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- Posts: 8
- Joined: Mon Mar 11, 2019 7:19 pm
This has also posed some challenges for me as I am used to small injection volumes (less than or equal to 15 uL) and being able to easily calibrate over 11 points and 6 orders of magnitude. In the case of this Beckman system and manual injections, I am injecting 200 uL on a 20 uL loop with a mobile phase composition of Water/ACN. Due to the large injection volumes, this makes calibrating to high concentrations seem non-ideal as the standards I have access to come in MeOH. I don't like the idea of shooting 200 uL of MeOH into such a mobile phase as the one that I am using (though I never saw issues with small injection volumes '15 uL' on this same mobile phase). I haven't tried such large injections with mismatch solvent composition as the mobile phase and I would rather avoid any potential issues and waste of expensive CRM.
The requirement for large injection volumes made me realize why my prior colleague had been doing single-point calibrations... Long story short this brings me to my question. I have been completely stumped trying to understand the validity of the previously implemented calculations for % analyte in sample.
The quick and dirty:
1. 2.5 g sample is dissolved into 50 mL ACN.
2. A 500 uL aliquot of sample-containing-solution is then dissolved into 10 mL of ACN.
3. 200 uL of sample is injected.
4. The resulted Area count for the peak in question is used to back-calculate to the original concentration of analyte in sample: [(100)/(2.5 g)]*[(Response)/(RF)] ; where RF is the response factor obtained for a SINGLE calibration point, for example: Response (Area of peak) of 100 ppm standard divided by 0.1 mg/mL = RF for that analyte.
Though this may not be how I would go about setting up calibrations and back-calculations for determining analyte concentrations samples, but I sure want to understand it! I cant quite wrap my head around the above calculation. Additionally, I was hoping this factor of 100 (one of the four components to the calculation) is somehow incorporating the dilution during sample prep but I just cant seem to make any sense of it. At the end of the day this colleague of mine has decades upon decades more experience than I do and I am not doubting his technique. I am just new to doing "single-point" calibration and am still unsure of its validity, though I have observed good linearity in response factor for the analyte in question over various concentrations.
Thank you to anyone who took the time to read this, I look forward to any and all replies!