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A. Liquid reagent requirements
Reagents such as methanol, acetonitrile must be chromatographically pure, other reagents should be chromatographically pure, and the reagent label should be labeled with chromatographic or HPLC. Water is ultrapure water or directly purchase purified water of guaranteed quality.1.5ml screw chromatography vials
B. Mobile phase configuration considerations
In general, the mixing of the solvent is carried out in a volume ratio (V/V) or a weight ratio (W/W). The volume of the solution varies with temperature, so the mobile phase is mixed and arranged in a weight ratio, and the repeatability of the sample measurement is good, but the operation is cumbersome, and usually it is mostly volume mixing.
There are three ways to mix the volume, such as 50% acetonitrile in water:
Mode (1): 500 ml of acetonitrile can be taken in a measuring cylinder, and 500 ml of water can be taken, and the two liquids are fully shaken and mixed in the bottle.
Mode (2): You can take 500ml of acetonitrile into a 1L volumetric flask and then make up to volume with water.
Mode (3): You can take 500ml of water into a 1L volumetric flask and then make up to volume with acetonitrile.
The composition of the mobile phase obtained by these three methods is different, and the retention time will not be the same even if other factors are not considered. In general, it is recommended to use the method (1) to configure the mobile phase.
C. Problems needing attention in the preparation of buffered salt mobile phase 1.5ml screw chromatography vials
The salt reagent used to configure the buffer salt should also be chromatographically pure, and the sample should be accurate. In order to meet the requirements, the balance of the appropriate range should be selected and the balance should be corrected before use every day. After being configured as required, it must be filtered through a 0.45μm or 0.22μm filter membrane and ultrasonically degassed for more than 20min.
Note: The buffer salt should be used with a new one.
D. When to adjust the pH of the mobile phase
When separating weak acid (3 ≤ pKa ≤ 7) or weak base (7 ≤ pKa ≤ 8) samples by reversed-phase chromatography, by adjusting the pH of the mobile phase, the dissociation of the sample components is inhibited, and the components are added in the stationary phase. The technique of retention and improvement of peak shape is called reverse phase ion suppression technology.1.5ml screw chromatography vials
(1) For weak acids, the lower the pH of the mobile phase, the larger the k value of the component. When the pH value is much smaller than the pKa value of the weak acid, the weak acid mainly exists in molecular form; when analyzing the weak acid sample, usually in the mobile phase A small amount of weak acid is added, and 50 mmol/L phosphate buffer and 1% acetic acid solution are usually used.
(2) For weak bases, the opposite is true. When analyzing a weak base sample, a small amount of a weak base is usually added to the mobile phase, and a 50 mmol/L phosphate buffer solution and a 30 mmol/L triethylamine solution are usually used.
The addition of organic amines to the mobile phase can weaken the strong interaction between the alkaline solutes and the residual silanol groups, reducing or eliminating peak tailing. Therefore, in this case, an organic amine such as triethylamine is also called a tailing agent or a tailing agent.1.5ml screw chromatography vials
Note: The pH adjustment should not exceed the pH tolerance of the column.
This is the end of the introduction of How to configure liquid chromatography mobile phase. I hope it can help you.
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