Chromatography Forum

dear friends,
i am developing a method for levidopa carbidopa and entacapon combine tablet. the peaks for levidopa and carbidopa are quite close and the resolution is not reproducible all the time. i am using bffer ph3 and acn gradient for 30 min. right now i am trying inertsil ph-3 column. can anybody suggest me how to inprove the resolution and which column is best for non retain compounds.
thanx in advance to all

Go for example at:
www.chrombook.merck.de

look for applications number:

021391
021375

both of them can be twicked a little to be even shorter
hope this helps.

for future needs, ask all your column vendors to provide you with applications CDs. you will find a very broad library which will be very handy in the future.

dear friends,
i am developing a method for levidopa carbidopa and entacapon combine tablet. the peaks for levidopa and carbidopa are quite close and the resolution is not reproducible all the time. i am using bffer ph3 and acn gradient for 30 min. right now i am trying inertsil ph-3 column. can anybody suggest me how to inprove the resolution and which column is best for non retain compounds.
thanx in advance to all

For resolution

try using a column length of 250 mm as it provides a delayed elution and a particle size of 5 micron.

resolution can be achieved by having a step by step increment in ur % B.

by adjustment of flow rates.

by achieveing a balanced wavelength for your two combination of drugs or by wavelength programming.

What kind of retention do you have? What is resolution between and cardidopa. I assume that this two are a critical pair because you should not have a problem retaining entacapone.
Check this method for the retention of DOPA:

http://hplcmethods.com/compound_101.php

Levidopa and carbidopa have different structures, hydrophobicity and pKa you can use these differences in mixed mode chromatography. Retention of DOPAs can be from 3 minutes to over 30 minutes depending on the mobile phase.
For UV you can use sulfuric, phosphoric acids or corresponding buffers
For LC/MS/ELSD you can use ammonium formate, ammonium acetate, formic acid and TFA.

Regards,

Vlad

:

dear friends,
i am developing a method for levidopa carbidopa and entacapon combine tablet. the peaks for levidopa and carbidopa are quite close and the resolution is not reproducible all the time. i am using bffer ph3 and acn gradient for 30 min. right now i am trying inertsil ph-3 column. can anybody suggest me how to inprove the resolution and which column is best for non retain compounds.
thanx in advance to all

thankx everybody again,
the retention time of levidopa and carbidopa are 2.5min and 3.9. the initial buffer acn ratio is 98:2 and i am using 250 mm column. will it be helpful to start with 100%water?
with regard
dhiren

Void for 250 mm column at 1 ml/min is approx. 2.5 minutes. One of your peaks is in void/ not retained. 100% vs. 98% is not going to change a lot. Any matrix effect will interfere with your first peak (2.5 minutes). May be AQ type of column will help a little.

I am not familar with your analytes but as general, you can try the following to help holding:
1) some columns are designed for very hydrophilic compounds;
2) use methanol instead of ACN;
3) use ion pairing reagents with long alkyl chain, high concentration, and a right pH if necessary.
good luck,