1. Brief development of liquid chromatography theory The separation principle of chromatography is: when the components dissolved in the mobile phase (mobile phase) pass through the stationary phase, due to the interaction with the stationary phase (adsorption, distribution, ion attraction, exclusion, affinity) The size and strength are different, and the residence time in the stationary phase is different, so that it flows out from the stationary phase successively. Also called chromatographic method and chromatography. Chromatography was first discovered by Russian botanist Tswett when he studied the separation of plant pigments with calcium carbonate in 1906. Chromatography was named after it. Later on this basis, paper chromatography, thin layer chromatography, gas chromatography, and liquid chromatography were developed. The beginning of liquid chromatography is to use a large-diameter glass column to transport the mobile phase with liquid level difference at room temperature and normal pressure. It is called classic liquid chromatography. This method has low column efficiency and long time (often several hours) ). High performance liquid chromatography (High performance Liquid Chromatography, HPLC) is based on the classic liquid chromatography method, and it was developed rapidly in the late 1960s by introducing gas chromatography theory. The difference between it and classic liquid chromatography is that the filler particles are small and uniform. The small particles have high column efficiency, but they will cause high resistance, and the mobile phase needs to be transported with high pressure, so it is also called high pressure liquid chromatography (High Pressure Liquid Chromatography, HPLC) . It is also called High Speed ​​Liquid Chromatography (HSLP) due to its fast analysis speed. Also known as modern liquid chromatography. Second, the characteristics and advantages of HPLC HPLC has the following characteristics: High pressure—pressure can reach 150 ~ 300Kg / cm2. The pressure drop per meter of the chromatographic column is above 75 Kg / cm2. High speed—flow rate is 0.1 ~ 10.0 ml / min. Efficient-up to 5000 trays per meter. Up to 100 components can be separated simultaneously in one column. High sensitivity—The sensitivity of the ultraviolet detector can reach 0.01ng. At the same time consume less sample. Compared with classic liquid chromatography, HPLC has the following advantages: Fast speed-usually analyze a sample within 15 ~ 30 min, some samples can be completed within 5 min. High resolution—choose stationary phase and mobile phase to achieve the best separation effect. High sensitivity-UV detector can reach 0.01ng, fluorescence and electrochemical detector can reach 0.1pg. The column can be used repeatedly—a single column can be used to separate different compounds. The sample volume is small and easy to recover-the sample is not destroyed after passing through the chromatography column, and can be collected or prepared for single component. 3. Classification by chromatography According to the physical state of the two phases, it can be divided into: gas chromatography (GC) and liquid chromatography (LC). Gas chromatography is suitable for separating volatile compounds. According to different stationary phases, GC can be divided into gas-solid chromatography (GSC) and gas-liquid chromatography (GLC), among which GLC is the most widely used. Liquid chromatography is suitable for the separation of low-volatility or non-volatile substances with poor thermal stability. LC can also be divided into liquid-solid chromatography (LSC) and liquid-liquid chromatography (LLC). In addition, there is supercritical fluid chromatography (SFC), which uses supercritical fluid (a phase between gas and liquid) as the mobile phase (commonly used CO2), because of its large diffusion coefficient, it can quickly reach equilibrium Therefore, the analysis time is short, especially suitable for the resolution of chiral compounds. According to the principle, it is divided into adsorption chromatography (AC), partition chromatography (DC), ion exchange chromatography (IEC), exclusion chromatography (EC, also known as molecular sieve, gel filtration (GFC), gel permeation chromatography ( GPC) and affinity chromatography. (In addition to electrophoresis.) According to the operation form, it can be divided into paper chromatography (PC), thin layer chromatography (TLC), and column chromatography. 4. Principle of chromatographic separation HPLC is divided into liquid-solid adsorption chromatography, liquid-liquid partition chromatography (normal phase and reverse phase), ion exchange chromatography, ion pair chromatography and molecular exclusion chromatography according to different separation mechanisms. 1. Liquid-solid chromatography uses a solid adsorbent, and the principle of separating the separated components on the chromatographic column is based on the difference in the fixed relative component adsorption force. The separation process is an equilibrium process of adsorption-desorption. The commonly used adsorbent is silica gel or alumina with a particle size of 5 ~ 10μm. It is suitable for separating components with a molecular weight of 200 ~ 1000, and most of them are used for non-ionic compounds. Ionic compounds are prone to tailing. Commonly used to separate isomers. 2. Liquid-liquid chromatography uses a stationary phase formed by coating a specific liquid substance on the surface of a support or chemically bonding to the surface of the support. The separation principle is based on the difference in the solubility of the separated components in the mobile phase and the stationary phase. The separation process is a distribution balance process. Coated stationary phases have good inertness; the mobile phase must be saturated with the stationary phase in advance to reduce the loss of stationary phase from the surface of the support; changes in temperature and the difference between different batches of mobile phase often cause changes in the column; in addition, they exist in the mobile phase The stationary phase also complicates the separation and collection of samples. Because it is difficult to avoid the loss of fixing liquid, the coated stationary phase has been rarely used. Currently, chemically bonded stationary phases are commonly used, such as C18, C8, amino columns, cyano columns, and phenyl columns. Liquid-liquid chromatography can be divided into normal phase chromatography (NPC) and reverse phase chromatography (RPC) according to the different polarities of the stationary phase and mobile phase. Normal phase chromatography uses a polar stationary phase (such as polyethylene glycol, amino and nitrile bonded phase); the mobile phase is a relatively non-polar hydrophobic solvent (alkanes such as n-hexane, cyclohexane), often Add ethanol, isopropanol, tetrahydrofuran, chloroform, etc. to adjust the retention time of the components. Commonly used to separate medium polar and strong polar compounds (such as phenols, amines, carbonyls and amino acids). Reverse phase chromatography generally uses a non-polar stationary phase (such as C18, C8); the mobile phase is water or buffer, often adding methanol, acetonitrile, isopropanol, acetone, tetrahydrofuran and other water-miscible organic solvents to adjust the retention time . It is suitable for separating non-polar and weak polar compounds. RPC is the most widely used in modern liquid chromatography. According to statistics, it accounts for about 80% of the entire HPLC application. With the rapid development of column packing, the application range of reversed-phase chromatography is gradually expanding, and it has been applied to the analysis of some inorganic samples or samples that are easily dissociated. In order to control the dissociation of the sample during the analysis, a buffer solution is commonly used to control the pH of the mobile phase. However, it should be noted that the pH value used for C18 and C8 is usually 2.5 ~ 7.5 (2 ~ 8). Too high a pH value will dissolve the silica gel, and too low a pH value will make the bonded alkyl group fall off. It has been reported that new commodity columns can be operated in the pH range of 1.5 to 10. Comparison between normal phase chromatography and reverse phase chromatography Normal phase chromatography reverse phase chromatography Stationary phase high to medium to low Low polarity of mobile phase ～ medium-medium ～ high The elution order of the components is smaller, the polarity is washed out first, and the polarity is washed out first. 3. Ion exchange chromatography Buffers are often used as mobile phases in ion exchange chromatography. The retention time of the separated component in the ion exchange column is not only related to the effect of the component ion and the ion exchange group on the resin, but also affected by the pH value and ionic strength of the mobile phase. The pH value can change the degree of dissociation of the compound, which in turn affects its interaction with the stationary phase. If the salt concentration of the mobile phase is large, the ionic strength is high, which is not conducive to the dissociation of the sample, which causes the sample to flow out faster. Ion exchange chromatography is mainly used to analyze organic acids, amino acids, peptides and nucleic acids. 4. Ion pair chromatography The ion-pair reagents commonly used for the analysis of alkaline substances are alkyl sulfonates, such as sodium pentane sulfonate, sodium octane sulfonate, etc. In addition, perchloric acid and trifluoroacetic acid can also form a strong ion pair with a variety of alkaline samples. The analysis of acidic substances commonly used tetrabutyl quaternary ammonium salt, such as tetrabutyl ammonium bromide, tetrabutyl ammonium phosphate. Ion pair chromatography is commonly used on ODS columns (ie C18). The mobile phase is methanol-water or acetonitrile-water, and 3 ~ 10 mmol / L of ion pair reagents are added to the water for separation within a certain pH range. The measured component retention time is related to ion pair properties, concentration, mobile phase composition and its pH value, and ionic strength. 5. Exclusion chromatography Travel Bag,Overnight Bag,Travel Backpack,Best Travel Backpack Ningbo Fineweather International Trade Co., Ltd. , https://www.nbfwsbag.com
As can be seen from the above table, when the polarity is medium, there is no obvious boundary between normal phase chromatography and reverse phase chromatography (such as amino bonded stationary phase).
The stationary phase is an ion exchange resin, which is usually a polymer skeleton formed by crosslinking styrene and diethylene, and a carboxyl group, a sulfonic acid group (called cation exchange resin) or a quaternary amino group (anion exchange resin) is attached to the aromatic ring at the end of the surface. The principle of separating the separated components on the chromatographic column is that the ionizable ions on the resin are reversibly exchanged with the ions of the same charge in the mobile phase and the ions of the measured component, and each ion and ion exchange group has different charge attraction And separation.
Also known as even ion chromatography, it is a branch of liquid-liquid chromatography. It forms a neutral ion-pair compound based on the measured component ion and the ion-pair reagent ion, and the solubility in the non-polar stationary phase increases, thereby improving the separation effect. It is mainly used to analyze acid and alkali substances with high ionic strength.
The stationary phase is a porous filler with a certain pore size, and the mobile phase is a solvent that can dissolve the sample. Small molecular weight compounds can enter the pores and have a long residence time; large molecular weight compounds cannot enter the pores and flow out directly with the mobile phase. It uses the difference in the molecular sieve's ability to exclude components with different molecular weights to complete the separation. Commonly used to separate polymer compounds, such as tissue extracts, peptides, proteins, nucleic acids, etc.