How to trouble shooting none peaks

[creageng]

Hi,there:
I new to LC. I run my samples which should contained enough target compound,with the new replaced column. My samples did not run before. And conditions and phase were followed by the papers.
But no peaks coming out. Baseline fluctuating +-5. I also did not see the injection peak.Shoud that always appear?
Many problems may have:
1) column leak?
2) needle problems?
3) my lamp dead?

Could you guyes tell me the most likely culprit? In which priority,I should trouble shooting this problem. Any procedule I can use to diagnosis this possibility?
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Agilent 1100 HPLC system
Column: Phenomenex (Torrance, CA) Curasil PFP 15 cm, 2 mm, 3 µm column
Injection volume: 10uL
Mobile phase: water-acetonitrile started 35% ACN increasing this linear to 60% in 6 min. Both solvents contained 200 µL/L trifluoroacetic acid
The wavelength 304 ± 4 nm
==============


thank you
creageng

[tom jupille]

If only some peaks are missing, the general troubleshooting procedure is given here:
http://www.lcresources.com/resources/TSWiz/hs90.htm

In this case (no peaks at all), any of the causes you list could be the problem. If it were my problem I would check the following:

1. Look at the waste line from the system to confirm that solvent is flowing. Collect solvent for a few minutes in a graduated cylinder to confirm that the flow rate is approximately correct. If it is not, re-prime the pump or find the leak.

2. If you have flow, check that the detector lamp is on (the Agilent software should confirm this).

3. You have already changed the column, so I doubt this is the problem. The column should have come with a manufacturer's test procedure. Run that to confirm the column is still good.

[creageng]

1) I let the flow out of the detection into the cylinder, 0.5mL/min rate 20min, the total volum 10+-0.1mL.Look fine.
Although I notice a mobile liquid drop out every ca 2min at the inlet of the column. I skewed tightly,it does not help.
2) lamp in on. But I dont know the detection desity.
3) In one vial I put 1mL 30% ACN,program sample 9 times 100uL 0.5mL/mL 65%ACN 10min. It should left 0.1mL,but it turn out ca 350uL left.Signal Baseline from 1-7 run , look fine +-1mA.But In 8th and 9th,8min weide peak over 1000mA for 4 channel I selected:256nm,287nm,304nM,396nM.

Any help?
thanks
creageng

[tom jupille]

That is progress:

1. You are getting flow through the system. The leak at the inlet is another problem, but that should not be completely eliminating response

2. How do you know the lamp is on? And to proceed further, you will need to know the sensitivity setting of the detector (how many absorbance units = full scale?). Typical for fairly concentrated samples would be 0.5 AUFS (absorbance units full scale). You may have to do some searching in the software to figure out how many volts = full scale and how many AU = 1 volt.

[At this point, if I had to guess, I would suggest that your detector setting is not sufficiently sensitive]

3. It sounds like you are getting injections. Your injector needle is probably not reaching down to the bottom of the vial, and you are injecting only air for runs 7 and 8. As with the leak at the column head, this is a minor problem, but should not cause total lack of sample for the first few injections.

Next thing to do: Set your detection wavelength to 215 nm and inject 10 microliters of 0.01N nitric acid. Nitrate ion is unretained by a reversed-phase column and absorbs strongly in the low-UV, so you should see a fairly big peak at about 0.5 to 0.6 minutes.

If you do not, you need to contact Agilent and run their qualification procedure to check that the instrument is functioning properly.

[creageng]

2,my systems detector showed mA.I don't know whether it can be changed to V
3,I run the standard soluctioin (65% ACN),the peaks and respective retention time is ok.
But my sample did not have any peaks.I believe my sample is ample enough,is that my running conditons is so low that the colomn retained all the compoun?
dount?

Any other suggestions

[tom jupille]

probably "milli-absorbance units".

If you ran a standard and got the expected results, that confirms that your system is running correctly.