Wako Pure Chemical Industries, Ltd.Wako Pure Chemical Industries, Ltd. - Laboratory Chemicals
Home > Products > Analysis and Environment > Wako FAQ -Chromatography- Q7

Wako FAQ -Chromatography- Q7


FAQ
Q7: Is column performance improved by narrowing the inside diameter of a column?



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.
Comparison of Number of Theoretical Plates, Column Pressure with Same Column Volume
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.
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 [2]. 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.








INDEX
Q1Which reference standard do you recommend for use to check performance of a column?
Q2Is there any simple method to change the solvent concentrations in the mobile phase once prepared?
Q3How are ODS columns, Wakosil-II HG, AR and RS, used in different situations?
Q4Ion pair reagents for low wavelength are on the market. What effects and differences do they show with actual samples?
Q5We wish to fractionate by HPLC. How can we determine the optimal separating conditions?
Q6We wish to perform fractionation by the reverse phase flash chromatography. How can we determine the optimal separating conditions?
Q7Is column performance improved by narrowing the inside diameter of a column?
Q8We are examining mobile phase conditions of HPLC.We intend to examine by changing solvents.Is there any method to estimate the elution profile in advance?
Q9We are examining the conditions of a mobile phase in the HPLC. We would like to know how to regulate retention of a sample that has a readily dissociating functional group.
Q10I have used a semi-microcolumn (2φ mm) but cannot obtain improvement of sensitivity generally offered.
I wonder what is the cause as I am sure to have used the system applicable to microcolumns..
Home > Products > Analysis and Environment > Wako FAQ -Chromatography- Q7