The Principle and Cleaning Method of NH2 HPLC Columns
NH2 HPLC columns refer to the chromatographic columns that use silica gel as matrix carrier to bond amino groups. They are usually used for the analysis of carbohydrate compounds, and the mobile phase is usually methanol, acetonitrile and buffers salt solution.
HPLC columns can be divided into analytical and preparative types according to their applications, and their size specifications are different:
1. Conventional analytical column (constant column), inner diameter 2~5mm (commonly used 4.6mm, domestic 4mm, and 5mm), column length 10~30cm;
2. Narrow bore (also known as the thin bore, semi-microcolumn), the inner diameter of 1 ~ 2mm, column length of 10 ~ 20cm;
3. Capillary column (also known as microcolumn), inner diameter 0.2~0.5mm;
4. Semi-preparative column, inner diameter > 5mm;
5. Laboratory preparation column, inner diameter 20~40mm, column length 10~30cm;
6. Production preparation column inner diameter up to several tens of centimeters.
The inner diameter of the column is generally determined by the length of the column, the particle size of the filler, and the flow rate of the fracture, in order to avoid the wall effect.
The cleaning method of NH2 HPLC columns
Foe columns used under positive phase condition, refer to the cleaning method of silica gel columns. For columns used under reverse phase condition, refer to the cleaning method of C18 HPLC columns.
The principle of NH2 HPLC columns
Simply, the principle of HPLC column is Based on RP principle. The separation of component molecules on the amino column mainly depends on the hydrogen bond force, and the orientation force and the induction force in the van der Waals force.
The separation principle of the HPLC columns is to use the components in the sample to have different partition coefficients between the mobile phase and the liquid phase of the stationary phase. Under the carrier gas, the components are repeatedly distributed between the two phases, because the component’s force in the column is different. After a certain column length, it is separated and flows out of the column.
It is the physical properties of the target molecules that determine what the most suitable HPLC column is for analyte separation in the lab. When selecting the HPLC column, the key characteristics you should pay attention to include hydrophobicity or hydrophilicity, intermolecular forces, intramolecular forces, and size.
In the molecular properties of the target molecules, multiple differences can be exploited by HPLC column separations. And the analyte elution profile is determined by the structure and chemistry of the HPLC column packing (stationary phase).
From the capillary to process scale, we can divide the HPLC column into different sizes. The quantity of sample loaded onto a column and the sensitivity of separation indicate the internal diameter and volume of an HPLC column. The column’s internal diameter can affect the separation profile. When you use gradient elution, the smaller internal diameter will increase separation and detection sensitivity. For analytical separations, we are always trying to find the balancing point between sensitivity and the sample volume loaded onto a column.
Regeneration of NH2 HPLC columns
Under abnormal circumstances, a series of remedial measures are taken to improve the column efficiency and prolong the service life.
NH2 HPLC columns are mainly used to separate optical isomers and substances with the large polar proportion that can’t be separated by reversed phase column, and analyze monosaccharides and hydrocarbon compounds with the features of weak retention, high polarity, unstable, easy hydrolysis under acidic conditions.
