Eric de Witte, Norbert Reuter*, Global Technical Support for CSD, Middelburg, The Netherlands
The lifetime of HPLC columns is determined by various factors. Injection of dirty or polluted samples often causes a decrease in lifetime of the HPLC columns. This article gives some guidance on how to protect your HPLC column and in case of pollution how to regenerate your reversed phase HPLC column.
Most contaminations of HPLC columns are caused by the injection of dirty samples onto the HPLC columns. For example, very hydrophobic sample matrices like oil, highly aromatic materials and waxes can stick to reversed phase packing materials and change their characteristics. Biological fluids such as serum or plasma containing proteinaceous materials can adsorb to the packing surface.
After a column is contaminated, its chromatographic performance can be different from that of an uncontaminated column, which can result in shifting retention times and changing selectivity’s. Also contaminated columns often show backpressure problems.
The best way to avoid contamination of your HPLC column is to limit the introduction of matrix components onto the column. So, this needs adequate sample preparation before the injection of the sample onto the column.
- Filtration of the sample by using syringes filters or Captiva filtration products is a possibility to avoid the introduction of small particles onto the column
- Solid Phase Extraction, liquid/liquid extraction and solid supported liquid/liquid extractions are very efficient techniques to get rid of a broad range of matrix components, which would otherwise contaminate your HPLC column
- The use of a guard column is always recommended to protect the analytical column and to increase the lifetime your HPLC column
After you have taken care of good sample clean up, it is still possible that contaminants are introduced onto the HPLC column. We will discuss some practical ways to try to return a bonded silica-based column to or nearly to its original state.
Different types of Matrix components:
Matrix components are of no interest to the analyst. Roughly matrix components can be divided into three groups.
- Very polar components (like salts), which elute from a Reversed Phase column without or very little retention. These components usually don’t cause much problems
- Matrix components, which have intermediate retention. These components elute slowly from the column and appear as wide peaks (sometimes in the next run), baseline disturbance or drift of the baseline.
- Matrix components, which are adsorbed or absorbed strongly to the column, like fatty components, lipids or hydrophobic proteins. The mobile phase composition never becomes strong enough to elute these components. These matrix components will cause problems. They usually accumulate at the top of the column. They can act as a new stationary phase, causing a change of selectivity and shifting retention times for the components of interest.
If sufficient contamination occurs, backpressure problems or complete blockage of the column can be the result.
Washing and regeneration of bonded silica columns:
It is essential to know the nature of the contaminants and find the right solvent that will remove them.
When contamination is the result of strongly retained substances from repeated injections, a simple washing process to strip these components can often restore column performance. Sometimes after isocratic operation, flushing a column with about 20 column volumes of the organic solvent (Solvent B in binary Reversed Phase system) is sufficient to remove the contaminants.
Sometimes, the strong solvent component of the mobile phase is insufficient to remove column contaminants. In that case a series of stronger solvents will be necessary to clean the column. If the contaminants are non-biological, then the user can pass one or more additional organic solvents through the column.
The wash solvents, which are used, increase in their solvent strength often ending with a solvent that could be very apolar (like hexane), which helps to dissolve very non-polar substances like oils and lipids. The Agilent User’s Manuals for Normal Phase, Reversed Phase and Polar Bonded columns gives a possible combination of solvents, which goes from methanol, isopropanol, dichloromethane, hexane back to isopropanol, methanol.
It is important to ensure that each solvent in the series is miscible with the next solvent. Isopropanol is often used as an intermediate solvent because it is miscible with organic solvents like hexane as well as with aqueous solutions.
When mobile phases with buffer solutions are used, it is important not to increase the organic solvent content too fast, to avoid precipitation of the buffer salts, which can result in problems like plugged column frits, but also plugged tubing or pump seal failure, it is important first to remove the buffer part of the mobile phase by replacing the buffer with water.
Because contaminants are usually at the head of the column, it is recommended to reverse the column during washing of the column.
In case biological materials such as plasma and serum have built up on the column chromatographers must use a somewhat different cleaning process. In most cases 100 % organic solvents like acetonitrile and methanol do not dissolve peptides and proteins and hence are ineffective for cleaning reversed phase columns. In this case mixtures of organic solvents with buffer, acids and sometimes ion-pairing reagent can be more effective. Initially, flushing a column with mobile phase that has a somewhat higher percentage of the stronger solvent should be attempted. In the literature you also find descriptions of somewhat more exotic cleaning procedures for this type of contamination, which go beyond the scope of this article.
Normally users tend to wait with the washing of the column until they see unusual behavior. However, an increased built up of contaminants will make it more difficult to clean the column. For this reason, if you know that you are subjecting your reversed phase column to dirty samples, it is recommended to clean your columns on a regular base, to increase the lifetime of the column.