Size column packing

Separation of enantiomers of etodolac using two different derivitization agents and three chiral stationary phases has been studied [24]. Etodolac was converted to its anilide derivative with either 1,3-dicyclohexyl-carbodiimide or l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. Etodolac, derivatizing agent, aniline, and dichloromethane were allowed to incubate for 30 minutes, which was followed by addition of 1 M HC1. The organic layer was removed, washed, dried, and then injected into normal phase or reverse phase HPLC. The HPLC system consisted of a 250 x 4.6 mm (5 pm particle size) column packed with chiral stationary phases, and detection was effected by the UV absorbances at 254 and 280 nm. Separation of etodolac enantiomers was achieved on only one of the stationary phases when using 20% 2-propanol in hexane as the mobile phase at a flow rate of 2.0 mL/min. 

Two classes of micron-sized stationary phases have been encountered in this section silica particles and cross-linked polymer resin beads. Both materials are porous, with pore sizes ranging from approximately 50 to 4000 A for silica particles and from 50 to 1,000,000 A for divinylbenzene cross-linked polystyrene resins. In size-exclusion chromatography, also called molecular-exclusion or gel-permeation chromatography, separation is based on the solute s ability to enter into the pores of the column packing. Smaller solutes spend proportionally more time within the pores and, consequently, take longer to elute from the column. 

The effective interfacial area depends on a number of factors, as discussed in a review by Charpentier [C/j m. Eng.J., 11, 161 (1976)]. Among these factors are (1) the shape and size of packing, (2) the packing material (for example, plastic generally gives smaller interfacial areas than either metal or ceramic), (3) the liquid mass velocity, and (4), for smaU-diameter towers, the column diameter. 

The explicit form of those equations that satisfy the preliminary data criteria, must then be tested against a series of data sets that have been obtained from different chromatographic systems. As an example, such systems might involve columns packed with different size particles, employed mobile phases or solutes having different but known physical properties such as diffusivity or capacity ratios (k"). 

The advantages of monosized chromatographic supports are as follows a uniform column packing, uniform flow velocity profile, low back pressure, high resolution, and high-speed separation compared with the materials of broad size distribution. Optical micrographs of 20-p,m monosized macroporous particles and a commercial chromatography resin of size 12-28 p,m are shown in Fig. 1.4. There is a clear difference in the size distribution between the monodispersed particles and the traditional column material (87). 

A trend in chromatography has been to use monosized particles as supports for ion-exchange and size-exclusion chromatography and to minimize the column size, such as using a 15 X 4.6-mm column packed with 3-/rm polymer particles for size exclusion chromatography. The more efficient and lower back pressure of monosized particles is applied in the separation. 

Size exclusion was first noted in the late fifties when separations of proteins on columns packed with swollen maize starch were observed (Lindqvist and Storgards, 1955 Lathe and Ruthven, 1956). The run time was typically 48 hr. With the advent of a commercial material for size separation of molecules, a gel of cross-linked dextran, researchers were given a purposely made material for size exclusion, or gel filtration, of solutes as described in the classical work by Porath and Flodin (1959). The material, named Sephadex, was made available commercially by Pharmacia in 1959. This promoted a rapid development of the technique and it was soon applied to the separation of proteins and aqueous polymers. The work by Porath and Flodin promoted Moore (1964) to apply the technique to size separation, gel permeation chromatography of organic molecules on gels of lightly cross-linked polystyrene (i.e., Styragel). 

Select the appropriate chromatographic column or columns. Choose a column packing with a pore size that will resolve the molecular size range of the sample. 

Small particle size resins provide higher resolution, as demonstrated in Fig. 4.41. Low molecular weight polystyrene standards are better separated on a GIOOOHxl column packed with 5 /u,m resin than a GlOOOHg column packed with 10 /Ltm resin when compared in the same analysis time. Therefore, smaller particle size resins generally attain a better required resolution in a shorter time. In this context, SuperH columns are best, and Hhr and Hxl columns are second best. Most analyses have been carried out on these three series of H type columns. However, the performance of columns packed with smaller particle size resins is susceptible to some experimental conditions such as the sample concentration of solution, injection volume, and detector cell volume. They must be kept as low as possible to obtain the maximum resolution. Chain scissions of polymer molecules are also easier to occur in columns packed with smaller particle size resins. The flow rate should be kept low in order to prevent this problem, particularly in the analyses of high molecular weight polymers. 

The injection volume should be kept as small as possible to attain maximum resolution in analyses. This is particularly important in analyses on columns packed with small particle size resins such as SuperH. Injection volumes of 0.1 % or less of the total column volume are recommended on SuperH columns. A few times larger injection volumes may be applied to other series of H type columns. 

The sample concentration also should be kept as low as possible, particularly in analyses of polymers on columns packed with small particle size resins. The maximum sample concentration to achieve maximum resolution decreases as the sample molecular weight becomes higher and the resin particle size becomes smaller. It is usually in the range of 0.05-5 mg/ml, depending on the sample molecular weight and resin particle size. 

With soft gels, column packing has often been plagued with such problems as inferior reproducibility and excessive time requirements. These problems are alleviated with physically stable Toyopearl HW media. However, an improperly packed column can have significantly reduced efficiency. The two key variables for the successful packing of Toyopearl HW media, packing velocity and column size, have been evaluated to determine the optimal packing conditions. 

Shodex has a wide variety of columns for organic GPC using organic solvents. The columns are packed with porous styrene-divinylbenzene copolymer gels especially developed for GPC use. Five types of standard-size GPC columns packed with different solvents are available. Downsized GPC columns are also available. 

---