Some samples still cannot be directly injected after pretreatment. Derivatization is required to make compounds without UV absorption or fluorescence detectable by UV or fluorescence detectors.

Improves sensitivity
Enhances separation (quality change)

Possible issues include sample loss, contamination, incomplete reactions, or multiple by-products, which may affect accuracy or introduce errors.

Sample Filtration

LC samples must be uniform and particle-free; particles may damage the injector or block the column. Steps include:

Shake the sample before injection and check for particles, turbidity, or emulsions
If particles are observed, filter the sample using a membrane that can retain ≥0.15μm particles

Potential issues: sample contamination, adsorption reducing component concentration, and solvent evaporation causing errors.

Extraction Methods

Extraction separates analytes from co-solvents or removes interfering substances (e.g., proteins). Organic solvents should be low-toxicity, volatile, low in impurities, and dissolve the sample well.

Common solvents: diethyl ether, ethyl acetate, dichloromethane, chloroform, benzene, or mixtures.

After extraction, samples can be injected directly or concentrated to increase analyte concentration and sensitivity.

Water-Soluble Sample Extraction

Acidic components and salts: extract impurities with organic solvent, adjust to acidic, then extract or inject; or dry under N₂ and dissolve in appropriate solvent before injection.
Basic components and salts: extract impurities with organic solvent, adjust to basic, then extract or inject; or dry under N₂ and dissolve in appropriate solvent before injection.
Neutral components: extract impurities with organic solvent, then analyze directly by reversed-phase chromatography.

Fat-Soluble Sample Extraction

Extract with organic solvent or inject directly; or dry under N₂ and dissolve in appropriate solvent before injection.

Contamination Sources

Potential sources of contamination in analysis:

Contamination Type	Source	Prevention Measures
Environmental	Harmful gases, dust, aerosols	Keep instrument room isolated, clean; install AC; control humidity <70%; prevent moisture, corrosion, vibration
Containers	Glass, ceramic, quartz, plastic labware	Select appropriate containers per sample type; clean thoroughly
Reagents	Non-chromatography-grade reagents	Use chromatography-grade, high-purity, or analytical-grade reagents

Standard Solution Preparation

Prepare internal standard solution first; use same batch in standards and samples to reduce errors
Substances must be chromatography-grade and stable
Store standard solutions in brown bottles at low temperature
For vitamins, use brown volumetric flasks or protect from light

Common Problems in Sample Pretreatment and Solutions

Problem	Cause	Solution
Unrelated peaks in chromatogram	Filter contamination	Soak filter in sample solvent and test injection Change filter type Use alternate cleaning techniques
Low peak response (especially low-concentration samples)	Adsorption on filter surface	Change filter type Process all samples under same conditions Use alternate cleaning techniques
Low recovery	Incomplete extraction	Increase extraction time, use hot solvent Adjust cleaning method
Broadened peaks, shortened column lifetime	Sample interference and contamination	Improve cleaning method
Poor precision	Incomplete recovery	Optimize or replace derivatization, separation, extraction, or other conditions Use automated handling to improve precision

Extremely High/Low Temperature and Light Exposure Effects on Sample Stability: Theory & Methodolog

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