LC-MS Contamination? Another Possible Cause. Are your Mobile Phase Bottles and Pick up Filters Clean ?

One of the more common LC/MS problems I am asked to help solve deals with contaminated LC-MS or LC/MS/MS systems. Over time, many systems will become contaminated with a wide variety of plasticizers, detergents, salts, metals and ion pairing agents that routine source cleaning will not remove. Often, these compounds are introduced to the system through the tools used (e.g. pipettes) chemicals, solvents, mobile phase additives or even the samples themselves. "Dirty" samples sometimes persist inside the system long after the analysis work is complete, leaving material in poorly maintained injection valves but also through the use of poorly washed / contaminated and fouled HPLC columns. Even the modern inline HPLC vacuum degasser has proven to be a source of contamination. 

In addition to the above mentioned sources of contamination, another more obvious source of contamination should always be addressed early in the process of cleaning the system. Specifically, the glass mobile phase bottles and the associated solvent pickup tubing and solvent pickup filters used with them. Contamination in these areas may directly infuse the system with undesirable material. Good cleaning and maintenance practices must be maintained to reduce this source of potential contamination. 

As a general guideline, we shall not place our mobile phase reservoir bottles in any type of dishwasher or wash them using any dish soaps. These may leave a residue easily detected by even the weakest mass spectrometer. Avoid contamination by purchasing high quality glass bottles with vented caps to keep dust out. If rinsing with organic solvents (and/or freshly prepared and filtered high resistance water) does not clean them, you can try a Nitric Acid rinse (up to 30%) followed by a neutralizing wash in 2M Sodium hydroxide. Follow-up with many rinses of HPLC Grade water (or LC/MS grade), oven drying, then re-fill with an appropriate mobile phase. Don't forget to replace those solvent pickup filters too. While many 316 SS pickup filters can be cleaned, most of the sintered glass style filters are designed to be disposed of (not cleaned or put in an ultrasonic cleaner!). So periodically dispose of the glass types and install new filters and fresh mobile phase into those recently cleaned bottles (before you start looking for the source of contamination in the more expensive parts of the instrument, clean or replace the filters). - Please don't re-contaminate an expensive HPLC or LC/MS system and invalidate your methods and data because you skipped replacing a $10 part. Keep commonly used spare parts in-stock and always maintain a clean system.

The Three Most Common HPLC Questions and How To Solve Them

The three most common HPLC related questions I am asked each week can be summarized below. Test your basic chromatography knowledge. Before reading the answers, see if you can answer them correctly on your own.

• "What Is Causing the HPLC Baseline, Pressure or Peak Retention Time(s) To: Wander, Change, Drift, Vary or be Unstable?"

• "How Should I Wash or Regenerate My HPLC Column?"

• "How Can I Tell if the Sample is Retained On the HPLC Column? or What Does It Mean When No Chromatography Took Place?"

Let us address each question in order and attempt to provide accurate answers (I have included links after each question to articles with more detailed explanations).

What Is Causing the HPLC Baseline, Pressure or Peak Retention Time(s) To: Wander, Change, Drift, Vary or be Unstable?

• Retention times must be reproducible from run to run.The causes of an unstable baseline and/or changing peak retention time(s) are often related. Common reasons include: Column temperature fluctuations, inadequate mobile phase mixing or degassing, leaks, dirty column, sample overload, lack of pH or buffering control (weakly ionizable samples can be very sensitive to changes). *Full Article link with detailed answers, here.

How Should I Wash or Regenerate My HPLC Column?

Note: Before proceeding with any column regeneration or cleaning procedures, always refer to the specific advice provided by the column manufacturer. Approved maintenance and cleaning instructions can often be found in the product guide or booklet which comes with the new column. Additional information can be found on the vendor's website or by contacting them directly.

• Two issues must be addressed to answer these types of questions. (1) Always wash your column with a specific column wash solution which is stronger than your analysis solution. The use of a stronger solution (In this context, "stronger" means better at dissolving the samples and faster at eluting them from the column) as the wash solution requires regular use to maintain the column. Failure to regularly wash your column may result in compounds accumulating on the column over time (fouling the column) resulting in poor reproducibility, higher back-pressures, contamination and/or poor peak shape. (2) Next, always wash your column after each analysis. This should be a separate step, not incorporated into your analysis method. The analysis method should not include the column re-equilibration steps at all. A second, separate wash method should always follow each analysis method which includes the rinsing of the column with a "stronger" solution for an adequate period of time, then adjustment back to initial conditions where re-equilibration can take place to get it ready for the next analysis run. These are fundamental guidelines of good method development and follow well established principles. Developing methods in this way should increase the lifetime of your columns and improve the reproducibility of results obtained (better %RSD run-to-run).

For more information on washing bound proteins off RP HPLC columns, please refer to this linked article found here.

How Can I Tell if the Sample Is Retained On the HPLC Column? or What Does It Mean When the Sample Comes Out At or Near the Column Void Volume?

• Chromatography is a tool which when used properly adds one or more additional dimensions of physical or chemical characterization information to your analysis data. It does so first by using on-column RETENTION. Samples must be run under conditions which allow the material to interact with the chromatography support for a period of time. We define this time as the retention time. A sample which does not interact at all with the column support material will elute off the column early (and not be retained) at the "column void time" (or column dead time). We refer to this void time as the "T zero" time. When a sample elutes at or near the T zero time, no chromatography has taken place and no method has been developed. It is as if the HPLC column was not used. How do you know what the "T zero" time is (it will be different for different methods)? You must first calculate the HPLC column's dead volume. Once you know the column dead volume and flow rate, you can calculate the T zero time. A scientifically valid HPLC method will include conditions which retain the sample on the column for a long enough period of time to insure that it is interacting with the support. This allows for separation from other compounds to take place and is the purpose of chromatographic resolution. Without this retention mechanism, you are just flow-injecting the sample past the column and skipping all chromatography. It would be far simpler to just place the sample in a spectrophotomer cell as no retention or additional data would be obtained using that technique.

• When first learning liquid chromatography, two of the very first calculations you must learn to use in HPLC are: Column Dead Volume (aka: Column Void Volume) and the K prime of a sample (aka: Peak Capacity Factor). Do you know how to calculate these? They are calculated and reported for each method used. You should be able to tell anyone who asks you what the values are for each method. A chromatographer must know and understand them before using an HPLC system or running a method. They are also critical to method specificity and proper validation. Here are links which after reading and practicing, should make you an expert in these two fundamental calculations. 

"Determination of HPLC Column Dead Volume / Dead Time (T zero)";

"K Prime (also known as: Capacity Factor or Retention Factor): One of the Single Most Important HPLC Parameters of All".

So, how did you do answering these basic questions? If you have put in the needed study time and practical experience to learn and use these fundamentals of high-performance liquid chromatography, then you should have been able to easily provide correct answers to all three questions. If not, then it is time to go back and study up on those basic liquid chromatography texts and article links, plus get more supervised hands-on time with the instruments.

HPLC Retention Time Drift, Change, Area Variability or Poor Reproducibility. Common Reasons for it.

Retention times and area measurements must be reproducible from run to run. When problems are observed, late, early or variable retention times (and/or peak area values) may be observed. Variation outside of acceptable limits indicates a problem with the sample preparation, method design, function of the HPLC system or a lack of training. Here are several commonly observed reasons why sample (or standard) peak retention times or peak area values may not be reproducible:

(1) TEMPERATURE FLUCTUATIONS:

To obtain reproducible results, the temperature of the HPLC column must be kept constant or controlled during each analysis. Laboratory room temperatures can vary up and down 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. Optical (Light) based detectors (i.e. 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 fully 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 sleeves 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. Method analysis temperature should be constant from run to run, not a variable. Be sure and document the temperature selected as part of your method. 

(2) INADEQUATE MOBILE PHASE MIXING:

Both high pressure (with separate pumps) and low pressure pumping (one pump with a proportioning valve module) systems depend on efficient mixing to accurately meter the requested mobile phase composition. 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 Retention time reproducibility. If your mobile phase composition changes, then the chromatography will change too (e.g. evaporation of the more volatile organic phase from an open bottle may result in a change in composition). "Mixing" is often accomplished directly in a mixer installed in the flow path of an HPLC pump (For more info, please read this article on selecting a mixer). The associated noise and ripple of incomplete mixing can reduce the limit of detection (LOD) and increase integration error. 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, flow instability 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. Monitor the baseline for drift, ripple and artifacts in real time to spot problems and make adjustments to correct them. 

(3) INADEQUATE MOBILE PHASE DEGASSING:

For the best results, continuously degass your mobile phase. Reducing the amount of gas in solution will also improve signal to noise levels of detection, reduce Retention time 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 to your system and methods. Maintain and Repair them just as you do for your other instrument modules. Gas bubbles may cause the inlet or outlet check valves to malfunction (get stuck), baseline noise spikes to appear randomly, flow rates and/or back pressure values 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. This can be accomplished through stand alone vacuum degassing modules or through gentle helium gas sparging. 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 continuously. The mobile phase solution starts to re-absorb gas as soon as you stop sonicating the solution. This results in continuous baseline drift.

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: Always degas your mobile phase solutions.

 

(4) SYSTEM LEAKS or FLOW RATE INSTABILITY:

If the peak retention times have increased over time, one possible reason for this change could be a leak. If your flow rate is reduced by a leak, then the retention times will be longer. Always be alert to this pattern of change and check for any signs of leaks on a regular basis. If you find a leak, do not use the HPLC system until it has been repaired. If there is no leak, then the flow rate may not be what you think it is. 

When the actual flow rate is in question, start by checking it manually (never trust the instrument's display screen or the software value for flow rate. Measure it). An easy way to measure the flow rate involves timing the amount of liquid that exits the HPLC detector line after a defined period of time. For example: If your flow rate is set at 1.000 ml/minute, measure the time it takes to fill a 10ml graduated cylinder. It must take exactly 10.00 minutes.

Inadequate degassing, sticking check valves and/or incorrect solvent compressibility values may also cause flow instability.

(5) COLUMN FOULING: 

One of the most common reasons for changes in retention times or area values of well established peak(s) are due to column contamination and fouling of the support material (or of the inlet frit, guard column). The most common reason for this to happen is due to a lack of column flushing or washing after each analysis (esp when running only isocratic methods). Samples that have been poorly prepared, not filtered or were sourced from a complex matrix (i.e. clinical samples) often contain many compounds which are in-addition to the compound of interest. These materials can be retained on the column and not eluted off during each analysis. They build up over time and cause all kinds of strange problems, including changing retention times, new peaks seen and poor overall or wide peak shapes. 

Gradient analysis provides an opportunity to make sure you use a strong enough mobile phase to elute everything off the column during the run. Make sure you ramp up to a high enough concentration of solvent and use a "hold time" to insure this.

Isocratic analysis is a worst case situation for this to occur as the mobile phase is not ramped up to a strong solvent at the end of the method to push off any late eluters, Instead, they accumulate on the column. 

If you use isocratic methods to analyze samples, then you must follow each analysis run with a second, and separate from your analysis method, "column only wash step". This method does not inject any sample. Instead, it uses a strong wash solution which is compatible with your column AND is well known to dissolve any accumulated material into solution and elute it all off the column. For NP applications an alcohol (e.g. MeOH) may be suitable for this job and for RP applications ACN or even MeCl may be be appropriate. Check with the column manufacturer to find out which wash solutions should be used (do not guess, base it on actual sample solubility). 

(6) SAMPLE OVERLOADING (or too large an Injection volume/concentration for the column): If you inject (load) more sample than the column can hold (as determined by a proper loading study), then the peak that results will often be broader in width with more tailing (from diffusion). This will result in a peak which elutes later than expected, fouls the column and results in poor reproducibility.  Be sure to inject the sample dissolved in the mobile phase (or a solution that is weaker than the mobile phase).

(7) SAMPLE INJECTION VOLUME VARIATION: The injection volume used must be appropriate for the type of injector used. All injectors have a stated range in which they are most accurate. Make sure you are injecting within this ideal range and not at the extreme ends of the range (larger error). Manual injectors with fixed loops should be overfilled (3x) for best results. Autosampler vials must be correctly chosen to be compatible with the injector used, contain an excess of liquid and have a loose cap to prevent evaporation or a vacuum from forming inside the vial. *Test injector accuracy and reproducibility separately, at the volume used for your analysis, as part of your method development review. *Review my article on HPLC injectors for more information.

(8) Changes in the pH OF the MOBILE PHASE: Samples containing ionizable compounds are strongly effected by the pH of your mobile phase. Solutions should be prepared fresh, each day (*acids in solvent may change over time). Buffer capacity is often overlooked (the ability to resist pH change). It is highest at the pKa of the acid/base. Try to work within ±1 pH unit of the buffers pKa value for the best pH control of the mobile phase. If your mobile phase is buffered too far away from its pKa, then poor peak shape or variable retention times are often the result. Note: Weakly ionizable samples can be very sensitive to changes of as little as 0.1 pH unit.