Problems with Kinetex C18, 1.7 µm

[Mattias]

Does anyone else have problems with the Kinetex C18, 1.7 µm columns?

When the columns are new, the backpressures are different from column to column. Some give 7000 psi, some give 9000 psi (in my method). The 9000 psi columns usually gives poor peak shapes (splitting) already from the first injection, while the 7000 psi columns can stay fine for many hundreds of injections.

I have exchanged all fittings now to Viper, but it doesn't help. When I get a bad column I can always take an old column and get back the good chromatography again (using the same mobile phase and fittings).

One more strange thing: I accidentally hooked up a 2.6 µm Kinetex the other day. The plate counts were actually slightly better than for a (good) 1.7 µm column... What is going on?

[cody84]

I had similar problems with the 2.6. Pressure was acting funny from run to run and between columns. I was told a better method to wash it and to use the security guard cartridges (sample injection based problem was suspected). I will be trying this solution out on Thursday.

Did you try the test kits on both columns before your own method?

[Klaus I.]

Does anyone else have problems with the Kinetex C18, 1.7 µm columns?

When the columns are new, the backpressures are different from column to column. Some give 7000 psi, some give 9000 psi (in my method). The 9000 psi columns usually gives poor peak shapes (splitting) already from the first injection, while the 7000 psi columns can stay fine for many hundreds of injections.

I have exchanged all fittings now to Viper, but it doesn't help. When I get a bad column I can always take an old column and get back the good chromatography again (using the same mobile phase and fittings).

One more strange thing: I accidentally hooked up a 2.6 µm Kinetex the other day. The plate counts were actually slightly better than for a (good) 1.7 µm column... What is going on?

1) Sometimes I have also observed that some columes (not Kinetex) have an unusual high back-pressure (above 20 % higher than other same brand and in fact same packing series) and bad performance. IMHO there is sometimes a problem during the packing of the columns.
You can check the back pressure during factory testing mentionend on the test certificate and ask the manufacturer for a refund.
BTW: The usable (allowed) maximum pressure of Kinetex 2.6 µm is specified to 600 bar (8700 psi). Only for columns with an ID of 2.1mm, Phenomenex have raised the maximum allowed pressure to 1000 bar (15000 psi).

2) The performance of the 2.6 µm Kinetex or other fused-core columns is nearly in the range of a 1.7 µm column with totally porous particels. In my experience, the 1.7 µm Kinetex works only better, when one use very high flow rates, but actually the possible usable back-pressure is limited by the instruments. IMHO the 1.7 µm Kinetex works fine, when one uses a short column length, high temperatures and the maximum back pressure. Please take in mind, that I have never compared this results with a 2.6 fused-core column using pressures above 600 bar (8700 psi).

Edit: Typo corrected - of course 1000 bar are about 15000 psi; Thanks DR

[Mattias]

Thanks,

It has to be a manufacturing problem of the 1.7 µm Kinetex C18 columns, and this is quite bad since we would like to use this method for QC release in the near future.

I have tested several of the 2.6 µm columns, and so far all have worked fine. Maybe the 2.6 µm Kinetex C18 are more consistent?

A question related to this: How much revalidation do you think would be necessary if I switch from 1.7 to 2.6 µm (but keep all other parameters intact)? I am of the opinion that only a small comparative study would be needed, but others in my company thinks that a full new ICH validation is necessary.

(The method will be used for release of phase III material in the near future, so it is full GMP)

[cody84]

Particle size can be reduced by up to 50% according to USP chromatography chapter. Doesn't say about increasing.

Do a risk assessment. As long as resolution is still intact there is no reason to fully revalidate. Same phase, same everything...makes NO sense to revalidate if they know ANY chemistry.
Or adjust parameters so chromatography is exactly the same.

[Klaus I.]

I have tested several of the 2.6 µm columns, and so far all have worked fine. Maybe the 2.6 µm Kinetex C18 are more consistent?

Maybe you had only bad luck with the 1.7 µm columns, and statistically there are no reasons for general suspections against the 1.7 µm Kinetex columns. I don't know, but I have observed this pressure problems (also during factory tests) also with traditional 3 µm or 5 µm columns.

A question related to this: How much revalidation do you think would be necessary if I switch from 1.7 to 2.6 µm (but keep all other parameters intact)? I am of the opinion that only a small comparative study would be needed, but others in my company thinks that a full new ICH validation is necessary.

(The method will be used for release of phase III material in the near future, so it is full GMP)

This is a long time back and I don't have the knowledge of the actual gold standard in method validation und regulatory matters. But a revalidation (including documentation) of a "known" method should be done in one week by 1.1 technicians, when good protocols and procedures exist. With one additional week, a huge amount of old samples is reanalyzed.

A "small" comparison study needs also its time, but this also depends on the experience of the technicians and the existing procedures.

[HPLCaddict]

Particle size can be reduced by up to 50% according to USP chromatography chapter. Doesn't say about increasing.

Do a risk assessment. As long as resolution is still intact there is no reason to fully revalidate. Same phase, same everything...makes NO sense to revalidate if they know ANY chemistry.
Or adjust parameters so chromatography is exactly the same.

ACK. As long as the chemistry (stationary phase) stays the same, you should get away with a small amount of work. I also think you should do a risk assessment stating that by changing particle size only the resolution of the chromatographic method should change. Then redo the selectivity part of the validation to show that 1) there's still enough resolution and 2) selectivity of the columns actually is equal (i.e. no retention time changes - wouldn't be the first time that different particle sizes of the same column actually gave slightly different selectivities ).
Explaining this matter to your regulatory people will pe a pain in the a**, though

[Consumer Products Guy]

I'm curious to know if you tried backflushing any of those "high pressure" columns, then installing them and seeing if pressure and peak shape are then OK.

[Mattias]

The method contains a SST test where I inject a standard spiked with 9 impurities. The demand is that all 9 components must be baseline separated. I also inject a 0.1% solution of the standard where the S/N value must be >10. So in a way, I am validating the methiod everytime I run it. I am trying to convince QA that it is enough if I compare SST data for three batches of 1.7 µm and three batches of 2.6 µm columns.

If I put a 1.7 and a 2.6 µm chromatogram in overlay, it is impossible to tell them apart by the eye.

Regarding the bad columns: I have tried to backflush them. No difference what so ever. They are bad from the start. Maybe they would work OK for other methods, I do not know (I do not have time to run test kits).

[Consumer Products Guy]

Regarding the bad columns: I have tried to backflush them. No difference what so ever. They are bad from the start. Maybe they would work OK for other methods, I do not know (I do not have time to run test kits).

I would contact Phenomenex technical service about those columns then.

[Klaus I.]

The method contains a SST test where I inject a standard spiked with 9 impurities. The demand is that all 9 components must be baseline separated. I also inject a 0.1% solution of the standard where the S/N value must be >10. So in a way, I am validating the methiod everytime I run it. I am trying to convince QA that it is enough if I compare SST data for three batches of 1.7 µm and three batches of 2.6 µm columns.

If I put a 1.7 and a 2.6 µm chromatogram in overlay, it is impossible to tell them apart by the eye.

Regarding the bad columns: I have tried to backflush them. No difference what so ever. They are bad from the start. Maybe they would work OK for other methods, I do not know (I do not have time to run test kits).

And looking on the factory test certificate, there was nothing exceptionally?

[Mattias]

I have not found any trends in the certificates!

I am really sick and tired of the whole thing right now...

The whole story:
I developed the method and validated it with three different batches of 1.7 µm columns. It was performed on two different Acquity systems and one Ultimate system and three different analysts. Everything looked fantastic with the lowest USP resolution of the 10 peaks of 3.0 in all runs. The robustness testing showed that I could change pH and acetonitrile concentration in a wide range without any significant loss of resolution.

The method is transferred to QC (in another country) without any problems. They buy a large number of columns for the process validation. Only a few works. Peaks are splitted or very broad. They record the backpressure, and we find that only columns with low initial backpressure works for the application in their lab.

I travel to the site and find that the capillaries and fittings that they use are in terrible shape. I exchange everything to Viper fittings. We test another new column from their stock, and it doesn't work. I then install a >500 injections old column from my lab and it works beautifully again.

So it looks as it is the columns that is the problem, but I am still not convinced. Could it be minor changes in the column installation/equilibration that decides if the column will have high backpressure/bad performance?

The product is a sterile filtered 100 µg/ml solution of a 1000 Da peptide in 5% mannitol. The method uses 19% acetonitrile in 2.5 mM ammonium acetate buffer pH 5. The injection volume is quite large for a 2.1 mm column (20 µl), but I would expect full peak compression since the product does not contain any organic.

[Mattias]

Hooked up a Acquity C18, 2.1x150 mm, 1.7 µm today.

The backpressure was 9000 psi, so it is actually comparable to the Kinetex 1.7µm column. The plate count was also very close. Slightly longer retention times compared to the Kinetex column, but the selectivity was strikingly similar.

I though the whole idea of core-shell was to get lower pressure and better efficiency? Is the whole thing just a commercial trick?

Anyhow, I might have found a way out of the problems. To QA it is much better to move to another C18 1.7 µm, than to stay with the current material and use 2.6 µm. Science?

[Klaus I.]

Hooked up a Acquity C18, 2.1x150 mm, 1.7 µm today.

The backpressure was 9000 psi, so it is actually comparable to the Kinetex 1.7µm column. The plate count was also very close. Slightly longer retention times compared to the Kinetex column, but the selectivity was strikingly similar.

I miss Dr. Uwe Neue really , he was was the authoricy to describe your observations. But I will try also to answer you questions (Please keep in mind, that I am only a simple operator of LC/MS systems), Backpressure is depending mainly on the particle-size. So different 1.7 µm particles should have a comparable backpessure, even when 1.7 µm totall porous particels are compared with 1.7 µm fused-core particles.

I though the whole idea of core-shell was to get lower pressure and better efficiency? Is the whole thing just a commercial trick?

I haved asked many times the representatives about the real clue (?, sorry I'm not a english native-speaker) about the fused-core story, but I have never received a reasonable answer.

Most robably the 2.6 µm fused-core particels act almost like 1.7 µm total-porous particles, but without the increase on back-pressure (my own measurments will confirm this.)
You may have the same effiency, but you have a signifcant lower back-pressure.

For the actually smalles Kinetex particle size, we do not have the adequate instruments, because back-pressure will be too high for our instrements, to use these small particles in the correct way.

[carls]

Mattias,

I'm sure you are aware of this but even a UHPLC system must be optimized in order to realize the improvement in performance provided by the highest effieciency columns available today. The QC results for the columns in question provide a good estimate of the performance under realistic conditions. These results are generated on standard UHPLC instruments (>100 instruments) so users should not have a problem reproducing these results when using similar equipment. If you are unable to reproduce the QC results then the system must be optimized. Unfortunately, many UHPLC system are not capable of showing the true performance of these ultra-high effieciency sorbents without careful optimization.