In this project, we will be looking at a study by Agilent Technogies, Inc. that describes the detection of carbendazim and 3 other fungicide residues in Orange Juice, and HPLC coupled to Mass Spectrometry (MC) was the detection method of choice.
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| In this study, the detector used in this image would be a Mass Spectrometer. |
How HPLC works:
In our case, what we need for our detection would be a Normal Phase HPLC
In a normal phase HPLC, we use a NON-POLAR mobile phase and a POLAR stationary phase. Firstly, we have 2 types of mobile phase 1) 0.1% formic acid in WATER and 2) 0.1% formic acid in METHANOL.
So in our solvent reservoir, we need 0.1% formic acid, water and methanol.
During the actual run of the analysis, the solvent are run through a vacuum degasser, where the solvent are removed of dissolved gas that cause inconsistent flowrate which could affect the results, into the quarternary pump where the different solvents are mixed to give the desired mobile phase that would pass through into the column. At this point the autosampler, the injection system, would introduce 5µm of our samples or standards into the column together with our mobile phase which is a packed column of C18 on 2.7µm spherical silica particles. At the end, we will use a mass spectometry detection method.
How Mass Spectrometry works:
The molecule is first ionised to give it a positive charge and then accelerated to have a constant kinetic energy for all molecules. Along the path which the molecules are travelling, they are deflected with a magnetic field. The strength of this deflection depends on the size of the molecules and also the charge that it obtained when it was earlier ionised, so the most deflected molecules are those with a large molecular weight and those with more charge. The detection of the ions is done by detecting the neutralised electric current.
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