Rest the Sampe or Addict the Collumn??

[Leonno85]

Hello everybody....bringing one more question for you...

I got a HPLC method for progesterone....seems very good...Rt = 4 min, no co-elution, nice simmetry. All the standards in linearity assay went good, with the same characteristics. It was on last week.

Here's the method:

ACN/Water 70:30
Lichrospher C18, 5um, 250 x 4,6, Merck
1 mL/min
Oven 30ºC

Yesterday (five days later) I tried to run out a solubility assay using progesterone in different media...pH 1,2; 4,5; 6,8 and different concentrations of SLS in water (0,1 - 1,0%). I took care on dilute my sample in mobile phase, in order to decrease pH and surfactant effect on the collumn. Very well...progesterone peaks eluted on 6-8 min, a great variability on the retention time. Although peaks look good (simmetry, etc) I thought i missed this assay.

Today, i tried to run the samples again. And surprise: the peaks "returned" to Rt = 4 min....I confess I don't understand what happens...is it possible that the samples need to "rest" (stabilize) before injection, or yesterday the collumn was getting used of progesterone?

I am really confused about this....since now, thank for answers...

Have a nice day!

Leonno,
São Paulo
Brazil.

[Gerhard Kratz]

What is the pH of your sample? Different to your solubility tests? What Buffer you used? How you stored your column in the five days, in mobile phase?

[Leonno85]

Buffers:

pH 1,2 = simulated gastric fluid (USP, 2010)
pH 4,5 = acetate buffer
pH 6,8 = phosphate buffer

About sample pH, i didn't measure it We add progesterone in 20mL of ACN, then complete to 100 mL of water.

Anyway, I'll try to make a sample and check it.

Note: progesterone pKa (basic) = -4,8; (acid) = 19. So progesterone isn't ionizable on physiological pH range...

Thank you!!

[HPLCaddict]

I got a HPLC method for progesterone....seems very good...Rt = 4 min, no co-elution, nice simmetry. All the standards in linearity assay went good, with the same characteristics. It was on last week.

Here's the method:

ACN/Water 70:30
Lichrospher C18, 5um, 250 x 4,6, Merck
1 mL/min
Oven 30ºC

Rt of 4 minutes under these conditions corresponds to a retention factor of roughly 0.5-0.6. That doesn't look "very good" to me. I don't know if this is the source of your problem, but usually you shoout for a retention factor of at least 2.
Did you use the same mobile phase for all of these runs or did you use fresh mobile phase every time? ACN will evaporate over time...

Just as a side note, I wonder how many millions of hours of working time have been lost in all the labs around the world by the infamous "250x4.6mm, 1mL/min"