How can I better separate compounds using reverse phase chromatography? - WKB70232

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ANSWER

Below are some general tips that could help improve resolution:　

1. Temperature can help resolve compounds. For example, the ACQUITY BEH C18 particle is very durable and can withstand temperatures up to 80 °C at low pH and and up to 60 °C at high pH. Try changing in increments of 5 degrees. Sometimes lowering temperature can give better separation. 　

2. Try running a shallower focused gradient where the peaks of interest come out. 　

3. Changing your organic modifier can sometimes give changes in separation.　

4. pH can have an impact on separation. For example, the ACQUITY BEH C18 is very pH tolerant from pH 1-12.　

5. Increase the resolution capabilities by going to a longer or smaller particle size column. The larger your resolution coefficient (Length/Particle diameter - L/dP), the greater the ability for the column to separate the compounds. 　

6. Optimizing flow rate can help separate compounds. 　

ADDITIONAL INFORMATION

Bioanalysis Method Development Wall Chart:

Efficient Reversed-Phase Method Development Strategy Wall Chart: