HPLC SOLVENT COMPRESSIBILITY - REVISITED

 Twelve years ago I published a short article here (HPLC PUMP SOLVENT COMPRESSIBILITY VALUES) which described the importance of setting the correct solvent compressibility values in the HPLC pump's table. Developing HPLC methods which exhibit smooth, stable baselines, with little measurable signal artifacts (e.g. spikes, noise, oscillation) and minimal pressure fluctuations help insure reliable, repeatable methods. Taking steps to insure that the LC pump operates is setup properly for the method are part of following good chromatography fundamentals

Over the past month I consulted for three different clients who needed help in troubleshooting various "pump stability problems". In all three cases, each HPLC system showed extreme pump pressure cycling, cavitation, noise and instability over time. Pressure fluctuations of 10% (or in one case, 10-30% Ripple values) were observed in several different HPLC methods that were used. One of the very first areas to check for problems with pump pressure instability is mobile phase degassing.  

Proper operation of the HPLC pump requires that efficient degassing of all mobile phases is performed before the liquids enter the pump head. 

Failure to properly degas liquids often results in pump cavitation, check valve sticking and baseline instability. An Inline vacuum degasser or continuous Helium sparing should be used to degas all mobile phase solution for use in HPLC (not sonication or vacuum filtration which perform poorly to solve degassing issues). 

In one of the three cases, the HPLC degasser was found to be broken and long overdue for service. Cleaning and servicing the degasser cured the problem and the method that once showed pressure ripple of >10% now shows no baseline disturbances and very low ripple of ~0.1% at ~ 70 bars system pressure. 

Before I was called in to assist each client, the clients had replaced numerous parts, including: pump seals, check valves, mixers, solvent frits and still had the same baseline instability issues (no change). As recommended by me, two of the clients had their very old degassers cleaned and serviced (as they were long overdue for service), but still had some baseline and pump instability (servicing the degassers improved the baselines, but the pump was not running as it should). In both cases, the cause for the remaining pump instability was quickly identified by me on-site (many problems can be quickly diagnosed on-site).

• The client had incompatible solvent compressibility values stored as part of their HPLC methods. This resulted in huge baseline disturbances, spikes, cavitation and occasional loss of prime. 

One of the clients normally ran methods containing high percentages of ACN (with some water) for their sample methods, but a few months earlier had switched to running with gradients containing high percentages of methanol. The solvent compressibility values stored in their system were appropriate for WATER, but they never updated them when they used the same method file to run samples in mostly methanol solutions (which need different compressibility values). Though they all had been using HPLC for many years, they had not received basic HPLC instrument training to know how to adjust and optimize these and other important instrument settings for EACH method (they were overwriting each new method, a common new user mistake, when making changes). Once we changed the method's solvent compressibility value to a more compatible one (in their case, for methanol), the baseline smoothed out in just a few minutes and all of the pressure instability issues went away (*they had replaced several thousand dollars worth of perfectly functioning parts trying to solve this issue before I arrived). Professional training in how to use and operate any HPLC instrument should always include how to set and optimize the compressibility value(s). Make sure you know how to incorporate the correct value in each new method that you create. Always spend up-front time to optimize each method for the application before you use it to analyze real samples. The initial time spent getting everything to run smoothly and reliably will improve overall accuracy plus save money and time.

• Note: In a low-pressure HPLC single-head pumping system with multi-position solvent selector valve (e.g. Most ternary or quaternary systems) one value is allowed, but in a true, dual-head binary pumping system each of the two pump-heads may have a separate field to input the solvent compressibility values.

The importance of inputting the correct and applicable solvent compressibility value(s) into the pump's settings, for each solvent used is one of many steps in creating an optimized HPLC method. There are no universal values, but the instrument manufacturer will have included a generic value in the pump's compressibility settings field. Should you use this generic value?  What are the chances that a randomly selected value used as a 'place holder' in the software is the correct value for your method?  Just as with flow rate, solvent composition, run time, stroke volume, wavelength etc., entering (and saving) the correct solvent compressibility value into EACH method helps to optimize the pumping performance. You will want to select an appropriate value FOR EACH AND EVERY HPLC METHOD YOU CREATE and use (and be sure to save the method with a unique name). Start by loading your HPLC method into the system, then look at the solvent compressibility value(s) used. Are they correct? Change the value(s) shown to values that are appropriate for your method. It is OK to experiment and try different values (we encourage it!). Monitor the S/N levels of the baseline noise for comparison. The instrument manufacturer should provide a table of suggestion solvent compressibility values for use with their system [For HP/Agilent systems, you can see an example table at the link I provided in the first paragraph of this article or review the operator's manual for more information].

• A related article to this one that you may find useful is; "Diagnosing & Troubleshooting HPLC Pressure Fluctuation Problems (Unstable Baseline)";
  

A Case of Changing Solution pH. Formic Acid Stability in Solution (Methanol)

Real life examples help to better illustrate problems that I am called in to troubleshoot for clients. As a professional scientific consultant, many of my clients have spent months (sometimes years) trying to solve an analytical problem on their own before I am brought in to make the diagnosis and propose a solution. Many years of working in a wide range of scientific fields allows me to identify problems quickly and efficiently saving clients the most money and allowing them to resume work on their projects.

This was the case during a recent consult for a major cannabis testing laboratory. They were having a great deal of difficulty obtaining reproducible results for their analytical testing screens (14 compounds in their analysis with a need for repeatable and accurate results). Variations from 25% to 50% were observed run-to-run over the course of seven days. They assured me they were doing everything in the same way. To begin the troubleshooting process, we started by looking at the actual data gathered and the actual method(s) used to acquire the data. These were evaluated to see if they followed good practices and techniques, also to make sure they had SOP's in place which were clear. Good SOP's must include enough detail to allow anyone reviewing them to prepare samples, standards and/or solutions in the exact same way. Additionally, the HPLC instrumentation was checked and tested to verify it was performing as designed.

After reviewing their training and methodologies on-site, a number of areas of concern were quickly identified. One of the most likely reasons for the variation in values over time was found to be caused by a common mistake in the preparation of mobile phase solutions for the HPLC system. To save time, the client's scientists prepared all organic solvent solutions in advance (~ one month or more), then filtered and stored them at room temperature. For example, their solutions of 0.1% formic acid in HPLC grade Methanol were pre-mixed and stored in glass one liter bottles. These bottles were then put aside, for an average of one month before use. This finding proved key as someone with proper HPLC training would be aware of a well known problem when formic acid is left in pure organic solvent, especially methanol, over time (less so with ACN). Briefly, the formic acid content degrades quickly over time and is often found to be only half of what it was initially after just three or four days (If you have not done so already, this is a simple and useful experiment to run in your lab, monitoring the acid level by titration, not with a pH meter, over time at room temperature in methanol)! This degradation continues over time reducing the amount of acid in solution. If the acid is added to the solution to enhance ionization (i.e. LC-MS; LC-MS/MS) or provide acidification to maintain the sample in a fully ionized form, then as the level of acidification decreases, so does the solution's ability to maintain it. In other words, your HPLC method may change over time (resulting in an in-valid method).

•  I have always promoted the importance of making and using freshly prepared mobile phase solutions (daily), especially where any aqueous solutions are used (to prevent degradation of additives and/or bacterial or fungi growth). However, this precaution does not normally apply to many pure organic solvents, but there are a few very important exceptions to this, formic acid and methanol in this example. 

Changes were made to their SOP's to insure that future solutions of formic acid in methanol were not prepared in advance, but instead, fresh on the day needed only. This coupled with a few basic improvements to their column washing, equilibration and overall training resulted in %RSD of only 0.3% for future analysis runs.

 
As a side note, I have been asked why solutions of formic acid in methanol are sold commercially for HPLC use? I have no answer to this, but respectfully remind everyone that just because something is offered for sale, does not mean it should be purchased. Ask yourself if the item is appropriate for your application? It may not be suitable for your use or application. 

BTW: Please be sure to flush your HPLC system of all organic acids (e.g. acetic, formic) after use and do not leave them in the HPLC system overnight. Even 1% levels of organic acids may be corrosive to stainless steel. 

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)".

Column Temperature in HPLC / UHPLC / LC-MS

Let us not forget the role of temperature in liquid chromatography. Just as mobile phase composition changes are used to develop better methods, column temperature is an important chromatography variable which must be addressed. I would like to call to your attention to a few different ways temperature can change your chromatography in this "hint and tip".

(1) Stability & Reproducibility of the Method: 
Maintaining a stable column temperature during a separation is important. Excellent temperature stability can lead to a high degree of reproducibility (*Their are of course many other factors to consider as well). For a typical analysis, temperature stability of 1.0 °C / hour (over the course of the analysis) is usually enough. If you are not using a thermostatted column compartment to perform your chromatography you may have already noticed the hour-to-hour or day-to-day fluctuations which can result from running samples under ambient temperature conditions. The normal changes in room temperature can be several degrees C over an eight hour period. These types of temperatures changes can make it impossible to achieve reproducible results for some samples. It is for this reason that it is critical that you include some type of temperature control as part of your method. Always record the temperature at the start and end of each run and include this data with your report. Most of the automated chromatography data systems provide this data as standard today and it is very valuable in reproducing the data as well as for troubleshooting, if needed.

(2) Back Pressure:
Column back pressure is directly changed by temperature. As the temperature rises, the column back pressure decreases. As the temperature decreases, the back pressure increases. This can be a useful variable when working with some of the newest sub-two micron particles on the market. The very high back pressures produced by these particles can be significantly reduced by increasing the column temperature [See "Pressure Drop Across an HPLC Column" http://www.hplctools.com/Tip%20114%20Pressure%20Drop%20Across%20an%20HPLC%20Column.htm]. 

When practical, try experimenting with your method by increasing the temperature, in increments of 5°C, to measure the change. You may discover an improved method with lower back pressures, a shorter run time and sharper peaks.

(3) Viscosity: 
Viscous mobile phase systems can take advantage of using higher temperatures to reduce the overall system back pressure. Since efficiency often improves with higher temperatures a double bonus of higher efficiency (sharper peaks) and lower back pressure can be achieved just by increasing the column temperature (peaks sometimes change elution order too so use standards to check this). 

(4) Practical Considerations:
Their are limits to using higher temperatures in chromatography which must be respected. The stability and solubility of your sample, the boiling point of your solvent, the maximum temperature setting of your column heater (mobile phase, flow cell and the rest of the HPLC system) and the stability of your column over time will determine how far you can safely push this.

(5) Specifications: 
One other issue worth mentioning here is that many traditional silica columns can loose their bonded phase at temperatures above 60°C. Some specialty silica phases (i.e. Waters XBridge & Zorbax StableBond) have temperature ratings to ~ 90°C. The more exotic non-silica based supports (e.g. Zirconium, graphitized carbon and/or PSDVB) often provide poor efficiency compared to the silica based products, but can handle temperatures in excess of 100°C. 

*Always consult with the column and/or instrument manufacturer to determine what the correct and safe operating conditions are before using any instrument, column or chemical.