Various chromatographic elution rules, concise and to the point

HPLC HPLC is divided into normal-phase and reversed-phase chromatography according to the relative polarity of the stationary phase and the mobile phase.

In the normal phase, the polarity of the mobile phase is smaller than that of the stationary phase, the polar components are retained, and the peak sequence is non-polar - > weak polar - > polarity.

The polarity of the reversed-phase chromatography mobile phase is greater than that of the stationary phase, the stationary phase is commonly used C8-C18, the non-polar components flow out last, the peak order: polarity-> weak polarity-> non-polarity, reversed-phase chromatography because it has no strong effect with the components, the components will not stay in the polluted column for a long time, and the reversed-phase chromatography uses water as the bottom solvent, and other weakly polar and non-polar solvents such as acetonitrile and other organic substances are mixed to change the polarity of the mobile phase to achieve the purpose of separation, and the ultraviolet absorption and containment wavelength of water is low, which will not interfere with the detection, and the water is easy to obtain cheaply.

There is also a point to note: the separation of impurities in a large number of substances should be let out of the small content first, if the large amount of components flow out first, it will drag the tail and cause impure impurity separation.

GC gas chromatography has nothing to say, the principle is similar, the main thing is the retention time, peak width, and correction factor.

Capillary electrophoresis, the general capillary wall is modified to be negatively charged, the electroosmotic flow rate is greater than the electrophoresis rate, positive and negative ions, neutral molecules move to the cathode, and the detection order is: positive ions-> neutral molecules-> negative ions.

What is said upstairs is not accurate, positive and negative phase chromatography.

In the liquid phase, there are only two types involved, which are liquid-solid and liquid-liquid.

High performance liquid chromatography.

There are four main types, as follows.

1。Liquid-solid adsorption chromatography. The stationary phase is a solid adsorbent, which is separated according to the difference in the adsorption of substances in the stationary phase. The stronger the adsorption, the k value.

The larger it is, the longer it will be retained.

2。Liquid-liquid partition chromatography. As the name suggests, it applies a stationary solution to the support body as a stationary phase, and its separation principle is similar to that of liquid-liquid extraction.

and thus obey the law of distribution. Solubility in fixative solution.

Large, large k-value, long retention time.

For reversed-phase chromatography, the less polar the species, the greater the polarity of the mobile phase and the longer the retention time. The less polar the substance, the less polarity the mobile phase and the shorter the retention time. For highly polar substances, the polarity of the mobile phase has little effect on its retention time.

Normal-phase chromatography is the opposite.

3。Ion-exchange chromatography. It is the reversible exchange between ionizable ions on ion exchange resins and analytes with the same charge, because the ions under test have different affinities on different exchangers.

The stronger the affinity, the greater the k value, and the longer the retention time.

4。Exclusion chromatography. The stationary phase is a porous gel, with pores inside, and molecules larger than the pores cannot enter the stationary phase, flowing directly from the surface, with almost no retention, and small molecule substances can freely enter and exit the pores without being hindered at all, and the retention time is long.

A medium-sized volume molecule is somewhere in between. The separation order is only related to the size of the molecule.

When using high-performance liquid chromatography for analysis, two elution methods are commonly used, one is isocratic elution and the other is gradient elution.

In chromatographic separations with isocratic elution, the composition of the mobile phase composed of different solvents, such as the polarity, ionic strength, and pH of the mobile phase, remains unchanged throughout the separation. In chromatographic separations with gradient elution, the composition of the mobile phase containing two or more solvents of different polarities changes continuously or intermittently during the elution process, during which the polarity of the mobile phase can be adjusted to improve the resolution between the components in the sample.

When gradient elution is used, strongly retained impurities that interfere with the separation are also cleared from the column in a shorter period of time, leaving the column clean for the next analysis. Gradient elution generally refers to the linear change of the composition of the mobile phase with the extension of the analysis time, that is, linear gradient elution, which can be used in reversed-phase and normal-phase high-performance liquid chromatography and ion-pair chromatography.

According to the polarity of your sample composition, it also depends on whether you use a forward or reversed-phase column, if you use a reverse column (C8, C18, etc.), and the polarity of the column packing is weak, then the substance being analyzed is the first peak with the highest polarity; If a forward column (CN, NH2) is used, and the column packing material is more polar, then the substance being analyzed is the first peak with less polarity.

The principle of liquid chromatography is mostly partition chromatography, that is, after the sample is adsorbed by the stationary phase (column packing), in the process of mobile phase elution, the polarity of the sample is different, and the concentration of dissolution in the mobile phase is also different, some can be miscible, some are not, and some have a certain solubility. It is possible to separate according to this principle. After a long interaction with the stationary phase, it is eluted, and the non-adsorbed ones are eluted first with the flow of the mobile phase.

The higher the RF value, the farther the compound travels on the mobile phase, indicating that the compound is eluted first, followed by the mobile phase earlier, i.e., the larger the RF value, it is eluted first, and conversely, the smaller the RF value, it is eluted later. Got it

Gradient elution is performed with a weakly polar solvent A and a strongly polar solvent B.

Principle of gradient elution:

The mobile phase is composed of several solvents of different polarities, and the polarity of the mobile phase is changed by changing the proportion of each solvent in the mobile phase to the silver composition, so that each flow has a suitable capacity factor k for the group width and disorder of each flow'and to achieve optimal separation of all components of the sample species in the shortest possible time.

Gradient elution features:

Increase column efficiency and improve detector sensitivity when the sample is k-high in one of the peaks'value and k of the last peak'Gradient elution is particularly effective when values differ by a factor of tens to hundreds. In gradient elution, a constant flow pump must be used to ensure a stable flow rate, otherwise it is difficult to obtain reproducible results.

Advantages of gradient elution:1Shorten the analysis cycle; 2.Improves separation capacity; 3.Peak shape is improved with little tailing; 4.Increased sensitivity. However, it can sometimes cause baseline drift.

Gradient elution is suitable for analyzing complex components that are not easily separated.