Sample Preparation for Analytical Characterization

To make a solution, one must consider the solubility of the substance that is being measured. The compound of interest must dissolve in the solvent in order to make a solution. Solubility is a factor of intermolecular interactions of the analyte with the solvent and can often be manipulated by changing the type of the solvent or the pH.

The first step in making a sample is choosing proper glassware and making a solution. Most samples in the liquid phase are made in volumetric flasks. Volumetric flasks are made to contain a certain volume of liquid at a given temperature (normally 20 °C), and are calibrated to be accurate less than 0.02% if they are class A glassware. Volumetric flasks are much more accurate for measuring liquids than graduated cylinders.

To make a solution of a solid, the solid must first be accurately massed with a calibrated scale. However, the mass of some reagents and precipitates can change because they are hygroscopic and adsorb water. If the reagent has adsorbed water it is impossible to use the non-hydrated molecular weight to obtain the correct number of moles. To remove adsorbed water, solids that are thermally stable are dried in an oven at ~110 °C. Solid reagents and precipitates are then stored in a desiccator containing a desiccant that adsorbs any water present.

If the sample to be diluted is a liquid a pipette is normally used to measure it. A glass transfer pipette is typically calibrated to deliver one accurate volume and the last drop stays in the pipette and should not be blown out. A measuring pipette will have multiple markings on it — similar to a burette — and is less accurate but more versatile than a transfer pipette. Smaller volumes can be measured using variable micropipetters, with disposable plastic tips, and these are available in volumes from 1–5,000 µL. Micropipetters should be calibrated every 6 months in order for them to maintain accuracy. If plastic is an issue, small microsyringes can also be used to measure out volumes in the microliter range.

After a solution is made there are other elements of sample preparation that may be pertinent. Any sample with solid remaining in the liquid should be filtered. Traditional filtration uses a setup with a filter paper that sits in a fritted glass funnel on top of a filter flask with an arm where vacuum can be pulled. This type of filtering is used to collect a precipitate in experiments such as gravimetric analysis. Smaller samples that are to be analyzed can be cleaned up via syringe filtering where the sample is loaded in a syringe and then passed through a polymer filter with down to 0.2-nm resolution. Additionally, spin filters are available where the sample is loaded in a microcentrifuge tube with a filter, the tube is placed in a centrifuge, and the filtered liquid is at the bottom after centrifugation. Spin filters are also used to concentrate larger analytes, such as proteins. Syringe and spin filters are useful to filter out contaminants and other solids that might interfere with the instrument or measurement. The type of filtration used depends on the amount of sample and the size of the solid that needs to be filtered out.

Sample preparation can also involve extracting or preconcentrating a sample. When studying metal ions, chelation can be used for selective extraction. Metal ions will bind to a chelating agent and then the chelated complex can be extracted out. Masking agents are used before chelation to bind a specific metal ion which is then not chelated by the chelating agent. A demasking chemical reaction is used to release the specific metal ion back in solution. Masking allows a more specific sample preparation and protection of certain metal ions.

Solubility is the amount of substance that will dissolve in a liquid. Generally, if less than 0.1 g will dissolve in 100 mL of solvent a substance is considered to be insoluble. Solubility depends on intermolecular interactions with the analyte and thus, the general rule in solubility is "like dissolves like". Polar substances tend to dissolve well in polar solvents while nonpolar analytes dissolve well in nonpolar solvents. Solubility of solids in a liquid is generally greater at higher temperatures, because of the added energy and molecular motion.

Chelation is accomplished by multidentate ligands that have multiple binding sites for a molecule. The most common chelating agent for metal ions is ethylenediaminetetraacetic acid (EDTA), which is hexadentate and binds through 2 nitrogen and 4 oxygen atoms. It has 6 acidic protons that it can lose upon metal-EDTA complex formation. The formation constant for binding is pH specific and the pH is often adjusted to adjust the specificity of the chelation reaction.

Because EDTA can complex with many different metals, masking is needed in order to perform analysis of a specific metal. Before the addition of the chelating agent, a masking agent is added to protect the ion of interest from reacting with the EDTA. The formation constant for the masking agent-metal complex must be greater than the formation constant for the EDTA-metal complex so that the EDTA will not react. For example, fluoride masks Al³⁺ and Fe³⁺. Cyanide is another common masking agent that does not react with Mg²⁺, Ca²⁺, or Pb²⁺ but does react with other metals such as Cd²⁺, Hg²⁺, Fe²⁺, Fe³⁺, and Ni⁺. Cyanide can form a toxic gas at low pH so it should always be used in a solution above pH 11. Demasking releases the masked metal ion; for example cyanide can be demasked by a chemical reaction with formaldehyde. Masking and demasking allow selectivity for measuring components of complex mixtures.

Spin filters are often used in biological analyses to clean up samples. If cellular debris from cell lysis is a problem, then the sample can be spin filtered and the filtrate at the bottom will be free from particles. If you wish to concentrate a protein or other bigger analyte, a filter with a small pore membrane can be used that the protein cannot pass through. After spin filtering the smaller molecules will be in the filtrate at the bottom and will be discarded. When the filter is inverted and spun again in another tube it can be released from the filter and collected in a concentrated form. Syringe filters are often used to remove dust particles and other small particles from chromatography samples, as the particles could clog the column and cause problems with the instrument.

EDTA is often used for titrations to determine metal contents. The number of moles of EDTA added equals the number of moles of metal. Chelation is also used for extractions in trace metal analysis. Chelating a metal will neutralize the charge and allow it to be extracted into an organic solvent if the chelating agent has a hydrophobic group. Masking prevents a metal from being chelated and therefore from being extracted. This method can be used for sample cleanup or preconcentration of trace metals.