Help me seperate these peaks (pics)

[firework1]

This is N-(4-hydroxyphenyl) retinamide standard. I am using 15cm C18 column, 1ml/min, 80/20 ACN/H2O. The peaks are sort of broadened, and I could not seperate them, though I did not expect to see so much impurities in standard. I tried to move them to a shorter time and squeeze imputity peaks into one, but did not help. I also acidified mobile phase with 2% Acetic acid, not much difference.

Anyone has some ideas?

Thanks.

[Bryan Evans]

A couple of options:
1. Decrease particle size (use 3um if you are currently using 5um)
2. Try switching to MeOH (use 90:10 MeOH:H2O for similar retention)
3. Look at sample diluent and ensure its similar to solvent strength as
mobile phase
3. Use a stronger solvent for B (e.g. THF)
4. Go to normal phase - (e.g. bare silica particle).

[philippem]

Hi Firework ,

There are many other things you can try:
change solventcomposition , more water will increase your retention time and the separation,
use methanol instead of acetontirle to change the selectivity
use longer column
use another stationary phase, either C18 or CN or ????
use smaller particles (3 um instead of 5um)

that is method development


by the way, what is the claimed purity of your standard ?

regards

philippe
.

[firework1]

Thanks guys

The purity on C of A is : >95%

[Chandresh K. Soni]

U can Also use Phenyl Column with Gradient Conditions.
Mobile Phase is ACN : Buffer ( 0.02M Ammonium Acetate and pH 4.65 Adjusted by Acetatic Acid.) :: 20 : 80 to 40 : 60 in 30 Minute.
it may be Possible to Solve your Problem.

[AdrianF]

The following reference gives conditions 80:18:2 ACN:H2O:CH3COOH
15cm SymmetryC18 3.5u at room temp. 0.7ml/min
http://www.nant.org/dl/PubsUpdate-20050 ... a_2004.pdf

What type of C18 are you using?

Try different temperatures, cooling can sometimes improve resolution.

[firework1]

I am using exactly the same column as in this artical, their 4-HPR peaks are broadened as well. I found out with or without acetic acid does not make much difference. Appearently, they did not have much impurities as I do, I have difficulty seperating impurities from 4-HPR

Anyway, I will try bring up column temp, if this does not work, I will switch to MeOH. I do not think they will let me buy another column.

I will let you know.

[unmgvar]

firework1

have you tried a gradient?

[firework1]

I have thought about gradient. The dilema here is, I want move these peaks to shorter time so that I could have better peak shape; but I also want to have better seperation. Would you suggest a gradient that serves both purposes? I am using 80:20 ACN:H2O

firework1

have you tried a gradient?

[Uwe Neue]

I would first try methanol, then temperature. Solvent changes tend to give larger differences in selectivity.