No Peaks on HPLC

[kristind858]

Hello,
I have looked up this topic on this forum and have taken some steps to try an resolve the issue. I am still having trouble. I am not seeing any peaks when injecting with or without a column. We were recently experiencing some issues with retention time of our Vitamin D3 peak. Our service engineer came out and changed the GPV, flow cell gasket, circuit board, and a few other consumbles. He checked the connections to the detector and they are ok. I am thinking there has to be air in the system, possibly the flow cell. All I get it noise from the mobile phase.

I have checked the injector volume and it is injecting the correct amount. The lamp is still good and all of the diagnostic tests are checking out. I have tried to flush the system with water, followed by MeOH. Any other suggestions of what may be causing this? Thank you in advance!

[HPLC chemist]

Vitamin D has a good UV spectrum but the concentration commonly found in a sample are about a factor of 10X too low for quantitation by a PDA (thus MS)! What is your expected concentration?

[HPLC chemist]

Here is an HPLC column you can use, but note they are using a MS detector and they did ot mention the sample matrix or extraction method.

http://www.restek.com/chromatogram/view/LC_CF0644

[kristind858]

Hello,
We are using the L8 aminopropylsilane column. Concentration of stock is around 3 mg/ml. Concentration of working standard is around 3 mcg/ml. We have not had a problem in the past. Even without a column, I am getting to signal. Thank you for your quick reply!

[HPLC chemist]

You should see a peak. Because it is a 'fat soluble vitamin' Vitamin D will elute 10-15 minutes on a C18 column flowing 95% Methanol. What is your mobile phase?

[HPLC chemist]

You may have a 'bubble' somewhere. Try flowing 5 mL/minute of 100% Methanol with the column offline.

With the column online, reverse it and flow 1 mL/Minute of 95% IPA then 95% Methanol (this will eliminate dirt). Be sure to filter both mobile phases (0.22 um).

[HPLC chemist]

Verify on a spectrophotometer that you have a maximum peak at 265 nm in your Vitamin D solution. That is you have Vitamin D in the solution.

[BostonFSE]

--try injecting higher conc closer to stock with a union, the peak should fly out
--try editing the method with different mobile phase lines (C and D, instead of A and B)
--I would have injected caffeine and ran both isocratic and a simple gradient method (water A 60%, B MeOH 40% to mimic your method. This would prove hardware and mcgv is ok as both A and B solenoids would be tested
--Isolate hardware from method/column issue, start simple
--rebuild method from scratch


good luck

[kristind858]

Hello,
My mobile phase is:

99% n-hexane
1% IPA
Isocratic - Flow 1.0 ml/min


I created another method to run lines C and D and still I am getting no Vitamin D3 peak. Wavelength is set to 265 nm. I flushed all lines with methanol at 5 ml/min. How long would you recommend the lines be flushed for? I have not tried cleaning the column yet because I am getting no Vitamin D3 peak without the column. When I did try running the method with the column online, the pressure was normal (~560 psi). Obviously still no Vitamin D3 peak since it doesn't even show up without column.

I know there could still possibly be a small internal leak which even diagnostic tests will not show. I might have to resort to collecting the HPLC waste and testing this for Vitamin D3 to see if it is making it through the system or if it is getting caught somewhere in the system. Any other suggestions? Thanks for your help!!

[kristind858]

UPDATE: We found out the injector was picking up the sample but was not aligned properly to inject into the system. We can now see our analytes for both CoQ10 method and Vitamin D3 method, however both analytes peaks are half the height and half the area they once were before we had all these HPLC issues.

Can someone please tell me what could be causing this half area/half height?

[BostonFSE]

Your service engineer changed alot of parts unrelated to your problem and could have injected caffeine with a restrictor capillary, found no peak and identified the sampler as the issue in 10 minutes

1. Check the instrument configuration so that the sample loop volume, needle and other sampler related hardware match what you actually have on the sampler.
2. If you are using inserts in your vials, remove them and use a regular 2 ml vial, this will test if your needle is not fully going all the way down into the insert.
3. Prime your syringe, air bubbles cause area reproducibility
4. Check the septa once it is pierced by the needle, it should be like a bullseye in the middle, if not, have the fse do a needle alignment
Is this a Standard sampler or well plate sampler?
5 If all this fails, PM the sampler (rotor seal change in particular)

Maintenance videos:
http://www.chem.agilent.com/cag/cabu/lc ... tosamplers

[kristind858]

Thank you so much for your help! My service engineer is new to the job so this may explain why he had issues troubleshooting. Please see my answers below:

1. Check the instrument configuration so that the sample loop volume, needle and other sampler related hardware match what you actually have on the sampler.

I will double check this tomorrow.


2. If you are using inserts in your vials, remove them and use a regular 2 ml vial, this will test if your needle is not fully going all the way down into the insert.

We do not use inserts.

3. Prime your syringe, air bubbles cause area reproducibility

We do this numerous times.

4. Check the septa once it is pierced by the needle, it should be like a bullseye in the middle, if not, have the fse do a needle alignment

We see the piercing of the septa. I also did a injection accuracy check using MeoH and its density to ensure the syringe was picking up the correct volume.


Is this a Standard sampler or well plate sampler?

Standard sampler.

Please let me know your thoughts. Thanks again

[lmh]

Embarrassingly, one of the commonest reasons why people in our lab fail to get peaks is that they've put 200uL in the bottom of a 2mL vial and the needle hasn't gone down far enough to touch the liquid.

[kristind858]

Hello Lmh,
I bet it has happened to a lot of people We always make sure the vial is filled to the top. We are actually getting our standard peaks now for various methods but the peak height and peak area are half of what they used to be. It is making our detection of low level Vitamin D3 (roughly 7.5 mcg) difficult because the peak height used to measure around 5500 pA. It now mostly gets lost as noise since the peak height has decreased.

[Perreman]

Do you have about the same S/N ratio as before? In that case, have a look if your software has some sort of gain-factor to be set.Usually these exist on ELSD or CAD detectors and I don't know of UV detectors having them, but it might be worth a look.