Peak shape problems are difficult to assess and correct. Because peak shape problems may not adversely affect the resultant data, determine if the analytical results are truly compromised before troubleshooting.it peaks or peaks with shoulders may be caused by a blocked frit, defective guard column, or column void. Begin by replacing the first frit downstream from the injector, as this is the most likely place for particles to accumulate. It is recommended that a 0.2 - 0.5 μm in- line filter be placed immediately after the injector to trap mobile phase particulates. If this does not correct the problem, replace the guard column. If problems persist, it may be time to replace the column. If only one or two peaks in the chromatogram show splitting, this may be the result of a non-resolved or interfering compound. The mobile phase composition should be altered in an attempt to resolve the mystery peak. Sample preparation procedures should also be reviewed to determine if the interference can be removed prior to analysis. Broad peaks or fronting, often accompanied by shifts to shorter retention times indicate sample overload. Dilute the sample by a factor of 10 to 20 and reinject, making sure that the system settings have been changed accordingly. Sharper peaks throughout the chromatogram confirm a sample overloading problem. Adjust the method and sample preparation procedure to avoid similar problems in the future.If early eluting peaks are affected more than later peaks, the problem may be related to extra-column effects or injection solvent problems. Extra-column effects are caused by excessive system dead volume. Inspect the system tubing to ensure that it is the proper internal diameter. Examine the flow cell to confirm that it is the proper volume, and check the data handling system for the proper sampling-rate setting. If the problem persists, check the injection solvent strength and sample size. A strong injection solvent can cause the sample to move quickly through the first portion of the column while it re-equilibrates with the mobile phase, thereby broadening or even splitting peaks. It is best to inject the sample in the starting mobile phase to avoid this type of problem. Band spreading often results from column degradation or contamination. If the column provides low efficiency, try cleaning it with a strong solvent before replacing the column. If the column is functioning properly, the problem may be related to a partially plugged in-line filter, an injector or detector problem, or incorrectly installed tubing. These problems will likely also produce other symptoms such as too-high pressure, leaks, or flow variation.Tailing peaks are usually the result of secondary retention interactions between basic functional groups in the analystes (amino groups for example) and unbonded exposed silanol groups on the column packing material. Peak tailing caused by this type of interaction can be minimized by using mobile phase modifiers such as triethylamine (TEA) at low concentrations of 20 to 30 mM. Using these types of mobile phase modifiers will irreversibly alter the column and render it unusable for other assays not requiring similar modifiers. Ion-pairing reagents, sample derivatization and strong buffers can also be employed to reduce peak tailing. As an alternative, new ultrapure base-deactivated columns are now available. These columns do not require mobile phase modifiers in order to provide good peak shapes for basic compounds. Ghost peaks are spurious peaks of unknown origin. One possible cause of ghost peaks is a difference between the amount of dissolved air in the sample and the mobile phase. This problem can be corrected by degassing both the mobile phase and sample by using an in-line vacuum degasser. When using UV detection, ghost peaks are more prevalent and typically larger at lower wavelengths. It is also useful to exchange acetonitrile (ACN) for methanol in the mobile phase since oxygen has lower solubility in ACN. Ghost peaks can also result from refractive-index effects, column contamination, mobile phase contamination, contaminated autosampler blank or wash solution, or microbial growth in a mobile-phase component or solvent filter.
