Baseline problems are indicated by a change in the appearance of the baseline. This change may occur even when no sample has been injected. Baseline noise can result from problems with the HPLC instrumentation, external environmental sources, or from the separation process itself. Locating the origin of the problem begins with determining which of these three factors is involved. The first step is to set the flow rate to zero and see if the problem disappears. If the problem is related to the chromatographic process, the baseline should be straight. If the problem is related to the instrument or environmental sources, the noise will continue.If the baseline noise is random or spiking and stops when the flow is turned off, the problem is most likely caused by bubbles in the detector cell or a contaminated HPLC column. Bubbles in the detector cell can often be removed by using a back-pressure regulator on the outlet of the flow cell. A slight positive pressure at the detector outlet causes gases to remain in solution until the mobile phase has cleared the detector cell. Often a short coil of small-diameter tubing at the detector outlet will suffice. THIS I HAVE PERSONALLY OBSERVED IN MY PROFESSIONAL LIFE THAT SMALL DIA. TUBING AT DETECTOR OUT LET WORKS...........Increasing flow rate also helps to purge bubbles from the detector cell by increasing the system pressure and forcing them back into solution. In either case, be careful not to exceed the maximum allowable pressure of the flow cell. Thoroughly degassing mobile phase solvents will also help prevent gas bubbles from forming.Cyclical baseline noise can result from small variations in mobile phase composition generated by solvent mixing in a gradient system. This is most likely to occur when the percentage of one solvent in the programmed mixture is low, 5% or less. Observe whether the period of the baseline noise corresponds to the period of the piston cycle in the low-flow pump of a high-pressure-mixing system, or to cycling of the mixing valve to the low-percentage solvent in a low-pressure mixing system. If the noise is due to solvent mixing, it can best be eliminated by premixing the mobile phase for an isocratic separation or by revising the mobile-phase components and program to avoid gradient mixing ratios of less than 5%. PRE MIXING WORKS WHEN THERE IS NO DEGASSER OR EXTERNAL PURGING GAS AVAILABE............OBSERVED IN WATERS MANUAL PREPARATIVE HPLC SYSTEM...............Other methods for reducing compositional noise include using mobile phase components that are more transparent at the wavelength being monitored and installing a larger-volume mixing chamber which will act as a more effective filter to damp out low-frequency variations in mobile phase composition.A wavy baseline is sometimes the result of contaminants or very late eluting components from the guard or analytical column. This may also result from contaminants in the mobile phase itself. Flush the column with a strong solvent to remove any contaminants, and always use pure HPLC grade water and solvents in the mobile phase.If the baseline noise continues under zero-flow conditions the problem may be related to a defective detector lamp or an environmental problem.
Rapid noise spikes may indicate the detector lamp needs replacement.
Most UV lamps have a lifetime of about 1000 hours of use and should be replaced after this period. BUT I HAVE USED WATERS 2998 PDA DETECTOR WITH LAMP HAVING MORE TAHN 4011 HRS LIFE AND STILL PRODUCING REPRODUCIBLE RESULTS IN QUALITATIVE ANALYSIS, QUANTIFICATION YET NOT TRIED ON THAT SYSTEM.........Baseline noise can also be caused by external environmental factors. If the noise appears at regular intervals, check for other equipment and devices that may be on the same electrical circuit and disconnect them or reroute them to other available circuits. Also, make sure the cases of all components of the HPLC system are connected to a common ground.
Baseline Drift
Baseline drift generally results from interferences in the mobile phase, contaminant buildup on the column, or changes in temperature. If the drift is repeatable between chromatographic runs, then the problem is probably related to the mobile phase. In gradient chromatography, a changing baseline during the course of a run is common. This is especially true when using UV detection at low wavelengths because of differences in absorbance of the different solvents used. Attempt to select solvents where the absorbances are balanced as closely as possible.If the baseline continues drifting randomly, then the problem may be coming from contamination building up on the column. If this is suspected, try flushing the column with a strong solvent. If the problem continues, replace the column and review the chromatographic and sample preparation procedures.If the drift is cyclic between operating sessions, then the problem may be related to changes in ambient air temperature which affect the chromatographic separation. Changes in laboratory air conditioning and heating often affect baseline drift and the separation itself. The most effective way to correct these problems is to thermostatically control column temperature. Drift can also result from the detector itself. Be sure the detector is warmed up sufficiently before performing chromatography. Check the detector manufacturer’s specification for drift, and be sure the lamp is not nearing the end of its expected lifetime.
