Column Selection for the Reversed-Phase Separation of Proteins (original) (raw)
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Separation of Peptides and Proteins by Reversed Phase HPLC
During this presentation we will show the influence of some chromatographic variables on the retention of protein test mixtures during reversed phase liquid chromatography. A discussion on the type of chromatographic interaction is included. Finally we will present data on the quantitative reproducibility of the analysis of proteins by gradient elution RPLC.
Journal of Chromatography A, 2014
Influence of acid concentration in the mobile phase on protein separation was studied in a wide concentration range using trifluoroacetic acid (TFA) and formic acid (FA). At low, 0.001-0.01 (v/v%) TFA concentration and appropriate solvent strength proteins elute before the column's dead time. This is explained by the proteins having a structured, but relatively extended conformation in the eluent; and are excluded from the pores of the stationary phase. Above ca. 0.01-0.05 (v/v%) TFA concentration proteins undergo further conformational change, leading to a compact, molten globule-like structure, likely stabilized by ion pairing. Proteins in this conformation enter the pores and are retained on the column. The results suggest a pore exclusion induced separation related to protein conformation. This effect is influenced by the pH and type of acid used, and is likely to involve ion-pair formation. The TFA concentration needed to result in protein folding (and therefore to observe retention on the column) depends on the protein; and therefore can be utilized to improve chromatographic performance. Conformation change was monitored by circular dichroism spectroscopy and mass spectrometry; and it was shown that not only TFA but FA can also induce molten globule formation.
Proceedings of the Estonian Academy of Sciences, 2015
The separation of high-molecular compounds is very difficult, if possible at all, under isocratic conditions. For this gradient elution is needed. The theory of gradient elution for small molecules is well established; however, its applications to reversed-phase gradient separations of biopolymers are not straightforward because of specific problems, such as slow diffusion, limited accessibility of the stationary phase for larger molecules, or possible sample conformation changes during the elution. We used high performance liquid chromatography to investigate the reversed-phase chromatographic behaviour of 14 proteins. The first step was the determination of the experimental data, and then these data were used to predict gradient retention times. A water-organic solvent-trifluoroacetic acid system was used to examine the influence of experimental parameters. The chromatographic results from four C18-chain-length supports were comparable.
Journal of Pharmaceutical and Biomedical Analysis, 2012
In the pharmaceutical field, there is considerable interest in the use of peptides and proteins for therapeutic purposes. There are various ways to characterize such complex samples, but during the last few years, a significant number of technological developments have been brought to the field of RPLC and RPLC-MS. Thus, the present review focuses first on the basics of RPLC for peptides and proteins, including the inherent problems, some possible solutions and some directions for developing a new RPLC method that is dedicated to biomolecules. Then the latest advances in RPLC, such as wide-pore core-shell particles, fully porous sub-2 m particles, organic monoliths, porous layer open tubular columns and elevated temperature, are described and critically discussed in terms of both kinetic efficiency and selectivity. Numerous applications with real samples are presented that confirm the relevance of these different strategies. Finally, one of the key advantages of RPLC for peptides and proteins over other historical approaches is its inherent compatibility with MS using both MALDI and ESI sources.
High performance liquid chromatography (HPLC) is employed with its various elution systems for the fractionation (by isocratic or gradient elution) of peptides and proteins. Lichrosorb Diol (pore size 100 Ǻ) was chosen for normal partition chromatography of proteins. More details about these separations are illustrated in the current research. The chromatographic capacity and its resolution are investigated and the guidelines are widely defined. Reverse-phase (RP) (more suitable with Lichrosorb RP-8) sorbent was partially dissolved during elution with n-propanol (˂ 40 vol. /vol.) and lyophilized during fractionation. An outstanding resolution of these compounds was seen both at pH 4.0 and 7.5 under room temperature and low flow rate at linear gradient of n-propanol. Selective adsorption had been initiated at pH < 4 and peak broadening was observed when salts eliminated from the eluents. It is suggested by the results of this paper, the use of normal phase with Lichrosorb Diol for...
Journal of Chromatography A, 2010
Caustic regeneration procedures are often used in chromatographic purification processes of peptides and proteins to remove irreversibly bound impurities from the stationary phase. Silica-based materials are the most commonly used materials in reversed phase chromatography of peptides. Their limited chemical stability at high pH can be, however, problematic when high pH column regeneration (i.e. cleaning in place) is required. The effect of cleaning in place on the surface chemistry of the stationary phase has been investigated using the Tanaka test. It has been shown that the high pH treatment does not significantly affect the hydrophobicity of the material, but it strongly increases its silanol activity. A representative peptide purification process has been used to investigate the impact of cleaning in place on the separation performance. It has been shown that the caustic regeneration increases the peptide retention at high pH (pH 6.5), due to the interactions between the peptide and the negatively charged silanol groups. These unwanted interactions reduce the separation performances by decreasing the selectivity between the late eluting impurities and the main peptide. However, it has been shown that the effect of the silanol groups on the peptide adsorption and on the separation performance can be minimized by carrying out the purification process at low pH (pH ∼ 2). In this case, the silanol groups are protonated and their electrostatic interactions with the positively charged analyte (i.e. peptides) are suppressed. In these conditions, the peptide adsorption and the impurity selectivity is not changing upon high pH column regeneration and the separation performance is not affected.
Biomedical Chromatography, 2014
Although the efficient separation of intact protein mixtures is extremely difficult, reversed-phase chromatography is an important technique for performing quantitative, accurate and reproducible proteomics analyses. Here, we show that, despite the operating constraints of conventional high-performance liquid chromatography, such as column temperature, operating pressure and separation time, comprehensive separation of fluorogenic derivatized intact proteins could be achieved with high resolution and separation efficiency. First, amylin was chosen as a model peptide and used to estimate the separation efficiency with respect to column temperature and flow rate, as indicated by peak capacity. Then, an extract of human primary hepatocytes was used to model complex component mixtures and the separation conditions were optimized. The effects of mobile-phase pH, the separation time and the column length were also investigated. Consequently, more than 890 peaks could be separated efficiently in the extract, which is 1.5-fold greater than when using conventional conditions. Finally, it was demonstrated that both longer separation time and column length contributed greatly to the effective separation of the protein mixture. These results are expected to provide insights into the separation of intact proteins. Copyright
Reverse-Phase Chromatography RP-HPLC for Peptides
Reverse-phase high performance liquid chromatography (RP-HPLC) is an extremely useful tool for analytical biochemists. However, unlike small molecule HPLC, separations of proteins and peptides are nearly always performed under gradient conditions. There are other differences that one needs to be aware of in order to develop RP-HPLC separations of proteins and peptides as efficiently as possible. The general guidelines given in this short article may help reduce your method development time. RP-HPLC of complex peptide or protein mixtures remains a general method of choice because of the resolution it provides. Unlike small organic molecules whose chromatographic behavior is described by a finite partitioning equilibrium between the stationary phase and the mobile phase, proteins and peptides typically do not exhibit such an effect. Instead, they exhibit an adsorption phenomenon in which the polypeptide is adsorbed onto the stationary phase and elutes only when the solvent strength of the mobile phase is sufficient to compete with the hydrophobic forces keeping it there. For this reason, elution of peptides or proteins from reverse-phase supports is by gradients of increasing solvent strength. When run under isocratic conditions, peaks for proteins and peptides are typically much broader than their small molecule counterparts.
1996
In this study, reversed-phase HPLC (RPC), particle beam FT-IR spectrometry, and capillary electrophoresis (CE) were employed to ascertain the dynamic conformational structure empirically of various forms of bovine ribonuclease A (RNase A) to investigate the interdependence of protein conformation and RPC separation. RNase A was analyzed in four conformational states: native, partially denatured, completely unfolded, and completely unfolded and disulfide-reduced (denatured). Each form was analyzed individually and produced similar multizoned RPC profiles composed of several low-response peaks before the primary peak. Particle beam FT-IR spectrometry results from the RPC fractions of the partially denatured and unfolded forms were identical to one another and exhibited a loss of ordered structure. The infrared spectra of the RNase A that was introduced into the HPLC in the denatured and reduced form showed an excess of-sheet content, particularly the primary peak. We believe this to be a non-native form of RNase A. CE migration times of RNase A samples that were reduced in 2-mercaptoethanol and unfolded in increasing concentrations of urea increased with the degree of unfolding. Mobilities of RNase A RPC fractions were compared to those of the urea unfolded samples. The migration time of the primary RPC fraction of the denatured form showed an intermediate migration rate between that of the native, spherical geometry and denatured, open geometry forms. It is unclear whether this-structure formed during column propagation or upon elution. Examination of Protein Structure by Reversed-Phase HPLC (RPC). Separation of the components of complex biological mixtures, which commonly contain species with widely varying molecular weights and chemical compositions, can be accomplished effectively and rapidly with the use of RPC. 1-3 RPC is a powerful analytical tool that enables separation of peptides and proteins that have only minor differences in their amino acid composition. 3 Application of RPC has extended into the areas of protein folding/unfolding, purification, and structure/function
Journal of Chromatography A, 2005
This article describes a new complementary peptide separation and purification concept that makes use of a novel mixed-mode reversedphase/weak anion-exchange (RP/WAX) type stationary phase. The RP/WAX is based on N-(10-undecenoyl)-3-aminoquinuclidine selector, which is covalently immobilized on thiol-modified silica particles (5 m, 100Å pore diameter) by radical addition reaction. Remaining thiol groups are capped by radical addition with 1-hexene. This newly developed separation material contains two distinct binding domains in a single chromatographic interactive ligand: a lipophilic alkyl chain for hydrophobic interactions with lipophilic moieties of the solute, such as in the reversed-phase chromatography, and a cationic site for anion-exchange chromatography with oppositely charged solutes, which also enables repulsive ionic interactions with positively charged functional groups, leading to ion-exclusion phenomena. The beneficial effect that may result from the combination of the two chromatographic modes is exemplified by the application of this new separation material for the chromatographic separation of the N-and C-terminally protected tetrapeptide N-acetyl-Ile-Glu-Gly-Arg-p-nitroanilide from its side products. Mobile phase variables have been thoroughly investigated to optimize the separation and to get a deeper insight into the retention and separation mechanism, which turned out to be more complex than any of the individual chromatography modes alone. A significant anion-exchange retention contribution at optimal pH of 4.5 was found only for acetate but not for formate as counter-ion. In loadability studies using acetate, peptide masses up to 200 mg could be injected onto an analytical 250 mm × 4 mm i.d. RP/WAX column (5 m) still without touching bands of major impurity and target peptide peaks. The corresponding loadability tests with formate allowed the injection of only 25% of this amount. The analysis of the purified peptide by capillary high-performance liquid chromatography (HPLC)-UV and HPLC-ESI-MS employing RP-18 columns revealed that the known major impurities have all been removed by a single chromatographic step employing the RP/WAX stationary phase. The better selectivity and enhanced sample loading capacity in comparison to RP-HPLC resulted in an improved productivity of the new purification protocol. For example, the yield of pure peptide per chromatographic run on RP/WAX phase was by a factor of about 15 higher compared to the standard gradient elution RP-purification protocol.