Simple idea of ​​choosing a liquid chromatography column
1. Determine the purpose of separation Determine if your application requires high resolution, short analysis time, high sensitivity, long column life, low operating costs, and more. 2. Assess the chemical properties of the analyte The chemical properties of the analyte, such as chemical structure, solubility, stability, and the like, are assessed. 3. Choose the right column Understand the physical and chemical properties of chromatographic packings. A filler matrix Silica gel matrix: high purity, low cost, high strength, easy chemical modification, but limited pH range. Most silica matrix fillers are stable between pH 2 and 8, but the specially modified silica bonded phase can be stabilized at pH 1.5-10. B particle shape Most modern HPLC fillers are spherical particles, but sometimes irregular particles. Spherical particles provide lower column pressure, higher efficiency and stability, and longer column life when using high viscosity mobile phases; irregular particles have a large specific surface area and relatively low cost. C particle size The smaller the particle size, the higher the column efficiency and the higher the degree of separation, but at the same time it leads to a higher column pressure drop. A 1.5-3 μm filler is selected to solve some complex samples, UPLC can use a 1.5 μm filler; another 10 μm or larger particle size filler is used as a semi-preparative or preparative column. D carbon content The carbon content refers to the proportion of the bonded phase on the surface of the silica gel, which is related to the specific surface area and the degree of bonding coverage. High carbon content increases column capacity, resolution, and analysis time for complex samples requiring high resolution; low carbon content analysis time is short, exhibits different selectivity for rapid analysis of simple samples and requires high water mobile phase Conditional sample. Generally, the carbon content of C18 ranges from 7-19%. E pore size and specific surface area The HPLC adsorption medium is a porous particle, and most of the reaction surface is in the pores. Therefore, molecules must enter the pores to be adsorbed and separated. F pore volume and mechanical strength Kong Rong, also known as "hole volume." Refers to the size of the void volume of the unit particles. It is a good response to the mechanical strength of the filler. A filler having a large pore volume is slightly weaker in mechanical strength than a filler having a small pore volume. Fillers with a pore volume of 1.5 mL/g or less are mostly used for HPLC separation, while fillers with a pore volume greater than 1.5 mL/g are used for size exclusion chromatography and low pressure chromatography. G-end capping Blocking reduces the tailing peaks of polar basic compounds due to interaction with bare silanol groups. Unblocked bonded phases produce different selectivities relative to the capped bonded phase, especially for polar samples. Chromatographic packing classification (bonded phase) The 1 s silica column is suitable for the separation of low molecular weight compounds that are soluble in organic solvents. The separation on the silica gel column is related to the orientation, type and amount of functional groups of the compound in the sample. In preparative chromatography, normal phase chromatography has great advantages because the purified sample only needs to evaporate the solvent to obtain a pure compound, unlike time-consuming freeze-drying like reversed-phase chromatography. 2 ODS/C8/C4 and other reversed phase columns ODS uses a monofunctional bonding agent to achieve high density bonding coverage under an optimized condition of an octadecyl bonded phase silica adsorbent, a durable small molecule HPLC column, C18 column often with water/methanol or water/ The acetonitrile mobile phase is used in combination to provide excellent separation ability for small molecules, proteins and peptides with different hydrophobicities. C8 is the same as the C18 column, and the sample is separated by a hydrophobic interaction mechanism, but the retention capacity is reduced. The C8 column is a good choice when the sample is too polar or too hydrophobic to be chromatographed on a C18 column. Some polar compounds have better performance on C8 columns. Due to the good surface coverage of C8 bonded phases, vitamins, antibiotics and basic drugs are usually separated on the C8 column better than the C18 column, and the peak shape More symmetrical. 3NH2 is an aminopropyl bonded silica adsorption phase that can be used for both normal phase analysis and reverse phase analysis. The selectivity of the amino stationary phase 4 diphenyl Suitable for compounds containing double bonds and aromatic groups. Compared to the C18 and phenylhexene bonded phases, rather than relying on the hydrophobic retention mechanism, the diphenyl bonded phase relies primarily on the difference in the π-electron structure of the compound to achieve separation. If you need to increase the selectivity of aromatic and double bond compounds in your analysis, increase resolution and increase sample throughput. 5 cyan column The cyanopropyl bonded silica adsorption phase is unique in that it can be used in both normal and reverse phase separation modes. When an aqueous organic mobile phase is used, the elution order of the analyte is consistent with the reverse phase mode: the polar compound elutes faster and the non-polar compound is more retained. Therefore, the cyano column can analyze compounds which are too hydrophobic for the C18 column and have poor solubility in organic solvents. When a cyano column is used as a normal phase column, the principle of separating compounds is based on polar interactions. Shanghai Jiapeng Technology Co., Ltd.
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Polymer matrix: Wide pH range, stable temperature (high temperature can reach 80 degrees or more), and low mechanical strength.
The pore size and specific surface area are two concepts that complement each other. The pore size is small and the specific surface area is large, and vice versa. The specific surface area is large, the reaction between the sample and the bonded phase is increased, the retention, the loading amount and the separation degree of the complex component are increased; the specific surface area is small, the equilibrium time is fast, and the gradient analysis is suitable.
Compared with C18, there is a big difference, which makes it possible to achieve ideal analysis results for polar combinations that are extremely difficult to analyze. In addition, the amino column exhibits higher separation efficiency for saccharide compounds than the ion gel column. A typical mobile phase in such applications is acetonitrile/water. The separation in normal phase mode is similar to unbonded silica gel, but the gradient equilibration takes less time and lasts longer.
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