Bonded-Phase Chromatography
Bonded-phase chromatography chemically bonds a stationary phase to a support material for enhanced stability and separation efficiency. This technique, used in HPLC and other chromatographic methods, offers improved reproducibility and selectivity, making it valuable for various analytical applications.
Bonded phase chromatography is a liquid-liquid chromatography method in which a stationary phase is covalently bound to a carrier particle, which overcomes the mechanical shock in the distribution chromatography, the constant loss of the stationary phase, and the gradual change of the nature of the column.
It is a liquid chromatography using a chemically bonded phase as a stationary phase, which is formed by chemically reacting an organic molecule to the surface of a carrier (silica gel) by a covalent bond to form a uniform, firm monolayer.
The separation mechanism is both adsorption and distribution. For most bonded phases, the distribution mechanism is dominant. Typically, the carrier of the chemically bonded phase is silica gel, and the surface of the silica gel has a silanol group, ≡Si-OH, which reacts with a suitable organic compound to obtain a chemically bonded phase of various properties.
On the surface of the silica gel, due to the steric structural disorder of the bonding groups, all 6 silanol groups per square nanometer for chemical bonding are not able to participate in the bonding reaction. The ratio of the number of silanol groups participating in the reaction to the total number of silica groups is referred to as the surface coverage of the stationary phase.
When the surface coverage of the ODS bond is 98%, that is, 2% of silanol groups remain, and the partitioning action is dominant. When the surface coverage of ODS is 50%, there is both distribution and adsorption.
A bonded phase refers to an organic functional group (such as C18, C8, CN, or NH₂) that is chemically attached (bonded) to the silica surface of the chromatographic column.
| Bonded Group | Name | Polarity | Typical Use |
|---|---|---|---|
| C18 | Octadecylsilane (ODS) | Non-polar | Reverse-phase separations |
| C8 | Octylsilane | Moderately non-polar | Reverse-phase with shorter retention |
| CN | Cyano | Polar | Normal or reverse-phase chromatography |
| NH₂ | Aminopropyl | Polar | Sugar analysis, HILIC, weak anion exchange |
How It Works:
- Stationary Phase: A silica-based support coated with a chemically bonded layer.
- Mobile Phase: A solvent or mixture of solvents that carries the sample through the column.
- Separation Mechanism: Compounds interact differently with the bonded phase based on polarity, hydrophobicity, or specific interactions, leading to separation as they move through the column at different rates.
Advantages of Bonded Phase Chromatography
1. Eliminate the surface-active points on the support and remove some possible catalytic activities;
2. Good thermal stability; the high temperature can increase the mass transfer rate, thereby improving the column efficiency, such as the application in small peptide drugs.
3. Resistant to solvent washing, the stationary phase will not be lost during use (this is also relatively speaking, the stationary phase will still be lost after a long time or beyond the scope of use).
4. Flexible surface modification, easy-to-obtain repeatable products (control the quality of silica gel and bonding process).
5. The sample load is large, which is about an order of magnitude higher than that of ordinary silica gel.
6. The residual effect of the solvent is small, and the gradient elution balance is fast.
Here we have a quick start for filler and model selection based on bonded phase chromatography.
There are several types of bonded phases in HPLC, each designed for specific separation characteristics. For highly polar samples, use C18, C8, amino, and cyano bonded phase silica gel fillers or butyl, octyl, phenyl bonded phase agarose and dextran fillers, or polymer fillers, reversed-phase Separation. Such as C18-Universal HPLC Column, and C8-Universal HPLC Column.
For medium and low polarity samples, normal phase separation or hydrophilic chromatography separation using amino group-containing, diol-based, and surface-modified silica-bonded silica gel fillers.
For high molecular weight samples, size exclusion (gel filtration) is performed with agarose and dextran or silica gel packing, such as Normal Phase Silica SPE Cartridges.
Specific structural samples, optional filler matrices including agarose and dextran, containing metal polymerization, activation coupling, small ligand affinity, antibody affinity, pigment affinity, bonding phase, and chiral groups, crowns, Polymer-bonded phase polymer packing, separated by affinity chromatography.
The ionizable sample, the ion exchange method, and the filler matrix were separated and purified by agarose and dextran containing an SP.Q.CM.DEAE bonded phase, silica gel, and a polymer epoxy resin by ion exchange.
In summary, the bonded phase in HPLC is a crucial element that determines the separation characteristics of analytes. The specific chemistry of the bonded phase interacts with the sample components to achieve the desired separation and analysis outcomes.
Click here to know more about the Characteristics of High-Performance Liquid Chromatography
