As the inside diameter of the column decreases, the number of theoretical plates is improved. That is to say that the peak height is increased and effective sensitivity is
increased when the same flow rate is used to equal the elution time of the peak.
|Semi-microcolumns and microcolumns generally show the following usefulness.
1) Only a small amount of a mobile phase is used.
2) Analysis is performed only with a small amount of a sample.
3) Detection sensitivity is increased.
On the other hand, it is essential that the HPLC unit (accuracy in flow rate of a pump, injector, detector, piping, etc.) is applicable to microcolumns.
A column with a smaller inside diameter is more affected by the system and careful handling is required.
As the question this time asks whether or not "performance is improved", we introduce data related.
Fig. 1 shows numbers of theoretical plates and pressures in columns that have nearly the same column volume but different inside diameters.
However, column length, optimum flow rate (exhibiting the maximum number of theoretical plates)
suited to the inside diameter of each column and the amount of sample injected are not taken into considerration in these data. However, you can see that, even taking the effects
of flow rate shown in Fig. 2 into consideration, the number of theoretical plates tend to increase with narrowing of the inside diameter of the column if the volume is the same.
This means that
3) the detection sensitivity is increased and
2) analysis can be performed with a small amount of a sample.
The extracts obtained by the method shown in Fig. 1 were purified according to the above procedure. The situations in the procedures (i), (iii) and (v) are shown in Figs. 2, 3 and
4, respectively. Wakosil 5C18 that has the same basic characteristics as Wakosil 40C18 was used as the HPLC filler for examination of conditions and the eluent was exchanged
every doubling (about 200 mL) of the column volume by the multistep elution method. The components in the respective fractions obtained showed excellent correlation with the
results of examination of conditions by the HPLC and separation has been achieved as targeted. It is also possible to fractionate as a single component according to procedure .
Chromatograms of the purified substances obtained finally by the purification of No. 5 fraction by there-flash method without using a HPLC filler suited to high-accuracy
purification (such as Wakosil Prep series) are shown in Fig. 5.
|In Fig. 1, the column pressure increases with narrowing of the inside diameter showing impression of difficulty in using.
However, even columns of different inside
diameters show the same column pressure when they are used at the optimal flow rate provided that they have the same length.
The optimal flow rates in the columns of different
inside diameters are almost the same as the linear velocity of the mobile phase flowing in the columns.
When a column is used at the optimal flow rate, the column pressure is equal but the flow rate depends on the inside diameter.
Thus, a thick column needs a large volume of the
mobile phase while a small amount of the mobile phase is sufficient for a narrow column. In addition, sensitivity can be improved several times compared to the conventional
size by setting optimal conditions taking the sample diluent, sample concentration, amount of injection, etc. into consideration. Finally, we introduce comparison data
for separation of 16 polyaromatic hydrocarbons (PAHs) in Fig. 3.
|* Standard column joint types are Waters (W) type and Du Pont (D) type. Please make inquiry for other types.