Chromatography Forum

Hello!
I have two question I appreciate if you can help me!
I am developing a HPLC method and I´m using a buffer ph 5.8, I inject the samples direct from the dissolution media (pH 1.2), Can the column damage?

If I want to save solvents (acetonitrile), Can I let the instrument running all nigth with flow 0.1 water:methanol 70:30? What do you recommend?

For your first question, no you won't damage your column (unless for some reason the volume of your loop is too high in relation to the volume of your column as it is sometimes the case in nano chromatography).

I see no reason really to have the instrument running overnight at all. Once you clean your column you can stop your flow.

In theory, injection of a solution at pH 1.2 can damage the column if it's a classical column (pH stability between 2 and 7) but it depend on the column (check your column stability in the manufacturer description, some columns are stable at pH 0 or 1). It also depends on the injection volume, if you want to keep your column a long life time,...

Usually, the best practice is to inject your sample in the same solvent or same pH than the mobile phase but it's not always needed. If it's possible it should be recommended to adjust the pH to 5.8 before injection (also perhaps to have a reproductible retention time if the molecule or the column bonding is ionizable).

"Can I let the instrument running all nigth with flow 0.1 water:methanol 70:30?"

Yes absolutely, personnaly I use 0.05 ml/min with minimum 20% of organic (to avoid microorganism growth).

Kwet,

Would you please clarify your reasoning for running mobile phase - f. ex. 0.05 mL/min through the night?

Best Regards

Insufficient information, as with most posts.

If you have a high concentration of an acetate buffer at pH 5.8, it is very unlikely that you will have a problem, unless your injection volume is outlandishly high. The injection of of a reasonable volume of the acidic sample will for the most part get you to a pH around 4 or so. If you have a phosphate buffer at pH 5.8, your true pH will shoot to down to around 2 or less, and some columns may not like this situation.

Also, I agree with Danko: from the standpoint of the column there is no reason to keep the flow going overnight.

We used to never stop the flow of our HPLC's, we just let run at minimum flow rate when no sample are to be injected. It's an advice given by the manufacturer to prevent some problems when you turn on the HPLC again. For exemple, the seal can dry and be more brittle; solvents can evaporate and concentrate impurities (in check valves for exemple),...
I don't really know if it's usefull to act this way, we just follow the advice. Perhaps it's only helpfull if we don't have analysis to do for a quite long time.

There are many HPLC columns which are very stable at low pH. This is a request for LC-MS application. I will suggest you the SiliaChrom SB, this column is great for low pH (0.8 at 70C). I used this column in my lab for very low pH without any problem.

Charles
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Thank you for your replies!

Uwe Neue, the volumen of injection is 10 ul I think that is no problem. But what do you think?

SiliCycle

The manufacter said that the column is stable between pH 1 to 10.

I have to say that I didn´t wash the column, I only flush it all nigth with water methanol. Now the peaks are no symetrics, what can I do?

The SiliaChrom SB is stable to pH 1.0 to 7.0. Higher pH, needs other column type. I used the SiliaChrom SB C18 (or C8) without any problem at low pH in my lab. Your agressive mobile phase can stay a while in the HPLC column, not necessary to flush it.

Charles

You do need to wash the column with higher % of organic solvent (does your gradient/method goes up to high % of organic solvent)?

Try to regenerate the column by using one of the washing/regeneration procedures that have been previously been proposed in this forum and see if that solves the problem...

Koko, you missed the most important point: what is the buffer at pH 5.8?

Considering that the column that you are using is good to pH 1, I do not see a reason to worry, no matter what you do.

Another missing information. What is the mobile phase flow? 10 µl injection and 2 ml/min flow in a 4,6 mm I.D. column would make no harm to the column. 10 µl injection and 0,2 ml/min flow in a 2,1 mm I.D. column can be a problem.

We always leave 0,05 ml/min flow for mobile phases containing buffers. Stopping flow of such a mobile phase causes crystalisation of buffers on pump pistons or pump seals. This way the pistons are being damaged faster and we had to change the pistons very often. Now we don't need to change the pistons at all (almost).

SiliCycle;
I know that the movil phase can stay a while in the column, but I have never known how much time, I let the column with movil phase only for a few hours.

Kostas Petritis:

About the gradient, is an isocratic method, Buffer:MeOH 70:30.

Uwe Neue:

I´m using a buffer Na2HPO4-NaH2PO4 5mM


Tomasz

The flow is 1 ml/min, 10 uL injection volume in a 4.6 mm ID column, I think there is no problem.

Thank you everyone I flush the column with MeCN 100% about 1 h at flush 0.8 ml/min, and seems that solves the problem. You helped me a lot with your advices.

I using SiliaChrom SB with pH=1 without any problem (my own column is keeping in TFA pH=1.0 for long period). Now on the HPLC market, you have some great column working at low pH. Most of the time is better to buy this kind of column instead to stop and go your application

Charles

Silicycle -

Do you have any data supporting durability down to pH = 1?
Imtakt's data shows 0.1% TFA = 2, and 0.5% TFA = 1.5

To get any kind of durability at pH 1.5 takes some pretty special bonding...