Adam Borton
Student #7695780
Lab B04, Rotation 1, Room 216, Locker #867
Experiment 3: Spectroscopic Analysis of Spinach
Experiment Date: 8:30 AM Oct. 8th, 2014
Submission Date: October 13th, 2014
Purpose Statement:
The purpose of this lab was to measure the absorption spectrum of spinach extract using a
spectrophotometer. The second half of the lab consisted of determining the concentration of iron
(III) in various solutions and the concentration of iron within spinach. Based on the readings in
the introduction to the lab, I am going to predict that spinach extract will fall somewhere within
the 400-500 nm range for peak absorbency. This is based on the graphs of the 3 compounds
(chlorophyll a and b, and beta-carotene) which all exhibit peaks within this range of wavelengths
(1).
Results:
Spectrum of Spinach Extract
3.5
3
Absorbance
2.5
2
1.5
1
0.5
0
400
450
500
550
Wavelength (nm)
600
650
700
Figure 1
Table 1: Concentration of Fe (III) and corresponding wavelength
Solution:
Concentration of Fe (III) (mol/L)
Absorption
1 mL FeCl3 : 9 mL HCl
0.00005
0.247
2 mL FeCl3 : 8 mL HCl
0.00010
0.359
3 mL FeCl3 : 7 mL HCl
0.00015
0.556
4 mL FeCl3 : 6 mL HCl
0.00020
1.009
5 mL FeCl3 : 5 mL HCl
0.00025
1.145
Correlation between Concentration of Fe (III) and Absoption
1.4
1.2
Absorption
1
0.8
0.6
y = 4892x - 0.0706
R² = 0.9512
0.4
0.2
0
0
0.00005
0.0001
0.00015
0.0002
Concentration of Fe (III) in mol/L
Figure 2
0.00025
0.0003
Calculating Concentration of Iron (III) in Spinach Extract using line of best fit:
Obtained using data from Figure 2
Y = absorbance = 0.289
Y = 4892x – 0.0706
0.289 = 4892x – 0.0706 + 0.0706
0.289 + 0.0706 = 4892x
0.3596 / 4892 = 4892x / 4892
X = 0.000073507
Concentration = 0.000073507 mol/L
n = CV
n = number of moles, C = concentration, V = volume
n = 0.000073507 mol/L x 0.02 L
n = 0.00000147 mol
Molar mass of Fe = 55.85 g/mol
Mass of Fe in spinach = 55.85 g/mol x 0.00000147 mol
Mass of Fe in spinach = 0.000082099 g
Per 100g of spinach: Final Answer in mg of Fe per 100g of Spinach
= 100g x 0.000082099g
= 0.0082099 g
= 8.21 mg
Sample Calculation for Determining Mass of KSCN:
Find Moles using data from Lab Manual
Molar concentration = 1.5 mol/L, Volume = 0.10 L
Moles = 1.5 mol/L x 0.10 L
= 0.15 mol
Find Mass using data from Lab Manual and Periodic Table
Molar Mass of KSCN = 97.19 g/mol
Mass = 97.19 g/mol x 0.15 mol
= 14.5785 g
Discussion:
Based on the data recorded in Part A of Laboratory 3, spinach extract appears to absorb
strongly at 435 nm. The wavelength of purple light is about 435 nm (2). This suggests that purple
light is absorbed by the spinach extract since the results of the spectrophotometer indicate a high
absorbance at this wavelength. The graph is lowest through the 500-600 nm range. This
corresponds with the wavelength of green to yellow light which suggests this colour of light is
not absorbed by the spinach extract very well. The graph peaks again around the 670 nm range
but does not reach absorbance levels of that of the purple light spectrum. 670 nm corresponds
with the wavelength of red-orange light (2).
When comparing the graphed data recorded by the spectrophotometer to that of
chlorophyll a, chlorophyll b and beta-carotene, there is a strong resemblance between our
laboratory data and chlorophyll a. Both graphs have peak absorbance levels around 435 nm
which is within the spectrum of purple light. Both graphs also peak again around 670 nm, the
wavelength of red-orange light. Based on the strong resemblance between the spectrum observed
in Laboratory 3 with that of chlorophyll a, the chlorophyll a compound is likely present within
the spinach extract. Although all 3 compound graphs have a similar shape (1), chlorophyll a is
the most similar to the spectrum viewed in the lab.
When the near-UV laser pen was shone on the spinach extract the leading edge of the
cuvette gave off a purple-red colour. The light coming from the pen was a light blue colour.
When the cuvette was viewed from the opposite side of the laser pen, the blue hue of the laser
pen could be observed. The purple hue given off by the spinach extract is due to the emission of
photons. Electrons become excited when exposed to light, causing them to reach higher energy
levels. When they begin to relax and drop back down to their ground state, they emit some of the
energy as light (1). In the case of the spinach extract, purple light was observed when the laser
pen was shone on the cuvette because the wavelength of purple light (approximately 435 nm) is a
peak where the spinach extract has a high absorbency value, causing this wavelength of light to
be emitted.
When compared to a reliable source, my value indicated for the amount of iron in mg per
100g of spinach does not match. I understand that values for mg of Fe in 100g of spinach will
vary but the answer I recorded seems way more off than it should be. I would conclude it is
inaccurate and thus unreliable. The recorded value for the amount of iron in mg per 100g of
spinach is 2.71 mg (3). I do not think that all spinach has that exact amount of iron but is very
close. I would conclude that similarly to portions of the periodic table, this value is an average of
the amount of iron found in 100g of spinach. The growth of spinach in the natural environment
has many environmental factors acting upon it which could influence the content of the spinach.
This is why I would suggest the recorded number is an average value based on many samples.
When preparing the solutions for the 5 test tubes, an error was made in the
measurements. Instead of using the burette to deliver each volume of FeCl3 and HCl, a graduated
cylinder was used for test tubes 1-4. This was simply due to human error and not observing the
proper procedure as closely as it should have been. Because graduated cylinders are not as
accurate as burettes, the results tabulated for this section may be slightly off due to the accuracy
of measurements. The values graphed in Figure 1 (Absorbency vs. Wavelength) were obtained
by Krystyna Koczankski. The data did not transfer properly on to my USB stick so she supplied
me with the needed file for Part A of the experiment.
Conclusion:
By using the spectrophotometer in the laboratory, we were able to find the absorption
spectra of spinach extract. Based on this data and data given in the introduction of the lab, the
compound chlorophyll a is most likely present within spinach. I am fairly confident in the end
results of part A because the absorbance spectrum observed matched the recorded absorbance
spectrum for chlorophyll A and generally followed the same shape for all 3 compounds (1). I am
not very confident in my values calculated for Part B because they do not match the recorded
values listed in reliable literature (3).
References:
1. Koczanski, Krystyna; Xidos, James D. Chem 1300 Laboratory Manuel; UMSU Copy Centre:
Winnipeg, MB, Canada, 2014, pp 67-70.
2. Olmsted, John III; Williams, Greg; Burk, Robert C. Chemistry, 1st Canadian ed.; John Wiley
and Sons Ltd: Mississauga, Canada, 2010, pp 1078.
3. CRC Handbook of Chemistry and Physics, 92nd ed. Haynes, W.A., Ed.; CRC press: Boca
Raton, FL, 2011; Chapter 7, pg 59.