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. It is also a good idea to review training in the correct operation of the RI detector too. Learning to correctly operate the flush and optional recycle valves on these detectors is critical to their operation. Failure to properly flush the reference cell before each analysis with fresh mobile phase may lead to baseline changes or artifacts.

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

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: "Noise" is a relative term, often w/o meaning. You should always describe it scientifically, measure and compare the signal to noise ration (S/N) of the baseline vs the peak plus note any cyclical patterns (useful in troubleshooting).

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; poor mixing; 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. Do not start an analysis until the baseline is stable.

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. Troubleshooting should be done on-site, not over the web or telephone.

TEMPERATURE FLUCTUATIONS:

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.

INADEQUATE MOBILE PHASE MIXING:

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. 

INADEQUATE MOBILE PHASE DEGASSING:

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 degas 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.

FLOW CELLS:

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.

Reference: http://hplctips.blogspot.com/2014/01/diagnosing-troubleshooting-hplc.html

Diagnosing & Troubleshooting HPLC Pressure Fluctuation Problems (Unstable Baseline)

Few things in chromatography are more frustrating than dealing with large pressure fluctuations (>1% ripple). If the pump pressure is unstable, and fluctuating up and down, then it will negatively impact your ability to analyze, measure and integrate sample peaks in a reliable manner. A smooth, flat baseline is needed to run and develop methods, collect the data (peaks), integrate and report the results which are reproducible. Baseline instability during an analysis may lead to the entire analysis being declared invalid.

So what causes the HPLC pressure to sometimes fluctuate in a wild manner up and down on your HPLC system? Unfortunately, many things... Most result from poor training, incorrect operation techniques, but some are maintenance related so be sure and keep your chromatograph in excellent condition. Maintain a logbook for each instrument and record what types of maintenance and service have been performed over-time, with the date and list of parts used/replaced. Additionally, maintain a preventative maintenance schedule (e.g. every six months) to inspect and clean the entire HPLC system to check condition, verify operation and minimize unproductive down time. 

HPLC Pump or System Pressure Fluctuation Causes and Solutions:

• Air / gas In the Liquid or Mobile phase (Failure to Degas Mobile phase OR loose fittings) --- Air gets into the system due to a leak or from gas trapped in the mobile phase. Find and correct the cause of the leak and/or degas the mobile phase (use continuous Vacuum degassing or a Helium sparging system only). Leaks are the most common cause of instability, but insufficiently degassed solution is a close second. Make sure your degasser is working 100% correctly (they require professional servicing every 5 years). HPLC pumps require degassed mobile phase for reliable operation.

• Loss of Prime. Improper Priming of the System --- Failure to flush ALL of the lines with freshly degassed mobile phase, before use (every day), will often result in all kinds of instability problems until all of the old gas-filled mobile phase has bee purged from the system. *This could take many column volumes of liquid. Make sure you account for any vacuum chamber volume too. Properly prime the pump heads before use.

• Sticking Check Valve(s) --- If air is exiting the pump outlet, the pump will not function properly. Both Inlet and Outlet valves should be inspected. Remove and clean the check valve(s). Be sure the pump is fully primed with liquid as the check valve might just have an air bubble in it (common on Waters, Thermo and Shimadzu systems). Sometimes sonication of the valve for ten minutes in a beaker containing warm solvent does the trick (e.g. MeOH or IPA/Water). Though very rare, ACN has a bad reputation for polymerizing in solution. If the system has sat unused for a long time OR was not properly flushed out when last used, it is possible that particulate matter may clog the flow path. Small sticky particles may form (ACN polymerization) and cause the check valve to stick inside the housing (use fresh, filtered solvents only to prevent these problems). Clean and inspect any suspect valve first. Replacement of the check valve may be needed in some cases to restore operation. Note, this problem of "sticking" check valves is most likely to be an issue in HPLC pumps with mechanical (gravity or spring) check valves with ruby balls, not modern style active inlet check valves ("AIV") which are electromechanical (solenoid valves) and are very reliable, much less susceptible to these problems. In any case, verify operation of all valves while under pressure (backpressure is needed for them to function correctly).
  

• Worn Pump Piston Seals --- Commonly observed as rapid up/down spiking on all channels and an inability to maintain or produce backpressure (the pump will often prime with no problem, as this is done at low-pressure). Run a formal pump high-pressure leak test at max pressure to confirm (remove the column and replace with a calibrated backpressure restriction line for all testing). Clean pistons and replace piston seals to repair (you should have spare pistons and seals on hand). *Seals are a maintenance item so expect them to wear out and need regular replacement.

• Flow rate too low (may be inappropriate for system). Running at a flow rate that is below the optimum range of the specific instrument (i.e. System rated for 200 to 2,000 uL/min, but run at 100 to 200 uL/min or at the limit of the range) may result in an unstable baseline. The cause may be due to pump cavitation, loss of prime, non-optimized piston stroke volume.

• HPLC System Back-pressure too low to maintain prime in system. Most types of analytical HPLC systems require a minimum system back-pressure of 40 or more bars to maintain enough pressure (mechanical compression) on the component parts to run in a reliable fashion (*Water's Article number: 32564 states the back-pressure must be at least 1000 psi for their Alliance systems). Too low a pressure often results in a loss of prime, cavitation, mixing problems, turbulence and poor reproducibility. Correct sizing of column, particle size, flow rate and mobile phase composition should all take into account achieving enough back-pressure on the system to maintain a stable baseline throughout the entire analysis. Monitor the system back-pressure at all times for stability. High quality research grade HPLC systems are often capable of maintaining stable isocratic flows with less than 1% ripple and 0.2% ripple common ("ripple" is a term we often use to describe the pump's pressure output over time relative to the baseline (S/N)).

• Mixing Problem (gradient or isocratic online mixing) --- If your active mixer or proportioning valve (AKA: Gradient valve) is defective or dirty, then one or more of your mobile phase channels may not be getting to the pump. Air would most likely be mixing with the mobile phase causing the unstable flow. Clean or replace the valve. Note: Always try flushing the gradient valve with pure IPA, then DH20 for about twenty minutes. This sometimes restores operation by wetting and flushing the internal seals (which may dry out).

• Wrong Pump Solvent Compressibility Settings --- In HPLC we routinely subject different liquids to very high pressures which can result in measurable liquid compression. The degree of actual compression for each liquid varies, but the modern HPLC pump can compensate for this to improve the accuracy of the mixing and flow delivery.  Most pumps provide for user adjustable solvent compressibility values. If the value input varies a great deal from the actual liquid in the system, then it can result in pressure fluctuations. Example: Water has a value of 46, but Methanol 120. Using the wrong value can cause instability.  

• Poor Solubility, Mobile Phase  --- Sometimes the mobile phase which has been prepared (or mixed online) is not 100% soluble. This could be due to an inorganic salt additive which has not gone into solution or failure to fully mix and filter the mobile phase before use. Ultrasonication, a bit of heat and stirring for 20 minutes can help to get everything dissolved. 

• Dirty inline filter --- A fouled or partially plugged filter can disrupt the normally smooth flow into a turbulent one. Some are installed as part of the pump (i.e. HP/Agilent brand pumps) and should be changed out every month (Yes, for the PTFE frit, replace it once a month with a new one). Other systems use these pre-filters downstream of the pump before the injector. Clean or replace all filters frequently. If used in your system, these are regular maintenance items and should be part of a general 'PM' program.

• Dirty Solvent Pickup Inlet Filters: These can become obstructed or fouled over time (esp. if used with aqueous solutions!). Just as with any built-in filter, the multiple solvent inlet pickup filters should be cleaned or replaced on a regular basis to prevent particulate or any material which may contaminate or restrict the flow path from entering the system. Mobile phase pickup filters are often 10 to 20 um and connect to the bottom of the low pressure (e.g. Teflon) solvent lines in each bottle. If you use 316 Stainless steel filter (recommended for organic solvents), they should be removed, cleaned in an ultrasonic bath, rinsed and replaced monthly. If you use sintered glass or other disposable type filters (often used with aqueous solutions), they should be disposed of on a regular basis and replaced with new ones (replacement, not cleaning is recommended because sintered glass can not be sonicated and should be disposed of to prevent bacterial, mold or fungal contamination). A quick way to check if one filter is causing the pressure to fluctuate is to remove the filter from the one line, then re-test the system. If the problem goes away, then returns when you re-install the filter back on the line, the filter may be obstructed (replace it),

The above list includes some of the most common reasons for unstable baselines. Other non-pump related causes would include a bad / old detector lamp(s) or contaminated mobile phase. To find the cause, test and verify the operation of each component part of the HPLC. Troubleshooting Advice: Test one part at-a-time, before moving to the next part. Never assume anything, test, re-test and verify or prove at each step.