The chromatographic columns can be divided into two categories: packed columns and open-tube columns and mostly are made of metal or glass. It could be straight, U-shaped, and coil tube, etc. Liquid chromatography usually uses packed columns. The separation effect of the chromatographic column depends on the selected stationary phase, and the preparation and operating conditions of the chromatographic column.
As an efficient method of separation analysis, chromatography’s core is to separate. Thus, the column responsible for separation is the heart of the chromatographic system, which requires high column efficiency, good selectivity, and fast analysis speed. Commercially available various particulate fillers are porous silica gel and silica-based bonded phase, alumina, organic polymer microspheres (including ion exchange resins), porous carbon, etc.
The particle size is generally 3,5, 7,10μm, etc., the theoretical value of column efficiency can reach 50,000~160,000/m. For general analysis, only 5000 plates are needed; for homologous analysis, 500 plates are needed; for difficult-to-separate substance pairs, up to 20,000 columns can be used, so generally, a column length of about 10~30cm can be used to meet the needs of complex mixture analysis.
Speaking of the column efficiency, there are many influencing factors that will have an effect on it, both inside and outside the column. Besides the small dead volume outside the column, a reasonable column structure (to minimize the dead volume outside the packed bed) and packing technology are required, so as to get the best efficiency for the column.
The packing conditions cannot be guaranteed the same even though adopting the most advanced packing technology. The part close to the pipe wall is relatively loose, easy to produce channeling, and the flow rate is faster, which affects the flow shape of the flushing agent and widens the band. This is the pipe wall effect. This tube wall area is about 30 times the thickness of the particle diameter from the inside of the tube wall. Generally speaking, the extra-column on column efficiency has a much better effect than the tube wall.
Selection of liquid chromatography column
Before choosing a liquid chromatography column, it’s better for the operator to learn more about your samples and impurities, their type, structure, polarity, acidity and alkalinity, molecular weight, etc. It would increase working efficiency and save much unnecessary trouble.
1. If the sample is polar and weakly acidic, you can choose C18 to detect under the condition of 100% acidic aqueous solution, that is, choose a column that withstands 100% pure water and retains very good polar compounds.
2. If the sample is too polar or too acidic, you can choose CN, NH2, or silica gel column, HILIC (hydrophilic chromatography), and also use C18+ strong anion pair reagent or strong anion exchange chromatography column (The disadvantage is that ion-pair reagents take a long time to equilibrate and require more precise pH of the mobile phase. Otherwise, it is difficult to repeat the experiment. In addition, ion-pair reagents are difficult to wash off. Basically, chromatographic columns using ion-pair cannot be used in other experiments)
3. If the sample is alkaline, you can choose a high-purity silica gel column (high-purity silica gel lacks metal impurities, and silica end groups are blocked) or some modified C18 flash columns (such as polar intercalation technology or alkaline deactivation technology, etc.) They will reduce the tailing of basic compounds, and generally choose to do it under neutral or partial alkali conditions because this can increase the retention of basic samples.
4. If with too strong polarity or basicity, choose a C18 column with a wide PH value to detect at a high pH value (the advantage is that the method development is simple, but the disadvantage is that few choice and high price) or use HILIC chromatography column (silica gel column is used under reversed phase conditions, which is also a classic method for detecting basic samples), select strong ion exchange column (the disadvantage is that it cannot be used to analyze other samples, and the pH of the mobile phase is more precise, otherwise it is difficult to repeat the experiment) C18+ strong anion pair reagent or strong anion exchange chromatography column is also used.
Features of FLASH columns
The linear gradient was transformed into a step gradient by flash chromatography, and the elution conditions were optimized near the elution peak. First, use a small column to purify a small portion of the sample. Once the run is complete, select the optimize button, and then select the target peak. A new and optimized one is automatically generated based on the gradient elution method. Select a new chromatographic column and collection holder, and the separation method is automatically adjusted according to the size of the new column and is ready to run.
The flash columns have high sample load, stability under low pH conditions, excellent peak shape, and outstanding column efficiency. The details are as follows:
1. Excellent chromatographic peak shape The FLASH column adopts a new type of bonding and end-capping technology, which can provide the chromatographic peak shape, greatly improving the accuracy of resolution and quantitative analysis of acid, alkali, and neutral compounds under low and medium pH conditions.
2. Excellent low pH stability Compared with other brands of silica gel columns, the stability of fast liquid chromatography columns under low pH conditions makes the column life longer.
3. Outstanding column efficiency So far, the advanced ultra-pure silica packing, combined with new bonding and end-group tail-capping technology, ensures that the fast liquid chromatography column has high efficiency and higher sensitivity.
4. Good batch reproducibility Fast liquid chromatography columns have reached the industry’s high level of inter-column and batch-to-batch reproducibility.
5. Excellent MS compatibility The fast liquid chromatography column is compatible with mass spectrometry and has the characteristics of sharp peak shape, excellent selectivity, higher peak capacity, and lower bonded phase loss. In addition, the resolution and low back pressure of the fast liquid chromatography column save the cost and time of analysis.