Application of chromatographic column maintenance in high pr

As a separation technology and method, spectroscopy has a history of more than 100 years since the beginning of this century, and now it has become an important branch of analytical chemistry. In the new era, people are facing the challenge of rapid development in the fields of information science, life science, material science, environmental science, etc. the demand for talents in these fields has become a crucial factor for the country's high development. Chromatography is an indispensable means and tool for life science, material science and environmental science. According to the latest statistics, among all kinds of analytical instruments in the world, the total marketing volume of gas chromatograph and liquid chromatograph accounts for 25% - 30%.
With the development of science and technology, chromatography technology has been further developed, and new combined technology has been applied. With the development of biomedicine, high sensitivity and high selectivity methods are required for qualitative and quantitative research of research objects.
Related properties of 1 column
Column packed with 1.1 column. Most of them are made of metal or glass. There are straight tube shape, coil shape, U-tube shape and so on.
1.2 classification: chromatographic columns can be divided into packed columns and open tubular columns. Packed column is usually used in liquid chromatography. The separation effect of chromatographic column depends on the selected stationary phase, as well as the preparation and operation conditions of chromatographic column. In short: common packing: ODS / C18, octy / C8, hexyl / C6, butyl / C4, methyl / C1, sax, SCX, phenyl, amino / NH2 and cyano / CN / nitrile. Common packing of adsorption column: silica gel column
The selection of chromatographic column will directly affect the separation of components in the mixture. For example: for the packed column, there are many stationary phases to choose and the column capacity is large. However, due to the limitation of column length, the separation capacity is limited, and it is mainly used for the separation of simple compounds.
2. Maintenance of chromatographic column
2.1 maintenance of chromatographic column. When we use a new column, we should conduct a performance test on our own liquid chromatograph, that is, to determine the column efficiency by using the test conditions and samples in the inspection report attached to the column. In addition, the chromatographic column should be tested frequently in the future. The main reasons for this phenomenon are: (1) insoluble matter in solvent; (2) insoluble matter in sample; (3) insoluble substance in pump injector; (4) formation of insoluble substance in column. If the chromatographic column is blocked, we can repair the column. First of all, after using saline buffer solution (such as ion pair reagent), wash it with solvent with strong solubility (such as water). Secondly, the detector should be disconnected first, and then the solvent with strong solubility for the substances from the sample should be washed. For the reversed-phase chromatographic column, methanol, acetonitrile, tetrahydrofuran, chloroform, heptane and so on can be used. Under this condition, the pH of the solvent should be avoided to be lower than 2 or higher than 8. Finally, if the chromatographic column pressure still does not drop after the above-mentioned repair, disconnect the detector, place the chromatographic column in the opposite direction, and then check the column pressure. If the pressure drops steadily, keep the chromatographic column connected in the opposite direction, and rinse with a flow rate lower than 0.5 ml / min for one hour. If the pressure does not drop, it depends on whether the built-in filter is blocked. If it is blocked, it should be flushed or replaced. If the filler at the inlet end is blocked, the column can not be completely recovered. Only by removing part of the filler at the inlet end and refilling, can the Limited column efficiency be restored. And the repair thickness is 2-5mm.
2.2 maintenance of chromatographic column. Before using the chromatographic column, it is better to test the performance of the column and keep the results as a reference for evaluating the performance changes of the column in the future. However, it should be noted that the performance of the column may be different due to the difference of sample, mobile phase, column temperature and other conditions; in addition, the column performance test is conducted according to the conditions in the ex factory report of chromatographic column (the conditions used in the factory test are the best conditions), and only in this way can the measured results be comparable. After testing the chromatographic column before use, we need to maintain the chromatographic column according to different conditions: ① the maintenance of the reversed-phase chromatographic column after every experiment: use buffer or mobile phase containing salt, after the experiment, use 10% methanol / water to wash for 30 minutes, wash off the salt in the column, and then wash it with methanol for 30 minutes. Note: do not wash the column with pure water, but add 10% methanol into the water to prevent the filler from collapsing. ② Long term preservation of chromatographic column: if the chromatographic column is to be preserved for a long time, it must be stored in a suitable solvent. The reversed phase column can be stored in pure methanol or acetonitrile, the normal phase column can be stored in pure hexane after strict dehydration, and the ion exchange column can be stored in water (containing preservative sodium azide or thiomersal), and the plug provided when purchasing a new chromatographic column is plugged. The best storage temperature is room temperature.
It can be summarized as follows: ① the column should not be collided, bent or strongly vibrated under any circumstances; ② when the column is connected with the chromatograph, the valve or pipeline must be cleaned; ③ attention should be paid to the degassing of the mobile phase; ④ avoid using high viscosity solvent as the mobile phase; ⑤ the sample should be purified; ⑥ strictly control the injection volume; and (7) clean the injection after the end of the analysis every day The residual sample in the valve; 8) after the analysis and determination every day, the column should be cleaned with appropriate solvent; 9) if the analytical column is not used for a long time, it should be stored and sealed with appropriate organic solvent.
Application of 3 column maintenance in high pressure liquid chromatography
High performance liquid chromatography (HPLC) is a separation and analysis technology developed in the late 1960s. It is an important means of modern separation and determination. Since its advent, it has been widely used in biochemistry, medicine and clinical analysis due to its high separation efficiency, fast analysis speed, good detection sensitivity and analysis of thermally unstable physiologically active substances with high boiling point but can not be gasified. High performance liquid chromatography (HPLC) column is a consumable. With the increase of using time or injection times, the peak height, peak width and column efficiency will decrease. Regular thorough cleaning and regeneration are required. Different cleaning methods of chromatographic column are different, such as:
3.1 the reversed-phase column was respectively washed with methanol: water = 90:10, pure methanol (HPLC grade), isopropanol (HPLC grade), dichloromethane (HPLC grade) and other solvents as mobile phases. Each mobile phase flowed through the column volume not less than 20 times of the column volume, and then washed in the reverse order.
3.2 normal phase column Use n-hexane (HPLC grade), isopropanol (HPLC grade), dichloromethane (HPLC grade) and methanol (HPLC grade) as mobile phases respectively. Each mobile phase flows through the chromatographic column at least 20 times of the column volume (isopropanol has high viscosity, which can reduce the flow rate and avoid high pressure). Note that the order of using solvents should not be reversed. After washing with methanol, the reverse order should be followed Flush to n-hexane, all mobile phases must be strictly dehydrated.
3.3 if ion exchange column is used in buffer solution for a long time and sample injection, the ion exchange capacity of chromatographic column will decrease. Washing with dilute acid buffer solution can regenerate cation column, otherwise, washing with dilute alkali buffer solution can regenerate anion column.
In addition, the solvent that can dissolve the pollutants in the column can be selected as the mobile phase for washing in the positive and reverse directions. If the column is reversed, it can be adjusted back, but the support in the column may collapse. If the column has to be reversed, the column should not be reversed.
Strengthening the maintenance of chromatographic column is of great significance for the application of chromatographic column in high pressure liquid chromatography. The standard operating procedures for chromatographic column management are formulated and strictly implemented. Each newly purchased chromatographic column must be registered in a register, including: production company, product name, model, specification, purchase time, start-up time, unit price, column protection solution, etc. The chromatographic columns were classified and stored in silica gel column, chemically bonded silica gel column, cyano or amino column, ion exchange resin column and gel column. Each column was labeled with labels. The column instructions are packed into a compact bag and attached to the register. For each chromatographic column used, a label should be attached on the chromatographic column, indicating the date of use. Each column should be maintained every two months, especially for some columns with less use frequency, because it is easy to dry the column due to long storage time. The maintenance record should be made every time. For those columns that have failed, make a record of their discontinuation, and do not discard them at will. If the fillers of the existing columns need to be filled, the fillers of the stored columns can be used. In the quality inspection department of the production unit, the column with long service time and low column efficiency can be used to detect the intermediate of the product.
It is of great significance to strengthen the maintenance of chromatographic column for the development of high-pressure liquid chromatography. High pressure liquid chromatography system includes chromatographic column. Therefore, strengthening the maintenance and maintenance of chromatographic column is conducive to the development of high-pressure liquid chromatography and promote the development of medical undertakings in China.
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