With the development of HPLC technology, especially the continuous improvement of the high selectivity, sensitivity, and throughput requirements for complex samples in separation analysis, it has greatly promoted the development of new chromatographic packing materials (stationary phases). The reason is that the core of HPLC is the chromatographic stationary phase, and the separation efficiency is closely related to the structure, physical and chemical properties of the stationary phase. Therefore, continuous research and improvement of the preparation process have greatly improved the performance of separation materials, making them have high permeability, high mechanical strength, good biocompatibility, and separation efficiency.
So far, a variety of chromatographic stationary phases have been successfully prepared, classified as follows:
The commonly used chromatographic stationary phase matrices include:
Silica gel stationary phase, polymer microsphere stationary phase, and metal oxide microsphere stationary phase.
1.Silica gel microspheres as a stationary phase.
Due to the large number of reactive silanol groups on the surface of silica gel, and the advantages of its strong matrix, porous structure, and good chemical stability, silica gel is a relatively ideal material for chromatographic stationary phase matrix. Silica gel microspheres not only have efficient and selective characteristics, but also have a large surface area and good chromatographic performance.
Therefore, silica gel microsphere stationary phase is currently the most widely used liquid chromatography packing material, especially for efficient separation and analysis of small organic molecules, and occupies the vast majority of market share. However, the chemical stability of silica gel matrix is relatively low and it can only be operated within the pH range of 2-8, otherwise hydrolysis will occur, which will reduce the service life of the chromatographic column. In addition, the remaining silanol groups exhibit the so-called “secondary effect”, which particularly causes tailing of peaks for basic compounds and to some extent limits the application of silica gel microsphere stationary phase in the analysis of basic compounds.
2. Polymer microsphere stationary phase.
Although silica gel stationary phase has advantages such as high column efficiency and good separation performance, it is still the mainstream of commercial chromatographic columns at present.
This type of packing material has two obvious drawbacks:
01
The range of suitable mobile phases is narrow and generally used within the pH range of 2-8.
02
The surface residual silanol groups result in poor separation of basic substances.
Organic polymer microspheres with sufficient mechanical strength can overcome the above-mentioned drawbacks of silica gel packing materials. At the same time, due to their better biocompatibility, they are more suitable for applications in life sciences and other fields. Polymer microsphere stationary phase has made significant progress so far, and some have reached a fairly high level of commercialization. However, this type of stationary phase still has some shortcomings, such as low mechanical strength, easy swelling, high mass transfer resistance, and low column efficiency, which limit their applications in certain aspects.
3. Metal oxide microsphere stationary phase.
To overcome the inherent drawbacks of silica gel and polymer microsphere stationary phases, researchers are continuously developing new matrix materials. The separation mechanism of aluminum oxide is quite complex. Currently, surface covered and surface bonded butyl aluminum oxide have been applied in chromatographic analysis and have shown good performance, but they still cannot replace silica gel. Zirconia and modified zirconia have both high mechanical strength like silica gel and excellent chemical stability like polymer matrices. Therefore, the study of zirconia as an HPLC packing material has aroused great interest in the chromatography field, mainly because zirconia has good alkali resistance, and its pH range can reach 1-14.
Additionally, it has other advantages:
01
High mechanical strength and can withstand high temperatures.
02
Controllable particle and pore sizes, suitable for separation of biotechnological products.
03
In normal-phase chromatography, it exhibits good peak symmetry due to no adsorption of basic functional groups such as amino groups.
04
No need for bonding when used as an ion-exchange chromatography packing material.
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