GC-MS Contamination Identification and Sources

Over the past few months I have received a number of questions regarding the identification and possible sources of contamination observed in GC-MS systems (GC/MSD, EI). Contamination can be sourced to the instrument itself or to materials used in the preparation of samples. We shall review a few of the most common types of contamination observed and how you can use the MS detector data to identify them in this article.

Air Leaks / Contamination: The GC-MS detector operates under vacuum conditions so air must be kept to a minimum detectable level. 

• Common Sources: Improper fitting connections; improper fitting materials/age; damaged or worn vacuum seals. 
• Identification: High background noise levels (poor S/N of stds); Peaks associated with air (i.e. m/z 18, 28, 32, 44).

Acetone Cleaning Fluid Contamination: Often used to clean the metal parts of the source, acetone may appear in the signal. Look for peaks at m/z 43 & 58.

Dimethylpolysiloxane Contamination/Bleed: Silicone from the various seals and septa may appear and depending on the chemical source, are often detected at m/z 147, 207,221, 281,295,355 & 429. *Always use high quality seals & inject blanks often to check for contamination.

Plasticizer Contamination: Many plastics are used in the seals found in the GC-MS instrument. These plastics may eventually bleed into the system and be detected (due to normal wear, heat stress or even poor sample preparation). It is critical to identify which ones result from worn out seals vs. use of plastics as part of improper sample preparation procedures (e.g. improper handling techniques, glove material, plastic sample tubes, washing glassware with soaps etc). One of the most common peaks observed will be m/z 149 (Phthalate).

Diffusion Pump Oil Fluid Contamination: Turning off the carrier gas flow may allow for some of the diffusion pump oil to back-stream. *Maintain carrier gas flow when this system is ON. If diffusion pump oil has entered the source, you should observe a strong signal peak (e.g. m/z 262, 446 for the oil).

Foreline Pump Oil Fluid Contamination: Look up the specific chemical composition of the oil used to obtain applicable m/z values to check for (e.g. hydrocarbons, m/z 69 ...).

*Avoid GC-MS contamination and trouble by first receiving the proper training to operate, use and maintain the GC-MS system. This includes making sure the entire system is fully maintained, seals changed, performance monitored regularly, insuring all of the vacuum pump oils are changed on a regular schedule and always use high quality replacement parts. Maintenance is something that most users can perform themselves, especially if they have completed a formal hands-on training class using their own GC-MS system (Most manufacturers offer basic on-site Maintenance classes).

Troubleshooting HPLC Gradient Valve / Proportioning Valve / MCGV GPV Leaks. How to Identify Them.

HPLC pumps which utilize low-pressure mixing VALVES are known by names such as: "Ternary" (3-solvents) or "Quaternary" (4-solvents) pumps.These types of HPLC pump configurations use a single, high-pressure pump head coupled to a multi-port / proportioning valve and represent some of the most popular and versatile pump configurations offered. Featuring random access to multiple solvent bottles (more than two is always better), lower operating costs and less maintenance work provides you with one of the best platforms to develop new methods on. I highly recommend them for most, but not all, HPLC applications (vs. Dual pump, high-pressure "Binary Pumps").

• If your HPLC system utilizes a single, high-pressure pump head coupled to a multi-port valve, then please remember that in addition to pump head maintenance, regular maintenance of the multi-port / proportioning valve is also required.

A few weeks ago I was hired by well known Pharma company to solve a gradient method problem that I was told has stumped their best scientists for almost one year. The client presented me with their validated UHPLC method which suddenly developed a shift in retention time of all peaks. The shift was significant, about 10% of the previous values over a 20 minute run, and had been observed on two different, but similarly configured HPLC systems in their lab. Changing the column to a new one showed no change on either HPLC system. They were out of ideas.

• Before I reveal the cause of the trouble, let us briefly think about what types of changes can result in a small, repeatable shifts of peak retention times. Four common ones that come to mind are: 

(1) Flow Rate changes;

(2) Column Temperature changes;

(3) Column Fouling;

(4) Mobile phase composition changes. 

Start the troubleshooting by ruling out the easy causes first (#1, 2 and 3 above).  

• (1) Flow Rate: When the actual flow rate is in question, start by measuring it manually Never trust the instrument's display screen value or the software's 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, then using water, measure the time it takes to fill a 10mL graduated cylinder to the 5 mL line. It must take exactly 5.00 minutes (= 1.00 mL/min). Run this flow test on each pump channel.
  
• (2) Temperature: The HPLC method should be run under controlled column temperature conditions. Verify this. Retention times are a function of temperature (i.e. cooler temps usually result in longer retention times, warmer = shorter). The temperature should be stable (~ 1 or 2 degrees C).
  
• (3) Column Fouling: To prevent fouling, wash the HPLC column with a solution that is STRONGER than the mobile phase after each analysis. Use fresh, clean solutions. Verify that the samples are dissolved in the mobile phase (100% dissolved) and filtered before injection. Verify that the injection volume is less than ~3% of the column volume and the concentration of the sample is not too high (avoid saturating or overloading the column). Solubility is very important for both the sample and any additives used in the mobile phase (to prevent precipitation). Anything that "fouls" the column support will directly effect the retention times and often the peak shape too. Be aware of these causes and take action to avoid them.  *Replacing a suspect column with a new one is often an inexpensive way of troubleshooting a "peak" problem. Always have a NEW spare column on hand for testing. *Columns are consumable items.
  

• (4) Mobile Phase: Changes to the actual amounts of additives, pH or final composition of the mobile phase may impact peak retention times (sometimes, the peak shape too). After all, the final composition used was developed for the purpose of establishing a reliable and reproducible method of analysis. It must be controlled. We must take steps to insure the mobile phase preparation and delivery are accurate. Always prepare fresh solutions each day (esp. all aqueous solutions!). pH values may change after a few days (e.g. even in MeOH / acidic solutions), bacteria/mold/algae grow quickly in many solutions, even in the refrigerator, so only prepare what you need for the day. Evaporation of more volatile solvents (in pre-mixed solutions) can change their actual concentration (always protect them from heat and evaporation).
  

*There is another way that the mobile phase composition can change which often goes unseen. It can change during delivery to the column. The HPLC's low pressure proportioning valve that allows us to easily select and use different solvents can develop small internal leaks, resulting in valve cross-flow leakage. This cross-flow leakage allows liquid (or air, if the line is not connected) to be drawn out of one channel and into another, changing the actual mobile phase composition. This happens because the valve seals, esp if they have been left unused for a long time, can change shape (e.g. shrink) and begin to leak over time. Often the amount of leakage is very small (ul/min), but depending on the method, a small change may result in a significant change to the chromatography.

I reviewed the client's method parameters and concluded that the method met good chromatography fundamentals. Checking the flow rate (using a graduated cylinder) confirmed the flow rate was accurately shown. A review of their mobile phase preparation procedures and methods also appeared OK. Degassing of mobile phase and column temperature were also satisfactory. 

As I looked more closely at the two running HPLC instruments they used, I began to quickly zero in on the most likely problem. 

• A long stream of air bubbles were observed exiting the HPLC pump's gradient valve leading into the high pressure pump head, but no air bubbles were seen exiting the degasser's outlet line (IOW: The vacuum degasser may or may not be the cause, though it is critical to insure the degasser is clean and fully serviced before use. Have the degasser professionally serviced first before proceeding with troubleshooting. Using a damaged degasser will make it difficult to use the pump or run any valid tests as degassed solution is needed). This was observed on several of their HPLC systems, including the two used for this method. The fittings connecting the lines from the degasser module to the valve were correctly connected (as a loose connection would cause air to leak in and must be quickly ruled out). 

The cause was from one or more of the unused gradient valve positions leaking air into the flow path, changing the mobile phase composition. Of four possible mobile phase lines available (A,B,C,D), the client only had two lines connected to mobile phase bottles (A,B) with the remaining two lines left open to the air. The internal valve seals in the unused 'C' and 'D' valve positions had deformed, shrinking in size, sticking,leaking, allowing air to flow into the mobile phase on one of the channels. This resulted in a change of the organic composition % used in the method (due to a cross-flow leak), changing the peak retention times (as the actual mobile phase composition used in their gradient was different). I directed the HPLC pump's outlet line to waste, placed all of the solvent pickup bottle lines (A,B,C,D) in a beaker filled with IPA and allowed the pump to run pure IPA at 1 mL/min across each channel, one-at-a-time (100%), for ~ 20 minutes to re-hydrate the internal gradient valve seals. This was repeated with each valve position, then all of the lines were placed in fresh mobile phase solution, primed and flushed. The system was restarted and the method now ran showing the expected peak retention times. Instructions were provided which included regularly using all of the channels and valve positions plus flushing weekly to maintain valve operation. Use ALL of the lines and flush the valve(s) through all positions, one-at-a-time, on a regular basis. If prolonged flushing with pure IPA does not fix the leak, then it is time to replace the valve. All valves eventually wear out and must be included in maintenance inspections and checks. This is especially true when you purchase your HPLC system at an auction or from an 'equipment' reseller. Never assume that the 10+ year old HPLC valve is OK. Test it first (e.g. Acetone tracer test).

 

Acetone Tracer Test: If you suspect that a cross-flow leak exists on a gradient valve, then one method I use to check for leakage is to mix up a "Tracer" solution of pure organic (often ACN) that has 1% Acetone mixed in (for RP methods). Remove the column and replace with a restriction capillary. Place the tracer solution on the valve position you suspect may be leaking at an appropriate flow rate and set it for 0%. Run one of the other channels with 100% (pure ACN in this example) and monitor the UV (265nm) for the presence of acetone. If the acetone leaks into the channel you are using, it will be easy to observe on the UV trace. So called "bubble" tests (introducing and monitoring the position of a gas bubble into the low pressure solvent line) are not reliable leak detection methods for small leaks. Use a tracer such as acetone to find the leaking channel(s). You can read more about these types of Valve Leakage tests in this article (Click Here).

Repair Corrupted Windows 7, 8 & 10 System Files Using the System DISM Tool

Time to share another useful Microsoft Windows command-line utility tool. If you have experienced the "Blue Screen of Death", a crash, service pack installation failure, windows update failures or observed general Windows system file corruption error messages, then this utility tool might be useful to you.

Many common Windows Update errors which result from new system file corruption can be corrected using the Windows DISM.exe tool. Failed application or update installs often result in corruption of some of the system files. These utilities are designed to detect corrupted file and repair them. It targets the currently running operating system for repair. The DISM tool must be run from the Windows Command prompt (cmd), with an account that has administrator privileges using " Run as administrator ".

DISM = Windows "Deployment Image Servicing and Management" tool.

• Before using any software utility program, make sure you first have permission and authorization to do so. Most Importantly of all: Back up your system programs and any data files before using any utilities such as this one. Create a Restore Point to protect the basic settings too. Do this now. Take precautions before using any utility programs and do so at your own risk. You are responsible for your data, programs and computer.
  

• Please make sure you have reviewed my earlier article on how to run the System File Checker (SFC) tool first. The SFC tool scans Windows operating system files for corruption AND restores any found corrupted files, all automatically! The SFC often quickly corrects many system errors and I always run that utility first. DISM is more thorough, but takes more time to run.

• Before using the DISM (or SFC) utilities, set a new restore point using the very useful "Restore Point" feature found in Windows (discussed in an earlier post). Make sure you have enough time available for the computer to run this utility (overnight is best). Once started, it will show a progress bar. Do not interrupt the process.
  

To run the DISM.exe utility, close down all applications for now. Make sure your account has Administrator privileges, then open up the Command Prompt using the " Run as administrator " option (you must do this so the system32 path is selected). 

At the command line prompt, Type the command line below (make sure to include the spaces, exactly as shown): 

 DISM /Online /Cleanup-image /Restorehealth  

 Optionally, for some O/S enter:  DISM.exe /Online /Cleanup-image /Restorehealth 

The screen should show " Deployment Image Servicing and Management Tool " with the version #. Image Version plus some [ ] showing the progress. When it is finished, it should report that "The operation completed successfully". Type 'EXIT' to close the Command Prompt screen, then Reboot your computer.

 

 

References:

1.  Microsoft Windows Support Page:

Fix Windows Update errors by using the DISM or System Update Readiness tool 

 

2.  If you encounter "error 87", then please confirm you have: typed the command line as shown; are running the cmd "as administrator"; have applied all new or pending Windows Updates and have run the SFC tool first. If you continue to see the error, then refer to Microsoft's knowledge base for additional tips.

Speed Up HPLC Analysis Time Using Higher than "Normal" Flow Rates with SMALLER Particles

Column efficiency (as described by Van Deemter) in HPLC is largely a function of dispersion, column particle size and the flow rate of the mobile phase.After a column has been selected, the Flow rate should be optimized for all methods (start with the nominal linear velocity). Once the optimum flow rate range is achieved, little to no advantage in analysis time or solvent savings is found by increasing it (as column efficiency normally decreases at higher flow rates).

From a practical point of view, columns packed with porous 3 to 5 micron diameter supports show only small differences in efficiency as the flow rate is varied above the initial, optimum level (linear velocity). Running at too low a flow rate serves no purpose, increases dispersion/diffusion and delays the peaks from eluting off the column in a timely manner. Higher rates often decrease column efficiency. Once the flow rate has been set within the 'optimized zone', it no longer becomes a variable in HPLC method development. 

Many ~ 3 micron supports do demonstrate some ability to maintain optimum efficiency at slightly higher flow rates (e.g. with linear velocities > 1 mm/second), but significant advantages in using higher flow rates to save time and solvent are not obvious unless the particle size is reduced further. 

With the much smaller diameter ~ 2 micron particles, column efficiency can be further optimized using higher than "normal" flow rates on standard columns. Columns packed with these smaller porous particles show optimized flow rates at much higher linear velocities (e.g. 2x normal or ~ 2 mm/second for standard analytical sized columns, but experiment using 2 to 5x the normal linear velocity to compare results). 

• For example: If your method currently runs at 1.000 mL/min, you may be able to run the same method at 2.000 mL/min OR if your method currently runs at 0.200 mL/min, you may be able to run the same method at 0.400 mL/min or higher using one of the 2.5 or smaller particles. 

This increased efficiency coupled with proper optimization of the HPLC's flow path to reduce dispersion, allows for a doubling of the flow rate without a loss of efficiency (or loss of resolution). Depending on the scaling used, a two-fold savings in analysis time over conventional methods using larger particles may be observed. There may be a corresponding increase in system back-pressure too (* if only the particle size is changed and the column dimensions are unchanged). *Some of this can be countered using proper scaling of the column dimensions too). 

NOTE: Do Not Optimize HPLC Methods for "Pressure". This goes against basic chromatography fundamentals. Back Pressure is a result of pushing mobile phase through the tubing and column and is not a method development tool or variable. As mobile phase composition changes, so does the pressure. Flow rates should be stable. Work within a pressure range that is high enough to permit the pump(s) to function properly, but below the point in which frictional heating interferes with the method.

Optimization of method resolution, overall analysis time and solvent usage should be considered. The increased efficiency gained from the smaller particle size supports also allows for scaling down the column dimensions (i.e. length, ID or both) too, though a trade-off between overall column efficiency vs. analysis time and/or too high a back-pressure must be addressed to optimize the method and meet the application goals.

Summary: HPLC analytical column flow rate is often ignored in method development (* esp after it has been adjusted to the initial optimum, often 1.0 mL/min for a 4.6 mm ID column), but IF you are using porous HPLC particles that are smaller than 3.5 micron diameter, please be sure to investigate if you should re-optimize the flow rate used in your method / application so you can take advantage of any increases in column efficiency and/or scaling. As with ALL applications using these very small particles, pre-optimization of the HPLC flow path is often needed to achieve many of the available benefits.