Wako Pure Chemical Industries, Ltd.Wako Pure Chemical Industries, Ltd. - Laboratory Chemicals
Home > Products > Analysis and Environment > Wako FAQ -Chromatography- Q10

Wako FAQ -Chromatography- Q10


FAQ
Q10: I 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.



We are introducing usefulness of semi-microcolumn such as follows compared to the use of a column (4.6φ mm) with some precautions.

    1) Only a small amount of a mobile phase is required.
    2) Analysis is performed only with a small amount of a sample.
    3) Detection sensitivity is increased.

However, in the actual practice, some precautions are needed in hardware and software aspects.

Concerning hardware aspects, you seem to have used a system applicable to microcolumns from the content of your question. However, as another precaution, peak shapes and retention times vary whether an autoinjector is used or the sample is injected manually.

In Fig. 1, we show the examples of analysis of polyaromatic hydrocarbons (PAHs). Use of an autoinjector results in poor finish of a peak and prolongation of the retention time of a component eluted in the early stage. These are attributed to the volume of a sample loop attached to the autoinjector indicating that even a factor not raising issues in the ordinary use may become problematic in the use in the range of low flow volume. In view from software, the key point is “which solvent a sample is dissolved in and how much it is injected” and these effects are also visible remarkably in the peak shapes of components eluted in the initial stage.
◆Fig. 1 Effect of the sample injection method◆
Fig.1_Effect of Sample Injection Method

In Fig. 2, differences in chromatograms when PAHs are dissolved in solvents of different polarities and injected in the same volume are shown. Dissolution of a strongly hydrophilic and freely water-soluble sample in water or an nitial solvent of a mobile phase is not so problematic. However, when a strongly hydrophobic sample such as PAHs is dissolved in a solvent with more potent elution capacity than the mobile phase, importance is attached to “which solvent the sample is dissolved in” and dissolution in a potent elution solvent in particular must be avoided as it may lead to broadening of a peak. Even when a sample is dissolved in the same solvent, injection of a minimum amount results in improvement of a peak shape.

◆Fig.2 Effect of the sample diluent and the amount of injection◆
Fig.2_Effect of Sample Diluent & Amount of Injection
As mentioned above, we explained precautions during use of a semi-microcolumn. Semi-microcolumn is extremely useful analytical tool provided that it is used with thorough understanding of its characteristics and differences from general-purpose columns.









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- Q10