Chromatography Forum

Jentle man,

Anlyte peak is showing the low Plate count(1800) and fronting with the following chromatographic conditions.

I have changed the mobile phase (TFA, Phosphate buffer, Ammonium acetate and also with ion par reagents) but still it same.

Same results was obtained with the different columns of different brands of C18, C8(ACE, Symmetry, Intertsil, HILIC, Zorbax etc.)


Current Method:
Column : Symmetry C18, 3.5µm, 150 mm x 4.6 mm or equivalent
Flow rate : 1.0 mL /min.
Wavelength : 254 nm
Column temperature : 25°C
Injection Volume : 20 µL
Data acquisition time : 10 minutes.

Mobile phase preparation:
Mobile phase-A:
Add 1 mL of Ortho-phosphoric acid to 1000 mL of Milli-Q water and filter the mobile phase through 0.45µ nylon membrane filter and degas it.
Mobile phase-B:
Acetonitrile
Pump the mobile phase A and B at the ratio of 55:45 %v/v.

Diluent: Acetonitrile


Please suggest to get sharpness and minimize the fronting.

One of the possible causes of the peak shape disturbance is the fact that you're using RP stronger eluting diluent than your actual mobile phase. Try to dissolve your standards / samples in diluent that has at most the same composition with your mobile phase, or use low organic phase diluent (if possible) to eliminate this possible influence factor.

Regards

Another possibility is overloading the column. The concentration of your samples may be too high. A quick check can be done by reducing the injection volume to see if peak shape improves.

If you're using anything like a modern HPLC system (Waters 2690, Agilent 1100, etc), then 20ul of ACN is too much for a 150x4.6mm column running 45% ACN. If preparing your sample according to Zokitano's advice is problematic for whatever reason, try simply injecting less of it. I have to do this frequently due to matrix constraints and find that a 5-10ul injection volume generally works quite well. The chromatography doesn't suffer, however care should be taken to be sure that you're not leaving junk on the column (i.e. rinse it with a high concentration of organic).