Cleaning and regeneration of reversed-phase column in high performance liquid chromatography

Reverse-phase chromatography is by far the most widely used technique in high-performance liquid chromatography, mainly because it is suitable for the analysis of most non-polar substances and many ionizable and ionic compounds. Most stationary phases used in reversed-phase chromatography are naturally hydrophobic substances. Therefore, analytes are separated according to the size of their hydrophobic interaction with the stationary phase, and mechanisms containing hydrophobicity can also be separated in the same retention manner.
There are also a few substances on the stationary phase, such as mixed phases (such as phenyl-hexyl), end-blocking and non-terminal-blocking species, and polar intercalating phases-also present on these bonded silicas. There are also many fillers used for reversed-phase chromatography, including polymers, coated with silica gel and alumina, inorganic-organic mixtures, coated zirconia, and graphitized carbon. Different types of stationary phases have their own advantages and disadvantages.
Reversed-phase chromatography columns use a variety of mobile phases and additives to have many applications. Some techniques use additives to modify or modify the surface of the filler. Sometimes these additives may contaminate the bonded phase surface. The surface of silica gel has some other chemical properties because of its hydrophobic bonding phase. Residual silanol is present in all silica-bonded fillers. These silanol have weak acidity, so they can combine with some analytes matrix components, especially alkaline components. Because the pKa value of silanol is about 4.5, ionization can occur under neutral pH conditions, so electrostatic interaction with cations is likely to occur. The older A-type silica gel can accommodate high concentrations of metal ions (sometimes 100 ppm or more), and this can make the surface of the silica gel more acidic or even cause metal chelation or remove some compounds. Residual silanol is even more annoying on non-terminally sealed silica bonded short-chain bonded phases such as C2 or C4.
Users must be aware of the special properties of the stationary phase surface they are using and possible analyte- stationary phase surface interactions, so that they can take into account possible matrix interactions when using reversed-phase methods. For example, very hydrophobic sample matrices such as corn oil, highly aromatic substances, and waxes can adhere to the stationary phase filling surface and change their properties. Biological fluids containing protein substances can also be adsorbed on the filling surface. Although analysts do their best to protect HPLC columns, certain analyte-matrix contamination can adversely affect the stationary phase.
When the column is contaminated, its chromatographic behavior will be slightly different from the uncontaminated column. Contaminated columns can create backpressure problems. The contaminated reversed-phase column must be cleaned and regenerated to restore the original operating conditions. This part of "Column Observation" will discuss possible ways to restore the column to its original or almost original state. Because bonded silica columns are the most popular, I focus on this column. Finally, I will discuss other cleaning steps for reversed-phase columns.
What causes contamination of the reversed-phase column? Usually, the sample contains something that is not of interest to the analyst. Salts, lipids, lipid-containing substances, humic acids, hydrophobic proteins, and other biological substances are some substances that may interact with the HPLC column during use. These substances have a smaller or larger retention value than the analyst's target. Substances with smaller retention values, such as salts, are generally flushed out of the column at empty volume. The interference of these non-target products can be detected by the detector and can form a tailing peak. The former is rare. > Chromatographic peaks, bubbles, baseline shift up or negative peaks. If the sample components are strongly retained in the column and the mobile phase solution components are not sufficient to elute these substances, the substances adsorbed on the surface of the column will usually accumulate on the column head after multiple loadings. These behaviors can usually only be discovered through parallel experiments. Samples with moderate retention values ​​can be eluted slowly and exhibit broad peaks, baseline disturbances, or baseline drift.
Sometimes these adsorbed sample components accumulate to a certain extent enough to allow them to start to form a new stationary phase. Analytes can form a certain separation mechanism with these impurities. The retention time will fluctuate and the tailing will appear. If enough pollution occurs, the back pressure of the column can exceed the maximum pressure the pump can withstand, making the column inoperable and creating empty volume at the blockage.

Cleaning the silica gel bonded column The key to regenerating a contaminated liquid chromatographic column is to know the nature of the contaminant and find the appropriate solvent to remove it. If contamination is caused by the accumulation of strongly retained substances during repeated injections, using simple steps to remove these contaminants can often restore their chromatographic behavior. Sometimes, after multiple operations, the column is washed with 90 to 100% of solvent B (the stronger solvent in the dual-solvent reversed-phase system) to flush 20 volumes to remove contaminants.
For example, the remaining lipids in the column can be used with non-aqueous solvents such as methanol, acetonitrile, and tetrahydrofuran.
If you are using a buffer system, do not switch directly to a strong solvent. Suddenly switching to a high-concentration organic solvent may precipitate the buffer in the HPLC flow system, which may cause greater problems such as clogged column heads, blocked pipes, and pump Leakage, damage to the piston or malfunction of the injection valve shaft. The unbuffered mobile phase should be used first (ie, change the buffer to water). After flushing 5 to 10 volumes, replace the strong solvent.
Sometimes, strong solvents cannot wash away contaminants remaining on the column. Then a stronger solvent or a series of solvents is necessary to clean the column. If the contaminant is a non-biological substance, the user can skip one or more additional organic solvents to remove the contaminant. There are many combinations of solvents and solvents. The recommended solvent system can be found on the column manufacturer's website.
In general, all cleaning methods have a similar form. The solvents used increase with the strength of the solvent, often the last solvent is very hydrophobic (such as ethyl acetate or even hydrocarbon

This article comes from the cleaning and regeneration of reversed-phase columns in high-performance liquid chromatography | Scientific Instrument Online Original Link: http: //

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