Content1. What is HPLC system?
1.1 HPLC principle
1.2 HPLC diagram
1.3 Composition of HPLC system2. HPLC vs. FPLC
2.1 What is FPLC chromatography?
2.2 Similarities and differences between HPLC systems and FPLC systems3. How to buy HPLC system?
What is HPLC system?
High-performance liquid chromatography system (HPLC system) is a laboratory instrument that applies the principle of high-performance liquid chromatography. It is mainly applied to analyze organic compounds with a high boiling point that are not volatile, unstable by heat, and large molecular weight.
High-performance liquid chromatography (HPLC) is an important branch of chromatography. It has a wide range of applications and is not limited by the volatility and thermal stability of the analytes. Almost all compounds, including high boiling point, polar, ionic compounds, and macromolecules, can be analyzed and determined by HPLC, thus making up for the shortcomings of gas chromatography. About 20% of the known organic compounds can be analyzed by gas chromatography, while 80% need to be analyzed by high-performance liquid chromatography. HPLC is characterized by high separation efficiency, fast analysis speed, good detection sensitivity, and the ability to analyze and separate thermally unstable physiologically active substances with high boiling points that cannot be vaporized. HPLC is widely used in life science, environmental research, pharmaceutical research, food science, and pharmaceutical research.
The separation principle of chromatography is that when the components dissolved in the mobile phase pass through the stationary phase, due to the different size and strength of the action with the station phase, which can be adsorption, distribution, exclusion, affinity, and different retention time in the stationary phase, thus successively flowing out from the stationary phase.High-performance liquid chromatography
is a method based on classical liquid chromatography in which liquid is used as the mobile phase and a high-pressure infusion system is used to pump mobile phases such as single solvents with different polarities or mixed solvents and buffers of different proportions into a column equipped with a highly efficient stationary phase of very fine particles. By using this method, the components in the column are separated and then detected by the detector, thus enabling the analysis of the sample.
Composition of HPLC system
The HPLC system
generally consists of a chromatographic pump, sampler, column, detector, and data processing system. Among them, the chromatographic pump, column and detector are the key components. In addition, the gradient elution device, online degasser, autosampler, pre-column or protection column, column temperature controller, and so on, can be configured according to the needs of modern HPLC instruments and microcomputer control systems for automated instrument control and data processing. Preparatory HPLC instruments
are also equipped with automatic fraction collection devices.
A. Chromatographic pump
Chromatographic pump is one of the most important components of HPLC system
. Chromatography pumps are divided into constant pressure pumps and constant flow pumps according to the factor of constant output liquid. The performance of the pumps directly affects the quality of the whole system and the reliability of the analysis results. Chromatography pumps are divided into pneumatic pumps and mechanical pumps according to the mode of operation. Among the mechanical pumps, they can be subdivided into screw drive syringe pumps, single piston reciprocating pumps, double piston reciprocating pumps, and reciprocating diaphragm pumps.
a. Performance comparison of different high-pressure pumps
|Name||Constant voltage/constant current||Pulse||Mobile phase replacement||Gradient elution||Recirculation||Cost
|Pneumatic amplification pump||Constant voltage||No||Inconvenience||Two pumps required||No||High
|Screw drive syringe pump||Constant current||No||Inconvenience||Two pumps required||No||Medium
|Single piston reciprocating pump||Constant current||Yes||Convenience||Yes||Yes||Low
|Double piston reciprocating pump||Constant current||Small||Convenience||Yes||Yes||High
|Reciprocating diaphragm pump||Constant current||Yes||Convenience||Yes||Yes||Medium
B. Degassing device
Bubbles in the mobile phase solution are due to the presence of dissolved oxygen or mixed air. Once air bubbles enter the detector, they will appear as sharp noise peaks on the chromatogram. Small bubbles slowly accumulate and become large bubbles, which enter the flow path or the column and slow down the flow rate of the mobile phase or cause the flow rate to become unstable, resulting in a fluctuating baseline. To make matters worse, once air bubbles enter the column, it is time-consuming to expel them. In fluorescence detection, dissolved oxygen can also quench fluorescence. Dissolved gases may also cause oxidation of some samples or change the pH of the solution. At present, off-line ultrasonic oscillation degassing, online inert gas bubble purge degassing, and online vacuum degassing are usually used to degas the mobile phase of liquid chromatography.
C. Gradient elution device
HPLC is available in both isocratic and gradient elution. Isocratic elution is to keep the composition of the mobile phase constant during the same analysis cycle and is suitable for samples with a small number of components and little difference in properties. Gradient elution is to control the composition of the mobile phase, such as the polarity, ionic strength, and pH of the solvent, within one analysis cycle and is used for complex samples with a large number of components and different properties. The advantages of using gradient elution are shorter analysis time, better separation, improved peak shape, and higher detection sensitivity, while the disadvantage is that it often causes baseline drift and reduces reproducibility. Gradient elution consisting of two solvents can be mixed to any degree, resulting in a variety of elution profiles, namely linear-gradient, concave gradient, convex gradient, and step gradient.
HPLC systems are usually equipped with a six-way injection valve. The injection method of the six-way valve has two types: partial liquid loading method and complete liquid loading method.
a. Partial loading method injection
The injection volume should be no more than 50% (up to 75%) of the volume of the quantification ring and requires each injection volume to be accurate and identical. The accuracy and repeatability of this method of injection is determined by the proficiency of the syringe sampling, and easy to produce the peak spreading caused by the injection.
b. Complete liquid loading method injection
The injection volume should be no less than 5~10 times the volume of the quantification ring (at least 3 times), so as to completely replace the mobile phase in the ring, eliminate the wall effect and ensure the accuracy and repeatability of the injection.
E. Separation system
The separation system includes the column, the guard column, and the column temperature chamber.
Chromatography is a means of separation and analysis. Separation is the core of the technique, so the column, which is responsible for the separation, is the heart of the chromatographic system. A chromatographic column consists of a column tube, pressure cap, ferrule (sealing ring), sieve plate (filter plate), joint, screw, etc. The chromatographic packing is the material particles, usually spherical particles of 5-10 particle size, which are used to fill the column after the preparation process. Chromatographic columns can be divided into analytical and preparative types according to their uses, and the size specifications are also different.
The function of the protective column is to prevent the contamination of the column by the strongly adsorbed impurities, thus prolonging the life of the column. For different types of columns, the protective columns with the corresponding packing should be selected. Pay attention to the expiration date of the column core, which should be replaced regularly.
The purpose of the column temperature chamber is to maintain a constant temperature of the column to avoid changes in retention time and to ensure the stability of the separation.
The detector is a device used to continuously monitor the composition and content changes of the effluent after separation by the column and is one of the three key components of the HPLC instrument. The detector of HPLC requires high sensitivity, low noise, wide linear range, good repeatability, and a wide range of applications.
G. Data processing system
The operations of collecting, storing, displaying, printing, and processing test data so that the separation, preparation, or identification of samples can be carried out correctly are the functions of the data processing system.
2. HPLC vs. FPLC
What is FPLC chromatography?
FPLC is also known as fast protein liquid chromatography. The principle of FPLC is similar to that of high performance liquid chromatography. It is a modern liquid chromatography developed by introducing gas chromatography theory from classical liquid column chromatography, and reforming the phase body with a high-pressure infusion pump, high sensitive detector, gradient elution device, automatic collection device, and microcomputer.
FPLC system is specially designed for the separation of proteins, peptides, and polynucleotides, which is an important innovation of FPLC in recent years. It not only maintains the characteristics of HPLC such as rapid and high resolution but also has the characteristics of large column capacity, high recovery efficiency, and less deactivation of biological macromolecules. Therefore, it has been widely used in the separation of proteins, peptides, and oligonucleotides in recent years.
Similarities and differences between HPLC systems and FPLC systems
|HPLC||Both are equipped with infusion pumps, highly sensitive detectors, gradient elution devices, automatic collection devices, and computer control, and both are characterized by rapidity, high resolution, and high separation efficiency.||a. The HPLC piping system is mostly made of metal.|
b. Mobile phase is driven by reciprocating pumps or diaphragm pumps.
c. It is characterized by high pressure, small column inner diameter, and high analytical accuracy.
d. The maximum flow rate usually does not exceed 20 mL/min (some preparative HPLC can reach 300 mL/min).
e. Sometimes requires the use of a certain concentration of organic solvent, and is suitable for microsample detection or preparation of small amounts of purification.
|HPLC can analyze low boiling point low relative molecular mass molecules, high boiling point medium molecules, high molecular organic compounds (including polar and non-polar), ionic inorganic compounds, and thermally unstable biomolecules.
|FPLC||a. The piping system of FPLC is composed of high-pressure resistant plastic tubes.|
b. The mobile phase is mostly driven by a plunger pump.
c. It has the characteristics of medium and low pressure and large inner diameter of column.
d. Flow rate range 0-100 mL/min (maximum flow rate up to 1,000 mL/min).
e. The analytical accuracy is not as high as that of HPLC, and only conventional inorganic salt solutions are used.
f. It is suitable for large-scale purification of samples.
|FPLC is specifically designed for the separation and purification of proteins, peptides and polynucleotides.
How to buy HPLC system?
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