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loss and peak broadening occurs for the largest molecule. If pore size can be optimized for the largest target molecule in the sample, it should be adequate for smaller molecules if retention is adequate as well. Determin- ing which solute is the largest may not be easy for unknown samples, but this is where SEC experiments can be helpful.


Size-exclusion chromatography


Much can be learned about solute size and how much access molecules have to the pore environment by performing size-exclusion experiments before conducting analytical experiments. SEC can also be valuable for troubleshooting after poor RP column performance has been observed. For this, an SEC column with a pore size similar to the high-resolution analytical column should be chosen.


The principles of SEC are described by the simple equations shown below. Additional details are available in Ref. 4.


VM VR


= V0 = V0


+ VP + KSize VP


Total column volume, VM particles, V0


a size constant, KSize , added to the volume inside the pores, VP


(1) (2)


, is the sum of the column volume outside the . Eq. [2] introduces


for large molecules. Thus, the observed elution volume, VR to or less than VM


, which ranges from one for small molecules to zero , can be equal


, but should never be greater unless solute is retained by the stationary phase. Small molecules should be free to move within all available pore space and therefore elute very near the total liquid volume of the column (VM as the solvent front.


), which is referred to in small-molecule HPLC


A low-MW marker is normally included in SEC calibrations to establish VM


. As molecules increase in MW and size, they gradually become more confined until they are totally excluded from all pores and elute at V0 excluded volume. V0


, the


is also called interstitial volume or the volume around the outside of the column particles. Nothing should elute sooner than V0


,


and everything should elute within the total volume of the column. SEC columns are defined by a calibration curve that typically shows a linear range of about two orders of magnitude, depending on the pore-size dis- tribution of the column particles. Only solutes that elute within the linear range of the calibration plot should be used for size estimation.


Klein and Treichel5 used the cylindrical pore-size model to arrive at the


geometric equation shown in Figure 2 to describe how solute-radius to pore-radius (a/r) affects the (pore) distribution constant, KOC


. A small


value for a/r (radius-ratio) is desirable for HPLC because it allows ad- equate stationary-phase access. Klein’s occupational constant is closely related to KSize


radius-ratio values approaching zero and a KSize


in Eq. [2]. Small molecules such as uracil would have approaching one for


almost any column pore size. They can be employed as markers for total column volume because they are free to travel nearly anywhere mobile phase is located and have access to the stationary phase for optimum retention. On the other hand, large molecules such as monoclonal antibodies have much larger radius-ratio values and may lose contact with the stationary phase unless a larger-pore column is installed. A radius-ratio value of 0.1 is shown at the vertical line in Figure 2, where


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