WorkBeads™ 40 Butyl SH is a resin for hydrophobic interaction chromatography (HIC) designed for research and industrial scale purification of proteins, peptides, plasmids, and oligonucleotides by utilizing the difference in their surface hydrophobicity. It’s often used to complement other techniques that separate according to either charge or size.
The functional ligand of WorkBeads 40 Butyl SH is n-butyl thioether. Since butyl is a very hydrophobic linear chain, minimal mixed-mode interactions are expected. The resin is optimized to offer reliable binding performance. The property of high-resolution separation, in combination with low backpressure, allows both capture and polishing purification applications in standard bioprocess columns. The resin is also available in several different ready-to-use prepacked column sizes, such as GoBio™ Mini 1 mL and 5 mL, GoBio Screen 7 x 100 (3.8 mL), GoBio Prep 16 x 100 (20 mL) and 26 x 100 (53 mL) as well as GoBio Prod columns starting at 1 L.
Hydrophobic interaction chromatography (HIC) separates molecules according to differences in their surface hydrophobicity through a reversible interaction between the molecules and the hydrophobic surface of the HIC resin. A high salt concentration enhances the interaction, and a low salt concentration weakens the interaction. The extent of the reversible interaction between the molecule and the hydrophobic surface of a HIC resin depends on the properties of the HIC resin and target molecule, and the running conditions, such as the salt concentration. The principle for molecule adsorption to HIC resins is orthogonal to ion exchange and size exclusion chromatography.
In HIC, the molecules to be separated are usually loaded onto the column under conditions of a high salt concentration, which promotes the exposure of hydrophobic regions and increased hydrophobic interactions. As the sample is applied, molecules with higher hydrophobicity tend to bind more strongly to the hydrophobic ligands, resulting in their retention on the column. The degree of retention depends on the strength of the hydrophobic interaction, which is influenced by factors such as salt concentration, types of salt, pH, and temperature.
WorkBeads are agarose-based chromatographic resins manufactured using a proprietary method that results in porous beads with tight size distribution and exceptional mechanical stability. Agarose-based matrices have been successfully used for decades in biotechnology purification, from research to production scale, due to their exceptional compatibility with biomolecules, including proteins, peptides, nucleic acids, and carbohydrates. WorkBeads resins are designed for separations requiring optimal capacity and purity.
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Hydrophobic resins have been meticulously formulated with a specialized focus on facilitating intermediate purification processes. These processes aim to eliminate host-cell contaminants from partially purified targets through the employment of hydrophobic interactions, be they robust or subtle in nature. Within the realm of protein and nucleic acid purification, there exists a selection of methacrylate-based beads expressly tailored to harness hydrophobic interactions. These beads prove to be valuable assets in purifying compounds characterized by regions featuring hydrophobic interactions of varying strengths.
Hydrophobic interaction chromatography (HIC)is a widely used method in the separation of macro-biomolecules such as proteins and peptides based on the hydrophobicity difference on the molecule surface. The biggest advantage HIC resins enjoy is their mild interaction with proteins, which helps to keep the natural structure and bioactivity of macro-biomolecules to the greatest extent.
The adsorption/separation approach (sample loading with high salinity and elution with low salinity) and chromatography principle help to make HIC resins an ideal supplementary chromatography method, as well as the adjustment technology between individual operational units. HIC resin is suitable for the separation of proteins with high hydrophobicity and excellent tolerance towards high salinity, serving as an efficient tool in the purification of macro-molecules including antibodies, vaccines, recombinant proteins, interferon, and hormones, particularly in the removal of aggregates.
Hydrophobic interaction chromatography serves as an effective method for the separation of proteins characterized by varying degrees of surface hydrophobicity. In this chromatographic process, proteins interact with a hydrophobic ligand present on the Hydrophobic Interaction Chromatography (HIC) resin within a binding buffer featuring a notably high concentration of salt.
Upon the reduction of the ionic strength of the buffer, a reversal of the hydrophobic interaction ensues. Consequently, the protein exhibiting the lowest degree of hydrophobicity is the first to be eluted. Subsequently, proteins with increasing hydrophobicity are sequentially eluted, with the most hydrophobic variant requiring a more significant reduction in salt concentration to effectively reverse the interaction and achieve elution.
The following are the main characteristics of hydrophobic interaction resins:
● Matrix and particle size
Similar to IEX resins, HIC resins are obtained by coupling hydrophobic ligands on the matrix. Bestchrom HIC resins fall into four categories, specifically, Bestarose FF(Fast Flow agarose matrix, 90 μm), Bestarose HP(High-Performance agarose matrix, 34 μm), Diamond (High rigidity agarose matrix, 90 μm) and Diamond Mustang(High Performance and high rigidity agarose matrix, 40 μm).
Thanks to their agarose-made matrix, Bestchrom HIC resins retain excellent hydrophilicity and porousness of natural polysaccharides. Among the four types of HIC resin, Diamond and Diamond Mustang enjoy better mechanical and chemical stability due to their high rigidity agarose matrix. Generally speaking, HIC resins enjoy different merits including high selectivity(due to smaller bead size) as well as tolerance towards high flow rate(due to highly rigid matrix), enabling their application over the whole process of protein chromatography from capture, intermediate purification to polishing phase.
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● Levels of hydrophobicity
The key to successful HIC resin selection lies in the ligands with appropriate hydrophobicity. In other words, proteins with strong hydrophobicity require resins with weak hydrophobicity, and vice versa. The different hydrophobicity levels of common HIC ligands are as follows: Butyl-S < Butyl < Octyl < Phenyl. Phenyl ligands enjoy the strongest hydrophobicity, which can be categorized into high substitute groups(High Sub, HS) and low substitute groups(Low Sub, LS);
Octyl and Butyle are aliphatic hydrophobic ligands, among which, Octyl ligands enjoy stronger hydrophobicity than Butyl ligand due to its longer chain. Whereas, Butyl-S HIC resin possesses the weakest hydrophobicity, making it suitable for the purification of molecules with strong hydrophobicity at low salinity conditions. When exploring suitable chromatography methods, it is recommended to start with resins with the strongest hydrophobicity and make sure of the applicability of HIC resins before optimizing resin combinations.
1. What is a HIC resin?
Hydrophobic resins or HIC resins are the stationary phase of liquid chromatography. HIC resins are composed of a porous matrix, such as agarose beads coupled to a ligand. Hydrophobic resins are available in bulk or in prepacked columns for the capture and purification or polishing of biomolecules like antibodies or plasmids.
2. What are the different hydrophobic ligands?
Common ligands used during HIC include phenyl, butyl, and octyl groups. Bestchrom offers a range of hydrophobic resins that use these ligands.
3. How do I know which HIC resin to choose?
Select hydrophobic resins based on the hydrophobicity of your target biomolecules. We offer screening and selection kits to help you choose an appropriate resin for high flow, capacity, and resolution.
Do you need hydrophobic resins? We are a professional chromatography resin manufacturer and exporter, specializing in providing high-quality hydrophobic interaction resins to overseas customers. If you need help or services from us, please send an or visit our website.
Reference
Mahn A. Hydrophobic interaction chromatography: fundamentals and applications in biomedical engineering[M]. INTECH Open Access Publisher, .
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