Chromatography Forum

Hi, all
I have a question quite urgent. I'm a new beginner in HPLC with a old machine, that is definitely worse than a new machine when you get no information about it. Before the analysis, I tried to check the status of it. Although I faced some problems with that machine: noisy in the baseline, high pressure, it all comes from the old column. After I purchase a new column, it seems to be OK. The column is Lichrosorb Si 60, 4.6 mm ID, 125mm, normal phase column, the guard column are coated with same material. My compound is Canthaxanthin (carotenoid). The new problem comes, the parameters I set as below:
Flow rate: 1ml/min
UV/Vis: 470nm
Mobile phase: n-hexan/acetone 86:14, V/V
Test solution: 1mg Canthaxanthin in 100ml Mobile phase.
The problem is I get very low peak height (0,0002 or 0,0003 volts)after 2 min, and then there is no peak any more.
I took off the column and tried to inject directly into the UV detector, the peak appeared and the peak height is around 0.2 volts.It seems my detector is ok.
I guess the problem maybe the concentration of test solution is too high. but I'm not so sure...
And today I tried to repeat the experiment at 0.5 ml/min flow rate, the only peak I mentioned above becomes more sharp and seems to be the peak of my compound, but still very low peak height, (Apr.0,006 volts, 10 times more than before),is my column has some problem? I hope not, it's a new one. and after this peak, there is a small negative peak, how can I do with that? Actually, I think the peak height even it is very low, but still make sense for my work, at least I get a peak, but negative peak, what can I do?
Thank you all

I suspect that what you are seeing is not your compound but rather the equilibrium disturbance caused by your injection. Your analyte is probably still stuck on the column.

Think of it this way: a 125mm x 4.6 mm HPLC column contains about 1 - 1.5 mL of liquid mobile phase. At 1 mL/min, that means that an unretained peak will come out at about 1 - 1.5 minutes (that's called the "dead time" of the column, and is usually symbolized by t0). A good rule of thumb for quantitation is that any peak of interest should come out somewhere between 2-times- and 11-times-t0 (actually, the FDA recommends a minimum of 3X).

When you inject a sample that does not exactly match your mobile phase, you disturb the equilibrium of the system; it's not unusual to see baseline perturbations as a result. The 3X t0 rule of thumb allows sufficient time for those perturbations to settle down so you can do effective quantitation.

I would try a series of experiments increasing the acetone concentration until you see your actual compound coming out (be prepared to see a lot lf garbage first as all the sample currently stuck on the column comes off!).

Thanks, Tom
I still not so clear about "increasing the aceton concentration", you mean the mobile phase, or the injection? Or should I try to inject pure aceton, so it may helps to bring the stuck compound goes out. Whether it is ok for my column? I have no experience to take care of it, but I hope it will not be distroyed by my mistake.
One more thing I forget to mention is the peak (flow rate 0.5 ml/min) comes out around 4-5 min. You really think it's not the peak I want if I have a pure compound?

I still not so clear about "increasing the aceton concentration", you mean the mobile phase, or the injection?

The concentration of acetone in the mobile phase. In normal-phase chromatography (which is what you are doing), hexane is a "weak" solvent; acetone is a moderately strong solvent. The more strong solvent in your mobile phase, the earlier your analytes (peaks) come out.

You really think it's not the peak I want if I have a pure compound?

Have I ever lied to you?