To Have A Comprehensive Understanding Of HPLC Columns
1.History of HPLC columns
In the mid 1870s, HPLC instruments began to appear. The main filler: 10 μm amorphous silica particles. In the late 1970s, reversed-phase liquid chromatography was developed. In the 1980s, HPLC was widely used for the separation of compounds. The main filler: spherical silica gel with a particle size of 5-10 μm. In the early 1990s, high-purity silica gel with a particle size of 5 μm, the so-called B-type silica gel, was developed and became the standard for fillers in this industry. This B-type silica gel contains trace amounts of metal. In the late 1990s, in order to meet the needs of rapid separation, 3 μm or 3.5 μm spherical silica gel was developed, and its function and performance gradually gained people’s approval and acceptance. In the early 21st century, in order to meet the ultra-fast separation requirements, fillers with a particle size of less than 2 μm were developed, and monolithic columns, inorganic and organic hybrid silica gels were developed.
At present, the popular HPLC silica gel matrix filler for analysis on the market is mainly type B silica gel.
2.Classification of chromatography
Liquid chromatography separation methods can be roughly divided into normal phase chromatography, reversed phase chromatography, ion exchange chromatography, hydrophobic interaction chromatography, volume exclusion chromatography, affinity chromatography and chiral chromatography. In high performance liquid chromatography, normal phase chromatography and reversed phase chromatography are the most widely used, and reversed phase chromatography is the most important.
3.Principle of chromatographic separation
Separation principle of normal phase chromatography: According to the difference of the solute polarities, the solutes are separated on the adsorbent.
Separation principle of reversed-phase chromatography: The solutes are separated according to the differences in the partition coefficients between the mobile and stationary phases due to the different solute hydrophobicity.
4.Silicone matrix and polymer matrix
Two popular fillers are made of silicone matrix material and polymer matrix material with the following features.
4.1 Silicone matrix material
1. The most widely used matrix material
2. High mechanical strength and high specific surface area
3. Can use direct and extensive surface bonding reactions to meet the needs of normal phase, reversed phase, ion exchange, hydrophobic interaction chromatography and volume exclusion chromatography.
4. High efficient
5. Good reproducibility
6. PH scope of application ph2-8
7. Has a surface active and acidic silicon base that easily causes tailing of strongly alkaline substances
Influence of Silicone Shape
1. Amorphous silicone: Original HPLC packing; Particle size> 5μm; Poor bed stability; Small specific surface area; Cheap
2. Spherical silica gel: Modern HPLC packing; Small particle size (5μm, 3μm, <2μm); Reproducible; Good stability; High separation efficiency
Influence of silica purity
The purity of silica gel is most important for the separation of highly polar compounds. Previously low-purity silica gels are called A-type silica gels). A-type silica gels are prepared from metal silica gel salts, have a high metal content, and can be used to separate neutral compounds from non-ionic compounds.
Silica gel with high purity and weak acidity (called B-type silica gel). This silica gel is prepared by a metal-free process and contains only a trace amount of metal. It is recommended for the separation of ionic compounds and ionizable compounds, especially alkaline. Metal impurities cause asymmetric or tailing peaks in the separation of integrator and basic compounds.
4.2 Polymer matrix material
1. Crosslinked styrene, divinylbenzene and methacrylic acid esters
2. Good PH stability: PH1-13
3. Surface chemical treatment can be performed in a wide range to meet the needs of reversed-phase chromatography, ion-exchange chromatography, hydrophobic interaction chromatography, and size exclusion chromatography.
4. Better peak shape for strongly alkaline substances at moderate pH
5. The volume of the filler varies with the mobile phase, such as expansion or contraction.
6. Low separation efficiency compared to silica gel
7. Poor reproducibility
5.Comparison between HPLC and classic liquid chromatography
I. High pressure: When the liquid mobile phase passes through the chromatographic column of a HPLC, it will encounter great resistance. To quickly pass through a HPLC column, high pressure must be applied to the carrier liquid.
2. High speed: The analysis speed is fast, and the carrier liquid speed is much faster than traditional liquid chromatography. Generally speaking, the analysis of HPLC samples can be completed within 15-30 minutes, and some samples can be completed within 5 minutes, usually less than 1 hour.
3. High efficiency: The choice of stationary phase and mobile phase can achieve the best separation effect, which is several times more than that of industrial distillation column and gas chromatography.
4. High sensitivity: UV detector can reach 0.01ng.
5. Wide application: Use HPLC to analyze more than 70% of organic compounds, especially suitable for the separation and analysis of compounds with high boiling points, large molecules, strong polarity, and poor thermal stability.
6. The HPLC column can be reused: one column can separate different compounds.
7. Small sample size and easy recovery: The sample will not be damaged after passing through the HPLC column, and a single component can be collected or prepared.