Translator for HPLC HINTS and TIPS for Chromatographers

Saturday, September 6, 2014

Common Causes of Baseline Noise in HPLC, UHPLC.

Achieving a flat baseline which does not exhibit spikes, ghost peaks, drift or wander in an unpredictable manner should be a primary goal when performing HPLC analysis or developing methods. Methods which result in flat baselines and have well defined, sharp peaks allow for accurate sample area integration. Integration algorithms perform poorly in quantifying peaks on sloped, drifting or noisy baselines. Excessive baseline noise contributes to many problems, including poor quantitation, high %RSD errors, peak identification errors, retention time variation and many other critical problems. Properly developed HPLC methods are reproducible methods which apply and utilize good chromatography fundamentals. 

Note: A lack of proper training in the operation of the HPLC system, improper start-up or poor quality maintenance of the chromatograph (Examples: failure to degas and purge the system lines before use; an air bubble stuck in a check valve, a bad detector lamp or a leak will often result in baseline noise) are the main causes of noise. Your HPLC system must be optimized for your specific application. Be sure and allow time for the mobile phase to reach full equilibration with the system before starting any analysis.

In this article, we will discuss how temperature fluctuations, inadequate mixing, inadequate degassing and flow cell contamination can result in excessive baseline noise. We will provide suggestions on how to reduce or eliminate these problems.

To obtain reproducible results, the temperature of the hplc column must be kept constant during each analysis. Laboratory room temperatures often vary by several degrees during the course of one day and these changes will often change the retention characteristics of the sample(s). The 'On' and 'Off' cycling of power from an air conditioner or heating unit will often cause the baseline to drift in a cyclical manner, up and down, during the day (this can often be seen as a clear sine wave pattern when you zoom-in to study the baseline trace over time). Temperature also changes the refractive index of the mobile phase. Light based detectors (UV/VIS, RI...) will show this change as drift up or down). In some cases, a temperature change of plus or minus one degree C from run-to-run can cause changes in retention times which effect reliability of the method. 

To reduce temperature fluctuations, you must control the temperature of the column and mobile phase (if applicable) during the analysis. This is most commonly done by: (a) using equilibrated mobile phase at the start of the day or analysis, (b) keeping the interconnecting lines as short as possible (esp. any which exit the column and go to detectors/flow cells), (c) insulating any stainless steel lines with plastic tubing to reduce heat loss and (d) using a thermostatted column compartment to maintain the column at a single set temperature throughout the day. Control of the column temperature will remove 'temperature' as a variable from your analysis. Temperature should be a constant run to run, not a variable. Be sure and document the temperature selected as part of your method.

The associated noise and ripple of incomplete mixing can reduce the limit of detection (LOD) and increase integration error. Both high pressure (with separate pumps) and low pressure pumping (one pump with a multi-channel proportioning valve) systems depend on efficient mixing to reduce noise. For gradient analysis, failure to completely mix the mobile phase solution before it enters the HPLC column often results in excessive baseline noise, spikes and poor reproducibility. "Mixing" is often initially accomplished by combining the flow paths of more than one solvent channel together, using a multi-channel gradient valve or tubing. Mixing also performed directly in a mixer installed in the flow path of an HPLC pump. This mixer is often a static mixer (a simple 'Tee', a tube filled with baffles, a frit or beads, valve orifice or microfluidic device) of low volume design for chromatography use, but allows adequate mixing of the liquids within a prescribed flow rate range. The best mixers incorporate longitudinal and radial mixing in-line. A mixer with too low a volume or of insufficient design can result in poor mixing of the mobile phase (note: incorrect solvent compressibility settings can also cause mixing and noise problems too). To reduce mixing problems, first insure that the mobile phases used are fully soluble with each other. Next, make sure that any mixer used is appropriate for the flow rates and volumes you will be using. If needed, run a gradient valve test to insure that each valve channel is working properly, not leaking or introducing any cross-flow leakage to another channel. Monitor the baseline for pressure stability (% ripple), drift and artifacts  (e.g. spikes) in real time to spot problems and make adjustments to correct them. 

For the best results, continuously degas your mobile phase. Reducing the amount of gas will also improve signal to noise levels of detection, reduce drift and reduce pump cavitation. If you are using an electronic vacuum degassing module, make sure it is maintained and working 100%. A faulty degasser may cause more damage (contamination) to your system and methods. Maintain and Repair them just as you do for your other instrument modules. Gas bubbles may cause check valves to malfunction (get stuck), baseline noise spikes to appear randomly, flow rates and/or pressures to become irregular, detector outputs to show high levels of noise (from air in the flow cell) and also cause the loss of prime or cavitation in pumps. To achieve the best balance of low noise levels and high reliability, both aqueous and organic mobile phases should be fully degassed before and during use. This can be accomplished through stand-alone inline vacuum degassing modules or through gentle continuous helium gas sparging (*Helium makes an excellent choice of gas as it is not soluble in the mobile phase. Never use Nitrogen or Argon gas, they are soluble in the liquid!). In all cases, degassing must be continuous (not just done one time). Continuous degassing reduces cyclical noise and signal variations. For this reason, I do not recommend using ultrasonic baths to degass mobile phase solutions as these are not used in a continuous mode. The mobile phase solution starts to re-absorb gas as soon as you stop sonicating the solution. This results in continuous baseline drift (up and down).
Removal of gasses is critical to the function of a modern HPLC pumping system. The liquids used are compressed to very high levels which forces out solubilized gas from the solutions. This is best accomplished before the liquid is transferred into the pump. These gas bubbles must be minimized to achieve desirable baselines. *Even if you use a high pressure pumping system, an inline degassing system reduces the amount of noise and baseline drift. Properly maintain and service your degasser to insure compliant operation. IOW: Whichever method you use, always degas your mobile phase solutions.

One other less common cause of baseline spikes and random noise is due to either a dirty flow cell (i.e. the windows) or an air bubble trapped inside the flow cell. If the flow cell is suspected of having one of these problems, then it should be carefully rinsed or flushed out with an appropriate mixture of suitable solutions to expel the air bubble or remove the contamination. If possible, keep a spare, 'known good' flow cell on hand to swap out for troubleshooting purposes. This can help to quickly determine where the problem is. This flow cell must be the exact same size and type (volume and path length) for this purpose. If the cell's windows are contaminated and flushing does not restore them, then many manufacturer's offer kits which allow you to replace the windows and gaskets used. Warning: When attempting to clean or repair any flow cell, be sure and work within the manufacturer's operational specifications for the specific flow cell. Some flow cells are not designed to withstand even very low back pressure and damage can result if you exceed their maximum pressure or chemical rating.

Many other types of problems not mentioned in this short article can also cause baseline noise. For example, a sticking inlet or outlet valve on the pump, worn piston seals, worn out detector lamp(s) or detector electrode (EC) can induce noise. In all cases, the cause must be investigated in a logical, step-wise manner. Demonstrate what is working and rule out items one-by-one.