High performance liquid chromatography

Chromatography with a liquid mobile phase is called liquid chromatography. The method of transporting the mobile phase by atmospheric pressure is classical liquid chromatography, which has low column performance and long separation period. High performance liquid chromatography (HPLC) is a chromatographic method developed on the basis of classical liquid chromatography.

Compared with the classic liquid chromatography, high-performance liquid chromatography has the following main advantages: 1 Application of a very fine particle size (generally less than 10μm), regular uniform phase, low mass transfer impedance, high column efficiency, high separation efficiency 2 The mobile phase is delivered by a high-pressure infusion pump, and the flow rate is fast. The analysis of a typical sample takes several minutes. The analysis of a complex sample can be completed in several tens of minutes. 3 A highly sensitive detector is widely used, which greatly improves the sensitivity. At present, many different stationary phases have been developed, and there are many different separation modes, so that the application range of high-performance liquid chromatography is continuously expanding. The following describes the relevant knowledge, new methods and techniques of HPLC, and its application in drug analysis.

First, the classification of high-performance liquid chromatography by the separation mechanism can be divided into the following categories: (A) adsorption chromatography (adsorption chromatography) to the adsorbent as the stationary phase chromatography method known as adsorption chromatography. The most used adsorption chromatographic stationary phase is silica gel, and the mobile phase generally uses a mixed solvent of one or more organic solvents. In adsorption chromatography, different components are separated due to the difference in the adsorptive force of the stationary phase. The more polar the component, the stronger the adsorption of the stationary phase, the longer the retention time. The greater the polarity of the mobile phase and the stronger the elution force, the shorter the retention time of the components.

(II) Liquid-liquid chromatography Liquid-liquid partition chromatography consists of a stationary phase and a mobile phase, which are incompatible with each other. When separated, the components dissolve in two phases. The distribution coefficient (K) is separated and different. Currently widely used chemically bonded stationary phases are made by bonding the functional groups of the fixative to the carrier. Chromatographic methods using chemically bonded stationary phases (abbreviated as bonded phase chromatography) can be explained by the principle of partition chromatography. Bonded phase chromatography occupies an extremely important position in HPLC and is the most widely used chromatographic method. According to the polarity of the stationary phase and the mobile phase, partition chromatography can be divided into normal phase chromatography and reversed phase chromatography.

1. Normal phase chromatography Chromatography in which the polarity of the stationary phase is greater than the polarity of the mobile phase is known as normal phase partition chromatography, abbreviated as normal phase chromatography. The polar chemically bonded stationary phase, such as cyano-bonded silica gel and amino-bonded silica gel, is a commonly used stationary phase for normal phase chromatography. The mobile phase for normal phase chromatography is generally a less polar organic solvent. In normal phase chromatography, the less polar component elutes first because the K value is smaller and the more polar component elutes. Normal phase chromatography is used for the separation of polar and moderately polar molecular species dissolved in organic solvents.

2. Reversed phase chromatography (reversed phase chromatography) Partition chromatography in which the polarity of the mobile phase is greater than the polarity of the stationary phase is known as reversed phase partition chromatography, abbreviated as reversed phase chromatography. Reversed-phase chromatography uses a non-polar stationary phase. The most commonly used non-polar stationary phase is octadecylsilane-bonded silica gel, and octyl-silane-bonded silica gel. The mobile phase is usually a mixed solvent of water and methanol, acetonitrile or tetrahydrofuran. In reversed-phase chromatography, very large fractions flow out of the column first because the K value is smaller, and the less polar fractions flow out. The proportion of organic solvents in the mobile phase increases, the mobile phase decreases in polarity, and the elution power increases. Reversed phase chromatography is currently the most widely used high performance liquid chromatography.

(III) Ion Exchange Chromatography (ionexchange chromatography) Ion pair exchange chromatography is a chromatographic method in which the ion exchanger is used as the stationary phase, and the components are separated due to the different affinity of the ion exchanger. The column packing contains polar ionizable groups, such as carboxylic acid, sulfonic acid, or quaternary ammonium ions. At a suitable pH, these groups will dissociate and attract oppositely charged species. Ionic substances can be separated because they react with the column packing.

The different components of the sample are separated by the difference in ion exchange equilibrium constants. The mobile phase of ion exchange chromatography is generally a buffer solution with a certain pH, and sometimes a small amount of organic solvents such as ethanol, tetrahydrofuran, acetonitrile, etc. are added to increase the solubility of the components in the mobile phase. The pH of the mobile phase influences the exchange capacity of the ion exchanger. For weakly acidic or slightly alkaline separated components, the pH of the mobile phase will also affect its ionization conditions. The pH of the mobile phase must be such that the components to be separated are in a dissociated state before they can be separated. Ion exchange chromatography is used to separate components that are in a dissociated state under the conditions of the assay, such as acidic or basic compounds. Reversed-phase ion-pair chromatography is widely used in drug analysis, such as alkaloids, sulfonamides, This method can be used for the analysis of certain antibiotics and vitamins.

(4) Space exclusion chromatography (chromatography) Space exclusion chromatography is also called gel chromatography. Its stationary phase is a porous material with a range of pore sizes, ie a gel. The separated components are separated due to the difference in the size of the molecular space. When components are carried into the column by the mobile phase, large molecules cannot enter the pores on the surface of the stationary phase, and the retention time is the shortest as the mobile phase passes directly through the column. Smaller molecules can enter the cavity, leaving longer pathways in the column and longer retention times. The smaller the size of the molecule, the more holes that can be accessed, the longer the path taken, and the longer the retention time. Therefore, in the gel chromatography, in a certain range, the retention time of molecules with different volumes is different, so as to achieve the purpose of separation. Gel chromatography is mainly used to separate high molecular compounds such as proteins and polysaccharides. Because of the relationship between molecular weight and molecular volume, gel chromatography can also be used to determine the molecular weight of a component.

(5) Affinity chromatography (lighperformanceaffinitychromatography) affinity chromatography is a chromatographic method that uses or simulates the specific interaction between biomolecules to separate and analyze certain substances from biological samples. The specific interactions between biomolecules include antigens and antibodies, enzymes and inhibitors, hormones and drug and cell receptors, vitamins and binding proteins, and specific affinity between genes and nucleic acids. The stationary phase of affinity chromatography is made by linking the ligand to a suitable carrier, and separates the various substances in the sample and the affinity of the ligand. When the sample solution passes through the chromatographic column, the substance X to be separated and the ligand L form an XL complex, which is bound to the stationary phase. Other substances flow out of the column directly due to lack of affinity with the ligand, and are combined with a suitable mobile phase. The substance to be separated is eluted, and if a certain concentration of acetic acid or ammonia solution is used as the mobile phase, the affinity of the substance to be separated and the ligand is reduced, the complex is dissociated, and the purified substance is eluted.

HPAC can be used to separate, purify, and measure biologically active substances. It can also be used to study intermolecular interactions and mechanisms in organisms.

(6) Chiral chromatography Many organic drugs have an asymmetric carbon atom in the structure, which is also called a chiral carbon atom. A drug with a chiral carbon atom has a spin. A pair of enantiomers with different stereostructures often have different efficacy and side effects. For example, the antihypertensive drug а-methyldopa is S-(-); if it is chloramphenicol (containing two chiral carbon atoms), only the D-(-) isomer is effective. The L-() isomer is completely ineffective. The two enantiomers of thalidomide (reaction stopped) have similar potency for sedation in mice, but only levorotatory isomers have embryotoxic and teratogenic effects. Therefore, the separation of enantiomers has important implications in terms of drug preparation and quality control. The physicochemical properties of the enantiomers under the common conditions are the same, so separation of the enantiomers needs to be performed under chiral conditions. The chromatographic method for the separation of enantiomers is called chiral chromatography. Chiral chromatography is divided into indirect method and direct method. The indirect method is to react the enantiomers with certain chiral derivatization reagents to convert them from enantiomers into diastereomers, and then use their differences in physicochemical properties to separate them with general chromatographic conditions. The direct method does not require a derivatization reaction, and the separation is directly performed using a chiral chromatographic column or a chiral mobile phase. This method is widely used. The direct method is mainly described below.

1. The Chiral Stationary Phase (CSP) The enantiomer before the resolution of the chiral drug is usually present as a mirror image, ie, in the form of a racemate. It cannot be resolved by conventional methods of analysis and preparation. An asymmetric (ie, chiral) environment needs to be introduced to form an enantiomer (sample), chiral agent (eg, stationary phase), and chiral origin that are to be resolved. Diastereomeric complexes. In order to form such a molecular complex, there is a mutual interaction between the molecules to maintain the spatial orientation of the molecule. Dalgliesh believes that there must be at least three forces, one of which is stereoselective, which can be either attractive or repulsive. This is the "three-point interaction" theory. If one of the enantiomers happens to be paired with these three interaction points, the interaction is stronger and the retention time is longer. However, due to the different spatial configurations, the other enantiomers cannot be completely matched, the interaction is relatively weak, and the retention time is short, so that they can be separated. The role of the stationary phase can be hydrogen bonding, dipole-dipole interaction, π-π interaction, electrostatic interaction, hydrophobic interaction, or steric interaction.

L.1 Common chiral stationary phases are the following.

(1) Pirkle-type chiral stationary phase The Pickle-type chiral stationary phase was developed by the American scholar Pirkle and mainly consists of π-alkaline (electron-fighting) chiral stationary phase and π-acidic (electron-absorbing) chiral Stationary Phase. In the process of separation, π-π charge transfer interaction occurs between the compound and the stationary phase. Such stationary phase is a charge transfer type chiral stationary phase.

(2) Protein Chiral Because phased proteins are macromolecules composed of amino acids of chiral subgroups, protein chiral stationary phases are made by binding proteins to silica gel via amino acids. The commonly used chiral stationary phase of proteins is the chiral stationary phase of bovine serum albumin (RSA) and human blood a1-acid glycoprotein (AGP). Chiral AGP, Resolvosil, and EnantioPac are commercially available.

Protein chiral stationary phases have a wide range of applications and good results, but the stationary phase has a smaller column capacity. The commonly used elution system is a phosphate buffer solution (PH 4-7), an ionic strength of 0-500 mmol, and an organic modifier of not more than 5%. When the enantiomers of acidic and basic pours are resolved using a chiral stationary phase of the protein, a small amount of ion pair reagents such as N,N-dimethyloctylamine, tert-butylamine hydrobromide and caprylic acid can be added to the mobile phase. Get the ideal separation result.

(3) Polysaccharides Chiral stationary phase Polysaccharides The most widely used chiral stationary phase is cyclodextrin. Cyclodextrin (CD) is a type of cyclic oligosaccharides, a chiral polymer material. According to the number of glucose units in the molecule, cyclodextrins can be divided into three classes: alpha, beta, and gamma. They consist of 6, 7, and 8 D-glucopyranoses, respectively. CD is attached to the surface of silica gel through silane chains, respectively. It constitutes a cyclodextrin chiral stationary phase. The cyclodextrin molecule is tapered into a barrel shape. The diameter of the lumen is determined by the number of glucose molecules that make up the cyclodextrin. For example, the commonly used β-cyclodextrin consists of seven glucose molecules with a lumen diameter of 0.8 nm.

When separation is performed with a cyclodextrin chiral stationary phase, first the separated components are required to enter caves to form clathrates, and the retention time depends on whether the components can enter the cave and its degree of closeness. Cyclodextrins There are also multiple chiral centers on the ring that can selectively interact with the enantiomers, resulting in separation of the enantiomers with different retention. For example, metal organic complexes such as ferrocene, amino acids, and alkaloids have been successfully separated using a β-cyclodextrin chiral stationary phase.

In addition to cyclodextrins, the chiral stationary phases of polysaccharides can also be made from cellulose, amylose, such as cellulose triacetate chiral stationary phase, cellulose tribenzoic acid acetic chiral stationary phase and cellulose amino Formate chiral fixation equal.

(4) Crown ether type chiral stationary phase crown ethers are similar to cyclodextrins and are chiral oligosaccharides. They are cyclic compounds containing ether bonds and have a crown-like structure. The outer layer is lipophilic. In the ethylene group, the inner layer of the ring is an electron-rich heteroatom, such as oxygen, nitrogen, sulfur, and the like. The commonly used 18-crown-6 derivatives are bonded to a polystyrene backbone or silica gel to form a crown ether chiral stationary phase. When the enantiomers were separated on a crown ether chiral stationary phase, the different enantiomers were separated due to the different stability of the host-guest complex formed with the crown ether ring cavity. Introduction of the double-naphthyl group on the crown ether can form a "chiral wall" and increase the stereoselectivity of the stationary phase. The protonated amino compounds, especially the amino acid enantiomers, can be well separated on the crown ether chiral stationary phase.

In addition to the above chiral stationary phase, there are also chiral stationary phase and ligand exchange chiral stationary phase.

2. Chiral mobile phase (chiral mobile phase, CMP) resolution Chiral mobile phase resolution is the addition of chiral reagents to the mobile phase, the use of chiral reagents and enantiomers with different stability constants or conjugates The difference in distribution on the stationary phase is separated. In recent years, the chiral mobile phase splitting method has been widely used in the resolution of drug enantiomers. The biggest advantage of this method is that it can use ordinary non-chiral stationary phase without derivatizing the sample. The chiral additive itself can flow out and can also be replaced. At the same time, the additive has a wide variable range and good stability. , And the price is cheaper.

(1) Ligand exchange type chiral additive The ligand exchange type chiral additive consists of a chiral ligand and a salt containing a divalent metal ion. Chiral ligands are mostly optically active amino acids and their derivatives. They chelate with divalent metal ions and are distributed in the mobile phase. When they meet the enantiomers to be separated, they form complexes. The separation between the mobile phase and the stationary phase is achieved. The mechanism of separation is generally believed to be that the chelate formed by the ligand and the metal ion is adsorbed on the stationary phase to form a dynamic chiral stationary phase. Can also be seen as chiral ligands, metal ions and enantiomers formed complexes with different stability constants to produce separation. Using a 5 μm particle size C18 phase, aspartame as CMPA, complexed with copper sulphate to determine the concentration of D- and L-pipecolinic acid in urine of patients with hyperkalemia.

(2) Cyclodextrin Additives Cyclodextrins have a certain degree of solubility in water. Cyclodextrins are used as chiral additives and added to the mobile phase. The separation mechanism is the same as that of the cyclodextrin chiral stationary phase, but the retention behavior is exactly the opposite. The stronger the stability of the inclusion complex formed between the enantiomer and the cyclodextrin, the faster the column exits the mobile phase and the shorter the retention time.

(3) Chiral organic compounds that dissociate from the additive can interact with reagents that contain complementary charges to generate electrically neutral ion pairs. For the separation of charged enantiomers, chiral ion pair reagents can be added to the mobile phase. The enantiomers and ion pair reagents form an electrically neutral ion pair. The ion pairs are distributed between the stationary phase and the mobile phase. The coefficients are different and separated. The commonly used chiral ion pair reagents are (+) 10-camphorsulfonic acid, quinine and the like.

Second, high performance liquid chromatography High performance liquid chromatography mainly by the infusion system, the sample system, the column system, the examination system, the data recording processing system and so on.

(I) The mobile phase of the infusion pump HPLC is delivered by high-pressure pump. There are different types of infusion pumps. According to the nature of infusion, they can be divided into constant-pressure pump and constant-current pump. At present, the plunger reciprocating pump is used more often, and the plunger reciprocating pump is a constant flow pump. The mobile phase can be delivered at a set flow rate. The flow rate is not affected by the column resistance, can be kept constant, and can be easily cleaned and replaced. Since the plunger-type reciprocating pump is used to close the liquid by the reciprocating movement of the plunger, the pulsation of the infusion liquid is large. At present, double pump compensation methods are often used to reduce pulsation. Double pump compensation is to use two pumps at the same time to work, and the two pumps can be connected in series or in parallel to pull, alternately send liquid and compensate each other to achieve the purpose of reducing pulsation.

(b) Sampler The sampler is the device that sends the sample to the column. It is generally required that the sample injection device be of good sealing property, small dead volume, good repeatability, ensure the center sample injection, and has little influence on the pressure and flow rate of the chromatographic system when injecting. There are injection valve and automatic sample feeding device. In addition to using the automatic sample introduction device, six-way injection valves are used for manual injection. In the working state, the instrument system is in a high pressure state. With the help of the six-way injection valve, continuous flow injection under normal pressure is realized.

Six-way injection valve can be divided into stator and rotor two parts, a total of six ports, it is called six-way injection valve. The rotor is adjusted by a wrench and can be placed in the "load" and "injection" positions, respectively. When the wrench is set to the "Injection" position, the flow path of the injection valve changes, the mobile phase passes through the sample tube, and the injected sample is brought to the column for analysis. Six-way injection valve has the advantages of convenient use, accurate sample injection and so on. The sample loop has 10μl, 20μl, 50μl and other different volumes, which can be matched. The sample loop can be used not only for storing liquids but also for measuring the volume of the sample solution. After the sample loop is filled, the injection volume is the volume of the sample loop. This injection mode is called “full cycle injection” or “quantitative loop injection”. The accuracy of the injection with the loop is good. This method should be used when using the external standard method.

(C) Column chromatography is the core of chromatographic separation. In order to ensure high column efficiency and long service life, commercial columns manufactured by specialized chemical plants are generally used. Column tubes are mostly made of stainless steel and contain a fixed phase. Chromatography length is generally 10 ~ 30cm, internal diameter of 2 ~ 5mm.

Chemically bonded phases are widely used stationary phases, among which octadecylsilylsilica gel (ODS) is the most widely used, ODS is a non-polar chemically compatible phase, and octane silane bonds are also included. Silica gel, etc. Non-polar chemical phase is used for reversed-phase chromatography. The moderately polar chemically bonded phases have the same phenyl chemical linkages, the cyano chemically bonded phase and the amino chemically bonded phase are commonly used polar chemically bonded phases, and the polar chemically bonded phases are generally used for normal phase chromatography. The most used stationary phase in adsorption chromatography is silica gel. Regardless of their own column or purchase of commercial columns, the use of indicators can include the column pressure under a certain experimental conditions, theoretical plate height and theoretical plate number, symmetry factor, capacity factor and the repeatability of the selectivity factor.

(d) The detector sample is separated by a chromatographic column before entering the detector for detection. According to the scope of application, the detectors can be divided into two types: general type and exclusive type. The exclusive type detector can only detect certain properties of certain components. The UV detector (UVD) and the fluorescence detector (ED) belong to this category. In the class, they only respond to components that have ultraviolet absorption or fluorescence emission; general-purpose detectors detect the properties that typical substances have, refractive index detectors (RID), evaporative light scattering detectors (ELSD), and the like.