HPLC Workflow
Sample Preparation Technical Library
- PDF: Millex Filter Finder Wall Chart
- PDF: Samplicity Filtration System Brochure
- Sample Preparation for Liquid Chromatography
- PDF: LC-MS Contaminants Technical Bulletin
Application Notes
- PDF: Multisample filtration of small volumes using the Samplicity® System
- PDF: Low-extractable Millex Samplicity® filters are ideal for preparing samples for sensitive HPLC detectors
- PDF: Filtration of viscous / particle-laden samples using the Samplicity® Filtration System
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Featured Application: Avoid, Identify and Minimize LC-MS Contaminants
Sample preparation for HPLC is particularly important when you use a sensitive downstream detection method, such as mass spectrometry (MS). Liquid chromatography-mass spectrometry (LC-MS) is widely used to analyze complex samples, such as plasma, urine, food samples, and wastewater. Of the key parameters determining LC-MS success (sensitivity, resolution, reproducibility, efficiency, selectivity, speed, column lifetime), sensitivity, reproducibility, and column lifetime are negatively affected by the presence of contaminants.| Parameters negatively affected by contaminants | What it is | How to minimize contaminants |
|---|---|---|
| Sensitivity | Lowest level of analyte detectable above background; sensitivity is reduced by ion suppression | Wash columns to mitigate column bleeding |
| Effective sample preparation | ||
| Use certified mobile phase | ||
| Reproducibility | Can mean column-to-column reproducibility or run-to-run reproducibility |
Prepare sample with devices that do not introduce extractable impurities |
| Run sufficient controls to verify run-to-run stability within a batch | ||
| Remove sample components that interfere with separation, ionization, and fragmentation | ||
| Use high-quality HPLC columns | ||
| Column Lifetime | Number of injections on a column without change in selectivity and efficiency | Eliminate sample contaminants that adsorb strongly or ionize easily. Avoid polymers that, when fragmented, result in multiple peaks of varying m/z. |
| Fully elute/clean column after each sample | ||
| Use guard column and proper sample preparation (such as centrifugation, filtration, and extraction) to remove particles and extend column lifetime. |
Sample Components That Can Interfere with LC-MS Results
Metabolites, detergents, salts, buffer components, and counterions are among the components of complex samples that can interfere with LC-MS performance. Some biological matrices, such as plasma, contain high amounts of phospholipids. If not removed prior to chromatography, separating phospholipids from analytes of interest can require long chromatography run times and high concentrations of organic solvents. Furthermore, phospholipids can build up on analytical column, and unexpectedly elute in future runs. Drug formulation agents, such as polysorbitans and polyethylene glycol, can also interfere and cause ionization suppression.Besides sample-derived contaminants, additional sources of contamination are sampling devices, solvent impurities, containers, sample preparation devices, volatile organics introduced as a result of handling personal care products, and even columns themselves. Plasticizers from labware can interfere with LC-MS, resulting in the need to lengthen the chromatography run in order to resolve these peaks from analyte peaks.
How to Quickly Identify Sources of LC-MS Contamination
Analysis software supporting many LC-MS instruments may help you identify signals in your mass spectrum that are likely to represent contamination. We have also compiled a list of common mass spectrometry contaminants, their monoisotopic masses, and the sources of these contaminants.Browse this list in the appendices of our technical bulletin, "LC-MS Contaminants."
