1. What is a liquid chromatograph (HPLC)?
High-performance liquid chromatography (HPLC) is an efficient and accurate analytical instrument, which is widely used in chemistry, pharmacy, food, environment and other fields. It carries samples through liquid mobile phase, realizes separation by adsorption, distribution and ion exchange of different substances on stationary phase, and makes quantitative and qualitative analysis by combining with detector.
Compared with traditional column chromatography, HPLC has the characteristics of high separation efficiency, high sensitivity and rapid analysis, and has become an indispensable tool in modern laboratories.
2. The basic working principle of HPLC
The core principle of HPLC is based on the difference of partition coefficient between stationary phase and mobile phase, so as to realize separation. The basic process is as follows:
Sample injection system: inject the sample into the mobile phase (usually solvent).
Infusion pump: provide stable high pressure (usually 150-400 bar) to make the mobile phase pass through the chromatographic column at a constant flow rate.
Chromatographic column: It is filled with stationary phase (such as C18, silica gel, etc.), where different components in the sample are adsorbed, desorbed or distributed.
Detector: Detect the separated components, and the common types include UV-Vis, FLD, MS, etc.
Data processing system: record and analyze data such as peak area and retention time for quantitative and qualitative analysis.
3. Main types of HPLC
According to the different separation mechanisms, HPLC can be divided into the following main types:
Scope of application of type separation principle
Reversed-phase liquid chromatography (RP-HPLC) is a nonpolar stationary phase (such as C18), and the polar mobile phase is the most commonly used, which is suitable for most organic compounds.
Normal phase liquid chromatography (NP-HPLC) polar stationary phase (such as silica gel), nonpolar mobile phase is suitable for fat-soluble and less polar compounds.
Ion exchange chromatography (IEC) is suitable for ionic compounds (such as amino acids and nucleic acids) based on ion exchange.
Size exclusion chromatography (SEC/GPC) is suitable for the separation of macromolecules such as protein and polymers based on molecular size.
Hydrophilic interaction chromatography (HILIC) polar stationary phase is suitable for the analysis of polar compounds, sugars and polar metabolites.
4. Key components of HPLC
(1) infusion pump
Provide stable high pressure to ensure that the mobile phase passes through the chromatographic column at a constant flow rate. The accuracy of modern HPLC pump can reach 0.1%.
(2) chromatographic column
Material: stainless steel or PEEK (corrosion resistance)
Stationary phase: C18 (reversed phase), silica gel (normal phase), ion exchange resin, etc.
Particle size: usually 3-5 μm, which affects the separation efficiency.
(3) detector
UV-Vis detector: most commonly used, suitable for compounds with ultraviolet absorption.
Fluorescent detector (FLD): High sensitivity, suitable for specific fluorescent substances.
Mass Spectrometer (MS): provides molecular weight and structure information, and is suitable for complex samples.
(4) Sampling system
The automatic sampler can improve the analysis efficiency and reduce human error.
5. Application fields of HPLC
HPLC is widely used in many industries because of its high precision and sensitivity:
(1) Pharmaceutical industry
Drug purity detection
Metabolite analysis
Separation of traditional Chinese medicine components
(2) Food testing
Pesticide residue analysis
Testing of additives (such as preservatives and pigments)
Analysis of nutritional components of food
(3) Environmental monitoring
Water quality analysis (such as heavy metals and organic pollutants)
Detection of soil pollutants
(4) Biotechnology
Purification and analysis of protein
Nucleic acid isolation
(5) Chemical industry
Analysis of petrochemical products
Characterization of polymer materials
6. HPLC vs. Other chromatographic techniques
Comparison item HPLC gas chromatography (GC) Supercritical fluid chromatography (SFC)
Mobile phase liquid gas supercritical fluid (such as co)
Suitable for samples with high boiling point, macromolecules, volatile thermally unstable substances and moderately polar and thermally stable substances in low boiling point compounds.
High separation efficiency
Analysis time is long, short and medium.
7. Future development trend
Ultra-Qualcomm HPLC: shorten the analysis time and improve the detection speed.
Combined technologies, such as HPLC-MS and HPLC-NMR, can improve the qualitative ability.
Green HPLC: adopt a more environmentally friendly solvent system (such as aqueous mobile phase)
Intelligence and automation: AI optimization method development, reducing manual intervention
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