Principle of High Performance Liquid Chromatography Analysis of the use and principle of High Performance Liquid Chromatography
High Performance Liquid Chromatography (HPLC) is one of the most effective and widely used methods of separating, analyzing, and purifying organic compounds (including inorganic substances that can be transformed into organic compounds through chemical reactions).
In the known organic compounds, about 80% can be separated and analyzed by HPLC, and because of the mild conditions of this method, without destroying the sample, it is particularly suitable for high boiling point, difficult to gasification volatility, poor thermal stability of organic compounds and life substances. HPLC system is generally composed of an infusion pump, injector, chromatographic column, detector, data logging and processing devices.
Among them, the infusion pump, column, detector is the key part. Some instruments also have gradient elution device, online degasser, autosampler, and column or protection of the residence, column temperature controller, etc., modern HPLC instrument has a microcomputer control system, automated instrument control and data processing.
Preparatory HPLC instrument is also equipped with automatic fraction collection device. At present, the common HPLC instrument manufacturers abroad are Waters, Agilent (formerly HP), Shimadzu, etc., the domestic Shanghai Wufeng Scientific Instruments Co.
First, the infusion pump 1. Pump structure and performance of the infusion pump is one of the most important components of the HPLC system. The performance of the pump directly affects the quality and reliability of the analysis results.
The infusion pump should have the following performance: ① flow stability, its RSD should be less than 0.5%, which is related to the accuracy of qualitative and quantitative; ② flow range is wide, analytical type should be in the range of 0.1 ~ 10ml/min continuous adjustment, the preparation type should be able to reach 100ml/min; ③ output pressure is high, it should be able to achieve a general of 150-300KG/CM2: ④ liquid cylinder volume is small; ⑤ good sealing performance, durable, and the liquid cylinder is small; ⑤ good sealing performance, good sealing performance, good sealing performance, good sealing performance, durable, and the liquid cylinder is small. ⑤ good sealing performance, corrosion resistance. There are many types of pumps, according to the nature of infusion can be divided into constant pressure pumps and constant flow pumps.
Constant flow pumps can be further divided into spiral injection pumps, plunger reciprocating pumps and spacer reciprocating pumps according to their structure. Constant pressure pump by the column Yin influence, the flow is not stable; spiral pump cylinder is too large, these two pumps have been eliminated at present the most widely used is the plunger reciprocating pump.
Plunger reciprocating pump cylinder volume is small, up to 0.1ml, so easy to clean and replace the mobile phase, especially suitable for recirculation and gradient elution; change the motor speed can easily adjust the flow rate, the flow rate is not affected by the column pressure; pumping pressure up to 400KG/CM2. ADW main disadvantage is the output of the pulse is larger, now colorful double pump system to overcome.
Dual pumps can be divided into parallel and tandem according to the connection mode, generally speaking, parallel pump flow reproducibility is better (RSD of 0.1% or so, tandem pumps for 0.2 ~ 0.3%), but there are more chances of failure (due to more check valves), the price is more expensive. Second, the feeder general HPLC analysis commonly used six-way injection valve (to the U.S. RHEODYNE company's 7725 and 7725I type is the most common), the key components by the circular sealing gasket (rotor) and fixed base (stator).
High-pressure resistant (35~40MPA), accurate sample intake, good repeatability (0.5%), easy to operate. There are two types of six-way valve feeding methods: partial liquid loading method and complete liquid loading method.
① Partial liquid filling method, the sample volume should be no more than 50% of the volume of the quantitative ring (up to 75%), and requires each time the sample volume is accurate and the same. The accuracy and repeatability of this method of sampling is determined by the injector sampling proficiency, and easy to produce the peak broadening caused by sampling.
② completely filled with liquid injection method, the injection volume should be not less than 5 to 10 times the volume of the quantitative ring 9 at least 3 times, so as to completely replace the quantitative ring and the mobile phase, to eliminate the wall effect, to ensure the accuracy and repeatability of the injection. Third, the chromatographic column chromatography is a means of separation and analysis, separation is the core, so bear the role of separation of the chromatographic column is the heart of the chromatographic system.
The requirements of the column are high column efficiency, good selectivity, fast analysis speed. Commercially available for HPLC a variety of particulate packing good porous silica gel and silica gel as the substrate of the bonding phase, alumina, organic polymer microspheres (including ion exchange resins), porous carbon, etc., and its particle size is generally 3,5,7,10UM, etc., the theoretical value of the column efficiency of up to 5 ~ 160,000/m.
For general analysis, only 5000 plates are needed; for homologue analysis, as long as 500 can be used; for more difficult to separate the material pairs can be used as high as 20,000 columns, so the general length of the column of about 10~30CM can meet the needs of complex mixture analysis. Column efficiency is affected by factors inside and outside the column, in order to achieve the best efficiency of the column, in addition to the column outside the dead volume should be small, do not have a reasonable column structure (as far as possible to reduce the dead volume outside the packed bed) and loading technology.
Even with the best loading technique, the filling situation is always different in the center of the column and along the wall, and the parts near the wall are more loose and prone to groove flow, with faster flow rate, which affects the flow pattern of the flushing agent and broadens the spectral band, which is the wall effect. This pipe wall area is about 30 times the thickness of the material diameter from the pipe wall inward.
In general, the liquid chromatography system, the effect of the column effect on the column efficiency is much greater than the wall effect. Fourth, the detector HPLC detector is divided into two categories: general-purpose detectors and specialized detectors.
1. General-purpose detectors can continuously measure all the changes in the characteristics of the effluent of the chromatographic column, usually using differential measurement method, such detectors, including oscillometric refractive detector, dielectric constant detector, conductivity detector, etc., the general-purpose detector is widely used, but due to the response to the mobile phase, it is susceptible to changes in temperature, changes in the mobile phase and the components of the impact of changes in the noise and drift are larger, and lower sensitivity, can not be used with the mobile phase, and can not be used with a special detector, so it is not necessary to use a special detector. Lower sensitivity, can not be eluted with a gradient. 2. Specialized detector to measure the separation of the sample components of a certain characteristic of the change.
These detectors are sensitive to a physical or chemical property of a component of the sample that is not present in the mobile phase, or at least not under operating conditions. This type of detector includes ultraviolet detectors, fluorescence detectors, radioactive detectors, and so on.
How does HPLC work?
The working principle of HPLC; the high-pressure pump sends the mobile phase from the reservoir into the column through the injector, and then out of the detector's outlet, at which point the entire system is filled with the mobile phase. When the sample to be separated from the injector into the mobile phase flowing through the injector will be brought into the column for separation, separation of different components in order to enter the detector, the recorder will enter the detector to record the signal to get the liquid chromatogram.
High-performance liquid chromatography is based on classical chromatography, citing the theory of gas chromatography, in terms of technology, the mobile phase is changed to high-pressure delivery, the column is a special method of filling with small-size filler, so that the column efficiency is much higher than the classical liquid chromatography (the number of plates per meter can be up to several tens of thousands or hundreds of thousands), and at the same time after the column connected to the detector with high sensitivity, which can be used to carry on the continuous detection of the effluent. The column is connected to a high sensitivity detector for continuous detection of the effluent.
Extended Information
HPLC is equipped with either a high-pressure binary pump or a low-pressure quaternary pump, and the stroke volume of the pump, as well as the size of the mixer volume, will have an impact on the baseline noise level of the chromatogram, especially in the case of gradient elution. In general, the smaller the stroke volume of the pump and the larger the volume of the mixer, the smaller the pulse caused by the infusion, the higher the responsiveness to changes in the gradient, and the smoother the baseline,
In the application of binary pumps, it is important to note that, when the proportion of one of the mobile phases in the binary mixture is less than 5%, especially in the chiral splitting of some pharmaceutical intermediates and end-products by using positive isotope elution, it will be more likely to cause noise in the baseline. When using positive isocratic elution for chiral splitting of some pharmaceutical intermediates and end-products, it is better to use single pump premixing. Avoid due to the pump in the low proportion of the pump liquid precision is relatively poor, and lead to the chromatographic baseline stroke-related peaks,
Reference source; Sogou Wikipedia - High Performance Liquid Chromatography
The basic working principle of high performance liquid chromatography
The basic working principle of high performance liquid chromatography
High performance liquid chromatography system consists of a liquid reservoir, pumps, injectors The mobile phase in the reservoir is pumped into the system by a high-pressure pump, and the sample solution enters the mobile phase through the injector, and is loaded into the column (stationary phase) by the mobile phase. As the components in the sample solution have different partition coefficients in the two phases, when they move relative to each other in the two phases, after repeated adsorption-desorption partitioning process, the components have a big difference in the speed of movement, and they are separated into individual components in order to flow out from the column, and when they pass through the detector, they flow out from the column. When it passes through the detector, the concentration of the sample is converted into an electrical signal and transmitted to the recorder, and the data is printed out in the form of a graph.
What is the principle of HPLC
Principle: The mobile phase in the reservoir is pumped into the system by a high-pressure pump, and the sample solution enters the mobile phase through the injector, and is loaded into the column (stationary phase) by the mobile phase. As the components in the sample solution have different partition coefficients in the two phases, the components are separated into individual components in order to move out of the column. In the relative movement of the two phases, after repeated adsorption-desorption distribution processes, each component produces a large difference in the movement speed.
Separated into individual components sequentially out of the column, through the detector, the sample concentration is converted into an electrical signal transmitted to the recorder, the data can be printed out in the form of a graph, so that researchers can analyze.
Expanded Information:
① High pressure: the mobile phase is a liquid, flow through the column, the resistance is greater, in order to be able to quickly pass through the column, the carrier liquid must be high pressure. ② High speed: fast analysis, fast flow rate of the carrier liquid, much faster than the classic liquid chromatography, usually analyze a sample in 15 ~ 30 minutes, some samples can even be completed in 5 minutes, usually less than 1 hour.
③ High efficiency: high separation efficiency. Fixed phase and mobile phase can be selected to achieve the best separation effect, which is many times higher than the separation efficiency of industrial distillation column and gas chromatography.
④High sensitivity: UV detector up to 0.01ng, injection volume in the μL order. ⑤ Wide range of applications: more than 70% of organic compounds can be analyzed by HPLC, especially for the separation and analysis of compounds with high boiling point, large molecules, strong polarity, and poor thermal stability, showing advantages.
⑥ Column can be used repeatedly: a column can be used to separate different compounds ⑦ Sample volume is small, easy to recover: the sample is not destroyed after the column, you can collect a single component or do the preparation. In addition, high performance liquid chromatography column can be used repeatedly, the sample is not destroyed, easy to recover and other advantages, but there are also shortcomings, compared with gas chromatography, each has its own strengths and complement each other.
The disadvantage of high performance liquid chromatography is that there is an "off-column effect". Between the injection and the detector, in addition to any dead space outside the column (injector, column joints, connecting tubes and detection cell, etc.), if the flow pattern of the mobile phase changes, any diffusion and retention of the separated substances will significantly lead to peak broadening and a reduction in column efficiency.
HPLC detectors are not as sensitive as gas chromatography. The column used for HPLC is very thin (1~6 mm), and the particle size of the stationary phase used is also very small (a few μm to tens of μm), so the mobile phase is subject to a great deal of resistance to flow in the column, and the flow rate of the mobile phase is very slow at atmospheric pressure, which makes the column efficiency low and time-consuming.
In order to achieve rapid and efficient separation, it is necessary to apply high pressure to the mobile phase to accelerate its flow rate in the column. For this purpose, a high pressure pump is used for high pressure infusion.
High-pressure, high-speed is one of the characteristics of high-performance liquid chromatography. high pressure pumps used in HPLC should meet the following conditions: a. flow rate is constant, no pulsation, and a large range of adjustment (generally 1 ~ 10 mL/min); b. solvent corrosion resistance; c. a high infusion pressure; for general separation, the pressure of 60 * 10 ^ 5 Pa to meet the requirements of the high-efficiency separation, requires 150 to 300 * 10 ^ 3 Pa. To reach 150~300*10^5Pa.
1) Reciprocating plunger pump When the plunger is pushed into the cylinder, the check valve at the outlet of the pump head (upper part) opens, and at the same time, the check valve for the entry of the mobile phase (lower part) closes, and a small amount of fluid is output at that time. Conversely, when the plunger is pulled outward, the check valve for the entrance of the mobile phase opens, and the check valve for the exit closes at the same time, and a certain amount of the mobile phase is sucked into the cylinder by its reservoir.
This pump is characterized by a continuous supply of a constant volume of mobile phase independent of slight changes in resistance throughout the rest of the chromatographic system. (2) pneumatic amplification pump Its working principle is: the pressure of p1 low-pressure gas to promote the large area (SA) piston A, then in the small area (SB) piston B output pressure increases to p2 of the liquid.
The pressure increases the multiple depends on the area ratio of the two pistons A and B. If the ratio of the area of A and B is 50 : 1, then the pressure is 5 * Pa of gas can be obtained for the pressure of 250 * Pa of the output liquid. This is a constant pressure pump.
References:
How does an HPLC meter work?
High performance liquid chromatography is based on classical chromatography, citing the theory of gas chromatography, in terms of technology, the mobile phase is changed to high-pressure delivery (the highest delivery pressure of up to 4.9?107Pa); the column is a special method of packing with small particle size filler, which makes the column efficiency is much higher than that of the classical liquid chromatography (the number of plates per meter can be up to several tens of thousands or hundreds of thousands); at the same time after the column connected to a At the same time, the column is connected to a high sensitivity detector, which can continuously detect the effluent.
Features 1. High pressure: liquid chromatography with liquid as the mobile phase (called carrier liquid), the liquid flow through the column, resistance is greater, in order to quickly through the column, must be applied to the carrier liquid high pressure. In order to pass through the column quickly, high pressure must be applied to the carrier liquid, which is generally 150~350*105Pa.
2. High speed: The flow rate of the mobile phase in the column is much faster than that of classical chromatography, which is generally up to 1~10 ml/min. The analytical time required by HPLC is much less than that of classical liquid chromatography, which is generally less than 1 h.
3.
3. High efficiency: recently researched many new stationary phase, so that the separation efficiency is greatly improved. 4. High sensitivity: high performance liquid chromatography has been widely used in high sensitivity detector, further improving the sensitivity of the analysis.
Such as fluorescence detector sensitivity up to 10-11g. In addition, the sample size is small, generally a few microliters.
5. Wide range of adaptability: gas chromatography and high-performance liquid chromatography comparison: gas chromatography has a good ability to separate, high sensitivity, fast analysis, easy to operate and so on, but by the technical conditions of the limitations of the boiling point of too high a substance or a poor thermal stability of the material is difficult to apply the gas chromatography for analysis. High-performance liquid chromatography, only requires that the sample can be made into a solution, without the need for gasification, and therefore not subject to the limitations of the volatility of the sample.
For the high boiling point, poor thermal stability, relative molecular weight (greater than 400 or more) of the organic matter (these substances accounted for almost 75% of the total organic ~ 80%) in principle, can be applied to high-performance liquid chromatography for separation, analysis. According to statistics, in the known compounds, can be analyzed by gas chromatography accounted for about 20%, and can be analyzed by liquid chromatography accounted for about 70 ~ 80%.
High-performance liquid chromatography can be divided into high-performance gel chromatography, hydrophobic high-performance liquid chromatography, reversed-phase high-performance liquid chromatography, high-performance ion-exchange liquid chromatography, high-performance affinity liquid chromatography, as well as high-performance focusing liquid chromatography and other types according to the nature of its stationary phase. The principle of separating or analyzing various compounds by different types of HPLC is basically similar to that of the corresponding general liquid chromatography.
The difference is that high performance liquid chromatography is sensitive, fast, high resolution, good reproducibility, and must be carried out in the chromatograph. The main types of high-performance liquid chromatography and its separation principle According to the different separation mechanisms, high-performance liquid chromatography can be divided into the following main types: 1 . Liquid-liquid Partition Chromatography (Liquid-liquid Partition Chromatography) and Chemically Bonded Phase Chromatography (Chemically Bonded Phase Chromatography) The mobile phase and the stationary phase are both liquids.
The mobile phase and the stationary phase should be immiscible with each other (different polarity, to avoid the loss of the stationary liquid), and there is a clear interface. When the sample enters the column, the solute is distributed between the two phases.
When equilibrium is reached, it obeys the following equation: where cs - solute concentration in the stationary phase; cm - solute concentration in the mobile phase; Vs - volume of the stationary phase; Vm - volume of the mobile phase. llpc has a similarity with gpc in that the order of separation depends on K , the component with large K has a large retention value; however, there are also differences, in GPC, the mobile phase has little effect on K, LLPC mobile phase has a large effect on K.
a. Normal Phase liquid chromatography (Normal Phase liquid chromatography): the polarity of the mobile phase is less than that of the stationary liquid. b. Reverse Phase liquid chromatography (Normal Phase liquid chromatography): the polarity of the mobile phase is less than that of the fixed liquid. b. Reverse Phase liquid Chromatography: The polarity of the mobile phase is greater than the polarity of the stationary liquid.
c. Disadvantages of liquid-liquid partitioning chromatography: Although the polarity requirements of the mobile phase and the stationary phase are completely different, the stationary liquid is still slightly dissolved in the mobile phase; the mechanical impact of the mobile phase passing through the chromatographic column will cause the loss of the stationary liquid. Chemical bonding stationary phase (see later), developed in the late 1970s, can overcome the above disadvantages.
It is now widely used (70~80%). 2 . Liquid - solid chromatography The mobile phase is a liquid, and the stationary phase is an adsorbent (such as silica gel, alumina, etc.).
This is based on the different adsorption of substances to separate. The mechanism is: when the sample into the column, solute molecules (X) and solvent molecules (S) on the surface of the adsorbent active center of the competition adsorption (not into the sample, all the active center of the adsorbent adsorption of the S), can be expressed as follows: Xm + nSa ====== Xa + nSm where: Xm - solute molecules in the mobile phase; Sa - solvent molecules in the stationary phase; Xa - solvent molecules in the stationary phase; Xa - solute molecules in the stationary phase. solute molecules in the stationary phase; Sm - solvent molecules in the mobile phase.
When the adsorption competition reaction reaches equilibrium: K=[Xa][Sm]/[Xm][Sa] where: K is the adsorption equilibrium constant. [Discussion: the larger K, the larger the retention value.
] 3 . Ion-exchange Chromatography IEC uses an ion exchanger as the stationary phase.
IEC is based on the reversible exchange of ionizable ions on the ion exchange resin with solute ions of the same charge in the mobile phase, and separates these ions on the basis of their different affinities for the exchanger. [X-] = KX [-R4N+Cl-]/[Cl-] [Discussion: relationship between DX and retention value] Any substance that can be ionized in a solvent can usually be separated by ion exchange chromatography. 4 . Ion Pair Chromatography (Ion Pair Chromatography) Ion Pair Chromatography (IPC) is the process of controlling the retention behavior of solute ions by adding one (or more) ions with opposite charge to the solute molecule (called pairs of ions or counter ions) to the mobile or stationary phase, so that they can be combined with the solute ions to form a hydrophobic pair of ionic compounds.
The principle can be expressed in the following formula: X + aqueous phase + Y - aqueous phase === X + Y - organic phase where: X + aqueous phase - mobile phase to be separated from the organic ions (but also cations); Y - aqueous phase - the mobile phase of the opposite charge of the ion pairs (such as tetrabutylammonium hydroxide, hexadecyltrimethylammonium hydroxide, etc.); X + Y - the formation of.
Liquid chromatograph use and working principle
Working principle: the mobile phase through the infusion pump flow through the injection valve, mixed with the sample solution, flow through the column, adsorption in the column, separation, and finally each component was transformed into a signal by the detector, and the corresponding peaks appeared on the chromatographic workstation.
The use of liquid chromatography: first of all, the sample pretreatment, and then injection, injection is complete, cleaning the injection port, after each analysis, cleaning the channel, and finally shut down the instrument.
Expanded Information:
The basic theory used in liquid chromatography: tower plate theory and rate equations are also basically the same as the gas chromatography, but due to the gas chromatography in the liquid instead of gas chromatography gas as the mobile phase, and the nature of the liquid and gas are not the same. In addition, the instrumentation and operating conditions used in liquid chromatography are also different from those of gas chromatography, so there are some differences between liquid chromatography and gas chromatography.
The following are the main forces: ① Different operating conditions and application scope For gas chromatography, it is heated operation. Can only analyze the operating temperature can be vaporized without decomposition of the material, the high boiling point compounds, non-volatile substances, thermally unstable compounds, ionic compounds and polymer separation, analysis is more difficult, resulting in a certain degree of limitation of its application, according to statistics, only about 20% of the organism can be analyzed by gas chromatography.
The liquid chromatography is room temperature operation, not subject to sample volatility and thermal stability of the limitations, it is very suitable for relative molecular weight, difficult to vaporize, not easy to volatilize or heat-sensitive substances, ionic compounds and polymers separation and analysis, accounting for about 70% of the organic matter ~ 80%. ② liquid chromatography can complete the difficult separation work a. Gas chromatography mobile phase carrier gas is chromatographically inert, basically not involved in the distribution of the equilibrium process, and the sample molecules do not have an affinity effect, the sample molecules mainly interact with the stationary phase.
And in liquid chromatography, the mobile phase liquid also competes with the stationary phase for sample molecules, adding a factor to improve selectivity. Two or more liquids in different ratios can also be selected as mobile phase to increase the selectivity of the separation.
b. Liquid chromatography stationary phase types, such as ion exchange chromatography and exclusion chromatography, etc., as the analysis, the choice is large; and gas chromatography is not possible. c. Liquid chromatography is usually operated at room temperature, lower temperature, generally conducive to the selection of chromatographic separation conditions.
③ Since the diffusivity of liquids is 105 times smaller than that of gases, the mass transfer rate of solutes in the liquid phase is slow, and the out-of-column effect is particularly important; whereas in gas chromatography, the dilatation caused by the out-of-column region is negligible. ④ In liquid chromatography, the preparation of samples is simple, and the recovery of samples is easier, and the recovery is quantitative, suitable for large quantities of preparation, but liquid chromatography still lacks a universal detector, together with more complex and expensive.
In practical application, these two techniques are complementary to each other. In summary, liquid chromatography has the advantages of high column efficiency, high selectivity, high sensitivity, fast analysis, good repeatability, wide range of applications, the method has become one of the main means of modern analytical techniques.
At present, it is widely used in chemistry, chemical industry, medicine, biochemistry, environmental protection, agriculture and other scientific fields. High-performance liquid chromatography is widely used, almost all over the quantitative and qualitative analysis of various fields.
(1) Separation of mixtures High performance liquid chromatography only requires that the sample can be made into a solution, not subject to the limitations of the volatility of the sample, the mobile phase can be selected from a wide range of stationary phase, a wide range of species, and thus can separate the thermally unstable and non-volatile, dissociated and non-dissociated and a variety of molecular weight ranges of the material. The high resolution and sensitivity achieved by HPLC, in combination with sample pretreatment techniques, allows for the separation and simultaneous determination of substances with very similar properties, and the separation of trace components in complex mixtures.
And with the development of stationary phase, but also in the full maintenance of biochemical substances under the conditions of the completion of their separation. (2) Biochemical analysis Due to the high performance liquid chromatography has a high resolution, high sensitivity, fast speed, the column can be repeatedly used, the effluent components are easy to collect and other advantages, and therefore is widely used in biochemistry, food analysis, pharmaceutical research, environmental analysis, inorganic analysis and other fields, and has become the most promising method to solve the problem of biochemical analysis.
(3) Instrumental coupling High-performance liquid chromatography and structural instrumentation coupling is an important direction of development. High-performance liquid chromatography and mass spectrometry technology has been generally valued, such as the analysis of carbamate pesticides and polynuclear aromatic hydrocarbons: high-performance liquid chromatography and infrared spectroscopy coupled to the development of a very fast, such as in the analysis of environmental pollution in the determination of hydrocarbons in water and so on.
References:
What is the principle of liquid chromatography? What is it used for?
The principle of liquid chromatography: The mobile phase in the reservoir is pumped into the system by a high-pressure pump, and the sample solution enters the mobile phase through the injector, and is loaded into the column (stationary phase) by the mobile phase. As the components in the sample solution have different partition coefficients in the two phases, the components are separated into individual components by a large difference in the speed of movement, after repeated adsorption-desorption partitioning in the two phases for relative motion. After repeated adsorption-desorption distribution process, each component has a large difference in the speed of movement, and is separated into individual components sequentially out of the column, and when it passes through the detector, the concentration of the sample is converted into an electrical signal and transmitted to the recorder, and the data is printed out in the form of a graph.
Mainly used for high boiling point, difficult to gas compounds of the mixture through the column core eluent and to achieve separation. Used in biochemistry, biomedicine, environmental chemistry, petrochemical and other departments.
Extended information on liquid chromatography according to the stationary phase is liquid or solid, and is divided into liquid-liquid chromatography (LLC) and liquid-solid chromatography (LSC). Modern liquid chromatograph consists of high-pressure infusion pump, injection system, temperature control system, column, detector, signal recording system and other components.
Compared with the classical liquid column chromatography device, it is characterized by high efficiency, rapidity and sensitivity. High-performance liquid chromatograph mainly has a feeding system, infusion system, separation system, detection system and data processing system.
The injection system generally uses a diaphragm injection injector or a high-pressure injection booth to complete the injection operation, and the injection volume is constant. This is useful for improving the reproducibility of the analyzed samples.
Infusion system The system consists of three parts: high pressure pump, mobile phase reservoir and gradient meter. The general pressure of the high-pressure pump is l.47~4.4X10Pa, and the flow rate is adjustable and stable. When the high-pressure mobile phase passes through the chromatography column, it can reduce the diffusion effect of the samples in the column, and it can accelerate its moving speed in the column, which is beneficial for improving the resolution, recovering the samples, and maintaining the biological activity of the samples.
Separation systemThe system includes a chromatographic column, connecting tubes and a thermostat. Chromatographic column is generally 10 ~ 50cm in length (need to be two connected, can be added between the two a connecting tube), the inner diameter of 2 ~ 5mm, made of "high-quality stainless steel or thick-walled glass tubes or titanium alloy and other materials, living inside the diameter of 5 ~ 10μm particle size of the stationary phase is equipped with a (consisting of substrate and fixed liquid).