can anyone explain this?

[mas23]

I have a compound that dimers to form an impurity which has 2 COOH groups. My issue is that I am trying to develop an impurities method that can properly quantitate this impurity amoung many others. I have the cromatography sorted out for the main compound and the rest of the impurities but am having trouble with the dimer impurity. The system right now is as follows:

Atlantix T3 150 x 4.6, 3 um
MPA: 0.1% TFA in water
MPB: 0.1% TFA in THF
Column Temp: 45C
Sample Concenration: 1 mg/mL
injection volumn: 10 uL
diluent: 1:1 water:ACN
Gradient:
0-25% B in 5 min
25-45% B in 20 min

The dimer peak requires a very steep slope to get a sharp peak and the Main peak requires a shallow gradient slope to provide seperation with similar impurities.

My problem is that even though I have got the dimer impurity peak to sharpen up with the steep gradient slope it still tails badly. It looks like a baseline shift occurs when the dimer elutes off of the column and never comes back down. I have also tried increasing the TFA concentration to 0.4% and I am still seeing the tailing however the peak is very sharp. Any words of advise are much appreciated!!!



Thank you in advance!!

[unmgvar]

first be very careful of using a 0.4% TFA conc.
check the pH, see that you are not below the column specs

how long do you re-equilibrate the gradient?
what is the peak at the beginning of the chromatogram?
from your chromatogram it looks like you do not leave enough time for gradient equilibration back to starting conditions

[mas23]

I am using 0.1 % TFA usually. My re-equilibration time is 10 min with a flow rate of 1 ml/min. This is a scaled chromatogram and the peak at the front is the peak that is the subject of this post.
Thanks!

[carls]

The time scale is illegible. Is the first peak (the dimer right?) eluting at the void time/volume of the column? If so, its unretained and there's nothing you can do to improve its peak shape until you get the compound retained. Also, the injection solvent (50/50 ACN/H2O - no TFA?) is very strong for your starting mobile phase conditions (0% THF?).

If you cannot retain the analyte with 0% THF then RP is not the right separation mode or a an ion pairing agent such as tetrabutylammonium/tetrapentylammonium (hydroxide or phosphate depending on desired pH - TCI or Sigma) or hexyltriethylammonium Regis) should be added.

[mirac_han]

I have a compound that dimers to form an impurity which has 2 COOH groups. My issue is that I am trying to develop an impurities method that can properly quantitate this impurity amoung many others. I have the cromatography sorted out for the main compound and the rest of the impurities but am having trouble with the dimer impurity. The system right now is as follows:

Atlantix T3 150 x 4.6, 3 um
MPA: 0.1% TFA in water
MPB: 0.1% TFA in THF
Column Temp: 45C
Sample Concenration: 1 mg/mL
injection volumn: 10 uL
diluent: 1:1 water:ACN
Gradient:
0-25% B in 5 min
25-45% B in 20 min

The dimer peak requires a very steep slope to get a sharp peak and the Main peak requires a shallow gradient slope to provide seperation with similar impurities.

My problem is that even though I have got the dimer impurity peak to sharpen up with the steep gradient slope it still tails badly. It looks like a baseline shift occurs when the dimer elutes off of the column and never comes back down. I have also tried increasing the TFA concentration to 0.4% and I am still seeing the tailing however the peak is very sharp. Any words of advise are much appreciated!!!

[img]http://i53.tinypic.com/2itk3ft.jpg[/im

Thank you in advance!!

ı thınk your sample solvent is strong than your mobile phase. first you change your sample solvent. you use low quantity of organic ın your sample solvent. and second ı thınk you change your graiyent slope and you give a reequliribilation your coloum. example 10 minutes initial gradiyent line

[unmgvar]

you should try another buffer
T3 is supposed to help in those cases
but maybe another polar embedded column would work best for you, they are after all mixed modes columns anyway
or you should look for a mixed mode column with a stronger polar group

[mas23]

Hey thanks everyone for replying. The peak does not come off in the void but does require a steeP slope for any kind of acceptable peak shape. This is not the method's finished product and I am starting at 0%B so I can have a steep slope for the two peaks that elute off at the lower % organic. I thought about the diluent strength and unfortunately the later eluding impurities will not dissolve in anything less that 50% ACN. THF seems to degrade the compounds in solution but not on column so I cannot use it as part of the diluent. The frustrating part about this separation is that I had it all figured out until the client threw this impurity into the mix...

The steep slope needs to finish at 25% organic so I can shallow out the slope for the crux of the impurities. This is also the first time that I have had to use THF as the organic phase but unfortunately it is required to get any kind of selectivity between the main peak and two of it's impurities which only differ in the number of CH bonds that are present. I have never seen a peak do this before and that is what is stumping me.

[Hollow]

what about the pKas of the dimers?
Why don't you add the TFA also to your sample?
I guess this could be an equilibration issue between the different (protonated) forms of the dimer. If so, a variation of the column temperature should have some impact (either positive or negative...).

And how does a blank chromatogram look like?

[mas23]

At hollow:

Unfortunately I do no have access to the impurity in purified form, just as an impurity in the final product which makes it tough. So the pka of the dimer is unknown. A blank chromatogram does not have a baseline shift at all so it is due entirely to the impurity.

[mas23]

Ok, new plan for Monday

Add TFA to diluent, And experiment with column temperature to see how it affects the peak shape of the dimer impurity. If anyone is interested I will re post with results. I do think that the impurity is between it's pkas though.

[unmgvar]

how bad does the peak looks when you use an isocratic step in the beginning?

[Hollow]

just some more, even "crazy", thoughts

what if you decrease the injcetion volume (or dilute your sample more)? will the "peak" shape gets better (->overload effects as allready mentioned, maybe focus on the dimer at this step)

Is your dimer still soluble under the inital conditions or does it precipitate (in the pores?) and then is hardly eluted again? (->increase the inital %B, ->s.a. unmgvar's post)

Any kind of interaction ("polymerization") reactions with ??? (itself?, stationary phase?...)

How much is your system's dwell volume (gradient delay)?
What are the RT of your peaks (can't identify it on your pic).