Dear all,
I am building calibration curve of IAA to quantify this molecule in seaweed.
I did a uv spectra to see where IAA have the stronger absorbance, I prepared a IAA:ACN 50ug/ml solution. I observed two zones where the signal is more absorbed between 257 and 290 nm-1 (peak 280 nm-1) and between 190 and 224 nm-1 (peak 220, stronger than first one).
So I started to build my curve injecting my standard in my hplc system (BC 126 gold system+ 166 uv detector), I set-up my instrument on 220 nm-1 and phase A is H2O+1%CH3COOH and B is ACN+1%CH3COOH. the elution was 75:25 mixture like what I found in literature.
I was waiting for a signal around 12min but I didn't observed anything, so I was starting to check other wavelength, and I found only signal around 250nm-1.
The question is: Why do not I see peak in hplc-uv curve? Can be a fault of the machine? How can I check?
You should give full name for IAA and solvent name in which you have followed UV spectra????
To rule out instrument issues, you should have a standard compound or mixture which has known to work well. The manufacture often has a column, solvent system, and can provide a test sample, or list how to make a test sample, to verify the instrument is working. Run this test to verify the system is working. If it doesn't work, it is easier to troubleshoot the system instead of wondering if the problem is with a new sample or the instrument.
Poor method! While running a spectrophotometric scan of the solution is nice and demonstrates that IAA is present, it also demonstrates that IAA is completely retained by the HPLC column (which you did not describe) under those chromatographic conditions. Remember, you are developing a concentration partition between the mobile phase and the stationary phase (the column).
In this case you can increase the concentration of the organic phase (ACN in this case) or use a small amount of a stronger solvent (I would suggest 5% IPA increments) in order to 'boot' the IAA off the column.
Sorry I was not clear and I did an error, I would to write 25 (water) :75 (acetonitrile). I took this value from a literature work, but I have the strong doubt about a fail in the instrument, because is quite old and without maintenance from a lot of time. My column is a C18 phenomex.
Today I did a test. I inject a solution of toluene and benzene in methanol to measure the signal. I obtained signal until 240 nm-1, when I put the lamp at 220 and 190, I didn't obtain signal, just noise. The mobile phase was 50:50 acetonitrile:water.
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