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Analytical Chemistry

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Sample Preparation for Analytical Characterization

Sample Preparation for Analytical Characterization


Source: Laboratory of Dr. B. Jill Venton - University of Virginia

Sample preparation is the way in which a sample is treated to prepare for analysis. Careful sample preparation is critical in analytical chemistry to accurately generate either a standard or unknown sample for a chemical measurement. Errors in analytical chemistry methods are categorized as random or systematic. Random errors are errors due to change and are often due to noise in instrument. Systematic errors are due to investigator or instrumental bias, which introduces an offset in the measured value. Errors in sample preparation are systematic errors, which will propagate through analysis, causing uncertainty or inaccuracies through improper calibration curves. Systematic errors can be eliminated through correct sample preparation and proper use of the instrument. Poor sample preparation can also sometimes cause harm to the instrument.


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.

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1. Making a Solution from a Solid

  1. Choose the correct glassware to make solution.
  2. Clean the glassware thoroughly via an acid bath of 1% HCl or HNO3 along with soap to remove any impurities (safety warning: with any strong acid use gloves, goggles, and other appropriate personal protective equipment).
  3. Rinse the glassware several times with distilled water. Dry in an oven if needed.
  4. To make a solution from a solid, mass out the correct amount of solid.
  5. Put the solid in the volumetric flask and then fill about ¾ full with solvent.
  6. Swirl to fully dissolve the solid before filling the volumetric flask fully.
  7. Fill the volumetric flask to the line. The meniscus should just touch the fill line. Then invert the flask several times with the cap on to further mix if necessary.

2. Making a Solution from a Liquid

  1. Choose the correct glassware to make solution. To deliver a liquid using a transfer pipette, fill the pipette to the line using a pipette bulb.
  2. Release the liquid into the volumetric flask for making the solution. Do not blow out the last drop.
  3. Fill the volumetric flask to the line so the meniscus touches the line. Mix the solution by inverting several times.

3. Filtering

  1. For a filter flask setup, place a piece of filter paper on the fritted glass filter.
  2. Attach the fritted glass filter to a filter flask.
  3. Attach a vacuum to the arm of the filter flask. A trap can also be used to prevent any liquid from going into the vacuum.
  4. Turn on the vacuum and pour the sample through the filter paper.
  5. Filter until a dry powder is left. Continue to dry the sample in an oven if a dry precipitate is desired.
  6. To syringe filter, add the sample to a clean syringe with a Luer lock end.
  7. Screw the syringe filter into the Luer lock. Push the plunger on the syringe and collect the liquid after the filter.
  8. For a spin filter, pre-rinse the filter with buffer or ultrapure water.
  9. Insert the spin filter into a microcentrifuge tube.
  10. Load the sample on top of the filter and cap the tube.
  11. Put the tube into a centrifuge, making sure to balance it properly with another tube on the other side, and centrifuge for 10–30 min, depending on the type of spin filter.
  12. Remove the filter and the liquid in the bottom is the filtered solution.
  13. If the sample cannot go through the membrane — such as a big protein — it will remain at the top of the filter. In this case, turn the filter over, put it in a new tube, and spin again. This will produce a concentrated sample.

4. Masking and Chelating

  1. For masking and chelating, adjust the pH to an appropriate value depending on the formation constants of the masking agent and the chelating agent.
  2. Add the masking agent to the solution and allow it to react for at least 10 min with the metal ion of choice.
  3. Add the chelating reagent. For EDTA, it typically forms a 1:1 complex with the metal ion, so add as many moles of EDTA as metal that will be chelated.
  4. After chelation, demask by adding a chemical that will react with the masked metal ion. The masked substance can then be analyzed or recovered by precipitation.

Proper sample preparation is a critical first step in all types of chemical analyses.

Suitable sample preparation is essential to reduce the potential for error. Error can be mitigated in several ways: from the selection of proper glassware to attention to significant figures in calculations.

For many analytical instruments a uniform solution of the sample of interest must first be prepared. The process of dissolution — where a solute dissolves in a solvent — enables the formation of a homogeneous solution that can then be withdrawn for use in one or more analyses.

However, the dissolved sample often requires additional preparation steps before it is ready for analysis. These techniques such as filtration, extraction, or chelation may also be performed prior to analysis.

This video will demonstrate some key steps in proper sample preparation for subsequent chemical use.

Errors can be classified as either systematic or random. Random errors are associated with unexpected change, such as environmental conditions like wind.

Systematic errors are associated with experimenter or instrument bias. These errors in sample preparation can be avoided by ensuring that both the procedure and the devices — such as a balance or pipette — are used properly.

When preparing a sample solution the choice of solvent is important, and can be based on the requirements of the instrument being used. Some instruments require aqueous solvents while others require organic. It is necessary for the sample to dissolve in the selected liquid. Solubility is a factor of intermolecular interactions of the analyte with the solvent, and can often be manipulated by changing the type of solvent, temperature, or pH.

A sample solution must have an accurate concentration. To prepare the solution, the solid sample is weighed on an analytical balance rather than a standard top loading balance for improved accuracy. If the solid sample is hygroscopic and contains moisture, it may need to be dried in an oven or desiccator before weighing.

If the sample is liquid, it can be measured by weight or volume. When using volume measurements, be sure to use a volumetric flask as errors can be introduced when using other measurement glassware such as a volumetric cylinder with large graduations. Alternatively, a glass volumetric pipette can be used. These are typically calibrated to deliver one accurate volume with the last drop remaining in the pipette.

In order to prepare the solution, the accurately measured sample is dissolved in a volumetric flask. Initially, use less than the final volume of solvent to dissolve the sample. After mixing add additional solvent carefully to bring the solution to its final volume.

After dissolving most of the sample into the solvent there may still be solids present that need to be removed prior to analysis. These unwanted solids can be removed by filtration in order to avoid damaging the instrument.

In some cases, a dissolved metal must be bound to another compound — called a chelating agent — in order to be detected. This process is called chelation. When unwanted species also bind to the chelating agent, they must be masked.

This is done by adding a masking agent which inhibits chelation of certain metals. This prevents the unwanted metals from being detected. Demasking of the interfering compounds can then be performed to release the metal ions and enable their analysis.

Now that the basics of sample preparation have been outlined, let's take a look at how it is performed in the laboratory.

To begin, select an appropriately-sized volumetric flask.

Soak the volumetric flask and stopper in 1% hydrochloric acid to remove any adsorbed cations. Remove the flask and stopper after soaking overnight.

Next, wash the volumetric flask and stopper with soap and deionized water, then rinse thoroughly.

Dry the flask and stopper in a drying oven until completely dry.

Once the flask is cool, weigh the required sample and add it to the flask. Record the mass of the sample used.

Add about ¾ of the solvent volume to the flask, put the stopper in place, and gently swirl to dissolve the solids.

Add the remainder of the solvent until the meniscus touches the calibration mark on the flask. Stopper the flask and invert it several times to thoroughly mix.

To remove undissolved solids load the sample into a syringe, place a syringe filter on the tip, and press the plunger to push out the sample through the filter. The collected sample is now fully prepared and ready for analysis.

If the sample to be analyzed is a liquid rather than a solid, it can be measured volumetrically using a pipette. Starting with a clean volumetric flask and stopper, add an appropriate volume of the sample to the flask and record the volume.

Add the solvent and finish preparing the sample as is done for solid samples.

If the dissolved sample requires metal chelation and masking, such as in a complexometric calcium analysis in the presence of iron, adjust the sample to an appropriate pH by adding base.

Add the cyanide masking agent to protect the iron from chelation. Allow it to react for at least 10 min.

Add EDTA to chelate the calcium and allow it to mix. Use enough to form a 1:1 complex. The sample is now ready for calcium determination.

To use the same sample for iron analysis, demask the iron by adding formaldehyde and mix. The sample is now ready for iron analysis.

Sample preparation is an important step in almost every experiment and analytical method used by chemists.

Industrial flue gas can be used as a source of carbon dioxide for culturing microalgae. In order to understand whether heavy metal contamination from the gas is a problem, the algae grown in this example were analyzed for heavy metal content.

After the growth period the algae were collected from the bioreactor fluid by centrifugation and freeze dried prior to beginning the sample preparation process.

The dried algae were digested using heat, nitric acid, and hydrogen peroxide in order to prepare a homogeneous solution suitable for chemical analysis. In this example, the content of heavy metals in dried algae was analyzed using inductively coupled plasma-mass spectrometry, or ICP-MS.

The technique simultaneously detected 12 metals in the sample without interference, and revealed that metal contaminants from the flue gas ended up in the algae biomass.

Proper sample preparation is also important when studying complex materials such as soil using atomic absorption spectroscopy.

In this experiment, a sample of dried soil was first weighed on an analytical balance. It was then added to a digestion tube with 1:1 water and concentrated nitric acid using a pipette.

After several digestion steps the sample was filtered to remove any solids, then was collected in a volumetric flask. Additional water was added to dilute the solution for analysis.

Lipid polyesters constitute the structural components of parts of cell walls. In order to study these chemicals using GC-MS, plant tissue was first collected and weighed.

After various processing and drying steps, a 1:1 mixture of toluene and heptane was added to dissolve the dry sample.

The vial was inserted into the automatic loading tray of a GC-MS for analysis.

You've just watched JoVE's introduction to Sample Preparation. You should now understand the basics of preparing solid and liquid samples for subsequent analysis.

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Applications and Summary

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.

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Sample Preparation Analytical Characterization Chemical Analysis Error Reduction Glassware Selection Significant Figures Dissolution Homogeneous Solution Filtration Extraction Chelation Sample Preparation Techniques Random Errors Systematic Errors Environmental Conditions Experimenter Bias Instrument Bias Solvent Choice

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