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Wako FAQ -Chromatography- Q6

Q6: We wish to perform fractionation by the reverse phase flash chromatography.
How can we determine the optimal separating conditions?

Flash chromatography, which is a very effective and simple purification method, offers separation and purification at high accuracy in a short period of time compared to conventional open chromatography.

With regard to separating conditions, TLC is performed frequently for normal phase systems and separating conditions can be set up easily from Rf values.

In the reverse phase flash chromatography, on the other hand, it is effective to set up conditions by the HPLC, especially by the gradient method not readily affected by the particle size. While the conditions of separation and purification differ between fractionation of all components and fractionation of a specific component alone, the procedure for setting is the same as shown in the following.

[1] Fractionation by flash chromatography
(i) Confirmation of elution patters of all components
(ii) Exploration of gradient conditions best suited to fractionation of a specific component if it is desired.
(iii) Confirmation of chromatography pattern by the multistep elution method assuming flash chromatography
(iv) Scale-up to flash chromatography
(v) Confirmation of fractions.

[2] High-accuracy purification procedure (re-flashing, HPLC fractionation, etc.) Fractionation of all components of powdered rhubarb using FLASH 40S System (filler: Wakosil 40C18) (BIOTAGE) is explained as an example.
Figure 1: Outline of Operating Procedure
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 the re-flash method without using a HPLC filler suited to high-accuracy purification (such as Wakosil Prep series) are shown in Fig. 5.
Reverse phase flash chromatography is especially effective means when components freely soluble in water such as powdered rhubarb in the present case are handled. The characteristics of Wakosil 40C18 are introduced together with Wakosil 25C18, the same reverse phase filler for fractionation, in p.2 for your reference.

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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