Source
r/labrats•
Sam__Mule

Need help proving my HPLC doesn’t have enough precision to consistently run the same sample within 2% of itself at values of 1-5ppm

Hi lab rats, i need help proving/explaining to a group of non-scientific corporate people that my HPLC machine simply can’t consistently measure the same sample within 2% of itself.

The 2% parameter was arbitrarily set some years ago by a manager who no longer works here, and in my opinion it was not a realistic benchmark. Now it’s my task to prove in plain and clear terms why it’s not. I’m planning on just doing a simple experiment to show this, but some math/literature evidence would help too.

Im using a 1100 series Agilent HPLC and measuring levels of a compound in citrus that is typically around 2-5 ppm. We report the data to the 3rd significant digit.

I want to point out that I’m the only HPLC technician in the company, and i’ve already found MANY things in my 1 year here that we’ve had to change, so i wouldn’t be surprised if we needed to change the significant digits in our data, etc.

If you all have some suggestions, i would love to hear them! Thanks.

Posted Jul 9, 2020, 3:44 PM UTC

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PeachFuzzRump•

Hey, some quick questions about your systems and methods because I run a handful of LC/MS systems and my systems are extremely consistent but I'm not clearly understanding what the 2% you mentioned is looking at.

What sort of work are you doing, what sort of column and mp? What signals are you detecting? (DAD, ELSD, MS)?

Full disclosure, I'm not working with 1100s. I'm mostly on 1200s and infinity IIs in the Agilent department.

Feel free to DM me directly.

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Sam__MuleOP•

Hi!

So i’m doing a simple SPE extraction with ACN and Water, then running the sample, which is just citrus juice. column is a C18 reverse phase, MP is 40 ACN/60 Water, signal is VWD at one wavelength

We cross test occasionally with other labs, but they send me samples that are around 1 ppm, and 2% variance at 1 ppm is hard to hit consistently.

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PeachFuzzRump•

Sorry for all of the questions I just want to make sure if I give any advice or references they apply to the issue you're having :)

Just to make sure I'm clear, you guys aren't using any modifiers in your mobile phase?

I'm assuming you're using a column compartment or column heater to keep the temp consistent.

Addressing the chromatography, I've never used a VWD but the principle is the same as DAD. How wide are your peaks? What's the peak shape like? You're using an isocratic method, what's your normal rt (this shouldn't be proprietary, but if it is just let me know). Are you guys running standards?

Edit: Sorry about replying to a different thread, Reddit mobile is not great.

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Sam__MuleOP•

not a problem, i appreciate it.

No modifiers in mp, column is in a compartment, kept at 40 celsius. Peak width is about .05 minutes, Peak shape is good, doesn’t have any conflicting peaks, but it gets worse around 1 ppm, signal to noise isn’t great at that level. rt is around 2.5 minutes.

I run a 5 point curve every day (lowest point is 1.4 ppm) and run one of the points at beginning and end of every run as a standard. We accept a range of plus/minus 10% on the standard... so idk why they expect a plus/minus of 2% on the messier sample... just realized this lol, that will probably be a good argument. I use a known sample as well as spiking a sample

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PeachFuzzRump•

The ppm are you referring to concentration? I do a lot of MS so at first I was thinking you were talking about mass accuracy I was so confused lol!

But the standards should be the only evidence you need to show that your system is not capable of maintaining precision or accuracy depending on what you're having issues with.

Do you calculate percentage by ((theoretical rt -actual rt )/theoretical rt)*100?

I'

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Sam__MuleOP•

Yes concentration sorry!

Not sure what you’re asking about the rt. We use area under the curve, and when we calculate the variance on sample we test it 2x and do (A-B)/B

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PeachFuzzRump•

Ah ok so you're looking at abundance not rt! Got it. You're having no issue with rt's staying consistent.

Two other things to check for consistency. How old is your lamp how often do you have change it. And how often do you change your MP?

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Sam__MuleOP•

Um we have lab advisor application that basically just tells us when to replace the lamp, it’s running right now so i can’t check, but it’s like 2,000 hours i think? or 20,000? also, the MP is in a 1L bottle and lasts like 4 or 5 days usually before it needs to be refilled

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TurdsofWisdom•

Do you degas the mobile phase as you prepare it, or consistently with helium bubbled through?

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