[hplcnewuser1]

Hi, I am trying to purify a peptide using agilent 1100 hplc. I swapped the analytical c18 column 25mc x 4.6mm 5um with a preparative c18 column 15cm x 22mm 5um. Unfortunately Agilent 1100 pump only goes up to 5ml/min and the purification column specs are at 10ml/min. I am OK with 5 mL or less just trying to understant how can a system perform at 10 ml/min when max is 5 mL/min?

I decided to shot 100uL of 1% peptide sample in water and run a H2O/ACN gradient at 3ml/min, only to find out the flow cell is leaking. Is it normal for flow cell to leak at 3ml/min?

How can I convert the agilent 1100 to purification column? How can I know when to collect the fraction if the flow cell leaks after 3ml/min?

Could I inject up to 1ml with agilent 1100 but the sample loop is only 900uL capacity. Is this going to cause a problem?

[Arkcon]

Hi, I am trying to purify a peptide using agilent 1100 hplc. I swapped the analytical c18 column 25mc x 4.6mm 5um with a preparative c18 column 15cm x 22mm 5um. Unfortunately Agilent 1100 pump only goes up to 5ml/min and the purification column specs are at 10ml/min. I am OK with 5 mL or less just trying to understant how can a system perform at 10 ml/min when max is 5 mL/min?

It appears you have an analytical model.  You will have to ask Agilent if they have a higher flow pump head to replace your current one.  You will lose some analytical flow control, however.

I decided to shot 100uL of 1% peptide sample in water and run a H2O/ACN gradient at 3ml/min, only to find out the flow cell is leaking. Is it normal for flow cell to leak at 3ml/min?

No.  All of the pressure should be at the head of the column, the exit of the column, and the flow cell should be at very low pressure, less than 100 psi (or 6-8 bar, as Agilent prefers to use,) which is the maximum a flow cell can take. If you sent high flow through without a column, you may have sent too much pressure through, and damaged the flow cell.  Otherwise, you have some plumbing work to attend to.

How can I convert the agilent 1100 to purification column? How can I know when to collect the fraction if the flow cell leaks after 3ml/min?

You've done all you can, changing the column, its time to get in touch with Agilent.  You're not going to get anything if the system has a leak, the sensors will see the liquid sooner or later, and shut the system down.

Could I inject up to 1ml with agilent 1100 but the sample loop is only 900uL capacity. Is this going to cause a problem?

Interesting.  You seem to have a mid-range loop.  My analytical system only injects 100 uL max.  I don't see how being 9/10's of the way there is a major problem.  However, you can have a larger loop installed.  You will lose analytical accuracy with a larger loop.

You have switched column lengths, so any analytical work you've done for this peptide with the 25cm x 4.6mm will not scale to your 15cm x 22mm, but I don't know if that's part of your plan.  Just a heads up.  There are dozens of flow rate calculators online, they can tell you how to scale your gradient and flow when you switch columns.  If the silica formulation is exactly the same, you may get good results.  Or at least, a good starting point.

[Babcock_Hall]

Just out of curiosity are you using an ion pairing reagent and if so, which one?  Are you using partial-loop (partial-fill) injection?   We often do analytical and preparative HPLC work on proteins, but not very frequently do we work with peptides.

[hplcnewuser1]

yes it is analytical, not that i knew the difference until now.
Pressure was 50 bar for preparative column. Very good to know we have bad flow cell. Now I can ask my supervisor for new flow-cell. I will definitely use one of those calculators for converting flow rate.
I have the same injector but if you go to injection/configuration and change seat capacity from 2.3 to 1400, it will let you set the injection 1000. I changed seat capacity to 400 and I am injecting 500ul now. Hopefully it will not cause any issue.

Babcock,
I am using 0.1%tfa water A and 0.1%tfa 90%acn B. analytical column is zorbax 300sb C-18, preparative is vydac c-18 10um pore size. Similiar except for lenght and width no ion  pairing or partial loop injection.
I am tryign to purify out the tfa which is left over after peptide synthesis as well as few fractions which are partial synthesis.

First time doing purification HPLC, Thanks for all your answers!