By Anonymous on Saturday, August 23, 2003 - 08:12 pm:
I am developing a HPLC method for a basic compd. using the following conditions:
Column : C18
MobilePhase :Octane sulphonic acid disodium salt:Acetonitrile(66:34)
Column Temp : 40deg C
Flow Rate : 1 mL/min
Elution Time : ~12.0 min
Sample Preprepation : all samples and standards prepared in phosphate buffer: methanol(3:1)
Initiial validation on linearity(50-150%) and recovery worked perfectly well. Both Linearity and recovery were carried out using different batch column and different HPLC. However the same method did not work as validation progresses. Retention times as well as areas(different areas from formerly) were not consistent using both the above HPLCs. Both the same and new columns were tried but failed. There is nothing wrong with the HPLCs and columns as other types of runs were later carried out with them and worked perfectly well.
Does anyone know why?
By Anonymous on Saturday, August 23, 2003 - 10:05 pm:
As an initial guess, you probably did not equilibrate long enough either at first, or later. Octane sulphonic acid is an ion-pairing agent, columns take a long time to equlibrate when using this modifier.
By Anonymous on Sunday, August 24, 2003 - 02:43 pm:
Do I understand this correctly? There is no buffer in your mobile phase?
By Anonymous on Sunday, August 24, 2003 - 06:27 pm:
I would like to clarify the following:
Mobile Phase : octane sulphonic acid disodium salt (pH3.0): Acetonitrile (66:34)
Sample Extraction Solvent : Phosphate Buffer(pH2.7):Methanol (3:1)
The column was being equilibrated for more than a day.
By Anonymous on Monday, August 25, 2003 - 06:35 am:
1: The OSA is probably not going to give you enough buffering capacity.
2: I use OSA in several ECD methods, it can take several days to really bring the column into equilibration.
3: Why are you using OSA?
4: Is your column oven working correctly?