1. How to choose a liquid chromatograph
A good quality liquid chromatography system should be considered from the following aspects:
One. The main technical indicators are excellent:
The first is how to look at the indicators. There are many indexes of liquid chromatographs, including pumps, detectors, chromatographic columns, etc. We think it depends on the main technical indicators. According to national standards, the main indicators of the instrument are noise, drift, minimum detection concentration, qualitative and quantitative repeatability, etc. These indicators should be placed in the system and viewed in the circuit to compare. It is necessary to connect all the unit devices, such as connecting the chromatographic column, the injection valve, and the mobile phase. Because you can only analyze after you are connected in the analysis, instead of just using a detector or a pump.
Then on this basis, we will compare these main indicators.
1. Noise refers to high-frequency noise and irregular fluctuations in the baseline caused by electrical components of the instrument, temperature fluctuations, linear pulses of voltage, and other non-solute effects. The size of noise is directly related to the detection sensitivity of the instrument. The greater the noise, the lower the detection sensitivity. For the detection of low-level samples, the noise of the instrument should be as low as possible, otherwise the excessive noise will cause unstable baseline and even affect the analysis results.
2. Minimum detection concentration (minimum detection limit)
It is an important parameter reflecting the sensitivity of the instrument. CL = 2 × Nd × C / H (CL: minimum detection concentration Nd: noise C: sample concentration minimum detection concentration H: sample peak height) can be seen from the above formula, the minimum detection concentration is proportional to noise, the greater the noise The larger the minimum detection concentration, the lower the sensitivity. Some manufacturers avoided this indicator, indicating that they were reluctant to compare noise based on the minimum detection concentration.
3. Drift refers to the distance that the baseline drifts from the origin within a period of time after the instrument is stable. It is usually used to measure the stability of the instrument. High-quality instruments can be stabilized in a relatively short period of time, thereby improving analysis efficiency to a certain extent.
4. Qualitative and quantitative repeatability is mainly an indicator to assess the stability of the instrument, which is very important for the analysis of samples. The stability of a good instrument should be very good, which requires the consistency of the retention time and content of multiple injections. The results of this can be convincing.
Some friends may think that these indicators are all detectors. That's right, but the conditions mentioned above are to be placed in the entire circuit and system to be compared. For example: the pulsation of the pump will directly affect the noise index, and the flow accuracy and precision index of the pump, as well as the poor sealing performance will also affect the relevant index. So look at the indicators systematically. For example, in the published indicators of some companies, the conditions for writing noise and drift indicators are empty pools or some simply do not write. This index only evaluates the optical and electrical characteristics of the UV detector, which is far from the actual situation. It does not test the pressure pulsation of the pump, the damping of the liquid flow circuit and the performance of the UV detector flow cell. Therefore, we think that some of the real level of the instrument can be reflected from the above main indicators.
The first is how to look at the indicators. There are many indexes of liquid chromatographs, including pumps, detectors, chromatographic columns, etc. We think it depends on the main technical indicators. According to national standards, the main indicators of the instrument are noise, drift, minimum detection concentration, qualitative and quantitative repeatability, etc. These indicators should be placed in the system and viewed in the circuit to compare. It is necessary to connect all the unit devices, such as connecting the chromatographic column, the injection valve, and the mobile phase. Because you can only analyze after you are connected in the analysis, instead of just using a detector or a pump.
Then on this basis, we will compare these main indicators.
1. Noise refers to high-frequency noise and irregular fluctuations in the baseline caused by electrical components of the instrument, temperature fluctuations, linear pulses of voltage, and other non-solute effects. The size of noise is directly related to the detection sensitivity of the instrument. The greater the noise, the lower the detection sensitivity. For the detection of low-level samples, the noise of the instrument should be as low as possible, otherwise the excessive noise will cause unstable baseline and even affect the analysis results.
2. Minimum detection concentration (minimum detection limit)
It is an important parameter reflecting the sensitivity of the instrument. CL = 2 × Nd × C / H (CL: minimum detection concentration Nd: noise C: sample concentration minimum detection concentration H: sample peak height) can be seen from the above formula, the minimum detection concentration is proportional to noise, the greater the noise The larger the minimum detection concentration, the lower the sensitivity. Some manufacturers avoided this indicator, indicating that they were reluctant to compare noise based on the minimum detection concentration.
3. Drift refers to the distance that the baseline drifts from the origin within a period of time after the instrument is stable. It is usually used to measure the stability of the instrument. High-quality instruments can be stabilized in a relatively short period of time, thereby improving analysis efficiency to a certain extent.
4. Qualitative and quantitative repeatability is mainly an indicator to assess the stability of the instrument, which is very important for the analysis of samples. The stability of a good instrument should be very good, which requires the consistency of the retention time and content of multiple injections. The results of this can be convincing.
Some friends may think that these indicators are all detectors. That's right, but the conditions mentioned above are to be placed in the entire circuit and system to be compared. For example: the pulsation of the pump will directly affect the noise index, and the flow accuracy and precision index of the pump, as well as the poor sealing performance will also affect the relevant index. So look at the indicators systematically. For example, in the published indicators of some companies, the conditions for writing noise and drift indicators are empty pools or some simply do not write. This index only evaluates the optical and electrical characteristics of the UV detector, which is far from the actual situation. It does not test the pressure pulsation of the pump, the damping of the liquid flow circuit and the performance of the UV detector flow cell. Therefore, we think that some of the real level of the instrument can be reflected from the above main indicators.
two. Easy to operate:
The ease of operation is very important for both novices and mature users. The simpler the operation, the better the analysis efficiency and the powerful help for future analysis method development. The WS-100 workstation software of Shanghai Wufeng Scientific Instrument Co., Ltd. has realized truly intelligent operation. It abandons the idea that the workstation software can only perform data processing in the past, and is the first to launch a software that integrates control and post-processing in China, which brings great convenience to users in operation and use.
The ease of operation is very important for both novices and mature users. The simpler the operation, the better the analysis efficiency and the powerful help for future analysis method development. The WS-100 workstation software of Shanghai Wufeng Scientific Instrument Co., Ltd. has realized truly intelligent operation. It abandons the idea that the workstation software can only perform data processing in the past, and is the first to launch a software that integrates control and post-processing in China, which brings great convenience to users in operation and use.
three. Systematic, overall development here refers to the overall development of the instrument, which is a complete system. This phenomenon is currently on the market, saying that my pump is imported, or how good the detector is, what many imported parts are used for assembly, and so on. In fact, this is a misunderstanding. Liquid chromatography is a complex system, not developed as a whole. There is no match between the various indicators, software and hardware, hardware and hardware, etc., the overall level will not be high, and it is not burdensome to users in terms of after-sales service. responsible.
The main advantages of the overall development are: unified technical indicators, communication coordination of each unit of the instrument, the ability to establish an overall digital evaluation system, which reflects the technology and development strength of the enterprise.
The products of well-known foreign instrument manufacturers are also developed as a whole, so they can guarantee the overall level of the instrument.
The main advantages of the overall development are: unified technical indicators, communication coordination of each unit of the instrument, the ability to establish an overall digital evaluation system, which reflects the technology and development strength of the enterprise.
The products of well-known foreign instrument manufacturers are also developed as a whole, so they can guarantee the overall level of the instrument.
four. Stable and reliable, and the low failure rate is known to everyone, so I won't say much.
2. The relationship and meaning between the minimum detection concentration of the liquid chromatograph and the optical path (volume) of the flow cell
When some domestic instruments published their technical indicators, most of them only wrote "noise and drift" indicators. Users feel that the indicators are similar, but they ignore the other two more important indicators: "Minimum detection concentration and optical path". What is the role of these two indicators? What do they mean?
Here to explain:
The minimum detection concentration is to test the sensitivity of the instrument. If the minimum detection concentration value is large, the sensitivity of the instrument is small, and it cannot reflect the real noise and drift level. For example: the minimum detection concentration of our company is less than 1 × 10-8g / ml (naphthalene / methanol solution), while some instruments are: 4 × 10-8g / ml (naphthalene / methanol solution). This means that our instrument has high sensitivity and can detect even smaller amounts of samples. Similarly, if the instrument of our company adjusts the minimum detection concentration to be the same as that of other products, it also shows that we can do 4 times lower noise and drift than other products.
It can be seen from this formula:
Minimum detection concentration = 2 × instrument noise × injected sample concentration / sample peak height value
What does the light path represent? Let's first look at the following formula, Beer's law:
A = log (I0 / I) = εCL
A = log (I0 / I) = εCL
A is the absorption rate; I0 represents the light intensity of the reference cell; I is the light intensity of the sample cell; ε is the molar absorption coefficient; C is the sample concentration; L is the optical path of the flow cell.
It can be seen that at the same concentration of the "C" sample, the larger the optical path of the "L" flow cell, the greater the "A absorption rate" of the instrument. The smaller the sample concentration that can be detected in this way. Why do some manufacturers make the optical path of the flow cell of the instrument very small, and some only have: 3.5mm, 4.5mm or 5.5mm, instead of our company's 8mm. This is because these manufacturers cannot effectively reduce the "noise and drift level" of the entire system, and can only sacrifice the optical path of the flow cell, that is, the detection sensitivity and minimum detection concentration of the instrument, to achieve the purpose of reducing the "noise and drift level" . In popular parables: HPLC is a stereo, and the optical path is the volume control key of the stereo. The smaller the optical path, the lower the volume. The noise and distortion of the ear by the ear (can be understood as the noise and the instrument) Drift). But it also shows that the manufacturing level of the overall system of these instruments is not high.
3. The requirements of liquid chromatograph for mobile phase
1. First of all, it must be of HPLC grade. I believe everyone has done it.
2. Degassing. Since the mobile phase contains a small amount of air, the pressure of the pump will generate bubbles in the flow cell, which will have a certain impact on the analysis, such as increased noise and even mask the signal peak. Therefore, the mobile phase must be degassed by ultrasound for more than 30 minutes before use.
3. Filter. Because there is very small waste in the mobile phase, if it is not filtered, it will occasionally get stuck in the one-way valve, resulting in pressure fluctuations. Many users do not pay much attention to this point, and do not know what caused the pressure fluctuations.
2. Degassing. Since the mobile phase contains a small amount of air, the pressure of the pump will generate bubbles in the flow cell, which will have a certain impact on the analysis, such as increased noise and even mask the signal peak. Therefore, the mobile phase must be degassed by ultrasound for more than 30 minutes before use.
3. Filter. Because there is very small waste in the mobile phase, if it is not filtered, it will occasionally get stuck in the one-way valve, resulting in pressure fluctuations. Many users do not pay much attention to this point, and do not know what caused the pressure fluctuations.
4. The influence of air bubbles on the detection in the high performance liquid chromatography system and its solution
When we conduct liquid chromatography analysis, we sometimes encounter such a problem: there are bubbles in the flow path of the system.
The presence of air bubbles will cause sharp noise peaks on the chromatogram, and in severe cases will cause a decrease in analytical sensitivity; when the air bubbles become larger and enter the flow path or column, the flow rate of the mobile phase will become slow or unstable, causing the baseline to fluctuate.
There are three main reasons for the above phenomenon: one is that the mobile phase solution often forms bubbles due to the dissolved oxygen or mixed with air; the second is that the air in the flow path cannot be driven out when the system starts to work; the third is when the sample is injected When inadvertently mixed with air.
In order to avoid such problems, the degassing of mobile phase must be emphasized during the actual analysis of liquid chromatography.
Commonly used degassing methods are as follows:
1. The helium blowing degassing method uses helium gas, which has a lower solubility in liquid than air, at a pressure of 0.1 MPa, and the mobile phase is passed into the mobile phase at a flow rate of about 60 ml / min for 10-15 min to drive off the dissolved gas. This method is used in all solvents, and the degassing effect is better, but because the price of helium is more expensive in China, this method is used less;
2. Heating and reflux method This method has good degassing effect;
3. The vacuum degassing method can use a micro vacuum pump at this time, and the dissolved gas can be removed by reducing the pressure to 0.05-0.07MPa. Obviously, using a water pump to connect the suction filter bottle and the G4 microporous glass funnel can complete the dual tasks of filtering mechanical impurities and degassing together. Since the vacuum will cause the composition of the mixed solvent to change, this method is suitable for degassing a single solvent system. The multi-solvent system should be degassed in advance and then mixed to ensure that the ratio after mixing remains unchanged.
4. Ultrasonic degassing method It is the most widely used degassing method in the laboratory at present. Place the prepared flow connection with the container into the ultrasonic water degassing for 10-20min. This method is easy to operate and can basically meet the requirements of daily analysis.
5. The online vacuum degassing method connects the vacuum degassing device to the liquid storage system in series and combines with the membrane filter to realize continuous vacuum degassing of the mobile phase before entering the infusion pump. The degassing effect of this method is significantly better than the above methods, and is suitable for multi-solvent systems.
Second, before the liquid chromatography system starts to work, you can use a syringe to connect the drain valve of the constant flow pump, draw in the mobile phase, and drive the air in the flow path clean.
Third, before injecting the sample, pay attention to exhaust the air in the sample syringe.
The presence of air bubbles will cause sharp noise peaks on the chromatogram, and in severe cases will cause a decrease in analytical sensitivity; when the air bubbles become larger and enter the flow path or column, the flow rate of the mobile phase will become slow or unstable, causing the baseline to fluctuate.
There are three main reasons for the above phenomenon: one is that the mobile phase solution often forms bubbles due to the dissolved oxygen or mixed with air; the second is that the air in the flow path cannot be driven out when the system starts to work; the third is when the sample is injected When inadvertently mixed with air.
In order to avoid such problems, the degassing of mobile phase must be emphasized during the actual analysis of liquid chromatography.
Commonly used degassing methods are as follows:
1. The helium blowing degassing method uses helium gas, which has a lower solubility in liquid than air, at a pressure of 0.1 MPa, and the mobile phase is passed into the mobile phase at a flow rate of about 60 ml / min for 10-15 min to drive off the dissolved gas. This method is used in all solvents, and the degassing effect is better, but because the price of helium is more expensive in China, this method is used less;
2. Heating and reflux method This method has good degassing effect;
3. The vacuum degassing method can use a micro vacuum pump at this time, and the dissolved gas can be removed by reducing the pressure to 0.05-0.07MPa. Obviously, using a water pump to connect the suction filter bottle and the G4 microporous glass funnel can complete the dual tasks of filtering mechanical impurities and degassing together. Since the vacuum will cause the composition of the mixed solvent to change, this method is suitable for degassing a single solvent system. The multi-solvent system should be degassed in advance and then mixed to ensure that the ratio after mixing remains unchanged.
4. Ultrasonic degassing method It is the most widely used degassing method in the laboratory at present. Place the prepared flow connection with the container into the ultrasonic water degassing for 10-20min. This method is easy to operate and can basically meet the requirements of daily analysis.
5. The online vacuum degassing method connects the vacuum degassing device to the liquid storage system in series and combines with the membrane filter to realize continuous vacuum degassing of the mobile phase before entering the infusion pump. The degassing effect of this method is significantly better than the above methods, and is suitable for multi-solvent systems.
Second, before the liquid chromatography system starts to work, you can use a syringe to connect the drain valve of the constant flow pump, draw in the mobile phase, and drive the air in the flow path clean.
Third, before injecting the sample, pay attention to exhaust the air in the sample syringe.
5. Daily maintenance and precautions of liquid chromatography system (Part 1)
Instrument installation conditions and precautions
The laboratory bench on which the instrument is installed should be level, stable, and have sufficient space for placement. Avoid installing the instrument in a place with corrosive gas, a lot of dust or a strong magnetic field, otherwise it will affect the normal operation of the instrument and shorten the life of the instrument.
Because the solvent used in the instrument is flammable and toxic, the room where the equipment is installed should be well ventilated and should have a device to exhaust the air, otherwise it may cause poisoning or physical discomfort, and may also cause fire.
More than four three-core power sockets (220V, 5 ～ 10A) should be installed and must have a special ground
Because the solvent used in the instrument is flammable and toxic, the room where the equipment is installed should be well ventilated and should have a device to exhaust the air, otherwise it may cause poisoning or physical discomfort, and may also cause fire.
More than four three-core power sockets (220V, 5 ～ 10A) should be installed and must have a special ground
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