SEC columns, high-speed

Figure 2 Chromatogram from conventional SEC column compared to high-speed SEC column tested on an identical instrument with polystyrene standards in tetrahydrofuran (THF). (See Color Plate Section at the end of this book.)...

There have been several approaches to overcome the traditionally slow SEC separations, which are caused by the diffusion processes in SEC columns. Most of them are column-related (see High-Speed SEC Columns, Small Particle Technology, and Smaller SEC Column Dimensions ) one utilizes the column void volume (cf. Overlaid Injections ), while another replaces separation with simplified sample preparation (see Flow Injection Analysis ). Cloning existing methods and instrumentation is also reviewed with respect to the potential time gain (see Cloning of SEC Systems ). Benefits and limitations of each method are summarized in Table 1. 

Time requirements of SEC experiments can be reduced substantially by using high-speed SEC columns. The availability of high-speed columns allows an increase in SEC separations by a factor of 10 and run times of 1 min are possible. Precision and accuracy of results are comparable with existing methods. Existing methods and instrumentation can still be used with highspeed columns. 

Fig. 1 Chromatogram of conventional SEC column (right part) compared to high-speed SEC column (left part) tested on identical...

Kilz, P. Methods and Columns for High Speed SEC Separations. In Handbook for Size Exclusion Chromatography and Related Techniques, Wu, C.-S., Ed. Marcel Dekker New York, 2002, in press. 

Modern SEC columns are packed with material other than polystyrene gels, such as porous silica particles or highly cross-linked styrene-divinylbenzene copolymers. Because of improvements in speed and resolution, the term SEC is sometimes replaced by the term high-performance size-exclusion chromatography (HPSEC). 

When a drop (water) falls to a flat interface (benzene-water) the entire drop does not always join the pool (water). Sometimes a small droplet is left behind and the entire process, called partial coalescence, is repeated. This can happen several times in succession. High-speed motion pictures, taken at about 2000 frames per second, have revealed the details of the action (W3). The film (benzene) ruptures at the critical film thickness and the hole expands rapidly. Surface and gravitational forces then tend to drag the drop into the main pool (water). But the inertia of the high column of incompressible liquid above the drop tends to resist this pull. The result is a horizontal contraction of the drop into a pillar of liquid above the interface. Further pull will cause the column to be pinched through, leaving a small droplet behind. Charles and Mason (C2) have observed that two pinches and two droplets occurred in a few cases. The entire series of events required about 0.20 sec. for aniline drops at an aniline-water interface (C2, W3). 

The pore volume of the column packing has been shown to be one of the major factors influencing peak resolution in SEC. True high-speed separations, with good resolu-... 

The time gain of high-speed columns can open up SEC methodology for... 

SFC uses the same stationary phases as FIPLC. Selectivity is similar but not identical. One of the greatest differences from n-FIPLC is in speed. SFC optimum flow is inherently three to five times faster, while peak shapes are often significantly better. Unlike n-HPLC, SFC reequilibrates after passage of only a few column volumes. Overall, SFC is often much more than 10 times faster than FIPLC. A fast chiral SFC separation is shown in Figure 4. Note that the column is not high speed but a standard 4.6x250 mm column with 10 pm particles. Some industrial pharmaceutical companies have dropped HPLC for chiral analysis and use SFC for less polar solutes, and high-performance capillary electrophoresis (HPCE) for water-soluble solutes. Unlike HPCE, SEC is scalable. 

Figure 1. High-speed isoeratic separation of a five-component synthetic mixture. Packing Hypersil S-pm, Column I.D. 0.26 cm. Column length 2.50 cm, Mobile phase 2.2% methyl acetate in n-pentane, Linear velocity 3.3 cm/sec. 1 p-xylene, 2 anisole, 3 nitrobenzene, 4 acetophenone, 5 dipropyl phthalate. 

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