Do you know the maintenance methods of Welch columns? Some people may use the wrong way to flush columns unconsciously, so it is recommended to learn some practical methods for flushing and regeneration of reversed-phase column as follows.

Introduction of Reverse-Phased Column

Reversed-phase chromatography is by far the most widely used technique in high performance liquid chromatography (HPLC). The reason for its popularity is that it can be applied to the analysis of most non-polar compounds, weak polar compounds and ionic compounds. Most of the stationary phases used in reversed-phase chromatography are naturally hydrophobic groups, which are separated due to the different interactions between the substances to be tested and the stationary phase.

How are Reverse-Phased Columns Contaminated?

The most commonly used reversed-phase column is the silica based column. There are much residual silanol on the surface of the silica matrix. Fig. 1 shows the various possible forms of silanol groups. In a weak acidic environment, these silanol groups can interact with specific compounds and matrix components, especially alkaline compounds, resulting in the adsorption of alkaline compounds on the silica surface. At the same time, the remaining heavy metal ions in the silica gel will introduce more acidic substances into the surface of the silica gel. In addition, silica gel may interact with metal chelates or detergents.

Fig.1 The existing state of silanol

Sample matrix, such as corn oil, wax and senior aromatic compounds will adhere to the surface of reversed-phase packing materials and change its properties. Biological samples containing proteins will also be adsorbed on the surface of the packing materials particles. Even use the method of sample pretreatment to avoid pollution of foreign substances, matrix of the object to be tested will still be adsorbed on the surface of column eventually. The chromatographic behavior of the contaminated column will be different from that of the uncontaminated column.

Usually the sample matrix contains contaminants that are not pertinent to the target analytes; For example, salts, esters, aliphatic compounds, organic acids, hydrophobic proteins, and other biological components are all substances that may interact with the stationary phase. The retention capacity of these contaminants may be greater than that of the target analyte. Once these unexpected contaminants are detected by the detector, the chromatogram will manifest as peak bifurcation, baseline fluctuations, and even negative peaks.

If the retention capacity of the contaminants on the column is so strong that the elution capacity of the mobile phase is insufficient to flush them out, the contaminants adsorbed on the column will accumulate after injecting for many times. When enough contaminants accumulate, they will behave like modified packing materials. The interaction between the target analytes and these contaminants will affect their original separation mechanism, leading to fluctuation of retention time and peak tailing. If there are more contaminants, the pressure may rise to very high level or even exceed the maximum column pressure.

Flush Reversed-Phase Column

Forward use and back flushing

Since most of the contaminants which have strong retention ability usually appear at the column-head, therefore, back flushing can greatly reduce the distance that contaminants need for leaving column. Because the pore size of the frits used at both ends of Welch’s column is the same, there will be no case where the packing materials is flushed out during backflushing. Most modern stainless steel columns are packed at higher pressure than normal, so the column bed is usually unaffected by back flushing.

The frequency of flushing columns depends on the number of the strongly retained materials which enter the column. Since reversed-phase columns can tolerate a high degree of contamination before they show signs of a decrease in resolution or release abnormal substances, users usually don’t flush columns until the column is observed to be abnormal. But the accumulation of contaminants will make cleaning more difficult. For this reason, if some of the sample matrix is complex, it is recommended to regularly flush the column. The higher the frequency of flushing the column, the simpler the flushing procedure required.

Common daily flushing

Abnormal flushing

When the column has problems of increased pressure, abnormal peak shape, decreased column efficiency and resolution.

Flush the Remaining Proteins of Reversed-Phase Column

If the contaminants are biological, such as plasma, serum, proteins, and peptides which accumulate in the column, pure organic solvents such as acetonitrile or methanol are often difficult to dissolve these contaminants. However, organic solvents mixed with buffer salts, acids, or ion pairs can flush out biological contaminants.

If the column is contaminated by biological samples, it is recommended to use acetonitrile: water: trifluoroacetic acid (50/50/0.1) as the mobile phase, back flush with 60 times of the column volume at the flow rate of preparing samples. Equilibrate the column with the initial mobile phase before injecting samples, then the separation capacity of the biological samples can be restored.

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