Sample preparation for Normal and Reversed
The sample preparation method for normal
and reversed phase analysis is as follows:
* 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.
(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
(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.
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.
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
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
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.