Problems with injections

[nellermann]

Hello all,

I just had a 500 uL loop installed on my dionex autosampler and for most of my methods it works fine. Most of my methods are injecting less than 100uL.

I really needed the new loop for my vitamin D method which I am currently developing and it seems that my peak size does not correlate with what I'd expect by injecting 500uL compared to the peak size I was getting when I only injected 100uL.

Would the diluent have anything to do with it? My diluent is a mixture of cyclohexane-n-heptane (50/50).

I just don't understand why the peak size is 35x less than what I expected.

Thanks for any help you can give me.

Natalie

[sepscientologist]

35x less than expected... so the 500 ul injection is 7x smaller than the 100 ul injection?

[LCbob]

First off I will assume the 35x sensitivity is a typo.

Make sure you are not overloading the detector.
It could be the diluent, what is your mobile phase?

[nellermann]

Actually, now that I go back and look at it, I'm not sure where I got the 35x. More like 69x.

I injected 100uL on the old injection loop and got an area of 13.8809.(mV*min)
Then on the new loop, I injected 500uL and only got 1.0700.
Shouldn't I have gotten around 69?

This was only my second time using the new loop and since then have had no problems with any of my other non-vitamin D runs. I just tested it and it was running fine with a 100% water diluent.

The diluent for the vitamin D's was mostly the cyclohexane-n-heptane that I use also for mobile phase.

[sepscientologist]

I wonder if they accidentally gave you a 5 uL loop? That would fit well with your data. When they
say it is 100 uL that is the nominal volume. The actual volume can by 50% different from that
based on the ID spec of tubing suppliers. It's usually much closer than 50%.

Try looking at the ID of the tubing. Compare to some known ID tubings and then calculate the
actual volume for both of your injection loops. Curious to see what they are.

[HW Mueller]

Am I starting to hallucinate? Or did nellerman say that he saw the problem only twice and not with aqu. solvent?

[nellermann]

It's definately a 500uL loop. I tested the loop yesterday with an aqueous diluent and it worked fine.

Again, my question is, could it be the diluent that I use for the vitamin D's?

[LCbob]

Hi
Just to recap

In normal phase mode you get significantly less loading on a 500ul loop compared to a 100ul loop? But in RP mode the loading makes sense, i.e. 5x more for the 500ul compared to the 100ul loop?

What instrument do you have?
Is the diluent exactly the same as the mobile phase, i.e. any modifier in there?
Are there any extra peaks in your chromatograms or does your solvent front look any different from each injection?

[DSP007]

Hi!
1) You Diluent of sample? Also hexane (ciclohexane, heptane) or not?
2) Mount of peak isn,t trim? (as variant Bloking integrator pen in right side paper, ADC overflow memory, T% light of solution many more 99%)

[unmgvar]

what instrument is it?
Summit, ultimate?

precise injection volume depends mainly on 2 parameters of speed and time
what speed have you set up for the syringe? decreasing speed is important for viscous liquids and preventing bubble creation
what is the draw delay you have set? this let's the needle in the vial waiting, letting the sample to go further up the needle into the loop. the more viscous the sample, the more time needed

since you are doing a normal phase application you need to make sure also that you have changed all the liquids to what suits for your application
in the summit, the mobile phase is in the needle so that is not a problem
for the ultimate you need to make sure you have changed the autosampler liquid, use something close to your mobile phase, or at least iso-propanol.

[HW Mueller]

Discussions on two initial trials with shaky error assessment? I would recommend an old fashioned systematic check, not forgetting the possibility of laminar flow effects.

[nellermann]

Hi
Just to recap

In normal phase mode you get significantly less loading on a 500ul loop compared to a 100ul loop? But in RP mode the loading makes sense, i.e. 5x more for the 500ul compared to the 100ul loop?

What instrument do you have?
Is the diluent exactly the same as the mobile phase, i.e. any modifier in there?
Are there any extra peaks in your chromatograms or does your solvent front look any different from each injection?

First paragraph is correct.

I have a Dionex Ultimate 3000 autosampler with an a fisher pump and old milton roy detector.

Yes, the diluent is the same as the moble phase. There are no extra peaks and the solvent front looks the same, just the peak of interest is smaller.

[nellermann]

what instrument is it?
Summit, ultimate?

precise injection volume depends mainly on 2 parameters of speed and time
what speed have you set up for the syringe? decreasing speed is important for viscous liquids and preventing bubble creation
what is the draw delay you have set? this let's the needle in the vial waiting, letting the sample to go further up the needle into the loop. the more viscous the sample, the more time needed

since you are doing a normal phase application you need to make sure also that you have changed all the liquids to what suits for your application
in the summit, the mobile phase is in the needle so that is not a problem
for the ultimate you need to make sure you have changed the autosampler liquid, use something close to your mobile phase, or at least iso-propanol.

Hello,

The diluent and mobile phase is 50/50 cyclohexane/n-heptane. The draw delay was 3000 ms and draw speed was 5.0 uL/s.

Autosampler is an ultimate. Do you mean, the rinse for the autosampler syringe needs to similar to the mobile phase, because currently I've been using 50/50 Methanol/water for all methods.

Thank you everyone for all your helpful comments. I plan on running vitamin D's next week and hope to be more successful. This was only my third time running vitamin D's, but the first time running it with the new syringe. The two other times running with a 100uL syringe, I had excellent results/recovery.

[remesquaddie]

I sit possible you're losing sample in the low pressure path somewhere? Try using just your mobil phase for injections looking for leaks.
The manufacturers of the injectors normally recommend a load of 3 times the sample loop for reproducibility. Tried that yet?

quote="nellermann"]Hello all,

I just had a 500 uL loop installed on my dionex autosampler and for most of my methods it works fine. Most of my methods are injecting less than 100uL.

I really needed the new loop for my vitamin D method which I am currently developing and it seems that my peak size does not correlate with what I'd expect by injecting 500uL compared to the peak size I was getting when I only injected 100uL.

Would the diluent have anything to do with it? My diluent is a mixture of cyclohexane-n-heptane (50/50).

I just don't understand why the peak size is 35x less than what I expected.

Thanks for any help you can give me.

Natalie[/quote]

[remesquaddie]

You may also have an air bubble in teh low pressure path, try PURGING a couple of times.

Hello all,

I just had a 500 uL loop installed on my dionex autosampler and for most of my methods it works fine. Most of my methods are injecting less than 100uL.

I really needed the new loop for my vitamin D method which I am currently developing and it seems that my peak size does not correlate with what I'd expect by injecting 500uL compared to the peak size I was getting when I only injected 100uL.

Would the diluent have anything to do with it? My diluent is a mixture of cyclohexane-n-heptane (50/50).

I just don't understand why the peak size is 35x less than what I expected.

Thanks for any help you can give me.

Natalie