Flame Emission Spectroscopy

Spectroscopy is used to find out the concentration of a solution by determining how much light is absorbed. The intensity of light emitted from a flame of a specific wavelength is used to determine the quantity of an element in a sample. The identity of the element is given by the wavelength of the atomic spectral line in the emission spectrum and the intensity of light represents the number of atoms in the element.

Flame test for metal ions

Introduction

When heated strongly, different metal ions produce different flame colours. When ions are heated at high temperatures, some electrons absorb enough energy to allow them to get to higher energy levels. The element is now known to be in an ‘excited state’ as the configuration is now unstable. However, as the electrons fall to their normal energy levels due to instability, the energy that was absorbed is emitted as electromagnetic energy and can be seen in the form of light, therefore elements can be identified from the light produced. Using flame tests, unknown metal ions can be tested and depending on the flame colour produced, can be identified. (Braid, Clayton, Falkner, Faudemer, Henderson, Howe, Jordin, Kordan, Scott, Train, 2018)

Materials and method

Safety: Wear safety glasses and a lab coat at all time. When Bunsen burner is not being used, leave on the orange flame.

Chemicals: 6 unknown metal cation samples

Apparatus: Nichrome wire loop, Bunsen burner, heat-proof mat

Method: A clean wire loop was dipped into a solid sample of the compound being tested. The loop was then heated onto the edge of the blue flame from a Bunsen burner, which is placed on the heat-proof mat in case of spillage of the metal cations. Observing the colour of the flame produced, results were recorded. Repeat for each compound, making sure the wire loop is clean for accurate and reliable results.

Results

Unknown compounds

Flame colour produced

1

Red

2

Yellow

3

Lilac

4

Orange-red

5

Green

6

Blue green

 

Conclusion

Unknown sample

Flame colour

Ion present

1

Red

Lithium, Li+

2

Yellow

Sodium, Na+

3

Lilac

Potassium, K

4

Orange-red

Calcium, Ca2+

5

Green

Barium, Ba2+

6

Blue green

Copper, Cu2+

Using the flame colour produced, we can identify the metal ions present in the unknown sample. For example, sample 1 produced a red flame colour, indicating that lithium is present as each metal ion produces a different flame colour.

Discussion

Possible errors during this experiment may have included the wire loop not being fully cleaned. In order to test the cleanliness of the loop, the wire loop can be dipped into hydrochloric acid then rinsed with distilled water. When the clean wire loop is held into the flame, you can tell if it is not thoroughly cleaned as a colour in the flame would be produced. Another limitation may be that low concentrations of ions are unable to be detected. Also, concentrations of the metal ion cannot be identified with this method- only what metal ion is present.

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In real life application, the flame test can relate to fireworks, which contain an explosive powder, as well as specific chemicals for their colours. When heated, the explosion of the firework produces gases and the electrons get ‘excited.’ As the electrons return to their normal energy levels (their ground state), the emit coloured light depending on the chemical. For example, greens from barium.

Determining the concentration of copper sulphate using colorimetry

Introduction

Colorimetry is the measure of colour to a quantitative measure and can be used to work out the concentrations of coloured solution, such as copper sulphate which is a shade of blue.

It works by choosing a colour for the filter which is complimentary to the solution. Light passes through this colour filter and the cuvette, which contains the solution. Less light should leave the sample than the amount of light that entered, giving measurements of absorbency. These results can then determine how concentrated a solution is.

Materials and method

Safety: Wear eye protection and a laboratory coat at all times during the investigation.

Chemicals: 24.9g of copper sulphate (s), distilled water

Apparatus: scales, beaker, volumetric flask, stirring rod, x6 test tubes, test tube rack, 10cm3 graduated cylinder, 10cm3 pipette, pipette pump, x6 cuvettes, colorimeter machine

Method: Using the scales, 24.9 grams of solid copper sulphate was weighed and dissolved in 75cm3 distilled water. This took a few minutes to happen. The solution was then transferred to a volumetric flask and another 25cm3 distilled water was added to make it up to 100cm3. This was labelled as 1 mole solution of copper sulphate. Different dilutions of the solution were made up in 6 different boiling tubes using a 10cm3 pipette. (see appendix A) Using different cuvettes for each different dilution of the copper sulphate, the cuvette was filled, starting with the most dilute, and absorbance was measured in the colorimeter at 600nm. Each dilution was tested 3 times for reliability and the absorbency was recorded. The mean was then worked out for each dilution. The absorbency of an unknown concentration of copper sulphate was then measured and the concentration was estimated by using the results already and the line of best fit on the graph drawn up of the results. (see appendix B)

 

Results

Results table to show the absorbency (%) of the different concentrations of the copper sulphate solution (m) and the mean result.

Concentration of solution (m)

0

0.2

0.4

0.6

0.8

1.0

Absorbency (%)

1

0.01

0.24

0.35

0.54

0.77

0.91

2

0.00

0.27

0.46

0.65

0.79

0.92

3

0.01

0.27

0.48

0.64

0.78

0.92

Mean (%)

0.006

0.26

0.43

0.61

0.78

0.92

 

Graph (see appendix B)

The results show that as the concentration of the solution of copper sulphate increases, so does the absorbency of the solution, showing they are directly proportional. The more concentrated a solution is, the more light it absorbs. The unknown concentration could therefore be measured using the calibration curve produced on the graph (see appendix B).

Conclusion

Colorimeters are a useful method as they are inexpensive. However, control variables must be regulated, for example the cuvette cannot be scratched as less light will be let through affecting the results.

Chromatography

Chromatography is the technique used to separate chemicals in a mixture so you can identify specific substances. There are two main principles in chromatography. Firstly, the mobile phase, which is where molecules can move, meaning it must be a liquid or gas. Secondly, the stationary phase where molecules are unable to move, meaning it must be either a solid or a liquid with solid support.

The distance each substance in the compound moves depends on its solubility in the mobile phase and its retention by the stationary phase. Substances more soluble in the mobile phase travel further than substances that are more strongly absorbed to the stationary phase. The different components of the compound are separated by their differences in solubility and retention. (Braid, Clayton, Falkner, Faudemer, Henderson, Howe, Jordin, Kordan, Scott, Train, 2018)

Thin layer chromatography (TLC) of amino acids

Introduction

The mobile phase in TLC is a liquid solvent and the stationary phase is a thin layer of silicon dioxide or aluminium dioxide attached to a glass or metal plate.

Materials and method

Safety: Wear gloves and safety glasses at all times because of the ethanoic acid, which can cause severe burns and eye damage.

Chemicals: arginine (A), leucine (B), glycerine (C), unknown mix (X), ethanoic acid

Apparatus: TLC glass plate coated with silica, pencil, 3 pipettes, beaker, forceps, watch glass, fume cupboard

Method: A glass plate is coated with silica. A line is drawn using the pencil and rule around 1cm from the bottom of the TLC plate, the baseline. A small drop of the mixture to be separated is put on the baseline labelled X using a pipette, alongside known amino acids labelled A-C, using a separate pipette per amino acid to prevent contamination. The spots are left to dry. The plate is placed into a beaker with a small volume of ethanoic acid (so it is below the baseline) using forceps or the mobile phase. The ethanoic acid is not placed higher than the baseline as otherwise it may dissolve the samples away. The top of the beaker is covered with a watch glass to reduce loss of solvent. As the solvent moves up the plate, it will carry the substances in the mixture with it. The beaker is left until the solvent has almost moved to the top of the plate. The plate is then removed from the beaker and a pencil is used to mark how far the solvent travelled before it has evaporated. This line is called the solvent front. The plate is placed in a fume cupboard to dry to prevent any toxic fumes spreading in the room. A chromatogram is formed and the positions of the chemicals on the chromatogram are used to identify the name of the chemicals. As amino acids have no colour, a revealing agent called ninhydrin is sprayed on the chromatogram.

Results

Amino Acids

Solvent distance (cm)

Spot distance (cm)

Rf

Arginine

7.30

1.7

4.29

Glycerine

7.30

1.5

4.86

Leucine

7.30

1.8

4.05

Unknown

7.30

1.7

4.29

 

(See Appendix C)

Rf value= distance travelled by spot

                    distance travelled by solvent

The Rf value helps identify the name of specific chemicals. To measure how far the spot has travelled from the base line to the vertical centre of the spot. To measure the distance travelled by the solvent measure from the baseline to the solvent front.

Rf values are properties of chemicals and to identify a chemical, the standard table of Rf values can be used. (see Appendix D) Or known compounds already tested can be used. However, chromatography can be significantly affected by change in temperature or composition of the TLC plate, meaning the results will show different Rf values.

The results show that the unknown mixture is likely to be arginine amino acid or contain arginine amino acid as both have identical Rf values.

Conclusion

Overall, TLC is a simple process and has a short development time and can help to identify individual chemicals within a compound. Also, it is relatively cheap when compared to other chromatography techniques. However other factors such as temperature can affect the results of the chromatogram and the detection limit is a lot higher as TLC is unable to support lower detection limits. 

Another form of TLC is two-dimensional thin layer chromatography (2D-TLC), which

Paper chromatography to separate the pigments in a leaf

Introduction

The pigments found in leaves can be separated using paper chromatography and identified using the Rf values. Pigments found in leaves can include carotenes, chlorophylls, which are the pigments of photosynthesis and xanthophylls.

Materials and method

Safety: Wear eye protection and a lab coat at all time. Propanone is highly flammable (keep away from sources of ignition) and an irritant (wear gloves.)

Chemicals: propanone, sand

Apparatus: pestle and mortar, chromatography paper, forceps, beaker (100cm3), small capillary tube, pencil, ruler, Sellotape, cut up dark leaves

Method: Filling the mortar to 2cm depth and adding a pinch of a sand and six drops of propanone, the mixture is grinded for approximately three minutes with a pestle until a paste is formed. 3cm from the bottom of a strip of chromatography paper, a line is drawn. This is the baseline. Using the fine glass tube, liquid from the leaf extract is added to the centre of the line. The spot is left to dry, and another spot is added on top to build up a concentrated small spot of the leaf extract. The chromatography paper is placed into the beaker of propanone using the forceps. The propanone level is lower than the baseline. The beaker is left until the propanone has travelled near the top of the paper and then removed from the beaker. A pencil is used to mark the solvent front and the chromatogram is left to dry.

Results

Pigment

Colour

Rf value

Chlorophyll a

Olive-green

0.45

Chlorophyll b

Yellow

0.65

Xanthophylls

Yellow

0.71

Carotene

Yellow orange

0.95

The different pigments are carried along at different rates as they are not equally soluble. The yellow spot is carotene as it travels the furthest due to the higher Rfvalue, showing it is the most soluble, whereas chlorophyll a travelled the least furthest, demonstrating lower solubility.

Conclusion

The test was made to be accurate by ensuring gloves were worn in order to prevent contamination and the solvent is kept below the baseline so the solvent and samples didn’t mix as the results would then be unreliable. Also, the capillary tube enabled the spot to be concentrated but not too big.

Gas chromatography (GC)

Gas chromatography is the easiest way to separate volatile liquids to identify chemicals. The stationary phase is a solid or a solid coated by a viscous liquid, such as an oil, packed into a long column, which is coiled to save space and built into an oven. The mobile phase is an unreactive carrier gas like nitrogen. The sample is vaporised and passes through the oven as a gas.

The retention time is measured by how long the substance takes from being injected into the column to being recorded at the other end. It depends on how much time the substance spends moving along with the carrier gas and how much time it spends absorbed in the viscous liquid. Generally, each separate component has a specific time retention, allowing them to be identified. To ensure accuracy, a known sample can be tested to allow for comparisons.

In real life application, GC is used to find the level of alcohol in blood or urine and to find the proportions of esters in oils used in paints. (Braid, Clayton, Falkner, Faudemer, Henderson, Howe, Jordin, Kordan, Scott, Train, 2018)

Gas chromatography-mass spectrometry (GC-MS) separates a sample using GC however, the separated substances are entered into a mass spectrometer, which produces a mass spectrum for each component. This can be used to identify each separate substance to show what the sample consists of. A computer is then able to use a database to identify substances within the sample. This is more accurate than average GC as some chemicals may have similar retention times, making it difficult to identify specific substances.

Column chromatography (CC)

CC is mainly used for purifying an organic compound by separating it from unreacted chemicals. For the stationary phase a glass column is packed with a solid, absorbent material, called a slurry. For example, aluminium oxide coated with water. The mixture to be separated is added to the column and drains into the slurry. For the mobile phase, a solvent is run slowly but continuously through the column. The substances of the compound separate as the mixture is washed down the column, depending on how soluble they are in the mobile phase and how strongly they are absorbed in the stationary phase (retention).

The longer a component spends dissolved in the mobile phase, the quicker it travels down the column whereas is a substance takes a long time to be absorbed in the stationary phase, the slower it moves down the column. Therefore, a more soluble substance will move quicker through the column.

When the sample reaches the end of the column it is collected and can be identified based on the time it took to travel through the column by using the retention time or using mass spectroscopy. (Braid, Clayton, Falkner, Faudemer, Henderson, Howe, Jordin, Kordan, Scott, Train, 2018)

High performance liquid chromatography (HPLC)

Instead of using a solvent, HPLC forces the sample through a column under gravity under high pressures of up to 400 atmospheres, making this technique much quicker. The high-pressure column is the stationary phase and the solvent is known as the mobile phase. (Smith, 2018)

Depending on the polarity of the solvent and the stationary phase, there are two different forms of HPLC. The normal phase consists of a column filled with small silica particles and a non-polar solvent, such as hexane. As the sample passes through the column, polar compounds stick longer to the polar silica than non-polar compounds, resulting in non-polar substances passing through the column more quickly. This is known as normal phase HPLC. On the other hand, the silica can be modified to make it non-polar by attaching long hydrocarbon chains to the surface. In this case, a polar solvent, such as a mixture of water and methanol, is used. There will be strong attractions between the polar solvent and polar molecules in the mixture as it is passed through the column and less attraction between the modified non-polar silica and the polar molecules in the solvent. Polar molecules will move with the solvent, whereas non-polar compounds will form attractions with the hydrocarbons attached to the silica because of van der Waals dispersion forces. Therefore, the non-polar molecules will spend less time in the solvent, meaning they will move more slowly through the column than polar molecules. (Clark, 2016)

HPLC produces a chromatogram from results of a detector, which shows peak points of the separation of components. If there is already a known sample, the different substances can be identified, however if the sample is completely unknown, the peaks just show the number of components in the sample. Retention time is the time interval between sample injection and the maximum of the peak. A substance with a high retention time has a high degree of absorption in the stationary phase.

HPLC allows you to use a smaller sample for the column packing material, giving a greater surface area for interactions between the stationary phase and the substances flowing through, so substances of the sample can separate more easily. This method is also more sensitive to other chromatography techniques. (Clark, 2016) In real life application, HPLC is used in pharmaceuticals and forensics.

Spectroscopic Methods

Spectroscopy provides qualitative and quantitative analysis. To identify unknown compounds, the sample is exposed to electro-magnetic radiation.

Infrared Spectroscopy (IR)

Infrared is the region of the electromagnetic spectrum that is light with a longer wavelength and lower frequency than visible light. In infrared spectroscopy, a beam of IR radiation is passed through a sample of a chemical, resulting in the IR radiation being absorbed by covalent bonds in the molecules, which increases their vibrational energy. When the frequency of the IR is the same as the vibrational frequency of a bond, absorption happens. Bonds between different atoms absorb different frequencies of IR radiation and bonds in different places of a molecule also absorb different frequencies. For example, the O-H bond in an alcohol absorbs different frequencies to an O-H bond in a carboxylic acid. The wavenumber is used to measure the frequency. (Libre Texts, 2019)

An IR spectrometer produces a graph to show the frequencies of radiation that a bond is absorbing, allowing you to identify functional groups in a molecule. Peaks on an IR spectra are shown upside down but it’s the peak that would show where the radiation is absorbed. The entire wavelength range can be measured using a Fourier transform instrument followed by generating an absorbance spectrum. (Braid, Clayton, Falkner, Faudemer, Henderson, Howe, Jordin, Kordan, Scott, Train, 2018)

Nuclear magnetic resonance (NMR)

NMR is a technique to provide information about a chemical structure of a compound by applying magnetic field to nuclei that behave as magnets because of its charge and spin. When a nucleus is placed in an NMR chamber and an external magnetic field is applied, the nucleus can orient itself with the magnetic field, resulting in a low energy state. If the nucleus orients itself to the opposite direction of the magnetic field, it is in a high energy state.

Some nuclei may be surround by electrons that shield the nuclei from the magnetic fields. These are called shielded nuclei and it causes them to not change their orientation due to diamagnetic shielding effect. However, nuclei not shielded from electrons are called deshielded electrons and they can change their orientation with expose to an external magnetic field.

However, when the shielded and deshielded nuclei are exposed to radiofrequency radiations, they can absorb energy from radiations and go into a high energy state. If both kinds of nuclei are in the same state, they are in resonance. However, deshielded nuclei need more energy to reach resonance and the shielded nuclei will need less energy to reach resonance. Therefore, nuclei in different environments need different amounts of energy to bring both into resonance. (A Level Chemistry, n.d.)

The NMR spectrum produced a signal showing the energy needed to bring the nuclei to resonance. In real life application, NMR is used in medical examinations of the human body to diagnose conditions. This is called an MRI scan and can provide detailed images of the body, including soft tissues.

Mass Spectroscopy (MS)

A mass spectrometer can give information about the relative atomic mass of an element and the relative abundance of its isotopes or the relative molecular mass.

There are four main things that happen when a sample is put into a time of flight mass spectrometer.

Firstly, the sample must be ionised before it enters the mass spectrometer by either electrospray ionisation, which involves dissolving the sample in a solvent and forcing it through a nozzle at a high pressure. A high voltage is then applied to the sample, resulting in each particle gaining a H+ ion. When the solvent is removed, a gas of positive ions is left. On the other hand, you can use electron impact ionisation, where the sample is vaporised, and high energy electrons are fired using a electron gun. One electron is knocked off each particle, resulting in +1 ions.

These positive ions are then accelerated by an electric field, which provides the same kinetic energy to all the ions. Now, the ions enter a space with no electric field so they drift through at the same speed they left the electric field. Lighter ions now drift at a faster speed. As the lighter ions travel at higher speeds, they reach the detector quicker than heavier ions, meaning the detector records how long each ion took to pass through the spectrometer. The data can then be used to work out mass and charge values needed to produce a mass spectrum. Braid, Clayton, Falkner, Faudemer, Henderson, Howe, Jordin, Kordan, Scott, Train, 2018)

Analysing a mass spectrum

A mass spectrum is the chart produced by a mass spectrometer. If the sample was an element, each line on the mass spectrum represents a different isotope of the element.

The Y-axis shows the abundance of ions as a percentage and the height of the peak for an element would represent the relative isotopic abundance. The X-axis show the mass/charge ratio, which is shown as m/z.

Appendices

Appendix A- Ratio of copper sulphate to water in each test tube during preparation.

Mole

1.0

0.8

0.6

0.4

0.2

0.0

Amount of copper sulphate

10cm3

8cm3

6cm3

4cm3

2cm3

0

Volume of water

0

2cm3

4cm3

6cm2

8cm3

10cm3

Appendix B- Graph of results, determining the unknown concentration of copper sulphate using the calibration curve

Appendix C- Photograph of thin layer chromatography investigation results

Appendix D- Rf values of amino acids

(El-Desoky. (n.d.) Amino acids mobility. (online) Available at: https://www.researchgate.net/figure/Mobility-of-amino-acids-in-terms-of-R-F-values-using-different-mobile-phases_tbl1_260231027 Accessed on: 17.06.2019)

References

1)     Braid, K. Clayton, E. Falkner, M. Faudemer, K. Henderson, G. Howe, E. Jordin, P. Kordan, R. Scott, S. Train, B. eds. (2018) A-Level Chemistry. Newcastle upon Tyne: CGP, pp 173

2)     Braid, K. Clayton, E. Falkner, M. Faudemer, K. Henderson, G. Howe, E. Jordin, P. Kordan, R. Scott, S. Train, B. eds. (2018) A-Level Chemistry. Newcastle upon Tyne: CGP, pp 386

3)     Braid, K. Clayton, E. Falkner, M. Faudemer, K. Henderson, G. Howe, E. Jordin, P. Kordan, R. Scott, S. Train, B. eds. (2018) A-Level Chemistry. Newcastle upon Tyne: CGP, pp 513

4)     Braid, K. Clayton, E. Falkner, M. Faudemer, K. Henderson, G. Howe, E. Jordin, P. Kordan, R. Scott, S. Train, B. eds. (2018) A-Level Chemistry. Newcastle upon Tyne: CGP, pp 511-512

5)     Clark, J. (2016) HPLC. (online) Available at: https://www.chemguide.co.uk/analysis/chromatography/hplc.html Accessed on: 18.06.2019

6)     Smith, C. (2018) Advantages and Disadvamtages of HPLC. (online) Available at: https://sciencing.com/disadvantages-advantages-hplc-5911530.html Accessed on 18.06.2019

7)     Libre Texts. (2019) Infared: Theory. (online) Available at: https://chem.libretexts.org/Bookshelves/Physical_and_Theoretical_Chemistry_Textbook_Maps/Supplemental_Modules_(Physical_and_Theoretical_Chemistry)/Spectroscopy/Vibrational_Spectroscopy/Infrared_Spectroscopy/Infrared%3A_Theory Accessed on: 18.06.2019

8)     Braid, K. Clayton, E. Falkner, M. Faudemer, K. Henderson, G. Howe, E. Jordin, P. Kordan, R. Scott, S. Train, B. eds. (2018) A-Level Chemistry. Newcastle upon Tyne: CGP, pp 256-257

9)     A Level Chemistry. (n.d.) NMR Spectroscopy. (online) Available at: https://alevelchemistry.co.uk/notes/nuclear-magnetic-resonance-spectroscopy/ Accessed on 19.06.2019

10) Braid, K. Clayton, E. Falkner, M. Faudemer, K. Henderson, G. Howe, E. Jordin, P. Kordan, R. Scott, S. Train, B. eds. (2018) A-Level Chemistry. Newcastle upon Tyne: CGP, pp 27