How to Choose the Suitable C18 HPLC Column?

When performing preparative HPLC, it is necessary to select columns with the highest possible purity and a large number of compounds that can be obtained simultaneously. Silica matrix-filled preparative HPLC column is widely used in synthetic chemistry, natural substance chemistry, etc. Reversed-phase preparative HPLC columns utilize different chemically bonded phases to maintain peak patterns and achieve high-purity refinement.

According to statistics, nearly 80% of organic and inorganic substances can be separated by high-performance liquid chromatography, of which the C18 HPLC column in reversed-phase chromatography is the most commonly used column in HPLC.

The column is used every day, and today HAWACH will discuss the C18 column selection. Here are five basic principles and two main considerations for the selection of the C18 HPLC column.

Basic principles of C18 column selection

  • The selection of packing material with an alkylation treatment seal can prevent the trailing phenomenon of alkaline compounds.
  • Use columns with high carbon content to increase the retention value.
  • Use shorter columns (e.g. 15cm, 7.5cm).
  • Use packing materials with small particle sizes.
  • Use columns with large pore-size packing for components with high molecular weight.

Effect of packing of C18 column on a chromatographic column

There are 2 main considerations for the selection of C18, namely the influence of column packing and column specifications on the chromatographic column. The main physical properties of the packing include the following: granularity, pore size, pore volume, bonded phase chemistry, carbon content, and alkylation treatment.

1. Granularity refers to the size of the particle diameter of the column packing. For the particle size marked on the column, it is an average value. Such as particle size “5μm” is not all the particles in the column packing diameter is 5μm, there is actually a particle distribution. This degree of distribution has an important role in the column back pressure and column efficiency.

Generally speaking, the smaller the average particle size and the smaller the particle distribution, the higher the column efficiency and the higher the inverse pressure.

2. Pore size refers to the pore gap between filler particles and it refers to the average pore size of the packing.

The average pore size distribution of spherical packing is relatively narrow after loading the column, the column bed structure is uniform, the column efficiency is high, and the reproducibility is good.

The average pore size distribution of amorphous packing is wider, the column bed structure is not uniform, the linear velocity of the mobile phase is not uniform, and the spectral band is broadened.

The average pore size has a large impact on the separation of large molecules, and there may be molecular exclusion effects or adsorption effects in the separation of samples containing larger molecules, thus affecting the recovery and accuracy of quantification. Therefore, when using reversed-phase chromatography to separate samples such as proteins or peptides, a reversed-phase column packing with a large pore size (e.g., 30 nm) should be considered.

The pore volume, as a parameter of silica gel porosity, can be used as a reference when separating and analyzing larger molecular compounds, and the reversed-phase column packing with a larger pore volume should be selected.

3. Chemical bonding phase packing occupies an extremely important position in high-performance liquid chromatography. It can be bonded to organic groups of greater polarity, using a less polar solvent as the mobile phase. C18 columns are based on silylated bonding (Si-O-Si-C), and this type of bonding reaction is now commonly used.

4. Carbon content, i.e., the carbon content in the filler. The conventional measurement technique is to heat the filler until the carbon-hydrogen bond is broken 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 bonds or by increasing the bond density.

An increase in carbon content increases the retention value of the column. The chromatographic behavior of the bonded phase is related to the bonding density, but also to the density of the silica gel and the surface area of the packing. The higher the density of the packing, the more silica gel is required to fill the column and the higher the carbon content of the column.

If the column is filled with 2 fillers of different densities with the same carbon content, the retention behavior will be significantly different. Therefore, it is not enough to predict chromatographic behavior by carbon content alone.

5. The C18 silylation reagent is a large molecule larger than 2 nm and therefore creates severe steric hindrance with the C18 silylation reagent that is already bonded to the adjacent silanol group. The result is that there are large amounts of residual silanol groups on the silica surface that do not react with the silylation reagent, and these polar silanol groups can interact with basic compounds under certain chromatographic conditions to cause peak trailing, which can affect quantitative analysis results. These problems can be overcome to some extent by alkylation treatment.

Alkylation is an independent reaction done on the bonded phase to reduce the silanol groups on the silica surface. Alkylation treatments use reagents with small molecules (e.g., trijasilane) that have much less spatial site resistance than the C18 group.

Most stationary phases have only 30% of the binding sites that can be covered. Coverage of up to 50% has been reported with certain extremely active chemical reagents and special reaction conditions.

A good understanding of the physical properties of the silica-bonded phase will help in the selection of a suitable column for the reaction in HPLC. While on the surface C18 columns appear to have the same chemical functional groups, in reality, the performance of different brands of C18 columns may vary greatly, resulting in different separation results.

Effect of C18 column specification on a chromatographic column

The choice of column packing is related to the possibility of chromatographic separation, while the choice of column specification directly affects the analysis speed, separation capacity, detection capacity, and solvent consumption per analysis.

There are two factors to describe the column specification: the length and inner diameter. Generally speaking, the column’s inner diameter does not affect the relationship between separation and analysis time. Today, column technology has developed to the point that columns with different internal diameters can have the same performance. Columns with different internal diameters have their own characteristics, and for the same analysis time and separation, columns with larger internal diameters consume more solvent than those with smaller internal diameters.

On the other hand, a smaller I.D. column requires less sample volume for the same detection signal. Although increasing the column length improves the separation, the resistance also increases and the inlet pressure must be increased. The column pressure is the main obstacle to both increasing the separation and reducing the analysis time. Separation, analysis time, and column pressure are mutually dependent, and if one of them is selected, the third factor is also selected. Long columns can give high separation and short columns can provide fast separation, so we can choose the right column according to the sample situation.

For more information about the C18 HPLC columns, welcome to contact HAWACH.