# Analysis and Application of Chemistry: Solutions and Spectroscopy

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8th Feb 2020 Chemistry Reference this

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Abstract

In analytical chemistry, precise preparation of solutions is key to determining concentration of any unknown compounds. This is particularly important in many areas such as chemistry, pharmaceuticals and in research laboratories where it is vital for known concentrations of chemicals to be produced and used. In this laboratory study, an unknown concentration of CuSO4.5H2O was examined using UV-Vis spectrophotometric method at 640nm to measure the absorbance if standard in a known concentration. This was used to determine the unknown by using the principles of Beer-Lambert theory (

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). The results show that the unknown equal to 0.5M solution, which was later prepared in larger amount by diluting the stock with the fresh solution prepared. Interestingly, this method was able to provide the exact concentration of the unknown and the technique was found to be rapid, precise and sensitive to determining concentrations. Thus, I suggest that concentrations of compounds that are soluble in water could be accurately determined by UV-Vis spectrophotometric techniques.

INTRODUCTION

The aim of analytical chemists is to identify and understand substances and how they behave in different conditions. There are diverse areas where an analytical chemist may work. Drug formulation and development, quality control, forensic analysis, and toxicology are only a few examples (PROSPECTS n.d.).

This study used ultraviolet and visible (UV-Vis) spectrophotometry to establish the concentration of a given CuSO4 solution and create more of the same concentration. Beer-Lambert’s law also plays an important role spectrophotometry as it relates absorbance directly to concentration. Therefore, it is important to understand the definition of concentration.

The concentration of a solution is the quantity of a solute contained in a quantity of solvent. There are many units for concentration, but the most common unit is molarity (M). Molarity is defined as the number of moles of solute present in 1L of solution.

This is important in chemistry as concentration determines how often molecules collide in solutions and therefore indirectly determines rates of reactions and conditions at equilibrium. In determining concentration, accurate measurement is fundamental and selection of the most appropriate measuring tools has to be taken into consideration to their degree if accuracy.

Spectroscopy is a technique that measures the interaction between molecules and electromagnetic radiation. The human eye is only sensitive to a small proportion of the whole electromagnetic spectrum, between 380 and 780nm and within this area, we can only perceive the colours of the rainbow from red through violet. UV-Vis spectroscopy uses ultraviolet and visible light in the wavelength range between 200 and 780nm (Roberts et al. 2018).

Molecules and atoms exist in several defined energy levels. To change energy levels, the absorption of a unit of energy (in this case, a photon) is required. Photons are used to promote electrons from the ground state to an excited state, as shown in Figure 1 below (Schmid 2001).

Figure 1. Excited states of photons (Wikipedia 2018)

It is known that light is a form of radiation, which is a form of energy. It can be measured by calculating the wavelength (λ) (Figure 2) and the frequency (v). These terms are related so that:

$c=\mathit{v\lambda }$

where c is the velocity of light in a vacuum.

Figure 2. Wavelength from crest to crest (Illustrated Glossary of Organic Chemistry n.d.)

The energy of a photon absorbed during a transition from one energy level to another is given by the equation:

$e=\mathit{hv}$

where h is known as the Planck’s constant and v is the frequency of the photon. Therefore, when these two equations are combined:

$E=\frac{\mathit{hc}}{\lambda }$

Hence, the shorter the wavelength, the greater the energy of the photon (Esfandiary and Middaugh 2012).

The strength of absorbance and the wavelength of absorption of any molecule in a substance not only depend on the chemical nature but it also depends on the molecular environment of its chromophores (Roberts et al. 2018, Schmid 2001).

METHODOLOGY

Part A. Determination of the concentration of a Copper (II) Ion Solution  Different dilutions of the stock solution of copper (II) sulfate was prepared in water from the dilution equation;

The concentrations were 1M, 0.5M, 0.25M, 0.1M and 0.05M. Absorbances of the solutions were taken at 640nm against a blank of deionised water in triplicate by using UV-Vis spectrophotometer. The wavelength of 640nm was identified to be the max absorbance (λmax) and a calibration curve was produced from the mean readings.

Part B. Preparation of a Copper (II) Ion solution from crystallised CuSO4.5H2O  The concentration of the unknown solution was calculated from the calibration curve’s equation;       $y=2.1606x$

to give 0.530mol/L. The mass of crystallised CuSO4.5H2O needed in 10mL of deionised water from 0.530mol/L was calculated to be 1.32g.

Part C. Preparation of a Copper (II) Ion solution by dilution of a stock CuSO4 solution  A 10mL copper (II) sulfate solution with the concentration of 0.530mol/L was prepared from the dilution equation. The volume required of the 1M stock solution was determined to be 5.3mL and 4.7mL of deionised water was added to obtain 10mL of the final concentration.

Table 2. Volumes of stock CuSO4 needed to make the final volume calculated by using the dilution equation.

 Concentration (mol/L) Mean Absorbance (nm) 1 2.156 0.5 1.088 0.25 0.543 0.1 0.218 0.05 0.106 0 0 Unknown 1.140

Mol/L = moles per litre       nm = nanometres

Table 1. Mean absorbances of CuSO4 (stock solution) in different concentrations in Part A

RESULTS

 Concentration (mol/L) CuSO4 (mL) dH2O (mL) 1 6 0 0.5 3 3 0.25 3 3 0.1 2.4 3.6 0.05 3 3

Mol/L = moles per litre   mL = millilitres  dH2O= deionised water

Part A of this study is based on the simple UV-VIS spectrophotometric scanning of copper (II) sulfate in deionised water solvent to determine its absorbances at different concentrations (Table 1). A calibration curve (Figure 3) was produced from these readings, which allowed for the concentration of the unknown CuSO4 in Part B to be calculated.

Figure 3. Mean Absorbances of Copper (II) Sulfate in Different Concentrations

After the concentration of CuSO4 (0.530M) was found in Part B, the mass of CuSO4.5H2O needed was calculated (1.32g) and a solution was prepared. While Part C was the preparation of a CuSO4 solution by the dilution of the stock solution given in Part A by using the dilution equation.

The dilutions of stock solution were prepared in deionised H2O by using the dilution equation from part A (table 2), with the final volume being 6mL.

At the lowest concentration of 0.05mol/L, the mean absorbance was 0.106nm. At 0.1mol/mL, the mean absorbance roughly doubled and gave the reading of 0.218nm (table 1). As the concentration doubled, the absorbances also doubled (figure 3). The calibration curve provided consistent linearity (R2) with equally high confidence at all the points along the graph (shown in yellow in figure 3).

The equation produced from the calibration curve (figure 1); $y=2.1606x$

was used to find the concentration of the unknown solution in part B, it was calculated to be 0.530mol/L and this concentration was used to produce a copper (II) sulfate solution in part C of the methodology.

DISCUSSION

Knowing the concentration of any substance is important in analytical chemistry. Over the years, UV-Vis spectrophotometry has evolved considerably from the 1930s.

UV-Vis spectrophotometry came about when scientists researching vitamins found that several vitamins, especially vitamin A, can absorb ultraviolet light. This boosted the American government’s curiosity in measuring vitamin content in their soldier’s rations and thus the research on UV-Vis culminated in the launch of UV-Vis spectrophotometers commercially in the 1940s (Buie 2011).

For this present study, UV-Vis spectroscopy was used to determine the concentration of the given CuSO4 solution and to produce more solution of that same concentration.

Preparation of solutions plays an important role in any analytical study. An accurate standard will give accurate results. Optimising serial dilutions and the machine being used to best fit the study will also contribute to reliable readings. In this study, the UV-Vis spectrophotometer was optimised to find the maximum absorbance within the range of the concentrations. By doing this, the machine gave concrete readings and resulted in the R2 = 1 (figure 3) and this gives an extreme confidence in the results.

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Deciding on which solvent to use is also important in producing accurate results in spectroscopy. In this study, deionised water was used as the solvent. The effect on the absorption spectrum of a compound, when diluted in a solvent, will differ depending on the chemical structures (Sancho et al. 2011). It is known that H2O is polar and CuSO4 is ionic in nature. Therefore, when copper (II) sulfate is dropped in water, it dissociates into positively charged copper (II) ions and negatively charged sulfate ions. This will result in a fully dissolved crystal which means that no CuSO4 was wasted and better readings will be detected.

The concentration of any substance in solution can be efficiently and accurately determined by absorbance measurements. So instead of plotting a calibration curve to find the concentration of the solution from part B, the Beer-Lambert’s law equation can be used. Absorbance (A) is related to the intensity of the initial light (I0) and light after (I) flow through the solution by equation [1]:

and the absorbance depends linearly on concentration, according to the Beer-Lambert law, equation [2]:

In equation [2], $l$

is the pathlength in cm, $c$

is the molar concentration and $\epsilon$

(L mol-1 cm-1) is the molar absorptivity coefficient. Therefore, the concentration of any substance in solution can be determined directly from its absorbance using equation [2] (Swinehart 1962).

The Beer-Lambert law relates to the calibration curve’s equation (figure 3) where y is the absorbance, a is the sum of the constant and pathlength and $x$

is the concentration. Therefore, this explains the results above (figure 3) as to why concentration is directly proportional to absorbance.

UV-Vis spectroscopy has many applications but there are two major measurement techniques, quantitative analysis, and qualitative analysis. However, quantitative analysis is preferred as other techniques will have to be coupled with UV-Vis for an accurate qualitative analysis. UV-Vis technique can be used for rate measurements which is a measurement of the change in concentration of a participant in the reaction as a function of time. A great example of this is the study for enzyme catalysis (Schmid 2001). The number of enzymes cannot be measured directly but their properties of catalysis allow their estimation from the speed of their reaction.

Another application is for analysis of mixtures. When there are several components which absorb radiation at the same wavelength, their absorbances add together and the absorbance of the sample will not be proportional to the concentration of one component. In this case, a chemical reaction is a common method to change the required component by adding a chemical reagent which reacts with it specifically to form a highly absorbing compound. This will avoid interference between compounds and the analysis is reduced to a simple case and the sensitivity is improved (Sawyer, Heineman and Beebe 1984).

Although UV-Vis spectroscopy has a lot of uses, it has its own limitations and the Beer-Lambert law is only true for low concentrations. For absorbances more than 2, the concentration will start to deviate from linearity (Bibby Scientific n.d., Schmid 2001).

CONCLUSION

UV-Vis spectrophotometry has advanced considerably from its origin in the 1930s. It is now a very accurate, rapid, simple, reliable, sensitive and economical technique to utilise for determining the concentration of a given substance. There are many new applications for UV-Vis spectrophotometry and as well as rate measurements of enzyme catalysis and analysis of mixtures, UV-Vis spectrophotometry can also be used to monitor processes in biological and food samples. The results are precise and is in agreement with the Beer-Lambert theory.

REFERENCES

Abstract

In analytical chemistry, precise preparation of solutions is key to determining concentration of any unknown compounds. This is particularly important in many areas such as chemistry, pharmaceuticals and in research laboratories where it is vital for known concentrations of chemicals to be produced and used. In this laboratory study, an unknown concentration of CuSO4.5H2O was examined using UV-Vis spectrophotometric method at 640nm to measure the absorbance if standard in a known concentration. This was used to determine the unknown by using the principles of Beer-Lambert theory (

). The results show that the unknown equal to 0.5M solution, which was later prepared in larger amount by diluting the stock with the fresh solution prepared. Interestingly, this method was able to provide the exact concentration of the unknown and the technique was found to be rapid, precise and sensitive to determining concentrations. Thus, I suggest that concentrations of compounds that are soluble in water could be accurately determined by UV-Vis spectrophotometric techniques.

INTRODUCTION

The aim of analytical chemists is to identify and understand substances and how they behave in different conditions. There are diverse areas where an analytical chemist may work. Drug formulation and development, quality control, forensic analysis, and toxicology are only a few examples (PROSPECTS n.d.).

This study used ultraviolet and visible (UV-Vis) spectrophotometry to establish the concentration of a given CuSO4 solution and create more of the same concentration. Beer-Lambert’s law also plays an important role spectrophotometry as it relates absorbance directly to concentration. Therefore, it is important to understand the definition of concentration.

The concentration of a solution is the quantity of a solute contained in a quantity of solvent. There are many units for concentration, but the most common unit is molarity (M). Molarity is defined as the number of moles of solute present in 1L of solution.

This is important in chemistry as concentration determines how often molecules collide in solutions and therefore indirectly determines rates of reactions and conditions at equilibrium. In determining concentration, accurate measurement is fundamental and selection of the most appropriate measuring tools has to be taken into consideration to their degree if accuracy.

Spectroscopy is a technique that measures the interaction between molecules and electromagnetic radiation. The human eye is only sensitive to a small proportion of the whole electromagnetic spectrum, between 380 and 780nm and within this area, we can only perceive the colours of the rainbow from red through violet. UV-Vis spectroscopy uses ultraviolet and visible light in the wavelength range between 200 and 780nm (Roberts et al. 2018).

Molecules and atoms exist in several defined energy levels. To change energy levels, the absorption of a unit of energy (in this case, a photon) is required. Photons are used to promote electrons from the ground state to an excited state, as shown in Figure 1 below (Schmid 2001).

Figure 1. Excited states of photons (Wikipedia 2018)

It is known that light is a form of radiation, which is a form of energy. It can be measured by calculating the wavelength (λ) (Figure 2) and the frequency (v). These terms are related so that:

$c=\mathit{v\lambda }$

where c is the velocity of light in a vacuum.

Figure 2. Wavelength from crest to crest (Illustrated Glossary of Organic Chemistry n.d.)

The energy of a photon absorbed during a transition from one energy level to another is given by the equation:

$e=\mathit{hv}$

where h is known as the Planck’s constant and v is the frequency of the photon. Therefore, when these two equations are combined:

$E=\frac{\mathit{hc}}{\lambda }$

Hence, the shorter the wavelength, the greater the energy of the photon (Esfandiary and Middaugh 2012).

The strength of absorbance and the wavelength of absorption of any molecule in a substance not only depend on the chemical nature but it also depends on the molecular environment of its chromophores (Roberts et al. 2018, Schmid 2001).

METHODOLOGY

Part A. Determination of the concentration of a Copper (II) Ion Solution  Different dilutions of the stock solution of copper (II) sulfate was prepared in water from the dilution equation;

The concentrations were 1M, 0.5M, 0.25M, 0.1M and 0.05M. Absorbances of the solutions were taken at 640nm against a blank of deionised water in triplicate by using UV-Vis spectrophotometer. The wavelength of 640nm was identified to be the max absorbance (λmax) and a calibration curve was produced from the mean readings.

Part B. Preparation of a Copper (II) Ion solution from crystallised CuSO4.5H2O  The concentration of the unknown solution was calculated from the calibration curve’s equation;

$y=2.1606x$

to give 0.530mol/L. The mass of crystallised CuSO4.5H2O needed in 10mL of deionised water from 0.530mol/L was calculated to be 1.32g.

Part C. Preparation of a Copper (II) Ion solution by dilution of a stock CuSO4 solution  A 10mL copper (II) sulfate solution with the concentration of 0.530mol/L was prepared from the dilution equation. The volume required of the 1M stock solution was determined to be 5.3mL and 4.7mL of deionised water was added to obtain 10mL of the final concentration.

Table 2. Volumes of stock CuSO4 needed to make the final volume calculated by using the dilution equation.

 Concentration (mol/L) Mean Absorbance (nm) 1 2.156 0.5 1.088 0.25 0.543 0.1 0.218 0.05 0.106 0 0 Unknown 1.140

Mol/L = moles per litre       nm = nanometres

Table 1. Mean absorbances of CuSO4 (stock solution) in different concentrations in Part A

RESULTS

 Concentration (mol/L) CuSO4 (mL) dH2O (mL) 1 6 0 0.5 3 3 0.25 3 3 0.1 2.4 3.6 0.05 3 3

Mol/L = moles per litre   mL = millilitres  dH2O= deionised water

Part A of this study is based on the simple UV-VIS spectrophotometric scanning of copper (II) sulfate in deionised water solvent to determine its absorbances at different concentrations (Table 1). A calibration curve (Figure 3) was produced from these readings, which allowed for the concentration of the unknown CuSO4 in Part B to be calculated.

Figure 3. Mean Absorbances of Copper (II) Sulfate in Different Concentrations

After the concentration of CuSO4 (0.530M) was found in Part B, the mass of CuSO4.5H2O needed was calculated (1.32g) and a solution was prepared. While Part C was the preparation of a CuSO4 solution by the dilution of the stock solution given in Part A by using the dilution equation.

The dilutions of stock solution were prepared in deionised H2O by using the dilution equation from part A (table 2), with the final volume being 6mL.

At the lowest concentration of 0.05mol/L, the mean absorbance was 0.106nm. At 0.1mol/mL, the mean absorbance roughly doubled and gave the reading of 0.218nm (table 1). As the concentration doubled, the absorbances also doubled (figure 3). The calibration curve provided consistent linearity (R2) with equally high confidence at all the points along the graph (shown in yellow in figure 3).

The equation produced from the calibration curve (figure 1);

$y=2.1606x$

was used to find the concentration of the unknown solution in part B, it was calculated to be 0.530mol/L and this concentration was used to produce a copper (II) sulfate solution in part C of the methodology.

DISCUSSION

Knowing the concentration of any substance is important in analytical chemistry. Over the years, UV-Vis spectrophotometry has evolved considerably from the 1930s.

UV-Vis spectrophotometry came about when scientists researching vitamins found that several vitamins, especially vitamin A, can absorb ultraviolet light. This boosted the American government’s curiosity in measuring vitamin content in their soldier’s rations and thus the research on UV-Vis culminated in the launch of UV-Vis spectrophotometers commercially in the 1940s (Buie 2011).

For this present study, UV-Vis spectroscopy was used to determine the concentration of the given CuSO4 solution and to produce more solution of that same concentration.

Preparation of solutions plays an important role in any analytical study. An accurate standard will give accurate results. Optimising serial dilutions and the machine being used to best fit the study will also contribute to reliable readings. In this study, the UV-Vis spectrophotometer was optimised to find the maximum absorbance within the range of the concentrations. By doing this, the machine gave concrete readings and resulted in the R2 = 1 (figure 3) and this gives an extreme confidence in the results.

Deciding on which solvent to use is also important in producing accurate results in spectroscopy. In this study, deionised water was used as the solvent. The effect on the absorption spectrum of a compound, when diluted in a solvent, will differ depending on the chemical structures (Sancho et al. 2011). It is known that H2O is polar and CuSO4 is ionic in nature. Therefore, when copper (II) sulfate is dropped in water, it dissociates into positively charged copper (II) ions and negatively charged sulfate ions. This will result in a fully dissolved crystal which means that no CuSO4 was wasted and better readings will be detected.

The concentration of any substance in solution can be efficiently and accurately determined by absorbance measurements. So instead of plotting a calibration curve to find the concentration of the solution from part B, the Beer-Lambert’s law equation can be used. Absorbance (A) is related to the intensity of the initial light (I0) and light after (I) flow through the solution by equation [1]:

and the absorbance depends linearly on concentration, according to the Beer-Lambert law, equation [2]:

In equation [2],

$l$

is the pathlength in cm,

$c$

is the molar concentration and

$\epsilon$

(L mol-1 cm-1) is the molar absorptivity coefficient. Therefore, the concentration of any substance in solution can be determined directly from its absorbance using equation [2] (Swinehart 1962).

The Beer-Lambert law relates to the calibration curve’s equation (figure 3) where y is the absorbance, a is the sum of the constant and pathlength and

$x$

is the concentration. Therefore, this explains the results above (figure 3) as to why concentration is directly proportional to absorbance.

UV-Vis spectroscopy has many applications but there are two major measurement techniques, quantitative analysis, and qualitative analysis. However, quantitative analysis is preferred as other techniques will have to be coupled with UV-Vis for an accurate qualitative analysis. UV-Vis technique can be used for rate measurements which is a measurement of the change in concentration of a participant in the reaction as a function of time. A great example of this is the study for enzyme catalysis (Schmid 2001). The number of enzymes cannot be measured directly but their properties of catalysis allow their estimation from the speed of their reaction.

Another application is for analysis of mixtures. When there are several components which absorb radiation at the same wavelength, their absorbances add together and the absorbance of the sample will not be proportional to the concentration of one component. In this case, a chemical reaction is a common method to change the required component by adding a chemical reagent which reacts with it specifically to form a highly absorbing compound. This will avoid interference between compounds and the analysis is reduced to a simple case and the sensitivity is improved (Sawyer, Heineman and Beebe 1984).

Although UV-Vis spectroscopy has a lot of uses, it has its own limitations and the Beer-Lambert law is only true for low concentrations. For absorbances more than 2, the concentration will start to deviate from linearity (Bibby Scientific n.d., Schmid 2001).

CONCLUSION

UV-Vis spectrophotometry has advanced considerably from its origin in the 1930s. It is now a very accurate, rapid, simple, reliable, sensitive and economical technique to utilise for determining the concentration of a given substance. There are many new applications for UV-Vis spectrophotometry and as well as rate measurements of enzyme catalysis and analysis of mixtures, UV-Vis spectrophotometry can also be used to monitor processes in biological and food samples. The results are precise and is in agreement with the Beer-Lambert theory.

REFERENCES

• Bibby Scientific (n.d.) JENWAY 7315 Operating Manual [online] available from <http://www.jenway.com/adminimages/Jenway_7310_7315_Spectro_Manual_Italian_LO.pdf> [10/04 2018]
• Buie, J. (2011) Evolution of UV-Vis Spectrophotometers
Learn the History of UV-Vis Spectrophotometers [online] available from <https://www.labmanager.com/lab-product/2011/07/evolution-of-uv-vis-spectrophotometers#.W7S83mhKjIU> [7/11 2018]
• Esfandiary, R. and Middaugh, C. R. (2012) ‘Ultraviolet Absorption Spectroscopy’. in Analysis of Aggregates and Particles in Protein Pharmaceuticals. ed. by Mahler, H. C., and Jiskoot, W. Canada: A JOHN WILEY & SONS, INC., PUBLICATION, 171-200
• Illustrated Glossary of Organic Chemistry (n.d.) Wavelength [online] available from <http://www.chem.ucla.edu/~harding/IGOC/W/wavelength.html> [10/05 2018]
• PROSPECTS (n.d.) Analytical Chemists [online] available from <https://www.prospects.ac.uk/job-profiles/analytical-chemist> [10/04 2018]
• Roberts, J., Power, A., Chapman, J., Chandra, S., and Cozzolino, D. (2018) ‘The use of UV-Vis Spectroscopy in Bioprocess and Fermentation Monitoring’ 4 (18), 1-2
• Sancho, M. I., Almandoz, M. C., Blanco, S. E., and Castro, E. A. (2011) ‘Spectroscopic Study of Solvent Effects on the Electronic Absorption Spectra of Flavone and 7-Hydroxyflavone in Neat and Binary Solvent Mixtures’ (12)
• Sawyer, D., Heineman, W., and Beebe, J. (1984) Chemistry Experiments for Instrumental Methods. 1st edn: John Wiley and Sons
• Schmid, F. X. (2001) ‘Biological Macromolecules: UV-Visible Spectrophotometry’. in Encylopedia of Life Sciences. ed. by AnonLondon, UK: Macmillan Publishers, 1-4
• Swinehart, D. F. (1962) ‘The Beer-Lambert Law’ 39 (7)
• Wikipedia (2018) Excited State [online] available from <https://en.wikipedia.org/wiki/Excited_state> [10/05 2018]

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