by Richard A. Henry and Stephanie A. Schuster AL
How to Avoid Size Mismatch Between Solutes and Column Pores for Optimum HPLC Performance
Improved large-molecule separations are achieved when samples are matched with the correct column pore sizes. During large-molecule separations, overlap of size-exclusion chromatography (SEC) with phase interaction modes (such as reversed-phase chromatography [RPC], normal-phase chromatography [NPC], and hydrophilic interaction chromatography [HILIC]) cannot be avoided; therefore, a better under- standing of how SEC impacts high-resolution HPLC is needed. Data from these authors and others suggest that the diameter of target molecules should be no larger than 10% of the mean pore diameter to minimize pore crowding, maintain adequate retention, and avoid significant loss of efficiency and resolution. This article describes a new approach for selecting optimum column pores for separating large molecules while maintaining high performance.
Introduction While superficially porous particles (SPPs) and fully porous particles
(FPPs) using silica substrates have been enhanced for small-molecule HPLC analysis, optimum particle designs and modifications are still evolving for analyzing large molecules. Depending on the application, SPP silica can be optimized by changing pore diameter of the porous layer, layer thickness, and overall particle diameter. The ability to control and optimize porous layer thickness and particle size independently is attractive for large-molecule separations.
A 3.4-µm, 400-Å SPP-type particle developed by Kirkland for proteins and other large molecules is shown in Figure 1B next to a 2.7-µm 90-Å SPP (Fig- ure 1A). Figure 1C shows pore-size characteristics of several commercial SPPs used for HPLC separations. Some porosity is necessary in HPLC to provide adequate surface area for loading stationary phase and creating a viable two-phase distribution system within the column, but pores should also be large enough to allow access to the stationary phase by larger solutes. Commercial silica columns typically have a wide pore distribution range to permit a single column to separate a range of different molecular sizes. When molecular size varies significantly within the sample, some molecules may show performance loss due to mismatch of larger solutes with smaller-particle pores. For maximum performance, method develop- ment for different-sized molecules requires agreement between column pore-size range and the molecular sizes within the sample. Ideally, all solutes should be able to enter the pores, diffuse rapidly, and not interfere in any way with the separation process.
Small molecules typically have diameters of 5–10 Å that can fit into most particles with 90-Å average pore diameter. Pores in the 90–120 Å
AMERICAN LABORATORY 22
Figure 1 – Differences in morphology for Fused-Core particles (Advanced Materials Technology, Inc., Wilmington, DE) optimized for A) small mol- ecules, 2.7 µm; 90 Å, and B) large molecules, 3.4 µm; 400 Å. C) Pore-size ranges of Fused-Core particles for different-sized solutes.
range do not normally confine small molecules, allowing them to diffuse freely within the pore space. Pore structure is seldom considered during small-molecule method development and is not deemed important for separation; yet pore size and distribution may become key performance factors if small molecules in the sample significantly exceed 10 Å.
A particle with an average pore size of 160 Å is shown in Figure 1C. For sample molecules in the 2000-Da range, it might be necessary to move from 90 Å to a particle with larger-pore diameter to relieve confinement and regain high performance. Pores in the 150–200 Å range usually work well up to about 15,000 Da before performance loss is observed. Par- ticles with mean pore diameters of 400 Å and 1000 Å should be ideal for solutes with molecular weight (MW) between 15,000 and 500,000 Da or larger. The effects of column pore size mismatch were described by Kirk- land et al.,1–3
regarding performance of 400-Å and 1000-Å SPP particles.
Molecular-weight data is readily available, but molecular size and shape are more important in determining solute behavior toward pore structure and for selecting optimum HPLC column pore size. If samples contain a wide-solute MW and size range, screening different RP columns with a range of pore sizes can establish the point at which significant retention
JUNE/JULY 2017
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56