GPC of small molecules

In particular, for copolymers this required an orthogonal coupling of one GPC to another to achieve the desired cro fractionation before application of dual detectors. This method is really a new polymer analysis member of a family of approaches developed in the literature which we are now terming "Orthogonal Chromatogr hy . It not only provides both a cro fractionation approach for copolymers and a new way of determining the GPC s "imperfect resolution" it also enables separation mechanisms previously reserved for the liquid chromatography of small molecules to be used for polymer analysis. 

Column Efficiency. The peak capacity (13) for a GPC column used in the analysis of small molecules is related to the number of theoretical plates (N) according to ... 

For the optimal application of GPC to the separation of discrete small molecules, three factors should be considered. Solvent effects are minimal, but may contribute selectivity when solvent-solute interactions occur. The resolving power in SMGPC increases as the square root of the column efficiency (plate count). New, efficient GPC columns exist which make the separation of small molecules affordable and practical, as indicated by applications to polymer, pesticide, pharmaceutical, and food samples. Finally, the slope and range of the calibration curve are indicative of the distribution of pores available within a column. Transformation of the calibration curve data for individual columns yields pore size distributions from which useful predictions can be made regarding the characteristics of column sets. 

Since there is no selectivity (a) involved in GPC, the ability to separate a pair of compounds depends upon the calibration curve and the efficiency of the column(s). One measure of the ability of the GPC column(s) to separate is the peak capacity of the column, which is defined as the number of resolvable peaks per chromatogram, n. The peak capacity (16) for a GPC column used in the analysis of small molecules is related to the number of theoretical plates (N) according to the equation... 

The objective of this experiment is to calibrate a gel permeation chromatographic column and to separate a series of small molecules by GPC. Additionally, this experiment will demonstrate the usefulness of a spectrophoto-metric and RI detector in GPC preparative separations. 

If the sample is readily soluble in the mobile phase, GPC is unmatched by any other mode of chromatography for simplicity, since the entire analysis is accomplished in a column volume. The time and effort required to develop a separation is less than any other mode of HPLC. It can be of immense value in the purification or organic and inorganic synthesis reaction mixtures, purification of natural products extracts, and for the rapid clean-up of extracts (from plants, insects, soil, etc.) prior to the assay of small molecules. Aqueous size separation is referred to as gel filtration chromatography and is very useful for protein separations and the analysis of water-soluble polymers. 

The sfff technique is versatile it can be used to study both soUds and solutions, no standards are required for cahbration, and it is a nondestmctive technique. Low shear forces are employed and minimal absorption occurs. Hence, sample degradation does not take place. The technique cannot, however, be routinely used for molecular weight characterization of small molecules. Newer variations of sfff include the development of thermal fff (73), whereby a temperature gradient is placed between two parallel metal blocks to allow the particles to migrate toward the wall. This approach is said to allow for the molecular weight characterization of polymeric materials which are much smaller in molecular size. Advantages of this technique over gpc He in the absence of conventional band broadening effects. 

Although SynChropak GPC supports have excellent efficiencies for small molecules at various flow rates, macromolecules, because of their low diffusion constants, exhibit band spreading when linear velocities are increased. This effect increases with molecular weight, as seen in Fig. 10.11 (4). It should be noted that proteins are usually homogeneous in size and thus yield better efficiencies than polymers, which are usually heterogeneous. For preliminary analy-... 

Most GPC columns are provided with vendor estimates of the plate count of the column and a chromatogram of a series of test peaks. These plate count estimates are usually obtained using small molecule analytes that elute at the total permeation volume (Vp) of the column. The Gaussian peak shape model... 

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. 

Size exclusion chromatography (SEC), also known as gel permeation chromatography (GPC), was used for the separation and fractionation of macromolecules on an analytical and preparative scale [17]. The separation occurs predominantly by the hydrodynamic volume of the macromolecules in solution, however, in some cases the polarity of the molecules can also influence the retention times. Like HPLC, the SEC technique is generally very reproducible with regard to its elution times (typically < 1 h) and hence can be used for automated synthesis. But because the cost for an automated SEC system is high, it must be considered as a serial separation technique. In addition, larger scale separations > 100 mg, usually require repetitive injection of small aliquots. 

Since the limit of detection for small molecule ligands, with modern ESI-Tof mass spectrometers, is approximately 0.05 pM, the concentration of the protein-ligand complex prior to the GPC spin column treatment must be about 0.25 pM. For initial protein and ligand concentrations >5 pM, this corresponds to values <20 pM, as indicated in Fig. 2.3. This is a desirable region for the GPC spin column studies, since one wants to be certain to detect ligands from the stronger as well as the weakest ligand binders. 

The GPC spin column/ESI-MS screening protocol rapidly analyzes the ability of small organic molecules to bind non-covalently to target protein molecules. 

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