trait
1. high pressure: liquid chromatography takes liquid as the mobile phase (called carrier liquid), and the liquid flows through the chromatographic column, which is subject to great resistance. In order to pass through the chromatographic column quickly, high pressure must be applied to the carrier liquid. Generally, it can reach 150 ~ 350× 105 Pa.
2. High speed: the flow rate of mobile phase in the column is much faster than that of classical chromatography, which can generally reach 1 ~ 10 ml/min. The analysis time of high performance liquid chromatography is much less than that of classical liquid chromatography, generally less than1h.
3. High efficiency: Recently, many new stationary phases have been developed, which greatly improves the separation efficiency.
4. High sensitivity: High sensitivity detector has been widely used in high performance liquid chromatography, which further improves the sensitivity of analysis. For example, the sensitivity of fluorescence detector can reach10-11g. In addition, the sample volume is small, usually a few microliters.
5. Wide scope of application: Comparison between gas chromatography and high performance liquid chromatography: 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 high boiling point or poor thermal stability by gas chromatography due to technical conditions. High performance liquid chromatography only requires that the sample can be made into solution without gasification, so it is not limited by the volatility of the sample. In principle, high-performance liquid chromatography can be used to separate and analyze organic compounds with high boiling point, poor thermal stability and large relative molecular weight (above 400) (these substances account for almost 75% ~ 80% of the total organic compounds). According to statistics, about 20% of the known compounds can be analyzed by gas chromatography and 70 ~ 80% by liquid chromatography.
According to the properties of its stationary phase, HPLC can be divided into high performance gel chromatography, hydrophobic high performance liquid chromatography, reversed-phase high performance liquid chromatography, high performance ion exchange liquid chromatography, high performance affinity liquid chromatography and high performance focused liquid chromatography. The principle of separating or analyzing various compounds by different types of high performance liquid chromatography is basically similar to that of the corresponding ordinary liquid chromatography. The difference is that HPLC is sensitive, rapid, high resolution and good repeatability, and it must be carried out in a chromatograph.
Main types of high performance liquid chromatography and its separation principle
According to the different separation mechanisms, HPLC can be divided into the following main types:
1. Liquid-liquid partition chromatography and chemically bonded phase chromatography.
Both mobile phase and stationary phase are liquids. Mobile phase and stationary phase are immiscible with each other (polarity is different to avoid the loss of stationary liquid), and there is an obvious interface. When the sample enters the chromatographic column, the solute is distributed between the two phases. When equilibrium is reached, the following formula is observed:
Where CS is the solute concentration in the stationary phase; Cm-concentration of solute in mobile phase; Vs- volume of stationary phase; VM- volume of mobile phase. LLPC is similar to GPC, that is, the separation order depends on k, and the greater the k, the greater the retention value of the components. However, there are also differences. In generalized predictive control, the flow has little influence on K, while the LLPC flow has great influence on K. ..
A. Normal phase liquid chromatography: the polarity of mobile phase is less than that of stationary phase.
B. reversed-phase liquid chromatography: the polarity of the mobile phase is greater than that of the stationary phase.
C. Disadvantages of liquid-liquid partition chromatography: Although the polarity requirements of mobile phase and stationary phase are completely different, a small amount of stationary phase is still dissolved; The mechanical impact of mobile phase passing through chromatographic column will cause the loss of stationary liquid. The chemically bonded stationary phase (see below) developed in the late 1970s can overcome the above shortcomings. Now it is widely used (70~80%).
2. Liquid-solid chromatography
The mobile phase is liquid, and the stationary phase is adsorbent (such as silica gel and alumina). ). This separation is based on the different adsorption of substances. The mechanism is that when the sample enters the chromatographic column, solute molecules (X) and solvent molecules (S) compete to adsorb the active centers on the surface of the adsorbent (when no sample is injected, all the active centers of the adsorbent adsorb S), which can be expressed as:
Xm + nSa ====== Xa + nSm
Among them: Xm-solute molecules in mobile phase; Sa-solvent molecules in stationary phase; Solute molecules in Xa-stationary phase; Sm-solvent molecules in mobile phase.
When the adsorption competition reaction reaches equilibrium:
K=[Xa][Sm]/[Xm][Sa]
Where: k is the adsorption equilibrium constant. [Discussion: The greater the k, the greater the retention value. ]
3. Ion exchange chromatography
IEC uses ion exchanger as stationary phase. IEC is based on reversible exchange between ionizable ions on ion exchange resin and solute ions with the same charge in mobile phase, and these ions are separated according to their different affinities with the exchanger.
Taking the 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-]
[Discussion: Relationship between DX and Reserved Value]
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 (called counterions or counterions) with opposite charges to solute molecules in the mobile phase or stationary phase, so that they can combine with solute ions to form hydrophobic ion-pair compounds, thus controlling the retention behavior of solute ions. Its principle can be expressed by the following formula:
X+ water phase +Y- water phase = = x+Y- organic phase
Wherein: X+ organic ions (or cations) to be separated in water phase-mobile phase; Y- water phase-ion pairs with opposite charges in mobile phase (such as tetrabutyl ammonium hydroxide, cetyltrimethyl ammonium hydroxide, etc.). ); Ion pair compounds formed by x+y.
When equilibrium is reached:
KXY = [X+Y-] organic phase /[ X+] water phase [Y-] water phase.
According to the definition, the distribution coefficient is:
DX= [X+Y-] organic phase /[ X+] water phase = KXY [Y-] water phase.
[Discussion: Relationship between DX and Reserved Value]
Ion-pair chromatography (especially reversed-phase chromatography) solves the separation problem of mixtures that were difficult to separate in the past, such as acids, bases, ionic and nonionic mixtures, especially some biochemical samples such as nucleic acids, nucleosides, alkaloids and drugs.
5. Ion chromatography
Ion exchange resin is used as stationary phase, and electrolyte solution is used as mobile phase. In order to eliminate the interference of strong electrolyte background ions in the mobile phase, a suppression column was set up and a conductivity detector was used as a general detector. The reaction principle of sample components on separation column and inhibition column is the same as that of ion exchange chromatography.
Take anion exchange resin (R-OH) as stationary phase to separate anions (such as Br-) as an example. When the anion Br- to be detected enters the chromatographic column with the mobile phase (NaOH), the following exchange reaction occurs (elution reaction is the reverse process of exchange reaction):
Suppress the reaction on the chromatographic 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 very low conductivity through the inhibition column, which eliminates the influence of background conductivity; The anion Br- in the sample is converted into the corresponding acid H+Br-, which can be sensitively detected by conductivity method.
Ion chromatography is the best method for anion analysis in solution. It can also be used for cation analysis.
6. Spatial exclusion chromatography.
Spatial exclusion chromatography uses gel as stationary phase. It is similar to molecular sieve, but the aperture ratio molecular sieve of gel is much larger, usually between several nanometers and several hundred nanometers. The separation of solute between two phases is not through the difference of their interaction forces, but through the molecular size. The separation is only related to the pore size distribution of gel and the hydrodynamic volume or molecular size of solute. After the sample enters the chromatographic column, it flows with the mobile phase outside the gel and in the gap near the hole. In the sample, some molecules that are too large can't enter the gel pores and are excluded, so they directly pass through the chromatographic column and first appear on the chromatogram, and some very small molecules can enter all the gel pores and penetrate into the particles. These components have the largest retention value on the chromatographic column and finally appear on the chromatogram.
Gas chromatography is a kind of chromatography. There are two phases in chromatography, one is mobile phase and the other is stationary phase. If liquid is used as the mobile phase, it is called liquid chromatography, and gas is used as the mobile phase, it is called gas chromatography.
Because of the different stationary phases used, gas chromatography can be divided into two types: gas-solid chromatography with solid adsorbent as stationary phase and gas-liquid chromatography with carrier coated with stationary liquid as stationary phase.
According to the principle of chromatographic separation, gas chromatography can also be divided into adsorption chromatography and partition chromatography. In gas-solid chromatography, stationary phase is adsorbent, gas-solid chromatography belongs to adsorption chromatography, and gas-liquid chromatography belongs to partition chromatography.
Gas chromatography belongs to column chromatography according to the chromatographic operation form, and can be divided into general packed column and capillary column according to the thickness of the chromatographic column used. The stationary phase is usually packed in glass or metal tube with an inner diameter of 2 ~ 6mm. Capillary column can be divided into hollow capillary column and packed capillary column. Hollow capillary column is to directly coat stationary liquid on the inner wall of glass or metal capillary whose inner diameter is only 0. 1 ~ 0.5 mm, while packed capillary column is developed in recent years. It is made by filling some porous solid particles into a thick-walled glass tube, and then heating and stretching to make a capillary tube with an inner diameter of 0.25 ~ 0.5 mm generally.