Dear Sirkiran Kota, Thank you for your suggestion. I equillibrate the column normally for 1 hour and my run time of experiment is 15 mins ( last analyte eluted in 10 min in first day experiment, 12 min in 2nd day experiment and 13.5 min in 3rd day experiment). I will try with increasing run time of my experiment.

Dear Colleagues,

Which sort of compound do you isolate with this column and buffer ?

You said in title "basic compound" pKa = 9.5. So, at pH = 7, your compound is not charged ?

Is there an influence of the lenght of your column (150 mm) compared to the strenght of "sticking" of your compondon on the column, and the retention time observed ?

In general, changing in retention time quite rapidly can be due to a general 'unstability' of your system (changing in compound composition, temperature, in column equilibrium with time ...).

Best regards

Didier J

Thank you Didier Jambou and Siakiran Kota. Explaining little bit deeply about my experiment, I have four compound to seperate, the first peak appears in retention time 3.5, 2nd peak in 7.5, 3rd peak in 8.5 and last peak in 12 (around and this peak fluctuate). I have developed the organic solvent and buffer concentration (20:80) and pH to be 7. If I increase organic solvent, the resolution will not be good and first peak elute faster as well as if I change the pH, there will be mergining of peaks. Also, regarding equillibration time and other constants mobile phase composition, pH are fixed.

Actually I am doing a pharmacokinetic study of drug and its metabolite by designing suitable dosage form to human. So, I am in stage of method development and validation in HPLC.

Dear Krishnasarma Pathy, the temperature has great effect in the resolution of my analytes. Normally, the retention time decreases with increasing the temperature. But for ionizable basic compound like mine, the retention time increases with increase in temperature, so I do not prefer high temperature (so I adjusted to 30). The reason for increasing the retention time with increasing temperature is due to lowering of effective pKa value with increasing temperature that can lead to decrease in solute ionization (Handerson-hasselbach equation) and atypical increase in retention at higher temperature. Please correct if I am wrong.

Dear Sabin Shakya ,

I better understand your system, and wiht these new informations, I could do extra comments:

- you are in the case that I had experimented in the past (see publications), when I had validated a system to separate 8 prostaglandins and leukotrien from biological fluids (cell culture medium then blood).

When you have a lot of compound on a column, and necessity to all separate them, you have a "contradiction" between the fact: the best separation, but not a too much long retention time. And if a compound "sticks hard " to the column in your conditions (the longer retention time), so there is a greater incertitude for the retention time than for the first peak with more reason than that. And the peak is less thin than the first ones. The essential is to be sure , by standards and control,s that it's always the compound you want.

- the second thing, and it joins another discussion in this topic, is the use of a "lipophilic" column for charged compounds, as these are more adapted to hydrophobic molecules, anhd in your case, these are not the best column for the best results. Perhaps are you obliged to use this system (more complicated) , but did you test other colums (ie -NH2 or others hydrophilic systems)

I used in the past C18 colum, because leuko and prostagalandin are hydrophobic, with a mobile phase acetonitril/water, at room temperature, and the RT were realtively stable, even at 30 min, but I used a 30 cm column.

Best regards

Didier

Thank you Didier Jambou sir

Just curious. Have you ever checked if the mobile composition might by varying (ie not consistent)? In their troubleshooting booklet, Waters technicians say, if you make an error of 1% the retention time changes by 5-15%. You can also watch an important podcast on this issue from Agilent I have attached the links. Their podcasts are very good and have helped me a lot in the technical part of HPLC.

http://www.chem.agilent.com/en-US/products-services/Columns-Sample-Preparation/LC-LC-MS-Columns/Pages/tshooting_retention_time.aspx

http://www.waters.com/webassets/cms/library/docs/wa20769.pdf

How about using the buffer with the pH more than 9.5?

If you require only phosphate buffer try with phosphate buffer pH 8, or else try with a buffer with pH more than 9.5.