Size Exclusion-Reversed Phase

FIGURE 8.6 Schematic diagram of a 2D SEC x RPLC instrument. Reprinted from Evans, C. R. and Jorgenson, J. W. (2004) Anal. Bioanal. Chem. 378,1952-1961, with kind permission of Springer Science and Business Media. 

FIGURE 8.7 2D chromatogram of a SEC x RPLC separation of a tryptic digest of ovalbumin. Reproduced with permission from Opiteck et al. (1997), copyright 1997, American Chemical Society. 

The SEC x RPLC approach was also used to separate intact proteins rather than peptides, in a collaborative effort with Arthur Moseley at Glaxo Wellcome, Inc. (Opiteck et al. 1998b). To optimize the size exclusion separation for proteins, six columns with 125 A pores were exchanged for a series of eight columns with 250 A pores or a combination of six columns with 250 A pores and six columns with 450 A 

J30S Ril 30S Ribo Si D-ribose binding HDEA prec. 

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Separation by HPLC typically falls into one of six categories chiral, ion exchange, ion pair, normal phase, reversed phase, or size exclusion. Reversed phase is the most common type employed, and although various detection principles can be used in HPLC, UV is the most typical. 

Purity and impurities Size exclusion (SDS-PAGE) (ELISA) Size exclusion Reversed phase (SDS-PAGE)... 

J. Ruiz Encinar, L. Ouerdane, W. Buchmann, J. Tortajada, R. Lobinski, J. Szpunar, Identification of water-soluble selenium-containing proteins in selenized yeast by size-exclusion-reversed-phase HPLC/ICPMS followed by MALDI-TOF and electrospray Q-TOF mass spectrometry, Anal. Chem., 75 (2003), 3765-3774. 

Glaze, W. H., Jones, P. C., and Saleh, F. W. (1981). Size exclusion, reverse phase, and weak anion exchange chromatography of natural organics in water. In Advances in the Identification and Analysis of Organic Pollutants in Water, Vol. 1 (L. H. Keith, ed.). Ann Arbor Science, Ann Arbor, MI, 120 pp. 

SAMPLE EEW (g/eq) EPOXY % TGMDA (HPLC) SIZE EXCLUSION/REVERSE PHASE VISCOSITY (CP) 50°C... 

Size-exclusion Reversed-phase Reversed-phase ion-pair Ion-pair partition Ion-exchange Normal-phase Charge-transfer Salting-out Ligand-exchange Chelation Affinity... 

Size exclusion, reversed-phase, paired ion reverse-phase, and ion exchange (cation and anion) HPLC have been used for selenium speciation. Size exclusion, also used in combination with affinity chromatography, has been used for the determination of selenoproteins and studies of interactions of selenium with proteins in the body. Ion-exchange and reversed-phase HPLC are often used for separation... 

J. Identiilcation of Water-Soluble Sele-niam-Contaming Prolems in Selenized Yeast by Size-Exclusion-Reversed-Phase HPLC-ICP-MS Followed by MALDI-TOF and ESI-Q-TOF-MS. Anal. Chem. 2003, 75,3765-3774. 

Separation can now be achieved by using any one and/or a combination of the various modes available. These modes include size exclusion, normal phase, reversed-phase, paired-ion reversed-phase and ion-exchange. The principles, theories and the... 

The slight hydrophobic character of organic size exclusion bonded phases may be used in a reversed-phase mode to retain proteins [44]. In a study where the concentration of trifluoroacetic acid (TEA) in the isocratic analysis of a peptide mixture on an SEC column was varied, the retention and the peak shapes of the components were affected differentially, yielding good resolution that was not by size [49]. 

This reversed-phase chromatography method was successfully used in a production-scale system to purify recombinant insulin. The insulin purified by reversed-phase chromatography has a biological potency equal to that obtained from a conventional system employing ion-exchange and size-exclusion chromatographies (14). The reversed-phase separation was, however, followed by a size-exclusion step to remove the acetonitrile eluent from the final product (12,14). 

Except for the high molecular weight range, nearly all substances can be separated by reversed-phase (RP) HPLC. The many different separation mechanisms in RP HPLC, based on hydi ophobic, hydi ophilic and ion-pairing interactions, and size exclusion effects together with the availability of a lai ge number of high quality stationary phases, explain its great populai ity. At present approximately 90% of all HPLC separations are carried out by reversed-phase mode of HPLC, and an estimated 800 different stationai y phases for RP HPLC are manufactured worldwide. 

Other groups have also used EC and CE to perform non-comprehensive multidimensional separations (15, 16). A three-dimensional separation was performed by Stromqvist in 1994, where size exclusion chromatography (SEC), reverse-phase HPLC, and CZE were used in an off-line manner to separate peptides (17). The most useful information gained from all of these non-comprehensive studies was knowledge of the orthogonality and compatibility of EC and CE. 

THREE-DIMENSIONAL SIZE EXCLUSION CHROMATOGRAPHY-REVERSE PHASE LIQUID CHROMATOGRAPHY-CAPILLARY ZONE ELECTROPHORESIS... 

Figure 9.10 Three-dimensional representation of the data volume of a tryptic digest of ovalbumin. Series of planar slices through the data volume produce stacks of disks in order to show peaks. Reprinted from Analytical Chemistry, 67, A. W. Moore Jr and J. W. Jorgenson, Comprehensive three-dimensional separation of peptides using size exclusion chromatogra-phy/reversed phase liquid chromatography/optically gated capillary zone electrophoresis, pp. 3456-3463, copyright 1995, with permission from the American Chemical Society.

M. Stromqvist, Peptide mapping using combinations of size-exclusion chromatography, reversed-phase chromatography and capillary electrophoresis , 7. Chromatogr. 667 304-310(1994). 

A. W. Moore, Jr and J. W. Jorgenson, Comprehensive three-dimensional separation of peptides using size exclusion chromatography/reversed phase liquid chromatography/ optically gated capillary zone electrophoresis . Anal. Chem. 67 3456-3463 (1995). 

Stopher and Gage used size-exclusion chromatography (SEL) coupled to reversed phase HPLC for the direct injection of plasma in the analysis of an antifungal agent, voriconazole (12). Their system consisted of three columns, i.e. first a size-exclusion... 

Figure 3 Reversed-phase chromatography of products after alkaline hydrolysis of /3-poly(L-malate), Discrete polymer products are formed, which differ in length by several units of L-malate. The absorbance at 220-nm wavelength was measured, (a) /3-Poly(L-malate) before hydrolysis, (b) After 10-min incubation in 20 mM NaOH at 37°C. (c) After 15 h in 20 mM NaOH at 37°C. (d) After I h in 500 mM NaOH at 100°C. High pressure chromatography (HPLC) on Waters reversed-phase Ci8- i-Bondapak. The methanol gradient (in water-trifluoro acetic acid, pH 3.0) was programmed as follows 0-40 min 0.3-23%, 40-47 min 23-40%, 47-49 min 40%, 49-54 min 40-0%. (d) Inset size exclusion chromatography after 3-min alkaline hydrolysis at pH 10.2. BioSil SEC 250 column of 300 mm x 7.8 mm size, 0.2 M potassium phosphate buffer pH 7.0.

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