Sample Handling
The first step in any analytical process is properly handling the initial sample. No matter the type of analysis being performed, the sample collected must be representative of the whole and remain unchanged from the time of collection to analysis. For liquids, solids, or gases, representative sub-samples must be taken following specific protocols to avoid contamination or degradation.
For liquid samples, aliquots should be pipetted or poured from the original sample container into clean testing vials or volumetric flasks. Care must be taken to gently mix any settled particles back into suspension before sampling. With solids, a minimum Sample Preparation is often specified and particles should be collected from different regions of the bulk material using a sampling probe or tool. Any large lumps should be broken up and thoroughly mixed. Gas samples may need to be collected directly into evacuated containers or sorption tubes at the point of generation.
Proper labeling of all sample containers with unique identifiers and metadata like collection date/time and location is critical. Samples should be stored in a manner that prevents further chemical or biological changes prior to analysis. This may involve refrigeration, freezing, or inert gas purging of the sample headspace. Documentation of the sampling method, conditions, and any pre-treatment is also important for establishing the sample provenance.
Sample Pre-treatment
In many cases, samples require some form of pre-treatment before analysis to make the target analytes easily accessible and compatible with the analytical method. Common pre-treatment steps include dissolution, extraction, digestion, and derivatization.
For dissolving solids, the appropriate solvent must be selected based on the chemical nature of the matrix and analytes. Agitation, heating, or sonication may aid in dissolving processes. Liquid-liquid extraction is often performed to separate and concentrate analytes from interfering matrix components when analyzing complex samples. Target compounds are transferred between immiscible liquid phases, typically using separatory funnels.
Digestion breaks down organic or inorganic matrices through acid treatment or oxidation to liberate elements or compounds of interest. Microwave-assisted, high pressure asher, or wet digestion are some common digestion techniques employed. Derivatization chemically modifies functional groups on analytes to improve volatility or chromophoric properties needed for certain instrumental methods like gas chromatography or ultraviolet-visible spectrophotometry.
Proper technique and documentation of pre-treatment conditions like temperature, pressures, times, and volumes used is necessary for method validation and quality control purposes. These preparation steps can significantly impact analysis accuracy if not standardized and controlled.
Sample Cleanup
Even after pre-treatment, samples may still contain residues or contaminants that could interfere with the chosen analysis or damage instrumentation. Cleanup procedures are thus required to isolate and concentrate the analytes while removing matrix components.
Solid phase extraction (SPE) cartridges packed with various sorbent chemistries allow selective retention of targeted compounds from liquids based on differences in partitioning properties or chemistries between analytes and interferences. Load, wash, and elution steps allow cleanup and analyte isolation. Automated SPE systems provide reproducible sample preparation with minimal technician time requirements.
Liquid-liquid extraction can also be employed as a cleanup technique taking advantage of differences in solubility between analytes and matrix components across aqueous and organic solvent phases. Multiple extractions may be needed for effective sample cleanup.
For solid sample extracts or digests, dispersive or liquid-liquid based partitioning allows removal of fat or pigment compounds through addition of salts, chelating agents, or other reagents that alter molecular associations or phase behavior. Size exclusion chromatography separates based on molecular size and is suitable for fractionating complex digests or isolates.
Regardless the cleanup method, method development and validation is essential to demonstrate effective isolation of analytes from interfering species without analyte losses. This ensures reliable quantitative results.
Sample Drying and Concentration
For instrumental analysis, liquid samples generally require removal of solvent before analysis or concentration to detectable levels. Drying and reconstitution processes can lose or alter some analytes requiring careful optimization.
Rotary evaporation is commonly used to remove organic solvents under vacuum at controlled temperatures, often into dryness. For thermally labile compounds, freeze-drying or centrifugal evaporation under inert gas may be preferable.
Concentration can be achieved through evaporation, solvent exchange to a smaller final volume, or application of a high velocity gas stream in devices like nitrogen blow-down evaporators. Micropipetting to predefined volume is ideal for accurate gravimetric sample loading onto prep plates or tubes.
Solids after extraction or digestion also require drying, usually in an oven at low temperature under vacuum or inert atmosphere. Drying time and temperatures need monitoring to prevent analyte losses from volatilization, decomposition or precipitation from solution.
Proper reconstitution or redissolution into an appropriate solvent is also important, often with brief sonication, to fully dissolve the sample matrix into a homogeneous solution for analysis. Inadequate drying or reconstitution can negatively impact performance.
In quality sample preparation free from contamination, degradation and incorporating standardized techniques is essential for producing reliable analytical results. Careful handling, pretreatment, cleanup and concentration maximizes recovery while minimizing matrix effects. This ensures regulatory and data quality requirements are satisfied.