Chromatography Forum

A reversed phase method I received for a peptide (Module phase 0.1% TFA ACN) called for injecting 200 uL of either standard or sample (Lysed cells) in 10% TFA. The column they used was a Spherisorb ODS2...

I have never ever injected anything in 10% TFA!!! Won't that concentration of TFA damage the column?

In addition I have no idea why they feel the need to use it!

Maybe the peptide stick to something without it?

Maybe because it is sample and not mobile phase is not exposed to that low pH long enough to cause an issue with the silica?

Maybe They don't know what they are doing?

So Is that likely to kill the column or is it OK to inject 10% TFA?

BTW as I only have UPLC columns I planned to redo the for a Waters HHS T3 C18... but as I said that sample prep has me concerned!

Thanks,
-Karen

I think the most likely answer is: "Maybe They don't know what they are doing?"

Even though your post is missing some critical basic HPLC information (see below), we would probably not recommend injecting such a solution into an HPLC system. TFA is a very strong and corrosive acid. Even 0.1% (commonly in use to provide ~ pH of 2.1) is often too strong a concentration for some applications.

Questions Asked/Answers Needed:, Two very important basic questions, (1) what is the injection volume; (2) what are the column dimensions (so we can calculate the column void volume). Depending on the injection volume and acid concentration (100x normal in this case!), if the injection volume is incredibly low relative to the column volume than the solution may be diluted sufficiently to not pose a threat to the Stainless steel and plastic parts of the HPLC flow path (and yes, the column support too). If the injection solution is quickly diluted in the flow path, it may not pose an issue, but we must consider both the concentration AND the chemical/physical properties of the solution too. While it may be possible to dilute the solution enough, the corrosive nature of the "slug" coming in contact with the parts in the flow path may result in damage.

In this case, the injection volume used would need to be extremely low (~ less than 0.1% of column volume, maybe less than 0.01%) to dilute it to a safe level, BUT using such a strong solution AND loading it into the injector flow path would likely result in damage to the system before the slug reached the column head (per manufacturer's spec, would probably damage the injector before it was fully diluted. The pH of the solution should not be below 2.5 or ~ 2. The extremely corrosive solution of 10% TFA would be expected to have a pH of ~ 0.45 !!! ).

Here are a few HPLC articles that may be of interest to you (and your client) which address some of your concerns.

"HPLC Injection Volume: What Should I Dilute It In and How Much Sample Can I Inject?"; https://hplctips.blogspot.com/2023/07/h ... uld-i.html

"Determination of HPLC Column Void Volume / Dead Volume, Dead Time (T zero)"; https://hplctips.blogspot.com/2011/05/d ... -time.html

I think the most likely answer is: "Maybe They don't know what they are doing?"
Questions Asked/Answers Needed:, Two very important basic questions, (1) what is the injection volume;

Injection Volume they used was 200 uL.
They started at 0% organic.

(2) what are the column dimensions (so we can calculate the column void volume).

4.6 X 250 mm...

Everything I know said it did not make sensate me ...but I could not think of a reason why they would do that if hey did not need to...

If they actually did it, I suspect their column lifetime was pretty short!

Thanks,
Karen

Wow, 200ul in a 4.6 x 250mm column.... That is around 7% of the column volume (Far too much). Based on the info provided, I do believe you have your answer as they have no formal HPLC training. While it may be OK for them to damage their HPLC system, please follow your instincts and do NOT damage yours.

HPLC Injector flow path damage is likely.

A reversed phase method I received for a peptide (Module phase 0.1% TFA ACN) called for injecting 200 uL of either standard or sample (Lysed cells) in 10% TFA.

In addition I have no idea why they feel the need to use it!

10 % TFA in the sample may be a result of adding TFA to the hydrolysate in order to inactivate the enzyme that could be used for the sample hydrolysis.

Wow, 200ul in a 4.6 x 250mm column.... That is around 7% of the column volume (Far too much).

If the sample was in water and they were injecting on RP at 0% organic mobile phase and then using a gradient, the injection volume could be OK because you are essentially lust concentrating your sample at the head ... I have done thing like that in the past for very dilute samples in aqueous media...

Based on the info provided, I do believe you have your answer as they have no formal HPLC training. While it may be OK for them to damage their HPLC system, please follow your instincts and do NOT damage yours.

Don't worry I'm not... I just wondered if it served some purse I was missing that could be dealt with another way...

I wondered if the 10% was typo, but even 1% makes no sense!

-Karen

Some basic HPLC fundamentals are being ignored... two big ones.
(1) 200 ul is too much for a 4.6 x 250mm column (with exception for running in displacement mode, but that is something we normally do in PREP LC not analytical LC). Such a large sample can not be loaded at the head of the column and will disperse out resulting in poor peak shape (and other issues, refer to article linked). "HPLC Injection Volume: What Should I Dilute It In and How Much Sample Can I Inject?"; https://hplctips.blogspot.com/2023/07/h ... uld-i.html
(2) Injecting a sample in a STRONG solution, relative to a weak mobile phase is also bad. Their sample is in 10% TFA solution which will probably cause peak shape issues and attack the injector flow path (causing damage $$$$). Most injectors are designed for a pH no lower than 2.5 and injector with PEEK rotor seals exposed to pH 0.5 (which many are today) would be destroyed.

As stated, they have no idea what they are doing.

... I have done thing like that in the past for very dilute samples in aqueous media...

I wondered if the 10% was typo, but even 1% makes no sense!
-Karen

10 % TFA in the sample may be a result of adding TFA to the hydrolysate in order to inactivate the enzyme that could be used for the sample hydrolysis.

or maybe they forgot to document some evaporation and reconstitution steps and their real samples contains much less TFA...

Only the authors will know (maybe...)