25


According to the Young-Laplace equation, the surface of the dried hydrophobic stationary phase requires very high pressure to drive the aqueous phase solvent into the pores contained on the surface. This equation relates the intrusion pressure to the surface tension of the water and to the contact angle of the water and air in the sorbent surface:


where ΔP is the intrusion pressure required to drive liquid into the pores, γ is the surface tension, d is the effective pore diameter, and θ is the contact angle made between water and air on the adsorbent surface. Since the contact angle made between 100% aqueous mobile phase and hydrophobic alkyl-bonded silica-gel pore is greater than 90°, therefore, θ from the above mentioned equation is smaller than 90° and ΔP should be a positive number, which means positive pressure is required to drive the aqueous mobile phase into the silica-gel pore. In regular liquid chromatography separation, the pressure driving aqueous mobile phase into the silica-gel pore is larger than the pressure required to drive the mobile phase fl owing through the whole chromatography column. So, when the separation is suddenly stopped, the pressure inside the silica-gel pore is greater than the pressure outside the silica-gel pore, as a result the aqueous mobile phase will be squeezed out from hydrophobic alkyl-bonded silica- gel pore. When this column is reused without special treatment, due to the dewetting status of the adsorbent pore, the interaction between the adsorbent and the analyte will be greatly reduced, resulting in a signifi cant loss of retention time for the analyte. However, when the stationary phase surface becomes more hydrophilic, the contact angle made between 100% aqueous mobile phase and hydrophobic alkyl-bonded silica-gel pore will be reduced. When this contact angle is smaller than 90°, which means θ is greater than 90°, ΔP should be a negative number. Under this condition, the pressure outside the silica-gel pore is greater than the pressure inside the silica-gel pore and the mobile phase will spontaneously enter the silica-gel pore. This is when the adsorbent pore is wetted.


The above theoretical analysis shows that for a conventional C18 column, hydrophilic modifi cations can be performed to the silica gel surface to improve the wettability in highly aqueous mobile phase [5], including


• non-endcapped, short-chain alkyl phases;


• hydrophilic, polar-endcapped, and polar-enhanced stationary phases; • polar-embedded alkyl phases; • long-chain alkyl phases; and • wide-pore-diameter phases.


Flash chromatography cartridges prepacked with a hydrophilic C18-bonded silica gel, in which hydrophilic cyano groups are introduced on the silica gel surface (as shown in Figure 3) have been prepared. The alkyl chains on the silica surface can be fully extended under highly aqueous conditions and the phase collapse/wettability phenomenon can be avoided. These modified C18 columns are called aqueous C18 columns, namely C18AQ columns (SepaFlash, Santai Technologies), are designed for highly aqueous elution conditions and can tolerate a 100% aqueous system. C18AQ columns have been widely applied in the separation and purification of highly polar compounds, including organic acids, peptides, nucleosides and water-soluble vitamins.


Figure 3. The schematic diagram of the bonded phases on the surface of silica gel in regular C18 column (left) and C18AQ column (right).


Desalting is a typical application of C18AQ columns in the Flash chromatography purification of samples, where the salt or buffer components in the sample solvent are removed to facilitate the use of the desalted sample in subsequent studies. In this application, a highly polar compound was used as the sample and purified on a C18AQ cartridge. The aqueous salty components contained in the raw sample were removed and the sample solvent replaced by organic solvent, thus facilitating the subsequent rotary evaporation saving both solvents and operating time.


Experimental Section Sample information


The sample was the fi nal product of a synthesis reaction which was shown in Figure 4.


Figure 4. The synthesis reaction formula of the sample.


As shown in Figure 4, in addition to the target product in the raw sample, there is also excess ammonium salt (tetrabutylammonium fluoride, MW 261) used in the synthesis reaction. The sample should be desalted in order to obtain the target product meeting the purity requirement. In chromatographic separation technology, gel filtration based on the principle of molecular sieving is generally used to desalt the macromolecular sample, including proteins, peptides, nuclei acids, etc. However, for the target product with MW of 252 in this post, it is a nearly impossible task for gel filtration to distinguish the target molecule from salt impurities with very similar MW. Other separation modes must be considered.


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