Column troubleshooting is usually performed by reference to the chromatogram and chromatographic peak. System operating parameters may also provide indicative troubleshooting information - the component perspective. It is always advisable to protect the analytical column from damage against particulates and strongly retained sample material.
These may eventually block the inlet frit of the column and occlude the packed stationary phase, resulting in increased backpressure and poor chromatographic peak shape respectively. Therefore, the installation of an in-line filter and/or a guard column in the system is strongly recommended.
Particulates and strongly adsorbed species can potentially come from:
- Piston seals
- The injector switching valve rotor seal
- Undissolved or precipitated buffer salts
- Contaminated sample matrices
Dedicating Columns
Once a column has been used for the separation of ‘real’ samples, then the selectivity offered by that column will necessarily differ from that of a new column of the same brand and part number. Columns can become chemically modified in a variety of ways once they have been used experimentally:
- Stripping of a column’s end-capping species may occur under low-pH conditions.
- Strongly retained sample material can bind irreversibly to the stationary phase surface, especially at the column inlet, causing a rise in system backpressure and a change in the chemical nature of the stationary phase.
- Analytes that are strongly basic in nature can bind irreversibly to free silanol groups on the column’s stationary phase. Performing separations at low pH can reduce the extent of silanol ionization, as they typically possess a pka of 3.8 - 4.1.
- As each HPLC method operates under slightly different conditions and is used for different sample types, then the column changes that can potentially occur will be unique for that method. Therefore, once a column has been used routinely for one application then it may no longer offer an identical separation to its new column equivalent, and chromatographic differences can then often be observed.
This may also be experienced when using the same column, but switching to a different method and analyte. Then it may be necessary to perform several “priming” injections with the new analyte prior to obtaining reproducible chromatography. Column equilibration times may also have to be extended, to accommodate the selectivity changes.
To minimize experiencing such variations it is recommended that a column be dedicated to a specific method and analyte type. The indiscriminate swapping of columns to develop methods may result in an unrepresentative chromatographic separation once a new column, which has never been exposed to the variety of sample types of its predecessor, is subsequently employed. Such variations can be relieved by the incorporation of a correct column-washing procedure.
ALSO READ: What is the Shelf Life for HPLC Columns?
In-line Filter
A common indicator of column failure is the gradual development of excessive backpressure. Such pressure increases typically result from the accumulation of particulate material on the inlet frit of the column, and one of the least problematic and inexpensive ways to extend a column’s life is to incorporate an in-line high-pressure filter between the autosampler and the column. In this location an in-line filter prevents particulates derived from both the sample and the autosampler injection switching valve rotor seal reaching the head of the column.
It is highly recommended that an in-line filter be incorporated in to each HPLC system, the only caveat being extremely low dead-volume systems, where the increase in extra column volume would contribute more significantly to chromatographic peak dispersion and band broadening. Apart from this scenario no appreciable change in the chromatogram will be observed.
The in-line filter can be either visually examined for contamination build up, or if the particulates are colorless, changed when the system backpressure has risen appreciably (Figure 1). In-line filters contain a frit that is most commonly 0.5 μm in porosity (0.2 μm are used for UHPLC applications). The porosity should be similar to, or smaller than, the frit porosity of your analytical column so that the frit catches any particles that would be likely to block the column frit.
Figure 1: Clean and dirty in-line filters.
Guard Columns/Disks
A guard column is a short column - typically 10-20 mm in length - that is packed with the same stationary phase material as the analytical column. If a cartridge system is being employed then an integral cartridge holder must also be used. Guard disks are small fritted disks, which when fitted into their holder are screwed directly into the analytical column. In contrast to the in-line filter, a guard column or disk should only be fitted to act as a chemical filter, ensuring the removal of any potentially strongly retained or aggressive material, and thereby preventing subsequent fouling of the analytical column.
In some instances, a guard column can provide sufficient protection for the analytical column to reduce or eliminate the need for any off-line sample clean-up procedure. However, as the guard column can potentially become contaminated with a variety of strongly retained material, it is an absolute requirement that when being flushed with a strong solvent it is done so offline from the analytical column, thereby preventing any such species from being inadvertently washed onto the head of the analytical column.
A guard column should not be used as a silica saturator column, as any void that subsequently develops in the guard column will compromise the chromatography (Figure 2). If a saturator column is felt necessary for a particular application i.e. high pH mobile phase, then it should be placed upstream of the injector with an in-line filter immediately afterward to retain the resulting small particulate material resulting from the subsequent silica dissolution.
Figure 2: Chromatograms illustrating the detrimental effect of the formation of a guard column void on the resulting chromatographic peak shape.
As with the incorporation of an in-line filter into the HPLC system, a guard column will also introduce an increase in extra column volume, which would contribute to chromatographic peak dispersion and band broadening in extremely low dead volume systems.
When do you replace a guard column? Three potential approaches can be used to determine when a guard column should be replaced:
- Waiting for the overall chromatographic separation to deteriorate is not recommended. It may be possible to monitor a pair of peaks in the chromatogram that are more poorly resolved than the pair of interest, making it possible to correlate a change in their critical separation with that of the peaks of interest. This would allow an informed judgment to be made as to how close the guard column is to potentially failing. This is illustrated in the chromatogram below, which shows the separation of a range of herbicide samples. The critical pair, the chromatographic peaks that are least resolved from one another, are labeled as 1 and 2. The chromatographic peaks of interest are labeled as 3 and 4. By monitoring the continual reduction in resolution between peaks 1 and 2 over time, a valued judgment can be made as to when to change the guard column without compromising the analysis with respect to the sample peaks of interest 3 and 4.
- Monitor the number of samples injected with the guard column in place before the system fails to deliver a satisfactory separation. Alter the method’s SOP so that the guard column is replaced after approximately 80% of its expected lifetime – be proactive rather than reactive.
- Monitor the guard column’s effective life in terms of the solvent volume used or the calendar time it has been installed.
Sample Clean-up
In the context of sample clean-up, it becomes a financial balancing act of weighing up the cost of such sample clean-up against the cost of column replacement. Consider the bioanalytical example of measuring the concentration of a drug substance in plasma. Protein precipitation by the addition of acetonitrile to the plasma, vortexing, centrifuging and injecting the supernatant will still produce a reasonably “dirty” sample, perhaps giving between 100-500 injections.
In contrast, a much cleaner sample can be obtained by using a solid phase extraction (SPE) procedure, enabling a column lifetime of between 1000-2000 injections. This whole SPE process and subsequent sample injection using a 96-well plate format could be automated. Whilst the gain in column lifetime would not in itself justify the cost of such an elaborate and expensive clean-up process, the method would benefit from improved reliability and if required for many years to come then this should be given due consideration.
Column Storage
To avoid contamination, store LC columns with end plugs securely fastened and be sure to include information describing the storage solvent.
For short-term storage, columns should be flushed with a solvent identical in composition to the most recently used mobile phase minus any buffered, acidic, or basic components.
For long-term storage, reversed-phase columns should be stored in 50% water/50% organic solvent (i.e. acetonitrile or methanol). Normal phase columns should be stored in a non-polar solvent (i.e. hexane). Ion exchange columns should be stored in methanol following flushing with water. HILIC columns should be stored in 80% acetonitrile/20% water.
Important: All buffers should be washed out of the column (use water) before flushing with acetonitrile as buffer salts are generally insoluble in acetonitrile and will precipitate and cause blockages in the column.
Column Replacement
By incorporating the points outlined above then the effective lifetime of any analytical column can be increased, ultimately however columns should be considered disposable items. How long a column lasts is determined by a number of interrelated factors e.g. buffer strength and pH, sample cleanliness, but typically between 500-2000 injections should be considered an acceptable range.
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Chromatography
