High Performance Liquid Chromatography (HPLC) is also known as “High Pressure Liquid Chromatography”, “High Speed Liquid Chromatography”, “High Resolution Liquid Chromatography”, “Modern Column Chromatography” and so on. High Performance Liquid Chromatography is an important branch of chromatography. It uses liquid as the mobile phase and uses a high-pressure infusion system to pump a single solvent with different polarities or mixed solvents and buffers in different proportions into the stationary phase. The chromatographic column, after the components in the column are separated, enters the detector for detection, so as to realize the analysis of the sample. This method has become an important separation analysis technology application in the fields of chemistry, medicine, industry, agronomy, commodity inspection and legal inspection.
HPLC working principle
Each group of the mixture is separated and purified in the chromatographic column through the interaction with the stationary phase and the mobile phase. The stationary phase is a very small porous particle material that exists in the chromatographic column. The mobile phase is forced to pass through under high pressure. A solvent or mixed solvent of the chromatographic column. Inject the sample with a syringe, mix the sample with the mobile phase through a valve connected to the sample loop, and then each component in the sample passes through the chromatographic column at different rates. Due to the different adsorption capacity between each component and the stationary phase, each group fractions flow out of the chromatographic column one by one, and the component concentration is converted into an electrical signal by a suitable detector and transmitted to the HPLC software of the computer. After the run is over, the chromatogram can be obtained in the HPLC software.
HPLC technical advantages
It has the characteristics of high pressure, high efficiency, high speed, high sensitivity and high selectivity.
Recommendations for HPLC experiment water
High performance liquid chromatography mobile phase water should be ultrapure water with a conductivity of 18.2 M/cm (25℃), and pass through a 0.22um terminal filter to remove particles, organics, inorganic ions, CO2 and other gases.
The reason why it is recommended to use first-grade water with low TOC content in liquid chromatography experiments is that the impurities in the water may:
- Pollutants will produce peaks during gradient operation, which will aggravate the difficulty of peak identification and quantification;
- When the standard solution is prepared with ultrapure water containing impurities, the identification and quantification of the sample will not be too accurate;
- It may damage the HPLC column
Recommended ultrapure water machine for liquid chromatography experiments
Heal Force Smart Mini ultra-pure water machine is produced by all-plastic molds. It has a beautiful appearance, small size, and does not occupy laboratory space. In order to reduce the possibility of generating TOC, the whole machine adopts a simple design to reduce the pure water pipeline in the host. The built-in 6.5L Mini pure water tank minimizes the possibility of microbial growth, and it is equipped with a UV ultraviolet light that degrades the TOC content. It can be used for tap water in and out of pure water and ultra-pure water, and it is cost-effective.
Precautions for HPLC daily operation
Several problems should be paid attention to when choosing mobile phase:
- Use high-purity reagents as mobile phase to prevent long-term accumulation of trace impurities from damaging the chromatographic column and increasing the noise of the detector.
- Avoid the interaction between the mobile phase and the stationary phase to reduce the column efficiency or damage the column.
- The sample should have suitable solubility in the mobile phase to prevent precipitation and deposition in the column.
- The requirements of the detector should also be met when the flow is the same. When using an ultraviolet detector, the mobile phase should not have strong ultraviolet absorption. Generally, the detection wavelength should be greater than the cut-off wavelength of the mobile phase.
- Prevent any solid particles from entering the pump body, otherwise it will wear the plunger, the sealing ring cylinder and the one-way valve. Therefore, the mobile phase should be filtered in advance, and Millipore filter membrane (0.22μm or 0.45μm) can be used for filtration. The inlet of the pump should be connected with a sand filter rod (or sheet), and the filter of the infusion pump should be replaced frequently.
- The mobile phase should not contain any corrosive substances, and the mobile phase containing buffer should not remain in the pump, especially if the pump is stopped overnight or longer. After the analysis work is completed, it must be thoroughly cleaned with pure water, and then replaced with a solvent suitable for column storage and pump maintenance (for reversed-phase bonded stationary phase, it can be methanol or methanol and water).
- When the pump is working, pay attention to prevent the mobile phase in the solvent bottle from running out, otherwise the empty pump will wear the plunger, seal ring or cylinder, and eventually cause liquid leakage.
- The working pressure of the infusion pump should not exceed the specified maximum pressure, otherwise it will deform the high-pressure sealing ring and cause liquid leakage.
Maintenance of UV detector:
- Bubbles entering the flow cell will interfere with the baseline and are extremely difficult to discharge. A fully degassed mobile phase should be used to reduce the generation of bubbles.
- The system should be flushed regularly to avoid drying up the flow cell.
- The deuterium lamp (light source) in the detector will reduce the intensity of the deuterium lamp and the noise will continue to increase with the increase of use time. Frequent opening and closing of the detector will also shorten the life of the detector. Generally, the opening interval is less than 4 hours, and it is not recommended to turn off the detector).
- Dust is easy to cover the optical components under the action of static electricity, which reduces the light transmission efficiency, increases light scattering, and affects the analysis effect. Therefore, the environment around the detector should be kept clean.