Influence of C18 Column Packing on Chromatographic Column

The physical properties of the column packing have an important influence on the chromatographic behavior of the packing. The main physical properties of the packing include the following: particle size, pore size, pore volume, bonded phase chemistry, carbon content, and alkylation treatment.

(1) Particle Size:

Smaller particle sizes provide higher surface area and can result in improved resolution and faster separations. However, smaller particles may also lead to higher backpressure in the chromatographic system.

The particle size refers to the size of the particle diameter of the column packing. In fact, the particle size marked on the column is an average value. For example, the particle size “5μm” does not mean that all the particles of the packing in the column have a diameter of 5μm, but there is actually a particle distribution degree. This degree of distribution plays an important role in column backpressure and column efficiency. Generally speaking, the smaller the average particle size, the smaller the particle distribution, the higher the column efficiency, and the higher the back pressure. At present, the particle size of C18 column packing is between 4 and 10 μm.

(2) Particle Shape:

The shape of the particles, such as fully porous or superficially porous (core-shell), can affect efficiency and pressure. Core-shell particles typically provide a compromise between efficiency and backpressure.

C18 Universal HPLC Columns

(3) Pore Size:

Pore size influences the retention and separation of compounds. Larger molecules may require larger pore sizes to prevent restricted access, while smaller pores can lead to increased retention of smaller molecules.

The pore diameter refers to the pore gap between packing particles. Generally speaking, the pore size refers to the average pore size of the packing. The average pore size distribution of spherical packing is relatively narrow after packing, the column bed structure is uniform, the column efficiency is high, and the reproducibility is good; the average pore size distribution of the amorphous packing is wide, the column bed structure is uneven, the linear velocity of the mobile phase is uneven, and the band is expanded width.

The average pore size has a greater impact on the separation of macromolecular compounds. There may be molecular exclusion effects or adsorption effects when separating samples containing larger molecules, which affects quantitative recovery and accuracy. Therefore, when using reversed-phase chromatography to separate samples such as proteins or peptides, a reversed-phase column packing with a large pore size (such as 30 nm) should be considered. The pore volume is used as a parameter of the porosity of silica gel and can be used as a reference when separating and analyzing larger molecular compounds. Choose a reversed-phase column packing with a larger pore volume.

(4) Column Length:

Longer columns generally provide better resolution but may require higher mobile phase flow rates, resulting in increased backpressure. Column length should be chosen based on the separation needs and system capabilities.

(5) Column Diameter:

The column diameter affects the column efficiency and the amount of sample that can be loaded onto the column. Smaller diameter columns generally offer better efficiency but may have limitations in sample loading capacity.

(6) Column Conditioning:

    • Proper conditioning of the C18 column before use is essential. This involves flushing the column with the mobile phase to remove impurities and ensure consistent performance.

(7) pH Stability:

C18 columns are sensitive to pH, and extreme pH conditions can lead to degradation of the stationary phase. It is important to operate within the recommended pH range specified by the manufacturer.

(8) Temperature:

Column temperature can affect separation efficiency and selectivity. Controlling the temperature within a specified range can optimize chromatographic performance.

(9) Mobile Phase Composition:

The choice of organic and aqueous components in the mobile phase can influence the selectivity and resolution of the separation. Optimization of the mobile phase composition is critical for achieving desired results.

(10) Sample Compatibility:

Consider the compatibility of the C18 column with the sample matrix. Some samples may contain impurities or components that can interact with the stationary phase, affecting separation.

(11) Backpressure:

Smaller particle sizes and longer columns can lead to increased backpressure. It’s important to ensure that the chromatographic system can handle the backpressure generated by the selected C18 column.

(12) Column Reversibility:

Some C18 columns may exhibit irreversibility after exposure to certain sample matrices or mobile phase conditions. Regularly inspecting and conditioning the column can help maintain its performance.

(13) Chemically bonded phase packing occupies an extremely important position in high performance liquid chromatography. It can bond with more polar organic groups and use a less polar solvent as the mobile phase. It is also possible to bond organic groups with less polarity and use a solvent with greater polarity as the mobile phase. The HPLC C18 chromatographic column is a silanized bonding type (Si-O-Si-C), and this type of bonding reaction is currently widely used. For example, octadecyl trichlorosilane reacts with fully porous silica gel M-Porasil-C18 to form an alkyl chemical bond phase, the trade name is M-Bondapak-C18.

(14) The carbon content is the carbon content in the packing. The traditional measurement technique is to heat the packing until the carbon-hydrogen bond breaks, and then calculate the carbon content by measuring the weight loss or the carbon dioxide formed. The carbon content can be increased by increasing the length of the carbon bond or increasing the bonding density. As the carbon content increases, the retention value of the column increases. The chromatographic behavior of the bonded phase is related to the bonding density, as well as the density of the silica gel and the surface area of the packing. The higher the density of the packing, the more silica gel required for packing and the higher the carbon content of the column. If the column is packed with two packings with different densities and the same carbon content, their retention behavior will be significantly different. Therefore, carbon content alone is not enough to predict chromatographic behavior.

(15) The C18 silylation reagent is a large molecule larger than 2 nm, so it will cause serious steric hindrance with the C18 silylation reagent that has been bonded to the adjacent silanol group. As a result, there are a large number of residual silanol groups on the surface of silica gel that did not react with the silanizing reagent. These polar silanol groups will interact with basic compounds under certain chromatographic conditions and cause peak tailing, which can affect the quantitative analysis. result.

These problems can be overcome by alkylation treatment to a certain extent. Alkylation is an independent reaction completed on the bonded phase to reduce the silanol groups on the surface of the silica gel. The alkylation treatment uses small molecules (such as trimethylsilane) reagents, and its steric hindrance is much smaller than that of C18 groups.

Most stationary phases have only 30% coverage of the bonding positions. According to reports, through some extremely active chemical reagents and special reaction conditions, the coverage can be as high as 50%. A good understanding of the physical properties of the silica bonded phase will help to select the appropriate column in the high performance liquid chromatography reaction. On the surface, although the chemical functional groups of the C18 column are the same, in fact, the performance of C18 columns of different brands may be very different, resulting in different separation results.

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