Sample Preparation for Analytical Characterization
Source: Laboratory of Dr. B. Jill Venton - University of Virginia
Sample preparation is the way in whicha sample is treated to preparefor analysis. Ca…
1. Making a Solution from a Solid
- Choose the correct glassware to make solution.
- 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).
- Rinse the glassware several times with distilled water. Dry in an oven if needed.
- To make a solution from a solid, mass out the correct amount of solid.
- Put the solid in the volumetric flask and then fill about ¾ full with solvent.
- Swirl to fully dissolve the solid before filling the volumetric flask fully.
- 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
- 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.
- Release the liquid into the volumetric flask for making the solution. Do not blow out the last drop.
- Fill the volumetric flask to the line so the meniscus touches the line. Mix the solution by inverting several times.
3. Filtering
- For a filter flask setup, place a piece of filter paper on the fritted glass filter.
- Attach the fritted glass filter to a filter flask.
- 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.
- Turn on the vacuum and pour the sample through the filter paper.
- Filter until a dry powder is left. Continue to dry the sample in an oven if a dry precipitate is desired.
- To syringe filter, add the sample to a clean syringe with a Luer lock end.
- Screw the syringe filter into the Luer lock. Push the plunger on the syringe and collect the liquid after the filter.
- For a spin filter, pre-rinse the filter with buffer or ultrapure water.
- Insert the spin filter into a microcentrifuge tube.
- Load the sample on top of the filter and cap the tube.
- 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.
- Remove the filter and the liquid in the bottom is the filtered solution.
- 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
- For masking and chelating, adjust the pH to an appropriate value depending on the formation constants of the masking agent and the chelating agent.
- Add the masking agent to the solution and allow it to react for at least 10 min with the metal ion of choice.
- 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.
- 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 ? 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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Q1: Why is proper sample preparation critical in analytical chemistry?
Proper sample preparation is essential to reduce errors and ensure accurate chemical analysis. Errors in sample preparation are systematic errors that propagate through analysis, causing uncertainty or inaccuracies in calibration curves. Poor preparation can also damage analytical instruments. Careful sample preparation ensures either a standard or unknown sample is correctly prepared for chemical measurement.
Q2: What is the difference between random and systematic errors in sample preparation?
Random errors result from unexpected changes, such as environmental conditions like wind, and are often due to instrument noise. Systematic errors stem from investigator or instrumental bias, introducing an offset in measured values. Systematic errors in sample preparation can be eliminated through correct procedures and proper use of instruments and devices like balances or pipettes.
Q3: How do you prepare a solid sample for analysis?
Weigh the solid sample on an analytical balance for improved accuracy. If hygroscopic, dry it in an oven or desiccator first. Dissolve the sample in a volumetric flask using less than the final solvent volume initially. After mixing, carefully add solvent to reach the calibration mark. Stopper and invert several times to thoroughly mix the homogeneous solution.
Q4: What role does solvent selection play in sample preparation?
Solvent choice is critical and depends on instrument requirements; some require aqueous solvents while others require organic. The sample must dissolve in the selected solvent. Solubility depends on intermolecular interactions between the analyte and solvent, and can be manipulated by changing solvent type, temperature, or pH to ensure proper dissolution.
Q5: When is chelation and masking used in sample preparation?
Chelation is used when a dissolved metal must be bound to a chelating agent like EDTA to be detected. Masking agents are added to inhibit chelation of unwanted metals, preventing their detection. Demasking can then release interfering metal ions for separate analysis. This technique is essential in complexometric analyses, such as calcium determination in the presence of iron.
Q6: How do you remove undissolved solids from a prepared sample?
Load the sample into a syringe and attach a syringe filter to the tip. Press the plunger to push the sample through the filter, which removes unwanted solids. This filtration step is necessary to prevent damage to analytical instruments and ensure the sample is fully prepared and ready for analysis.
Q7: What are the key differences between preparing solid and liquid samples?
Solid samples must be weighed on an analytical balance and may require drying before weighing. Liquid samples can be measured by weight or volume using volumetric flasks or calibrated glass pipettes. Both types are then dissolved in volumetric flasks with solvent and mixed to the calibration mark. Liquid samples skip the initial weighing step but follow the same dissolution and mixing procedures.