Flush the pumps and piping of the chromatograph thoroughly with the filtered and degassed mobile phase (without buffer salts) so that the system is free of bubbles. According to the flow direction of the mobile phase marked on the chromatographic column, connect the inlet of the chromatographic column with the pipe at the pump end and the outlet of the column is not connected.
Set the flow rate of the pump to 0.1mL/min or lower, and the flow rate slowly rises to the normal flow rate (t > 5min). When the solvent flows freely from the exit of the column, set the flow rate as 0, and connect the column exit with the pipeline at the detector end.
The chromatographic column was balanced with the mobile phase and normal flow rate of 10-30 times column volume. Amino column, cyano column can be used in the positive phase environment, but also in the inverse phase environment. When it is necessary to change from the reverse phase into the positive phase or the positive phase into the reverse phase, it is necessary to use 20-30 times the column volume of THF as the transition solvent to flush the system, otherwise, it is easy to make the one-way valve of the pump blocked, abnormal pressure phenomenon.
Notes for chromatographic column useMobile phase: the solvent must be HPLC grade and the reagent should be as pure as possible. There is no chemical reaction with the fixed phase, the viscosity is small, and the sample has an appropriate solubility, k is required in the range of 1 ~ 10 (available range) or 2 ~ 5(the best range), k value is too small, is not conducive to the separation of k value. Too large and may precipitate the sample in the mobile phase. The mobile phase must be filtered and degassing d before use to ensure that the solvents are miscible with each other. The mobile phase must match the detector. For example, when the UV detector is used, the solvent whose cut-off wavelength is greater than the detection wavelength cannot be used.
Trace impurities can greatly degrade the performance of the column, ensuring that solvents (buffers) are miscible with each other when the mobile phase needs to be replaced. If the solvent used and the column of the solvent is not miscible, the use of transition solvent, otherwise the column will produce permanent damage. The separation of salt or buffer from the mobile phase can also permanently damage the column. The solubility of the sample in the mobile phase should be checked before each sample injection, and the sample should be dissolved in the mobile phase whenever possible.
Fixed phase: the pH value of the mobile phase should be kept between 2.0 and 8.0 (unless otherwise specified); Use pre-saturated column and guard column if necessary; When using an amino column, avoid aldehydes and ketones in mobile phase and sample.
Column packing can be divided into the following three: high pH (>8.0), silica gel dissolution. If the pH of the mobile phase is close to 2.0 or 8.0, a saturated column is required. Silica gel column: high column efficiency, high mechanical strength, but sensitive to pH, low pH (<2.0) will cause the bonding phase to hydrolyze (bond removal of functional groups).
Organic polymer packing: in the wide range of stable residual silica hydroxyl effect is small, but its column efficiency is low. Mechanical strength is not high and may shrink or swell in different solvents. In the application process, the pressure must not exceed its maximum bearing range. And mobile phase loyalty, organic solvents such as methanol. The content of isopropanol ethanol should not exceed 10%.
A new type of organic-inorganic hybrid filler with two high – purity monomers. Synthetic high purity filler which combines the advantages of silica gel and an organic polymer. High efficiency, high mechanical strength, good stability, wide ph range. The silicon hydroxyl group can be effectively shielded for the most part. So that it has better retention of alkaline substances.
Back pressure and flow rate
Keep the back down to 3,500 psi. Avoid any sudden changes in stress. If the back pressure is high, the column can recoil, but the flow rate should be 1/5 to 1/2 of the normal flow rate. If the test pool needs to be degassed, use the back pressure governor. In order to extend the service life of the chromatographic column to the greatest extent, the velocity of flow can be adjusted according to the particle size of packing and the inner diameter of the chromatographic column. So that the back pressure does not exceed the limit.
Keep the back down to 3,500 psi. Avoid any sudden changes in stress. If the back pressure is high, the column can recoil, but the flow rate should be 1/5 to 1/2 of the normal flow rate. If the test pool needs to be degassed, use the back pressure governor. In order to extend the service life of the chromatographic column to the greatest extent, the velocity of flow can be adjusted according to the particle size of packing and the inner diameter of the chromatographic column. So that the back pressure does not exceed the limit.
Column temperature
Most of the separation is done at room temperature, and if the temperature in the lab stays the same, there will be no problems. But most work environments have temperature fluctuations. The retention time of temperature equivalence separation has an effect. Even under the condition of the constant temperature of day and night, the room temperature will change greatly due to the difference in temperature between day and night. Especially when large quantities of samples are analyzed, there will be large differences. The separation of compounds is achieved by controlling the temperature. The retention time and selectivity of the column change with the change of temperature, and the peak width also change.
Most of the separation is done at room temperature, and if the temperature in the lab stays the same, there will be no problems. But most work environments have temperature fluctuations. The retention time of temperature equivalence separation has an effect. Even under the condition of the constant temperature of day and night, the room temperature will change greatly due to the difference in temperature between day and night. Especially when large quantities of samples are analyzed, there will be large differences. The separation of compounds is achieved by controlling the temperature. The retention time and selectivity of the column change with the change of temperature, and the peak width also change.
Preservation of chromatographic column
The preservation condition of the column will affect the life of the column. The storage solution should contain at least 20% organic solvent to prevent the growth of microorganisms. Never use buffer to store chromatographic column, wash chromatographic column with 5 times column volume of mobile phase without a buffer to remove buffer and salt.
The preservation condition of the column will affect the life of the column. The storage solution should contain at least 20% organic solvent to prevent the growth of microorganisms. Never use buffer to store chromatographic column, wash chromatographic column with 5 times column volume of mobile phase without a buffer to remove buffer and salt.
没有评论:
发表评论