Stationary phases small molecule

Molecular exclusion chromatography is based on the inability of large molecules to enter small pores in the stationary phase. Small molecules enter these pores and therefore exhibit longer elution times than large molecules. Molecular exclusion is used for separations based on size and for molecular mass determinations of macromolecules. In affinity chromatography, the stationary phase retains one particular solute in a complex mixture. After all other components have been eluted, the desired species is liberated by a change in conditions. 

Gel filtration (size exclusion) Gel filtration chromatography (also called size exclusion chromatography) employs porous beads with a defined pore size distribution as the stationary phase. Small molecules can enter the entire intraparticular pore space and hence elute last, whereas large molecules are excluded from all pores and hence elute first. Molecules of intermediate size are found that can enter a certain... 

Size-exclusion chromatography (SEC) uses porous particles as stationary phases. Small molecules enter the pores, following a complex movement path thus, their migration along the column is slow. Large species (e.g., macromolecules) cannot readily diffuse into the pores thus, they move faster. 

Gel chromatography, as a separation method, is based on the size of molecules, which in turn determines the extent of their diffusion into the pores of gel particles packed as a stationary phase. Large molecules are excluded from most of the pores, whereas small molecules can diffuse further into the stationary phase. 

In 1964, Merries thought of using micelles as the big objects not retained or excluded by a GPC polymer phase. Small molecules are retained by the pore of the stationary phase. They are less retained when solubilized inside an excluded micelle. The idea was to measure solute affinity for micelles through solute retention times. This was the first time a micellar phase was used in chromatography [16]. This part of MLC history was already exposed in Chapter 3. Terabe and Okada developed a slightly different approach to model the small molecule and ion retention in micellar GPC [ 17, 18]. The equation is ... 

Shifts in the SEC fractionation range are not new. It has been known for decades that adding chaotropes to mobile phases causes proteins to elute as if they were much larger molecules. Sodium dodecyl sulfate (SDS) (9) and guanidinium hydrochloride (Gd.HCl) (9-12) have been used for this purpose. It has not been clearly determined in every case if these shifts reflect effects of the chaotropes on the solutes or on the stationary phase. Proteins are denatured by chaotropes the loss of tertiary structure increases their hydrodynamic radius. However, a similar shift in elution times has been observed with SEC of peptides in 0.1% trifluoroacetic acid (TEA) (13-15) or 0.1 M formic acid (16), even if they were too small to have significant tertiary structure. Speculation as to the cause involved solvation effects that decreased the effective pore size of the... 

Flow markers are often chosen to be chemically pure small molecules that can fully permeate the GPC packing and elute as a sharp peak at the total permeation volume (Vp) of the column. Examples of a few common flow markers reported in the literature for nonaqueous GPC include xylene, dioctyl phthalate, ethylbenzene, and sulfur. The flow marker must in no way perturb the chromatography of the analyte, either by coeluting with the analyte peak of interest or by influencing the retention of the analyte. In all cases it is essential that the flow marker experience no adsorption on the stationary phase of the column. The variability that occurs in a flow marker when it experiences differences in how it adsorbs to a column is more than sufficient to obscure the flow rate deviations that one is trying to monitor and correct for. 

The two examples of sample preparation for the analysis of trace material in liquid matrixes are typical of those met in the analytical laboratory. They are dealt with in two quite different ways one uses the now well established cartridge extraction technique which is the most common the other uses a unique type of stationary phase which separates simultaneously on two different principles. Firstly, due to its design it can exclude large molecules from the interacting surface secondly, small molecules that can penetrate to the retentive surface can be separated by dispersive interactions. The two examples given will be the determination of trimethoprim in blood serum and the determination of herbicides in pond water. 

The analysis demonstrates the elegant use of a very specific type of column packing. As a result, there is no sample preparation, so after the serum has been filtered or centrifuged, which is a precautionary measure to protect the apparatus, 10 p.1 of serum is injected directly on to the column. The separation obtained is shown in figure 13. The stationary phase, as described by Supelco, was a silica based material with a polymeric surface containing dispersive areas surrounded by a polar network. Small molecules can penetrate the polar network and interact with the dispersive areas and be retained, whereas the larger molecules, such as proteins, cannot reach the interactive surface and are thus rapidly eluted from the column. The chemical nature of the material is not clear, but it can be assumed that the dispersive surface where interaction with the small molecules can take place probably contains hydrocarbon chains like a reversed phase. 

Figure 4-2. Size-exclusion chromatography. A A mixture of large molecules (diamonds) and small molecules (circles) are applied to the top of a gel filtration column. B Upon entering the column, the small molecules enter pores in the stationary phase matrix from which the large molecules are excluded. C As the mobile phase flows down the column, the large, excluded molecules flow with it while the small molecules, which are temporarily sheltered from the flow when inside the pores, lag farther and farther behind.

In exclusion chromatography, the total volume of mobile phase in the column is the sum of the volume external to the stationary phase particles (the void volume, V0) and the volume within the pores of the particles (the interstitial volume, Vj). Large molecules that are excluded from the pores must have a retention volume VQ, small molecules that can completely permeate the porous network will have a retention volume of (Vo + Fj). Molecules of intermediate size that can enter some, but not all of the pore space will have a retention volume between VQ and (V0 + Fj). Provided that exclusion is the only separation mechanism (ie no adsorption, partition or ion-exchange), the entire sample must elute between these two volume limits. 

Other applications that utilize different types of reversed-phase columns in both dimensions have been advocated by Carr (Stoll et al., 2006) for metabolomics work in small-molecule separations. These stationary phases include a pentafluorophenyl-propyl stationary phase in the first dimension and a carbon-coated zirconia material stationary phase in the second dimension. A common mistake in 2D method development is to mismatch the solvent system the two solvent systems must be miscible as discussed below. 

The fourth type of mechanism is exclusion although perhaps inclusion would be a better description. Strictly, it is not a true sorption process as the separating solutes remain in the mobile phase throughout. Separations occur because of variations in the extent to which the solute molecules can diffuse through an inert but porous stationary phase. This is normally a gel structure which has a small pore size and into which small molecules up to a certain critical size can diffuse. Molecules larger than the critical size are excluded from the gel and move unhindered through the column or layer whilst smaller ones are retarded to an extent dependent on molecular size. 

Ctl is the mass transfer term and arises because of the finite time taken for solute molecules to move between the two phases. Consequently, a true equilibrium situation is never established as the solute moves through the system, and spreading of the concentration profiles results. The effect is minimal for small particle size and thin coatings of stationary phase but increases with flow rate and length of column or surface. 

Mass transfer (the C term), which involves collisions and interactions between molecules, applies differently to both packed and capillary columns. Packed columns are mostly filled with stationary phase so liquid phase diffusion dominates. The mass transfer is minimized by using a small mass of low-viscosity liquid phase. Capillary columns are mostly filled with mobile phase, so mass transfer is important in both the gas and liquid phases. A small mass of low-viscosity liquid phase combined with a low-molecular weight carrier gas will minimize this term. 

The stationary phase consists of porous polymer resin particles. The components to be separated can enter the pores of these particles and be slowed from progressing through this stationary phase as a result. Thus, the separation depends on the sizes of the pores relative to the sizes of the molecules to be separated. Small particles are slowed to a greater extent than larger particles, some of which may not enter the pores at all, and thus the separation occurs. The mobile phase for this type can also only be a liquid, and it too is discussed further in Chapter 13. The separation mechanism is depicted in Figure 11.11. 

Size exclusion chromatography. The separation occurs because the stationary phase particles are porous and the small molecules enter the pores and are slowed from passing through the column, while the large molecules pass through more quickly since they do not enter the pores. 

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