Column Choice for basic compound

[R&D-Chemist]

I am working on a basic compound method development at pH 2.5 with phosphate buffer (25mM) and ACN as mobile phases.
Column I choose was Atlantic dC18 150x4.6mm (pH range 2-7).
All initial runs gave plate count about 72000. After about 50-100 injections column plate count changed to about 17000. Except this, rest of the chromatography is good (Ex: All impurities retained well, good resolution etc)

Do I need to consider changing the column at this point?

Thanks in advance for your input.

[Uwe Neue]

If your separation is adequate, and the peak integration is OK, then do not worry about the loss of plates.

You need to consider column issues and changing the column only if either you get double peaks (column voiding) or if you have resolution/integration problems. Once you get to such a point, you can consider some column washing, but this is a lot of work and may not always be successful.

[R&D-Chemist]

Uwe- Thanks for the reply.

Other concerns I have - is this column eventually going to crash out with this chromatographic conditions? Or any troubles when it transferred into QC.

[Uwe Neue]

Read the C&U manual. The pH is adequate for this column. There are columns around with improved pH stability compared to the one that you are using (Atlantis T3, XBridge C18), but I do not see a problem with your pH of 2.5.

[R&D-Chemist]

Uwe,

I might to go with the same column what I am using now.

I choose the column (dC18) based on the Column C & U manual only. It is supposed to be a good fit for the use of phosphate buffer pH at 2.5.
But when had a talk with column mfg technical service about plate count drop led me to think this column might not be a good fit for the basic compounds. At this point I wanted to see some opinions about this issue.

Thanks for your input.

[carls]

A bit more information is required to give an opinion.

What is the maximum concentration of ACN used in the separation?

What is the cause of reduced plates, e.g. increased peak width, decreased retention?

Do all peaks show the same problem?

What is the tailing factor of the main peak before and after degradation?

What is the peak area %RSD for 10 injections of the same standard?

[R&D-Chemist]

Carls,

Here is the example of recent run:

RSD of 5 injections is 0.1%.
First Std Injection Plate count: 17198; Tailing: 0.94
Bracketing Std Plate count (After 15 injections): 14593; Tailing: 0.94
Plate count decreased due to the increased peak width: yes all the peaks show same problem.
I have gradient run, started with 95% buffer (MP A) and 5 % Buffer:Acteonitrile(MP B) (30:70) to 60%MPA&40% MPB

All my Initial runs have shown plate count about 72000 & Tailing about 1.4.
In this run, also observed some small peaks (unknown) have not been detected compare to the initial run.

Thanks

[Uwe Neue]

One of things worth pointing out is that plate counts in gradients are not valid plate counts, since they depend on the detailed execution of the gradient. They are only of some value, if you compare the exact same conditions on the exact same instrument. In general, I do not recommend to use plate counts in gradients. Such values are no supported by any theory.

[HW Mueller]

If there are some extra peaks appearing the first thouhgt is that the matrix is crudding the column, also causing the possible change in appearance. So one would need to clean the column periodically or/and clean up the act.

[carls]

RSD of 5 injections is 0.1%.
First Std Injection Plate count: 17198; Tailing: 0.94
Bracketing Std Plate count (After 15 injections): 14593; Tailing: 0.94
Plate count decreased due to the increased peak width: yes all the peaks show same problem.
All my Initial runs have shown plate count about 72000 & Tailing about 1.4.

Sounds like the inlet frit is fouled or the column is voided.

Have you tried a strong organic flush on this column?

If not, purge the column with 20 column volumes (1CV ~ 1.5mL) of 10/90 ACN/H2O to remove all buffer. Then run a gradient from 10/90 to 100% ACN over 10min @ 1mL/min and hold at 100% ACN for 10 CV. Next flush the column with 5 CV of 100% IPA at a low flow rate - be careful not to over pressurize the column as IPA gives VERY high back pressure. Next flush with 20 CV 20/80 MeOH/CH2Cl2. Flush again with 10 CV 100% IPA to remove all CH2Cl2 then flush with 5 CV 10/90 ACN/H2O.

Equilibrate with 10 CV inital mobile phase and see if perfomance has improved.

[R&D-Chemist]

Agree with Mueller & Carls

This is what noticed.

When I noticed that column performance is going bad, tried to regenerate the column as per the C&U manual.
Washed the column with: 100%DI-H20 →50:50 Water: Acetonitrile→ 100% ACN→THF→CH2Cl2→THF→CH2Cl2→THF-100%ACN. Still had the same results (Above results are after the column washes –posted on October 29th).
Again, Friday I washed the column with good amounts of 50:50 Water: Acetonitrile followed by 100% Acetonitrile until column pressure is ~1000psi with 1.0 ml flow (Initial pressure ~2200) and got back initial expected results.

As HW Mueller said, now I am considering implementing column wash process periodically.

Thank you for input.