Classification, Selection, and Maintenance of HPLC Columns

High-performance liquid chromatography analysis is an efficient, fast, and accurate separation and analysis method. The selection of liquid chromatography columns is significant for the separation of substances. This issue introduces how to classify, select, and maintain liquid chromatography columns.

A. Classification of chromatographic columns

1. Structure of chromatographic columns

HPLC chromatographic columns are composed of fillers, column tubes, pressure caps, ferrules (sealing rings), sieve plates (filter discs), joints, screws, etc., as shown in the figure below:

2. Classification of HPLC columns

Usually, they can be divided into Normal phase, reverse phase, ion exchange, and other chromatographic columns according to the separation mode. In daily analysis work, the proportion is as shown in the figure below:

3. Factors affecting the performance of chromatographic columns

The main factors affecting the performance of chromatographic columns are the properties of fillers and filling technology. The influence of filler factors on the performance of chromatographic columns:

4. Characterization of chromatographic column performance

The parameters that characterize the performance of chromatographic columns mainly include: theoretical plate number, asymmetry factor, selectivity for two different solutes, back pressure of chromatographic columns, reproducibility of retention time, bond and phase concentration, stability of chromatographic columns, etc.

5. Application of chromatographic columns with different fillers

Chromatographic columns also follow such a rule: structure determines properties, properties determines uses, and chromatographic columns with different fillers are suitable for different uses.

B. Selection of chromatographic columns

There are many factors to consider when selecting chromatographic columns, but they generally follow the following principles:

1. Pay attention to the methods in international and other literature

Select chromatographic columns with similar particle size, specific surface area, matrix surface characteristics, bonding type, etc. as those in national standards and other literature, or use the USP website and some chromatographic column selection software to select chromatographic columns with high similarity coefficients.

2. Selection of chromatographic columns for separation of new compounds

(1) Silica gel matrix fillers are suitable for most HPLC analyses, especially small molecule compounds;

(2) For high pH or alkaline compounds, high-end or special-end capped chromatographic columns need to be selected to improve peak shape and extend the service life of the chromatographic column;

(3) When achieving baseline separation of each peak, use as short a column as possible to shorten the analysis time;

(4) Separation using an isocratic method with a long column is better than separation using a gradient method with a short column, etc.

C. Maintenance of chromatographic columns

1. Before using the chromatographic column, carefully read the instruction manual of the chromatographic column, and perform the performance test of the column according to the conditions in the factory report of the chromatographic column. The results are archived as a reference for evaluating the performance of the column in the future;

2. Installing a protective column with the same filler is an important measure to protect the chromatographic column fully, especially when analyzing biological samples and complex mixtures such as Chinese patent medicines and traditional Chinese medicines;

3. When replacing and loading and unloading the column, the action should be gentle and the joints should be tightened appropriately to avoid strong mechanical vibrations that may cause gaps in the column bed;

4. When doing routine analysis, if the sample type is valid, try to use a dedicated column to extend its service life;

5. Degas and filter the mobile phase before use;

6. Avoid sudden changes in temperature and pressure. Sudden temperature changes will affect the filling condition in the column; sudden increase or decrease in column pressure will also cause the filling in the column to collapse, so the flow rate should be adjusted slowly;

7. The indicated pH range of the bonded silica gel column can reach 2-10. Try Not to exceed the pH stability range of the chromatographic column, especially in alkaline conditions, which seriously affects the service life;

8. When storing the chromatographic column, fill it with appropriate organic phases such as methanol, n-hexane, etc. as required. The column joints should be tightened to prevent the solvent from volatilizing and drying. The buffer solution should not be left in the column overnight or longer.