Like other conventional analytical instruments, in order to make HPLC work better and obtain reliable data during the experiment, you should first maintain it and make it in a healthy standby state, so that you can obtain ideal results smoothly when you use it for detection and analysis. Moreover, good operating habits can also prolong the service life of the instrument.
In daily use and maintenance, there are three main points: degassing, filtration and flushing. These three points belong to the most conventional operation requirements and are also essential processes in detection and analysis. The small editing meeting will be divided into three periods to explain to you. Today, we will take you to understand the specific causes and methods of degassing.
The existence of bubbles in the mobile phase is a common problem in the operation of HPLC system. Bubbles will cause the problem of pump output and false color harmonic peaks in the output results of the detector. Most bubble problems can be eliminated by degassing before using the mobile phase. The following is a brief summary of the main purpose of degassing:
- Prevent detection instability caused by changes in the amount of gas dissolved (in the liquid).
- Improve the reproducibility of retention time and chromatographic peak area.
- Prevent bubbles from causing spikes.
- Make the baseline stable and improve the signal-to-noise ratio.
- To prevent the failure caused by the generation of bubbles, differential refractive index detector: change the refractive index, UV detector (below 200nm): dissolved oxygen has absorption, fluorescence detector: dissolved oxygen has extinction effect.
- Reduce dead volume.
- Prevent filler oxidation.
As long as air remains dissolved in the mobile phase, bubble problems rarely occur. In principle, the manually equipped isocratic elution mobile phase can be used in the experiment without degassing, but the solution saturated with gas can be degassed only with a very small pressure drop. For example, when the mobile phase flows through the online filter of the solvent population, or when the mobile phase enters the detector solution pool with relatively low pressure. For this reason and in order to make the general HPLC operation reliable, we strongly recommend that all solvents used in RP chromatography must be degassed. Degassing will not cause many problems for normal phase HPLC, so degassing is optional when using normal phase chromatography. The amount of gas dissolved in the mobile phase to be removed varies according to the design of HPLC pumps. Some pumps can bear a large amount of gas dissolved in the mobile phase, while others need complete degassing to achieve reliable operation effect.
Common types of Degassing Method
- Vacuum degassing method: this method can use a vacuum pump to reduce the pressure to 0.05 ~ 0.07MPa to remove the dissolved gas. 60% – 70% of the gas dissolved in the mobile phase can be removed by vacuum degassing for 10-15 minutes. However, vacuum degassing will change the composition of mixed solvents, which will affect the reproducibility of the experiment, so it is mostly used for the simple analysis of single solvent system.
- Helium spray washing degassing method: helium spray washing is the most effective technology to remove the gas in the mobile phase. It mainly uses the characteristic that the solubility of helium in the liquid is lower than that of air. Under the pressure of 0.1MPa, it is introduced into the mobile phase storage container at the flow rate of about 60ml / min for 10 ~ 15min, which can effectively remove the dissolved air from the mobile phase and nearly 80% – 90% of the dissolved gas. Using an efficient distributed jet flow device, a volume of helium can remove almost all gases of the same volume from the mobile phase.
- Online degassing method: the main advantages of online degassing are simple operation, low fault and very effective
- Heating reflux method: this method has good degassing effect. However, there are still some deficiencies, that is, pay special attention to the cooling efficiency of the condensing tower during operation, otherwise the solvent will be lost and the proportion of mixed mobile phase will change.
- Ultrasonic degassing method: the most common degassing method in the laboratory. The main operation is to place the mobile phase to be degassed in the ultrasonic cleaner. The ultrasonic vibration time should not be too long to avoid the loss of volatile components caused by temperature rise, which is generally within 5min. However, compared with other degassing methods, it has the advantages of easy operation and short time. The disadvantage is that the degassing effect of this method is relatively poor.
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