Chromatography
The use of a Reverse Phase (C18AQ) Flash
Chromatography Cartridge in Sample Desalting Cong Zhang1
, Bo Xu1 , Andre Couture2 *
1: Santai Technologies Inc., 78 Qingyang Road, Xinbei District, Changzhou, Jiangsu Province, 213125, China 2: Santai Science Inc., 214 Brunswick, Pointe-Claire, Montréal, QC H9R 1A6, Canada *Corresponding Author:
andrecouture@santaitech.com
Reversed phase liquid chromatography (RPLC) is a widely used separation mechanism in liquid chromatography. RPLC has been used to separate numerous compounds, from organic acids with a low molecular weight (MW) to proteins with a MW of up to 150 kDa. However, analytes with good water solubility (i.e., hydrophilic or polar) exhibit poor retention on conventional C18 RP columns. In this case, the aqueous ratio in the mobile phase needs to be increased in order to enhance the interaction between the polar analyte and the hydrophobic stationary phase, thereby increasing the retention of polar analytes. The greater the polarity of the analyte, the higher the proportion of water required in the mobile phase. When regular alkyl bonded reversed phase columns e.g. octadecyldimethylsilane (ODS, C18) are used in pure aqueous or highly aqueous mobile phases for a long time, problems can occur including loss in retention time for the analyte and irreproducible separation results. This phenomenon is referred to phase collapse [1]. The classic explanation for this phenomenon is that the C18 stationary phase bonded to the surface of the silica gel changes the spatial arrangement of the carbon chain in highly aqueous mobile phase, that is, changes from perpendicular to the surface of the silica gel to lying fl at on the same plane as the silica surface (as shown in Figure 1). The change in the spatial arrangement of the stationary phase reduces its interaction with the analyte, thus reducing the retention time of the analyte and often causing it to be unretained.
case, the mobile phase no longer remains in the porous space of the stationary phase particles, so the chance of the stationary phase and the analyte coming into contact with each other is also reduced, which in turn causes the reduced retention time of the analyte. An experiment confi rmed that when the mobile phase in the C18 column was suddenly switched from a high proportion of organic solvent to 100% aqueous solvent, the volume of the mobile phase in the column was signifi cantly reduced, and the magnitude of the decrease in such volume was closely related to the loss of retention time for the analyte [3, 4].
Figure 1. Illustration of the classic explanation of phase collapse in reversed phase chromatography (reproduced from Ref.1). Shown are the confi gurations of long-chain bonded alkyl phases (a) in water- methanol mixtures and (b) in 100% water.
Researchers have conducted in-depth studies of the phase collapse phenomenon. The continuous accumulation of experimental evidence has led many researchers to accept another new theoretical explanation, that is, the loss in retention time is actually due to the ‘dewetting’ which occurs in the fi ne pores of the stationary phase particles [2]. In the new theoretical explanation (Figure 2), a high surface tension is generated between the aqueous mobile phase and the surface of the hydrophobic stationary phase. Therefore, the mobile phase is easily expelled from the porous space of hydrophobic stationary phase. In this
Figure 2. Illustration of a possible mechanism of pore dewetting for reversed phase chromatography (reproduced from Ref. 1). The analytes are properly retained when the alkyl chains on the stationary phase are properly solvated with pressure using a 100% aqueous mobile phase (a). When the fl ow has been stopped to allow expulsion of water from the pores, with fl ow resumed the pores are still dewetted and analytes cannot enter pores and have little or no retention (b).
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