Abstract
A combined experimental and theoretical study was performed to understand how the pore size of packing materials with pores 60–300 Å in size affects the separation of 5–50-mer oligonucleotides. For this purpose, we developed a model in which the solutes were described as thin rods to estimate the accessible surface area of the solute as a function of the pore size and solute size. First, an analytical investigation was conducted in which we found that the selectivity increased by a factor of 2.5 when separating 5- and 15-mer oligonucleotides using packing with 300 Å rather than 100 Å pores. We complemented the analytical investigation by theoretically demonstrating how the selectivity is dependent on the column's accessible surface area as a function of solute size. In the preparative investigation, we determined adsorption isotherms for oligonucleotides using the inverse method for separations of a 9- and a 10-mer. We found that preparative columns with a 60 Å-pore-size packing material provided a 10% increase in productivity as compared with a 300 Å packing material, although the surface area of the 60 Å packing is as much as five time larger.
Highlights
High-performance liquid chromatography (HPLC) has become a standard technique for separating biopharmaceutical compounds, with ion-pair reversed-phase liquid chromatography commonly being used [1,2]
We continue by discussing the analyticalscale separations of T5–T50 oligonucleotides in terms of retention and selectivity to find out how selectivity is dependent on pore size and how this is related to the change in accessible surface area
Determining the adsorption isotherm for methyl mandelate confirmed that the saturation capacity is linearly proportional to the accessible surface area
Summary
High-performance liquid chromatography (HPLC) has become a standard technique for separating biopharmaceutical compounds, with ion-pair reversed-phase liquid chromatography (ion-pairRPLC) commonly being used [1,2]. Several papers have studied the separation of nucleic acids using ion-pair-RPLC [3,4,5,6]. A useful strategy to improve separation performance in RPLC is to alter the column properties. The column properties that can affect the separation include the surface chemistry, particle size, pore size, and column dimensions, which can be scaled to enable improved separation efficiency or higher sample loads [7,8,9,10]. We recently published a study on the separation of 5-20 long homomer oligonucleotides where we compared the performance of three alkyl phases (C4, C8, C18) and one aryl phase (phenyl). Our findings showed that C4 was preferable in terms of selectivity when separating native oligonucleotides, using triethylammonium acetate (TEtAA) as ion-pairing reagent [10]
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