Chromatographic column technology began in the 1950s. With the development of packing and packing technology, chromatographic column technology has become increasingly mature and its functions have become more and more perfect. It has been widely used in life sciences, environmental protection, materials, food, drug development and other fields.
The liquid chromatographic column mainly plays a role in separating the detected substances in the chromatographic analysis system, just like the heart of the chromatographic system, but also a consumable product. In order to reduce losses, the maintenance of the chromatographic column is very important!
Common problems in the use of liquid chromatography columns include column connection, column activation, column use, column maintenance, column storage, etc.
1. Column connection
Column installation direction
The chromatographic column should be connected in the same direction and connected according to the direction instructions on the chromatographic column. Try to avoid the reverse connection of the chromatographic column!
There are two main problems with common chromatographic column connection. When the chromatographic column is installed, the length of the pipe extension joint is too long, which makes the screw thread shallow, which will lead to poor sealing and leakage, further causing baseline drift or reduced response; on the contrary, There will be a dead volume in the front section of the pipeline, causing the peak shape to broaden and the sensitivity to decrease.
The ideal joint connection should have the following characteristics: no dead volume between the pipeline and the interface; always avoid leakage under ultra-high pressure and high temperature; excellent long-term stability to prevent pipeline sliding; simple and easy to use.
The choice of pipeline is also very important. The most commonly used specifications for analytical liquid phase systems are pipelines with internal diameters of 0.12 and 0.17mm. When replacing the pipeline, first confirm the current pipeline specifications and replace the pipeline with the same inner diameter and length, otherwise the results before and after the replacement will be inconsistent, because the volume of the pipeline will affect the extra-column volume of the system, thereby affecting the peak shape and retention time. To
2. Reversed phase column activation equilibrium
1) First, use methanol or acetonitrile to flush approximately 20 times the column volume.
2) If the mobile phase contains buffer salts, use the same proportions of ultrapure water and organic phase as the initial ratio in the mobile phase to rinse for about 20 times the column volume, and then use the buffer salt-containing mobile phase to equilibrate and rinse about 20 times the column volume or more .
3) If the mobile phase does not contain buffer salts, you can directly use the mobile phase to equilibrate the column, about 20 times the column volume or more.
4) When the baseline and pressure are stable, test to determine whether the balance is fully balanced based on the reproduction of the continuous injection results. If it is not enough, the equilibrium time of the mobile phase can be extended.
3. Rinse and save the reversed-phase column
1) Use 50:50 methanol or a mixed solution of acetonitrile and water to wash 20-30 times the column volume;
2) Use pure methanol or acetonitrile to flush 20-30 times the column volume;
3) Tightly seal the plug on the column end joint before storage to prevent the packing from drying out.
4. Reverse phase chromatography column cleaning and regeneration
When cleaning or backflushing the reversed-phase chromatography column, flush the column with at least 30 column volumes of the following solvents:
Disconnect the chromatographic column from the detector, leave the line at the end of the chromatographic column, put it in the beaker that receives the liquid, first rinse with the mobile phase without buffer salt (water/organic phase), and then use 100% Rinse the organic phase (methanol and acetonitrile) to check whether the pressure returns to normal. If not, proceed to the next step.
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 is worth noting that whether using hexane or methylene chloride, it must be rinsed with isopropanol before use or before resuming the use of reversed-phase mobile phase.
About column backflushing
Although the column should not be backflushed easily, it is the most effective remedy when it is clearly known that the overpressure comes from the clogging of the sieve plate or column head contamination. Backflushing the chromatographic column can quickly flush out the particulate matter. In addition, it can also quickly flush out the column head to strongly adsorb contaminants. After the column is backflushed, it is best to still use it in a forward connection. However, backflushing can also bring negative effects, such as loosening of the column bed, changes in retention time, and backflushing of small particle size chromatographic columns may cause packing to flow out.
Among them, the chromatographic columns that can be backflushed include: chromatographic columns with a particle size greater than 2um (2.7, 3, 3.5, 4, 5μm, etc.); chromatographic columns that cannot be backflushed include: chromatographic columns with a particle size of less than 2um (1.8μm RRHD /RRHT; 1.9μm Poroshell).
5. Common problems in the use of chromatographic columns
The most common problems in the use of liquid chromatography columns include pH, temperature, solvent tolerance, pressure, samples, etc.
The use conditions of the chromatographic column shall not exceed the range recommended by the manufacturer, including the maximum pressure, pH range, water phase tolerance, column temperature, etc. When the test conditions are close to the limit value of the column usage range, the column life will be affected.
1. How to adjust the pH and temperature of C18 column to improve the resolution?
Answer: Optimizing resolution by adjusting pH and column temperature is a very important method in method development. Simply put, neutral or non-ionizable compounds are not sensitive to pH changes. For ionizable compounds, the reverse phase retention of the compound can be changed by adjusting the pH of the mobile phase and controlling the ionization state of the compound. Lowering the pH increases the retention of acidic compounds, while increasing the pH increases the retention of basic compounds. Adjust the pH to change the compound retention and optimize the separation between the components.
Generally, increasing the column temperature speeds up the mass transfer and reduces the retention. However, the retention of different compounds is sensitive to temperature changes. Therefore, the resolution can also be optimized by adjusting the column temperature and changing the retention time of each component.
2. What could be the reason for the total overpressure of the chromatographic column?
Answer: Overpressure generally means that there may be a blockage in the liquid flow path including the chromatographic column. It is necessary to perform a segmented investigation to determine the blocked location, and then investigate the possible cause of the blockage based on the blocked location.
If the chromatographic column is clogged, there are many common reasons, such as dirty samples, complex matrix and not good pretreatment, or pretreatment enters the liquid system and precipitates out, causing blockage or contamination (solution: strengthen sample pretreatment Treatment); overpressure of the chromatographic column or use beyond the pH range will cause the packing to fragment, and the debris particles will block the chromatographic column (solution: select the appropriate column according to the test conditions to avoid over-range use); caused by wear and tear of parts during the use of the instrument Blockage (solution: timely replacement of damaged parts), etc., will cause the pressure of the system column to rise.
3. What are the factors affecting the late peak time of C18 column? The peak time is delayed after a period of time. Is it related to the mobile phase?
Answer: The main factors affecting compound retention in liquid chromatography include: sample, chromatographic column, mobile phase (flow rate, composition, ratio, etc.), column temperature, etc. If the retention time drift is found during use, it is necessary to investigate from the following influencing factors: You can first check whether the pressure curve under the same conditions before and after the retention time drift recurs, so as to initially investigate the possible causes. If the pressure curve does not reappear, first confirm whether the test conditions have changed, check whether the flow rate, composition, ratio, etc. of the mobile phase have changed, whether there is a change in flow rate and proportion caused by leakage or air bubbles; samples that are sensitive to changes in mobile phase composition The method should ensure the reproducibility of each mobile phase preparation; check whether the chromatographic column is blocked or contaminated; whether the temperature control of the instrument is accurate, etc. There are many possible reasons, and the specific reasons need to be further investigated.
4. What mobile phase is the best storage for the column? Is it easy to dry with pure organic reagents?
Answer: The reversed-phase column can be stored in HPLC grade methanol or acetonitrile, pay attention to tightly connect the plug. Under normal circumstances, as long as the plug is blocked, the solvent is not easy to dry. Of course, adding 5%-10% water to the storage solvent is no problem.
5. Acetonitrile mobile phase is always easy to polymerize, is there any solution?
Answer: The polymerization of acetonitrile requires certain conditions and time. Be sure to use reliable HPLC-grade solvents and ensure that the solvents used are as fresh as possible. If it is an acetonitrile solvent that has been stored for a long time, it will be improved if it is filtered before use.
6. What liquid chromatography column is generally used for small molecule polar substances?
Answer: You can first try to use a column that can tolerate a high proportion of water phase and increase the mobile phase water ratio to enhance retention. If it is an ionizable compound, such as an acidic or basic compound, you can first try to increase the retention by adjusting the pH of the mobile phase in reversed phase mode. Acidic compounds need to lower the pH of the mobile phase, and basic compounds increase the pH of the mobile phase, depending on the pH conditions. Choose a column that can withstand it. If the retention in reversed-phase mode is still very weak after adjusting the pH, you can also consider using other retention mode columns, such as HILIC column, HILIC-Z, HILIC-OH5, or pure silica HILIC column, etc. You can also use ion exchange Chromatographic column or normal phase chromatography, etc.
7. What should I do if the column separation effect is poor?
Answer: The main reason for the decrease in the resolution of the chromatographic column is the decrease in the efficiency of the chromatographic column and the change in the selectivity of the chromatographic column. There are many reasons for the decrease in column efficiency. If the column efficiency is lost due to improper connection, just reconnect it correctly. If the column efficiency is reduced or the resolution is reduced due to the change of selectivity caused by column contamination in the use of the chromatographic column, you can try to clean and regenerate the column. If the column efficiency is reduced due to the damage of the chromatographic column itself, the resolution change is usually irreversible. You can only replace the chromatographic column, and try to avoid various operations that damage the column when using a new chromatographic column.