High-speed countercurrent chromatography
(High-speed Countercurrent CHROMatography, referred to as HSCCC), in 1982 by the National Institutes of Health Dr. Ito developed a new, continuous and efficient liquid-liquid chromatography technology, and other chromatographic techniques is that it does not require any solid state Carrier, thus avoiding the solid carrier surface reaction with the sample resulting in sample contamination, deactivation, denaturation and irreversible adsorption and other adverse effects. At the same time it also has a wide range of applications, fast, injection volume, low cost, high recovery rate advantages. Therefore, it has been widely used in the fields of biology, medicine, food, materials, cosmetics and environmental protection, especially in the field of separation and purification of active ingredients of natural products.
principle
High-speed countercurrent chromatography is a countercurrent chromatography method based on a one-way hydrodynamic equilibrium system, which was discovered by chance when studying the hydrodynamic balance of a rotating tube. When the coil rotates slowly, both phases in the coil are distributed from one end to the other. When one mobile phase is being eluted from one phase to the other, the remaining phase in the helix is about 50%, but this retention decreases as the mobile phase flow rate increases, causing the separation Reduced efficiency. But to speed up the spiral tube, the distribution of the two phases change. When the rotational speed reaches the critical range, the two phases will be completely separated along the length of the spiral tube. One of them occupies a section of the first end. We call this phase the first phase and the other section occupies the last section, called the trailing phase. High-speed countercurrent chromatography is the use of this one-way distribution of two-phase characteristics of the spiral in the high speed of rotation, if from the tail into the first phase, it will move through the tail phase to the head, Likewise, if the tail phase is fed from the head phase, it will move through the head phase towards the trailing end of the spiral. On detachment, one of the phases (stationary phase) is first injected into the spiral tube and the mobile phase is pumped from the appropriate end to carry an unlimited number of samples in the spiral tube. The faster the instrument, the more stationary phase retention, the better the separation effect, and greatly improve the separation speed, so called high-speed countercurrent chromatography.
Features
1. Wide range of applications, good adaptability.
Since the solvent system can have an infinite number of components and ratios, it can theoretically be applied to the separation of samples in any polarity range and has its unique characteristics in the separation of natural compounds. Since the stationary phase in the Teflon tube is liquid and does not require a solid support, the adsorption loss due to the use of a solid support in solid-liquid chromatography can be eliminated, which makes it particularly suitable for separating polar substances.
2. Easy to operate, easy to grasp.
The instrument is simple to operate and requires low pretreatment of the sample. General crude extracts can be prepared for separation or analysis.
3. High recovery rate.
The absence of a solid support eliminates the loss of the sample due to irreversible adsorption and degradation of the sample on the solid support, and the theoretical recovery of the sample is attainable. In the experiment as long as the adjustment of the separation conditions, generally have a high recovery rate.
Good reproducibility
If the sample does not have a strong surface activity, acid-base is not strong, even after multiple injections, the separation process remained stable, and the reproducibility is quite good.
5. High separation efficiency, large amount of separation.
Due to its different chromatographic separation methods, it can achieve gradient elution and reverse elution, and also can perform repeated injection, making it especially suitable for preparative separation. The product has high purity and large preparation volume.
Partition coefficient
The choice of solvent system is the key to the successful separation of the samples by the simultaneous selection of the two phases of the chromatographic separation process. The partition coefficients of the components in the sample determine the suitability of the solvent system. Therefore, the partition coefficient is determined by selecting the solvent system Important part. At present, the determination of partition coefficients using TLC, capillary electrophoresis, HPLC method, biological activity distribution ratio method and analytical HSCCC method.
Solvent system
The choice of solvent system is crucial for HSCCC separation. Unfortunately, the choice of solvent system so far has not been fully justified, but rather based on the actual experience gained. In general, the solvent system should meet the following requirements: The solvent system will not cause the sample decomposition or denaturation Sample components in the solvent system have a suitable partition coefficient, the partition coefficient is generally considered in the range of 0.2-5 is more appropriate , And the components of the distribution coefficient values should be sufficiently different, the separation factor is preferably greater than or equal to 1.5; solvent system does not interfere with the detection of the sample; in order to ensure the retention of the stationary phase of not less than 50% of the solvent system Stratification time does not exceed 30 seconds; the volume ratio of the upper and lower phase is appropriate, so as not to waste the solvent; as far as possible the use of volatile solvents to facilitate follow-up treatment to avoid the use of toxic solvents. According to the polarity of the solvent system can be divided into three categories of weak polarity, moderate polarity and strong polarity. The classic solvent systems are n-hexane-methanol-water, n-hexane-ethyl acetate-methanol-water, chloroform-methanol-water and n-butanol-methanol-water. In the experiment, according to the actual situation, the summary analysis and reference to the relevant monographs and literature, starting from the type of material to be separated to find similar separation examples, select the appropriate polarity of the solvent system, adjust the relative proportions of various solvents , The determination of the distribution coefficient of the target component, the final choice of a suitable solvent system.
Affect the separation factor
1. The retention of the stationary phase
In countercurrent chromatography, the amount of stationary phase remaining in the tube is an important factor affecting the resolution of the solute peak. High retention will greatly improve peak resolution.
The influence of the instrument on the retention value (external factor) Studies have shown that the ratio B between the radius of rotation r and the radius of revolution R of the helical tube support is a key factor that affects the retention of two mutually immiscible solvents in the rotating coil. A further increase in the value with a large diameter support will result in a one-way reversal of the hydrodynamic distribution of the hydrophilic solvent system; conversely, decreasing the value with a smaller diameter support will allow the single Reverse the direction of the fluid movement, and between the hydrophobic and hydrophilic solvents in the middle of the polar solvent, the distribution of the two phases will be affected by the centrifugal force conditions.
Effect of solvent system physical factors on retention (internal)
The physical factors of the solvent system such as the viscosity of the solvent have a great influence on the retention of the stationary phase. The low-viscosity solvent system is expected to retain the high stationary phase. The interfacial tension and the density difference between the two phases will delaminate the solvent near the critical point Time have a greater impact, generally to ensure that the stationary phase retention is appropriate, when the solvent system stratification is less than 30s.
2 speed of the impact
The rotational speed of the spiral tube on the two-phase solvent in the hydrodynamic equilibrium volume ratio, that is, the retention of stationary phase has a great impact, thus affecting the separation effect. Of course, not the faster the better, generally not good for the distribution of the solvent system, such as two-phase system with chloroform, but also fixed phase stratified relatively slow two-phase system, usually the higher the speed, The more easy to produce emulsification.
Effect of flow rate
The flow rate of the mobile phase also affects the distribution of the two phases. In general, the higher the mobile phase flow rate, the heavier the stationary phase, but the slower the flow rate will cause the separation time to be too long, resulting in a waste of solvent.
4. Temperature effects
Increasing the temperature The viscosity of the solvent can have a large effect. Generally, the higher the temperature, the higher the retention. The lower the temperature, the lower the retention. However, the general temperature can not be increased too much, because all organic solvents are used, Boiling point is very low.
5 associated detection technology
Currently, visible light detection, UV detection technology used in HSCCC more. But sometimes because the components of the visible or ultraviolet light without absorption, or the loss of the stationary phase led to the effusion emulsified, it can not be detected with common optical detector. Nowadays, there have been many studies on the interaction between HSCCC and mass spectrometry (MS), electron ionization mass spectrometry (EI-MS), chemical ionization mass spectrometry (FAB-MS), thermal spray mass spectrometry (TSP-MS) and electrospray ionization mass spectrometry (ESI-MS).
Technological development
In the 1960s, first in Japan, and later at the National Institutes of Medicine, an interesting phenomenon was discovered: immiscible biphasic solvents segregated in small, spiral-shaped tubes and Reverse convection between the two solvent phases can be achieved. Ito and its followers studied and designed a series of countercurrent chromatographic devices based on this principle. In the early days, a closed type of spiral planet centrifuge CPC (coil planet centrifuge) was used to separate dyes, proteins and cell particles . A few years later, Ito introduced the mechanism of circulation into the spiral column system, making it possible to achieve continuous elution, separation, detection and collection as well as modern chromatography, and to establish two basic circulation systems. Among them are the CCCs developed on the basis of the relatively simple hydrostatic equilibrium system HDES for analysis and separation, and are used for preparing the separated DCCC and the shift chamber CCC. On the other hand, based on the hydrodynamic equilibrium system HDES, a large preparative separation instrument under the action of gravity field and an analytical and semi-preparative separation instrument under the action of centrifugal force field are developed.
research Development
In recent years, solvent systems have become more and more widely used. Some have proposed the use of supercritical carbon dioxide as the mobile phase, using its advantages of high dispersibility, low viscosity, fluid properties and environmentally friendly solvents incomparable advantages of other compounds, as well as people The possibility of using refrigerant as a mobile phase is proposed. Others have also proposed the use of a three-phase solvent system for high-speed countercurrent chromatography, which allows good separation of samples in a wide range of polarities. The current three-phase solvent is only used for the separation of standard mixture,
Not yet used for the separation of specific natural products, I believe further development of complex natural products and the separation of drugs has great application prospects. The pH zone countercurrent chromatography is a newly developed preparative chromatography technique that increases the load capacity of a sample by more than 10-fold and allows highly concentrated substances at low levels. It is in the stationary phase and mobile phase by adding a pair of reagent - retention agent and eluent, the retention agent used to keep the sample components in the column, when the eluent containing mobile phase at a certain flow rate When passing through the stationary phase, the acid-base reaction eventually reaches equilibrium. The retention of the retention agent in the two phases is higher than that of the standard partition coefficient. The distribution coefficient of the retention reagent, the distribution coefficient of the solute and the difference between the calibration values determine the peak time of the solute. Depending on the Pka and the hydrophobicity of the different components To achieve separation. The chromatographic peaks are highly contiguous, rectangularly stacked, with very little overlap, much like the chromatographic peaks of alternative chromatograms.
Ion countercurrent chromatography is added to the appropriate phase in the stationary phase ligand, in order to improve the retention of solute in the stationary phase, improved peak resolution, has been widely used in natural medicine peptide components, alkaloids and amino acids and other separation.
Binary mode countercurrent chromatography elution with the same two-phase partitioned solvent system can be used both for normal phase elution and for reversed phase elution.
The combination of HSCCC and other technologies such as mass spectrometry is also a current research hotspot. It combines the diversity of HSCCC separation with the high-sensitivity detection and structural analysis of mass spectrometry. The prospect is very promising. In order to overcome the relative lag of HSCCC theoretical research, many researchers are engaged in theoretical research, trying to establish a sound theoretical basis to guide the choice of solvent system in order to make HSCCC develop from a separation technology as soon as possible to become a separation science.
HSCCC A unique liquid-liquid chromatographic technique without solid support is a practical separation and preparation technique for continuous and efficient separation using a wide range of solvent systems for the separation of samples of any polarity range from Micrograms, micro-liters of analysis isolated hundreds of milligrams, a few grams of preparation for purification, suitable for a large number of crude samples without strict intermediate separation, but also with mass spectrometer, infrared spectrometer and other analytical instruments Used for high-purity analysis, the application of a very broad prospect.
principle
High-speed countercurrent chromatography is a countercurrent chromatography method based on a one-way hydrodynamic equilibrium system, which was discovered by chance when studying the hydrodynamic balance of a rotating tube. When the coil rotates slowly, both phases in the coil are distributed from one end to the other. When one mobile phase is being eluted from one phase to the other, the remaining phase in the helix is about 50%, but this retention decreases as the mobile phase flow rate increases, causing the separation Reduced efficiency. But to speed up the spiral tube, the distribution of the two phases change. When the rotational speed reaches the critical range, the two phases will be completely separated along the length of the spiral tube. One of them occupies a section of the first end. We call this phase the first phase and the other section occupies the last section, called the trailing phase. High-speed countercurrent chromatography is the use of this one-way distribution of two-phase characteristics of the spiral in the high speed of rotation, if from the tail into the first phase, it will move through the tail phase to the head, Likewise, if the tail phase is fed from the head phase, it will move through the head phase towards the trailing end of the spiral. On detachment, one of the phases (stationary phase) is first injected into the spiral tube and the mobile phase is pumped from the appropriate end to carry an unlimited number of samples in the spiral tube. The faster the instrument, the more stationary phase retention, the better the separation effect, and greatly improve the separation speed, so called high-speed countercurrent chromatography.
Features
1. Wide range of applications, good adaptability.
Since the solvent system can have an infinite number of components and ratios, it can theoretically be applied to the separation of samples in any polarity range and has its unique characteristics in the separation of natural compounds. Since the stationary phase in the Teflon tube is liquid and does not require a solid support, the adsorption loss due to the use of a solid support in solid-liquid chromatography can be eliminated, which makes it particularly suitable for separating polar substances.
2. Easy to operate, easy to grasp.
The instrument is simple to operate and requires low pretreatment of the sample. General crude extracts can be prepared for separation or analysis.
3. High recovery rate.
The absence of a solid support eliminates the loss of the sample due to irreversible adsorption and degradation of the sample on the solid support, and the theoretical recovery of the sample is attainable. In the experiment as long as the adjustment of the separation conditions, generally have a high recovery rate.
Good reproducibility
If the sample does not have a strong surface activity, acid-base is not strong, even after multiple injections, the separation process remained stable, and the reproducibility is quite good.
5. High separation efficiency, large amount of separation.
Due to its different chromatographic separation methods, it can achieve gradient elution and reverse elution, and also can perform repeated injection, making it especially suitable for preparative separation. The product has high purity and large preparation volume.
Partition coefficient
The choice of solvent system is the key to the successful separation of the samples by the simultaneous selection of the two phases of the chromatographic separation process. The partition coefficients of the components in the sample determine the suitability of the solvent system. Therefore, the partition coefficient is determined by selecting the solvent system Important part. At present, the determination of partition coefficients using TLC, capillary electrophoresis, HPLC method, biological activity distribution ratio method and analytical HSCCC method.
Solvent system
The choice of solvent system is crucial for HSCCC separation. Unfortunately, the choice of solvent system so far has not been fully justified, but rather based on the actual experience gained. In general, the solvent system should meet the following requirements: The solvent system will not cause the sample decomposition or denaturation Sample components in the solvent system have a suitable partition coefficient, the partition coefficient is generally considered in the range of 0.2-5 is more appropriate , And the components of the distribution coefficient values should be sufficiently different, the separation factor is preferably greater than or equal to 1.5; solvent system does not interfere with the detection of the sample; in order to ensure the retention of the stationary phase of not less than 50% of the solvent system Stratification time does not exceed 30 seconds; the volume ratio of the upper and lower phase is appropriate, so as not to waste the solvent; as far as possible the use of volatile solvents to facilitate follow-up treatment to avoid the use of toxic solvents. According to the polarity of the solvent system can be divided into three categories of weak polarity, moderate polarity and strong polarity. The classic solvent systems are n-hexane-methanol-water, n-hexane-ethyl acetate-methanol-water, chloroform-methanol-water and n-butanol-methanol-water. In the experiment, according to the actual situation, the summary analysis and reference to the relevant monographs and literature, starting from the type of material to be separated to find similar separation examples, select the appropriate polarity of the solvent system, adjust the relative proportions of various solvents , The determination of the distribution coefficient of the target component, the final choice of a suitable solvent system.
Affect the separation factor
1. The retention of the stationary phase
In countercurrent chromatography, the amount of stationary phase remaining in the tube is an important factor affecting the resolution of the solute peak. High retention will greatly improve peak resolution.
The influence of the instrument on the retention value (external factor) Studies have shown that the ratio B between the radius of rotation r and the radius of revolution R of the helical tube support is a key factor that affects the retention of two mutually immiscible solvents in the rotating coil. A further increase in the value with a large diameter support will result in a one-way reversal of the hydrodynamic distribution of the hydrophilic solvent system; conversely, decreasing the value with a smaller diameter support will allow the single Reverse the direction of the fluid movement, and between the hydrophobic and hydrophilic solvents in the middle of the polar solvent, the distribution of the two phases will be affected by the centrifugal force conditions.
Effect of solvent system physical factors on retention (internal)
The physical factors of the solvent system such as the viscosity of the solvent have a great influence on the retention of the stationary phase. The low-viscosity solvent system is expected to retain the high stationary phase. The interfacial tension and the density difference between the two phases will delaminate the solvent near the critical point Time have a greater impact, generally to ensure that the stationary phase retention is appropriate, when the solvent system stratification is less than 30s.
2 speed of the impact
The rotational speed of the spiral tube on the two-phase solvent in the hydrodynamic equilibrium volume ratio, that is, the retention of stationary phase has a great impact, thus affecting the separation effect. Of course, not the faster the better, generally not good for the distribution of the solvent system, such as two-phase system with chloroform, but also fixed phase stratified relatively slow two-phase system, usually the higher the speed, The more easy to produce emulsification.
Effect of flow rate
The flow rate of the mobile phase also affects the distribution of the two phases. In general, the higher the mobile phase flow rate, the heavier the stationary phase, but the slower the flow rate will cause the separation time to be too long, resulting in a waste of solvent.
4. Temperature effects
Increasing the temperature The viscosity of the solvent can have a large effect. Generally, the higher the temperature, the higher the retention. The lower the temperature, the lower the retention. However, the general temperature can not be increased too much, because all organic solvents are used, Boiling point is very low.
5 associated detection technology
Currently, visible light detection, UV detection technology used in HSCCC more. But sometimes because the components of the visible or ultraviolet light without absorption, or the loss of the stationary phase led to the effusion emulsified, it can not be detected with common optical detector. Nowadays, there have been many studies on the interaction between HSCCC and mass spectrometry (MS), electron ionization mass spectrometry (EI-MS), chemical ionization mass spectrometry (FAB-MS), thermal spray mass spectrometry (TSP-MS) and electrospray ionization mass spectrometry (ESI-MS).
Technological development
In the 1960s, first in Japan, and later at the National Institutes of Medicine, an interesting phenomenon was discovered: immiscible biphasic solvents segregated in small, spiral-shaped tubes and Reverse convection between the two solvent phases can be achieved. Ito and its followers studied and designed a series of countercurrent chromatographic devices based on this principle. In the early days, a closed type of spiral planet centrifuge CPC (coil planet centrifuge) was used to separate dyes, proteins and cell particles . A few years later, Ito introduced the mechanism of circulation into the spiral column system, making it possible to achieve continuous elution, separation, detection and collection as well as modern chromatography, and to establish two basic circulation systems. Among them are the CCCs developed on the basis of the relatively simple hydrostatic equilibrium system HDES for analysis and separation, and are used for preparing the separated DCCC and the shift chamber CCC. On the other hand, based on the hydrodynamic equilibrium system HDES, a large preparative separation instrument under the action of gravity field and an analytical and semi-preparative separation instrument under the action of centrifugal force field are developed.
research Development
In recent years, solvent systems have become more and more widely used. Some have proposed the use of supercritical carbon dioxide as the mobile phase, using its advantages of high dispersibility, low viscosity, fluid properties and environmentally friendly solvents incomparable advantages of other compounds, as well as people The possibility of using refrigerant as a mobile phase is proposed. Others have also proposed the use of a three-phase solvent system for high-speed countercurrent chromatography, which allows good separation of samples in a wide range of polarities. The current three-phase solvent is only used for the separation of standard mixture,
Not yet used for the separation of specific natural products, I believe further development of complex natural products and the separation of drugs has great application prospects. The pH zone countercurrent chromatography is a newly developed preparative chromatography technique that increases the load capacity of a sample by more than 10-fold and allows highly concentrated substances at low levels. It is in the stationary phase and mobile phase by adding a pair of reagent - retention agent and eluent, the retention agent used to keep the sample components in the column, when the eluent containing mobile phase at a certain flow rate When passing through the stationary phase, the acid-base reaction eventually reaches equilibrium. The retention of the retention agent in the two phases is higher than that of the standard partition coefficient. The distribution coefficient of the retention reagent, the distribution coefficient of the solute and the difference between the calibration values determine the peak time of the solute. Depending on the Pka and the hydrophobicity of the different components To achieve separation. The chromatographic peaks are highly contiguous, rectangularly stacked, with very little overlap, much like the chromatographic peaks of alternative chromatograms.
Ion countercurrent chromatography is added to the appropriate phase in the stationary phase ligand, in order to improve the retention of solute in the stationary phase, improved peak resolution, has been widely used in natural medicine peptide components, alkaloids and amino acids and other separation.
Binary mode countercurrent chromatography elution with the same two-phase partitioned solvent system can be used both for normal phase elution and for reversed phase elution.
The combination of HSCCC and other technologies such as mass spectrometry is also a current research hotspot. It combines the diversity of HSCCC separation with the high-sensitivity detection and structural analysis of mass spectrometry. The prospect is very promising. In order to overcome the relative lag of HSCCC theoretical research, many researchers are engaged in theoretical research, trying to establish a sound theoretical basis to guide the choice of solvent system in order to make HSCCC develop from a separation technology as soon as possible to become a separation science.
HSCCC A unique liquid-liquid chromatographic technique without solid support is a practical separation and preparation technique for continuous and efficient separation using a wide range of solvent systems for the separation of samples of any polarity range from Micrograms, micro-liters of analysis isolated hundreds of milligrams, a few grams of preparation for purification, suitable for a large number of crude samples without strict intermediate separation, but also with mass spectrometer, infrared spectrometer and other analytical instruments Used for high-purity analysis, the application of a very broad prospect.
