Protein separation elution profile

The hydrophilic surface characteristics and the chemical nature of the polymer backbone in Toyopearl HW resins are the same as for packings in TSK-GEL PW HPLC columns. Consequently, Toyopearl HW packings are ideal scaleup resins for analytical separation methods developed with TSK-GEL HPLC columns. Eigure 4.44 shows a protein mixture first analyzed on TSK-GEL G3000 SWxl and TSK-GEL G3000 PWxl columns, then purified with the same mobile-phase conditions in a preparative Toyopearl HW-55 column. The elution profile and resolution remained similar from the analytical separation on the TSK-GEL G3000 PWxl column to the process-scale Toyopearl column. Scaleup from TSK-GEL PW columns can be direct and more predictable with Toyopearl HW resins. 

Figure 276 shows the results of an EDC conjugation study comparing a reaction done at pH 4.7 (A) to one done at pH 7.3 (B and C), with and without added sulfo-NHS (see Chapter 3, Section 1.2). The graphs show the elution profiles of a gel filtration separation after conjugation. In each case, a blank run done without the addition of EDC illustrates the separation of the protein carrier (the first peak) from... 

A large number of unnatural a-amino acids are commercially available. Many of them are more hydrophobic than natural amino acids. To detect and clearly separate hydrophobic PTH-amino acids using the sequencer, we have extended the elution time from a total of 22 min to 28 min prior to washing the column with 90% solvent B (88% acetonitrile/12% isopropanol, v/v) (Table I). To use the existing software in the protein sequencer to read all 20 PTH-natural amino acids automatically, the first 18 min of the gradient profile is identical to that recommended by the manufacturer. This ensures that the elution profile of natural amino acids remains unchanged. 

Due to the structural complexity of proteins, proper confirmation of product identity generally requires the use of several different techniques in parallel. For example, use of RP-HPLC and capillary electrophoresis in parallel provides a powerful means for proving identity of a particular peptide fragment since these two techniques exhibit independent elution profiles for typical peptide digests (1) with RP-HPLC separating on the basis of hydrophob-icity and capillary electrophoresis separating on the basis of charge. 

However, the spectra as well as the elution profiles may be very similar (i.e. closely related, ill-resolved solutes). Figure 5.38 shows an example of a result of PCA applied to a cluster of three peaks using LC-UV detection. The problem involves the separation of three proteins with very similar UV spectra. The chromatogram obtained is shown in figure... 

To obtain the separation needed for purification, a few initial runs are carried out to verify solubility and to get some idea of the complexity of the sample. Separation of the proteins can require modification of several variables, including the concentration range of the salt used for the gradient as well as the salt itself. For example, the range of salt concentrations used in the gradient can have a significant effect on the elution profile of a series of... 

The CCC fractions, HDL-LDL and VLDL-serum proteins, were each separately dialyzed against distilled water until the concentration of the potassium phosphate was decreased to that in the starting buffer used for the hydroxyapatite chromatography. These two fractions were concentrated separately by ultrafiltration. The concentrates of both fractions were chromatographed on the hydroxyapatite column. Fig. 4 shows the elution profile on hydroxyapatite obtained from the HDL-LDL fraction. A 1.4-mL volume of the concentrate was loaded onto a Bio-Gel HTP DNA-grade column (5.0 x 2.5 cm I.D.)... 

Fig. 3. Separation of serum proteins by gel filtration on Sephadex G-200. Elution was done with 0.1 M Tris-HCl (pH 8.0) containing 0.2 M NaCl. (A) Elution profile obtained from 12 ml of Trematomus borchgrevinki serum. (B) Elution profile obtained from 12 ml of human serum. Each tube contained 8 ml. The following abbreviations are used Alb., albumin Trans., transferrin 7-glob., 7-globulin o-/3 lip., a-fi lipoprotein. These areas apply also to (A), and areas I, II, III, and IV apply also to (B). From Komatsu et al. (1970b), reproduced with permission.

CPG surface. As appears from the comparison of the elution profiles illustrated in Fig. 21, the sorbent prepared on CPG with boron enriched surface (chromatogram B) allows to separate partly the proteins of fraction III eluted by (NH4)2S04 gradient, whereas the analogous material prepared from the initial CPG (with the surface not enriched in boron atoms) does not give a such possibility. 

Figure 4. Calculated elution profile to simulate a protein separation on Sephadex G-7S using equation (12). This simulation is based on parameter values for a 30 x 1.5 cm column with a flowrate of 1.77 ml/min and a longitudinal diffusivity of 0.01 cm2/min. The ratio of mobile phase volume to pore volume was 0.9, and the sample volume was 0.17 ml. Capacity factors for each of the solutes are 0, 0.5, and 1.1, respectively.

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