It sure would help to know what your actual sample and method are, but regardless of that information, the initial advice will be the same... you need to develop a method to resolve any impurity peak(s) away from the main peak. Without knowing anything about your sample or detailed method, we can not make any specific suggestions. Here are some general comments:

• Make sure your sample dissolves 100% in the mobile phase.
• You are using a 300 A large pore C4 column, so it sounds like your sample is large (>10,000 Mw). Try a large pore C8 or C18 column for better retention.
• Try several standard gradient runs to find the conditions which retain, hold, then elute the compounds apart. Try using neutral, basic and acidic modes (since we have no idea what you are analyzing).  Be sure and start with binary gradients of Water/ACN and also try Water/MeOH in all three modes (Water/MeOH has much better solubility characteristics than ACN). *Run 95% Water to 98% organic (not 100%).
• Be sure and flush the column with a STRONGER solution after each run.

Dear Rekha,

Assuming that you see your main peak and the impurity/s, will have to play around with parameters, such as column temperatures, pump flow rates and different types of gradients and proportions of the two different mobile phases.

Kind regards,

Ade Kujore

Marketing

Cecil Instruments Limited

Milton Technical Centre, Cambridge CB24 6AZ

United Kingdom

email:- [email protected]

telephone:- +44 (0) 1223 420821

fax.:- +44 (0) 1223 420475

web site:- www.cecilinstruments.com

I would assume that the impurity is close in structure and chemistry to the API.  Adjust your mobile phase, gradient ramp...

Change mobile phase B to 95% ACN.  Are you sure the impurity has to be protonated (requires formic acid)?

I am sad to hear that "the use of the AN is bad".   Thus, I have repeatedly been declaring that "please use the alcohol instead of AN".  

AN is surely the dangerous drug or reagent to the human health and also to the HPLC pump.   Further, AN+TFA system has always given low-recovery for thiol-molecules, although firstly I have published by this AN system (please see files isocra PTH,  and Isocratic Biotin Determination), and I must have to improve these separation methods to safer methanol and 2-propanol systems (please see files PTH Roma MeOH, and Biotin Determination 2-Propanol), respectively.   For protein and peptide analysis, please see file "Lysozyme by RP-HPLC" (Gradient elution method using 3-phases system of the acidic-phosphate-buffer (pH 2.0) / 2-propanol / ethylene glycol system).   This 3-phase system has been invented by German Drs. Wolfgang Mönch and Walter Dehnen (Universität Düsseldorf, Düsseldorf, Germany) giving high column-inlet pressure in commercial 25-cm length column.   Therefore, we have manually made a short column of 5-cm to reduce the column-inlet pressure (now 5-cm packed column can be obtainable commercially).    It is important that protein separation by RP-HPLC should be performed by the gradient elution and the short ODS column (similar to affinity column, which is designated as HPLC-Hydrophobic-Affinity column or HPHAC) by me, gives sufficient peak shape.   Then, even the small molecule of biotin-ADAM ester is able to be separated and determined by using short 3.3-cm-length avidin-affinity column (high-performance-avidin-affinity column; HPAAC; file Biotin Determination 2-Propanol).   

Dear Rekha,

There are certain parameters that you can change.

1) initially you decrease your flow rate to separate. it not happen then

2) you can change your gradient in your mathod or

3) you can change your organic phase from ACN to MeOH, or

4) if you are not able to separate after all above then you can change your column.

It sure would help to know what your actual sample and method are, but regardless of that information, the initial advice will be the same... you need to develop a method to resolve any impurity peak(s) away from the main peak. Without knowing anything about your sample or detailed method, we can not make any specific suggestions. Here are some general comments:

• Make sure your sample dissolves 100% in the mobile phase.
• You are using a 300 A large pore C4 column, so it sounds like your sample is large (>10,000 Mw). Try a large pore C8 or C18 column for better retention.
• Try several standard gradient runs to find the conditions which retain, hold, then elute the compounds apart. Try using neutral, basic and acidic modes (since we have no idea what you are analyzing).  Be sure and start with binary gradients of Water/ACN and also try Water/MeOH in all three modes (Water/MeOH has much better solubility characteristics than ACN). *Run 95% Water to 98% organic (not 100%).
• Be sure and flush the column with a STRONGER solution after each run.

Bill provided excellent suggestions above. I am curious about something else. How do you know that the impurity is ~ 2% if you did not separate the analyte from the impurity. What type of detector are you using?

thanks to all for your Valuable suggestion. we have tried with different solvent composition, columns, gradient, flow rate. But nothing worked out..

Dear Rekha,

What is the chemical nature of the analytes and impurity  you looking at and which type of detector are you using.

Kind regards,

Ade Kujore

Marketing

Cecil Instruments Limited

Milton Technical Centre, Cambridge CB24 6AZ

United Kingdom

email:- [email protected]

telephone:- +44 (0) 1223 420821

fax.:- +44 (0) 1223 420475

web site:- www.cecilinstruments.com

You will have to change the proportionality of concentration between the mobile phase and the stationary phase (column) because the impurity is so chemically and structurally similar to the API.  This means the HPLC column remains the same but you change the composition of the mobile phase over time (thus gradient).  Add Methanol (~5%) to mobile phase B and use a slow gradient ramp around the API.

Be sure you are using a C18 HPLC column that has a 'high carbon load (~12-18%)'.

A C4-300 does not have sufficient resolving power.  The 300 is the pore size (I presume you are separating a protein).  Use a 'Discovery' C18 that is endcapped (all unreacted silanols are blocked, thus the higher carbon load).

Using Column C18 will be best.. 

thanks to all for your valuable suggestions