High Pressure Liquid Chromatography Special Point
High Pressure
Liquid Chromatography The liquid is a mobile phase (referred to as a carrier liquid), the liquid flows through the column, and is affected by resistance Larger, in order to quickly pass the column, the carrier is applied to the carrier fluid. Generally up to 150 ~ 350 × 105Pa.
High Speed
The flow rate in the column is much more faster than the classic chromatography, generally up to 1 to 10 ml / min. The analysis time required for high performance liquid chromatography is much less than the classic liquid chromatography, generally less than 1 h.
High-efficiency
has recently studied many new fixed phase, which greatly improves the separation efficiency.
High sensitivity
High-performance liquid chromatography has widely used high sensitivity detector, further improves the sensitivity of the analysis. Such as fluorescent detector sensitivity can reach 10-11G. In addition, the sample is small, generally several microliters.
Compare the range
Gas chromatography and high performance liquid chromatography: Although gas chromatography has good separation ability, high sensitivity, fast analysis speed, convenient operation, etc. However, it is difficult to apply gas chromatography to substances that are limited by technical conditions, substances that are too high, and poor thermostability are difficult to analyze gas chromatography. High performance liquid chromatography, as long as the sample can be made into solution without gasification, it is not limited by the sample. For the high boiling point, the thermal stability is poor, the relative molecular weight (greater than 400 or more) organic matter (75% to 80% of the total number of substances) can be used in principle, isolated, analyzed. Separation, analysis. According to statistics, in a known compound, it can be used for 20% by gas chromatography, and from 70 to 80% of the liquid chromatographic analysis can be used.
Depending on the separation mechanism, high-performance liquid chromatography can be divided into several main types
liquid-liquid distribution chromatography and chemical bond chromatography
< P> The flow phase and the fixed phase are liquid. There is an obvious interface between the mobile phase and the fixed phase (polarity, avoiding the loss of fixed fluid). There is a significant interface. When the sample enters the column, the solute is allocated between two phases.a. Normal phase fluid - liquid distribution chromatography: The polarity of the mobile phase is smaller than the polarity of the fixed liquid.
b. inverted liquid - liquid distribution chromatography: The polarity of the mobile phase is greater than the polarity of the fixed liquid.
Liquid-liquid distribution chromatography disadvantage: Although the polarity requirements of the mobile phase are completely different, the fixing liquid still has a trace dissolution in the flow phase; the mechanical impact of the mobile phase through the column Force, it will cause a fixed fluid loss. The chemical bonding fixed phase developed in the end of the 1970s (see after seeing), can overcome the above disadvantages, the application is wide (70 ~ 80%).
Liquid-solid color spectrum
Flow phase is liquid, fixed to the adsorbent (such as silica gel, alumina, etc.). This is separated depending on the substance adsorption effect. Its action mechanism is: When the sample enters the column, solute molecules (X) and solvent molecules (s) compete for adsorbent surface activity center (not injection, all adsorbent activity center adsorbed S) It can be represented as follows:
xa + nsa ====== xa + nsm
: the solute molecule in XM-mobile phase;
SA - Solvent molecule in the fixed phase;
Xa - fixed phase solute molecule;
SM - solvent molecules in the flow phase.
When adsorption competition reactivity is balanced:
k = [xa] [SM] / [xm] [SA]
in: K is adsorbed Equilibrium constant. [Discussion: The larger K, the greater the reserved value. ]
Ion exchange chromatography
IEC is an ion exchanger as a fixed phase. IEC is an ion-free ion based on ion exchange resin to have a solute ion having the same charge in the mobile phase to perform reversibly exchange, which is separated by different affinity in exchange agents.
as an anion exchange agent as an example, its exchange process can be represented as follows:
x- (Solvent) + (resin-R4N + Cl -) === (resin-R4N + X -) + Cl- (in solvent)
When exchange of balance:
kx = [- R4N + X -] [cl -] / [- R4N + CL -] [x -]
The distribution coefficient is:
dx = [- R4N + X -] / [x -] = kx [-r4n + cl -] / [CL -]
[Discussion: DX and retention value]
Any substance that is ionization in the solvent can generally be separated by ion exchange chromatography.
ion pair chromatography
ion pair chromatography is an ion (or more) to flow with ions (referred to as ions or anti-ions) of the solute molecule charge. In the phase or fixed phase, it is made to form a hydrophobic ion to the compound with solute ions, thereby controlling the retention behavior of solute ions. The principle can be represented by the following formula:
x + water phase + Y-water phase === x + y - Organic phase
: x + water phase - mobile phase Separated organic ions (or cation);
Y-aqueous phase - flow phase with opposite charge (such as hydroxide, hydroxide, hydroxide trimethylammonium) );
x + y --- formed ion pair compound.
When the balance is loaded:
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: DX and retention value Relationship]
ionic chromatography (especially inverse phase), solving the separation problem of the mixture that is difficult to separate, such as acid, base, and ion, nonion mixture, especially some biochemical samples such as Isolation of nucleic acids, nucleosides, alkaloids, and drugs.
Ion chromatography
The ion exchange resin is a fixed phase, and the electrolyte solution is a mobile phase. In order to eliminate the interference of the conductance detector in the electrolyte background ions in the flow phase, the electrical conductive detector is set. The reactive principle of the sample component is the same as the ion exchange chromatography on the separation column and the inhibitory column.
is fixed as a fixed phase in anion exchange resin (R-OH), and the anion (such as BR-) is taken as an example. When the anion BR is to be tested with the flow phase (NaOH), the following exchange reaction occurs (the elution reaction is the reverse process of the exchange reaction):
inhibit the reaction in the column:
R-H ++ Na + OH - === R-Na ++ H2O
R-H ++ Na + Br - === R-NA ++ H + BR-
It can be seen by suppressing the eluate into water with small conductance value, the effect of this electrical conductivity is eliminated; the sample anion BR- is converted into corresponding acid H + Br -, can be sensitive to the use of electrical conductivity.
Ion chromatography is the best method for anion analysis in the solution. It is also available for cationic analysis.
Spatial Exhaust Chromatography
Spatial Exhaust Chromatography As a fixed phase, it is similar to the molecular sieve, but the pore diameter of the gel is large. Many, generally numbers of nanometers to hundreds of nanometers. The solute is not separated from the two phases of the interaction force, but separated by the molecular size. The separation is only related to the pore size distribution of the gel and the fluid volume or molecular size of the solute. After the sample enters the column, the outer gap of the gel and the hole acupoint can be flowed as the flow phase. Some of the molecules in the sample cannot be entered into the colloles, so it is directly passed through the column, first appearing on the chromatogram, some small molecules can enter all of the colloles and penetrate into the granules, these components The retention value on the column is the largest, and the last appears on the chromogram.