Yuzhong Technology - Industry Applications

Liquid chromatography (HPLC) is one of the commonly used testing instruments in analytical laboratories, and its application is becoming increasingly widespread. During the use of this instrument, various problems are inevitable and will directly affect the accuracy of the measured data and the normal operation of the instrument. If the operator can understand the causes of the faults, they can have a clear understanding of the methods to prevent and eliminate these faults, and can use the instrument correctly and maximize its performance. Today, we will share with you several issues that need to be noted when using a liquid chromatograph from the following aspects.

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1、 The issue of using test tubes

1. The cleanliness issue of test tubes. Liquid chromatography analysis is a highly sensitive analytical method, and the use of unclean test tubes can affect the accuracy of the test results. For example, when using methanol as a solvent to dissolve samples, the small test tube used has a rubber stopper as the lid. Therefore, there is a fixed retention time interference peak present during each injection. Later, it was confirmed that this interference peak is generated by the components dissolved by soaking the rubber stopper in methanol. After switching to a glass test tube, the interference peak was eliminated.

2. The problem of dissolution in plastic test tubes. In recent years, disposable plastic test tubes have brought great convenience to testers. However, it is important to pay attention to the phenomenon of organic solvents dissolving the test tube during use. When using this type of test tube to extract samples, some organic solvents (such as lvfang) may dissolve the tube wall, and these dissolved substances can sometimes generate signals on the detector, thereby interfering with the determination of the sample. At this point, the same experimental conditions can be used to test whether the extraction solution can generate interference signals on the detector when it does not contain the extracted substance. If there is indeed interference signal present, a glass test tube resistant to organic solvents can only be used.

3. The adsorption problem of the tested sample on the test tube wall. This issue should also be noted, otherwise it will also affect the accuracy of the test results. In therapeutic drug monitoring (TDM), some tested drugs such as amitriptyline and imipramine are prone to adsorb on the wall of glass test tubes. Therefore, polypropylene tubes should be used during operation. To prevent adsorption during extraction, 0.5% diethylenetriamine solution can be used as the extraction agent, which can effectively prevent adsorption.

2、 Problem with operating the injection valve

At present, the commonly used injection valve in analytical liquid chromatographs is the 7725 injection valve, which has a six way valve structure that makes the injection operation very convenient. However, improper use can also cause problems. For example, in the experimental process of liquid chromatography, there may sometimes be abnormal chromatographic peaks and poor reproducibility issues, which are mainly caused by improper operating methods. To solve such problems, the following aspects need to be addressed.

1. Control of injection volume. When using an injection valve for injection, the sample ring inside the valve is quantitative (the sample ring volume of a typical analytical injection valve is 20ul). Due to the radial velocity gradient of the sample solution injected into the injection valve in the pipeline of the sample ring during injection (i.e., the liquid flow velocity at the pipe axis is faster than that at the pipe wall). Therefore, in order to fill the sample ring with sample solution and accurately quantify it using an injection valve, the injection volume must be greater than twice the volume of the sample ring. If a syringe is used to control the injection volume, only half of the sample ring volume can be injected to prevent some samples from overflowing from the overflow tube and causing errors in quantitative analysis.

2. Cleaning issues with the injection valve. If there is residual sample from the last injection in the sample ring, it will inevitably contaminate the next injection sample. To prevent this phenomenon from happening, the following steps should be followed: a. The injection valve has two positions, INJECT and LOAD. Firstly, when in the LOAD position, use a syringe to inject the mobile phase into the injection valve for cleaning several times, with a dosage of approximately 40ul each time; b. Then, when the injection valve plate is manually pulled to the PROJECT position, clean the mobile phase again several times, with a dosage of 40ul each time; c. Finally, inject the sample into the injection valve.

Following the above steps can avoid contamination caused by the injection valve, thereby eliminating interference peaks and improving the accuracy of analysis results.

3. Blockage of the overflow pipe of the injection valve. Sometimes, the overflow pipe of the injection valve may become blocked, and when injecting the sample into the injection valve, the injection needle cannot be pushed. This malfunction is caused by blockage of the overflow pipe. The reason for blockage is mostly due to the use of salt solution as the mobile phase for dissolving the sample, and the salt crystallizes at the emptying port of the overflow pipe. At this point, a small beaker can be used to hold a small amount of distilled water and soak the overflow pipe slightly. The salt crystals at the port can be dissolved and the fault can be resolved. If the salt in the overflow tube can be washed out repeatedly with distilled water after each injection, this fault can be avoided.

3、 The problem of mobile phase (MOBLLE PHASE)

Methanol and acetonitrile are often used to prepare mobile phases in liquid chromatography analysis. The commonly used reagents in liquid chromatography are high-grade reagents, such as chromatographically pure reagents. When the requirements are not too strict, high-quality or even analytical grade reagents can also be used. UV detectors are commonly used in liquid chromatography analysis. Therefore, from the perspective of reducing baseline noise and improving analytical sensitivity, chromatography pure reagents with low UV absorption and low impurity content should be used.

1. Filtering of mobile phase. The prepared mobile phase must be filtered using a 0.5um pore size microporous membrane before use. This is because the solution contains many tiny particles that are difficult to detect with the naked eye. If they are not filtered out, they will clog the filters on the pump port and column head, thus blocking the normal channels of the mobile phase, increasing the resistance of the chromatographic column, increasing column pressure, and decreasing column efficiency. When encountering this situation, the filtered mobile phase should be used and the clogged filter should be removed and soaked in a 20% nitric acid solution for 20 minutes using an ultrasonic cleaning machine to remove blockages on the filter.

2. Degassing of mobile phase. The mobile phase must be degassed before use to remove as much gas as possible dissolved in the mobile phase. Otherwise, these gases will reduce the performance of the column packing and cause significant interference with the detector signal. There are various methods for degassing, such as ultrasonic degassing, vacuum degassing, nitrogen degassing, etc. Vacuum degassing method and nitrogen flow degassing method are currently the most commonly used degassing methods. After mixing water and methanol, a large number of bubbles will be generated. If used without degassing, the bubbles will enter the chromatographic column and detector, and will affect the normal operation of the analysis work.

4、 Use and maintenance of chromatographic columns

The chromatographic column is the most important component of liquid chromatography, and the performance of the chromatographic column plays a decisive role in determining whether the analyte can be well separated and determined. Therefore, in daily work, special attention should be paid to the correct use and maintenance of chromatography columns to extend their service life.

1. Use pre columns and protective columns. A pre column is installed between the pump and the injector, providing a balance for the mobile phase in the chromatographic column and preventing components or contaminants that can damage the column packing from entering the column. A guard column can prevent components that can be firmly adsorbed on the chromatographic column from entering the column, and the guard column should be the same as the packing of the chromatographic column. The pre column and protective column can be replaced frequently without the need for frequent replacement of the chromatographic column, which extends the service life of the chromatographic column.

2. Prevent gas from entering the chromatographic column. Some chromatographic columns (such as gel columns) do not allow bubbles to enter, otherwise the column efficiency will be reduced or even a small gas chamber that is difficult to expel will be formed. Therefore, in order to prevent bubbles from entering the chromatographic column, it is necessary to use a degassed mobile phase and strictly follow the following steps to install the chromatographic column. a. Remove the sealing screw at the entrance of the chromatographic column and observe for solvent leakage; b. If there is solvent leakage, the chromatography column can be connected to the pipeline to avoid the entry of bubbles; c. If there is no solvent leakage, it indicates that air has entered this end of the chromatographic column. At this time, the outlet end of the chromatographic column can be connected to the injection valve, and the chromatographic column can be flushed in the opposite direction with the mobile phase to remove the air inside the column. Using a small flow rate of 0.2ml/min to flush the chromatography column, if the solvent flow rate is too fast or the pressure suddenly increases, it will lead to a decrease in column performance; d. If the solvent flowing out does not contain bubbles, it means that the gas inside the column has been expelled. Then, connect the chromatography column in the correct direction so that the bubbles cannot enter the column.

3. Cleaning of chromatographic columns. In order to prevent the analyte and impurities from staying in the chromatographic column, the column should be cleaned promptly after each sample analysis is completed. Firstly, a solvent with strong elution ability for the tested sample should be used to elute the chromatographic column. Taking the commonly used reverse phase chromatography analysis method in analytical work as an example, because the substance that flows out first is a highly polar substance, 100% methanol or slightly less polar solvents such as isopropyl pure and tetrahydrofuran should be used to elute the less polar substance adsorbed in the column. The amount of eluent used is generally 20 times the volume of the column. If the mobile phase is a buffer solution, the chromatographic column should be washed with distilled water first to remove the salts inside the column, and then rinsed with a suitable solvent.

The gel column used in gel filtration chromatography usually uses buffer solution as mobile phase. After use, of course, it should be washed with distilled water. If it is a continuous operation, the buffer solution can be left in the column overnight, but the flow rate should be maintained at a low level (<0.5ml/min) to prevent the precipitation of buffer salts. If the mobile phase contains halides, even overnight, the chromatographic column must be washed clean with distilled water to prevent their corrosion to the column.

4. Storage of chromatographic columns. If the chromatographic column is not used temporarily, the following points should be noted when storing:

a. For short-term storage within a few days, the chromatographic column should be washed with solvent (for example, the gel column should be washed with distilled water), and then both ends of the chromatographic column should be sealed with sealing screws.

b. If the chromatographic column is not used for a long time, only the above methods can not be used to treat it. At this time, the chromatographic column should be filled with the solvent specified in the instruction manual of the chromatographic column. The reverse phase column generally uses methanol, the normal phase column can use n-hexane or heptane, and the gel column cannot use water. Because the growth of microorganisms in the column will reduce the column efficiency, at this time, the chromatographic column should be washed with 0.05% NaNs aqueous solution (preservative), and then the chromatographic column should be sealed tightly. When the chromatographic column is placed for a long time, it is necessary to seal both ends of the column tightly to prevent the drying shrinkage of the column packing caused by solvent evaporation, which can lead to a serious decrease in column efficiency.

c. The chromatographic column should be stored at room temperature. If placed in an environment below 0 ℃, the column will freeze, which will also lead to a decrease in column efficiency.

5、 The bimodal problem of chromatographic peaks

If impurities enter the chromatographic column after long-term use, it will cause the fixed phase at the inlet of the column to "plate" and collapse under the high pressure generated by the mobile phase, resulting in the normal chromatographic peaks of the analyzed sample components becoming bimodal. At this time, the chromatographic column can be repaired by the following method. Firstly, unscrew the fastening nut of the column head, and you will find that the fixed phase inside the column head has been compressed, and in severe cases, it can be retracted by more than 10mm. In this case, we can use a needle tip to remove the yellowed part of the mobile phase surface layer, and fill and compact the collapsed area with the same fixed phase. Then, tighten the fastening screws of the chromatography column, and the repair work is completed. When using the repaired chromatographic column to make samples again, the chromatographic peaks returned to normal.

The above briefly introduces the correct use and troubleshooting methods of chromatography from several aspects. In daily work, if these issues can be taken seriously and resolved in a timely manner, it will greatly extend the normal service life of the instrument and maximize its performance.