sample diluent for HPLC

[jaigodheja]

Is it necessary to use the same solvent as mobile phase which is used to dissolve the sample?

[jaigodheja]

My sample is dissolved in ethyl acetate. Can I use ethyl acetate as mobile phase in reverse phase HPLC?

[Blazer]

It's not necessary, but it is considered a best practice.

Note: my comment was made to the original post. Second post by the OP was merged into this thread.

[Consumer Products Guy]

Is it necessary to use the same solvent as mobile phase which is used to dissolve the sample?

Occasionally we did. Most times we did not.

For example, some consumer products dissolved well in methanol, some in DMF, etc., as compared to mobile phase Our mobile phases often were low pH water and ACN. The tricks with reverse phase when extracting into solvents "stronger" than the mobile phase include keeping the injection volume small, or diluting the strong extract with water to make it "weaker".

Posters here need to have more descriptive post titles !

[tom jupille]

Sorry if the sequence here is confusing. I merged two semi-duplicate topics but can't re-order the posts.

My sample is dissolved in ethyl acetate. Can I use ethyl acetate as mobile phase in reverse phase HPLC?

Reversed phase almost always uses water as the weak solvent. The strong (organic) solvent should be miscible with water. (Yes, non-aqueous-reversed-phase, aka "NARP", does exist but it is very rarely used).

[lmh]

The point is that if you inject in a solvent that is much stronger than the running solvent then the compounds of interest may not bind to the column straight away, because they are in eluting conditions. They will migrate into the column until they're diluted out (gradually) by the actual running sample, and then they will bind.
The worst case scenario is that they'll actually elute completely without binding. The more likely outcome, however, is that they'll have poor peak-shape and run earlier than expected.
Isocratic methods are extremely vulnerable because the running solvent will be only just strong enough for the analyte to bind, and because the retention time depends entirely on how fast the analyte moves along the column (so giving it a head-start will definitely mess up the retention time).
Gradient methods are more resilient, because if the gradient starts at 10% acetonitrile, the analyte doesn't actually start to elute until 40%, and you inject in 35%, then the analyte will bind fine. In fact if you inject in 60%, which is theoretically enough for a problem, the problem will actually be very small because the 10% starting-point of the gradient will dilute the sample to less than 40% very rapidly; the actual elution time depends mostly on the time that the gradient reaches 40%, not how far it happened to have got into the column before it was diluted (these are all just randomly-chosen example numbers, to illustrate what happens).
In gradient methods, if the injection solvent is too strong, then early peaks are misshapen and too early, while late peaks are often unaffected.
(Of course there is the opposite risk, usually in preparative methods, that injecting in 60% methanol using a gradient that starts at 10% might result in the sample precipitating and blocking the autosampler! This is rare in analytical methods)

[JI2002]

You probably cannot use UV as a detector if you choose to use ethyl acetate as mobile phase due to its high UV cutoff. Try to avoid it if you can.