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- Posts: 5
- Joined: Fri Nov 17, 2017 9:36 am
We have been doing a lot of runs recently trying to develop our method but keep coming into a certain problem with some samples but not others. The only relationship we can think of between the samples is the presence of acidic cannabinoids.
When ever we test products with THCa we get this giant broad band, see attached file Fig.1. At first, we thought this was due to overloading, so we diluted 100x instead of just 10x, and made a smaller injection of 2ul instead of 5ul, but we still get this broadband.
(Fig. 1)
We tried to vary the pH of our buffered water between 4.2-4.8 because we thought the broadening is happening with the acidic analytes but not the neutral ones, but we still get the 'hump back'.
We also tried to experiment with gradient to see what happens with a slow and fast gradient ( Fig 2,3).
(Fig. 2)
(Fig. 3)
We have a feeling the hump-back may also be 2 compounds co-eluting. There is a clear bump at one wave length, but not at another (see Fig 4.)
(Fig. 4)
When looking at the spectra (fig 5) although im sure its not pure, it appears to be one of our acidic analytes. We tried to run some standards we purchased to see what happens when we inject a known acidic cannabinoid compound and again we got issues with very broad bands.
(Fig. 5)
Fig 6 shows what we call the Batman.... we actually prepared a sample with CBD and CBD acid standards, same concentration (50uL aliquot of CBDa and 50uL aliquot of CBD). The large peak has a spectra that matches the known spectra for CBD, however our acidic analyte seems to have gone, or become batman!
(Fig. 6)
We currently use 25mM ammoniun Formate buffer in our water, pH to 4.3 with Formic Acid. We also tried putting a drop or formic acid in our Methanol to make 0.1% V/V and tried to see if we could resolve our issues, but no luck.
Our next step was to try 50mM buffer, and also start all over again with Acetonitrile as our B instead of methanol.
Now we're really quite lost as to where to go next. We feel like there is something happening to our acidic analytes, causing this broad hump back peak, but we're unsure what else to do. We are at our limit with flowrate, any higher and we are over pressure ( small bore colum 2.1mm x100mm, with 2.7um).
If anyone has any tips or suggestions, we would greatly appreciate some input.
Thanks in advance!