My mobile phases are methanol:acetonitrile:dichloromethane:acetic acid (50:44:3:3, v/v/v/v).
My samples are extracted with dichloromethane:methanol (2:1, v/v).
My standard is oryzanol. First, I need to prepare a stock solution and then further diluting it to make the standards (2000 ppm, 1000 ppm, 500 ppm, 100 ppm and 50 ppm).
So, my standard should be diluted in? Is it have to be dichloromethane and methanol as these are used to extract my samples?
Thank you in advance.
Since it seems you are working under isocratic conditions, you should dilute the standards in whatever solvent the samples are dissolved in.
Since solubility of the standard in the mobile phase is always an issue, use your mobile phase.
To minimize the unknown effect of different solvent, please use the same solvent condition for both sample and standard. Whatever it is.
Use the same solvent (mobile phase solution) for both sample and standard. Although others have mentioned this, I am adding a bit more info: loading a sample in a different solution may affect the RP phase column resin and will result in different binding and releasing conditions of the standard- hence the reason to stick with the same mobile phase.
In HPLC analysis, it is always advisable to prepare the solutions of both standard and sample in the mobile phase.
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