Solvents serve a dual purpose in chromatographic workflows, being utilized both during sample extraction and in the subsequent analytical analysis. In preparing samples for chromatography-mass spectrometry (LC-MS or GC-MS), the choice of solvent or solvent mixture is a critical factor that can directly impact the extraction efficiency, analyte solubility, and instrument compatibility. This is a guide to some of the most widely used solvent mixtures for MS sample preparation and analysis in modern analytical labs.
Why Use Solvent Mixtures?
Mixtures are much more commonly used in sample preparation procedures because they offer increased advantages over individual solvents. While pure solvents can be effective, mixtures offer greater flexibility in adjusting sample polarity, optimizing extraction, and ensuring compatibility with both the chromatographic system and the mass spectrometer [1]. The right combination of solvents can aid in enhanced sensitivity, improved peak shape, and facilitation of ionization, a process essential for effective MS detection [2].
Top Solvent Mixes in Chromatography-MS
1. Water–Acetonitrile (H₂O–ACN)
- Most common for LC-MS (especially in reverse-phase)
- Typical ratios: 95:5 to 5:95, often used in gradients throughout analytical run.
- Why it's popular: Acetonitrile is less polar than water, providing strong elution strength for non-polar and moderately polar compounds. This mix is highly compatible with electrospray ionization (ESI) and other MS techniques used with LC-MS [2, 3].
- Additives: These mixtures will often contain 0.1% formic acid, 0.1% trifluoroacetic acid , or another acidic solvent to aid in the ionization process and improve peak shape [2, 3].
2. Water–Methanol (H₂O–MeOH)
- Widely used alternative to acetonitrile
- Typical ratios: Similar to water–acetonitrile mixtures, ranging from 95:5 to 5:95
- Why it's popular: Methanol is more polar than acetonitrile and can be more suitable for some analytes and column types. It’s also cost-effective, readily available, and less toxic than acetonitrile [2].
- Additives: Formic acid or ammonium acetate are commonly added for enhanced MS response [2, 3].
3. Hexane–Ethyl Acetate
- Common in normal-phase chromatography, extractions, and thin layer chromatography (TLC).
- Typical ratios: 9:1 to 1:1, depending on analyte polarity
- Why it's popular: Hexane is non-polar, while ethyl acetate is relatively polar, making this mixture ideal for extracting a wide range of organic compounds in various procedures [4].
4. Chloroform–Methanol
- Used for lipid and metabolite extraction
- Typical ratios: 2:1 or 1:2, often followed by an addition of water to achieve phase separation.
- Why it's popular: This mixture efficiently extracts lipids and other hydrophobic biomolecules from biological samples, as lipids are soluble in these two solvents, whereas many other cellular components are not [5].
5. Specific Application Mixtures
- For specific applications: Solvent blends like isopropanol–water, or the addition of dimethyl sulfoxide (DMSO) or tetrahydrofuran (THF) can be used to improve solubility for more challenging analytes [2].
- For paper chromatography: Various mixtures such as n-butanol–water– with an added acid are used in this application [6].
How Are These Mixes Prepared?
- High-purity solvents: To avoid background noise and contamination use HPLC- or MS-grade solvents [7].
- Filtration and degassing: Filtering the mixture through a 0.2 µm membrane and degassing it (by sonication or helium sparging) helps to prevent air bubbles and particulates from interfering with chromatography.
- Additives: The addition of acids or buffers just before use can help to maintain stability and reproducibility of results [2].
Conclusion
Water–acetonitrile and water–methanol mixtures are the primary solvents used in analytical chromatography and mass spectrometry. They are typically supplemented with a small percentage of acid, such as formic acid or acetic acid, to enhance ionization [1, 2]. For extractions and sample cleanup procedures, hexane–ethyl acetate and chloroform–methanol are commonly used [4, 5]. The choice of solvent and its mixture ratios largely depend on the properties of the analyte of interest, the chromatographic method employed, and the requirements of the analytical instrument. With understanding and effective application of these common solvent mixtures, you can ensure robust, reproducible, and sensitive results in your chromatography–mass spectrometry workflows.
Citations:
- https://www.organomation.com/solvent-extraction-techniques#:~:text=Solvent%20Blends:%20Combining%20different%20solvents,of%20compounds%20from%20complex%20matrices
- https://www.waters.com/nextgen/us/en/education/primers/the-mass-spectrometry-primer/solvents-and-caveats-for-lcms.html
- https://www.ucl.ac.uk/chemistry/sites/chemistry/files/ms-hplc_solvents_and_mobile_phase_additives_for_ms.pdf
- https://community.wvu.edu/~josbour1/Labs/Exp%205%20-%20TLC%20-%20F17.pdf
- https://www.sciencedirect.com/topics/engineering/lipid-extraction#:~:text=The%20extraction%20of%20lipids%20with,of%20globules%20in%20the%20cytoplasm
- https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/paper-chromatography#:~:text=Solvents,as%20the%20eluent%20for%20chromatography
- https://www.organomation.com/solvents-in-sample-preparation-for-chromatography-and-mass-spectrometry