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Wako FAQ -Chromatography- Q4


FAQ
Q4: Ion pair reagents for low wavelength are on the market. What effects and differences do they show with actual samples?



As an example, there is an analysis method in which a quaternary ammonium salt is used as an ion pair reagent to analyze bromine in foods by HPLC at UV 205 nm. Use of a general ion pair reagent even though it is used routinely at 254 nm without any trouble, may result in high background with phenomena such as (1) disturbance of baseline, (2) appearance of contaminant peaks, and (3) no detection of a peak of the target substance when the concentration of the target component is low and measurement is performed at a low UV wavelength. You can see the necessity of ion pair reagents for low wavelengths (low UV) by comparison of chromatograms shown below.

Injection of 100 μL of 10 μg/mL KBr (left)Injection of 2 μL of 100 μg/mL KBR (right) Injection of 10μL of 10 μg/mL KBr
Fig. 1 Use of general ion pair reagent Fig. 2 Use of low UV type ion pair reagent

<Operating conditions>
Column: Wakosil-II 3C18HG (4.6φ x 50 mm)
Eluent:
(Fig.1) KH2PO4 (819 mg) + NaH2PO4(714 mg) + 0.5M Tetre-n-butylammonium
Phosphate Soln.(10 mL) in 1000 mL H2O
(Fig.2) KH2PO4 (819 mg) + NaH2PO4(714 mg) + 0.5M Tetre-n-butylammonium
Phosphate Soln.(Low UV Type 10 mL) in 1000ml H2O
Flow rate: 1.0mL/min. at 40°C
Detector: UV 205,201nm
Sample: Potassium Bromide in H2O








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.
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