At the 63rd ASMS, held in St Louis, MO, USA, Dr. Vivek Joshi, Principal Research Scientist at EMD Millipore, discussed everything you need to know to be an LC-MS expert. In an exclusive interview after his seminar, Dr. Joshi revealed how to avoid common mistakes when preparing samples for LC-MS and how to optimize your method to collect the highest quality data.
Read on for tips on sample preparation, streamlining your workflow and choosing the best column for the job.
Streamline your workflow
The three main points when looking at streamlining your LC-MS workflow are sample preparation, choice of mobile phase and choice of column. Neglecting any of these will negatively affect your data, but spending too long on them can slow you down. With run times becoming increasingly shorter, sample preparation can become the new bottleneck, hence it makes sense to do the minimum sample preparation which gives you the results you are looking for. Just by paying attention to quality of mobile phase ingredients, overall analytical performance can be improved. Finally selecting the right column type will provide the data a scientist is looking for.
Understand your sample
Before you start, it is very important to understand the type of analyte you are working with and its physical characteristics, as they will determine your method of sample preparation, the components of your mobile phase, and the type and selectivity of your column; for example, whether it is hydrophobic or hydrophilic, soluble or not etc. In some cases, you also need to think about other matrix components that are present in your sample and can interfere with the analyte of interest. Sample preparation and appropriate column selection can help with these interfering matrix components.
Optimize sample preparation
Once you have identified the needs of your analyte, you can start preparing it for analysis. The main goal of sample preparation is to bring sample to a form which is amenable to downstream HPLC analysis. That means at a bare minimum the sample is dissolved in a solvent and free of particles. However, depending on the analysis needs, more complex sample preparation will need to be done which not only removes the particles but also removes soluble interfering matrix components. Some parts of the sample preparation can be automated to reduce overall time required for sample preparation.
Filtration is one of the most common steps in the preparation of an analyte for LC-MS, usually to remove any particles from the sample. However, the buffer solution can also introduce particles, from undissolved salts, and other contamination can be introduced from the solvent used in the sample preparation or even during the LC-MS run. Although LC-MS grade solvents are filtered and packed under an inert atmosphere, the laboratories where they are used are seldom completely clean. Dr Joshi recommends filtering everything, from sample to mobile phase, instead of relying on the cleanliness of the tubes and bottles used during the solvent transfer.
Pick your filtration membrane wisely
With all the additional filtering you'll now be doing, it's more important than ever to use the appropriate filtration membrane. Each different process used by manufacturers gives membrane with slightly different morphologies and pore sizes, but Dr Joshi recommends a filter made from hydrophilic PTFE membrane. Hydrophilic PTFE has broad chemical compatibility with very low extractables and analyte binding, allowing the majority of the sample to be recovered and with minimum additional contamination.
Use the most appropriate mobile phase
When making up your mobile phase, always use the highest grade solvents and reagents possible. Buffer salts used in LC-MS work flow have to be volatile, as non-volatile salts can precipitate out in the MS. Every mobile phase component in your LC-MS run (water, solvents and buffer salts) contributes to your background signal, and a high level of background noise will negatively affect your limit of detection (LOD) and limit of quantification (LOQ).
Don't rely on manufacturers
However, even hyper- or LC-MC-grade solvents can contain some contaminants, and each manufacturer will use a slightly different method of production, which will vary the nature and levels of contamination. Some solvents, such as butanol, aren't available in the higher grades, so will need to be filtered and purified in the lab before your LC-MS can be carried out.
Once you have filtered your solvents completely, try to use them straight away. Storing them can lead to leaching of contaminants from the storage container. Incomplete removal of particles from the components of the mobile phase can also lead to increased back pressure during a HPLC run, which in turn can shorten the life of your column or lead to system shut down during a critical sample run because of excess backpressure.
Choose your column
As technology progresses from HPLC to UHPLC, the separation efficiency and lifetime of the column is increasingly affected by packing type. Monolith-type columns offer the highest efficiency with the lowest necessary sample preparation and the lowest back pressure. Their particle-free design is less likely to become blogged, improving their robustness and subsequently their lifetime. They are therefore ideal for high-throughput operation, improving the efficiency of any lab.
Learn more about HPLC and choosing a column with our exclusive Buying Guide.