Mobile phase in HPLC

[alleyoop]

Why are the sample solutions prepared with mobile phase?

Thanx a lot.

[MG]

Because the plug of sample solvent also acts as a mobile phase briefly at the head of the column. If the sample solvent is stronger (in terms of elution strength) than the mobile phase, some of your analyte can move down the column faster than it would otherwise, causing peak distortion or split peaks. So ideally your sample solvent should have the same or weaker eluting strength as your mobile phase (or your starting mobile phase in case of gradients).

If you are running a gradient and your injection volume is small enough, you can bend this rule and inject solvents stronger than your starting mobile phase, but how much you can bend it depends on your conditions and your analytes.

[unmgvar]

Hello alleyoop,

I will only had to MG's explanation that it is important to inject in the LC sample that need to have the same or weaker eluting power.

the sample preparation itself can be started by dissolving your matter into a 100% organic solvent (many times you cannot dissolve your sample directly with the mobile phase itself) and then to perform dilution with the mobile phase. Alternatavely the next step dilution can be simply to add the buffer compostion.
for examlpe you dissolve your sample in 100 % Acetonitrile and then you perform a dilution step of 40 ml into a 100ml volumetric flask and you had 60 ml buffer. this step is done because your mobile phase is actually 40%ACN and 60% buffer.