The Application of SepaFlash® Strong Anion Exchange Chromatography Columns in the Purification of Acidic Compounds

Background: ion exchange chromatography (IEC) is a kind of chromatographic method commonly used to separate and purify compounds in solution in the form of ions According to the different charge symbols of exchangeable ions, ion exchange chromatography can be divided into cation exchange chromatography and anion exchange chromatography In cation exchange chromatography, acidic groups are bonded on the surface of the separation medium, such as sulfonic group (- SO3H) in strong cation exchange (SCX), which dissociates H + in the solution, and the negative group - SO3 - contained in the body can adsorb other cations in the combined solution In anion exchange chromatography, basic groups are bonded on the surface of separation medium, such as quaternary amine group (- NR 3OH, where R is hydrocarbon group) in strong anion exchange (sax), which dissociates Oh in solution, and the positive Group-N + R 3 contained in the body can adsorb other anions in the combined solution, thus producing anion exchange Among the natural products, flavonoids have been paid more attention by researchers because of their role in the prevention and treatment of cardiovascular diseases Because the flavonoid molecules contain phenolic hydroxyl and are acidic, in addition to the normal phase and reverse phase chromatography, ion exchange chromatography is also an option for the separation and purification of these acidic compounds In flash preparation chromatography, the commonly used ion exchange separation medium is the bonded ion exchange group on silica gel matrix, the most commonly used include SCX (generally sulfonic) and sax (generally quaternary amine) The application of SCX separation column in the separation and purification of basic compounds was introduced in the application article "sepaflash ® strong cation exchange chromatography column application in the purification of basic compounds" published before Santai technology In this paper, the mixture of neutral and acid standards was used as samples to study the application of Sax column in the separation and purification of acid compounds Fig 1 Chemical structure of the packing surface bonding phase of Sax separation column in this paper The packing surface bonding phase of Sax separation column used in this paper is a quaternary group (see Fig 1 for the bonding phase structure formula), and the sample is a mixture of chromone and 2,4-dihydroxybenzoic acid (see Fig 2 for the sample structure formula) Dissolve the mixture sample in methanol, use a syringe to sample the sample solution on the flash separation column, and the flash separation and purification experimental parameters of the sample are shown in Table 1 Figure 2 Chemical structure formula of two components in the sample table 1 Sepabean ® machine t chromatographic column 4 g sepabelash ® standard series positive phase separation column (amorphous silica gel, 40-63 μ m, 60 μ m, order No.: s-5101-0004) 4 g sepabelash ® bonded phase series Sax separation column（ Amorphous silica gel, 40 - 63 μ m, 60 μ m, Order No.: sw-5001-004-ir) detection wavelength 254 nm; mobile phase solvent A: n-hexane; solvent B: flow rate of ethyl acetate 30 ml / min 20 ml / min injection volume 20 mg (mixture of component A and component B) elution gradient time (CV) solvent B (%) time (CV) Solvent B (%) 0.001.7 12 14.0 100 3.7 12 / / 16.0 100 / / 18.0 100 / / results and discussion First, the mixture sample is separated on a normal silica gel column, and two components in the sample are successively eluted from the separation column (as shown in Figure 3) Next, the Sax column was used to separate and purify the sample The acid component B in the sample was completely retained on the Sax column, and the other neutral component a (chromone) was eluted from the column gradually with the mobile phase (as shown in Figure 4) Figure 3 Separation spectrum of sample on normal phase silica gel column Figure 4 Comparison of separation spectrum of sample on Sax separation column Figure 3 and Figure 4 show that the peak shape of elution peak of component A in sample on two different separation columns is inconsistent In order to confirm the corresponding components of elution peak, we can use sepabean ® machine Full band scanning function built in the control software Open the experimental data of two separations respectively in the experimental record, drag the indicator line on the time axis (CV) in the separation map to the highest point and the secondary high point of the elution peak corresponding to component A, and the full band absorption spectrum at these two points will be automatically displayed under the separation map (see Fig 5 and Fig 6) Comparing the full band absorption spectrum data in the two experiments, we can see that component A is in the two experiments The spectral data in is consistent For the reason that the peak shape of the elution peak of component A on the two separation columns is inconsistent, we speculate that there may be some impurities in component A, and the chromatographic retention behavior of component A and impurities is different on the normal silica gel column and sax column, so the peak order is inconsistent, resulting in the difference of the peak shape of the elution peak Figure 5 Full band scanning spectrogram of component A and impurity on normal silica gel column 6 Full band scanning spectrogram of component A and impurity on Sax column If the product to be collected is neutral component A, the purification task can be completed by directly using Sax column for sample loading and elution If the product to be collected is acid component B, the method of "capture release" can be used, with only a little adjustment in the experimental steps: after sample loading and elution on the Sax column (using the organic solvent system of positive phase separation), switch the mobile phase to the methanol solution containing 5% acetic acid, use the acetate ion in the mobile phase to compete with component B and combine with the fixed phase on the Sax column The quaternary amine ion group on B is used to elute component B and obtain the desired target product See Figure 7 for the sample separation spectrum in the ion exchange separation mode Fig 7 The separation map of component B on Sax column eluted by ion exchange mode Therefore, by adopting different purification strategies, Sax column and normal phase silica gel column can achieve rapid separation and purification of acid or neutral samples In addition, due to the different separation modes, the peak shapes of the elution peaks of the components in the samples are different Using the full band scanning function built in sepabean ® machine control software, it is convenient to compare and confirm the characteristic absorption spectra of the elution components, so as to help researchers quickly judge the composition and purity of the elution components and improve the work efficiency Sepaflash ® Sax separation column series products launched by Santai technology have a variety of specifications (see Table 2) Table 2 Sepaflash ® Sax separation column parameters (packing: ultra pure irregularsax, 40 – 63 μ m, 60 Å ）
Item Number
Column Size
Flow Rate
(mL/min)
Max.Pressure
(psi/bar)
SW-5001-004-IR
5.9 g
10-20
400/27.5
SW-5001-012-IR
23 g
15-30
400/27.5
SW-5001-025-IR
38 g
15-30
400/27.5
SW-5001-040-IR
55 g
20-40
400 / 27.5 sw-5001-080-ir 122 g 30-60 350 / 24.0 sw-5001-120-ir 180 g 40-80 300 / 20.7 sw-5001-220-ir 340 g 50-100 300 / 20.7 sw-5001-330-ir 475 g 50-100 250 / 17.2 to learn more about sepabean ® machine Please visit the CBG online store at https://store.chembergo.cn/ for detailed specifications or ordering information of flash purification column.