Degassing the mobile-phase

Certain specific problems arise with the use of alkylamine-substi-tuted stationary phases. Since amines are readily oxidized, degassing the mobile phase and avoiding solvents that may contain peroxides, e.g., diethyl ether and tetrahydrofuran, are recommended. Samples or impurities in the mobile phase containing ketone or aldehyde groups may... 

With vacuum degassing, the mobile phase is stirred in a side-armed flask or mobile phase handling system under... 

This is a combination inlet filter (0.2-(im PTFE) and degassing Alter. An aspiration tube is connected through the off-center union, which pulls a vacuum through the Alter and degasses the mobile phase as it is pulled into the pump head. 

The effect of the mobile-phase composition on the operation of the different interfaces is an important consideration which will be discussed in the appropriate chapter of this book but mobile-phase parameters which affect the operation of the interface include its boiling point, surface tension and conductivity. The importance of degassing solvents to prevent the formation of bubbles within the LC-MS interface must be stressed. 

The mobile phase consisted degassed distilled water containing 1.0 grams/liter of Aeorosol(B)-OT and varying amounts of sodium nitrate, NaNOj. The detector was a DuPont Model 840 UV photometer with a fixed wavelength of 254 nm. 

Although the method is simple and straightforward, there are a few important points to consider. First, since 200 pL are being injected on to a 2-mm diameter HPLC column, compatible solvents must be injected on to the column and the amount of acetonitrile used in the prepared samples should be exactly as directed. Second, the temperature of the column and the use of a degassing system for the mobile phase are critical components required to guarantee reproducible chromatography. In addition, the standards should be stored in a refrigerator when not in use. 

Quenching Impurities in the mobile phase, particularly oxygen, may entirely quench the signal from low concentrations of fluorescent compounds (see solvent degassing). 

Method 3 was modified to an internal standard method into Method 5 by changing the bonded phase and the mobile phase composition. Biphenyl was used as an internal standard added into the reaction. Aliquots were withdrawn, diluted with degassed acetonitrile, and analyzed according to Method 5. This internal standard method, Method 5, was helpful in the optimization of the desired ris-1,2/1,4 product of the key step of the LANA reaction (scheme 5). 

For oxidative detection removal of dissolved air from the mobile phase is necessary to prevent air bubble formation at the column outlet, which disturbs the electrolysis process. Vacuum filtration usually is sufficient to remove enough air for bubble-free operation. However, air readily redissolves in the mobile phase. Continuous helium sparging is therefore the only effective degassing method for longer periods. 

Again, helium degassing as described above is an appropriate way to remove oxygen, especially when combined with heating (40 to 50°C) of the mobile phase. Special care must be taken that oxygen cannot diffuse back into the mobile phase replace PTFE tubing by steel tubing between mobile phase container and pump, and between column and detector. 

The HPLC pump draws the mobile phase from the reservoir via vacuum action. In the process, air dissolved in the mobile phase may withdraw from the liquid and form bubbles in the flow stream unless such air is removed from the liquid in advance. Air in the flow stream is undesirable because it can cause a wide variety of problems, such as poor pump performance or poor detector response. Removing air from the mobile phase, called degassing, in advance of the chromatography is a routine matter, however, and can be done in one of several ways 1) helium sparging, 2) ultrasonic agitation, 3) drawing a vacuum over the surface of the liquid, or 4) a combination of numbers 2 and 3. 

Left, Charlie Focht of the Nebraska State Agriculture Laboratory prepares the mobile phase for an atrazine assay. Note that the vacuum flask is positioned in an ultrasonic cleaner bath. Simultaneous vacuum filtration and sonication provide a more efficient means for degassing. Right, Charlie adjusts the flow rate setting on the HPLC pump. 

If the mobile phase contains liquids that are not certified as HPLC grade solvents, they must be filtered ahead of time as well as degassed. The reason is that the packed bed of finely divided stationary phase particles through which the mobile phase percolates is itself an excellent filter. Particles in the mobile phase as small as 0.5 jim in diameter can be filtered out. The result of this is a decreased effectiveness of the column with time and possibly a blocked flow path. Unfiltered samples also may contain particles and cause this problem. 

The problem is solved by prefiltering all mobile phases and samples before beginning the experiment. For mobile phases and large sample volumes, this involves utilizing a vacuum apparatus that draws the liquid through a 0.5-pim filter. Since such filtration involves a vacuum, the mobile phase is automatically degassed as well, so the filtration need not be a separate step. An efficient operation would be to filter a mobile phase with a vacuum apparatus while simultaneously sonicating. See Workplace Scene 13.1. 

Why do air bubbles form in the flow stream between the mobile phase reservoir and the pump when the mobile is not degassed ... 

The great versatility of HPLC lies in the fact that the stability of the chemically bonded stationary phases used in partition chromatography allows the use of a wide range of liquids as a mobile phase without the stationary phase being lost or destroyed. This means that there is less need for a large number of different stationary phases as is the case in gas chromatography. The mobile phase must be available in a pure form and usually requires degassing before use. The choice of mobile phase (Table 3.6) is influenced by several factors. 

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