Contamination is often one of the most frustrating and challenging issues to deal with in the lab. This is because it can be difficult to identify the source of the contamination, and in some cases, it can be even more challenging to rid the system of any contamination issues which can cause prolonged instrument downtime. Routine maintenance is an important part of keeping your instrument performing appropriately. Instrument parts from check valves to rotors, and everything in between play a crucial role in ensuring the instrument is running efficiently. It is also always best practice to be mindful of changing pipette tips and gloves, as well as washing hands and practicing basic lab cleanliness to minimize the risk of contamination.
In this blog, we take a deeper dive into some additional areas where contamination can occur, and how to diagnose and troubleshoot the issue. We will focus on four main areas to troubleshoot if you are dealing with analyte contamination: column, LC pumps/mobile phase, injector, and sample preparation materials.
Contamination of the Column (Carryover)
You may fear that your column is contaminated if you start noticing analytes where they should not be, like in blank or negative samples, but oftentimes this is just due to carryover. Although annoying, carryover can be very common and thankfully an easy fix in most situations. Carryover can occur for several reasons. First, if you are injecting too much sample, in terms of either volume or concentration, this could cause carryover into other samples in your batch. This can be easily overcome by decreasing your injection volume, or by diluting your sample before injecting. Additionally, you may need to add more time at the end of the method with a stronger solvent to fully flush the column before running the next sample. A quick way to diagnose if it is instrument or column contamination is to take the column and run the same method on a different instrument if one is available. If you see the contamination on a new instrument and you were not seeing it previously, then the contamination is likely from the column. If you do not see it on a new instrument, then the contamination is likely coming from somewhere on the original instrument. If another instrument is not available, try switching the column and running the method about 10 times with the highest calibrator. If the new column does not experience the contamination, then it is likely that the analytical column or guard column needs to be changed. If the new column also shows carryover, then the method needs to be adjusted to adequately flush the analytical column between injections. Additionally, you can try a different column stationary phase to see if the analytes have the same retention issues on a new phase.
Another reason there could be analyte carryover on your column is due to chelation. In some cases, analytes interact with the metal surface of the column, and this can cause the analyte to stick to the column and then elute later in the run or in another sample. An easy way to solve this problem is by using a column with inert hardware when running these challenging analytes. More information on inert column hardware can be found here.
Contamination of the LC Pumps/ Mobile Phases
Contamination can also occur in different parts of the LC system. Analyte contamination can be present if a mobile phase or autosampler rinse is contaminated. To avoid contaminating the mobile phase, it is always best practice to prepare the solvents in a clean lab space away from where any sample preparation or use of concentrated standards is occurring. It is also important to wear clean gloves when making the mobile phases and use clean pipettes and pipette tips. If contamination appears to be present in a mobile phase or instrument solvent, try moving the mobile phase to a different instrument where there was no previous contamination and see if the contamination occurs. If an additional instrument is not an option, there are other ways to determine if the mobile phase is the source of the contamination. To determine if the analyte contamination is coming your mobile phase, try following these simple steps:
- Let the column equilibrate for 5 minutes with your starting mobile phase conditions
- Inject a null injection
- Let the column equilibrate for an additional 5 minutes
- Inject another null injection
- Let the column equilibrate for 10 minutes
- Inject a third null injection
- Compare peak intensity of the null injections
If the intensity of the contamination peak increases as equilibration time increases, then it is likely due to contamination in the mobile phase. This can be a tricky contamination issue to address since the mobile phase touches all aspects of your instrument. For starters, it is recommended to change solvent lots as well as the lots of any additives that are used when making up the mobile phases. Outside of the solvents themselves, it is crucial to replace any glassware used when preparing these mobile phases and the mobile phase bottles themselves as analytes can strongly stick to the glass making it difficult to remove them even during cleaning. When replacing the glassware, it is also very important to change the mobile phase filter frits and the lines as these have been sitting in contaminated mobile phase for a prolonged period of time. Replacing all parts at the same time will minimize the risk of recontamination in the system. Replacements for mobile phase bottles and parts can be found here.
Contamination of the Injector, Needle, or Sample Loop
Contamination of the LC can also come from the injector. This can lead to an increase in contamination over time after many injections. The biggest cause of carryover in the injector is from analytes sticking to the needle and/or sample loop. To test if the contamination is coming from the injector, try injecting a solvent sample several times, increasing the injection volume for subsequent injections. If there is an increase in the contamination peak when increasing the injection volume, then it is likely a contamination in the injector. To address this source, you can start by changing the needle wash procedure. For example, you can change the volume used to perform the wash, or try including additives in the needle wash solvent, such as Medronic acid or Formic acid, that will help remove analytes. An additional part of the injector that can cause contamination is the sample loop. This can present as carryover and can sometimes be solved by flushing the loop. If changing the needle wash procedure and solvent does not solve the issue, it may be necessary to replace parts of the injector. The needle, needle seat, sample loop, and rotor can all be replaced and should be considered when troubleshooting injector contamination.
Contamination of Sample Preparation Materials
If none of the tips above helped to solve the contamination issue you are experiencing, it may help to take a step back before the samples get to the instrument and look at the materials used for sample preparation. If you use any types of solvents in the sample preparation, it could help to take an aliquot of each and test it using your analytical method. If you increase the injection volume of each sample and see the analyte recovery increase respectively, then the sample preparation solvent could be responsible for the contamination and should be replaced. If testing the solvents on the instrument is too time-consuming, replacing all solvents used at the bench top could also solve the problem if this is the source.
Outside of the tips recommended above, there have been other instances of contamination where the laboratory environment is to blame. LC-MS instruments are extremely sensitive and can detect traces of airborne contaminants if they settle near the instrument. If there is being any sort of sample preparation done with bulk materials, this should be done in a separate setting than where you house your instrument. For example, if a lab is also doing drug chemistry testing, these samples should be prepared in a separate lab from where the instruments are kept in order to avoid any chance of contamination from airborne contaminants. If a separate laboratory space is not possible, the mobile phases and instrument space should be protected as much as possible.
While contamination can be a frustrating issue to address, hopefully having some of these tips and tricks in your tool belt helps you to feel more comfortable when diagnosing and troubleshooting the issue. As always, we are here to help if you need further assistance through our technical assistance by emailing Technical Support!
