and program bellow:

Chromatographic condition for Risedronate impurity:
- Column: C18 (150x4.6-mm; 3-µm), Ecosil SH/, PN: AS1203-1546
- Detector: UV 263 nm
- Temperature: 40oC
- Flow rate: 1.0 mL/min
- Injection volume: 20 µL
Buffer solution pH 7.0: Dissolve 1.75 g dibasic potassium phosphate (K2HPO4—174.18) and 0.40 g edetate disodium (C10H14N2O8Na2·2H2O—372.2), 1.7 g tetrabutylammonium hydrogen sulfate in water sufficient 1000 mL. Adjust pH 7.0 by sodium hydroxide 1 N.
Gradient:
Time (minutes)/ % ACN/ % buffer pH 7.0
0 7 93
14 7 93
15 25 75
50 25 75
51 7 93
72 7 93

Diluent: Water

Thank you very much.
Try to increase the equilibration time in the end of the run by 10 or even 20 min. Ion-pair chromatography and gradient elution are a bad combination.
Alternatively, try to use the methods from BP without modifications. Isocratic elution is used in the original methods:
https://www.pharmacopoeia.com/monograph ... ablets.pdf
Dear Van,
as per my understanding you are working on the ion pair mobile phase which requires the more stabilization time as compared to normal reverse phase HPLC.
Normally Ion pair mobile phase used with isocratic mode and rarely with Gradient.
During column cleaning you need precautions due to TBAHS and Edetate.
Kindly search the following document on the net and read this one for column cleaning may be you will get better results.

COLUMN WATCH
` The Cleaning and
Regeneration of
Reversed-Phase HPLC Columns
Ronald E. Majors, Agilent Technologies, Wilmington Delaware, USA.elaware, USA.
[I Modified method from BP to combine 2 procedures A and B for just 1 running. But I think my column with this mobile phase containing both TBAHS and EDTA, they make the column degraded faster, so now I want to find a flushing method can remove EDTA and TBAHS out of C18 column.="vmu"]Try to increase the equilibration time in the end of the run by 10 or even 20 min. Ion-pair chromatography and gradient elution are a bad combination.
Alternatively, try to use the methods from BP without modifications. Isocratic elution is used in the original methods:
https://www.pharmacopoeia.com/monograph ... ablets.pdf[/quote]
[Yes I realized my problem is C18 column after running with the mobile phase containing ion pair EDTA and TBAHS. They are cause make my column degraded, so that, the next running station phase can not keep analytes. Therfore it make main peak (Risedronate) decrease retention time and other impurities overlap together, can not separate as initial running ="cksoni"]Dear Van,
as per my understanding you are working on the ion pair mobile phase which requires the more stabilization time as compared to normal reverse phase HPLC.
Normally Ion pair mobile phase used with isocratic mode and rarely with Gradient.
During column cleaning you need precautions due to TBAHS and Edetate.
Kindly search the following document on the net and read this one for column cleaning may be you will get better results.

COLUMN WATCH
` The Cleaning and
Regeneration of
Reversed-Phase HPLC Columns
Ronald E. Majors, Agilent Technologies, Wilmington Delaware, USA.elaware, USA.[/quote]
Below is the link of the document

https://www.chemass.si/ca_content/Clean ... olumns.pdf
This document provides a good procedure of column cleaning and restoration as per sample nature and also as per mobile phase which we used.
vannguyen wrote:
I Modified method from BP to combine 2 procedures A and B for just 1 running. But I think my column with this mobile phase containing both TBAHS and EDTA, they make the column degraded faster

Mobile phases with TBAHS and EDTA are used in the original BP methods as well. There are no problems with these mobile phases. Generally, pH 7.5 is not good for silica-based stationary phases (especially at higher temperatures such as 40 °C). However, the working pH range of your column declared by the manufacturer is 1 to 10.

Using your column, try first to reproduce the original isocratic methods from BP. If you succeed, there is no column degradation. Also you can check the column using the conditions of the test chromatogram provided by the column manufacturer.
Hi vmu,
I have already submitted for registration this method and if I change method, I have to re-submit and re-validation method. So that, I just want to find the proper flushing method for this column to can increase using lifetime of this column (current we use this column about 3 times after running). I requested support from the column manufacturer about this problem, but they recommend use another column, Ecosil EPS, but it have retention time differ to Ecosil SH column, I used.
Otherwise, I don't know, do anyone try BP method with Luna column (recommend from BP) and they have similar issue with me or not?
Thank cksoni,
I have read this guideline, and tried flushing some ways but they are not effective. And the next time, I plan to try with this flushing method below to see the column will regeneration or not.
1. 30V with MeOH: acetic acid 1% (7:93)
2. 20V with MeOH: acetic acid 1% (20:80)
3. 10V with MeOH: water (20:80)
4. 20V with MeOH: water adjust pH 7.5 by NaOH (20:80)
5. 30V with MeOH: water adjust pH 7.5 by NaOH (7:93)
6. 30V with MeOH: water (50:50)
7. 20V with MeOH (100)
Temperatrure: 45oC
Flow rate: 1.0mL/min
vannguyen wrote:
Hi vmu,
I have already submitted for registration this method and if I change method, I have to re-submit and re-validation method. So that, I just want to find the proper flushing method for this column to can increase using lifetime of this column (current we use this column about 3 times after running).

If you submitted your method for registration, you had validated it and had had no problems with it until certain time. Do you see the decrease in retention time during one sequence of samples or do you see the differences when you compare different sequences? Can you see stable retention time in a single sequence? How do you store the column between the sequences? Flush the column with water/ACN eluent increasing the ACN fraction up to 70-100 percent and store the column in this eluent.
Hi vmu, when I develop method, I realize that Risedronate compound is stable API, it almost does not degradation in all stress conditions (temperature, acid, base, peroxide, light). So that, we only control unspecified impurities. When we validated the method, we know retention time have gradually decrease, but at that time the column only run 1 or 2 times, so we do not see the unspecified impurities overlap until we use this column (third time) and continue run, the result showed % unspecified impurity higher than the first time running column with same sample (remark: first time running %impurity: 0.08%, third time: 0.2-0.3%). It is reason I think stationary phase have been locked by EDTA or TBAHS, making stationary phase can not have good separation.
My column after run with this MP, I flush column with ACN/MeOH/water and store it in ACN 100%.
Hi guys,,
After a run on a C18 column where the mobile phase contains Edetate ethylenediaminetetraacetic acid and TBAHS tetrabutylammonium hydroxide solution, individuals may be concerned about potential column fouling or degradation. They might contemplate the need for thorough cleaning procedures to maintain column performance and prolong its lifespan, ensuring accurate and reliable analytical results in subsequent runs. lic premium calculator
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