Help Lowering Retention Time

[DagOnChrom]

Hello,

My boss asked me to develop a HPLC method for three flea killers, PBO(Piperonyl Butoxide), Sumithrin and Nylar.

After much trial and error, I made a working method.

Mobile Phase: Acetonitrile/H20 75/25(v/v)
Column: Luna C18, 5u, 4.6 mm x 250 mm
Flow: 2.0 ml/min
Detector: UV at 254 nm

The retenion times for PBO is 6.228, Nylar is 6.858, I-Summithrn is 17.110, and II-Summithrn is 18.283 (all with good separation) The total run time is around 19 minutes.

My boss wants the total run time to be around 12 minutes. So I need to figure out how to get the retention time of summithrn down.

When I change the mobile phase to have more acetonitrile, it makes the total run time much lower, but PBO and Nylar peaks start to overlap each other. Same thing happens when I switch to a 150mm column.

Any ideas on what I should try next to lower the retention time would be greatly appreciated.

Thanks,
David

[tom jupille]

The most straightforward approach would be to simply increase the flow rate to 3 mL/min. That will cost you some resolution, but if you have some excess to begin with you should still be OK. Of course, you will run a higher pressure.

Or, use a 3-micron packing in a 150 mm column. That should keep the resolution about the same and shorten the run time.

Or, try increasing the temperature and running at a higher flow rate. The higher temperature should give you more plates and a lower pressure, but may also change the selectivity (peak spacing).

Or run a gradient.

Or redevelop the method using methanol or a different column (in hopes that the selectivity will be better; you can then trade off resolution for run time).

[Perreman]

I agree with Tom's suggestions.
I myself would probably try to crank up the flow rate and add a gradient step (up to 100 % Acetonitrile) when Nylar has eluted.

If you go for the column change, this chart might be of use http://www.waters.com/waters/promotionD ... d=10048475.

[Consumer Products Guy]

After much trial and error, I made a working method.

I hope there was scientific thought and your learnings put into each new try!

Column: Luna C18, 5u, 4.6 mm x 250 mm
Flow: 2.0 ml/min Detector: UV at 254 nm

That's an "old school" column dimension. If boss wants shorter run times, ask for a 3 micron column with narrower diameter, 4.6mm is large, noting your 2ml/min flow rate.

I was also at first going to suggest gradient elution, but then your re-equilibration time must be also taken into account, that's very important.

Other types of C18 column material - even from Phenomenex - can affect your separation. Since you've volunteered your 3 analytes, maybe their tech help can try out for you on their vast inventory of different types of columns.

Solvent change to methanol from ACN, or even a mixture of both, can affect separations. I'd also try 10 or 15 degrees lower column temperature, and higher, and see if that can help the separation too. Higher temperatures will lower solvent viscosity too, maybe allowing quicker runs. Also, a few percent of THF (unpreserved) modifier is real good for affecting separations. Columns from other vendors can be tried, each vendor offers many similar but different C18 columns. Maybe phenyl or other types could help here.

This is the fun stuff, enjoy.

[Vlad Orlovsky]

if you want to stay with the same column try to add 5-20% of THF, it will help you elute compounds faster.

[DagOnChrom]

Thanks for all the ideas.

I got it down to 10 minutes.

I ended up changing the flow to 2.5 ml/min and changing the column to a C18 3u 150mm 4.6mm column.

I would have tried using a gradient method, but my boss wants to keep it isocratic. I'll probably try a gradient when I'm bored on a weekend.

Once again, thanks. It would have taken a bunch longer without everyone's help.

[aniket1706]

Try using fused core columns which provide good resolution with shorter runtime. You can also use columns with less carbon loadying like C-4.