Sample preparation for Normal and Reversed Phase Chromatography

The sample preparation method for normal and reversed phase analysis is as follows:

Fundamental theory
* The sample should be dissolved in the eluent to be used and filtrated with 0.45 micron disposable filter. Then, it should be injected into HPLC system about 10 to 50 micro-L.
When the sample is dissolved in the eluent, good separation can be expected even for the sample whose peaks appear near Vo because the system peak will not appear near Vo. (When the column size is 4.6mmI.D. x 150 to 250mm length)
* Avoid overloading.
Too much sample should not be injected. If peak shape changes by diluting the sample 10 to 100 folds or by decreasing the injection volume to one fifth, there is a possibility of sample overloading.
* The injection volume of standards and that of actual samle should be the same. If they are different, retention times may be different.

Some troubles may happen when the actual sample is analyzed though the troubles never happen when standards are analyzed. In most cases, they are caused by inappropriate selection of the solvent in which the sample is dissolved.

Troubles
(a) Leading or tailing of peaks.
(b) Instablity of retention time.
(c) Split of peak top.
(d) Very broad ghost peak.
(When the ghost peak is too broad to be recognized as a peak, it may be recognized as the baseline noise.)
(e) Increase of column pressure.
(f) The above troubles happen just for a specific sample (or peak).
The way how to select the solvent in which the sample is dissolved is explained below.

Reversed phase
Consider the situation that acetonitrle/water = 50/50 is used as the eluent and sample is easy to be dissolved in acetonitrile. Of course, the best way is to dissolve the sample in the eluent. However, the sample cannot be dissolved in the eluent, what solvent should be selected to dissolve the sample? 100% acetonitrile? Or, acetonitrile solution less than 50%?
In this case, the first choice should be "acetonitrile solution less than 50%". The second choice is "100% water" and it can be used up to 500 micro-L. The worst choice is "100% acetonitrile". When the sample is dissolved in 100% actonitrile, there is a possibility that the sample educe in the column. Even if the sample is completely dissolved in 100% acetonitrile, it may educe in the column when the acetonitrile concentration decrease in the column. If such eduction takes place, first, trouble (a) and/or (b), and then, trouble (c) and/or (d) may happen. In the worst case, (f) may happen and it may damage the column. In case that the sample is a mixture, (g) may happen.
In order to judge whether sample eduction is taking place in the column, reduce the concentration of acetonitrile and increase the injection volume. For example, instead of injecting 25 micro-L of sample dissolved in 100% actonitrile, inject 125 micro-L of sample dissolved in 20% acetonitrile. If the trouble is solved, it is considered that sample eduction is taking place in the column. And, in such case, the selection 100% acetonitrile is inappropreate.

Normal phase
Consider the situation that 15% dichloromethane/hexsane is used as the eluent and sample is easy to be dissolved in dichloromethane. The best way is to dissolve the sample in the eluent.
If the sample cannot be dissolved in the eluent and 100% dichloromethane is selected as the solvent in which sample is dissolved, some trouble may happen.

In reversed phase or normal phase chromatography, usually, a mixture of some solvents is used as the eluent. The solvent in which the sample dissolves more easily advances the elution time. Before selecting a solvent in which sample is dissolved, it is recommended to change the eluent composition by 5 to 10% and check the elution time. Of course, it is neccesssary that the solvent can be dissolved in the eluent.