Chromatography Forum

Hopefully this is in the right section.
I'm working in a QC lab and looking for a little confirmation on what i'm doing.
I'm trying to come up with a very basic wash method for all of our hplc's. Currently we run a variety of tests and columns, and since the machines are run overnight, after a test is run, we setup a simple method to pass ACN:water or MeOH:water gradient through the column.

At the moment, this is as follows 50:50 for 5 minutes, 5:95 for an hour, 0:100 for an hour and 60:40 thereafter.
We have not started using this as yet, and its only setup to run on c8,c18 columns.
Is there a better way i could do this, bearing in mind that this is just a replacement to having the columns run mobile phase all night.
Is the gradient alright?

thanks

We oftentimes "wash out" columns after a sequence of isocratic assays is run (our gradient assays are "self-washing"). We program up to about 90% ACN for 15 minutes. I think you're overdoing things, but wouldn't hurt. In your case, where you do the same assay the next day, I would bring back the system to your starting mobile phase composition, but lower the flow down to a tiny flow, e.g. 0.05 ml/min. You'll need to lower your minimum pressure limit so your pump doesn't shut off, and you should turn your lamp off as well; you can make up and save a clean-out method and load that as last line in a sequence table.

Thanks,
At least i know now what direction to take.

What you are considering is a good idea, especially for isocratic separations. Here are a few additional thoughts:

1. If you have any buffers or dissolved solids in your mobile phase, get rid of them first. That is, pump only acn:water (no buffer) for a while, then do the cleaning.
2. Usually there is no need to do an intermediate wash. I usually go immediately to 100 % acn (or meoh).
3. For setting the times, think in terms of "column volumes" not actual time. The column volume is the "void volume" in the column (look for the "squiggles" in the baseline just after injection to get an idea). As a general rule, you would want to pump 10 - 15 column volumes through with your cleaning mobile phase. Then, about the same volume of your original mobile phase before you begin again. So, a short column will require less time than a longer one, even though you pump through the same number of column volumes.
4. You can also watch your detector baseline, and that may give you an idea of how much washing is needed. If you have an absorbance detector, set it to a low wavelength (e.g., 210 nm) and watch the baseline. When you no longer see changes/drifting, the system is probably clean. There can be a great difference in how much time this takes, depending on many factors. Similarly, the system is ready for analysis once the baseline returns to a normal, stable state.

.... 1. If you have any buffers or dissolved solids in your mobile phase, get rid of them first. That is, pump only acn:water (no buffer) for a while, then do the cleaning.....

Don't use too much of organic solvent to avoid precipitation of buffer. When we use buffers, first "washing" solvent is approx. 30%ACN/H2O.

"Don't use too much of organic solvent to avoid precipitation of buffer. When we use buffers, first "washing" solvent is approx. 30%ACN/H2O."

A good clarification; I agree. The amount of "organic" that your system can tolerate will depend on what is in the system now, and what kind of buffer you are using. Phosphates are likely to precipitate if the acetonitrile content gets too high.

A more general rule might be: first flush the system with the same organic/water composition as your mobile phase, but without the buffer present. So, if you are running 40% acetonitrile in a phosphate buffer, flush with 40% acetonitrile in water first, then increase the acetonitrile content.

If i was doing this separately for each method i could change up the composition of my wash. However, with the number of methods it would take me weeks to prepare and upload all these separate wash methods, and it would probably lead to a lot of confusion.
What i'm currently trying to do is get a very broad wash method that will clean the columns after 75%+ of all tests. There is little variation with our mobile phases, but i will look into possible precipitation problems and maybe reduce the initial wash ratio.

I think that broad (general) wash method that will clean major part of the columns is:

- 10-20 column volumes of mobile phase 30% ACN/H2O (if you are using buffers)
then
- 10-20 column volumes of mobile phase 70% - 90% ACN/H2O.
(it depends on nature of your analytes (their polarity and absorption on sorbent)
-Instrument shutdown (or 0.05-0.2 ml/min flow).

We use this method for all C8, C18 columns without problems. Simply add this method at the end of sequence table.