[Reader Insight] How to Select Sample Preparation Methods: A Summary of Experience

This article is written by an expert chromatographer under the pen name of Chromatography Mound. Welch Materials, Inc. is authorized to translate this article to English and publish it on behalf of the author.

Sample preparation is a critical step in analytical method development, closely tied to extraction efficiency, purification, separation performance, and matrix effects.

Selecting an appropriate preparation method can effectively reduce testing costs. In this article, the author summarizes methods for detecting various compounds and provides insights into special applications for sharing and exchange with analytical professionals.

Select suitable solvents based on molecular formulas and known properties of the analyte, or use a mixture of solvents. Detection instruments vary depending on the chosen solvent. Common solvents (with boiling points in °C) include:

• Liquid Chromatography: Water, methanol (64.7), ethanol (78), isopropanol (82.5), acetonitrile (81), acetone (56.5), salt buffers.
• LC-MS: Water, methanol (64.7), ethanol (78), isopropanol (82.5), acetonitrile (81).
• Gas Chromatography: Methanol (64.7), ethanol (78), isopropanol (82.5), acetonitrile (81), hexane (69), toluene (110), ethyl acetate (77), acetone (56.5), dichloromethane (39.8), chloroform (61.3).
• GC-MS: Hexane (69), acetone (56.5), ethyl acetate (77).

Advantages: Quick and simple preparation; broad range of solvent polarities available.
Disadvantages: Non-specific extraction; high impurity content; chromatograms with multiple peaks; strong matrix effects in mass spectrometry.

Add two immiscible solvents with significant polarity differences to separate water-soluble and oil-soluble substances. One phase serves as the extraction phase and the other as the impurity-removal phase. For instance, oil-soluble analytes can be extracted with hexane, followed by washing with water, acid, base, or saline solutions.

Advantages: Removes impurities with large polarity differences from the analyte.
Disadvantages: Non-selective solvents; e.g., oil-soluble analytes extracted with ether cannot be directly analyzed using liquid chromatography.

An extension of solvent extraction. After analyte extraction, the solution passes through a solid-phase extraction cartridge where analytes are adsorbed on a solid material, eluted with solvents of varying polarities.

Advantages: High selectivity and enrichment efficiency of SPE materials.
Disadvantages: For multi-component compounds, recovery rates may vary; potential for overload effects.

For low-polarity analytes requiring low-polarity solvents, solvent switching is performed due to unsuitable boiling points or polarities for liquid chromatography. Extracted and purified solvent is evaporated under nitrogen and reconstituted with highly polar solvents such as water, methanol, or acetonitrile.

Advantages: Enables LC analysis for low-polarity analytes unsuitable for direct methanol or acetonitrile extraction.
Disadvantages: Requires stable analytes; not suitable for volatile or easily oxidized substances.

Add a 1:1 mixture of water and acetonitrile. After extraction, add a salt pack to create a buffer system. Shake and allow phases to separate, with the acetonitrile phase containing the analyte. Widely applied for complex compound extractions.

Derivative Applications: Dissolve derivatization reagents in acetonitrile and mix with aqueous samples for reactions under water bath or ultrasonic conditions. Add salts to separate acetonitrile.

Advantages: Convenient for single-phase extraction or reactions; no solvent switching needed for LC or LC-MS analysis.
Disadvantages: Single organic phase may lead to low recovery rates for certain multi-component compounds.

Combine two miscible solvents with large polarity differences; after extraction, add an aqueous phase to induce separation. This method is suitable for low-polarity solvent extraction in GC or GC-MS analysis. One solvent serves as a diluent, the other as a derivatization solvent that dissolves easily in the added aqueous phase.

Example: Mix toluene with methanol containing acetyl chloride; after reaction, add Na₂CO₃ solution to remove byproducts and separate toluene from methanol.

Common purification agents include:

• PSA (Ethylenediamine-N-propylsilane): Removes fatty acids and organic acids, common matrix interferences in MS detection.
• C18 (Octadecyl Bonded Silica): Removes fats and low-polarity impurities.
• GCB (Graphitized Carbon Black): Removes pigments like carotenoids and chlorophyll, as well as planar impurities and sterols.
• EMR (Enhanced Matrix Removal): High selectivity for fat removal with minimal impact on analytes.
• Al-N (Neutral Alumina): Hydrogen bonding interactions remove acidic oxygen-based impurities.
• Silica Gel: Removes surfactants and highly polar impurities.