Chromatography Forum

I am looking for experts who can help answer my question.

I inject an analyte solution(Dissolve in mobile phase) in HPLC, which elutes at 2.8 min (tr); however the solvents(methanol and acetonitrile) also have the absorption peak at 4.5min, the same intensity for each injection. Does it mean that the analyte has no interation with stationary phase in the C18 column? The lineary and standard curve of the analyte meet the HPLC method validation criteria. Can I still use the method to assay the concentration of the analyte?

The HPLC condition is shown as following, RP-HPLC
C18 column,
mobile phase: Acetonitrile: buffer(TFA ph=3.0): methanol = 12:40:48;
Flow rate: 1ml/min;

Waiting for some response! Thanks a lot!!!!

If you are injecting a sample of analyte dissolved in mobile phase into a stream of the (same composition) mobile phase then I would expect little or no disturbance in the chromatogaphy when the injection elutes.

If your analyte elutes at 2.8 minutes and shows a concentration related response the late peak is not going to be due to your injection solvent mix (the solvents are not going to separate and and be retained by the column).

the fact that you are getting similar elution in each injection would lead me to suspect that something is getting into the system as the injector actuates. I would look at performing some maintenance on it.

Absolute retention time gives no indication if your analyte actually shows interaction with the stationary phase. The interesting value is the RETENTION FACTOR k. So, what's the retention factor? Alternateviley, what's the dimensions of the column you're using?

Absolute retention time gives no indication if your analyte actually shows interaction with the stationary phase. The interesting value is the RETENTION FACTOR k. So, what's the retention factor? Alternateviley, what's the dimensions of the column you're using?

The retention factor is 1.8, I am using 5micro, 220*4.6mm C18 column.

If you are injecting a sample of analyte dissolved in mobile phase into a stream of the (same composition) mobile phase then I would expect little or no disturbance in the chromatogaphy when the injection elutes.

If your analyte elutes at 2.8 minutes and shows a concentration related response the late peak is not going to be due to your injection solvent mix (the solvents are not going to separate and and be retained by the column).

the fact that you are getting similar elution in each injection would lead me to suspect that something is getting into the system as the injector actuates. I would look at performing some maintenance on it.

How to perform the maintenance? My analyte show concentration dependence at 2.8 min, the following 4.5min peak always show with same AU, I exclude all the contamination possibility, so does it mean the analyte elute with the solvent?

If you are injecting a sample of analyte dissolved in mobile phase into a stream of the (same composition) mobile phase then I would expect little or no disturbance in the chromatogaphy when the injection elutes.

If your analyte elutes at 2.8 minutes and shows a concentration related response the late peak is not going to be due to your injection solvent mix (the solvents are not going to separate and and be retained by the column).

the fact that you are getting similar elution in each injection would lead me to suspect that something is getting into the system as the injector actuates. I would look at performing some maintenance on it.

How to perform the maintenance? My analyte show concentration dependence at 2.8 min, the following 4.5min peak always show with same AU, I exclude all the contamination possibility, so does it mean the analyte elute with the solvent?

You will need to check the instument manual, I am not psychic (go figure!) so don't know what instrument you use. My suspicion is that a worn part is posibly allowing foreign material (lubricant?) into your system as it actuates resulting in the peak at 4.5 min with each injection.

If you are injecting a sample of analyte dissolved in mobile phase into a stream of the (same composition) mobile phase then I would expect little or no disturbance in the chromatogaphy when the injection elutes.

If your analyte elutes at 2.8 minutes and shows a concentration related response the late peak is not going to be due to your injection solvent mix (the solvents are not going to separate and and be retained by the column).

the fact that you are getting similar elution in each injection would lead me to suspect that something is getting into the system as the injector actuates. I would look at performing some maintenance on it.

How to perform the maintenance? My analyte show concentration dependence at 2.8 min, the following 4.5min peak always show with same AU, I exclude all the contamination possibility, so does it mean the analyte elute with the solvent?

You will need to check the instument manual, I am not psychic (go figure!) so don't know what instrument you use. My suspicion is that a worn part is posibly allowing foreign material (lubricant?) into your system as it actuates resulting in the peak at 4.5 min with each injection.

Or if the autosampler uses a rinse like some Waters or Shimadzu units there could be a contaminate in the rinse solvent.

You will need to check the instument manual, I am not psychic (go figure!) so don't know what instrument you use. My suspicion is that a worn part is posibly allowing foreign material (lubricant?) into your system as it actuates resulting in the peak at 4.5 min with each injection.

No injector I know of uses a lubricant. If it is an autosampler, then it could definitely be the wash solvent.

Otherwise, there is some contaminate in the solvent or the sample you're injecting. How large is the peak, what kind of response are we talking about? 500uau? 200mau? 4au?

If you are determining concentration by a peak area assay, I would be concerned that there are impurities coeluting and adding to your peak area if you are not getting any retention. Why not lower the strength of your mobile phase so that you get retention?

The retention factor is 1.8, I am using 5micro, 220*4.6mm C18 column.

I don't know how you got to that retention factor, but it's wrong. You're using a 220x4.6mm column (is it really 220mm? Not 250mm?) at a flow rate of 1mL/min - and your analyte elutes at 2.5 minutes. This means very little to no retention (no matter if the column is 220 or 250mm in length). The actual retention factor is close to zero. The first thing you should try is to get decent retention. "Decent retention" is isocratic runs usually means a retention factor between 2 and 10, meaning your analytes retention time should be at least threefold the deadtime. For a 250x4.6mm column at 1mL/min this would be in the range 7.5-8 minutes...