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Wako FAQ -Chromatography- Q5


FAQ
Q5: We wish to fractionate by HPLC. How can we determine the optimal separating conditions?



It is general to set up conditions using a column of analysis size and, then, shift to fractionation size because examination of optimal separating conditions directly with a fractionation column requires a large volume of sample and fractionation columns are expensive.

In this case, it must be noted that the conditions cannot be shifted smoothly if the particle size distribution of fillers differs between the analysis column and fractionation column. Although such problem does not occur if the same filler is used in the analysis and fractionation, use of the filler for the analysis size in the fractionation results in a high pressure restricting conditions.

On the contrary, even if the pressure is low, the advantages of HPLC fractionation are not utilized if the number of theoretical plates is small. That is to say that key to smooth transfer of conditions to a large size column is the use of a filler specific for fractionation that exhibits a certain level of separation performance and low pressure during use. Then, concerning the issues associated with transfer of conditions, parallel relationship is established for the amount of injection and the number of theoretical plates or separation capacity between columns of different diameters allowing smooth transfer of conditions provided that the linear velocity of the mobile phase is the same. This means that examination of optimal separating conditions using a column of analysis size allows transfer of conditions to a column of fractionation size.

Fractionation of arbutin is illustrated as an example of application of fractionation using a filler meeting the above conditions. It can be seen that the pressure is
controlled low, and the filler exhibits almost the same performance from a column 4.6 to 50 mm in inside diameter.

Analysis and fractionation of arbutin
Wakosil-II 5C18 RS Prep is a low-cost and high-performance fractionation filler 5 μm in particle size prepared for fractionation while retaining the advantages of RS filler for analysis. We offer wide ranges of fillers for fractionation, HG Prep, AR Prep and 5 SIL Prep, allowing smooth transfer to fractionation. Please ask for information.








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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