Principle of High Performance Liquid Chromatography

    High performance liquid chromatography is based on classical chromatography and cites the theory of gas chromatography. Technically, the mobile phase is changed to high-pressure delivery (the maximum delivery pressure can reach 4.9´107Pa); the chromatographic column is filled with small-particle fillers using a special method, so that the column efficiency is much higher than that of classical liquid chromatography (the number of plates per meter can reach tens of thousands or hundreds of thousands); at the same time, a high-sensitivity detector is connected after the column to continuously detect the effluent.

　　Features

　　1. High pressure: Liquid chromatography uses liquid as the mobile phase (called carrier liquid). When the liquid flows through the chromatographic column, it encounters great resistance. In order to pass through the chromatographic column quickly, high pressure must be applied to the carrier liquid, which can generally reach 150 to 350×105Pa.

　　2. High speed: The flow rate of the mobile phase in the column is much faster than that of classical chromatography, generally up to 1-10 ml/min. The analysis time required for HPLC is much shorter than that of classical HPLC, generally less than 1 hour.

　　3. High efficiency: Many new stationary phases have been developed recently, which greatly improve the separation efficiency.

　　4. High sensitivity: HPLC has widely adopted high-sensitivity detectors, which further improves the sensitivity of analysis. For example, the sensitivity of fluorescence detectors can reach 10-11g. In addition, the sample volume is small, generally a few microliters.

　　5. Wide range of application: Comparison between gas chromatography and HPLC: Although gas chromatography has the advantages of good separation ability, high sensitivity, fast analysis speed, and convenient operation, it is difficult to analyze substances with too high boiling points or poor thermal stability by gas chromatography due to technical conditions. High performance liquid chromatography only requires that the sample can be made into a solution without gasification, so it is not limited by the volatility of the sample. In principle, organic substances with high boiling points, poor thermal stability, and large relative molecular weight (greater than 400) (these substances account for almost 75% to 80% of the total number of organic substances) can be separated and analyzed by high performance liquid chromatography. According to statistics, among the known compounds, about 20% can be analyzed by gas chromatography, while about 70 to 80% can be analyzed by liquid chromatography.

　　According to the properties of its stationary phase, HPLC can be divided into high performance gel chromatography, hydrophobic HPLC, reversed phase HPLC, high performance ion exchange HPLC, high performance affinity HPLC and high performance focusing HPLC. The principle of using different types of HPLC to separate or analyze various compounds is basically similar to the principle of corresponding ordinary liquid chromatography. The difference is that HPLC is sensitive, fast, has high resolution, good repeatability, and must be performed in a chromatograph .

    The main types of HPLC and their separation principles

　　Based on the separation mechanism, HPLC can be divided into the following main types:

　　1. Liquid-liquid partition chromatography and chemically bonded phase chromatography. Both the mobile phase and the stationary phase are liquids. The mobile phase and the stationary phase should be immiscible (different polarity to avoid loss of the stationary phase) and have a distinct interface. When the sample enters the chromatographic column, the solute is distributed between the two phases. When equilibrium is reached, it obeys the following formula: Where, cs—concentration of solute in the stationary phase; cm—concentration of solute in the mobile phase; Vs—volume of the stationary phase; Vm—volume of the mobile phase. LLPC and GPC are similar in that the order of separation depends on K, and the component with a large K has a large retention value; but there are also differences. In GPC, the mobile phase has little effect on K, while the mobile phase in LLPC has a greater effect on K.

　　a. Normal Phase Liquid-Liquid Partition Chromatography: The polarity of the mobile phase is less than that of the stationary phase.

　　b. Reverse Phase liquid-liquid partition chromatography (Reverse Phase liquidChromatography): The polarity of the mobile phase is greater than the polarity of the stationary liquid.

　　c. Disadvantages of liquid-liquid distribution chromatography: Although the polarity requirements of the mobile phase and the stationary phase are completely different, the stationary liquid still dissolves in the mobile phase in trace amounts; the mechanical impact of the mobile phase passing through the chromatographic column will cause the stationary liquid to be lost. The chemically bonded stationary phase (see below) developed in the late 1970s can overcome the above disadvantages. It is now widely used (70~80%).

　　2. Liquid-solid chromatography

　　The mobile phase is liquid, and the stationary phase is adsorbent (such as silica gel, alumina, etc.). This is based on the different adsorption effects of substances to separate. Its mechanism of action is: when the sample enters the chromatographic column, the solute molecules (X) and solvent molecules (S) compete for adsorption on the active centers on the adsorbent surface (when no sample is injected, all active centers of the adsorbent adsorb S), which can be expressed as follows: Xm + nSa ====== Xa + nSm

　　In the formula: Xm--solute molecules in the mobile phase; Sa--solvent molecules in the stationary phase; Xa--solute molecules in the stationary phase; Sm--solvent molecules in the mobile phase.

　　When the adsorption competition reaction reaches equilibrium: K = [Xa] [Sm] / [Xm] [Sa] where K is the adsorption equilibrium constant.

    3. Ion-exchange Chromatography

　　IEC uses ion exchangers as stationary phases. IEC is based on the reversible exchange of ionizable ions on ion exchange resins with solute ions of the same charge in the mobile phase, and separation of these ions based on their different affinities with the exchangers.

　　Taking anion exchanger as an example, the exchange process can be expressed as follows:

　　X-(in solvent) + (resin-R4N+Cl-) === (resin-R4N+ X-) + Cl-(in solvent)

　　When the exchange reaches equilibrium:

　　KX=［-R4N+ X-］［Cl-］/［-R4N+Cl-］［X-］

　　The distribution coefficient is:

　　DX=［-R4N+ X-］/［X-］= KX ［-R4N+Cl-］/［Cl-］

　　Any substance that can be ionized in a solvent can usually be separated by ion exchange chromatography.

　　4. Ion Pair Chromatography

　　Ion pair chromatography is to add one (or more) ions with opposite charges to the solute molecules (called counter ions or counter ions) to the mobile phase or stationary phase, so that they combine with the solute ions to form hydrophobic ion pair compounds, thereby controlling the retention behavior of the solute ions. The principle can be expressed by the following formula: X+aqueous phase + Y-aqueous phase === X+Y-organic phase

　　In the formula: X+aqueous phase--the organic ions to be separated in the mobile phase (can also be cations); Y-aqueous phase--the ion pairs with opposite charges in the mobile phase (such as tetrabutylammonium hydroxide, hexadecyltrimethylammonium hydroxide, etc.); X+Y---the ion pair compounds formed. [page]

　　When equilibrium is reached: KXY = [X+Y-] organic phase/[X+] aqueous phase

　　[Y-] Water phase By definition, the distribution coefficient is:

　　DX = [X+Y-] organic phase / [X+] aqueous phase = KXY [Y-] aqueous phase

    Ion pair chromatography (especially reverse phase) solves the problem of separation of mixtures that were previously difficult to separate, such as acids, bases, and ions, non-ionic mixtures, especially the separation of some biochemical samples such as nucleic acids, nucleosides, alkaloids and drugs.

　　5. Ion Chromatography

　　Ion exchange resin is used as the stationary phase and electrolyte solution is used as the mobile phase. The conductivity detector is used as the universal detector. In order to eliminate the interference of strong electrolyte background ions in the mobile phase on the conductivity detector, an inhibition column is set. The reaction principle of the sample components on the separation column and the inhibition column is the same as that of ion exchange chromatography.

　　Take anion exchange resin (R-OH) as the stationary phase to separate anions (such as Br-) as an example. When the anion Br- to be measured enters the chromatographic column with the mobile phase (NaOH), the following exchange reaction occurs (the elution reaction is the reverse process of the exchange reaction):

　　Inhibit reactions occurring on the column:

　　R-H+ + Na+OH- === R-Na+ + H2O

　　R-H+ + Na+Br- === R-Na+ + H+Br-

　　It can be seen that the eluent is converted into water with a very low conductivity value through the suppression column, eliminating the influence of background conductivity; the sample anion Br- is converted into the corresponding acid H+Br-, which can be sensitively detected by the conductivity method.

　　Ion chromatography is the best method for the analysis of anions in solution. It can also be used for the analysis of cations.

　　6. Steric Exclusion Chromatography

　　Size exclusion chromatography uses gel as the stationary phase. It is similar to the function of molecular sieves, but the pore size of gel is much larger than that of molecular sieves, generally ranging from several nanometers to hundreds of nanometers. The solutes are not separated by the difference in their interaction forces between the two phases, but by the molecular size. The separation is only related to the pore size distribution of the gel and the hydrodynamic volume or molecular size of the solute. After the sample enters the chromatographic column, it flows through the gaps outside the gel and the holes with the mobile phase. Some molecules in the sample that are too large cannot enter the gel pores and are excluded, so they pass directly through the column and appear first on the chromatogram. Some very small molecules can enter all the gel pores and penetrate into the particles. These components have the largest retention values ​​on the column and appear last on the chromatogram.

The HPLC　　mainly consists of the sample injection system, the liquid infusion system, the separation system, the detection system and the data processing system. The following will describe their respective components and characteristics.

    1. Sample injection system

　　Generally, the injection operation is completed by using a diaphragm injection injector or a high-pressure injection room, and the injection volume is constant. This is beneficial to improving the repeatability of the analysis sample.

　　2. Infusion system

　　The system consists of three parts: a high-pressure pump, a mobile phase reservoir, and a gradient meter. The general pressure of the high-pressure pump is 1.47-4.4X107Pa, and the flow rate is adjustable and stable. When the high-pressure mobile phase passes through the chromatography column, it can reduce the diffusion effect of the sample in the column and speed up its movement in the column, which is beneficial to improving resolution, recovering samples, and maintaining the biological activity of samples. The mobile phase reservoir and gradient meter can change the mobile phase with the properties of the stationary phase and the sample, including changing the polarity, ionic strength, pH value of the eluent, or using competitive inhibitors or denaturants. This allows various substances (even if there is only one group difference or isomers) to be effectively separated.

　　3. Separation system

　　The system includes chromatographic columns, connecting tubes and thermostats. The chromatographic columns are generally 10 to 50 cm long (when two columns are needed, a connecting tube can be added between them), with an inner diameter of 2 to 5 mm. They are made of "high-quality stainless steel or thick-walled glass tubes or titanium alloys, etc., and contain a stationary phase (composed of a matrix and a stationary liquid) with a particle size of 5 to 10 μm. The matrix in the stationary phase is composed of a resin or silica gel with high mechanical strength. They are inert (such as the silica gel surface silica gel has been basically removed), porous (pore size can reach 1000?) and large specific surface area. In addition, their surfaces are mechanically coated (the same as the preparation of stationary phases in gas chromatography), or chemically coupled with various genes (such as phosphate, quaternary amino, hydroxymethyl, phenyl, amino or alkyl carbon chains of various lengths) or ligands of organic compounds.

　　Therefore, this type of fixed phase has good selectivity for substances with different structures. For example, after coupling pea lectin (PSA) on the porous silica surface, a glycoprotein in fibroblasts can be separated.

　　In addition, the stationary phase matrix particles are small, the column bed is easy to achieve a uniform and dense state, and the eddy diffusion effect is easy to reduce. The matrix particle size is small, the micropores are shallow, and the sample mass transfer in the micropore area is short. These are beneficial to narrowing the band width and improving the resolution. According to the column efficiency theory, the smaller the matrix particle size, the larger the theoretical number of tower plates N. This also further proves that the smaller the matrix particle size, the higher the resolution.

    Furthermore, the thermostat of HPLC can adjust the temperature from room temperature to 60°C, which can increase the efficiency of the chromatography column by improving the mass transfer rate and shortening the analysis time. [page]

　　4. Detection system

　　The three commonly used detectors in HPLC are UV detector, differential refractometer and fluorescence detector.

　　(1) UV detector

　　This detector is suitable for the detection of samples that have the ability to absorb ultraviolet light (or visible light). Its characteristics are: wide application (such as proteins, nucleic acids, amino acids, nucleotides, peptides, hormones, etc.); high sensitivity (the detection limit is 10-10g/ml); wide linear range; insensitive to changes in temperature and flow rate; and can detect samples eluted with gradient solutions.

　　(2) Differential refractometer

　　Any sample component with a different refractive index from the mobile phase can be detected using a differential refractometer. Currently, this detection system is mostly used for the detection of sugar compounds. This system is highly versatile and easy to operate, but has low sensitivity (the detection limit is 10-7g/ml). Changes in the mobile phase will cause changes in the refractive index. Therefore, it is not suitable for trace analysis or the detection of gradient elution samples.

　　(3) Fluorescence detector

　　For any fluorescent substance, under certain conditions, the fluorescence intensity of the emitted light is proportional to the concentration of the substance. Therefore, this detector is only suitable for the determination of fluorescent organic compounds (such as polycyclic aromatic hydrocarbons, amino acids, amines, vitamins and certain proteins, etc.), and its sensitivity is very high (the detection limit is 10-12 ~ 10-14g/ml), and it can be used for trace analysis and gradient elution work detection.

　　(4) Data processing system

　　The system can collect, store, display, print and process test data, so that the separation, preparation or identification of samples can be carried out correctly.