Content1. What is HPLC column heater?
1.1 HPLC working principle
1.2 What is retention time in HPLC2. Types of column chromatography
2.1 Different types of column chromatography3. Difference between HPLC and column chromatography
3.1 Liquid chromatography vs column chromatography4. How to buy HPLC column heater?
What is HPLC column heater?
Those who often do liquid phase analysis must be familiar with the column heater. This is because the column temperature chamber allows for more stable chromatographic retention without interference from temperature fluctuations, resulting in better chromatographic reproducibility. At the same time, it has better chromatographic separation efficiency and a more symmetrical peak shape and improves the mass transfer term of the Van Diemen equation. The HPLC column heater has the advantages of flexible placement, quick installation, easy observation, and a safe leak-proof design.
HPLC working principle
The components of the mixture are separated and purified in the chromatographic column
by interaction with the stationary and mobile phases. The stationary phase is a very small porous particle material present in the chromatographic column. The mobile phase is a solvent or mixture of solvents that is forced through the column under high pressure. The sample is injected with a syringe and mixed with the mobile phase through a valve connected to the sample loop, and the components of the sample are then passed through the column at different rates. Due to the different adsorption capacities between the components and the stationary phase, each component flows out of the column in turn and the appropriate detector converts the component concentration into a telecommunication number for transmission to the HPLC software of the computer. At the end of the run, the chromatogram is available in the HPLC software.
What is retention time in HPLC
The retention time in HPLC
is the time required for the sample to flow from the entry to the exit of the column. Different substances eluting on different columns with different mobile phases will have different retention times, so retention time is one of the more important parameters of chromatographic analysis.
Separated sample components from the beginning of the injection to the time after the column when the concentration of the component of the maximum value, that is, from the beginning of the injection to the peak of a component of the chromatographic time elapsed when the peak, known as the retention time
of this component. The retention time of HPLC
is expressed by RT, often in minutes (min) as the unit of time. The retention time is determined by the thermodynamic factors in the chromatographic process. Under certain chromatographic operating conditions, any substance has a defined retention time, which has the same role as the specific shift value and can be used as the basis for qualitative chromatographic analysis.
You should pay attention to retention time variation in HPLC
. In HPLC separation
, if the liquid chromatography retention time
is too short, it means that the separated substance is poorly retained on top of the stationary phase, and it is possible to interfere with the determination of the substance to be measured by mixing with impurities that are not retained. Of course, this is only a common situation when using detectors that are not very specific, such as UV and ELSD. However, if a detector with high specificities such as fluorescence or mass spectrometry is used, this interference will be greatly reduced or excluded.
Types of column chromatography
Chromatography, also known as liquid chromatography, and chromatographic analysis, is a separation and analysis method that has very wide applications in analytical chemistry, organic chemistry, biochemistry, and other fields. The common chromatographic methods include column chromatography, thin-layer chromatography, high-performance liquid chromatography, gas chromatography, and supercritical fluid chromatography.
Different types of column chromatography
A. Column chromatographic method
The original chromatographic method, which injects the stationary phase into a glass tube stuffed with cotton or filter paper at the lower end, spreads the stationary phase powder saturated by the sample on the top of the glass tube and elutes with the mobile phase. There are two common elution methods: one is top-down relying on the gravity of the solvent itself, and the other is bottom-up relying on capillary action.
There are also two different methods to collect the pure fraction after separation. One method is to accept the effluent solution directly at the end of the column, and the other method is to dry the stationary phase and then mechanically separate the individual bands and extract the component molecules by soaking the stationary phase in a suitable solvent.
Column chromatography is widely used for the separation of mixtures, including the separation of organic synthesis products, natural extracts, and biological macromolecules.
B. Thin-layer chromatography
Thin layer chromatography is a very widely used chromatographic method. In this chromatographic method, the stationary phase is coated on a metal or glass plate to form a thin layer, and the sample is spotted on one end of the plate with a capillary tube, pen, or other tools, after which the spotted end is immersed in the mobile phase and the mobile phase solvent is spread along the plate by capillary action.
Thin-layer chromatography is an inexpensive and easy-to-use method that is used for the crude measurement of samples and the detection of reaction processes in organic synthesis.
C. High-performance liquid chromatography (HPLC)
At present, high-performance liquid chromatography (HPLC) is one of the more widely used chromatographic analysis methods. A liquid chromatography system consists of a mobile phase reservoir bottle, infusion pump, injector, column, detector, and recorder, and its overall composition is similar to gas chromatography. The HPLC infusion pump requires a stable and balanced infusion volume; the injection system requires convenient injection and tight switching; as the viscosity of the liquid mobile phase is much smaller than that of the gas, the column of liquid chromatography is generally thicker and the length is much smaller than that of gas chromatography column to reduce the column pressure. HPLC is used in a wide range of applications, in almost every field of quantitative and qualitative analysis.
D. Gas chromatography
Gas chromatography is a method of separation by using gases such as helium or argon as the carrier gas (called mobile phase), and injecting the mixture sample into the column with filler (called stationary phase). The separated components are converted into electrical signals by a detector and recorded by a recorder.
E. Supercritical fluid chromatography
Supercritical fluid chromatography (SFC) is a chromatographic process that uses a supercritical fluid as the mobile phase to perform separation and analysis by relying on the solventizing ability of the mobile phase. It is a new technology developed and perfected in the 1980s. Supercritical fluid chromatography it easier to achieve higher column efficiency than liquid chromatography.
Difference between HPLC and column chromatography
Liquid chromatography vs column chromatography
A. Different types
a. High-performance liquid chromatography (HPLC)
a) Adsorption Chromatography
b) Partition Chromatography
c) Ion Chromatography
d) Size Exclusion Chromatography
e) bonded-phase chromatography
f) Affinity Chromatography
b. Liquid chromatographic method
a) The stationary phase of adsorption chromatography is the adsorbent, and the separation process of chromatography is carried out on the surface of the adsorbent, without entering the interior of the stationary phase.
b) In partition chromatography, both the mobile and stationary phases are liquids. The sample molecules quickly reach equilibrium partition between the two liquid phases and are separated using the difference in the partition coefficients of the components in the two phases, which is similar to an extraction process.
c) Ion exchange chromatography usually uses an ion exchange resin as the stationary phase. Generally, the sample ions are reversibly exchanged with the stationary phase ions, and the separation of chromatography is achieved due to the different exchange capacities of each component ion.
B. Different characteristics
a) High pressure. The mobile phase is a liquid, which is subject to high resistance when flowing through the column. To pass through the column quickly, high pressure must be applied to the carrier liquid.
b) High speed. Fast analysis and fast carrier fluid flow rate, much faster than classical liquid chromatography. Usually, it analyzes a sample in 15 to 30 minutes, and some samples can even be completed in 5 minutes. The analysis time is usually less than 1 hour.
c) High efficiency. High separation efficiency. The stationary 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 columns and gas chromatography.
b. Liquid chromatographic method
Using liquid as the mobile phase, the stationary phase can be in various forms, such as paper, thin plates, and filled beds.
|High-performance liquid chromatography (HPLC)||Liquid chromatographic method
|Due to the advantages of high resolution, high sensitivity, high speed, repeated use of columns, and easy collection of effluent components, high-performance liquid chromatography has been widely used in various fields such as biochemistry, food analysis, pharmaceutical research, environmental analysis, and inorganic analysis, and has become the most promising method for solving biochemical analysis problems.||Liquid chromatography is operated at room temperature and is not limited by the volatility and thermal stability of the sample. It is ideal for the separation and analysis of substances with large relative molecular weight, difficult to vaporize, not easily volatile or heat sensitive, ionic compounds and polymers, which account for about 70% to 80% of organic substances.
How to buy HPLC column heater?
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