Injecting
samples into the HPLC injector is very simple; this is done by manual syringe
or auto-sampler injection. The Rheodyne
sample loops and auto-sampler have fixed or variable volumes (0.1 – 100 μL) that
can inject into the HPLC column. The peak area and height increase in
proportion to the injection volume of the sample solution. When the injection
volume becomes too high the result in the peak is broadened and broadening this
peak may reduce the resolution between adjacent peaks. The injection volume
must be chosen in accord with the column geometry. We can use a volume between 1
and 5% of the column dead volume as the maximum injection load per column;
however, we have to determine this. The best injection volumes are related to
the volume of the HPLC column and, as a result, are dependent on the column
length and cross-sectional area.
Here
some approximate volume range of the sample component is mentioned if it is
prepared in the same mobile phase as used in the system.
Column ID
|
Length of Column
|
Injection Volume
|
2.1 mm
|
30 to 100 mm
|
1 to 3 µL
|
3.0 to 3.2 mm
|
50 to 150 mm
|
2 to12 µL
|
4.6 mm
|
50 to 150 mm
|
8 to 40 µL
|
10 mm
|
50 to 250 mm
|
40 to 100 µL
|
21.2 mm
|
50 to 250 mm
|
150 to 300 µL
|
30 mm
|
50 to 250 mm
|
300 to 700 µL
|
50 mm
|
50 to 250 mm
|
1000 to 2000 µL
|
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