Chromatography Forum

We tried to separate 2 substances from a dosage form using gradient elution. The problem is occurred to the latest substance that need stronger mobile phase. A ghost comes out and interfere the latest peaks. As the result, the calibration curve could not pass through zero point and bad recovery of the substance in the sample. Water for HPLC was used but the ghost still in there. It follows wherever the peak goes when the strength of mobile phase was changed. What should we do to expel the ghost away from main peak’s body?

Best regards,
Siswanto Tanuatmojo

Hello

Do a blank run, without injecting the sample.

If you still get the ghost peak, then it could be coming from the injector loop. Flush the injector loop with methanol or acetonitrile, followed by mobile phase and try a blank run again.

If you're still getting the ghost peak, check the solvents used for your mobile phase.

Hope this helps.

Does your mobile phase contains any additives? Does the ghost peak area constant or does the area change with different column equilibration times? If the area change, it is probably mobile phase contamination (either your water or any additives you may use).

Does your mobile phase contains any additives? Does the ghost peak area constant or does the area change with different column equilibration times? If the area change, it is probably mobile phase contamination (either your water or any additives you may use).

Ghost peaks always haunt!

Please elaborate this point. How does equilibration of column has correlation with ghost peak area?

Regards,

Amaryl.

Siswanto,

How does the ghost peak area responds to changing concentration of your injecting solution?

Regards,


Amaryl.

Amaryl,

Assuming that it is a contamination in mobile phase A (and has some affinity with the stationary phase) increasing the equilibration time will incrase the amount of the contamination that will be fixed in the stationary phase and then be eluted as a chromatographic peak. If this is the case, the longer the equilibration time, the highest the peak area of the "ghost" peak.

There is always the possibility that it will be a "system" peak but we do not have enough information for this (i.e. exact mobile phase and detector...).

Amaryl,

Assuming that it is a contamination in mobile phase A (and has some affinity with the stationary phase) increasing the equilibration time will incrase the amount of the contamination that will be fixed in the stationary phase and then be eluted as a chromatographic peak. If this is the case, the longer the equilibration time, the highest the peak area of the "ghost" peak.

There is always the possibility that it will be a "system" peak but we do not have enough information for this (i.e. exact mobile phase and detector...).

Hmm ...ok what if we suppose the impurity is in mobile phase. So ideally the mobile phase injection should give the ghost peak.

Am I correct?

Regards,

Amaryl.

There could even be possiblity of any excipient peak.

Amaryl.

I have run the system without any injection to check the baseline with or without salt in aqueous portion of mobile phase. Both chromatograms are same: a ghost stays on Peak 2’s retention time. So, I summarized that the ghost was coming from the water. When we used water for chromatography, the ghost was still in there with smaller area.

UV-Vis and DAD detectors are used for this method.

The mobile phase was made by first preparing a phosphate buffer solution of KH2PO4 and Na2HPO4 (pH 7.0, 0.02 M). This buffer solution was then mixed with acetonitrile in ratios of 93:7 (v/v) buffer–acetonitrile to yield Mobile Phase A. Mobile Phase B is 100% acetonitrile. The two-step gradient profile begins with a 12 min linear gradient from 100% Mobile Phase A to Mobile Phase A–Mobile Phase B (92:8, v/v). This is followed by a linear ramp over the next 16 min to Mobile Phase A–Mobile Phase B (38:62, v/v). Finally, the composition is returned to 100% Mobile Phase A over 2 min followed by a 5 min equilibration for a total run time of 35 min. The mobile phase flow rate is 1.0 ml/min, the column temperature is controlled at 35 °C, and the injection volume is 20 uL.

Then, we made modifications on second step gradient, i.e. change in slope, initial proportion, column temperature, flow rate, and/or pH. The ghost won't be expelled from the Peak 2’s body.

I suggest that you do as I recommended and increase the equilibration time (i.e. from 5 min to 10, 20, 30 min). just to make sure that it is indeed the water.

If it is the water, an easy fix would be to have your water (or mobile phase A) pass through a C18 column. As your impurity is quite hydrophobic, it will be stay in the stationary phase, so you will have contamination free water for your application...

I suggest that you do as I recommended and increase the equilibration time (i.e. from 5 min to 10, 20, 30 min). just to make sure that it is indeed the water.

If it is the water, an easy fix would be to have your water (or mobile phase A) pass through a C18 column. As your impurity is quite hydrophobic, it will be stay in the stationary phase, so you will have contamination free water for your application...

If impurity is made to stay on stationary phase by passing water, won't it elute out later when we run mobile phase? How will it help kostas!

Regards,

Amaryl.

I think what Kostas means is to put an old C18 column or guard cartridge in the line between the A pump and the mixer (this only works if you have a high-pressure mixing system).

Alternatively, you could pump your A mobile phase through a column or guard separately (off line) and use the collected solvent in your actual system.

A third alternative is to filter your A solvent through a membrane filter impregnated with C18 (3M make such filters, I think)

Tom is right.

I was referring to his second alternative...

Thanks got it.

Amaryl.

I have the same idea with Tom’s second option. I would rather to ‘purify’ water or buffer solution instead of Mobile Phase A. I will try it and report the result here.