Chromatography Forum

I am running isocratic HPLC using C18 column in 100uL ultrafiltrated serum sample. My target peak appears at 3.5min. To flush out possible dirty substances in the column, I continue the isocratic run with the mobile phase, i.e. KHPO4 buffered 15% ACN (pH 3.5) for 10 more minutes, but I see no peaks coming out using 333nm.

Can I stop the run earlier to shorten analysis time?

For isocratic analysis at 15% ACN, I washed the column after 6 runs with 5% ACN 10min, then gradually to 70% ACN and hold for 10 min.

My question is:
1. How often should I run the wash, every 6 runs, less or more?
2. What % organic and gradient program should I follow?

I appreciate any comment.

Hi Eddie,
It is ok to wash the column almost three times the RT of analyste. I wont prefer to shrten this run time because your sample matrix may be dirty (I dont have any idea about your sample preparation) in some case. Even though you do not gat any peaks , there might be substances which do not absorb UV or very weak absorbance at your set wavelenght.
regarding the washing step, try to start with 15% ACN to aviod sudden change in pressure which is not good if you are going to wash every after six injections. Then go up to 95% ACN if the coulumn allows. Have a look to the column user guideline as different column have different conditions. If you want, you also can continue to decrease the ACN from 95 % to 5% at the end of the wash and increase further to 15% which is your isocratic level. But always check the specification of the column and programe the washing method.

phosphate buffer will precipitate in 95% ACN;
not sure, but I think 70% ACN is the maximum

I am running isocratic HPLC using C18 column in 100uL ultrafiltrated serum sample. My target peak appears at 3.5min. To flush out possible dirty substances in the column, I continue the isocratic run with the mobile phase, i.e. KHPO4 buffered 15% ACN (pH 3.5) for 10 more minutes, but I see no peaks coming out using 333nm.

Can I stop the run earlier to shorten analysis time?

For isocratic analysis at 15% ACN, I washed the column after 6 runs with 5% ACN 10min, then gradually to 70% ACN and hold for 10 min.

My question is:
1. How often should I run the wash, every 6 runs, less or more?
2. What % organic and gradient program should I follow?

I appreciate any comment.

During a method Validation, I have always performed an experiment where I would extend the chromatogram run by at least X3. If I can't see any peaks during this time I can justify a reduction in the run time. For your material I would perform some runs lasting at least 30 minutes. If you dont see peaks coming off, you could reduce the time down to 6 minutes.

First, thank you all for your valuable comments.

If I can't see any peaks during this time I can justify a reduction in the run time.

At what wavelength? Is it right that a lower wavelength such as 210nm is more universal for dirty substances?

If you dont see peaks coming off, you could reduce the time down to 6 minutes.

I read from a website for basic HPLC design (can't remember the address) that as a rule of thumb, flush the column for an additional 10 min after the peak of interest comes out.
I don't know the rationale behind it.
If the total run time is 6min, should I carry out a wash frequently between analysis?
But how frequent?

I appreciate your further comments.

If my analyte peak eluted at 3.5 minutes isocratic, and at 333nm, and no matrix or other peaks were observed (and documented), we would likely shorten the isocratic run time to about 6 minutes.

Typically, we clean out the column (and some stuff cleaned off may not even absorb at 333nm, so you may not even "see" crap coming offf the column) after our sequence has completed, using something like 90% organic for 15 minutes.

If we do have later-eluting peaks not the analyte, we either extend the run time or install a gradient with a clean out program, but that also requires proper re-equilibration time. We do not (in my lab) try to adjust run times so that late-eluting peaks elute in a non-crucial region of the following chromatogram, some do.

Hi Friederike,
oops you are right, the phosphate may precipitate it.
Else, reverse the gardient i,e decrease the ACN and incresa it afterwards.

Many thanks to you all for pointing the way to solve my problem.

For your material I would perform some runs lasting at least 30 minutes.

I analyzed serum samples from 20 in-patients using an extended isocratic program (35 min). The longest time in which a peak appears (at 210 nm: I also need to monitor other wavelengths because I have to identify the unknown by UV spectrum) is 15 min. Therefore, if I use the current run time (13.5 min), the unknown peak will appear at 1.5 min of the following run, which does not elute with my target compound (RT 3.4 min).

Hence, I decided to shorten the run time slightly to 12.5 min.

Typically, we clean out the column (and some stuff cleaned off may not even absorb at 333nm, so you may not even "see" crap coming offf the column) after our sequence has completed, using something like 90% organic for 15 minutes.

In that case, do you mean I can do without the wash per every 6 runs?