Hi,
I am a graduate student using a Shimadzu HPLC with a PDA detector.
This instrument was newly installed. Only the tech at installation and myself have run samples.
I am using it for research on cannabinoids specifically CBD and THC and only running diluted standards right now to modify the method.
I am using water (0.1% formic acid) as mobile phase A, and acetonitrile (0.1% formic acid) as mobile phase b. The total run time is 10 min. I have a gradient that starts at 70% B for 3 min, then ramps up to 90% B for 2 min, hold for 1 min, then back down to 70% and hold for the rest of the time. The flow rate is 0.2 mL/min. My injection volume is 1 uL.
The column is a Shimadzu C18-120, 3 um 3.0 x 50 mm.
The PDA detector wavelength is from 190 nm to 600 nm and specific wavelengths are 210 nm and 220 nm.
During my first run I ran a CBD sample (CBD 50 ng/mL in acetonitrile). A mistake was made of not running a blank before the CBD sample :(
I ran several blanks after and still continue to see peaks.
I have remade the blanks many times in different vials, new solutions of blank.
I set up a run consisting of 75 blanks on a 2 minute "cleanout method" run where I was using 90% ACN for the full time. All the blanks in this batch had the same peak with a consistent intensity. (The intensity of the peak did not really decrease over the 75 injections).
I have also run Null injections and have gotten the same peak in those injections as well.
I switched mobile phases from ACN to methanol. When running the blanks with the methanol I still got the same peak. After reversing the column, running methanol through, then fixing the column I still got the same peak again.
First we had thought it was CBD that contaminated the instrument, but after switching the phases and running so many blanks we are not sure if this is something with the instrument that we should try to change/clean?
At this point I am not sure where this peak could be coming from and was looking for some advice/direction on what to try next!
I can provide additional information as needed!
try this
The column and autosampler were thermostated to 35 °C and 4 °C, respectively, for the RP-HPLC analysis. 5.0 μL was the injection volume, and the sample concentration was 4 mg/mL. Following this protocol, gradient elution was employed at a flow rate of 1.6 mL/min and UV detection was employed at 220 nm. Eluent mixtures consisting of acetonitrile plus 0.085% phosphoric acid (B) and water plus 0.085% phosphoric acid (A). 85% of B up to 7 minutes, 95% of B to 7.01 up to 8.00 minutes, and 70% of B up to 10 minutes are the gradient elution times. Previously, the eluent mixture was filtered using a Millipore system fitted with a nylon filter measuring 0.2 μm.
What is the retention time for the peak in acetonitrile? What is the retention time in methanol?
"Ghost" peaks come from contaminants. Null runs without injections showing the peak tends to rule out the injector as a source of the peak. The peak seems very reproducible.
One source of ghost peaks are contaminants in solvents. It could be the formic acid or water, you still saw the peak when you tried methanol which suggests the acetonitrile might not be the source.
If you post a chromatogram, it would be helpful.
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