How to correct the delay from detection to colletion in rpHPLC? - (Nov/27/2007 )
Hi guys,
Recently I used rpHPLC to purify my peptide. From the chromatograph I estimate I should have 9mg peptide in total, while I actually got only 4mg. I think I lost at least half of my peptide because of the delay from peptide elution detection to product collection. I notice that my collecting tubing is ~1.3m long and can contain ~1.3ml solution. It is very possible that I only collected half of my target elution peak. Here I have a few questions for you guys.
1. Does shortening the collecting tubing help?
2. I was told that the HPLC system itself was set some delay to correct the delay from detection to collection. If I shorten the tubing, I may collect the target peak too late and then I still lose sample. So I am thinking maybe I can use some colorimetric protein to test my HPLC system, in which I can see the colored protein come out. This way I will be able to tell how much delay my HPLC system has. Could you please recommend what colorimetric proteins should be proper to my testing purpose? Which companies have this kind of product?
3. Can you recommend some other way to test the delay?
Thank you very much!
You can calculate exact time delay from volumetric flow rate, tubing (internal) diameter and length.
Recently I used rpHPLC to purify my peptide. From the chromatograph I estimate I should have 9mg peptide in total, while I actually got only 4mg. I think I lost at least half of my peptide because of the delay from peptide elution detection to product collection. I notice that my collecting tubing is ~1.3m long and can contain ~1.3ml solution. It is very possible that I only collected half of my target elution peak. Here I have a few questions for you guys.
1. Does shortening the collecting tubing help?
2. I was told that the HPLC system itself was set some delay to correct the delay from detection to collection. If I shorten the tubing, I may collect the target peak too late and then I still lose sample. So I am thinking maybe I can use some colorimetric protein to test my HPLC system, in which I can see the colored protein come out. This way I will be able to tell how much delay my HPLC system has. Could you please recommend what colorimetric proteins should be proper to my testing purpose? Which companies have this kind of product?
3. Can you recommend some other way to test the delay?
Thank you very much!
1) shortening the tubing won't help because it is very low volume for its length.
2) colored compound (protein is not necessary, you are running rp) is a good idea but you have to watch carefully for the start of color to be accurate.
3) k.b.'s suggestion is good but you need to know the holdup volume of any module in between the monitor and the collector.
If you know the volume between detector and collector, it's very easy to correct.
let's say you collect 0.5 mL per tube, you know the molecule will not be in the tube in the collector when you will see the peak on the screen, but in the third one (1.3 mL later).
Did I answer?
mdfenko, I have a feeling he has tubing this long because he's using it as pressure dampener so there won't be much between monitor and collector.
I think there must be delay setting somewhere in the system. Even so simple devices as AKTAprime have it.
k.b., i wasn't refuting what you suggested, i agree with you. i was just adding other things ionchannelbk had to take into consideration when calculating the volume.
as an example, i have an aktapurifier. after the optical cell there is a conductivity probe, pH probe, flow restrictor and a switching valve before the final tubing to the collector (which has a drop holder syringe for fraction switching, i'm not sure if that is its actual name but it gets the function across).
as you can see, tubing volume is not necessarily the only consideration when calculating the delay.
mdfenko, that's ok, I understand you and agree.
Thank you very much, guys. Sorry not to reply in time; I have been too busy to check the forum. I agree with mdfenko that the delay may be not only caused by the collecting tubing, but other factors may count. Besides, the HPLC system may already set a delay there (But I did not find where to set it in the software.). This is why I prefer to use a colorimetric tracer to detect the exact delay. Do you guys have any this kind of colorimetric tracer (don't have to be some protein) for recommendation? K. B., why can a pressure dampener cancel the delay between monitor and colletor?
thanks again, guys.
you can try almost anything that has color (you may have to find conditions that will elute it from the column). maybe you can try bromphenol blue.
you should find the delay setting in the part of your program where you set up the fractionation.