How to Choose a Liquid Chromatography Column?
The simple idea of liquid chromatography column selection
1. Determine the purpose of the separation determine if your application requires high resolution, short analysis time, high sensitivity, long column life, low manipulative cost, and so on.
2. Assess the chemical nature of the analyte. Assess the chemical nature of the analyte. Such as chemical structure, solubility, stability, and so on.
3. Select the appropriate column to understand the physical and chemical properties of the chromatographic packing.
(II) Conditions for LC column selection
A. filler matrix
1, Silica gel matrix: high purity, low cost, high strength, easy chemical modification, but the pH range is limited. Most silica matrix packings are stable between pH 2-8, but the specially modified silica-bonded phase can be stabilized at pH 1.5-10.
2, The polymer matrix: the application of a wide range of pH value, temperature stability (high temperature can reach 80 degrees or more), mechanical strength is small.
B. Particle shape
Most modern HPLC packings are spherical particles, but are sometimes irregular particles. Spherical particles provide lower column pressure, higher efficiency and stability, and longer column life when using highly viscous mobile phases; irregular particles have a large specific surface area and a relatively low price. The
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 will lead to higher column pressure drop. A 1.5-3 μm filler was selected to solve some complex samples. UPLC can use 1.5 μm filler; another 10 μm or larger filler is used as a semi-preparative or preparative column.
D. Carbon content
The carbon content refers to the proportion of the bonding phase of the silica surface, which is related to the specific surface area and the bonding coverage. High carbon content improves column capacity, resolution and analysis time for complex samples requiring high resolution; low carbon analysis time is short, exhibits different selectivity, for rapid analysis of simple samples and requires high water activity phase Condition of the sample. The carbon content of general C18 varies from 7-19%.
E. Pore size and specific surface area
The HPLC adsorption medium is a porous particle with most of the reaction surface in the pore. Therefore, molecules must enter the pores to be adsorbed and separated.
The pore size and specific surface area are two complementary concepts. The small pore size has a large specific surface area, and vice versa. With a large specific surface area, it increases the reaction between the sample and the bonding phase, increases the storage, the amount of sample and the separation of complex components, and has a small specific surface area and a fast balance time, making it suitable for gradient analysis.
F. Hole capacity and mechanical strength
Kong Rong, also known as “hole volume.” Refers to the size of the void volume per particle. It can react well to the mechanical strength of the filler. The fillers with large pore volume are slightly weaker than the fillers with smaller pore volume. Fillers with a pore volume of 1.5 mL/g or less are mostly used for HPLC separation, and fillers with a pore volume of more than 1.5 mL/g are used for size exclusion chromatography and low-pressure chromatography.
G. End cap
Capping can reduce the tailing peak of polar basic compounds due to their interaction with the exposed silanol groups. The non-end-capping phase produces different selectivity with respect to the end-capping phase, especially for polar samples.
Liquid chromatography column classification
Columns can be divided into analytical and preparative types according to their use
1. routine analysis column (constant column), internal diameter 2 ~ 5mm, column length 10~30cm;
2. narrow bore columns, inner diameter 1~2mm, column length 10 ~ 20cm;
3. capillary column (also known as microcolumn), internal diameter 0.2 ~ 0.5mm;
4. semi-preparative columns, internal diameter> 5mm;
5. laboratory preparation column, internal diameter 20 ~ 40mm, column length 10 ~ 30cm;
6. production and preparation of column diameter up to tens of centimeters.