Liquid chromatography is the main method for testing the content of each component and impurities in raw materials, intermediates, preparations and packaging materials, but many substances do not have standard methods to rely on, so it is inevitable to develop new methods. In the development of liquid phase methods, the chromatographic column is the core of liquid chromatography, so how to choose a suitable chromatographic column is crucial. In this article, the author will explain how to choose a liquid chromatography column from three aspects: overall ideas, considerations and application scope.
A.Overall ideas for selecting liquid chromatography columns
1. Evaluate the physical and chemical properties of the analyte: such as chemical structure, solubility, stability (such as whether it is easy to be oxidized/reduced/hydrolyzed), acidity and alkalinity, etc., especially the chemical structure is the key factor in determining the properties, such as the conjugated group has strong ultraviolet absorption and strong fluorescence;
2. Determine the purpose of analysis: whether high separation, high column efficiency, short analysis time, high sensitivity, high pressure resistance, long column life, low cost, etc. are required;
- Choose a suitable chromatographic column: understand the composition, physical and chemical properties of the chromatographic filler, such as the particle size, pore size, temperature tolerance, pH tolerance, adsorption of the analyte, etc.
- Considerations for selecting liquid chromatography columns
This chapter will discuss the factors to be considered when selecting a chromatography column from the perspective of the physical and chemical properties of the chromatography column itself. 2.1 Filler matrix
2.1.1 Silica gel matrix The filler matrix of most liquid chromatography columns is silica gel. This type of filler has high purity, low cost, high mechanical strength, and is easy to modify groups (such as phenyl bonding, amino bonding, cyano bonding, etc.), but the pH value and temperature range it tolerates are limited: the pH range of most silica gel matrix fillers is 2 to 8, but the pH range of specially modified silica gel bonded phases can be as wide as 1.5 to 10, and there are also specially modified silica gel bonded phases that are stable at low pH, such as Agilent ZORBAX RRHD stablebond-C18, which is stable at pH 1 to 8; the upper temperature limit of the silica gel matrix is usually 60 ℃, and some chromatography columns can tolerate a temperature of 40 ℃ at high pH.
2.1.2 Polymer matrix Polymer fillers are mostly polystyrene-divinylbenzene or polymethacrylate. Their advantages are that they can tolerate a wide pH range – they can be used in the range of 1 to 14, and they are more resistant to high temperatures (can reach above 80 °C). Compared with silica-based C18 fillers, this type of filler has stronger hydrophobicity, and the macroporous polymer is very effective in separating samples such as proteins. Its disadvantages are that the column efficiency is lower and the mechanical strength is weaker than that of silica-based fillers. 2.2 Particle shape
Most modern HPLC fillers are spherical particles, but sometimes they are irregular particles. Spherical particles can provide lower column pressure, higher column efficiency, stability and longer life; when using high-viscosity mobile phases (such as phosphoric acid) or when the sample solution is viscous, irregular particles have a larger specific surface area, which is more conducive to the full action of the two phases, and the price is relatively low. 2.3 Particle size
The smaller the particle size, the higher the column efficiency and the higher the separation, but the worse the high pressure resistance. The most commonly used column is the 5 μm particle size column; if the separation requirement is high, a 1.5-3 μm filler can be selected, which is conducive to solving the separation problem of some complex matrix and multi-component samples. UPLC can use 1.5 μm fillers; 10 μm or larger particle size fillers are often used for semi-preparative or preparative columns. 2.4 Carbon content
Carbon content refers to the proportion of bonded phase on the surface of silica gel, which is related to specific surface area and bonded phase coverage. High carbon content provides high column capacity and high resolution, and is often used for complex samples requiring high separation, but due to the long interaction time between the two phases, the analysis time is long; low carbon content chromatographic columns have a shorter analysis time and can show different selectivities, and are often used for simple samples that require rapid analysis and samples that require high aqueous phase conditions. Generally, the carbon content of C18 ranges from 7% to 19%. 2.5 Pore size and specific surface area
HPLC adsorption media are porous particles, and most interactions take place in the pores. Therefore, molecules must enter the pores to be adsorbed and separated.
Pore size and specific surface area are two complementary concepts. Small pore size means large specific surface area, and vice versa. A large specific surface area can increase the interaction between sample molecules and bonded phases, enhance retention, increase sample loading and column capacity, and separation of complex components. Fully porous fillers belong to this type of fillers. For those with high separation requirements, it is recommended to choose fillers with large specific surface area; small specific surface area can reduce back pressure, improve column efficiency, and reduce equilibrium time, which is suitable for gradient analysis. Core-shell fillers belong to this type of fillers. On the premise of ensuring separation, it is recommended to choose fillers with small specific surface area for those with high analysis efficiency requirements. 2.6 Pore volume and mechanical strength
Pore volume, also known as “pore volume”, refers to the size of the void volume per unit particle. It can well reflect the mechanical strength of the filler. The mechanical strength of fillers with large pore volume is slightly weaker than that of fillers with small pore volume. Fillers with pore volume less than or equal to 1.5 mL/g are mostly used for HPLC separation, while fillers with pore volume greater than 1.5 mL/g are mainly used for molecular exclusion chromatography and low-pressure chromatography. 2.7 Capping rate
Capping can reduce the tailing peaks caused by the interaction between compounds and exposed silanol groups (such as ionic bonding between alkaline compounds and silanol groups, van der Waals forces and hydrogen bonds between acidic compounds and silanol groups), thereby improving column efficiency and peak shape. Uncapped bonded phases will produce different selectivities relative to capped bonded phases, especially for polar samples.
- Application scope of different liquid chromatography columns
This chapter will describe the application scope of different types of liquid chromatography columns through some cases.
3.1 Reversed-phase C18 chromatographic column
The C18 column is the most commonly used reversed-phase column, which can meet the content and impurity tests of most organic substances, and is applicable to medium-polar, weakly polar and non-polar substances. The type and specification of C18 chromatographic column should be selected according to the specific separation requirements. For example, for substances with high separation requirements, 5 μm*4.6 mm*250 mm specifications are often used; for substances with complex separation matrices and similar polarity, 4 μm*4.6 mm*250 mm specifications or smaller particle sizes can be used. For example, the author used a 3 μm*4.6 mm*250 mm column to detect two genotoxic impurities in celecoxib API. The separation of the two substances can reach 2.9, which is excellent. In addition, under the premise of ensuring separation, if rapid analysis is required, a short column of 10 mm or 15 mm is often selected. For example, when the author used LC-MS/MS to detect a genotoxic impurity in piperaquine phosphate API, a 3 μm*2.1 mm*100 mm column was used. The separation between the impurity and the main component was 2.0, and the detection of a sample can be completed in 5 minutes. 3.2 Reversed-phase phenyl column
Phenyl column is also a type of reversed-phase column. This type of column has strong selectivity for aromatic compounds. If the response of aromatic compounds measured by ordinary C18 column is weak, you can consider replacing the phenyl column. For example, when I was making celecoxib API, the main component response measured by the phenyl column of the same manufacturer and the same specification (all 5 μm*4.6 mm*250 mm) was about 7 times that of the C18 column. 3.3 Normal-phase column
As an effective supplement to reversed-phase column, normal-phase column is suitable for highly polar compounds. If the peak is still very fast when eluting with more than 90% aqueous phase in the reversed-phase column, and even close to and overlaps with the solvent peak, you can consider replacing the normal-phase column. This type of column includes hilic column, amino column, cyano column, etc.
3.3.1 Hilic column Hilic column usually embeds hydrophilic groups in the bonded alkyl chain to enhance the response to polar substances. This type of column is suitable for the analysis of sugar substances. The author used this type of column when doing the content and related substances of xylose and its derivatives. The isomers of a xylose derivative can also be well separated;
3.3.2 Amino column and cyano column Amino column and cyano column refer to the introduction of amino and cyano modifications at the end of the bonded alkyl chain, respectively, to improve the selectivity for special substances: for example, amino column is a good choice for the separation of sugars, amino acids, bases, and amides; cyano column has better selectivity when separating hydrogenated and unhydrogenated structural similar substances due to the presence of conjugated bonds. Amino column and cyano column can often be switched between normal phase column and reverse phase column, but frequent switching is not recommended. 3.4 Chiral column
Chiral column, as the name suggests, is suitable for the separation and analysis of chiral compounds, especially in the field of pharmaceuticals. This type of column can be considered when conventional reverse phase and normal phase columns cannot achieve the separation of isomers. For example, the author used a 5 μm*4.6 mm*250 mm chiral column to separate the two isomers of 1,2-diphenylethylenediamine: (1S, 2S)-1, 2-diphenylethylenediamine and (1R, 2R)-1, 2-diphenylethylenediamine, and the separation between the two reached about 2.0. However, chiral columns are more expensive than other types of columns, usually 1W+/piece. If there is a need for such columns, the unit needs to make a sufficient budget. 3.5 Ion exchange column
Ion exchange columns are suitable for the separation and analysis of charged ions, such as ions, proteins, nucleic acids, and some sugar substances. According to the filler type, they are divided into cation exchange columns, anion exchange columns, and strong cation exchange columns.
Cation exchange columns include calcium-based and hydrogen-based columns, which are mainly suitable for the analysis of cationic substances such as amino acids. For example, the author used calcium-based columns when analyzing calcium gluconate and calcium acetate in a flushing solution. Both substances had strong responses at λ=210nm, and the separation degree reached 3.0; the author used hydrogen-based columns when analyzing glucose-related substances. Several major related substances – maltose, maltotriose and fructose – had high sensitivity under differential detectors, with a detection limit as low as 0.5 ppm and a separation degree of 2.0-2.5.
Anion exchange columns are mainly suitable for the analysis of anionic substances such as organic acids and halogen ions; strong cation exchange columns have higher ion exchange capacity and selectivity, and are suitable for the separation and analysis of complex samples.
The above is just an introduction to the types and application ranges of several common liquid chromatography columns combined with the author’s own experience. There are other special types of chromatographic columns in actual applications, such as large-pore chromatographic columns, small-pore chromatographic columns, affinity chromatography columns, multimode chromatographic columns, ultra-high performance liquid chromatography columns (UHPLC), supercritical fluid chromatography columns (SFC), etc. They play an important role in different fields. The specific type of chromatographic column should be selected according to the structure and properties of the sample, separation requirements and other purposes.
Post time: Jun-14-2024