Chromatography Forum

Greetings

I am studing phlorotanins and have to analyse in an extract.
I bought a Waters spherisorb 5 micrm. 4.6X250 mm. silica (normal phase)cartridge. As I was looking for methods in the literature, it always refer to 2 micrL. loop and an injection volume of 2 micrL.

Is there a reason to the loop and the injection volume should be so low?
Can I use a 20 micrL. loop?

Thank you in advance for your answers

Best regards
Rui

I would suspect it may be to minimise the effect on the column eqilibrium caused by the sample injection. (larger injection volumes may require much longer post injection re-equilibration.

However, without knowing what mobile phase you're using and what solvent your samples are in I can't say for sure

Hi

Thank you for your reply.

The mobile phase is constituted by:

A) DCM, MeOH, H2O, AcOH (82:14:2:2 v/v/v/v)

B) MeOH, H2O, AcOH (96:2:2 v/v/v)

And the sample will be redissolved in H2O

I have a 2 micL. loop, but it is an internal loop and a have to replace the seal, unfurtunately the loop has 4 ports and my valve only have two, so I can´t insert it in the valve. We have others HPLC with 4 ports valves, but at this moment they are occupied, and has it is only for screening I thout that probably I can use the 20 micrL loop, but I have to be shure that it won´t damage the column.

Why don´t you just try it? Or, there is no possibility for partial filling? Or can you play with the sample solvent?
Why do you think you could ruin the column?

Well, this is better. At least we have some solid information on the mobile phase. You also seem to be running a gradient, but you kept this information still secret...

You dissolve the sample in water. This is usually a no-no in normal phase chromatography, since the injection of water will lead to peak distortion and - on the long run - to a deactivation of the column. However, your mobile phase contains some water. With other words, the person who developed the procedure was aware of these potential problems and added water to the mobile phase to develop and maintain an equilibrium with the water. The surface of the silica contains now a controlled amount of water, and good method reproducibility can be achieved with such a method.

Since you have a water-controlled surface, there is nothing wrong with injecting an aqueous sample. However, there can be other issues. There will be a limited solubility of your aqueous sample in the mobile phase, which contains a large proportion of DCM. It is possible that with a sufficiently large injection volume, you will get a plug of sample that migrates through the column without ever seeing the stationary phase. you need to watch out for such an event as you increase the sample volume. Also, pressure spikes are possible.

So, as Hans said: go for it! But also be aware that there may be some issues.

Hello

I will try it.

In all the papers I read they use small injection volume, so I thought that my injection volume could damage the packing, or as the analytes concentration is higher so they could be retained in the columns for a log period of time.

I will try and I will write giving news.
Thank you for your reply. Hope I can be helpfull some day

Best regards

Hello Uwe Neue

I am not hidding anything, this is a method from a paper I read, it´s public, we are only trying to adapt to our matrices. I didn´t thought that the information of a gradient was important. Thank you for your important infromation, maybe the dissolving the sample in DCM will the best way...

Best regards

Rui,

My comment about the "secret" was a little jab. But you still did not yet tell us anything about your gradient... It is not terribly important any more, but could have been important for answering your question or helping you in making a decision.)

The following statements are not just for you, but to everyone posting questions. Without knowing a lot of details, it is often not possible to make a recommendation, and many of us do not bother any more responding to questions with insufficient details. For example, going back to your original question: you can inject on any column any volume that you choose, even liters. On its own, the question can not be answered. JGK focused on the relevant parameters, which you then provided to a level that enabled an answer.

Remember and realize: if you did not get an answer to your question, the question may not have been comprehensible to others, who know nothing about the work that you are doing.

My apologies. You are absolutly right.

My problem is that sometimes I can´t understand and can't write perfectly english, so it becomes dificult for you to understand.
It is a Ph studant that is working on it, so i don´t have the toltal information with me, monday I will provide it for hepling us.

Thank you.

Your sample is dissolved in water, which is a (very) strong solvent in normal-phase LC. Injecting a solvent stronger than the mobile phase can cause serious peak shape problems (the strong solvent diluent "pushes" part of the sample down the column). If a strong-solvent diluent is absolutely required, the peak shape problem can be minimized by limiting the injection volume. I suspect that is the reason for the 2 microliter volume in your case.

Injection a larger volume will probably not harm the column, but may give you unacceptable peak shape. Try it and see what happens.

Hello again

The mobile phase is constituted by:

A) DCM, MeOH, H2O, AcOH (82:14:2:2 v/v/v/v)

B) MeOH, H2O, AcOH (96:2:2 v/v/v)

The sample will be redissolved in H2O

The gradient elution is: 0 min 100% A; 0-30 min 0-17.6% B ia A; 30-45 min 17.6-30.7% B in A; 45-50 min 30.7-87.8% B in A; 50-60 min 87.8% B in A; 60-80% min 87.8%-0% B in A; 80-105 min 100% A. The detection wavelength is 280 nm and the flow-rate is 1mL./min

Thank you all for your reply. We will try it and I will give news

Best regards