Buffer salts are often used to adjust the pH of the mobile phase during chromatographic analysis. Improper use of buffer salts can increase column pressure, decrease column efficiency and change the retention time of compounds on the column.
1) Elevated column pressure
Cause: Improper use of buffer salt leads to buffer salt precipitation, blocking the pores between the plug plate and bonded phase particles, impeding the mass transfer of the mobile phase, and causing the column pressure to rise.
2) Change in retention time of the same compound
Cause: If the sample is injected without being rinsed well, the compound’s retention time will change due to the salt contained in the column.
3) Decrease in column efficiency
Cause:
i) Some buffer salts can penetrate deep into the bonding phase and damage the silica matrix, resulting in the loss of the bonding phase from the column, loosening the column bed and decreasing the column efficiency;
ii) Condensation on the surface of the bonded phase makes it difficult to stretch the C18 carbon chain and reduces the retention ability of the substance, resulting in a decrease in column efficiency. Therefore, it is necessary to rinse the column after using buffer salts, and special attention should be paid when the concentration of buffer salts in water is considerable.
Proper use of buffer salts in the mobile phase
1. Pre-use treatment: Before using buffer salts as the mobile phase, the column needs to be rinsed with the mobile phase without buffer salts until the baseline is smooth. In principle, the mobile phase used for flushing contains the same proportion of water (or more water) as the mobile phase used for analysis, except that the mobile phase used for flushing does not contain buffer salts. Reason: Buffer salts are usually soluble in water and insoluble in organic solvents. If the mobile phase containing buffer salts (especially when the mobile phase is saturated with buffer salts) is used for analysis, if the proportion of water in the mobile phase used to preserve the column before the examination is relatively small and is not flushed out first, then the mobile phase used for the following sample will contain a large amount of organic solvent. The water proportion is insufficient to dissolve the buffer salts, and the buffer salts will be precipitated on the column and deposited. This may lead to damage, as mentioned above, to the column.
2. Post-use treatment: Rinse the column with a mobile phase containing the same proportion of water as that used for analysis (the only difference from the mobile analytical phase is that the mobile phase used for rinsing does not contain the buffer salt) for about 30 min until the baseline is smooth. If the column is not to be used for an extended period and is to be stored for a long period, an additional step of flushing with a pure organic solvent is required until the baseline is smooth.
There are several points to note when using buffers
1: Avoid using hydrochloride, which has a corrosive effect on the steel.
2: The buffer is best to be used now. Often the cushion is a good culture of bacteria, the next day or place for a long time experiment will have many strange phenomena occur.
3: After the experiment can not use organic solvents directly excessively, organic solvents will be at the precipitation of salts, resulting in the liquid path or column blockage, available 95:5 water methanol rinse.
4: Use buffers to keep abreast of the ph range so you have a good idea.
5: When cleaning the liquid path and column, there is a temperature control that can be heated to 30 degrees Celsius and easy to flush.
6: long time with the buffer solution should observe the joints with or without precipitation. If there is white salt precipitation, you can consider an inevitable cycle with 10% nitric acid to flush the liquid path (remove the column, go 30ml, and then flush with five times water) can avoid the blockage of the liquid way.
7: Select the buffer to use reliable reagents to avoid unnecessary trouble caused by impure salts.
If the proportion of organic solvents in the mobile phase is very high and is not used for flushing the buffer salts, it is not washed out. Usually, the C18 column is rinsed with 5%~10% methanol. First, it is possible to rinse out the buffer salt, and then the pure organic solvent is used to protect the column. The best way is to use the same concentration as the mobile phase without salt for cleaning. But it is just a little slower. Using water is for rapid replacement. Generally, within 15 minutes is best, and a flow rate of 0.8 is better. If you flush with pure water, it is easy to cause the loss of bonded carbon chains, and it is better to rinse with a 5%~10% methanol aqueous solution. You can use pure water instead of the buffer in the mobile phase, and the organic phase remains unchanged. It is more prudent to flush the column in this way. The role of pH and buffer salts in the mobile phase of liquid phase (LC) and liquid mass (LCMS) systems.
Chromatographic column anomalies and solutions
The column pressure is related to the morphology of the silica matrix (such as amorphous or spherical silica), particle size, packing synthesis conditions, column loading conditions, mobile phase used, and temperature during analysis. The column pressure of different manufacturers’ columns may vary. Under the same mobile phase and temperature conditions, the column pressure of new columns from other manufacturers may vary by 4 or 5 MPa, especially between low-end and high-end columns, and this difference is more prominent. This is determined by the silica matrix selected by the column manufacturer and its production conditions, and such differences exist every day. It should also be noted that the column pressure has a specific relationship with the column efficiency. Usually, the column pressure of the column with high column efficiency is relatively higher, but the column with high column pressure may have low column efficiency.
There are two forms of column pressure increase during the use of chromatographic columns.
The first is that the column pressure rises slowly as the use time increases, which is normal.
The second is that the column pressure suddenly rises much higher during use (under conditions where the mobile phase and temperature have not changed). This sudden pressure increase is usually caused by improper staff operation.
Causes:
1) The sample is too dirty and not filtered before use, resulting in the column sieve plate clogging.
2) the sample contains impurities that are not very soluble in the mobile phase and precipitate out after mixing with the mobile phase, resulting in clogging of the plunger plate.
3) Using buffer salts and mishandling them, the buffer salts precipitate out in the column and clog the pores between the stopper plate and the bonded phase particles.
Solutions For the second one, i.e., the sudden increase in column pressure, there are usually several solutions as follows.
1) Invert the column and flush it with a mobile phase containing a more significant proportion of water.
2) Remove the sieve plate at the inlet end of the column and place it in water and methanol for sonication or replace it with a new column frit. If the column efficiency does not change, but the column pressure is still high, the contamination of the packing at the inlet end should be considered. Therefore, in addition to removing the HPLC frit at the inlet end and sonicating it, it is necessary to dig out part of the packing at the inlet end and check the color of the packing before digging out the packing. If the color of the packing has changed, it should be dug out until white packing is seen.
The filler can be obtained from the outlet end of another scrap column of the same brand and type. The filler is made into a paste with organic solvents such as methanol and loaded into the gap, pressed and scraped flat, and then mounted on the sieve plate.
Column use experience talk
Before using a chromatographic column, it is advisable to perform a column performance test and save the results as a reference for future evaluation of column performance changes. However, it should be noted that: the column performance may vary due to the differences in the conditions of the used sample, mobile phase, and column temperature; in addition, the column performance test is done following the conditions in the factory report of the column (the conditions used in the factory test are the best conditions), only in this way, the measured results are comparable.
1, The sample pretreatment
a. It is best to use a mobile phase to dissolve the sample.
b. Use the column to remove the sample’s strong polarity or irreversible impurities adsorption with the column packing.
c, using 0.45µm filter membrane filtration to remove particulate impurities.
2, Formulation of the mobile phase
Liquid chromatography is the separation of sample components in the mass exchange between the column packing and the mobile phase, so the mobile phase must have the following characteristics.
a. the mobile phase has a specific sample solubility to ensure that the sample components will not precipitate in the column (or be retained in the queue for a long time).
b. The mobile phase is inert and does not react chemically with the sample (except in exceptional cases).
c. the viscosity of the mobile phase should be as small as possible to get a good separation effect when using a more extended analytical column; at the same time to reduce the column pressure drop to extend the service life of the liquid pump (can be used to increase the temperature method to reduce the viscosity of the mobile phase).
d. the physical and chemical properties of the mobile phase to adapt to the detector used. Such as the use of a UV detector, it is best to use the lower absorption of UV solvent formulation.
e., the mobile phase boiling point should be. Otherwise reasonable it is easy to produce bubbles, resulting in experiments that need to be carried out.
f. After the mobile phase is prepared, it must be degassed. Removal of trace gases dissolved in the mobile phase is beneficial to both the detection and prevention of trace oxygen in the mobile phase and the sample.
Caution.
a. For reversed-phase columns, a methanol system is recommended due to the cheapness of methanol (except when acetonitrile must be used).
b. For the normal phase column, petroleum ether or purified hexane with a boiling range of 30-60°C is recommended as the mobile phase, and no purified hexane should be used. Water is best to use ultra-pure water (resistivity more significant than 18 megohms). Deionized and double distilled water contain phenolic impurities, which may affect the analytical results.
c. Water-containing mobile phase is most * prepared before the experiment, especially in summer use of buffer solution as the mobile phase does not overnight. It is best to add sodium azide to prevent bacterial growth.
d. The mobile phase should be filtered with a 0.45 µm membrane to remove particulate impurities.
e. Use HPLC grade solvent to prepare the mobile phase. Using a suitable mobile phase can extend the column’s life and improve the column’s performance.
Purpose of flushing the column
As long as it is an organic solvent, the viscosity should be manageable because the organic solvent can prevent bacterial growth. The purpose of flushing the column is to prevent bacterial growth from blocking the instrument system and the column. Generally, methanol and acetonitrile flush with each other is no problem, but acetonitrile is more expensive than methanol. How to balance a new HPLC column!
Reasons for retention time variation :
| 1. Column temperature variation |
Column thermostat |
| 2. Insufficient equilibrium between isocratic and gradient |
Equilibrate the column with at least 10 times the column volume of the mobile phase |
| 3. Insufficient buffer volume |
Use >25mmol/L buffer |
| 4. Column contamination |
Column rinsed daily |
| 5. Change in column conditions |
Stabilize injection conditions and adjust the mobile phase |
|
6. Column reaches lifetime quickly |
Use of guard columns |
Column head collapse
During use, the packing material sinks, and a small space appears at the inlet of the column, making the separation poor.
Remedy:
- Remove the column head screw, find a similar packing, moisten it with methanol and add it to the column.
- Repeat several times.
- Put on the screw and rinse with solvent for 1-2 hours to make it balanced.
Summary
It is necessary to use buffer salt correctly, which can prevent buffer salt precipitation and also achieve the purpose of improving the lives of the column. We may summarize its use in one sentence: it should be filtered before use and rinsed after use.
Post time: Dec-31-2022
