High loss rate of liquid chromatography column? Five key mai

Chromatographic column technology began in the 1950s. With the development of packing and filling technology, chromatographic column technology is becoming more and more mature, and its function is also increasingly perfect. At present, it has been widely used in life science, environmental protection, materials, food, drug development and other fields.
As the heart of the chromatographic system, the liquid chromatography column plays an important role in the separation and detection of substances in the chromatographic analysis system. In order to reduce the loss, the use and maintenance of chromatographic column is very important!
The common problems in the use of liquid chromatography column include column connection, column activation, column use, column maintenance, column preservation, etc.
1. Column connection
Column installation direction
Chromatographic column installation should be connected in the same direction, and should be connected according to the direction indication on the column, so as to avoid reverse connection of chromatographic column!
There are two common problems in the connection of chromatographic column. When installing chromatographic column, the length of the pipeline extending out of the joint is too long, which makes the thread screw in shallowly, which will lead to poor sealing performance and liquid leakage, and further cause baseline drift or response reduction; on the contrary, dead volume will appear in the front section of the pipeline, leading to the broadening of peak shape and the decrease of sensitivity.
The ideal joint connection should have the following characteristics: there is no dead volume between the pipeline and the interface; always avoid leakage under ultra-high pressure and high temperature; excellent long-term service stability to prevent pipeline sliding; simple and easy to use.
Pipeline selection is also very important. The most commonly used specifications of analytical liquid phase system are 0.12 and 0.17 mm inner diameter pipelines. When replacing the pipeline, the specification of the current pipeline should be confirmed first, and the pipeline with the same inner diameter and length should be replaced. Otherwise, the results will be inconsistent before and after the replacement, because the pipeline volume will affect the volume outside the column of the system, thus affecting the peak shape and retention time.
2. Activation equilibrium of reversed phase column
1) First, wash with methanol or acetonitrile about 20 times the volume of the column.
2) If the mobile phase contains buffer salt, use ultrapure water and organic phase in the same proportion as the initial proportion of the mobile phase to wash the transition about 20 times of the column volume, and then flush the mobile phase with buffer salt about 20 times of the column volume or more.
3) If the mobile phase does not contain buffer salts, the mobile phase can be directly used to balance the chromatographic column, about 20 times the volume of the column or more.
4) When the baseline and pressure are stable, the test is conducted to determine whether the balance is sufficient, and the recurrence of continuous injection results shall prevail. If not enough, the equilibrium time of mobile phase can be prolonged.
3. Reverse column washing and preservation
1) Wash 20-30 times of column volume with 50:50 methanol or acetonitrile and water;
2) Wash 20-30 times of column volume with pure methanol or acetonitrile;
3) Seal the plug tightly to the column end joint before storage to prevent the packing from drying out.
4. Cleaning and regeneration of reversed phase chromatographic column
When cleaning or backwashing the reversed-phase chromatographic column, wash the column with at least 30 times the volume of the column with the following solvents:
Disconnect the chromatographic column from the detector, leave the pipeline at the end of the column, and put it into the beaker receiving the liquid. First, rinse with mobile phase without buffer salt (water / organic phase), and then wash with 100% organic phase (methanol and acetonitrile). Check whether the pressure returns to normal. If not, proceed to the next operation.
If the pressure does not return to normal, discard the column or consider cleaning with stronger conditions: 75% acetonitrile / 25% isopropanol, 100% isopropanol, 100% dichloromethane, 100% hexane.
It should be noted that both hexane and dichloromethane must be washed with isopropanol before use or before resuming the use of the reverse phase mobile phase.
About column recoil
Although the column should not be recoiled easily, backwash is the most effective remedy when it is clearly known that the overpressure comes from the blockage of sieve plate or column head contamination by particles. In addition, it can also quickly wash out the strong adsorbed pollutants on the column head. After the column recoil, it is better to connect the column head forward. However, backwash can also bring negative effects, such as loose column bed, change of retention time, backflush of small particle size column may lead to packing outflow, etc.
Among them, the chromatographic columns that can be recoiled include: chromatographic columns with particle size greater than 2um (2.7, 3, 3.5, 4, 5 μ m, etc.); and the chromatographic columns that can not be recoiled are those with particle size less than 2um (1.8 μ m rrhd / rrht; 1.9 μ M poroshell).
5. Common problems in using chromatographic column
The most common problems in the use of liquid chromatography column include pH value, temperature, solvent tolerance, pressure, sample, etc. The operating conditions of chromatographic column should not exceed the range recommended by the manufacturer, including maximum pressure, pH range, water phase tolerance, column temperature, etc. When the test conditions are close to the limit value of the column service range, the column life will be affected.
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A collection of questions
1. How to adjust the pH and temperature of C18 column to improve the separation?
A: optimizing the resolution by adjusting pH and column temperature is a very important means in the development of the method. In short, neutral or non ionizable compounds are not sensitive to pH changes. For the ionizable compounds, the reversed phase retention of the compounds can be changed by adjusting the pH value of the mobile phase and controlling the ionization state of the compounds. Decreasing pH can increase the retention of acidic compounds, while increasing pH can increase the retention of basic compounds. By adjusting the pH to change the retention of the compounds, the resolution between the components was optimized.
Generally, increasing the column temperature will accelerate the mass transfer and reduce the retention. However, the sensitivity of different compounds to temperature change is different. Therefore, the separation degree can be optimized by adjusting the column temperature and changing the retention time of each component.
2. What may be the cause of total overpressure of chromatographic column?
A: overpressure is usually due to blockage of the internal part of the liquid flow path, including the chromatographic column. It is necessary to conduct sectional investigation to determine the location of the blockage, and then to investigate the possible causes of the blockage according to the blocking position.
If the chromatographic column is blocked, there are many common reasons, such as the sample is dirty, the matrix is complex and has not been well pretreated, or after pretreatment, it enters the liquid phase system and precipitates again, resulting in blockage or pollution (solution: strengthen sample pretreatment); the use of overpressure or beyond the pH range leads to packing fragmentation, and debris particles block the column (solution) Methods: select the appropriate chromatographic column according to the test conditions to avoid over range use; blockage caused by wear debris of components during the use of the instrument (solution: timely replacement of damaged components), etc., will cause the pressure rise of chromatographic column in the system.
3. What factors affect the delay of peak time of C18 column? After a period of time, the peak time is delayed. Is it related to the mobile phase?
A: the main factors affecting the retention of compounds in liquid chromatography include: sample, chromatographic column, mobile phase (flow rate, composition, ratio, etc.), column temperature, etc. If the retention time drift is found in the use process, the following factors should be investigated: the possible causes can be preliminarily investigated by comparing whether the pressure curve under the same conditions before and after the retention time drift is repeated. If the pressure curve does not reappear, first confirm whether the test conditions have been changed, check whether the flow rate, composition and proportion of the mobile phase are changed, and whether there are changes in the flow rate and proportion caused by leakage or bubbles; for samples and methods sensitive to changes in composition of mobile phase, ensure the reproducibility of each preparation of mobile phase; check whether the chromatographic column is blocked and polluted; whether the instrument temperature control is accurate There are many possible reasons, and the specific reasons need further investigation.
4. What is the best mobile phase for chromatographic column preservation? Is it easy to dry with pure organic reagent?
A: the reversed phase column can be preserved with HPLC grade methanol or acetonitrile. Pay attention to the tight connection plug. Under normal circumstances, as long as the plug is plugged, the solvent is not easy to dry. Of course, adding 5% - 10% water to the preservation solvent is no problem.
5. The mobile phase of acetonitrile is always easy to polymerize. Is there any solution?
A: the polymerization of acetonitrile requires certain conditions and time. It is necessary to use HPLC grade solvent with reliable quality and ensure that the solvent used is as fresh as possible. If it is the acetonitrile solvent which has been stored for a long time, it can be improved by filtering it before use.
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6. What kind of liquid chromatographic column is commonly used for small molecular polar substances?
A: we can first try to use a column that can tolerate high proportion of water phase and improve the mobile phase water ratio to enhance retention. If it is an ionizable compound, such as acidic or alkaline compound, we can first try to increase retention by adjusting the pH of the mobile phase in the reverse phase mode. Acidic compounds need to reduce the pH of the mobile phase, and alkaline compounds should increase the pH of the mobile phase, and select the tolerable column according to the pH conditions. If the retention of reversed phase mode is still weak after adjusting pH, you can also consider using other retention mode columns, such as HILIC column, hilic-z column, hilic-oh5 column, HILIC column with pure silica gel, etc., or ion exchange column or normal phase chromatography.
7. How to deal with poor separation effect?
A: the main reasons for the decrease of column resolution are the decrease of column efficiency and the change of column selectivity. There are many reasons for the decrease of column efficiency. If it is the loss of column efficiency caused by improper connection, it can be connected correctly again. If the column efficiency is reduced or the resolution is reduced due to column contamination or selectivity change, the column can be cleaned and regenerated. If it is the damage of the chromatographic column itself, which results in the decrease of column efficiency and the change of resolution, it is usually irreversible. Only the chromatographic column can be replaced, and all kinds of operations that damage the column should be avoided when using the new column in the future.
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