Translator for HPLC HINTS and TIPS for Chromatographers

Saturday, February 10, 2018

HPLC Baseline Stabilization Tips for Refractive Index Detectors (RI or RID)

If you use refractive index detection (RID) for your HPLC samples, then you are already familiar with the very long equilibration periods needed to stabilize the system and associated baseline drift. Initial equilibration can take several hours. In fact, re-equilibration takes far longer to achieve with this detection mode than most others (i.e. UV/VIS, FLUOR, EC). While there is no quick cure for these delays, there are a number of things that you may be able to do to minimize or reduce these wait times. Here are a few to consider.


  • ROOM TEMPERATURE: Locate the RID in a quiet, stable location. If the room temperature in which your HPLC system with RID system is located fluctuates by even one degree C, that can effect the stabilization of the system. The ideal room to use RID will be away from any windows, drafts, doorways, direct sunlight and HVAC vents or ducts. It should be located in a quiet area away from people walking by it. All of these things can contribute to temperature instability, which is what you want to avoid.

  • INSULATION of HPLC CAPILLARY LINES: All of those stainless steel capillary lines leading from your column outlet to the RID's flow cell are loosing heat to the surrounding air (cooling). To reduce this thermal effect, insulate any metal lines with plastic tubing to reduce the heat loss. Most any type of laboratory grade, thick walled plastic tubing can be used. Pass the SS tubing through the plastic insulated tubing or use a section of split-tubing to cover it. Cover as much of the exposed tubing as possible, right up to the fittings. - Note: Sometime the HPLC system's solvent bottles may be subjected to varying temperature changes too. In these cases you can wrap the bottles with an appropriate insulating material to reduce the effects.
  • FLOW CELL TEMPERATURE: Modern RID units have a heated flow cell with thermostat to control the temperature of the flow cell. This helps stabilize the temperature inside the flow cell as well as minimize the unintended effect that the heat given off by the RID's electronics has on the temperature inside the flow cell. If the flow cell temperature does not stabilize, then the baseline will drift in response to it. For most methods, select a flow cell temperature which is at least 10 degrees C above ambient (since most of these units can heat only, not cool). Factor in any column temperature used too. If you are maintaining your column at 40C, then try to maintain your flow cell at the same temperature to minimize any differences. Feel free to experiment to find the best temperature for your flow cell. Try different temperatures (in 5 degree C intervals), wait for the system to fully equilibrate, then measure the baseline S/N ratio. You may find best results using different column and flow cell temperatures. Sometimes the room temperature effect can be countered by using an optimized flow cell temperature (higher or lower). Always factor in your mobile phase boiling point (b.p.) into your method and keep the column and flow cell temperatures well below the b.p.

  • DEGASSING / DECREASING DISSOLVED OXYGEN: Reduce and stabilize the amount of dissolved gas inside the mobile phase and you may achieve faster equilibration times with a RID. You do not need to remove all the dissolved gas (in fact, a reduction of 50% is often enough). The amount of dissolved gas inside the mobile phase effects the measured refractive index. As it changes, so does your baseline. High percentages of mobile phase dissolved gas = lower RI; Less dissolved gas = higher RI. Now water holds less dissolved gas than non-polar organic solvents (e.g. THF) so this effect is more pronounced when you are running non-aqueous GPC separations, but maintaining a stable dissolved gas level for all mobile phase types is important to reduce baseline drift. Stability is our goal. Continuous degassing of the mobile phase either through sparging with high purity helium gas (best for non-aqueous separations) OR using an inline vacuum degasser should provide you with a way to control the amount of dissolved gas in the solution and reduce drift.

These are a few of the factors which can effect the equilibration and drift times of an HPLC system equipped with a refractive index detector (RID). Careful selection of the instrument module's location, insulating the exposed capillary lines and bottles, optimizing the column and flow cell temperatures, maintaining a steady and controlled temperature in the environment, plus removing dissolved gas from the mobile phase may all contribute to more stable baselines and better quality peak integration.

Another article which may help you improve your analysis method can be found on this site. "Diagnosing & Troubleshooting HPLC Pressure Fluctuation Problems (Unstable Baseline)".

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