Reverse phase chromatography (RPC), as an efficient separation technology based on the principle of hydrophobic interaction, realizes the separation of peptides and proteins by virtue of the specific interaction between the stationary phase and the mobile phase. The retention behavior of polypeptides in RPC has significant particularity - solute retention is extremely sensitive to small changes in solvent strength, so gradient elution has become the preferred solution for complex polypeptide mixtures that cannot be separated by isocratic elution. At present, peptide reverse phase purification technology is widely used in drug research and development, biological activity research and other fields, and has become the "gold standard" of peptide purification. With the continuous development of technology, the reverse phase purification technology has been continuously improved in separation efficiency, purity, automation and other aspects, and is also developing in the direction of green, efficient and accurate.
I Selection of packing for reverse chromatography
The chemical properties (such as the type of bonding phase, carbon loading, end capping treatment) and physical structure (particle size, pore size, specific surface area) of the filler determine the hydrophobic interaction strength with the polypeptide. For example, C18 packing has strong hydrophobicity and is suitable for separating hydrophobic polypeptides; C4 packing has weak hydrophobicity and is suitable for macromolecules or polypeptides that are easy to aggregate. Selecting suitable packing can improve the separation and recovery. ; If the hydrophobicity is too weak, the separation effect is poor. According to the hydrophobicity, molecular weight and structural characteristics of the polypeptide, the appropriate stationary phase was selected. For example, for polypeptides with strong hydrophobicity, C18 filler can be selected; C4 filler can be selected for macromolecules or polypeptides that are easy to aggregate.
The degree of substitution of hydrophobic ligands on RPC medium is much higher than that on HIC medium. RPC is more suitable for the separation and purification of peptides and small molecular proteins with good stability in water organic solvent system. The elution conditions of HIC process are mild. Generally, the purpose can be achieved by reducing the salt concentration of eluent. RPC has a wide range of applications in protein purification. HIC process has mild elution conditions. Usually, the purpose can be achieved by reducing the salt concentration of eluent. It is widely used in protein purification.
1.c18 packing:
Bonded octadecyl is one of the most commonly used reverse phase fillers. It is suitable for the separation of non-polar to medium polar compounds, such as antibiotics, barbiturates, essential oils, steroids, surfactants, etc. It has strong hydrophobicity and strong retention ability, and can effectively separate hydrophobic compounds. .
2.c8 packing:
Octyl modification has weaker nonpolarity and retention ability than C18. . C8 packing has moderate retention capacity, fast separation speed and low back pressure, and is suitable for the rapid separation of moderately hydrophobic compounds. It is widely used for the fine purification of insulin and glp-1ra.
3.c4 packing:
Butyl modification, with weaker nonpolarity, is suitable for the separation of peptides, proteins and other macromolecules. Its moderate hydrophilicity makes it selective for hydrophobic amino acid residues, which can better separate peptides and proteins and reduce aggregation and side reactions. It is selective for peptides and proteins, can reduce aggregation and side reactions, and is suitable for the separation of biological macromolecules. Butyl groups bonded on Polymethyl propionate microspheres or agarose microspheres were used as hydrophobic chromatography packing, and saline system was used as mobile phase for the separation and purification of protein, antibody, fermented glp-1ra and insulin precursor.
4. polymer reverse packing:
Such as polystyrene divinylbenzene or polymethacrylate, which has a wide pH tolerance range (1-14), high temperature resistance and high pressure resistance, is suitable for macromolecular separation and separation under extreme conditions, but the column efficiency is relatively low. . With the development of technology, the particle size of ps/dvb porous microspheres has been comparable to that of silica gel microspheres. For example, the particle size of seplife RP lxms-10 is 10um, which has high column efficiency after column installation, and its widely used pH range (1-14), better stability and longer service life. It can replace the fillers such as silica gel microspheres C8 with the same resolution.
5. phenyl filler:
Phenyl modification on silica gel has π - π interaction characteristics and higher selectivity for aromatic compounds, which is suitable for the separation of compounds with phenyl ring structure, such as polycyclic aromatic hydrocarbons, aromatic drugs, etc. Like butyl filler, it is usually used as hydrophobic filler bonded on Polymethyl propionate microspheres or agarose microspheres, with saline system as mobile phase, and is widely used for the separation and purification of various biomolecules. For example, phenyl seplifeff has been used in large-scale industrial production for the separation and purification of recombinant human serum albumin.
6. cyano filler (CN):
Cyanopropyl bonded phase, featuring polar selectivity, is suitable for the separation of polar compounds, such as those with amine, alcohol, and acid functional groups. It improves the separation of analytes that exhibit weak interactions with non-polar stationary phases.
7.hlb packing:
Composed of an intertwined hydrophilic and hydrophobic structure, it exhibits broad retention capabilities for both polar and non-polar analytes. Commonly used in solid-phase extraction and liquid chromatography separation, it can simultaneously retain hydrophilic and lipophilic compounds, thereby enhancing analytical sensitivity and accuracy. The balanced hydrophilic and hydrophobic structure provides wide retention for analytes ranging from polar to non-polar, making it suitable for solid-phase extraction and liquid chromatography separation.
8.Graphitized Carbon Packing：
With strong surface retention capabilities, it can be used for the separation of geometric isomers and can operate under any pH and temperature conditions, although its range of applications is relatively narrow. Possessing strong retention, it is usable across all pH levels and temperatures but has a limited scope of application, primarily for separating geometric isomers.
9. alumina filler:
It can be used in mobile phase with pH up to 12, but it has strong interaction with basic compounds, and its application scope is limited. The
10. zirconia filler:
The polymer coated porous zirconia microsphere column has good chemical stability and mechanical strength when applied in the pH range of 1-14 and the temperature can reach 100 ℃. It has good chemical stability and mechanical strength, wide application pH range and high temperature tolerance, but relatively few commercial products.
Selection basis of reverse chromatography packing
In HPLC, rigid matrix with high mechanical strength should be selected; If the molecular weight of the substance to be separated is large and the sample volume is large, macroporous matrix should be selected, such as agarose gel; . The sample volume of HIC is affected by the concentration of components in the sample and the binding capacity of the medium. The diluted sample can be directly added without concentration.
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Polarity of the Analyte: For highly polar analytes, hydrophilic C18 or high-carbon-content C18 packings are suitable; for weakly polar analytes, C8 or C4 packings can be selected.
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Structure of the Analyte: For analytes containing aromatic structures like benzene rings, phenyl or pentafluorophenyl (PFP) packings are recommended. For analytes with specific functional groups, the choice of packing should be based on the properties of those groups.
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Molecular Weight: For analytes with a molecular weight greater than 5000, packings with a pore size larger than 150 Å are recommended; for those greater than 10,000, packings with a pore size larger than 300 Å should be chosen.
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Sample Complexity: For complex samples with multiple components, selecting packings with small particle sizes (e.g., 3 μm, 5 μm) can improve resolution.
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Mobile Phase pH Value: If the mobile phase has an extreme pH, acid- and alkali-resistant packings, such as polymer-based packings or hybrid organic-inorganic packings, are necessary.
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Separation Purpose: For analytical separations, high-efficiency silica-based packings are preferred. For preparative separations, high-capacity polymer-based packings or wide-pore packings are suitable.
II Mobile phase composition
The polarity of the mobile phase is determined by the ratio of organic solvents (such as acetonitrile or methanol) to water. The stronger the polarity (high aqueous phase ratio), the stronger the hydrophobic interaction between the peptide and the stationary phase, leading to longer retention times; the weaker the polarity (high organic phase ratio), the weaker the hydrophobic interaction, resulting in peptide elution. Furthermore, the pH of the mobile phase indirectly affects the hydrophobicity of peptides by influencing their charge state and conformation.
When separating and purifying large molecules (greater than 10 KDa), hydrophobic interaction chromatography media with a saline buffer system can be chosen as the mobile phase to avoid the use of organic solvents which might affect the structure and activity of biomolecules.
Optimizing the mobile phase composition enables effective separation and elution of peptides. For example, gradient elution (gradually increasing the organic phase ratio) can separate complex peptide mixtures; an appropriate pH (e.g., acidic conditions) can suppress the ionization of peptides and the stationary phase, reducing peak tailing and non-specific adsorption. The mobile phase composition, including the type of organic solvent, its ratio, and pH, should be optimized experimentally. For instance, for highly hydrophobic peptides, the organic phase ratio can be increased; for peptides prone to ionization, an acidic mobile phase can be selected.
III Column temperature
The increase of temperature can reduce the viscosity of mobile phase, increase the diffusion coefficient of polypeptide, accelerate the mass transfer speed and improve the separation efficiency. . Appropriately increasing the column temperature (such as 30-60 ℃) can shorten the separation time and improve the resolution, but too high temperature may lead to peptide degradation or stationary phase damage. For heat sensitive peptides, a lower temperature (e.g. 25-30 ℃) should be selected to retain activity. According to the properties of peptides and separation requirements, the appropriate column temperature was selected. For thermally stable peptides, a higher column temperature (such as 40-60 ℃) can be selected; For heat sensitive peptides, a lower column temperature (such as 25-30 ℃) can be selected.
IV. Sample Loading / Loading Capacity
Excessive sample loading may lead to overloading of the stationary phase, saturating the binding sites between the peptide and the stationary phase, which results in diminished separation efficiency. Additionally, an excessive amount of sample may precipitate at the column head or block the packing pores, affecting the uniform distribution of the mobile phase. Properly controlling the sample loading ensures separation efficiency and recovery rate. Generally, the optimal sample loading should be determined through preliminary experiments to avoid peak broadening, tailing, or reduced resolution caused by overloading. For example, for highly hydrophobic peptides, the sample loading should be appropriately reduced; for large molecules or cases where peptides are prone to aggregation, a lower sample loading is advisable.
V. Flow Rate
The flow rate affects the mass transfer equilibrium of peptides between the stationary phase and the mobile phase. If the flow rate is too fast, the contact time between the peptides and the stationary phase is insufficient, leading to incomplete separation; if the flow rate is too slow, it may cause peak broadening and extend separation time. Optimizing the flow rate can enhance separation efficiency and resolution. Generally, small-molecule peptides are more sensitive to flow rate and require a lower flow rate; large-molecule peptides or proteins are less sensitive to flow rate, allowing for an appropriately increased flow rate to shorten separation time. The flow rate should be selected based on the molecular weight and structural characteristics of the peptides.
VI. Sample Pretreatment
The solubility, purity, and stability of the sample directly affect the separation performance in reversed-phase chromatography. Poor sample solubility may lead to precipitation at the column head or blockage of the packing material; the presence of impurities may compete with the target peptide for binding sites, impacting separation purity. Proper sample pretreatment (such as filtration, solid-phase extraction, and pH adjustment) can enhance sample quality, reduce interference from impurities, and improve separation efficiency and recovery.
For example, dissolving the sample in water or acetonitrile containing 0.1% TFA can improve the solubility and stability of peptides. The optimal sample pretreatment method should be determined through preliminary experiments. For instance, for highly hydrophobic peptides, dissolving the sample in acetonitrile containing 0.1% TFA may be suitable; for large molecules or cases where peptides are prone to aggregation, methods such as solid-phase extraction or filtration can be chosen to remove impurities.
