Chromatography Characterization of Proteins using HPLC Columns

Protein purification and separation are vital for the characterization of the function, structure and interactions of the proteins. Popular chromatography techniques include size-exclusion, ion exchange, reversed-phase, hydrophobic interaction, HILIC, and mixed-modes.   These separation steps exploit differences in protein size, charge, polarity, and binding affinity. Typically, the separation of one protein from its variance in a mixture is the most difficult step. By choosing the right combination of separation modes in HPLC columns, an experienced scientist can do so in a relatively least-laborious way.

Reversed-Phase Chromatography (RP)

Most peptide and protein reversed-phase HPLC columns are silica-based particles bonded with C18, C8, and other functional groups. Their pore sizes are practically limited to the maximum of 300Å due to the natural of the silica material. Larger pores are available but they are relatively fragile. Nonetheless, these silica-based columns are the best choice when working with peptides and small proteins, providing excellent peak selectivity and efficiency.

Size-Exclusion Chromatography (SEC, GPC, or GFC)

Size-exclusion chromatography (SEC) is also known as gel permeation chromatography (GPC) when organic solvents are used as mobile phase, or gel filtration chromatography (GFC) when aqueous solvents are used. This chromatography method separates proteins and peptides according to their size (or more accurately according to their hydrodynamic diameter or hydrodynamic volume). Smaller proteins are able to enter the pores of the stationary phase, thus, have relatively long path to travel until reaching the detector. Larger proteins cannot enter the pores, thus, have shorter path to travel and elute out first. The average residence time in the pores depends upon the effective size of the proteins. It is generally a low-resolution chromatography technique with base-line separation possible if the molecular weights of the two proteins are about one order of magnitude different. It is also useful for determining the tertiary structure and quaternary structure of purified proteins, especially since it can be carried out under native solution conditions. Salt and organic modifiers are often added to minimize interaction of proteins to the stationary phase so a true MW determination can be achieved with a calibration curve.

Ion-Exchange Chromatography (IEX)

Ion-exchange chromatography is a process that allows the separation of amino acids, peptides, and proteins based on their charge. This chromatography technique uses charged stationary phase resins that interact with oppositely charged proteins or peptides. A salt gradient of increasing concentration is used to force out the analytes from the stationary phase. Less charged proteins and peptides have less interaction with the stationary phase, thus, would elute out first.

Hydrophobic Interaction Chromatography (HIC)

Hydrophobic interaction chromatography (HIC) is often used for macromolecule (protein, nucleotides…etc) purifications. It has both salt precipitation and adsorption-desorption chromatography characteristics. At high salt concentration, protein adsorption occurs to the hydrophobic stationary phase. As the salt concentration decreases over the gradient, proteins lose the adsorption and elute out. HIC is a sensitive technique that can determine one single amino acid modification in a very large macromolecule. HIC is also used frequently in conjunction with size-exclusion, ion-exchange, and reversed-phase chromatography techniques to determine post-translational modification of antibodies and other proteins. Typical samples are hydrophilic peptides, glycopeptides, phosphopeptides, protein digests…etc.

Hydrophilic Interaction Chromatography (HILIC or normal phase)

Hydrophilic Interaction Liquid Chromatography (HILIC) is a variant of normal-phase chromatography that contains some aqueous solvent in the mobile phase. This chromatography technique separates proteins, peptides, and amino acids based on polarity differences. It is often used in in-line electrospray-ionization mass spectrometry, because the high organic solvent content in the mobile phase often helps in increasing sensitivity.

Non-Porous are Faster than Porous Resins in Protein Ion-Exchange and HIC Separations

The high-speed non-porous ion-exchange columns are designed for high throughput of quality control, process monitoring, and other applications which need rapid scanning of complex samples. The major advantages besides speed are higher sensitivity, better resolution/ efficiency, and much less solvent consumption.

Inline Protein Removal System - ZirChrom ProTain

For in-line protein removal applications, ZirChrom ProTain (zircornia-based guard column) works in a three-fold way to bind proteins:  1. Ion exchange (neg/pos)  2. Ligand exchange (Lewis acid on zir/Lewis base of protein)  3. Hydrophobic/hydrophilic. This three-fold attachment of proteins to ProTain is very strong and is generally on/off (conditions get proteins off quickly OR retain them strongly no in-between), making it useful as a trap for contaminating proteins.  ProTain columns are compatible with all analytical columns you are currently using (silica-based, polymer-based, or hybrids). They are easy to use when the small molecule analytes are neutral or negatively charged. Positively charged small molecules can be retained by ProTain, but usually at a much weaker interaction than proteins. Appropriate conditions will need to be experimentally determined. Protein trapping capacity depends on the pH and buffer used, with the upper range of approximately 5 mg. Regeneration of ProTain columns are possible, however, most customers find it convenient to replace them with new cartridges to save time.
ZirChrom ProTain Inline Protein Removal Instruction

HPLC Columns for Large Proteins = *

Reversed Phase Chromatography
Polymer-based particles
Jordi Protein Peptide*
Silica-based particles
Agela Venusil ASB C18 (300A, low pH)
Agela Venusil ASB C8 (300A, low pH)
Agela Venusil XBP C18 (300A)
Agela Venusil XBP C1 (300A)
Agela Venusil XBP C4 (300A)
Agela Venusil XBP C8 (300A)
Agela Venusil XBP CN (300A)
Agela Venusil XBP Phenyl (300A)
Chromenta BB-C18 (300A)
Chromenta BB-C1 (300A)
Chromenta BB-C4 (300A)
Chromenta BB-C8 (300A)
Chromenta BB-CN (300A)
Chromenta BB-Ph (300A)
Nacalai Cosmosil C18-AR-300 (300A)
Nacalai Cosmosil C4-AR-300 (300A)
Nacalai Cosmosil C8-AR-300 (300A)
Nacalai Cosmosil Ph-AR-300 (300A)
Nacalai Cosmosil Protein-R (300A)
Dikma Bio-Bond C18 (300A)
Dikma Bio-Bond C4 (300A)
Dikma Bio-Bond C8 (300A)

Ion Exchange Chromatography
Anion Exchange Chromatography
Nacalai Cosmogel IEX Type Q* (1,000A)
Nacalai Cosmosil DEAE*
Nacalai Cosmosil QA*
Jordi Protein Peptide SAX*
PolyLC PolyWAX LP*
Sepax Proteomix WAX-POR*
Sepax Proteomix SAX-POR*
Cation-Exchange Chromatography
Nacalai Cosmogel IEX Type S* (1,000A)
Nacalai Cosmosil CM*
Nacalai Cosmosil SP*
PolyLC PolySULFOETHYL A (peptides)
PolyLC PolyCAT A*
Sepax Proteomix WCX-POR*
Sepax Proteomix SCX-POR*
Amphoteric Ion-Exchange Chromatography
Nacalai Cosmogel IEX Type M* (1,000A)
PolyLC PolyCATWAX*

Size Exclusion Chromatography (GFC)
Nacalai Cosmosil Diol-120-II
Nacalai Cosmosil Diol-300-II*
Sepax Zenix SEC (3um, 100-300A)
Sepax SRT SEC* (100-1,000A)
Sepax SRT-C SEC* (100-1,000A)
Sepax Nanofilm SEC* (150-950A)

Hydrophobic Interaction Chromatography
Silica-based particles
Nacalai Cosmosil HIC*
PolyLC PolyETHYL A*
PolyLC PolyMETHYL A*
PolyLC PolyPROPYL A*

Normal Phase / HILIC
Agela Venusil XBP CN (300A)
Agela Venusil XBP Diol (300A)
Agela Venusil XBP NH2 (300A)
Agela Venusil XBP Silica (300A)
Chromenta BB-NH2 (300A)
Chromenta BB-SiO2 (300A)
PolyLC PolyGLYCOPLEX
PolyLC PolyHYDROXYETHYL A

MIxed Mode (RP + IEX)
SIELC Promix SP (100A)
SIELC Promix AP (100 or 300A)
SIELC Promix MP* (300 or 800A)

Non-Porous Particles* - Ultra Fast LC
Ion Exchange Chromatography
Nacalai Cosmogel IEX Type Q-N*
Nacalai Cosmogel IEX Type S-N*
Nacalai Cosmogel IEX Type M-N*
Sepax Antibodix WCX*
Sepax Proteomix WAX-NP*
Sepax Proteomix SAX-NP*
Sepax Proteomix WCX-NP*
Sepax Proteomix SCX-NP*

Remove Proteins (In-Line)
ZirChrom ProTain*