Washing Reversed-Phase Silica-Based Columns

The answer to the following question was provided by LCGC’s “Column Watch” editor Ron Majors.

Q:What is the best way to regenerate a contaminated silica-based, reversed-phase HPLC column?

Ron Majors:The keys to rejuvenating a contaminated HPLC column are knowing the nature of the contaminants and finding an appropriate solvent that will remove them. When contamination results from the accumulation of strongly retained substances from repeated injections, a simple washing process to strip these contaminants can often restore column performance. Sometimes, after isocratic operation for a reversed-phase chromatography column, flushing it with 20 column volumes of 90–100% solvent B (the stronger solvent in a binary reversed-phase system) can remove the contaminants. As a rule of thumb, the column volume of a 4.6- X 250-mm column is roughly about 2.5-mL. To wash the column usewater-miscible, nonaqueous solvents such as methanol, acetonitrile, or tetrahydrofuran. If you are using a buffered aqueous mobile phase, do not jump immediately to the strong solvent. An abrupt change to high organic solvent content could result in buffer precipitation in the HPLC flow system, which could cause even bigger problems such as plugged frits, plugged connecting tubing, pump seal failure, a scratched piston or injection valve rotor failure. Instead, flush the column with a buffer-free mobile phase (that is, the same mobile phase but without the buffer salt). After flushing with 5–10 column volumes of buffer-free mobile phase, the stronger solvent then can be passed through the column.

Occasionally, the strong solvent component of a mobile phase is insufficient to remove the column contaminants. A stronger solvent or series of solvents will be necessary to clean the column. If the contaminants are nonbiological, then users can pass one or more additional organic solvents through the column to remove the undesired compounds. The solvents and solvent combinations that can be used are numerous. Visit one or more column manufacturers’ websites to see various recommended solvent systems.

Generally, all washing approaches follow a similar pattern. The wash solvents used are increased in their solvent strength, and for a reversed-phase column, often ending with a solvent that could be very nonpolar (for example, ethyl acetate or even a hydrocarbon), which helps to solubilize nonpolar substances such as lipids and oils. It is important to ensure that each solvent in the series is miscible with the next solvent. At the conclusion of the wash cycle, go backwards through an intermediately miscible solvent before returning to the original mobile-phase system. For example, isopropanol is an excellent solvent for this intermediate step because it is miscible with organic solvents such as hexane or methylene chloride and also is miscible with aqueous solutions. Because isopropanol is quite viscous, make sure that the flow rate is not high enough to cause pump overpressure. Also, if using a UV detector, avoid using solvents that absorb in the ultraviolet region of the spectrum because it may require a great deal of washing to remove all of the absorbing solvent to get a stable baseline.

A recommended column washing system for a typical bonded-silica column and a mobile phase without buffer salts is to use
• 100% methanol,
• 100% acetonitrile,
• 75% acetonitrile–25% isopropanol,
• 100% isopropanol,
• 100% methylene chloride, and
• 100% hexane.

When using methylene chloride or hexane, the column must be flushed with isopropanol before returning to an aqueous mobile phase because of solvent immiscibility. A minimum of 10 column volumes of each wash solvent should be passed through a column. For 250 mm X 4.6 mm analytical columns, analysts can use a typical 1–2 mL/min HPLC flow rate. To return to the original mobile phase, chromatographers can usually skip going through the entire series in reverse order. Using isopropanol as an intermediate solvent is recommended followed by mobile phase without buffer, then finally with the starting mobile-phase composition. Tetrahydrofuran is another popular solvent that can be used for cleaning contaminated columns. If users suspect severe fouling, they can mix dimethyl sulfoxide (DMSO) or dimethylformamide mixed 50:50 with water and pass them at flow rates less than 0.5 mL/min. The successful regeneration of a reversed-phase column can be a time-consuming process, and solvent washings can be programmed into a gradient system for overnight operation.

A question arises as to whether to reverse the HPLC column during the washing procedure. As most of the strongly held contaminants are usually at the head of the column, reversing the column can shorten the migration distance that the solubilized contaminants must travel to exit the column. As far as the packed-bed stability is concerned, most modern HPLC columns have been packed at a considerably higher pressure than the normal operating pressure; therefore, their beds should not be disturbed by the reversed flow. However, if a top frit is of a higher porosity than a bottom frit, this type of reversal could be detrimental. For example, if the bottom frit is of 2 μm porosity, it is usually sufficient to contain column packing with an average particle size of 5 μm (with a +/-2 μm particle size distribution). However, a manufacturer will sometimes put a larger-porosity frit at the top of the column to prevent plugging with sample or mobile-phase particulates. If the porosity of this frit is larger than that of the smallest particles in the particle-size distribution curve, some of the packing material could conceivably pass through the frit and be removed from the column, thereby creating a void. If a column has an arrow to recommend the direction of flow, I would consult the manual or instruction sheet, the manufacturer’s website or the technical support group before reversing the column to make sure that it is a safe practice. Whether you reverse the column or not, it is a good practice to disconnect the column from the HPLC detector so that contaminants or particulates lodged on the frit are not swept into the detector cell, where they can cause contamination.

The frequency of cleaning fouled reversed-phase columns depends upon how much unretained material has been injected onto the column. Because reversed-phase columns can sometimes withstand a great deal of contamination before resolution loss or elution of extraneous compounds, users tend to wait until they observe some unusual behavior. However, an increased build-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 columns to dirty sample matrices, I recommend cleaning your columns on a regular basis. The more frequent the cleaning, the less rigorous cleaning conditions you will need.

Of course, if one is always injecting samples that are “dirty” rather than having to do a drastic column cleanup procedure, why not use a guard column to collect contaminants? The guard column acts as a chemical filter and can periodically be replaced before the main analytical column suffers contamination. One must empirically determine the number of injections that a guard column can withstand. Alternatively, one could do a study to determine the sample capacity of the guard column but this requires additional work. Guard columns are generally much cheaper than the analytical columns that they serve.

Furthermore, good sample cleanup procedures can also minimize column cleanup requirements and also prolong column lifetime, Solid-phase extraction and liquid–liquid extraction are often used for sample cleanup.