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Quantitative Spectrophotometry Lilley Tran,

Krystal Moua,

Isaac Pelayo,

Vannessa Oyco

February 25, 2015

Introduction The spectrophotometer is a vital machine for researchers, scientists, and biological and chemical engineers in studying various substances. Understanding the function and output results of spectrophotometry can be essential for future studies involving light absorbance of a particular wavelength and the concentration of a sample. This lab discusses the important relationship between material concentration and the intensity of light over a function of wavelength (λ). The natural concentration of a unique sample solute diluted in a solution can be measured and observed using spectrophotometric techniques, the Beer-Lambert law, quantitative and qualitative analysis via calorimetry, and a calibration curve. A photometer at the end of the spectrophotometric machine significantly detects the measured absorbance and transmittance of light which can be plotted on a calibration curve. The main goal of this experiment was to calculate the concentration a known solution and unknown solution and the molar extinction coefficients ( ϵ ). The values were obtained through multiple steps and equations. In order to calculate the absorbance (A), the equation was A=2−log (T ) . In the equation, T represents the transmittance, which is the ratio of light that

is transmitted through the spectrophotometer. The equation for transmittance (T) is

T=

I ∙ 100 . In the equation, Io

I represents the intensity of the light and

I o represents the

light intensity that is passed through the solution that is from absorption. Transmittance is

presented as a percentage and absorbance is unitless. The Beer-Lambert law is represented as an equation that shows the relationship between the absorbance and transmittance. The equation is expressed as

A=ε cl , where ε represents the molar absorptivity, c as the concentration of

the compound inside the solution, and l is the length of the light that is passed through the sample. These equations are used to calculate the data in this experiment to get the final results.

Materials and Methods The materials that were used in this experiment consisted of 100-1000 µL pipette, 20-200 µL pipette, a beaker with water, five sample tubes, seven cuvettes,small and large pipette tips, one blue standard solution, and an unknown solution #4. A spectrophotometer was also used to measure the absorbance (AU) and peak (nm) of the diluted blue standard solution samples and the unknown solution #4. Each member in the group was also required to wear gloves for protection.

1. With the given blue standard solution, five different percentages were chosen to serve as the five diluted samples for the experiment. The percentages were 1%, 5%, 10%, 15%, and 20% which had to be calculated. 2. The total 1 mL volume in each mixture was combined with different amounts of water and standard blue concentration to achieve the correct % concentration. Alternating between pipettes, standard blue solution had to be mixed with water in 5 different sample tubes. All pipettes were disposed in an appropriate waste container. 3. Next, the 100-1000 µL pipette was used to transfer each of the 5 diluted solutions from the sample tube into 5 cuvettes. Since the total diluted solution in the cuvette was 1 mL, it was necessary to add another 1 mL of water to create a 2 mL solution.

4. With a new pipette tip and clean cuvette, 2 mL of the unknown solution was transferred to the cuvette which needed to be tested to compare with the 5 blue diluted solutions. 5. All 6 cuvette samples were placed into the spectrophotometer to record the absorbance. 6. Prior to testing the 6 samples, a blank cuvette of water was used to zero the spectrophotometer machine and normalize the readings. In addition, each cuvette had to be wiped with a Kimwipe to eliminate the oil left by fingerprints which would interfere with the data results. 7. The order in which the cuvettes were placed into the spectrophotometer went from the least concentrated to most concentrated; The purpose of this order was to prevent contamination with the strongest solution. The computer was able to record the absorbance and peak for each sample, along with a graph, including the unknown solution. 8. Since the solutions used in this experiment were non-hazardous, the solutions were able to be poured down the drain followed by large amounts of running water. The methods that were used in this experiment did not cause any significant errors in any of the steps that were done. Each step was done carefully with precision by each member. The instructions were followed thoroughly from the beginning to the end of the experiment.

Results and Discussion

Figure 1. Absorbance of green and blue standard concentration with a linear regression trendline followed by a generated slope intercept form.

With the spectrophotometric machine, absorbance values were measured for each blue and green standard solution with a range of concentrations from 1 to 20 (Table 1, Table 2). A scatter plot of the absorbance values over a function of concentration was graphed to create a linear regression trendline as seen in Figure 1. From the trendline, a slope intercept form equation, y=mx+b, was generated. Both trendlines result in a positive slope (m) which revealed the absorbance increased as the concentration of blue or green increased. The blue concentration produced a slope intercept equation of y=0.0279x+0.0248 and y=0.0951x+0.0283 for the green. The slope is equal to the molar extinction coefficient ( ϵ ) from the Beer Lambert’s Law (equation 1). At a given

wavelength of 1 cm, Therefore the green standard had molar extinction coefficient of 0.0951, and 0.0279 for the blue standard.

Figure 2 Spectrophotometry measurement of blue standard concentrates and unknown 4 Figure.2 reveals unknown solution #4 peaked at 411nm where as the blue standard concentrations peaked around 408 and 409 nm. By observance of the different lowest peaks between the blue standard and green standard, the unknown #4 was found similarly in the peak of the green standard. To confirm the findings, the calculated molar extinction concentration for the green standard and unknown measured absorbance was contributed to the Beer-Lambert’s Law equation to solve for the actual concentration. As a result, the calculated concentration was determined as 6.55% green standard in water solution. The unknown solution concentration was found to be accurately similar to the absorbance and concentration of the green standard solution using the molar extinction coefficient ( ϵ ) and Beer-Lambert’s Law (equation 1). This information was quite significant in that our unknown #4 had peaks, absorbance, and concentration within green standard margin values.

The possible propagation of errors that may have contributed to any data inaccuracy could have been from pipetting, material mishandling, and human error. Despite common lab errors, the calculated unknown concentration was found best fitted on the graphed absorbance relative to the concentration of the green standard solution.

Conclusion In conclusion, the quality of these results could be improved. The experiment only called for a single run but using the data collected from other groups one can compare results. Using the Beer-Lambert law and a calibration curve, a graph with several group data was created. The equation created from this collection of data can be used to generate the theoretical values of which all groups can compare. Compared to the values in this experiment to the theoretical values the results show a percent error range between 6.23% - 34.0%. 34% error is quite significant. More data would be able to elaborate as to whether outliers were present and had significant effects in determining the theoretical values and average slope. One possible way to see improvements for this set of results as well as the entire class would have been simply to require many more trials to provide a much more accurate representation of what the curve actually is.

Appendix: Figures, Tables, and Equations Table 1. Green standard concentration and measured absorbance Concentration (%)

Absorbance (Au)

2

0.17647

4

0.36864

6

0.54194

8

0.67765

10

0.91849

12

1.1281

16

1.5057

Table 2. Blue standard concentration and measured absorbance Concentration (%))

Absorbance (Au)

1

0.034776

4

0.25659

5

0.13719

5

0.13766

8

0.24156

10

0.27688

10

0.32882

12

0.25039

15

0.39914

15

0.53301

16

0.46946

20

0.5756

20

0.61999

Table 3. Group 2 Raw Data Sample

Concentration

Standard

Water

Wavelength

(%)

Solution

(micro L)

(nm)

(micro L)

Absorbance

1

1

20

1980

408

.0034776

2

5

100

1900

408

.13766

3

10

200

1800

408

.32882

4

15

300

1700

408

.53301

5

20

400

1600

409

.61999

blank

0

0

2000

0

0

411

.62304

Unknown #4

A=ε cl

Beer Lambert’s Law

Equation 1

Where: T

= percent transmittance

I

= intensity of transmitted light

IO

= intensity of incident light

A=2−log(T )

Equation 2

Where: A

= absorbance

T

= transmittance

A=ϵ cl

Where: A

= absorbance

Equation 3

ϵ

= molar extinction coefficient of absorbing molecules

c

= concentration of solute

l = path length of light through sample

Appendix: Contributions ● Lilley Tran Wrote the introduction of this document and did the calculation portion. ● Krystal Moua Wrote results and discussion of this document and did calculation portion. ● Isaac Pelayo Wrote conclusion and proofread this document. ● Vannessa Oyco Wrote materials and methodology and proofread this document.

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Krystal Moua,

Isaac Pelayo,

Vannessa Oyco

February 25, 2015

Introduction The spectrophotometer is a vital machine for researchers, scientists, and biological and chemical engineers in studying various substances. Understanding the function and output results of spectrophotometry can be essential for future studies involving light absorbance of a particular wavelength and the concentration of a sample. This lab discusses the important relationship between material concentration and the intensity of light over a function of wavelength (λ). The natural concentration of a unique sample solute diluted in a solution can be measured and observed using spectrophotometric techniques, the Beer-Lambert law, quantitative and qualitative analysis via calorimetry, and a calibration curve. A photometer at the end of the spectrophotometric machine significantly detects the measured absorbance and transmittance of light which can be plotted on a calibration curve. The main goal of this experiment was to calculate the concentration a known solution and unknown solution and the molar extinction coefficients ( ϵ ). The values were obtained through multiple steps and equations. In order to calculate the absorbance (A), the equation was A=2−log (T ) . In the equation, T represents the transmittance, which is the ratio of light that

is transmitted through the spectrophotometer. The equation for transmittance (T) is

T=

I ∙ 100 . In the equation, Io

I represents the intensity of the light and

I o represents the

light intensity that is passed through the solution that is from absorption. Transmittance is

presented as a percentage and absorbance is unitless. The Beer-Lambert law is represented as an equation that shows the relationship between the absorbance and transmittance. The equation is expressed as

A=ε cl , where ε represents the molar absorptivity, c as the concentration of

the compound inside the solution, and l is the length of the light that is passed through the sample. These equations are used to calculate the data in this experiment to get the final results.

Materials and Methods The materials that were used in this experiment consisted of 100-1000 µL pipette, 20-200 µL pipette, a beaker with water, five sample tubes, seven cuvettes,small and large pipette tips, one blue standard solution, and an unknown solution #4. A spectrophotometer was also used to measure the absorbance (AU) and peak (nm) of the diluted blue standard solution samples and the unknown solution #4. Each member in the group was also required to wear gloves for protection.

1. With the given blue standard solution, five different percentages were chosen to serve as the five diluted samples for the experiment. The percentages were 1%, 5%, 10%, 15%, and 20% which had to be calculated. 2. The total 1 mL volume in each mixture was combined with different amounts of water and standard blue concentration to achieve the correct % concentration. Alternating between pipettes, standard blue solution had to be mixed with water in 5 different sample tubes. All pipettes were disposed in an appropriate waste container. 3. Next, the 100-1000 µL pipette was used to transfer each of the 5 diluted solutions from the sample tube into 5 cuvettes. Since the total diluted solution in the cuvette was 1 mL, it was necessary to add another 1 mL of water to create a 2 mL solution.

4. With a new pipette tip and clean cuvette, 2 mL of the unknown solution was transferred to the cuvette which needed to be tested to compare with the 5 blue diluted solutions. 5. All 6 cuvette samples were placed into the spectrophotometer to record the absorbance. 6. Prior to testing the 6 samples, a blank cuvette of water was used to zero the spectrophotometer machine and normalize the readings. In addition, each cuvette had to be wiped with a Kimwipe to eliminate the oil left by fingerprints which would interfere with the data results. 7. The order in which the cuvettes were placed into the spectrophotometer went from the least concentrated to most concentrated; The purpose of this order was to prevent contamination with the strongest solution. The computer was able to record the absorbance and peak for each sample, along with a graph, including the unknown solution. 8. Since the solutions used in this experiment were non-hazardous, the solutions were able to be poured down the drain followed by large amounts of running water. The methods that were used in this experiment did not cause any significant errors in any of the steps that were done. Each step was done carefully with precision by each member. The instructions were followed thoroughly from the beginning to the end of the experiment.

Results and Discussion

Figure 1. Absorbance of green and blue standard concentration with a linear regression trendline followed by a generated slope intercept form.

With the spectrophotometric machine, absorbance values were measured for each blue and green standard solution with a range of concentrations from 1 to 20 (Table 1, Table 2). A scatter plot of the absorbance values over a function of concentration was graphed to create a linear regression trendline as seen in Figure 1. From the trendline, a slope intercept form equation, y=mx+b, was generated. Both trendlines result in a positive slope (m) which revealed the absorbance increased as the concentration of blue or green increased. The blue concentration produced a slope intercept equation of y=0.0279x+0.0248 and y=0.0951x+0.0283 for the green. The slope is equal to the molar extinction coefficient ( ϵ ) from the Beer Lambert’s Law (equation 1). At a given

wavelength of 1 cm, Therefore the green standard had molar extinction coefficient of 0.0951, and 0.0279 for the blue standard.

Figure 2 Spectrophotometry measurement of blue standard concentrates and unknown 4 Figure.2 reveals unknown solution #4 peaked at 411nm where as the blue standard concentrations peaked around 408 and 409 nm. By observance of the different lowest peaks between the blue standard and green standard, the unknown #4 was found similarly in the peak of the green standard. To confirm the findings, the calculated molar extinction concentration for the green standard and unknown measured absorbance was contributed to the Beer-Lambert’s Law equation to solve for the actual concentration. As a result, the calculated concentration was determined as 6.55% green standard in water solution. The unknown solution concentration was found to be accurately similar to the absorbance and concentration of the green standard solution using the molar extinction coefficient ( ϵ ) and Beer-Lambert’s Law (equation 1). This information was quite significant in that our unknown #4 had peaks, absorbance, and concentration within green standard margin values.

The possible propagation of errors that may have contributed to any data inaccuracy could have been from pipetting, material mishandling, and human error. Despite common lab errors, the calculated unknown concentration was found best fitted on the graphed absorbance relative to the concentration of the green standard solution.

Conclusion In conclusion, the quality of these results could be improved. The experiment only called for a single run but using the data collected from other groups one can compare results. Using the Beer-Lambert law and a calibration curve, a graph with several group data was created. The equation created from this collection of data can be used to generate the theoretical values of which all groups can compare. Compared to the values in this experiment to the theoretical values the results show a percent error range between 6.23% - 34.0%. 34% error is quite significant. More data would be able to elaborate as to whether outliers were present and had significant effects in determining the theoretical values and average slope. One possible way to see improvements for this set of results as well as the entire class would have been simply to require many more trials to provide a much more accurate representation of what the curve actually is.

Appendix: Figures, Tables, and Equations Table 1. Green standard concentration and measured absorbance Concentration (%)

Absorbance (Au)

2

0.17647

4

0.36864

6

0.54194

8

0.67765

10

0.91849

12

1.1281

16

1.5057

Table 2. Blue standard concentration and measured absorbance Concentration (%))

Absorbance (Au)

1

0.034776

4

0.25659

5

0.13719

5

0.13766

8

0.24156

10

0.27688

10

0.32882

12

0.25039

15

0.39914

15

0.53301

16

0.46946

20

0.5756

20

0.61999

Table 3. Group 2 Raw Data Sample

Concentration

Standard

Water

Wavelength

(%)

Solution

(micro L)

(nm)

(micro L)

Absorbance

1

1

20

1980

408

.0034776

2

5

100

1900

408

.13766

3

10

200

1800

408

.32882

4

15

300

1700

408

.53301

5

20

400

1600

409

.61999

blank

0

0

2000

0

0

411

.62304

Unknown #4

A=ε cl

Beer Lambert’s Law

Equation 1

Where: T

= percent transmittance

I

= intensity of transmitted light

IO

= intensity of incident light

A=2−log(T )

Equation 2

Where: A

= absorbance

T

= transmittance

A=ϵ cl

Where: A

= absorbance

Equation 3

ϵ

= molar extinction coefficient of absorbing molecules

c

= concentration of solute

l = path length of light through sample

Appendix: Contributions ● Lilley Tran Wrote the introduction of this document and did the calculation portion. ● Krystal Moua Wrote results and discussion of this document and did calculation portion. ● Isaac Pelayo Wrote conclusion and proofread this document. ● Vannessa Oyco Wrote materials and methodology and proofread this document.

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