Mr. Skumar,

You could try with UPLC.

in the question you mentioned that your compounds elute with your solvent peak which should be below 1 min retention. then you say the peak is at 6 mins this can not be your solvent peak. If your compounds elute with your solvent below 1 min then you it means you are not equilibrating your column properly.

The injection volume and flow rate suggest an analytical column of some sort. With typical column sizes, I'd expect the void elution time to be ~1 to 2 minutes, depending on the column size. So it seems there is some retention, since something comes out at 6 minutes. What is the actual column size?

I suggest starting at 5% organic solvent (for the aqueous runs) to avoid phase collapse, where the C18 strands interact with one another rather than the sample at low concentrations of organic solvent.

I also note that the sample was run 0 to 100% acetonitrile in methanol- first, the solvents are reversed for "non-aqueous reverse phase" since acetonitrile is less polar than methanol. Second, if the compound eluted in an aqueous system, it should elute at the void in this solvent system.

I'm wondering if the HPLC is running correctly. You should work with someone in your lab and determine the system is delivering the solvents correctly; run some standards first.

Thank you all.

Hello Ms. Ivanova ,

UPLC is a better alternative, but the facility is not available in my reach.

Hello Mr. Alkhdhairawi and Mr. Silver,

I missed to run a blank sample solvent. Since in the chromatogram of every sample, there was a single peak at around 6 min, I mistook it as solvent peak.

It is an analytical column of dimensions 4.6x250mm and the packing material is 5 micrometers C18.

I had found contradictory references on the polarity indexes of acetonitrile and methanol, where the acetonitrile is more polar than methanol. As was rightly pointed out, I will try 0-100% acetonitrile in methanol (also as the elution strength of acetonitrile is larger than methanol).

The HPLC and the column is working fine. I ran the standard compound in gradient 0-100% acetonitrile in water, the retention time was 33 min.

Also, one more question. Will acetonitrile, water or methanol show a solvent peak in the detection wavelength of 300nm?

I read that all solvents that you used show a peak at 6 minutes, including the methanol/acetonitrile gradient. Although "ghost peaks" do arise from solvents, I don't think they would all arise the same way from such a variety of solvents. The peak should be at the "void" in the all-organic solvent system if it showed retention in the aqueous systems. Likewise, if it were sample related, or carry-over from a previous sample, it should elute at the void as well. At 0.5 mL/min, something could be be getting into the system at about 3 mL away from the injector. Assuming about 1.5 mL for the column volume, that leaves 1.5 mL to the top of the column. Depending on the LC system, there could possible be something going on with the pumps an absorbing solvent getting in?). This is all guesswork on my part.

There are 4 pumps in the system out of which, two are functional. The other two has different solvents. The run time data shows proper working of those two functional pumps. I had purged them every time when the solvents are changed.

I could replace the other solvent reservoirs with mobile phase solvents and see the difference. But can the other non-functional pumps mess with the working mobile phases to result like this?

Also I had forgotten to mention. I had collected the 6 min fraction and checked with TLC. There were some compounds missing in the collected fraction when compared to the crude sample. Is it because of stronger bonding between compounds and the stationary phase.

attach your chromatogram.

20 uL is a rather large injection for a 0.5 mL/min flow rate. If you are injecting it in 100% methanol this becomes a serious issue within the column. Evaporate away the methanol and reconstitute your sample in 10% methanol/water or 20% methanol/water and use a 10 uL injection.

You don't tell us how fast your gradient is; that information would help.

5 uM columns are relatively low resolution columns. For a 4.6 mm column your optimum flow rate is going to be 1.0 to 1.2 mL/minute. At 0.5 mL/minute you are working in a very inefficient portion of the Van Deemter curve. This, coupled with a relatively low resolution column, is going to cause you serious problems. Raise your flow rate up to 1.0 mL/minute.

I agreed with the collegues above, and I ask you what solvent you use in the TLC? I wuold an isocratic run first, I think is the better way to develop a method. When this not work, then I would try a gradient. Another thing, when you runed another standard, you see a sinal in 6min? Sometimes, a contamination in the top of column may create a sinal like this. I hope helped.

I am attaching chromatograms of two different crude samples. Detector A is RID and detector B is UV-DAD. Kindly refer the chromatogram of detector B (detector A has some settings problem).The run time is 60 mins gradient of 0-100% MeOH in acetonitrile. The similar peaks are obtained for the other solvent systems mentioned above.

(I had also run the samples in 0-100% acetonitrile in methanol gradient as suggested, but there is no change in the peak pattern).

Hello Mr. Krause and Mr. Brito,

The speed of the HPLC gradient is 1.7% increase per min.

Solvent system of TLC (silica gel) is 20% ethyl acetate in hexane.

Thank you for the suggestions. I will look into the sample preparation, the flow rates and try isocratic runs.

From my point of view, you do not have a chromatographic process. Their compounds have no retention in this column, simple as that. Note that the volume of your column is somewhere around 2.8mL, running at 0.5 mL / min. it will take around 5,6min.

I think you have to review the characteristics of your compounds and look for another column. Notice that in MeOH / H2O there is still an apparent separation, the shoulders at the peak begin to stand out. Perhaps, as I had already quoted an isocratic run or even on plateaus, with a low concentration of MeOH or ACN in water. I hope helped.

Thank you very much.