In this chapter the most common problems that occur during the use of an HPLC column are summarized. Guide lines to overcome these problems are given.
Increasing back pressure
The backpressure of your HPLC instrument increases continuously and exceeds 10% of the original value.
The capillary tubing, the column or the detector cell is blocked.
A blockage can be caused by several things:
- Mechanical contamination coming from pump seals or the rotor seal of the injection valve
- Precipitation of buffer salts or precipitation of the sample
- Bacterial growth or contaminations in the mobile phase
First of all, locate the exact position of the blockage. To do this systematically, open all connections starting from the detector and then back to the injector while the pump is running. This will allow you to identify clearly where the blockage is located by observing where the pressure drop occurs. Replace the blocked parts if frits or meshes are causing a blockage please clean or exchange them.
Filter all mobile phases through a 0.45 μm filter before using in HPLC.
Always check the solubility of the buffer salts in the mobile phase you are using.
If you have particulate material in your sample, filter it.
Use pre- or guard column filters. On a long term basis, this is the most inexpensive way to go.
Decrease of Resolution or low efficiency
The resolution is continuously decreasing, peaks are loosing efficiency and beginning to show tailing, a shoulder or even double peaks.
The column is contaminated with strongly retained components. This contamination comes from either the mobile phase or the sample.
A channelling inside the column has occurred. This may have been caused by a mechanical shock to the column bed.
The column has a void at the inlet.
Regenerate the column according to the protocol in chapter 1.3 "Usage and Care". If there is channelling in the column, repair of the column is impossible.Please use a new column.
If there is a void in the inlet in the column, you can try to repair in the column. To do so, you have to remove the sieve sandwich and about 5 mm of the packing at the column inlet. Replace the removed packing material with the paste of Methanol and new paking material of the same type. Take away the exceeding material and close the column with a new sieve sandwich. Install the column in reversed direction. Please check that no packing material can be flushed into the flow cell. Column repair is not always successful. The procedure is also time consuming.
Use always a pre- or guard column. Strongly retained compounds will stay in the guard column and the analytical column is therefore be protected.
Avoid pressure shocks to the column. If you start running your HPLC equipment, start with a low flow rate and increase it slowly. After use, put the column back into the box to protect it of mechanical shocks.
Increasing back pressure in combination with low efficiency
After a few hours of use, the backpressure of the column increases continuously, even though you have filtered the eluent before usage.
You may be working above the upper pH limit of the column. Silica based columns are only stable up to pH 8. Above pH 8 silica starts to dissolve. During the dissolution smaller particles are formed and these particles generate a higher backpressure. After a while a large void will develop at the column inlet. This void causes asymmetrical and broad peaks.
If you have to work at higher pH values, install a saturation column between the pump and the injection valve. This column should be a short HPLC column or a guard column packed with the same phase as the analytical column. If available, the particle size of this packing should be larger than the packing in the analytical column. If a saturation column is installed in the system, the eluent will become saturated by the silica of the saturation column and inhibits dissolution of the packing of the analytical column.
Varying retention times
The retention times of the peaks in the chromatogram are varying or decreasing continuously. The original separation can not be achieved.
Normal Phase (NP) mode::
Varying retention times can be observed very often in normal phase chromatography. Water is the strongest solvent in NP mode. The water content of organic solvents will vary depending on the moisture in the air of the laboratories. Therefore, the retention time of some analytes is lower on a rainy day compared to a dry sunny day.
Reverse Phase (RP) mode:
In RP HPLC the variation of retention times is mainly related to problems with the HPLC pump. Very often the check valves of the pump may not be closing properly. If the retention time is decreasing continuously, it is a hint that you may be loosing the bonded functional groups of your stationary phase. If you are working with silica based RP packings below pH 2, the bonding of the RP packing may be hydrolyzed and the alkyl chains can be washed out.
Normal Phase (NP) mode:
To avoid varying retention times in the NP mode you should have add your mobile phase above your molecular sievesome molecular sieve to the mobile phase. Often the addition of 1% of a polar modifier like methanol helps to achieve stable retention times.
Reverse Phase (RP) mode:
In the RP mode with silica based packings, you should take care not to work outside pH range of 2 to 8. Many RP packings are loosing their bonding below pH 2. If you have to work below pH 2, you should use only columns that are recommended to work in these pH ranges (e.g. ProntoSIL C18 SH, ProntoSIL C18 ace-EPS).
The separation can not be achieved with a new column
You have developed a new HPLC method. After installing a new column you can no longer achieve the separation you had on the old column.
During method development, you are using several different solvents, modifiers and buffers. Some buffers, especially ion pair reagents, may be irreversibly adsorbed on the column packing. If you work even for a short period of time, the silica or the bonded phase may be affected. This may influence the selectivity of the column dramatically.It also may occur that the column you use for method development already has a history that you are unaware of. You may have found it somewhere in your laboratory.There might be some contaminations on this column coming from the matrix of earlier injections, or this column may not have been stored in a proper solvent and its selectivity is therefore modified.
If you are using a column that is packed with a stationary phase based on an old generation type A silica, difficulties in batch to batch reproducibility of the column might also be the reason for the irreproducible result.
Keep a column file and record time of usage, solvents and buffers used, storage solvent used, and the persons who have used the column are recorded. If you have a file like that, you can always find out what happened with the column before.
If possible you should always use a column with new generation based packing (high or ultra pure silica) to develop a new method. This type B silica based columns show a much higher batch to batch reproducibility.
A HPLC method that has worked for years is no longer working when using a
You have a HPLC method that runs for years in your QC laboratory without any problem. Suddenly, after installing a new column, the retention times and/or the selectivity changes and the results do not meet the specifications of the method.
This problem is not necessarily related to the column. If you install a new column, a new mobile phase is also often used. A change in buffer additives (especially in ion pair HPLC) may cause the problem.
First of all, check the HPLC instrument and the mobile phases. This can be done by running the system suitability test with the old column. At the second step run the same test with the new column. Now compare the results of both columns. If the new column shows different results, please contact your column manufacturer.
The efficiency of the column varies
The efficiency of the column is not constant. For one injection you achieve good efficiency for the next injection you get broad and unresolved peaks.
This effect is mainly related to an overloading of the column. The loadability of an HPLC column depends on the mass of stationary phase inside the column and the physicochemical properties of the packing material (specific surface area and carbon content). The volume loadability depends on the volume of the column (column dimensions). In addition to that, the loadability depends also on the adsorption enthalpy of the analytes.
To determine if the varying efficiency is caused by the loadability of the column, dilute the sample by a factor 2 and inject only half of the normal amount of the injection. Observing now higher efficiency by using the diluted sample, the reason for the low efficiency was overloading. The rule of thumb for the loadability limit of a standard HPLC column is 100 μg/g of stationary phase for mass loadability and 50 μl/g of stationary phase for volume loadability.
The peaks in the chromatogram are fronting. Retention times are shifting to lower retention.
In most of the cases, the origin of this phenomenon is a solvent strength effect. The solvent you are using to dissolve the sample has a higher solvent strength in comparison to the mobile phase you are using for the chromatography. (Example: Your chromatography runs in an eluent acetonitrile/water 20:80 but the sample is dissolved in 100% acetonitrile). If the origin of the fronting is a solvent strength effect, you should observe a decrease in fronting for peaks that are eluted later.
Fronting peaks can also be caused by channelling of the column at the column inlet. In this case, the fronting for all peaks will be more or less the same.
Always use the eluent or a weaker solvent to dissolve the sample. If the fronting comes from channelling, you have to use a new column. If the fronting caused by a void you can try to repair the column as described under “Decrease of Resolution or low efficiency”. This will help you for a few further injections but be aware that you have a need for a new column in a short time.
Spikes in the chromatogram
Spikes in the chromatogram are disturbing the data processing. They appear aperiodic.
There are several possible causes for spikes in the chromatogram. They may result from the electronic or from the column. If they come from the electronic, they mostly will occur if an electronic instrument in laboratory is switched on (e.g. refrigerator or oven). If they come from the column they are mostly bubbles out of the eluent because the eluent is not properly degased. In addition, columns that have dried-out can also cause spikes. If a HPLC column is really dry, the spikes in the chromatogram can last for hours.
To avoid electrically caused spikes use a mains or power rectifyer to connect your HPLC equipment to mains.
To avoid air bubbles from the eluent, degas the mobile phase properly before using in HPLC. The most efficient way for degasing is the use of an inline degaser or the continuous purging of the eluent with Helium. Columns have to be closed properly for storage after use. Therefore use the supplied column end stoppers to avoid a column dry-out. Most of the columns do not loose performance by drying out. It takes a long time to get such a column back to work since air bubbles will occur as spikes in the chromatogram mostly for several hours. If you have a dry column please flush it with isopropanol. Isopropanol has a low surface tension and is therefore the best solvent to remove the air from the column.
In application section, under "Trouble shooting in HPLC Column" link, most seen problems in columns are summirized. You can forward your problems which are not mentioned in this section to firstname.lastname@example.org .