Major Inconsistency between identical standards run on HPLC

[unclebenjaminjones]

I am working on a little RDX in wastewater testing project and wanted to see if anyone could help me with the following conundrum.

I ordered some commercially prepared calibration mix (8330 calibration mix #1 from Restek) which claims to have all components at a conc of 1000 ug/mL in ACN. When I prepare an RDX standard in ACN at a conc of 1,120 ug/mL, the resulting peak height and area are approx. 1/215th the size of the RDX peak found in the commercial cal mix. I made the standard by weighing 0.1120 g of RDX and diluting it in a 100 mL flask with ACN.

I feel like I'm going crazy. Is this possible (me going crazy)?

I am using an Agilent 1200 Series HPLC with a DAD. All instrument parameters are the same for these two shots.

[tom jupille]

Your math is right. So some possibilities:
- you botched the weighing (hey, it happens!)
- your RDX standard is no good
- Restek botched their weighing
- most of the solvent has evaporated from the Restek standard

If it were me, I'd prepare a fresh standard solution and see what happens.

[bisnettrj2]

Where'd you get neat RDX? Unless you're working directly with the DoD, that couldn't have been easy.

Did you filter your standard after prepping it? If so, you might have had losses on the filter. The following link contains a paper (http://www.seas.ucla.edu/stenstro/r/r38) where they referenced the paper below in regard to adsorptive losses on different filter types (pg 31-32 of the paper).

Jenkins, T.F ., Leggett, D .C ., Grant, C .L. and Bauer, C .F . (1986) . "Reversed-Phase HighPerformance Liquid Chromatographic Determination of Nitroorganics in Munitions
Wastewater," Analytical Chemistry, Vol . 58, No . 1, pp . 170-175 .

I haven't used that particular Restek mix, but the standards I normally get from them are usually accurate. I personally get my explosives calibration mixes from Accustandard.

[tadah]

Wikipedia displays this compound in ionic form. Did you purchase it as a salt?

[unclebenjaminjones]

I test munitions wastewater. The RDX was taken from production and I was told it was pure, dry, etc. Perhaps it is not as pure as I've been told. I just can't imagine that it is 1/215th the concentration of the commercially prepared standard. Can anyone who has experience with RDX production speak to the purity of 'normal' product?

[bisnettrj2]

If the production folks say it's pure, then hopefully you took Tom's advice and re-prepped your standard. And while I doubt Restek made that big of a mistake, you could purchase another standard from a different manufacturer to check the Restek mix. Here's another thought - that Restek mix has 7 components - are you seeing 7 peaks when you run the mix? If you aren't, then you might be co-eluting RDX and 1,3,5-TNB or RDX and HMX, and thus seeing an erroneously high peak in your mix as compared to your pure RDX standard.

[unclebenjaminjones]

I'm seeing all seven peaks in the calibration mix, so I don't think I'm co eluting. I'll do more asking about the purity of the RDX.

[unclebenjaminjones]

I also re prepped my standard with the same results as before.

[lmh]

You have to consider too that the standards are supposed to be 1.12mg/mL, so if your standard is pure and correct and Restek's is wrong, theirs must be just over 240mg/mL. I have no idea of the solubility of your material, but at this concentration you should be able to see the material if you evaporated only a few uL of Restek's standard. Good luck!

[bisnettrj2]

unclebenjaminjones:

Any luck on resolving this issue?

[unclebenjaminjones]

Yes. We have a degasser on order, but have not received it. I did some vacuum degassing on my mobile phase and following that things looked way more sensible. Perhaps I was reading a small peak or noise as my expected peak. I did not think the gas in the mobile phase would wreak such havoc. whatever the reason, I'm back on track.

[bisnettrj2]

unclebenjaminjones:

So, are you saying you really had no peak at all after prepping a ~1120 ppm RDX standard from the 'pure' material that the production folks gave you? Did you filter the standard after prepping it? Are you seeing any significant peaks anywhere in the chromatogram?