High performance liquid chromatography and its application in pharmaceutical analysis

Abstract: Chromatography with liquid as mobile phase is called liquid chromatography. The method of transporting the mobile phase at atmospheric pressure is classic liquid chromatography, which has low column efficiency and long separation cycle. High performance liquid chromatography (highperformanceliquidchromatography, HPLC for short) is a chromatographic method developed on the basis of classic liquid chromatography.

Compared with classic liquid chromatography, high-performance liquid chromatography has the following main advantages: ① The application of very fine particles (generally less than 10µm), regular uniform stationary phase, low mass transfer resistance, high column efficiency, high separation efficiency ; ② The high-pressure infusion pump is used to transport the mobile phase, and the flow rate is fast. The analysis of general samples takes several minutes, and the analysis of complex samples can be completed within tens of minutes; ③ The highly sensitive detector is widely used, which greatly improves the sensitivity. At present, a variety of different stationary phases have been developed, and there are many different separation modes, which make the application range of high performance liquid chromatography continue to expand. The following introduces the relevant knowledge of high-performance liquid chromatography, new methods and techniques, and applications in pharmaceutical analysis.

1. Classification

High performance liquid chromatography can be divided into the following categories according to the different separation mechanisms:

(1) Adsorption chromatography (adsorption chromatography) The chromatographic method with adsorbent as the stationary phase is called adsorption chromatography. The most used adsorption chromatography 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 different adsorption capacity of the stationary phase. The greater the polarity of the components and 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.

(2) Liquid-liquid chromatography (liquid-liquid chromatography) The stationary phase and mobile phase of liquid-liquid chromatography are two solvents that are incompatible with each other. During separation, the components dissolve into the two phases. The partition coefficient (K) is separated. Currently widely used chemically bonded stationary phases are made by bonding the functional groups of a fixing solution to a carrier. Chromatography using chemically bonded stationary phases (bonded phase chromatography for short) 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 chromatography method. According to the different polarities of stationary phase and mobile phase, partition chromatography can be divided into normal phase chromatography and reverse phase chromatography.

1. Normal phase chromatography (normalphasechromatography) The stationary phase polarity is greater than the polarity of the mobile phase partition chromatography is called normal phase partition chromatography, referred to as normal phase chromatography. Polar chemically bonded stationary phases such as cyano-bonded silica gel and amino-bonded silica gel are commonly used stationary phases for normal-phase chromatography, and the mobile phase of normal-phase chromatography is generally an organic solvent with a lower polarity. In normal phase chromatography, the less polar components flow out first due to the smaller K value, and the more polar components flow out later. Normal phase chromatography is used for the separation of polar and moderately polar molecular substances dissolved in organic solvents.

2. Reverse phase chromatography (reversed phase chromatography) The mobile phase polarity is greater than that of the stationary phase. Partition chromatography is called reversed phase chromatography, or simply reversed phase chromatography. Reverse phase chromatography uses a non-polar stationary phase. The most commonly used non-polar stationary phase is octadecylsilane-bonded silica gel and octylsilane-bonded silica gel. The mobile phase is usually a mixed solvent of water and methanol, acetonitrile or tetrahydrofuran. In reversed-phase chromatography, the largest component flows out of the column first due to the smaller K value, and then the component with lower polarity. The proportion of organic solvent in the mobile phase increases, the polarity of the mobile phase decreases, and the elution force increases. Reverse phase chromatography is currently the most widely used high-performance liquid chromatography.

(3) Ion exchange chromatography (ion exchange chromatography) Ion exchange chromatography is a chromatography method in which an ion exchanger is the stationary phase, and the components are separated due to the different affinity with the ion exchanger. The column packing contains polar ionizable groups, such as carboxylic acid, sulfonic acid or quaternary ammonium ions. At an appropriate pH, these groups will dissociate and attract oppositely charged substances. Since the ionic substance can react with the column packing, it can be separated.

The different components in the sample are separated due to the different ion exchange equilibrium constants. The mobile phase of ion exchange chromatography is generally a buffer solution with a certain pH value, 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 value of the mobile phase affects the exchange capacity of the ion exchanger. For the separated components with weak acid or basicity, the PH value of the mobile phase will also affect its ionization status. The PH value of the mobile phase must keep the components to be separated in a dissociated state before they can be separated. Ion exchange chromatography is used to separate components that are dissociated under measurement conditions, such as compounds with acidic or basic properties. Reverse-phase ion pair chromatography is widely used in pharmaceutical analysis, such as alkaloids, sulfonamides, This method can be used for the analysis of certain antibiotics and vitamins.

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

(5) Affinity chromatography (lighperformanceaffinitychromatography) Affinity chromatography is a chromatographic method that uses or simulates the specific action between biological molecules to separate and analyze some special substances from biological samples. The specific functions between biomolecules include specific affinity between antigen and antibody, enzyme and inhibitor, hormone and drug and cell receptor, vitamin and binding protein, gene and nucleic acid, etc. The stationary phase of affinity chromatography is made by attaching ligands to a suitable carrier, and uses the different affinity of various substances in the sample to ligands to achieve separation. When the sample solution passes through the chromatographic column, the substance X to be separated forms a XL complex with the ligand L, which is bound to the stationary phase, and other substances directly flow out of the column due to the lack of affinity for the ligand, and will be combined with a suitable mobile phase. For the elution of the substance to be separated, for example, a certain concentration of acetic acid or ammonia solution is used as the mobile phase to reduce the affinity of the substance to be separated and the ligand, dissociate the complex, and thereby elute the purified substance.

HPAC can be used for the separation, purification and determination of biologically active substances. It can also be used to study the interactions and mechanisms between molecules in organisms.

(6) Chiral chromatography (chiralchromatography) Many organic drugs have asymmetric carbon atoms in their structures, also known as chiral carbon atoms. Drugs with chiral carbon atoms have optical rotation. A pair of enantiomers with different stereo configurations often have different drug effects and toxic side effects. For example, the antihypertensive drug а-methyldopa is the S-(-) body; for example, chloramphenicol (containing two chiral carbon atoms), only the D-(-) isomer is effective. L- () Isomers are completely invalid. The two enantiomers of thalidomide (response stop) have similar potency for sedation in mice, but only the L-isomer has embryotoxic and teratogenic effects. Therefore, the separation of enantiomers is of great significance in the preparation and quality control of drugs. The physical and chemical properties of the enantiomers are the same under ordinary conditions, so the separation of the enantiomers needs to be carried out under chiral conditions. The chromatographic method for separating 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 transform them from enantiomers to diastereomers, and then use their differences in physical and chemical properties to separate them by general chromatographic conditions. The direct method does not need to be derivatized. It is directly separated by chiral chromatographic column or chiral mobile phase. It has many applications. The following mainly introduces the direct method.

1. Chiral Stationary Phase (CSP) The enantiomer before chiral drug resolution usually exists as a mirror image, that is, as a racemate. It cannot be resolved by conventional analysis and preparation methods. It is necessary to introduce an asymmetric (ie, chiral) environment to form an enantiomer (sample), chiral substance (such as stationary phase) and chiral source to be resolved Complexes of diastereomeric molecules. In order to form such a molecular complex, there must be a mutual force between the molecules to maintain the spatial positioning of the molecules. Dalgliesh believes that there must be at least three forces, one of which must be stereoselective, either attractive or repulsive. This is the "three-point interaction" theory. If one of the enantiomers is paired with these three points of action, the interaction is strong and the retention time is long. However, due to different spatial configurations, the other enantiomers cannot be completely paired, the effect 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 spatial interaction.

l.1 The commonly used chiral stationary phases are as follows.

(1) Pirkle type chiral stationary phase

The Pickle-type chiral stationary phase was developed by American scholar Pirkle. It mainly includes π-basic (electron-repelling group) chiral stationary phase and π-acid (electron-withdrawing group) chiral stationary phase. During the separation process, π-π charge transfer interactions occur between the compound and the stationary phase. Such stationary phases are charge-transferring chiral stationary phases.

(2) Protein chirality phasing

Proteins are macromolecular substances composed of amino acids with chiral subunits. Protein-based chiral stationary phases are made by bonding proteins to silica gel through amino acids. Commonly used protein-based chiral stationary phases are bovine serum albumin (RSA) and human blood a1-acid glycoprotein (AGP) chiral stationary phases, and commercial products include ChiralAGP, Resolvosil, EnantioPac, etc.

Protein chiral stationary phases have a wide range of applications and good results, but the column capacity of such stationary phases is relatively small. Commonly used elution system is phosphate buffer solution (PH4 ~ 7), ionic strength is 0 ~ 500mmol, organic modifier should not exceed 5%. When the enantiomers of acidic and basic pourables are resolved with a protein chiral stationary phase, a small amount of ion pair reagents, such as N, N-dimethyloctylamine, tert-butylamine hydrobromide and octanoic acid, can be added to the mobile phase, To obtain the ideal separation results.

(3) Polysaccharide chiral stationary phase

The most widely used polysaccharide chiral stationary phase is cyclodextrin. Cyclodextrin (CD) is a kind of cyclic oligosaccharide, which is a chiral polymer substance. According to the number of glucose units in the molecule, cyclodextrins can be divided into three categories: α, β, and γ. They are composed of 6, 7, and 8 D-glucopyranosides, respectively, and connect CD to the surface of silica gel through silane chains. This constitutes the cyclodextrin chiral stationary phase. The cyclodextrin molecules are in the shape of cone barrels, and the diameter of the inner cavity is determined by the number of glucoses that make up the cyclodextrin. For example, the commonly used β-cyclodextrin consists of 7 glucose molecules, and the inner cavity diameter is 0.8 nm.

When separating with a cyclodextrin chiral stationary phase, the first component is required to enter the human cave to form an inclusion compound, and the retention time depends on whether the component can enter the human cave and how tight it is. There are also multiple chiral centers on the ring, which can be selectively reacted with the enantiomers, which leads to the separation of the enantiomers. Such as β-cyclodextrin chiral stationary phase has successfully separated metallocene complexes such as ferrocene, amino acids and alkaloids.

In addition to cyclodextrin, the chiral stationary phase of polysaccharides can also be made of cellulose and amylose, such as cellulose triacetate chiral stationary phase, cellulose tribenzoate chiral stationary phase and cellulose amino group The formate chirality is fixed equal.

(4) Crown ether (Grownether) chiral stationary phase

Crown ethers are similar to cyclodextrins. They are chiral oligosaccharides. They are cyclic compounds containing ether bonds. They have a crown-like structure. The outer layer is a lipophilic ethylene group. The inner layer of the ring is electron-rich. Heteroatoms, such as oxygen, nitrogen, sulfur, etc. The derivative of 18-crown-6 is usually used, which is bonded to the polystyrene vinyl skeleton or silica gel to form a crown ether chiral stationary phase. When the enantiomers are separated with a crown ether chiral stationary phase, the different enantiomers are separated due to the different stability of the host-guest complex formed with the crown ether ring cavity. Introducing bis-naphthyl groups on crown ethers, bis-naphthyl groups can form "chiral walls" and increase the stereoselectivity of the stationary phase. Protonated amino compounds, especially amino acid enantiomers, can be well separated on the crown ether chiral stationary phase.

In addition to the above chiral stationary phases, there are also simulated enzyme chiral stationary phases and coordination exchange chiral stationary phases are equal.

2. Chiral mobile phase (chiralmobilephase, CMP) resolution method

Chiral mobile phase resolution method is to add chiral reagents to the mobile phase, and use chiral reagents to separate the enantiomers with different stability constants or the distribution of the conjugates on the stationary phase. In recent years, the chiral mobile phase resolution method has been widely used in the resolution of drug enantiomers. The biggest advantage of this method is that it can use ordinary achiral stationary phase, without derivatizing the sample, the chiral additive itself can be flowed out or replaced, and the variable range of the additive is wide and the stability is good , And the price is cheap.

(1) Ligand exchange chiral additives

The ligand-exchange chiral additive is composed of chiral ligands and salts containing divalent metal ions. 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 encounter the enantiomer to be separated, they form a complex, and then The mobile phase and the stationary phase are distributed to achieve separation. The mechanism of separation is generally considered to be that the chelate formed by ligand and metal ion is adsorbed on the stationary phase, forming a dynamic chiral stationary phase to play a role. It can also be considered that the complex formed by chiral ligands, metal ions and enantiomers has different stability constants to cause separation. Using 5μm particle size C18 stationary phase, aspartame as CMPA, complexed with copper sulfate, the concentration of D- and L-2-piperidine acid in urine of patients with hemolysin was determined.

(2) Cyclodextrin additives

Cyclodextrin has a certain solubility in water. Cyclodextrin is used as a chiral additive and added to the mobile phase. The separation mechanism is the same as the cyclodextrin chiral stationary phase, but the retention behavior is just the opposite. The greater the stability of the inclusion compound formed by the enantiomer and cyclodextrin, the faster the mobile phase exits the column and the shorter the retention time.

(3) Chirality is dependent on additives

Dissociated organic compounds can interact with reagents containing complementary charges to generate electrically neutral ion pairs. When separating charged enantiomers, chiral ion pair reagents can be added to the mobile phase. The enantiomers and ion pair reagents form electrically neutral ion pairs, and the ion pairs are distributed between the stationary phase and the mobile phase The coefficients are different and separated. Commonly used chiral ion pair reagents include (+) 10-camphorsulfonic acid and quinine.

2. High-performance liquid chromatograph High-performance liquid chromatograph is mainly composed of infusion system, sampling system, chromatographic column system, detection system, data record processing system, etc.

(1) Infusion pump

The mobile phase of high-performance liquid chromatography is transported by high-pressure pumps. There are different types of infusion pumps, which can be divided into constant pressure pumps and constant flow pumps according to the nature of the infusion. At present, plunger type reciprocating pumps are mostly used. Plunger type reciprocating pumps are constant flow pumps, which can deliver mobile phase at a set flow rate. The flow rate is not affected by the resistance of the column and can be kept constant. It is easy to clean and replace. Because the plunger reciprocating pump shuts off the liquid by the reciprocating motion of the plunger, the pulsation of the infusion is greater. At present, the method of double pump compensation is mostly used to reduce the pulsatility. Double pump compensation is to use two pumps to work at the same time. The two pumps can be connected in series or parallel to alternately send liquid and compensate each other to reduce the pulsation.

(2) Sample injector

A sampler is a device that sends a sample to a chromatographic column. It is generally required that the sampling device has good sealing, small dead volume, and good repeatability, to ensure that the center sample is injected, and the pressure and flow rate of the chromatography system are small when the sample is injected. There are two kinds of sampling valve and automatic sampling device. In addition to the use of automatic sampling devices, manual injection uses a six-way injection valve. In the working state, the instrument system is in a high-pressure state, with the help of a six-way sampling valve, non-stop sampling under normal pressure is achieved.

The six-way sampling valve can be divided into two parts, the stator and the rotor, with a total of 6 ports, so it is called the six-way sampling valve. The rotor is adjusted by a wrench and can be in the two positions of "loading" and "injecting" respectively. When the wrench is turned to the "injection" position, the flow path of the injection valve is changed, the mobile phase passes through the sample tube, and the injected sample is brought into the chromatography column for analysis. The six-way sampling valve has the advantages of convenient use and accurate injection volume. Sample loops are available in different volumes such as 10µl, 20µl, 50µl, etc. The sample loop can be used not only for storing liquid, but also for measuring the volume of sample solution. After the sample loop is filled, the sample is injected, and the injection volume is the volume of the sample loop. This injection method is called "full loop injection" or "quantitative loop injection". The precision of sampling with the loop is good, and this operation method should be used when the external standard method is used.

(3) Chromatography column Chromatography column is the core of chromatographic separation. In order to ensure the high efficiency and long service life of chromatographic columns, commercial columns produced by specialized chemical plants are generally used. The chromatographic column tubes are mostly made of stainless steel with a certain stationary phase inside. Chromatography is generally 10 to 30 cm long, with an inner diameter of 2 to 5 mm.

The chemically bonded phase is a widely used stationary phase. Among them, octadecylsilane-bonded silica gel (octadecylsilylsilicagel, ODS) is the most widely used. ODS is a non-polar chemically bonded phase, in addition to octylsilane bonded Non-polar chemical complex phases such as silica gel are used for reversed-phase chromatography. The medium-polarity chemical bonding phases have phenyl chemical bonding phases equal, cyano chemical bonding phases and amino chemical bonding phases are commonly used polar chemical bonding phases, and polar chemical bonding phases are generally used in normal phase chromatography. Silica gel is the most commonly used stationary phase for adsorption chromatography. Regardless of the chromatographic column packed by yourself or the commercial column purchased, the performance indicators include column pressure under certain experimental conditions, theoretical plate height and theoretical plate number, symmetry factor, capacity factor, and selectivity factor repeatability.

(4) After the detector sample is separated by the chromatographic column, it enters the side detector for detection. According to its scope of application, the detector can be divided into two categories: general-purpose type and exclusive type. The exclusive type detector can only detect a certain property of certain components. Ultraviolet detector (UVD) and fluorescence detector (ED) belong to this category. They only respond to components with ultraviolet absorption or fluorescence emission; universal detectors detect the properties of common substances, differential refractive index detectors (RID), evaporative light scattering detectors (ELSD), etc.

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