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

HPLC Solution Degassing, Sparging With the Wrong Gas (Gas Choice Matters)

The other day I took a call from a client whom explained they were having a number of problems with their HPLC pump. They felt that they were very experienced chromatographers whom had been unable to find the reason for why their pump flow stability was poor. It had very high ripple and noise. The pump had been fully serviced one month earlier and passed all qualification tests. Their UV/VIS detector appeared to work fine and was ruled out as being the problem early on. They used HPLC grade filtered solvents, operated at an appropriate flow rate, had a clean and tested column installed, always primed their pump before use each day and sparged each solvent reservoir with a low stream of continuous gas kept away from the solvent inlet lines. Everything seemed in order, but something was clearly wrong. Their vendor suspected the check valves were to blame so they purchased and installed new ones with no change. They still had an unstable flow rate under all conditions tested (pump pulsation of 5%). When I asked them if they had changed anything related to the HPLC system in the past few months I was reassured that nothing had been altered.


Often the best way to solve a problem is to start at the beginning. Take nothing for granted. This started as one of those many phone calls I receive where someone wants me to solve their problem over the phone and by not visiting their laboratory. Sometimes this is possible, but sometimes the problem is something that can only be seen by being physically present in their laboratory. I felt this was one of those times. So, they agreed to pay for a few hours of consulting time to have me come out and go over their system to find the problem. Once I arrived at the client's lab I quickly went over to inspect the layout of the equipment and check the tubing connections for the correct fittings and tightness. Next, I looked at the software parameters being used to operate the system. Some small issues were found, but not enough to explain the problem seen. I then looked at the physical output of the pump and detector to get a better idea of the period, cycle and type of noise seen. While I was reviewing the data and still looking over the system, I found the problem. The high pressure gas cylinder next to the instrument was labeled ARGON. Argon was being used as the sparging gas for the mobile phase instead of the more appropriate gas, Helium. They had in fact recently switched to argon gas because it was less expensive to use than helium. The person (their senior chemist) who had made this substitution was rewarded for his cost-cutting suggestion. Their choice of argon gas had of course cost them several weeks of down time while they tried to solve this problem on their own.... not much of a savings when you consider that! So, they had in fact caused the problem themselves, but were not aware of the fundamental reason why changing to argon gas was a very bad idea.

Why does the gas choice matter? For liquid chromatography applications we only use high-purity helium gas for sparging because it is one of the few inert gases which is the least soluble in water and mobile phase solutions. Gases such as argon and nitrogen ARE soluble in water and mobile phase solutions.  While they can be used to displace oxygen from air (great if you are making wine, but not so great if you are using the solution for HPLC), they infuse the liquid with gas (like a soda). Helium easily displaces air (oxygen and nitrogen) from solutions while not adding significant amount of dissolved gas to the solution. Helium is the least soluble and most inert gas to use. If we sparge with argon or nitrogen, then we infuse the solution with gas. This is the opposite of what we wish to accomplish by degassing our mobile phase. Please, if you wish to use the continuous gas sparging method to degass your mobile phase, then use high-purity helium gas only.

So I suggested that they replace their high pressure argon cylinder with a tank of high purity helium. Luckily they still had their original helium tank available so we hooked it back up. I sparged their mobile phase with the helium gas for about ten minutes then primed the pumps with the solution. The helium was left continuously flowing at a very low pressure (~ 2 psi) through a dedicated SS frit in the mobile phase. This keeps the level of helium in solution constant over time, resulting in stable baselines. After about five minutes the pump was running smooth and about as pulse free as you could hope for (0.1% pulsation). Lesson: Never assume anything and don't forget to make decisions which incorporate some basic scientific reasoning into them first.

INLINE HPLC DEGASSING MODULES

    INLINE HPLC VACUUM DEGASSER / DEGASSING MODULES:

Degassing your mobile phase is critical to maintaining a stable baseline without fluctuations. Loss of HPLC pump prime or cavitation, baseline instability and poor reproducibility often result when the mobile phase is not degassed. Modern electronic vacuum degassing systems are a great convenience (just plug them in). Changing heavy Helium gas cylinders, monitoring gas pressures and levels over time have been replaced in many labs with the sound of small gurgling vacuum pumps. While continuous helium sparging provides the best type of degassing for HPLC and benchtop ultrasonic baths are poorly suited to degassing HPLC mobile phases, vacuum degassers are widely used. 

• Since sonicators and in-line vacuum degassers do not specifically remove oxygen from the gas inside the liquid, continuous helium sparging should still be used for applications where low mobile phase oxygen levels are needed to maintain high-sensitivity (i.e. Fluorescence detection or very low UV detection). While the convenience gained through the use of these in-line vacuum devices can be great, there are some important downsides to this technology. One of them will be mentioned here.

Total Channel Volume: The volume of liquid contained in the tubing and vacuum chamber of EACH vacuum degassing channel can be enormous compared to the tiny volume often found in systems which utilized Helium sparging alone. When the system is turned off, this solvent often sits stagnant over night, allowing air to bleed in, and must be flushed out when the system is re-initialized for use the following day. *If you just switched bottles of mobile phase, then the old mobile phase is still inside the system and needs to be completely flushed out before use or you will have some very strange chromatographic results in the coming weeks! This process takes time and should be performed on every channel in your system, even if you are not going to use them all. How much volume you ask ?

Let’s look at the chamber volumes found in a few commonly used vacuum degassing units (You can look up the spec’s or measure it on your own).

• Agilent/HP Brand Degassing Module, Model G1322A; Each vacuum channel chamber has a volume of about 10 to 12 ml. The interconnecting tubing (solvent frit to bottle head, bottle head to degassing unit and degassing unit to pump inlet) can add another 10 mls more. In this example, we recommend that you flow at least 30 mls of the appropriate solvent through the line before use. This will insure that the channel is primed with fresh mobile phase. That step is for EACH channel so it is best to flush them one-at-a-time, in sequential order, to keep track of them.

• Agilent/HP Brand Micro Degassing Module, Model G1379A, G1379B or G4225A:  Each vacuum channel chamber has a volume of about 1 ml. The interconnecting tubing (solvent frit to bottle head, bottle head to degassing unit and degassing unit to pump inlet) can add another 10 mls more. In this example, we recommend that you flow at least 20 mls of the appropriate solvent through the line before use. This will insure that the channel is primed with fresh mobile phase (it is best to flush them one-at-a-time, in sequential order,100% A, 100% B ... to reduce miscibility concerns).

• Shimadzu DGU-20A3, DGU-20A5, DGU-20A3R & DGU-20A5R. Each vacuum channel in these modules has a volume of between 0.4 and 0.5 mL. We recommend that you flow at least 15 mls of the appropriate solvent through each line before use.

• Waters Alliance 2690, 2695, 2790, 2795, 1525, DG2 Systems and Acquity BSM/QSM. Each vacuum channel in these modules has a 0.5 ml volume. Because these are integrated degassers (exc for the DG2 stand-alone), the interconnecting tubing volumes are also low ~ 5 ml each. For this example, we recommend you flush each channel with at least 10 mls of mobile phase before use. Remember to flush them one-at-a-time, in sequential order, for best results.

NOTE: Some early models of HPLC vacuum degassing systems had individual chamber volumes of 30 to 50 mls each ! When you add the total tubing volume to that you end up with a flush volume of 40 to 60 mls per channel. These systems took a very long time to flush out.

To save time performing these initial “flush” steps, you should take advantage of the highest available flow rate setting of your pump. With the priming valve open (so the solvent goes to waste, not to the column), set the pump flow rate to 5 or 10 ml/min at 100% for the first channel (i.e. 'A') and you will be able to flush out a single channel in two or three minutes. Repeat for each of the other channels and then adjust the flow rate slowly back down to your normal equilibration flow rate before closing the prime purge valve again. Our lab performs this flushing procedure at the start of each day to insure the HPLC columns receive fresh solvent. This procedure should also be performed any time a mobile phase bottle is changed out to a different bottle or the liquid is "topped off" too.

Please keep in mind that running liquid at 10 ml/min during the flush phase in most analytical degassing modules will not result in very efficient degassing as degassing efficiency is a function of flow rate (surface area of liquid exposed over time. The lower the flow rate, the better the degassing). You will still need to wait a period of time until the new solvent is fully degassed once again, but at least you will know it is free of any older mobile phase. *Remember, everything you do before you start an analysis contributes to the final result.

•  Make sure that your degassing module is 100% fully functional. These modules usually wear out after between three to five years of normal use. We have found that some models like the Agilent G1379A and G1379B's may wear out sooner (~ 3 years). Problems with leaks, liquid exiting the vacuum pump exhaust line, bubbles, contamination and failed vacuum pumps are very common. These are maintenance items.  If the unit is repaired early, the cost may be much less than delaying repair so have the system properly tested and diagnosed at the first sign of trouble. Anytime the degasser's performance drops below the manufacturer's specification, your hear unusual noises and/or liquid is observed exiting from the vacuum pump's exhaust line, your entire HPLC system may fall out of compliance. You must repair the degasser module to use it. The degasser is an important component of your entire HPLC system so it must be maintained and serviced just like the other modules. RED or Yellow Error lights (on Agilent/HP models) or "Degasser hardware fault" error (Waters) on these modules usually indicates a need for professional service. 

  References:

    TROUBLESHOOTING INFO PAGES:  

• Waters Brand HPLC Vacuum Degasser Modules: 
  

• Agilent / HP Brand HPLC Vacuum Degasser Modules:
  

• Professional VACUUM DEGASER DIAGNOSTIC TESTING and REPAIR SERVICES (USA): Do you own an Agilent, HP, Shimadzu, Thermo, Dionex, Systec, IDEX or Waters brand HPLC degasser module which needs parts or service? These modules require service at least every 3 to 5 years. Chiralizer Services can professionally repair most HPLC degasser modules, with quick turnaround (1-2 days), for far less money than other companies. Don't buy another contaminated or broken degasser from one of the auction web sites (e.g. ebay etc). Save money and have your own HPLC vacuum degasser professionally cleaned and repaired, quickly by the experts at Chiralizer Services, LLC. Please refer to this link for more information: http://www.chiralizer.com/hplc-degasser-repair.html