Chromatography Forum

Dear members,
The retention time of our analyte is gradually decreased though other peaks (unretained peak and placebo's peaks) are remain stable. Why this problem could be occured? What should we do to resolve the problem?
Thanks in advance.

Best regards,
Siswanto

Can you give more details:

Column
Mobile phase
Type of compound being analysed

Hi,

What is the Pka of your moloecue and mobile phase which is currently.
There is chances of variation in RT at mobile phase of pH which equal to Pka of your molecule

One more is due sensitivity of only anlyte towards organic.


chandra

Because you said "placebo" sometimes in this kind of analysis the matrix contains a little bit of compound that is adsorbed stronger by the stationary phase. If you use an isocratic elution this compound will adsorb more and more blocking the stationary phase for the analyte. The effect is decreasing retention times of your compound. If you use gradient possibly the equilibration time is unsufficient. But the first effect I have seen for the analysis of Nitrates from foods on SCX column.

If you use an isocratic elution this compound will adsorb more and more blocking the stationary phase for the analyte.

I will try to add gradient steps to remove absorbed compounds if presence.

i'm having troubles trying to determine mecobalamine in mecobalamin injection. i modified the method from Shijiazhuang Institute Journal, using a Phenomenex Luna C18 (2) 100A 5U 250x4.60 mm column, mobile phase Methanol : Acetonitrile : KH2PO4 buffer 0.05M pH 4.0 (13:10:77), flowrate 1.5 ml/minute, injection volume 5 mcl, detection 264 nm, oven temperature 40ºC.
The problem is the retention time for Mecobalamin (at about 11.5 minute) decrease from time to time. i tried to inject my samples of Mecobalamin in injection and the placebo gives a peak, at about 9.5 minute. the retention times of the peak from the placebo is also decreasing, but it's not as extreme as the mecobalamin's peak.
what should i try next?

I would suspect that your analyte (mecobalamine) retention may be affected by very small variations in the buffer pH

Minor deviations in buffer pH may cuse appreciable shifts in RT.

How stable is your day to day pH meter calibration ?

in my opinion,there are four reasons for retention time delay as indicated above.
1.inaccurate pump flow
2.mobile phase
3.colum temp.
4.Column contamination

JKG has a good point. My first guess would be pH as well.
Buffers have been discussed here in the past. The three pKa values for phosphoric acid are:
pKa(1)=2.15
pKa(2)=7.2
pKa(3)=12.3

So the useful pH range for phosphate buffers are:
pH(1)=2.2±0.5 (1.7 to 2.7)
pH(2)=7.2±0.5 (6.7 to 7.7)
pH(3)=12.3±0.5 (11.8to 12.8)

The closer you stay to the pKa the better the buffer (higher buffer capacity) I personally try to stay around pKa±0.5 or closer but I think you might be able to get away with it if you stay around pKa±1.0
So phosphate is not useful as buffer at pH=4.0. Sodium acetate/acetic acid (pKa=4.8) is a much better joice.

JKG has a good point. My first guess would be pH as well.
Buffers have been discussed here in the past. The three pKa values for phosphoric acid are:
pKa(1)=2.15
pKa(2)=7.2
pKa(3)=12.3

So the useful pH range for phosphate buffers are:
pH(1)=2.2±0.5 (1.7 to 2.7)
pH(2)=7.2±0.5 (6.7 to 7.7)
pH(3)=12.3±0.5 (11.8to 12.

The closer you stay to the pKa the better the buffer (higher buffer capacity) I personally try to stay around pKa±0.5 or closer but I think you might be able to get away with it if you stay around pKa±1.0
So phosphate is not useful as buffer at pH=4.0. Sodium acetate/acetic acid (pKa=4. is a much better joice.

rhaefe is right, buffers should be within a pH range of pKa ± 1.0 ideally, so with a monobasic phosphate salt you are well outside this range. As suggested you could use either an acetate or a formate buffer system which would encompass pH 4 within this comfort zone.

my analyst reported that the retention is relatively stable after she changed to acetate buffer. thank you for any help.