According to the hardware module of liquid chromatograph, according to the sequence of mobile phase passing through: mobile phase solvent bottle → high pressure pump → injector → chromatographic column → detector → waste liquid. The correct maintenance of high performance liquid chromatograph in daily use and related precautions are described step by step.
1. Mobile phase solvent bottle
The solvent bottle is the starting point of the mobile phase, usually containing aqueous solution or organic phase solution.
1. Aqueous solution
For aqueous solutions, the primary problem is to prevent contamination. Although most of the water used in the liquid phase is sterilized, the vitality of bacteria is very strong. Under the appropriate temperature and light conditions, they will become active. If phosphate and other additives are added to the mobile phase, they will be even more powerful. Therefore, for solvent bottles, we have to do a very important job is to change the mobile phase frequently, often change often new.
2. Organic phase solution
For organic solutions, there is no need to worry about bacterial reproduction. However, the organic phase is easy to polymerize, especially when acetonitrile is easily polymerized under suitable light conditions, and some flocculent polymerization precipitates will appear in the bottle. In order to prevent polymerization, brown solvent bottle should be used when loading acetonitrile to avoid direct sunlight. When replacing acetonitrile, the remaining solution at the bottom of bottle should be discarded.
3. Cleaning and filtering
The filter head in the solvent bottle is used to prevent the particles and impurities in the solution bottle from entering the flow path system of the instrument. Its materials are usually divided into glass sintered quartz and stainless steel. If it is accidentally blocked, the mobile phase will not absorb liquid smoothly. Therefore, it must be cleaned. The glass material is usually made of dilute nitric acid, while the stainless steel material can be directly cleaned by ultrasonic cleaning.
4. Degassing of mobile phase
The purpose of degassing is to remove the dissolved gas in the mobile phase, make the infusion of chromatographic pump accurate, improve the reproducibility of retention time and chromatographic peak area, stabilize the baseline, increase the signal-to-noise ratio, prevent bubbles from causing spikes, so as to improve the performance of the detector, reduce the dead volume, prevent the oxidation of fillers, so as to protect the chromatographic column. At present, there are four commonly used degassing methods.
(1) Helium degassing method: using the solubility of helium in liquid is lower than that in air, continuous blowing helium degassing has better effect but higher cost;
(2) Heating reflux method: the effect is good, but the operation is complex, and there is toxic volatilization pollution;
(3) Vacuum degassing method: easy to remove organic phase;
(4) Ultrasonic degassing method: the mobile phase is placed in an ultrasonic container and vibrated by ultrasonic wave for 10-15min. This method is not effective, but easy to operate. In practice, the ultrasonic degassing method is simple and still widely used. Although this method sometimes causes the increase of gas solubility, it can basically meet the requirements of daily analysis.
2. Maintenance of high pressure pump
The pump is the core of liquid chromatography. The pump transports the mobile phase from the solvent bottle to the liquid flow system, and maintains the stability of flow and pressure under high pressure. The normal state of the high-pressure pump is the basis of accurate analysis of liquid chromatography, so we must pay attention to the maintenance of the pump.
In many cases, pump problems are reflected in the pressure, and pressure fluctuation is a common problem. The normal pressure fluctuation of the pump is usually less than 2%, and it is stable; the abnormal fluctuation is usually caused by bubbles and salt. If the bubble in the mobile phase is not removed by the degasser and reaches the pump, it will cause pressure fluctuation. Usually, we can clean the flow path and add the degassing flow again to solve the problem.
The fluctuation caused by salt is mainly due to the addition of buffer salt with high concentration in the mobile phase. When the salt containing mobile phase is mixed with the organic phase, the salt will precipitate slightly, resulting in abnormal pressure fluctuation. To solve this problem, it is necessary to reduce the salt concentration appropriately. If this kind of mobile phase must be used, it can be considered to add a certain proportion of water into the organic phase, and then appropriately increase the end point of the gradient.
3. Maintenance of injector
Another problem with the autosampler is the poor reproducibility of the peak area, which may be related to the sample taken by the autosampler. First, observe whether the liquid level of the sample is high enough to ensure that the sample can be absorbed by the injector. After eliminating this problem, check the settings of the autosampler. For some samples with high viscosity, reduce the sampling speed of the autosampler.
2. Fine operation
The operation points of the manual injector are roughly the same. The special flat head injection needle for liquid chromatography should be used. The needle should be inserted to the bottom when it is not in use. The needle should be cleaned before and after use.
4. Maintenance of chromatographic column
Chromatographic column is the key to the separation of compounds. The well maintained chromatographic column has a high number of plates, and the baseline of the instrument is stable. The chromatographic column is generally expensive, so the following matters should be paid attention to in the normal work:
(1) The column should not collide, bend or vibrate strongly. The valve or pipeline should be kept clean during installation;
(2) The mobile phase must be degassed before use, and high viscosity mobile phase should not be used or used less;
(3) When the sensitivity is satisfied, the injection volume should be as small as possible. If the sample is dirty, it should be purified or purified;
(4) After the analysis, the residual sample in the injection valve should be cleaned, and the chromatographic column should be cleaned with mobile phase or appropriate solvent;
(5) If the chromatographic column is not used for a long time, it should be preserved and sealed with appropriate organic solvent or supplemented with appropriate mobile phase regularly. The reversed phase column can be stored in pure methanol or acetonitrile, the normal phase column can be stored in pure hexane after strict dehydration, and the ion exchange column can be stored in water.
5. Maintenance of detector
At present, there are many kinds of detectors in the market, and each has its own characteristics. For the commonly used ultraviolet (VWD) / diode array (DAD) detectors, these two kinds of detectors are both UV type. Although the optical path design is different, the essential principle is the same.
1. Light source
The light source is a very important part of the detector. The light source has requirements on the emission energy. Once the energy is attenuated to a certain extent, there will be a series of problems that affect the use of the detector, such as increasing baseline noise and reducing sensitivity. Therefore, the light source is a consumable. Usually, the life span of UV lamp is 2000h. When the time limit is reached, we should pay special attention to the energy status of the lamp. We can judge it by the "lamp energy test" function in the instrument maintenance software. The test results will evaluate the energy of low, medium and high wavelengths respectively. Once the test results of a certain wavelength segment show failure, it means that the lamp needs to be replaced.
2. Detection cell
Another important part of the detector is the detection pool, also known as the flow pool. Usually, one of the most concerned problems is that the detection pool is blocked, because the detection pool is not very pressure resistant, so once it is blocked, it is likely to cause damage. In fact, the detection cell is not easy to be blocked because almost all the particulate impurities will be blocked by the chromatographic column, so the things blocking the detector are basically not from the sample, which is likely to be "generated" later, for example, the residual salt containing mobile phase in the detection cell leads to salt precipitation.
6 disposal of waste liquid
Most of the waste liquid of liquid chromatograph contains organic solvents, which will cause certain harm to human body and pollute the environment. It is strictly forbidden to dump at will and should be treated in time. At present, most laboratories in China send waste liquid to special waste liquid treatment station for centralized harmless treatment.
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1. Mobile phase solvent bottle
The solvent bottle is the starting point of the mobile phase, usually containing aqueous solution or organic phase solution.
1. Aqueous solution
For aqueous solutions, the primary problem is to prevent contamination. Although most of the water used in the liquid phase is sterilized, the vitality of bacteria is very strong. Under the appropriate temperature and light conditions, they will become active. If phosphate and other additives are added to the mobile phase, they will be even more powerful. Therefore, for solvent bottles, we have to do a very important job is to change the mobile phase frequently, often change often new.
2. Organic phase solution
For organic solutions, there is no need to worry about bacterial reproduction. However, the organic phase is easy to polymerize, especially when acetonitrile is easily polymerized under suitable light conditions, and some flocculent polymerization precipitates will appear in the bottle. In order to prevent polymerization, brown solvent bottle should be used when loading acetonitrile to avoid direct sunlight. When replacing acetonitrile, the remaining solution at the bottom of bottle should be discarded.
3. Cleaning and filtering
The filter head in the solvent bottle is used to prevent the particles and impurities in the solution bottle from entering the flow path system of the instrument. Its materials are usually divided into glass sintered quartz and stainless steel. If it is accidentally blocked, the mobile phase will not absorb liquid smoothly. Therefore, it must be cleaned. The glass material is usually made of dilute nitric acid, while the stainless steel material can be directly cleaned by ultrasonic cleaning.
4. Degassing of mobile phase
The purpose of degassing is to remove the dissolved gas in the mobile phase, make the infusion of chromatographic pump accurate, improve the reproducibility of retention time and chromatographic peak area, stabilize the baseline, increase the signal-to-noise ratio, prevent bubbles from causing spikes, so as to improve the performance of the detector, reduce the dead volume, prevent the oxidation of fillers, so as to protect the chromatographic column. At present, there are four commonly used degassing methods.
(1) Helium degassing method: using the solubility of helium in liquid is lower than that in air, continuous blowing helium degassing has better effect but higher cost;
(2) Heating reflux method: the effect is good, but the operation is complex, and there is toxic volatilization pollution;
(3) Vacuum degassing method: easy to remove organic phase;
(4) Ultrasonic degassing method: the mobile phase is placed in an ultrasonic container and vibrated by ultrasonic wave for 10-15min. This method is not effective, but easy to operate. In practice, the ultrasonic degassing method is simple and still widely used. Although this method sometimes causes the increase of gas solubility, it can basically meet the requirements of daily analysis.
2. Maintenance of high pressure pump
The pump is the core of liquid chromatography. The pump transports the mobile phase from the solvent bottle to the liquid flow system, and maintains the stability of flow and pressure under high pressure. The normal state of the high-pressure pump is the basis of accurate analysis of liquid chromatography, so we must pay attention to the maintenance of the pump.
In many cases, pump problems are reflected in the pressure, and pressure fluctuation is a common problem. The normal pressure fluctuation of the pump is usually less than 2%, and it is stable; the abnormal fluctuation is usually caused by bubbles and salt. If the bubble in the mobile phase is not removed by the degasser and reaches the pump, it will cause pressure fluctuation. Usually, we can clean the flow path and add the degassing flow again to solve the problem.
The fluctuation caused by salt is mainly due to the addition of buffer salt with high concentration in the mobile phase. When the salt containing mobile phase is mixed with the organic phase, the salt will precipitate slightly, resulting in abnormal pressure fluctuation. To solve this problem, it is necessary to reduce the salt concentration appropriately. If this kind of mobile phase must be used, it can be considered to add a certain proportion of water into the organic phase, and then appropriately increase the end point of the gradient.
3. Maintenance of injector
Another problem with the autosampler is the poor reproducibility of the peak area, which may be related to the sample taken by the autosampler. First, observe whether the liquid level of the sample is high enough to ensure that the sample can be absorbed by the injector. After eliminating this problem, check the settings of the autosampler. For some samples with high viscosity, reduce the sampling speed of the autosampler.
2. Fine operation
The operation points of the manual injector are roughly the same. The special flat head injection needle for liquid chromatography should be used. The needle should be inserted to the bottom when it is not in use. The needle should be cleaned before and after use.
4. Maintenance of chromatographic column
Chromatographic column is the key to the separation of compounds. The well maintained chromatographic column has a high number of plates, and the baseline of the instrument is stable. The chromatographic column is generally expensive, so the following matters should be paid attention to in the normal work:
(1) The column should not collide, bend or vibrate strongly. The valve or pipeline should be kept clean during installation;
(2) The mobile phase must be degassed before use, and high viscosity mobile phase should not be used or used less;
(3) When the sensitivity is satisfied, the injection volume should be as small as possible. If the sample is dirty, it should be purified or purified;
(4) After the analysis, the residual sample in the injection valve should be cleaned, and the chromatographic column should be cleaned with mobile phase or appropriate solvent;
(5) If the chromatographic column is not used for a long time, it should be preserved and sealed with appropriate organic solvent or supplemented with appropriate mobile phase regularly. The reversed phase column can be stored in pure methanol or acetonitrile, the normal phase column can be stored in pure hexane after strict dehydration, and the ion exchange column can be stored in water.
5. Maintenance of detector
At present, there are many kinds of detectors in the market, and each has its own characteristics. For the commonly used ultraviolet (VWD) / diode array (DAD) detectors, these two kinds of detectors are both UV type. Although the optical path design is different, the essential principle is the same.
1. Light source
The light source is a very important part of the detector. The light source has requirements on the emission energy. Once the energy is attenuated to a certain extent, there will be a series of problems that affect the use of the detector, such as increasing baseline noise and reducing sensitivity. Therefore, the light source is a consumable. Usually, the life span of UV lamp is 2000h. When the time limit is reached, we should pay special attention to the energy status of the lamp. We can judge it by the "lamp energy test" function in the instrument maintenance software. The test results will evaluate the energy of low, medium and high wavelengths respectively. Once the test results of a certain wavelength segment show failure, it means that the lamp needs to be replaced.
2. Detection cell
Another important part of the detector is the detection pool, also known as the flow pool. Usually, one of the most concerned problems is that the detection pool is blocked, because the detection pool is not very pressure resistant, so once it is blocked, it is likely to cause damage. In fact, the detection cell is not easy to be blocked because almost all the particulate impurities will be blocked by the chromatographic column, so the things blocking the detector are basically not from the sample, which is likely to be "generated" later, for example, the residual salt containing mobile phase in the detection cell leads to salt precipitation.
6 disposal of waste liquid
Most of the waste liquid of liquid chromatograph contains organic solvents, which will cause certain harm to human body and pollute the environment. It is strictly forbidden to dump at will and should be treated in time. At present, most laboratories in China send waste liquid to special waste liquid treatment station for centralized harmless treatment.
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