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High performance liquid chromatography 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 efficiency focusing liquid according to the nature of its stationary phase. Types such as chromatography. The principle of separating or analyzing various compounds by different types of high performance liquid chromatography is basically similar to the principle of the corresponding ordinary liquid chromatography. The difference is that HPLC vials is sensitive, fast, high resolution, and repeatable, and must be performed in a chromatograph.
The main types of high performance liquid chromatography and their separation principle
According to the different separation mechanism, high performance liquid chromatography can be divided into the following main types:
1 . Liquid-liquid Partition Chromatography and Chemically Bonded Phase Chromatography 2ML HPLC vials manufacturer
Both the mobile phase and the stationary phase are liquids. The mobile phase and the stationary phase should be incompatible with each other (different polarity, avoiding the loss of fixative) and have a distinct interface. When the sample enters the column, the solute is partitioned between the two phases. When reaching equilibrium, it is subject to the following formula:
Where cs - the concentration of the solute in the stationary phase; cm - the concentration of the solute in the mobile phase; Vs - the volume of the stationary phase; Vm - the volume of the mobile phase. LLPC has similarities with GPC, that is, the order of separation depends on K, and the large component retention value of K is large; but there are also differences. In GPC, the flow has little effect on K, and LLPC flow has a greater influence on K.
a. Normal Phase liquid Chromatography: The polarity of the mobile phase is less than the polarity of the fixative.
b. Reverse Phase liquid Chromatography: The polarity of the mobile phase is greater than the polarity of the fixative.
c. Disadvantages of liquid-liquid partition chromatography: Although the polarities of the mobile phase and the stationary phase are completely different, the fixative still has a slight amount of dissolution in the mobile phase; the mechanical impact of the mobile phase as it passes through the column causes fixation. Fluid loss. The chemically bonded stationary phase developed in the late 1970s (see below) overcomes these shortcomings. It is now widely used (70~80%).
2 . Liquid-solid chromatography 2ML HPLC vials manufacturer
The mobile phase is a liquid and the stationary phase is an adsorbent (such as silica gel, alumina, etc.). This is based on the difference in the adsorption of the substance. The mechanism of action is: when the sample enters the column, the solute molecule (X) and the solvent molecule (S) compete for adsorption on the surface active center of the adsorbent (when not injected, all adsorbent active centers adsorb S) Can be expressed as follows:
Xm + nSa ====== Xa + nSm
Where: Xm - the solute molecule in the mobile phase; Sa - the solvent molecule in the stationary phase; Xa - the solute molecule in the stationary phase; Sm - the solvent molecule in the mobile phase.
When the adsorption competitive reaction reaches equilibrium:
K=[Xa][Sm]/[Xm][Sa] 2ML HPLC vials manufacturer
Where: K is the adsorption equilibrium constant. [Discussion: The larger K is, the larger the retention value. ]
3 . Ion-exchange Chromatography
The IEC uses an ion exchanger as the stationary phase. The IEC is based on the reversible exchange of ionizable ions on an ion exchange resin with solute ions of the same charge in the mobile phase, depending on which ions have different affinities for separating them.
Taking an anion exchanger as an example, the exchange process can be expressed as follows:
X-(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 values]
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 the addition of one (or more) ions (called counterions or counterions) opposite the charge of a solute molecule to a mobile or stationary phase, which combines with solute ions to form a hydrophobic ion-pair compound. To control the retention behavior of solute ions. The principle can be expressed by the following formula:
X+aqueous phase + Y-aqueous phase === X+Y-organic phase Chromatography vials China
Where: X+aqueous phase-organic ions (also cations) to be separated in the mobile phase; Y-aqueous phase- oppositely charged ion pairs in the mobile phase (eg tetrabutylammonium hydroxide, hexafluoride) Alkyltrimethylammonium, etc.); X+Y---formed ion-pair compound.
When reaching balance:
KXY = [X+Y-] organic phase / [ X + ] aqueous phase [Y-] aqueous phase
By definition, the partition coefficient is:
DX= [X+Y-]organic phase/[X+]aqueous phase = KXY [Y-]aqueous phase
[Discussion: Relationship between DX and reserved values]
Ion-pair chromatography (especially reversed phase) solves the problem of separation of previously difficult-to-separate mixtures, such as acids, bases and ions, non-ionic mixtures, especially biochemical samples such as nucleic acids, nucleosides, alkaloids, and pharmaceuticals. Separation.
5 . Ion Chromatography
The ion exchange resin is used as a stationary phase, and the electrolyte solution is a mobile phase. The conductivity detector is used as a universal detector. In order to eliminate the interference of the strong electrolyte background ions in the mobile phase to the conductivity detector, a suppression column is arranged. The reaction principle of the sample components on the separation column and the suppression column is the same as that of the ion exchange Chromatography vials China.
An anion exchange resin (R-OH) is used as a stationary phase, and an anion such as Br- is isolated as an example. When the anion Br- to be tested enters the column with the mobile phase (NaOH), the following exchange reaction occurs (the elution reaction is the reverse of the exchange reaction):
Suppress the reaction that occurs on the column:
R-H+ + Na+OH- === R-Na+ + H2O
R-H+ + Na+Br- === R-Na+ + H+Br-
It can be seen that the inhibition of the column converts the eluent into water with a small conductance value, eliminating the influence of the background conductance; the sample anion Br- is converted into the corresponding acid H+Br-, which can be sensitive by conductance. Detection.
Ion chromatography is a good method for anion analysis in solution. It can also be used for cation analysis.
6 . Space exclusion chromatography (Steric Exclusion Chromatography vials China)
Size exclusion chromatography uses a gel as the stationary phase. It is similar to the role of molecular sieves, but the pore size of the gel is much larger than that of molecular sieves, typically several nanometers to hundreds of nanometers. Solutes are separated between the two phases not by their interaction forces, but by molecular size. 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 column, it flows with the mobile phase outside the gel gap and along the pores. Some of the too large molecules in the sample can not enter the pores and are excluded. Therefore, they pass directly through the column, first appearing on the chromatogram, and some small molecules can enter all the pores and penetrate into the particles. These components The retention value on the column is the largest and appears last on the chromatogram.
This is the end of the introduction of High performance liquid chromatography (HPLC)classification. I hope it can help you.
