HPLC Resolution

[cody84]

Hi, I'm having trouble separating some flavours and colours from a benzocaine peak. My mobile phase is 56:40:4 MeOH, water, acetic acid. I can't find the chemistry for any of the compounds except benzocaine. Any suggestions I'm kind of new to this.
Column: kinetix C18 2.6u 100x 4.6, 100A

Thanks

[cody84]

actually, are there any general guidelines to follow when optimizing an HPLC method? Specifically stability indicating methods.

[Bryan Evans]

Usual recommendation is for 2<k'<10
Changing buffer pH can change elution times of different solutes.

Just curious - what HPLC are you using? What is the current pressure of your system?

http://www.fda.gov/downloads/Regulatory ... 128204.pdf

[cody84]

I'll try changing the pH, do you think doing this through a gradient or changing the pH of the MP itself would work better. It would be kind of hard adjusting the pH after like 2 injections and preping the system.

Its a varian HPLC, PDA detector.

[cody84]

pressure was around 340 bar with 100% MP

[Bryan Evans]

Yikes! 340 Bar is craziness for this assay. You're putting unnecessary stress on your instrument.

Here are some suggestions:

1. Purchase a good book on HPLC and / or take a class on HPLC

2. Take the current column out of your instrument and set it aside.

3. Purchase a 5um particle size column from a reputable column / instrument manufacturer.
When you get comfortable with 5um, then you can start to look at high performance 3um columns.

4. Find a mentor / read the regulations ect. for stability indicating assays.

[cody84]

sorry that's 240 bar haha
and yea, I don't have the option of school or a mentor here. I'm trying to learn as I go for now. Have done plenty of routine HPLC and had a course in college, however there was never much on the chem involved.

I will look for some books though, have any good suggestions?
I heard there's a good one by Snyder.

[tom jupille]

I'll throw in a plug for our "Fundamentals of HPLC" web course, which will run on July 12, 14, & 16.
http://www.lcresources.com/training/trfund.html

[bisnettrj2]

viewtopic.php?t=12365

[cody84]

cool thanks!

[wburt]

Have you had any further success with your problem? I have several suggestions -
1) try a different type of column. Instead of a C18 column, try a phenyl column.
2) Use a longer column to obtain greater resolution. A longer column will produce a higher pressure, so you may need to adjust your flow rate.
3) Decrease your methanol some. This will usually increase your resolution. However, different compounds will elute at different rates and this may backfire.
4) Try acetonitrile instead of methanol. The solvent strength of ACN is stronger than methanol. A good starting point is about 45% ACN. You can adjust from there.
5) Have you tried a gradient separation?
6) What is your current column oven temperature? If you can increase your temperature, the peaks will move in closer, but they will also become more narrow and that may improve your resolution.

Another option is to try gas chromatography to analyze for the benzocaine.