This is the Part 6 of our following series “Q&A about Liquid Chromatography” that was published before. Which will cover some more Q&A, help you to know more about liquid Chromatography

Q1: The number of theoretical Plate decreased

A: It is normal that the separation performance of the column decreases gradually during use. However, if there is a sudden decrease or change, it is an abnormal phenomenon. At this time, the causes should be analyzed and targeted measures should be taken to avoid this situation, or the column regeneration should be carried out in time, so as to restore its performance as much as possible. There are many reasons for the decline of column efficiency, but the most basic reason is the change of packing characteristics or column bed structure caused by improper use.

  1. pH value of the mobile phase is the most important parameter in the use of the column. Chromatographic fillers based on silica gel are usually only suitable for use in the range of pH 2 ~ 8. Too low or too high pH value will cause the filler ligand to fall off or the matrix to dissolve. Sometimes, only the pH value of the sample solution will greatly damage the performance of the column.
  2. The substances with strong polarity or strong nonspecific adsorption in the nonspecific adsorption sample may be adsorbed on the surface of the filler to form a nonspecific adsorption layer, change the surface activity of the column, change the separation performance and reduce the column efficiency. For example, some plant pigments are often adsorbed on the column head, which deepens the color at the inlet end of the column and reduces the overall column efficiency; Some protein components will precipitate after stigma degeneration.
  3. Loss of packing, poor quality filter plate with improper manufacture, and the above pressure pulse, etc.
  4. Too long connecting pipeline with out of column effect, too large inner diameter of connecting pipeline, too large volume of detection cell, too large injection volume and so on will all lead to the decline of column efficiency.

Q2: The main causes and solutions of insufficient sensitivity of HPLC.


>Insufficient sample quantity. The solution is to increase the amount of samples.
>The sample did not flow out of the column. Change the mobile phase or column according to the chemical properties of the sample.
>The sample does not match the detector. Adjust the wavelength or change the detector according to the chemical properties of the sample.
>Detector attenuation too much. Adjust the attenuation.
>Detector time constant is too large. The solution is to reduce the time parameter.
>The detector pool window is polluted. The solution is to clean the tank window.
>There are bubbles in the detection cell. The solution is exhaust.
>The pressure measuring range of the recorder is improper. Adjust the voltage range.
>The flow rate of mobile phase is inappropriate. Adjust the flow rate.
>The detector and recorder exceed the correction curve. The solution is to check the recorder and detector and redo >the correction curve.

Q3: Is the flow phase in the column drained?

A: Many people who perform chromatographic separations have encountered situations where the mobile phase was not replenished promptly, and the pump drained the mobile phase from the solvent bottle, causing the HPLC system to stop working. Will this damage the column? Has the pump drained all mobile phases from the column? Is the column still functional? In fact, there is no damage to the column if the mobile phase is sucked dry by the pump. Even if the pump is filled with air, it will not pump air into the column. Because the pump can only transport liquid, but not air.

In contrast, it is also more likely to forget to cover the sealing caps at both ends of the column or the lid is too loose and the column dries out. Similarly, it is unlikely that the entire column will dry out. It is likely that only a few millimetres at both ends of the column will dry out because it takes quite a long time for the column to dry out because all the solvent has evaporated. Even if the column does dry out, it’s not necessarily hopeless. An attempt may be made to flush the column with a fully degassed, low surface tension solvent such as methanol to remove the gas. Lower surface tension helps to infiltrate the packing surface; The degassed solvent should be able to dissolve and remove trapped gas in the packing. It takes approximately one hour or more (at a flow rate of 1mL/min) for the column to be thoroughly infiltrated and returned to its normal state.

Q4: Effect of water quality on high performance liquid chromatography (HPLC)?

A: One of the most common problems in HPLC is the influence of pollutants in the solvent on the analysis results.
Particles: particles can damage pumps and syringes. Particles can also clog the column and melt it, which can lead to an increase in back pressure. The particles will also behave as another solid phase, which may change the composition of the sample.

Organic matter: The organic matter in ultrapure water may affect the resolution and integration of chromatographic peaks, may lead to ghost peaks, may also change the selectivity of stationary phase and affect the chromatographic baseline.

Ions: The change of ion concentration will affect the separation results, and some ions that can absorb UV will affect the peak. Some corrosive ions will also reduce the service life of accessories such as high-pressure infusion pump.

Colloid: The colloid will be irreversibly adsorbed on the stationary phase, affecting the separation effect of the column.
Microorganism: microorganism will block the chromatographic column, and its metabolites will increase organic matter, particles and other pollutants.

Bottled purified water and distilled water: At present, some purified water used in HPLC analysis. Because bottled purified water and distilled water are only ordinary pure water rather than ultrapure water, and the water still contains a small amount of ions, organics and other impurities, the preparation of liquid phase and eluent may not only pollute the expensive chromatographic column, but also affect the determination of flat baseline by HPLC.

Purification process of bottled purified water: It usually includes adsorption, filtration, reverse osmosis, etc., which has a good effect on the removal of particulate organic matter, but the removal of trace ions and organic matter can not meet the needs of high-sensitivity HPLC experiment. Moreover, because its packaging is usually PVC bottle, it has high oxygen permeability and certain chemical dissolution, which pollutes the purified water in the bottle, Especially after storage for a period of time, the water quality decreased more, so it was shown in the HPLC chromatogram. Although there was no specific absorption peak, there was serious unstable interference at the baseline.

Distilled water: Refers to pure water prepared by distillation. Distilled water can be divided into one and more times. After one-time distillation, the non-volatile components are removed from the container, and the volatile components enter the initial fraction of distilled water. Usually, only the middle part of the fraction is collected, accounting for about 60%. To obtain more pure water, alkaline potassium permanganate solution can be added to the primary distilled water to remove organic matter and carbon dioxide; Add nonvolatile acid (sulfuric acid or phosphoric acid) to make ammonia become nonvolatile ammonium salt. Through the HPLC detection of double distilled water, it is found that there are strong absorption peaks at 254 nm and 214 nm at 22 ~ 27 minutes, which indicates that there is organic pollution with strong hydrophobicity. The reason should be that the azeotropic phenomenon in the distillation process leads to the incomplete removal of some volatile organic compounds.

Ultra pure water machine: It integrates a variety of purification processes such as reverse osmosis, ion exchange, activated carbon adsorption, membrane filtration, ultrafiltration and UV oxidation. The conductivity of produced water reaches 18.2MΩ• cm. The produced water quality exceeds the national standard first-class water standard and is stable and measurable. Ultra pure water can be taken and used immediately without pollution due to storage. The water quality is guaranteed. Compared with bottled purified water or distilled water, It can better meet the needs of users for high-precision instrument analysis.

Q5: How to determine when to replace the Guard column?

A: Many experimenters consider replacing the guard column only when the peak bifurcation occurs when using the guard column, and the pressure rises beyond the alarm limit of the instrument. The analytical column is polluted because the guard column is not replaced for a long time, so the guard column needs to be replaced even according to the use situation.
The replacement frequency of the guard column is usually determined by experience and considered comprehensively according to each method / sample type, but many analysts will replace the guard column after completing a certain number of sample analysis or testing the performance indicators of some columns (such as increasing 10% peak broadening or tailing factor, or reducing 10% column efficiency (number of trays).

Q6: Leakage occurs in the use of guard column?

A: Leakage on the protective column when using the guard column, there are usually two places where the leakage is possible, one is the joint place, the other is the gap in the middle of the guard column; The joint leakage, refer to the above process. There are two possibilities for liquid leakage in the middle. One is that the guard column core may not be put to the end, because the peek at both ends of the guard column core may be slightly deformed during transportation. It is good to put it in carefully. The other is because the sleeve is hand tight, the surface of the hand tight with the grain, some customers think that as long as the hand can be tightened, but in fact, only by hand to tighten is not enough, need to use a wrench to slightly tighten, about the hand after the wrench to continue to twist 30 ~ 45 degrees Angle can be.

Q7: Guard Column Maintenance method.


  1. A new guard column core has no direction. From the first use, the guard column core gives direction, because solid particulate matter and strong retention matter always reach the guard column from the inlet end and begin to accumulate at this end. Therefore, the direction of the column core should be confirmed after it is used.
  2. When it is found that there are factors affecting the separation, such as too high column pressure, decreased column efficiency or decreased resolution in the analysis process, the guard column needs to be maintained. The maintenance methods are as follows:
    Mark the column sleeve to confirm the positive and negative directions of the guard column core; Remove the guard column, connect the guard column reversely without opening the ferrule (note that the chromatographic column cannot be connected at this time), wash it with methanol: water = 20 / 80 at the flow rate of 3ml / min for 10min, and then wash it with pure methanol at the flow rate of 3ml / min for 10min;
    After washing, connect the guard column directly, turn on the pump to remove the air in the guard column and connecting pipe, and then connect the chromatogram column for use as usual.

Q8: When should the pump seal ring be replaced?

A: Generally, when the chromatographic column pressure rises abnormally and the pump seal ring should be replaced regularly. It is recommended that each liquid chromatograph user establish a regular maintenance plan for the replacement of pump sealing ring. This should depend on the use of the pump and the composition of the mobile phase. Due to their easy wear characteristics, salt buffer greatly accelerates the wear of sealing ring and piston. When leakage is detected on the pump head under the piston, the retention time is unstable, or the system pressure is unstable, the sealing ring should be replaced. Please follow the following procedure to replace the sealing ring of the pump being maintained.

Q9: What is the difference between stigma collapse and phase collapse?


Stigma collapse : It refers to the looseness of column bed due to the loss of silica gel matrix or bonding phase in the filler after the chromatographic column has been used for a long time. Under the action of unidirectional pressure (the inlet end of chromatographic column bears most of the system pressure and the outlet end pressure is very small), the overall performance is the lack of filler at the inlet end of chromatographic column, and the gap of filler can be seen in serious cases.

Phase collapse: It refers to the phenomenon that the retention time of the conventional C18 column is gradually shortened after being washed with the mobile phase with high water content (> 90%) for a long time, and finally there may be no retention on the chromatographic column.

Q10: Are stigma collapse and Phase collapse maintained separately?


Stigma collapse: Generally, the chromatogram shows the decline of column efficiency, serious backward drag of peak shape, and sometimes serious forward drag. The collapse of the column head is mainly caused by the mobile phase and samples. If the pH exceeds the standard or is used near the critical point of the pH tolerance range of the chromatographic column for a long time at the flow rate of 1.0ml/min, it is easy to cause the collapse of the column head. In addition, the dissolution law of the silica gel matrix is that the control range of the mobile ph value is 2 ~ 8, and exceeding the range will cause serious damage to the chromatographic column. It is recommended to avoid the use of high concentration buffer salt, or high proportion of pure water phase, or the use of strong acid or strong alkaline chromatographic columns to ensure good tolerance of chromatographic columns.

Phase collapse: After the phase collapse of the chromatographic column occurs, it is usually washed continuously with pure acetonitrile or methanol to restore the chromatographic column with phase collapse to its original state. Complete recovery takes a long time, usually 30 minutes to 1 hour.

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