Regarding SEC, as molecules of various sizes flow through the column, molecules of small size flow slowly while penetrating deep into the pores in the packing material (Fig. 1, right), while molecules of large size flow out of the column without penetrating the pores (Fig. 1, left). As a result, the elution order from the column is faster for larger molecules and slower for smaller molecules. SEC is a separation mode that sieves molecules according to their size.
In SEC, as shown in Fig.1, the elution order from the column is faster for larger molecules and slower for smaller molecules. The calibration curve shows this relationship graphically. Fig.2 shows the relationship between retention time (also called elution volume) and molecular weight for SEC. When molecules larger than a certain size is separated by size exclusion chromatography, the retention time is the same because the substance passes through the pores of the packing material. This is called the exclusion limit. Conversely, molecules below a certain size completely penetrate the pores and tend to elute almost in the same position. This is called the permeation limit.
In order to obtain an accurate molecular weight distribution of polymer materials, it is necessary to set analytical conditions such that the measured polymer materials are eluted between the exclusion limit and the penetration limit.