An Analysis of the Amount of β-carotene in Carrot Samples

By: Roman Hodson (rh28397)

TA: Maggie Weber

May 7, 2015

1

Abstract

This experiment used UV-Vis spectrophotometry to determine if the amount of β-

carotene in non-organic and organic carrot samples decreased after being left to sit for a week in

a dark laboratory cabinet due to rotting. The data was inconclusive however, because the samples

were not properly dried before experimentation meaning they contained added water weight, so

less carrot was weighed out than expected. In addition, the calculated β-carotene concentrations

were less than the expected value of 74.06 ppm, which is most likely due to loss of β-carotene

during the extraction procedure.1 However, the experiment was successful in determining that β-

carotene absorbs at 449 nm, as reported by Biswas et. al.1 Therefore, the results are inconclusive

as they were not able to confirm or deny the hypothesis.

Introduction/Background

This experiment is concerned with determining the amount of β-carotene in organic and

non-organic carrot samples using UV-Vis spectrophotometry. The molecular structure of β-

carotene is provided in Figure 1.

Figure 1. β-carotene Structure2

As shown in Figure 1, β-carotene is a large, non-polar organic compound, classified as a

carotenoid, or a phytochemical.3 Carotenoids are red, orange, or yellow fat-soluble compounds,

mainly found in plants and vegetables.3

The β-carotene in carrots is what gives carrots their

orange color. β-carotene is referred to as a phytochemical when being described as a compound

which affects human health.4 As a phytochemical, β-carotene has antioxidant properties, and

2

diets which contain β-carotene have been shown to reduce the risk of certain diseases, such as

certain cancers and diabetes.4 In addition to reducing the risk of varying diseases, β-carotene

allows humans to access vitamin A. The structure of vitamin A is shown in Figure 2.

Figure 2. Vitamin A Structure5

Vitamin A is vital for human health, as it has important roles in embryonic development,

immune system health, eye development, and vision.6 The National Institutes of Health

recommends 0.05 mcg RAE of β-carotene, and 900 mcg RAE of vitamin A for males and 700

mcg RAE of vitamin A for females per day.7 Since β-carotene and vitamin A work in tandem to

promote good health, their consumption is crucial. Therefore, it is important to let the public

know the amount of β-carotene they are consuming in their diet, so they can decide if they need

to adjust their eating habits.

There was a method developed, reported by Biswas et. al, which used UV-Vis

spectrophotometry to determine the amount of β-carotene in carrot samples.1 UV-Vis

spectrophotometry is based on the concept that molecules with pi-bonding or non-bonding

electrons can absorb light in the visible and ultraviolet (UV) spectrum, which promotes their

electrons to higher energy levels.8 The energy of a photon is described by Equation 1

E = hν = ℎ𝑐

𝜆 (1)

3

where E is the energy of the photon (J), h is Planck’s constant (J s), ν is the frequency of light

(Hz), c is the speed of light (m s-1

), and λ is the wavelength of light (nm).8 A transition from a

lower energy level to a higher energy level can only occur if the energy of the photon equals the

difference in energy between the two energy levels.8 In addition, since the frequency of light is

invariant in a vacuum and wavelength is inversely proportional to energy, a smaller energy gap

transition corresponds to a longer wavelength of light. Conversely, the larger the energy gap

between energy levels, the shorter the wavelength of light which is required for excitation.8

Figure 3 shows the process of absorption.

Figure 3. Absorption9

Absorbance can be used as a spectroscopic technique to determine the concentration of a

species, using Beer’s Law. Beer’s Law states that the absorbance of a species is proportional to

its concentration, shown in Equation 2

A = εlc (2)

4

where A is absorbance, ε is the molar extinction coefficient (M-1

cm-1

), l is the path length of the

cuvette (cm), and c is the concentration of the species (M-1

). In addition, Beer’s Law is based on

the idea that the particles are infinitely far apart and do not interact with one another.8 This

previous statement means that the solution which contains the absorbing species must be

adequately dilute for it to be modeled using Beer’s Law. An important aspect of Beer’s Law is

the molar extinction coefficient, which describes how well a species absorbs light. Even if a

species is dilute in solution, if it has a large molar extinction coefficient it still has the ability to

absorb a large amount of light. Absorbance is measured as a function of the intensity of light,

shown in Equation 3

A = -log(𝐼

𝐼𝑜) (3)

where Io is the initial intensity of light, and I is the intensity of light it passes through the sample.

The value of absorbance is important for UV-Vis spectrophotometry, as it determines whether or

not the relationship between concentration and absorbance is linear. For example, if the

absorbance has a value of 1, 90% of the incoming light is being absorbed by the sample. This

high absorbance causes deviation from linearity, as the sample approaches optical saturation at

absorbance values greater than 1.10

Optical saturation is a phenomenon where there are equal

populations of molecules in the ground and excited states.10

As the absorbing species approaches

optical saturation, it can no longer absorb light in a linear fashion. Therefore, the amount of light

the species absorbs decreases with increasing absorbance values. A representative graph of

Beer’s Law and its deviations from linearity is provided in Figure 4.

5

Figure 4. Beer’s Law Representative Graph11

If the absorbance is too high, UV-Vis spectrophotometry cannot accurately determine the

concentration of a species in solution.

As stated previously, UV-Vis spectrophotometry uses absorbance to determine the

concentration of a species in solution. A diagram of a UV-Vis spectrophotometer is provided in

Figure 5.

Figure 5. UV-Vis Spectrophotometer12

As shown in Figure 5, the initial intensity of light decreases as it passes through the sample. An

absorption spectrum is made by comparing a known initial intensity of light to the intensity of

light after it passes through a sample.13

In addition, the UV-Vis spectrophotometer uses a range

6

of wavelengths to create an absorbance spectrum. This range of wavelengths corresponds to

varying photon energies, and therefore varying excited states, shown in Figure 6.

Figure 6. Absorption and Differing Excited States14

Using the knowledge that certain transitions only occur with the correct amount of energy, a

sample is exposed to a range of wavelengths. Allowing the sample to absorb various

wavelengths allows for the determination of what wavelengths cause excitation. Figure 7

provides an example of an absorbance spectrum.

7

Figure 7. Absorbance Spectrum9

Looking at Figure 7, it is evident that a species absorbs over a range of wavelengths rather than

just one wavelength. This range of absorbance, rather than just one absorbance peak, is due to

solvation energetics. In solution, the solute particles are surrounded by solvent particles which

help to dissolve the solute.15

However, not all of the solvent particles are arranged the same way

across the solution, which leads to a distribution of energy between the solute particles.15

Solvation energetics is demonstrated in Figure 8, with Figure 9 comparing a spectrum with and

without solvation energetics.

8

Figure 8. Solvation Energetics15

Figure 9. Absorption Spectrum with and without Solvation Energetics15

As shown in Figure 9, an absorbance spectrum without solvation energetics has sharp peaks

where the energy level transitions occur, rather than broad peaks. Solvation energetics can be

avoided if the sample is in the gas phase.15

However, in this experiment the sample is in the

liquid phase.

As stated previously, this experiment used UV-Vis spectrophotometry to determine the

amount of β-carotene in non-organic and carrot samples. The purpose of this experiment was to

determine whether the non-organic carrots or organic carrots contained the most β-carotene. In

9

addition, this experiment investigated if the amount of β-carotene in the non-organic and organic

carrot samples decreased due to rotting after being left to sit for a week in a dark laboratory

cabinet. It is expected that the amount of β-carotene will decrease after the samples are left to sit

out for a week in the dark, due to the carrot rotting.

Experimental

This experiment consisted of an extraction process, the creation of standard addition

solutions, as well as a spike recovery in order to determine the amount of β-carotene in non-

organic and organic carrot samples. A study by Biswas et. al reported that acetone works as an

extracting solvent for β-carotene.1 However, during the first three weeks of experimentation,

acetone proved to be an inadequate extracting solvent, as the solution became cloudy during the

dilution steps in the creation of standard additions solutions. Using acetone as a solvent most

likely extracted other compounds, such as various lipids, inherent in the carrot besides the β-

carotene. This extraction of additional compounds caused the solution to become cloudy upon

further addition of acetone. Therefore, hexanes was implemented as an extracting solvent, as

suggested in a study by Taungbodhitham et. al, which proved to be effective.16

In preparing for the extraction process, samples of non-organic and organic carrots were

finely ground using a blender the night before experimentation. During experimentation, three 12

g samples of non-organic and organic carrots were placed into 50 mL centrifuge tubes. Then,

these centrifuge tubes had 15 mL of hexanes added to them, and were centrifuged for 5 minutes

at 4000 revolutions/minute. While these samples were centrifuging, a 5 mg/ 50 mL stock

solution of β-carotene in hexanes was created. After centrifuging, the extracted samples were

10

pooled into two different beakers, denoting if they were non-organic or organic, and were then

filtered 4 times using vacuum filtration.

After the filtration steps, standard addition solutions were created. During this part of the

experiment, 4 standard solutions of 25x dilution were made in triplicate for each of the carrot

types, resulting in a 24 total standard addition solutions. The standard addition solutions were

created using 1.6, 0.8, 0.4, and 0.2 ppm additions of the stock β-carotene solution. After creating

the standard addition solutions, the samples were run through the UV-Vis spectrophotometer

over a wavelength range of 300-600 nm, using hexanes as a blank. The same procedure was

carried out for the week old carrot samples.

The next part of the experimental procedure consisted of spike recoveries in order to

determine the amount of β-carotene lost during the extraction step. For this procedure, 12 g of

non-organic and organic carrot samples had 1.6 ppm β-carotene added to them before

undergoing the extraction process described previously. After undergoing the same extraction,

filtration, and 25x dilution process, the spike recovery samples were run through the UV-Vis at a

wavelength range of 300-600 nm using hexanes as a blank.

Results and Discussion

This section does not include all of the data obtained during experimentation, but instead

contains representative Tables and Figures. Supplementary Tables and Figures are provided in

the appendix. In the context of this report, the word “fresh” indicates that the samples were

prepared the night before experimentation, and word “week old” indicates the samples that were

left to sit for a week in a dark laboratory cabinet. Figures 10-13 provide representative

absorbance spectra and standard additions curves for the fresh non-organic and organic samples.

11

In addition, Table 1 provides the statistical data obtained from the standard additions curves for

the fresh samples, with Table 2 providing the data in terms of weight percent.

Figure 10. Organic Sample A Absorbance Spectrum

Figure 11. Organic Sample A Standard Additions Curve

-0.05

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

400 420 440 460 480 500

Ab

sorb

ance

Wavelength (nm)

Organic Sample A

1.6 ppm

0.8 ppm

0.4 ppm

0.2 ppm

449 nm

y = 0.1768x + 0.0618 R² = 0.999

-0.2

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

-1 -0.5 0 0.5 1 1.5 2 2.5 3

Ab

sorb

ance

Concentration (ppm)

Organic Standard Addition A

12

Figure 12. Non-Organic Sample C Absorbance Spectrum

Figure 13. Non-Organic Sample C Standard Additions Curve

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

400 420 440 460 480 500

Ab

sorb

ance

Wavelength (nm)

Non-Organic Sample C

1.6 ppm

0.8 ppm

0.4 ppm

0.2 ppm

449 nm

y = 0.1881x + 0.0816 R² = 0.9998

-0.2

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

-1 -0.5 0 0.5 1 1.5 2 2.5 3

Ab

sorb

ance

Concentration (ppm)

Non-Organic Standard Additions C

13

Table 1. β-carotene Concentrations of Fresh Carrot Samples

Sample Average Conc

(ppm)

Standard

Deviation

%RSD Confidence

Interval

(ppm)

Non-Organic 10.45 0.54 5.15 10.45 ± 0.91

Organic 8.33 1.39 16.66 8.33 ± 2.34

Table 2. β-carotene Weight Percent Data of Fresh Carrot Samples

Sample Average Mass

(mg)

Standard

Deviation

%RSD Confidence

Interval (mg)

Weight

Percent (%)

Non-Organic 0.157 8 x 10-3

5 0.157 ± 0.014 1.31 x 10-3

Organic 0.125 2.1 x 10-2

17 0.125 ± 0.036 1.04 x 10-3

Looking at the data from Tables 1 and 2, the average concentration of β-carotene in the

non-organic samples is 14.1% of the expected concentration of 74.06 ppm, and the average

concentration of β-carotene in the organic samples is 11.2% of the expected concentration. This

difference in concentrations is most likely due to the loss of β-carotene during the extraction

steps. In reality, the average masses should be higher for the carrot samples, and therefore the

weight percent of β-carotene in the carrot samples should be greater than the values calculated.

However, looking at Figures 10 and 12, the sample absorbed at or near 449 nm, which was the

wavelength at which β-carotene absorbs, as reported by Biswas et. al.1

The following data is representative of the week old non-organic and organic carrot

samples. Supplementary Tables and Figures are provided in the appendix. Figures 14-17 show

the absorbance spectra and standard additions curves for the week old non-organic and organic

samples. In addition, Table 3 provides the statistical data obtained from the standard additions

curves for the non-organic and organic carrot samples, with Table 4 providing the data in terms

14

of weight percent. In addition, Figures 18 and 19 show the absorbance spectra of the spike

recovery samples.

Figure 14. Week Old Non-Organic Sample B Absorbance Spectrum

Figure 15. Week Old Non-Organic Sample B Standard Additions Curve

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

400 420 440 460 480 500

Ab

sorb

ance

Wavelength (nm)

Week Old Non-Organic Sample B 449 nm

y = 0.1186x + 0.1709 R² = 0.9768

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

-3 -2 -1 0 1 2 3

Ab

sorb

ance

Concentration (ppm)

Week Old Non-Organic B Standard Additions

15

Figure 16. Week Old Organic Sample A Absorbance Spectrum

Figure 17. Week Old Organic Sample A Standard Additions Curve

0

0.05

0.1

0.15

0.2

0.25

0.3

400 420 440 460 480 500

Ab

sorb

ance

Wavelength (nm)

Week Old Organic Sample A

1.6 ppm

0.8 ppm

0.4 ppm

0.2 ppm

449 nm

y = 0.1213x + 0.0589 R² = 0.9916

-0.2

-0.1

0

0.1

0.2

0.3

0.4

0.5

-3 -2 -1 0 1 2 3

Ab

sorb

ance

Concentration (ppm)

Week Old Organic A Standard Additions

16

Table 3. β-carotene Concentrations of Week Old Carrot Samples

Sample Average Conc

(ppm)

Standard

Deviation

%RSD Confidence

Interval

(ppm)

Non-Organic 21.32 13.57 63.65 21.32 ± 22.88

Organic 12.27 0.15 1.20 12.27 ± 0.25

Table 4. β-carotene Weight Percent Data of Week Old Carrot Samples

Sample Average Mass

(mg)

Standard

Deviation

%RSD Confidence

Interval (mg)

Weight

Percent (%)

Non-Organic 0.319 0.204 64 0.319 ± 0.343 2.7 x 10-2

Organic 0.184 2 x 10-3

1 0.184 ± 0.004 1.5 x 10-2

Looking at the data in Tables 3 and 4, it is apparent that the amount of β-carotene in the

carrot samples seem to have increased by sitting in the dark for a week. However, this previous

statement is most likely not the case. It is most likely that the apparent increase in β-carotene

concentration is due to water’s evaporating during the samples’ time in the laboratory cabinet.

During the week with the fresh samples, there would be added water weight to the samples

because the water would not have evaporated yet, and during the weighing process less carrot

would be weighed out compared to a sample with its water evaporated. In addition, the data from

the non-organic sample is not statistically reliable as it has a large standard deviation, and as

indicated by Table 4, an impossible confidence interval. This inconclusive data is most likely

caused by the results gathered from Figure 31 in the appendix, as the spectrum is not reliable.

However, this data does show that the β-carotene does absorb at a wavelength at or near 449 nm.

In addition, the results show that results closer to the expected concentration can be achieved if

17

the samples are dried before experimentation, because the dry samples do not carry the extra

water weight.

In addition to the standard addition solutions, spike recoveries were performed in order to

determine the amount of β-carotene lost during extraction. Figures 18 and 19 provide the spike

recovery spectra, and Table 5 provides the spike recovery concentration data. Also, Figure 20

provides the hexanes blank spectrum.

Figure 18. Non-Organic Spike Recovery Spectrum

-0.005

0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

0.04

400 420 440 460 480 500

Ab

sorb

ance

Wavelength (nm)

Non-Organic Spike Recovery

449 nm

18

Figure 19. Organic Spike Recovery Spectrum

Figure 20. Hexanes Blank Spectrum

-0.01

0

0.01

0.02

0.03

0.04

0.05

0.06

400 420 440 460 480 500

Ab

sorb

ance

Wavelength (nm)

Organic Spike Recovery

449 nm

-0.0004

-0.0002

0

0.0002

0.0004

0.0006

0.0008

0.001

0.0012

400 420 440 460 480 500

Ab

sorb

ance

Wavelength (nm)

Hexanes Blank Spectrum

19

Table 5. Spike Recovery Data

Sample Spike Concentration (ppm) Extraction Efficiency (%)

Non-Organic 45.0 24.1%

Organic 50.5 18.8%

In addition to determining the extraction efficiency, the limit of detection was determined to be 3

x 10-3

ppm, which was calculated in part using the blank absorbance spectrum provided in Figure

20. Looking at the data from Figures 18 and 19, the results show that β-carotene absorbs at 449

nm. However, the results from the spike recoveries are not conclusive, as the absorbance values

do not fall within the range of 0.1-1.0, as this previously stated range is optimal for determining

concentrations.10

In addition, these spike recoveries were performed with the fresh samples, and

therefore had added water weight to them, meaning that less carrot was weighed out than what

was recorded. These previously stated factors render the data from the spike recoveries

unreliable.

Conclusion

The data from this experiment is not conclusive as it is unreliable and could not be used

to confirm or deny the hypothesis. By not drying the fresh samples before experimentation, the

samples were incorrectly weighed, and therefore less β-carotene was extracted than planned,

which led to lesser β-carotene concentrations than the results from the week old samples. If the

samples were to have been dried before experimentation, the results possibly could have been

used to determine if the amount of β-carotene decreases over time. In addition, neither the fresh

nor the week old sample β-carotene concentrations were close to the expected value of 74.06

ppm, which is most likely due to loss of β-carotene during the extraction procedure. In addition,

the spike recovery data is not reliable either as it came from the fresh samples which had the

20

added water weight. However, this experiment was successful in determining that β-carotene

absorbs at a wavelength at or near 449 nm, as expected from the report by Biswas et. al.1

In

conclusion, the results from the experiment are inconclusive, and better data could have been

obtained by drying out the samples before experimentation.

References

1. Biswas, A.K., Sahoo, J., & Chatli, M.K. (2011). A simple UV-Vis spectrophotometric method

for determination of β-carotene content in raw carrot, sweet potato, and supplemented chicken

meat nuggets. Food Science and Technology, 44, 1809-1813.

2. Chemical Book. (n.d.). Beta-Carotene. Retrieved April 18, 2015, from

http://www.chemicalbook.com/ChemicalProductProperty_EN_cb4148267.htm

3. MAYO Clinic. (n.d.). Beta-Carotene. Retrieved April 18, 2015, from

http://www.mayoclinic.org/drugs-supplements/beta-carotene/background/hrb-20058836

4. American Cancer Society. (n.d.). Phytochemicals. Retrieved April 18, 2015, from

http://www.cancer.org/treatment/treatmentsandsideeffects/complementaryandalternativemedicin

e/herbsvitaminsandminerals/phytochemicals

5. Vision Training. (n.d.). Vitamin A. Retrieved April 18, 2015, from http://www.vision-

training.com/en/eNewsletter/Australia/Winter%2008/Dry%20eyes/Vitamin%20A.html

6. Oregon State University. (n.d.). Vitamin A. Retrieved April 18, 2015, from

http://lpi.oregonstate.edu/mic/vitamins/vitamin-A

7. The National Institutes of Health. (n.d.). Vitamin A Fact Sheet. Retrieved April 18, 2015, from

http://ods.od.nih.gov/factsheets/VitaminA-HealthProfessional/

8. Harris, Daniel C., Quantitative Chemical Analysis, 8th

Ed. W.H. Freeman and Company:

New York, 2010, pp. 394-400, 404.

9. UC Davis. (n.d.). Overview of Spectroscopy. Retrieved April 18, 2015, from

http://chemwiki.ucdavis.edu/Analytical_Chemistry/Analytical_Chemistry_2.0/10_Spectroscopic

_Methods/10A%3A_Overview_of_Spectroscopy

10. O'Haver, T. (n.d.). Instrumental Deviation from Beer's Law. Retrieved April 18, 2015, 2015,

from http://terpconnect.umd.edu/~toh/models/BeersLaw.html

21

11. SenseAir. (n.d.). Beer's Law [Image] Retrieved April 18, 2015, 2015 .

http://www.senseair.se/senseair/ research-development/technology/lambert-%E2%80%93-beers-

law/

12. Bio 750. (n.d.). Spectroscopy - UV-Vis [Image] Retrieved April 18, 2015, 2015 . Retrieved

from http://www.sci.sdsu.edu/TFrey/Bio750/ UV-VisSpectroscopy.html

13. UT Dept. of Chemistry (2015). CH376K Fluorimetry: Determination of Quinine in Tonic

Water & Urine.

14. UCLA. (n.d.). Infrared Spectroscopy. Retrieved April 18, 2015, from

http://www.wag.caltech.edu/home/jang/genchem/infrared.htm

15. Shear, J. (n.d.). Solvation Energetics. Retrieved April 18, 2015, from

https://utexas.instructure.com/courses/1128486/files/folder/Course%2520Notes?preview=35842

117

16. Taungbodhitham, A. K., Jones, G. P., Wahlqvist, M. L., & Briggs, D. R. (1998). Evaluation

of Extraction Method for the Analysis of Carotenoids in Fruits and Vegetables. Food Chemistry,

63(4), 577-584.

22

Appendix

1. Calculating concentration of β-carotene concentration organic sample A

Linear Fit: y = 0.1768x + 0.0618

x-intercept = 0.0618

−0.1768 = 0.349 ppm

Multiply by dilution factor: 0.349 ppm x 25 = 8.74 ppm

2. Calculating β-carotene average concentration of organic samples

Average concentration = 8.74+6.79+9.47

3 = 8.33 ppm

3. Calculating β-carotene standard deviation of organic samples

Standard deviation = √(8.74−8.33)2+ (6.79−8.33)2+ (9.47−8.33)2

2 = 1.39

4. Calculating %RSD of organic samples

%RSD = 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝑑𝑒𝑣𝑖𝑎𝑡𝑖𝑜𝑛

𝑎𝑣𝑒𝑟𝑎𝑔𝑒 × 100 =

1.39

8.33 × 100 = 16.66

5. Calculating confidence interval of organic samples

Confidence interval = average ± t𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝑑𝑒𝑣𝑖𝑎𝑡𝑖𝑜𝑛

√𝑛

Confidence interval = 8.33 ± (2.92)1.39

√3 = 8.33 ± 2.34 ppm

6. Spike Recovery Calculations (Non-Organic)

𝑝𝑝𝑚 𝛽 − 𝑐𝑎𝑟𝑜𝑡𝑒𝑛𝑒 𝑖𝑛 5 𝑚𝐿 𝑢𝑛𝑘𝑛𝑜𝑤𝑛 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛

𝑝𝑝𝑚 𝛽 − 𝑐𝑎𝑟𝑜𝑡𝑒𝑛𝑒 𝑖𝑛 5 𝑚𝐿 𝑠𝑝𝑖𝑘𝑒𝑑 𝑢𝑛𝑘𝑛𝑜𝑤𝑛 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛=

𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑝𝑒𝑎𝑘 𝑜𝑓 𝑢𝑛𝑘𝑛𝑜𝑤𝑛

𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑝𝑒𝑎𝑘 𝑜𝑓 𝑠𝑝𝑖𝑘𝑒𝑑 𝑢𝑛𝑘𝑛𝑜𝑤𝑛

Β-carotene spike: 1.6 ppm

Absorbance peak of unknown = 0.309

Absorbance peak of spiked unknown = 0.0347

𝑥

𝑥 + 1.6 𝑝𝑝𝑚=

0.309

0.0347

Solve for x

x = 1.8 ppm

multiply by dilution factor of 25

(1.8 ppm)(25) = 45.0 ppm

23

7. Calculating limit of detection (used slope of Non-Organic Samples Standard Additions Curve)

Standard deviation = 1.91 x 10-4

LOD = 3 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝑑𝑒𝑣𝑖𝑎𝑡𝑖𝑜𝑛

𝑠𝑙𝑜𝑝𝑒

Slope = 0.1771

LOD = 3 x 10-3

ppm

8. Calculating Weight Percent (Used Non-Organic Fresh Data)

Concentrations = 10.6, 9.84, and 10.8 ppm

Convert to mg

Mass = 10.6 ppm x (15 mL/1000) = 0.160 mg

Repeat for all concentrations

Find average mass

Average mass = 0.160 𝑚𝑔+0.147 𝑚𝑔+0.163 𝑚𝑔

3= 0.157 𝑚𝑔

Weight Percent = 0.157 𝑚𝑔

1200 𝑚𝑔 𝑐𝑎𝑟𝑟𝑜𝑡 x 100% = 1.31 x 10

-3 %

8. Supplementary Figures 21-36

Figure 21. Organic Sample B Absorbance Spectrum

-0.05

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

400 420 440 460 480 500

Ab

sorb

ance

Wavelength (nm)

Organic Sample B

1.6 ppm

0.8 ppm

0.4 ppm

0.2 ppm

449 nm

24

Figure 22. Organic Sample B Standard Additions Curve

Figure 23. Organic Sample C Absorbance Spectrum

y = 0.2111x + 0.0573 R² = 0.9999

-0.2

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

-1 -0.5 0 0.5 1 1.5 2 2.5 3

Ab

sorb

ance

Concentration (ppm)

Organic Standard Additions B

-0.05

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

400 420 440 460 480 500

Ab

sorb

ance

Wavelength

Organic Sample C

1.6 ppm

0.8 ppm

0.4 ppm

0.2 ppm

449 nm

25

Figure 24. Organic Sample C Standard Additions Curve

Figure 25. Non-Organic Sample B Absorbance Spectrum

y = 0.1972x + 0.0747 R² = 0.9893

-0.2

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

-1 -0.5 0 0.5 1 1.5 2 2.5 3

Ab

sorb

ance

Concentration (ppm)

Organic Standard Additions C

-0.05

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

400 420 440 460 480 500

Ab

sorb

ance

Wavelength (nm)

Non-Organic Sample B

1.6 ppm

0.8 ppm

0.4 ppm

0.2 ppm

449

26

Figure 26. Non-Organic Sample B Standard Additions Curve

Figure 27. Non-Organic Sample C Absorbance Spectrum

y = 0.1926x + 0.0758 R² = 0.9864

-0.2

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

-1 -0.5 0 0.5 1 1.5 2 2.5 3

Ab

sorb

ance

Concentration (ppm)

Non-Organic Standard Additions B

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

400 420 440 460 480 500

Ab

sorb

ance

Wavelength (nm)

Non-Organic Sample C

1.6 ppm

0.8 ppm

0.4 ppm

0.2 ppm

27

Figure 28. Non-Organic Sample C Standard Additions Curve

Figure 29. Week Old Non-Organic Sample A Absorbance Spectrum

y = 0.1881x + 0.0816 R² = 0.9998

-0.2

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

-1 -0.5 0 0.5 1 1.5 2 2.5 3

Ab

sorb

ance

Concentration (ppm)

Non-Organic Standard Additions C

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

400 420 440 460 480 500

Ab

sorb

ance

Wavelength (nm)

Week Old Non-Organic Sample A 449 nm

28

Figure 30. Week Old Non-Organic Sample A Standard Additions Curve

Figure 31. Week Old Non-Organic Sample C Absorbance Spectrum

y = 0.1298x + 0.0969 R² = 0.9893

-0.1

0

0.1

0.2

0.3

0.4

0.5

-2 -1 0 1 2 3

Ab

sorb

ance

Concentration (ppm)

Week Old Non-Organic A Standard Additions

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

400 420 440 460 480 500

Ab

sorb

ance

Wavelength (nm)

Week Old Non-Organic Sample C

1.6 ppm

0.8 ppm

0.4 ppm

0.2 ppm

449nm

29

Figure 32. Week Old Non-Organic Sample C Standard Additions Curve

Figure 33. Week Old Organic Sample B Absorbance Spectrum

y = 0.3881x + 0.144 R² = 0.7469

-0.8

-0.6

-0.4

-0.2

0

0.2

0.4

0.6

0.8

1

-2 -1.5 -1 -0.5 0 0.5 1 1.5 2Ab

sorb

ance

Concentration (ppm)

Week Old Non-Organic C Standard Additions

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

400 420 440 460 480 500

Ab

sorb

ance

Wavelength (nm)

Week Old Organic Sample B

1.6 ppm

0.8 ppm

0.4 ppm

0.2 ppm

449 nm

30

Figure 34. Week Old Organic Sample B Standard Additions Curve

Figure 35. Week Old Organic Sample C Absorbance Spectrum

y = 0.1396x + 0.0694 R² = 0.9906

-0.3

-0.2

-0.1

0

0.1

0.2

0.3

0.4

0.5

-3 -2 -1 0 1 2 3

Ab

sorb

ance

Concentration (ppm)

Week Old Organic B Standard Additions

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

400 420 440 460 480 500

Ab

sorb

ance

Wavelength (nm)

Week Old Organic C Sample

1.6 ppm

0.8 ppm

0.4 ppm

0.2 ppm

449 nm

31

Figure 36. Week Old Organic Sample C Standard Additions Curve

y = 0.1443x + 0.0706 R² = 0.9979

-0.3

-0.2

-0.1

0

0.1

0.2

0.3

0.4

0.5

-3 -2 -1 0 1 2 3

Ab

sorb

ance

Concentration (ppm)

Week Old Organic C Standard Additions