Chemical And Biomolecular Engineering

Department
Lafayette College
TITLE: Absorbance

SUBMITTED BY:

David Angel (A)

LAB PARTNERS: Brandy West (B), Gabby Montes (C)

DATE OF LAB WORK:

DATE DUE: October 17, 2015
SUBMITTED: October 17, 2015
COURSE:

DATE

CHE 312: Experimental Design I

SECTION: 3
INSTRUCTOR: Joe Woo
ABSTRACT:

The following report details the experimental data of the relationship

between absorbance and concentration for three different commercially
important dyes: FD&C Red # 40, D&C Blue # 1, and FD&C Yellow # 5, and
different mixture of them. It was concluded that the relationship between
the variables of interest agreed with the mathematical relationship known as
Beers law only for pure species solutions. Mixtures of the substances did
not agree with said relationship.

Abstract

The following report details the experimental data of the relationship

between absorbance and concentration for three different commercially
important dyes: FD&C Red # 40, D&C Blue # 1, and FD&C Yellow # 5, and
different mixture of them. It was concluded that the relationship between the
variables of interest agreed with the mathematical relationship known as
Beers law only for pure species solutions. Mixtures of the substances did not
agree with said relationship.
Introduction
The relationship between the absorption of radiant energy by an opaque
medium, more specifically a liquid solution of a solid, and the concentration
of the solid in solution was first studied by the German mathematician and
chemist August Beer. In 1852 he proposed the mathematical relationship
known as Beers law which states that: the absorptive capacity of a
dissolved substance is directly proportional to its concentration in
a solution1
A=Lc
(Eq. 1.1)
Where:
A absorbanc e

[=] AU

extinction coefficient [ ]

L path lengthof light

c concentration of solute

L
gcm

[=] cm
[=]

g
L

It is important to note that the molar extinction coefficient is a constant that

is dependent of the nature of the substance and the wavelength of the light
being measured.
1 http://0-www.britannica.com.libcat.lafayette.edu/science/Beers-law

For a solution of multiple components, Eq.1.1 extends to:

A = n , Lc n
i

(Eq. 1.2)
In other words, the absorbance at a given wavelength of the solution will be
given by the summation of the absorbance of the individual components of
the solution. For a given mixture if the extinction coefficient is known for
each component at different wavelengths then theoretically the
concentrations of each component can be found by applying the Beers law
relationship at a minimum of n wavelengths, where n is the number of
components in the mixture. And the wavelengths are the highest absorbance
wavelength for each dye. By picking the highest absorbance wavelength, the
highest signal to noise ratio is guaranteed.
This experiment has three main objectives:
1. To find the wavelength of maximum absorbance for three major
commercially important food dyes and compare the experimental
value to the value reported in the literature.
2. To find, by means of a regression, the relationship between the
absorbance of radiant energy and the concentration of solutions of
pure food dyes at each of the wavelengths of maximum absorbance
and explore whether Beers law is appropriately implemented for
dyes, and if so to determine the extinction coefficient of each of the
dyes at each of the wavelengths to further explore the application of
Beers law for mixtures of dyes.
3. To explore whether Beers law is an appropriate mathematical
expression for the relationship between the absorbance of the multicomponent solution at different wavelengths and the concentration of
each component.
Experimental Methods
Reagents:
Three commercially important food dyes where used in this experiment to
help meet the objectives outlined previously in the introduction section. The
dyes used were: the FD&C Red # 40, FD&C Blue # 1, and FD&C Yellow # 5.
All the dyes are food grade and manufactured by ROHA.
Apparatus:

The Genesis 2 Spectrophotometer was used to measure absorbance of

samples at different wavelengths. The machine had to be turned on 30
minutes previous to any measurement because the bulbs need time to warm
up

Figure 1. The front view of the Genesis 2 spectrophotometer.2

Figure
process
of the

2. The
diagram

measurement process inside of the Genesis 2 spectrophotometer. 3

2http://www.frankshospitalworkshop.com/equipment/documents/photometer/user_
manuals/Spectronic%20Genesys%20Spectrometer%20-%20User%20manual.pdf
3 Et. Al

Figure 2 shows the specific process by which the Genesis 2

spectrophotometer operates: Two lamps produce a beam of light that
contains all wavelengths. The beam then passes through a narrow slit and
only a wavelength of light is selectively let through to pass through the
sample. The reference detector measures the intensity of the beam of light
before it passes through the sample. The detector then measures the
intensity of the incoming light. Both of the intensity measurements are
compared to determine the absorbance of light.
Procedure:
The first step in the experimental procedure is to prepare a number of
samples of each dye and mixture of dyes. The first step in this process was
to prepare a large amount of a stock solution with a concentration near
0.02g/L of each dye. One serial dilution (refer to process for serial dilution in
the appendix) was then performed of each of the stock solutions by each of
the members of the team. The concentration of each of the resulting
samples are described in Table 1.
Table 1.
Red Dye
Sample
g/L
S1 (stock)
0.0217
S2
0.0109
S3
0.0054
S4
0.0027
S5
0.0014
S6
0.0007
S7
0.0003

Concentration of samples
Blue Dye
Sample
g/L
S1 (stock)
0.0222
S2
0.0111
S3
0.0056
S4
0.0028
S5
0.0014
S6
0.0007
S7
0.0003

Yellow Dye
Sample
g/L
S1 (stock)
0.0221
S2
0.0111
S3
0.0055
S4
0.0028
S5
0.0014
S6
0.0007
S7
0.0003

The next step in the experimental procedure was to determine the

wavelength of maximum absorbance for each dye, by testing a stock
solution. This was achieved by manually changing the wavelength of
measurement, and recording the absorbance at each of the wavelengths,
while the stock sample was in the chamber of the spectrophotometer.
Data of absorbance was then collected at a number of concentrations for
each dye in order to explore the relationship between the absorbance at
each of the three wavelengths determined previously and the concentration
of a sample of pure dye.

A number of samples were then created (refer to procedure to produce

samples of mixtures in the appendix) of mixture of dyes at different known
concentrations and then collect data of the absorbance at the three major
wavelengths. The concentrations of the samples tested can be found in the
appendix.
Safety:
FD&C Red # 40 In general has less health concerns than other azo dyes.
There have been claims that ingestion of the dye could be related to ADHD
but evidence is not substantial.
FD&C Blue # 1 was handled with care. It has the capacity to induce allergic
reactions in individuals with asthma.
FD&C Yellow # 5 was handled with care. At least 0.01% of the population is
sensitive to contact with the dye. Particularly individuals with asthma and
aspirin intolerance.
The general safety considerations were followed as well: when handling the
powdered form of the dyes gloves must be worn to avoid discovering allergic
reactions in group members. The powdered form of the dyes must not be
consumed or inhaled. If in contact with the dye group members must rinse
the affected area with water to avoid any possible complications.
Results and Discussion
The results of the measurement of the absorbance of the three different dyes
at different wavelengths is shown below. The stock solutions were used for
measurement.

a)

b)

c)
Figure 3. Scatter plots of absorbance of each dye at different wavelengths.
Figure 3 shows graphically the dependence of absorbance and wavelength of
light measured for the stock solutions of the three different dyes. In general,
the experimentally determined maximum absorbance of each of the dyes
was not too different from the value reported in the literature. Table 2 shows
the divergence of both values.
Table 2.
Dye

Wavelength
(nm)

Red
Blue
Yellow

495
630
430

Reported
Wavelength
(nm)
504
628
425

With the experimentally determined maximum absorbance wavelength data

reported in Table 2, experimental data of the absorbance of each of the dyes

at each of the wavelengths for different concentrations was collected. Tables

3 and 4 present the results of a linear regression between absorbance and
concentration for each of the dyes at each of the wavelengths with the
respective 95% confidence intervals. The raw measurement data and plots
of the regressions can be found in the appendix.

L
Red Dye gcm

L
Blue Dye gcm

L
Yellow Dye gcm

430
nm

6.721

(6.66437,
6.77733)

1.26

(1.22877,
1.29170)

22.36

(22.1403,
22.5747)

495
nm

21.49

(21.2720,
21.7149)

0.7209

(0.694012
,
0.747820)

1.956

(1.88262,
2.02973)

630
nm

0.1239

(0.08213
11,
0.165669
)

45.92

(45.3338,
46.5059)

0.05998

(0.005331
3,
0.125285)

Table 3

430 nm

Red Dye
0.0585

495 nm

0.0578

630 nm

0.0543

Blue Dye
0.0587
(0.05843,
0.05904)

Yellow Dye
0.0610
(0.0589,
0.0631)

(0.0557,
0.0599)

0.0563

0.0570

(0.05627,
0.05769)

(0.0538899
, 0.054681)

0.0565

0.0552

(0.0546134
, 0.055873)

(0.05798,
0.05905)

(0.056107
,
0.056628)
(0.0509,
0.0622

Table 4

Table 3 reports the slope of the linear regression while Table 4 reports the
intercept with the Y axis. Visually the plots of the linear regressions included
in the appendix show that the linear regression is a fairly accurate model for
the relationship between absorbance and concentration of the dyes. In fact,
the fit is so good that a residual analysis was not even necessary. However
Given that the samples measure where prepared by three different members, an analysis of
variation can be performed to evaluate differences in the data with respect to the person that
prepared it and to obviously the concentration. In the appendix box diagrams are reported that
visually show the small variances on the data. It can be seen that member C usually had higher
absorbance reading, however the deviations were really small.
A 2-way ANOVA was attempted, however because of the independence
between the measurements and the individual that performed the dilutions,
it did not give satisfactory results. For this reason, two 1-way ANOVA were
performed instead. The result of this calculations can be found in the
appendix.

After a close look of the ANOVA data we can conclude that the difference in the measurement
between group members was not statistically significant (P=1>>0.05), this is seen because the
confidence intervals overlap a considerable amount. The one way ANOVA between absorbance
and sample number shows that, to no surprise, the absorbance is highly dependent on the sample
concentration (sample number).
By examining the resulting mathematical expression for the linear
regressions we can observe that with the exception of having a small yintercept they agree with Beers law with the slope corresponding to the
extinction coefficient. By examining the y-intercepts magnitude for each of
the regressions we can observe that they are relatively constant, which
indicates that the intercept is the result of a systematic error in the
equipment. Therefore, for the application of Beers law to mixtures of the
dyes, the slope of the regressions were used as the extinction coefficient for
each dye at each wavelength and the intercept was ignored.
It is important to note that the 95% CI of the extinction coefficient of the yellow dye at 630nm
has 0 in it. Which means that for yellow dye at 630 nm concentration does not influence the
absorbance.

Five mixtures of known concentration were created by each member and the
absorbance at the three major wavelengths were measured. According to
Beers law for mixtures described in Eq.1.2:
A = n , Lc n
430

430

A = n , Lc n
495

495

A = n , Lc n
630

630

The expression then takes into account the absorbance of three components
at three different wavelengths. The resulting set of three equations has three
unknowns (concentrations of each component) that can be solved for. The
data for absorbance and a sample of the calculations of the predicted
concentrations can be found in the Appendix. Table 5 shows the comparison
between the actual concentration for each dye in each mixture and the
experimentally determined concentration:

Table 5
Concentrations
g/ L
Experime Beers
ntal
law
Red and Blue
\.,

%
error

Red

0.0014

0.0037

Blue
Yello
w

0.0014

0.0026

164.
3
85.7

0.0018

N/A

Red and Yellow

Red

0.0014

0.0036

157.
1

Blue
Yello
w

0.0012

0.0014

0.0044

N/A
214.
3

Yellow and Blue

0 0.0025
0.0014 0.0026

Red
N/A
Blue
85.7
Yello
135.
0.0014 0.0033
w
7
Red, Blue and Yellow (same
concentration)
133.
Red
0.0018 0.0042
3
Blue
0.0019
0.003 57.9
Yello
138.
0.0018 0.0043
w
9
Red, Blue and Yellow (strong
Green)
330.
Red
0.001 0.0043
0
Blue
0.005 0.0059 18.0
Yello
0.005 0.0065 30.0
w
By looking at the % errors for each of the dye concentrations reported in
Table 5 we can see that Beers law does not apply to the mixtures of dyes as
it applies to the pure dyes. The concentrations obtained by the calculation of
Eq.1.2 resulted in values for non-existent dyes in the mixture. In other words,
the dyes present in the mixtures generated the illusion of a third dye. It can
also be seen that the higher the concentrations of the dyes the more
accurate the prediction of Beers law, this is apparent from the results of the
strong green mixture.
Green is a particularly commercially important color. As can be seen from the
results in Table 5, a desired hue of green, which would theoretically need to
have a high concentration of red dye, can be obtained simply by combining
high concentrations of blue and yellow dye. This knowledge is potentially
important for the industry because it implies that there is no need to waste
red dye in the production of the desired color.

Sources of error:

The development of Beers law has been incredibly useful for industrial
applications in part because of its simple linear relationship between
absorbance and concentration. However, although simple, the measure of
absorbance is actually developed from a more complex logarithmic
relationship: transmittance. In other words:
Absorbance=2log 10 (Transmittance)
Given this logarithmic relationship, the linearity of absorbance breaks down
at high concentrations (low transmittances). Theoretically 4.0 AU the linear
relationship does not hold any longer4. Samples of high concentrations are
therefore a source of error.
For low concentration samples the sources of error include: light refraction,
reflection, and scattering. All of the previous effects will have the effect of
causing extra absorbance that is not caused by the sample itself.
Experimental improvements:
Possible experimental improvements would rely on reducing the sources of
errors. The first improvement would be to ensure that there were no dye big
particles that could scatter light. This could be achieved by mixing the
solutions thoroughly and even using finer dye powder. Big particles in
solution cause scattering of light which decreases the accuracy of the
measurements. By making sure the dyes are thoroughly dissolved with no
particles in solution the scattering will be avoided. The second possible
improvement is to be certain there is no cross contamination between
samples. This could be achieved by using different pipets every time.
Another possible improvement would be to use perfectly clean and flawless
cuvettes in the measurement to avoid scattering of light due to residue in
walls of the cuvette and the imperfections of the plastic.
Conclusions
Based on the experimental data obtained and presented in the previous
section, we can make a variety of conclusions. The first conclusion has to do
with the wavelength for maximum absorbance of each dye. It is clear from
looking at Figure 3 that the wavelength for maximum absorbance for each
dye differs from the wavelength reported in the literature. The exact reason
4 http://www.turnerdesigns.com/t2/doc/appnotes/S-0075.pdf

this is the case may have to do with the purity of the dye, possible
unaccounted contaminants, or an error in the function of the spectrometer.
The second conclusion has to do with the application of Beers law to pure
dyes. From figures #-# we can observe that the linear fit is more than
accurate for the dyes tested. The relationship found by the regression
analysis seems to be very precise, given that the 95% CI are so small. The
fact that all of the dyes systematically exhibited a similar y-intercept
probably has to do with a systematic measurement failure in the
spectrophotometer. In general Beers law seems to be a good fit for the data.
The third conclusion has to do with the absorbance of multi-component
solutions of dyes. By looking at Tables 5 we can see that the Beers law
prediction for the multi-component solution had a significant error is
therefore not a useful mathematical relationship to determine concentrations
of mixtures of dyes. The reason behind the inapplicability of Beers law
probably has to do with synergistic interactions between the dyes that
caused the absorptions to be higher than they would have been without said
interactions. Looking at Table 5 (strong green) for instance it can be seen
that the predictions for the concentration for blue and yellow did not have as
big an error as other samples. However the prediction for the concentration
of the red dye had a 330% error which suggests that the high concentrations
of yellow and blue interacted to produce the effect of the red dye being
present.

Literature Cited
Turner Deigns triology laboratory fluorimeter application note on
absorbance: http://www.turnerdesigns.com/t2/doc/appnotes/S-0075.pdf
Milton Roys Genesys 2 spectrophotometer operators manual:
http://www.frankshospitalworkshop.com/equipment/documents/photometer/u
ser_manuals/Spectronic%20Genesys%20Spectrometer%20-%20User
%20manual.pdf
Encyclopedia Britannicas entry on Beers law: http://0www.britannica.com.libcat.lafayette.edu/science/Beers-law

Appendix

Procedure to prepare a serial dilution set:

Serial dilutions of the stock solution must be prepared by following the next
steps:
1. mix 1 ml of stock solution with 1 ml of DI water to create S2.
2. mix 1 ml of the S2 with 1 ml of DI water to create S3.
3. Repeat 1-2 until S7 is obtained.
Producing samples of mixtures:
For mixtures of two dyes: combine S4 of one of the pure dye samples to the
S4 of another of the dyes.
For mixture of three dyes: combine S4 of two dyes then taking 1 ml of the
mixture and mixing it with S5 of the third dyes
Concentration dependence of absorbance of Red Dye at 430nm:

Red Dye Calibration Plot at 430 nm

Absorbance = 0.05851 + 6.721 Concentrations (g/L)
0.225

Regression
95% CI

0.200

S
R-Sq
R-Sq(adj)

Absorbance

0.175

0.0008904
100.0%
100.0%

0.150
0.125
0.100
0.075
0.050
0.000

0.005

0.010
0.015
Concentrations (g/ L)

0.020

Concentration dependence of absorbance of Red Dye at 495nm:

Red Dye Calibration Plot at 495 nm

Absorbance = 0.05783 + 21.49 Concentrations (g/L)
Regression
95% CI

0.5

S
R-Sq
R-Sq(adj)

Absorbance

0.4

0.0034909
100.0%
100.0%

0.3
0.2

0.1
0.0
0.000

0.005

0.010
0.015
Concentrations (g/ L)

0.020

Concentration dependence of absorbance of Red Dye at 630nm:

Red Dye Calibration Plot at 630 nm

Absorbance = 0.05429 + 0.1239 Concentrations (g/L)
Regression
95% CI

0.058

S
R-Sq
R-Sq(adj)

Absorbance

0.057

0.056

0.055

0.054
0.000

0.005

0.010
0.015
Concentrations (g/ L)

0.020

0.0006585
67.0%
65.2%

Concentration dependence of absorbance of Blue Dye at 430nm:

Blue Dye Calibration Plot at 430nm

Absorbance = 0.05874 + 1.260 Concentrations (g/L)
0.090

Regression
95% CI

0.085

S
R-Sq
R-Sq(adj)

Absorbance

0.080

0.0005074
99.7%
99.7%

0.075
0.070
0.065
0.060
0.000

0.005

0.010
0.015
Concentrations (g/ L)

0.020

0.025

Concentration dependence of absorbance of Blue Dye at 495nm:

Blue Dye Calibration Plot at 495 nm
Absorbance = 0.05637 + 0.7209 Concentrations (g/L)
0.075

Regression
95% CI
S
R-Sq
R-Sq(adj)

Absorbance

0.070

0.065

0.060

0.055
0.000

0.005

0.010
0.015
Concentrations (g/ L)

0.020

0.025

0.0004339
99.4%
99.4%

Concentration dependence of absorbance of Blue Dye at 630nm:

Blue Dye Calibration Plot at 630 nm

Absorbance = 0.05654 + 45.92 Concentrations (g/L)
1.2

Regression
95% CI

Absorbance

1.0

S
R-Sq
R-Sq(adj)

0.0094516
99.9%
99.9%

0.8
0.6
0.4
0.2
0.0
0.000

0.005

0.010
0.015
Concentrations (g/ L)

0.020

0.025

Concentration dependence of absorbance of Yellow Dye at 430nm:

Yellow Dye Calibration Plot at 430 nm
Absorbance = 0.06098 + 22.36 Concentrations (g/L)
0.6

Regression
95% CI

Absorbance

0.5

S
R-Sq
R-Sq(adj)

0.0034870
100.0%
100.0%

0.4
0.3
0.2
0.1
0.0
0.000

0.005

0.010
0.015
Concentrations (g/ L)

0.020

0.025

Concentration dependence of absorbance of Yellow Dye at 495nm:

Yellow Dye Calibration Plot at 495 nm

Absorbance = 0.05698 + 1.956 Concentrations (g/L)
Regression
95% CI

0.10

S
R-Sq
R-Sq(adj)

Absorbance

0.09

0.0011810
99.4%
99.4%

0.08

0.07

0.06
0.000

0.005

0.010
0.015
Concentrations (g/ L)

0.020

0.025

Concentration dependence of absorbance of Yellow Dye at 630nm:

Yellow Dye Calibration Plot at 630 nm

Absorbance = 0.05524 + 0.05998 Concentrations (g/L)
Regression
95% CI

0.058

S
R-Sq
R-Sq(adj)

Absorbance

0.057

0.0010486
16.3%
11.9%

0.056

0.055

0.054
0.000

0.005

0.010
0.015
Concentrations (g/ L)

0.020

0.025

Raw Experimental Data:

Red
wavelengt absorbanc
h
e
350
0.049
375
0.149
400
0.11
425
0.141
450
0.186
475
0.358
480
0.374

Maximum Absorbance
Blue
wavelengt absorbanc
h
e
350
0.025
375
0.117
400
0.086
425
0.045
450
0.003
475
0.023
500
0.016

yellow
wavelengt absorbanc
h
e
350
0.119
375
0.334
400
0.404
405
0.419
410
0.431
415
0.532
420
0.47

485
490
495
500
504
505
510
515
520
525
550
575
600
625
650
675
700
725
750
775
800

0.428
0.499
0.515
0.466
0.467
0.487
0.451
0.459
0.427
0.407
0.188
0.093
0.002
0.003
0
0
0
0
0
0
0

525
550
575
580
585
590
595
600
605
610
615
620
625
628
630
635
640
645
650
675
700
725
750
775
800

0.038
0.06
0.31
0.341
0.314
0.413
0.409
0.45
0.545
0.684
0.818
0.872
0.972
0.998
1.052
1.001
0.856
0.68
0.441
0.009
0.054
0.052
0.054
0.054
0.054

425
430
435
440
445
450
475
500
525
550
575
600
525
650
675
700
725
750
775
800

0.486
0.544
0.493
0.458
0.461
0.41
0.219
0.02
0
0
0
0
0
0
0
0
0
0
0
0

absorba
nce

Red
wavelgth
430 S1
S2
S3
S4
S5
S6
S7
concentration g/L
Dilution A
0.204 0.131 0.095 0.076 0.067 0.063 0.061
Dilution B
0.204 0.132 0.095 0.076 0.067 0.063 0.061
Dilution C
0.206 0.129 0.096 0.078 0.068 0.064 0.061

Red
wavelgth
495 S1
concentration g/L

S2

S3

S4

S5

S6

S7

absorbance

Dilution A
Dilution B
Dilution C

0.51
9
0.51
9
0.52
9

0.29
6
0.29
6
0.28
8

0.17
7
0.17
8
0.18

0.11
3
0.11
4
0.11
9

0.08
5
0.08
5
0.08
8

0.07
0.07
1
0.07
2

0.06
3
0.06
4
0.06
5

Red

absorbance

wavelgth
630 S1
S2
S3
S4
S5
S6
S7
concentration g/L
0.05
0.05
0.05
0.05
0.05
0.05
0.05
Dilution A
8
5
4
4
4
4
4
0.05
0.05
0.05
0.05
0.05
0.05
0.05
Dilution B
8
5
5
5
5
5
5
0.05
0.05
0.05
0.05
0.05
0.05
0.05
Dilution C
6
5
5
5
5
5
4

Blue

absorba
nce

wavelgth
430 S1
S2
S3
S4
S5
S6
S7
concentration g/L
Dilution A
0.087
0.73 0.065 0.062
0.06
0.06 0.059
Dilution B
0.087 0.073 0.065 0.062
0.06
0.06
0.06
Dilution C
0.086 0.073 0.066 0.063 0.061 0.059 0.059

Blue

absorbance

wavelgth
495 S1
concentration g/L

S2

S3

Dilution A

0.072

0.064

Dilution B

0.072

0.065

Dilution C

0.073

0.065

0.0
6
0.0
6
0.0
6

S4

S5

S6

S7

0.058

0.057

0.057

0.057

0.058

0.057

0.057

0.057

0.059

0.057

0.057

0.057

S7

Blue

absorban
ce

wavelgth
630 S1
concentration g/L
Dilution A
Dilution B

1.059
1.06

S2

S3

S4

S5

S6

0.583
0.584

0.309
0.31

0.181
0.181

0.117
0.118

0.086
0.086

0.0
7
0.0
7

Dilution C

1.083

absorban
ce

wavelgth
430 S1
concentration g/L
Dilution A
0.549
Dilution B
0.55
Dilution C

0.557

absorbance

wavelgth
495 S1
concentration g/L
0.10
Dilution A
1
0.10
Dilution B
1
Dilution C

0.1

0.586

0.317

0.0
7

0.183

0.118

0.085

Yellow
S2
S3

S4

S5

S6

S7

0.312
0.312

0.185
0.185

0.122
0.122

0.091
0.091

0.075
0.075

0.067
0.066

0.316

0.189

0.127

0.091

0.074

0.066

Yellow
S2
S3

0.08
0.07
5
0.07
7

S4

0.06
8
0.06
8
0.06
8

0.06
3
0.06
3
0.06
4

S5

0.06
0.06
1
0.05
9

S6
0.05
8
0.05
9
0.05
8

S7
0.05
7
0.05
7
0.05
7

absorbance

Yellow
wavelgth
630 S1
S2
S3
S4
S5
S6
S7
concentration g/L
0.05
0.05
0.05
0.05
0.05
0.05
0.05
Dilution A
8
7
5
6
6
5
4
0.05
0.05
0.05
0.05
0.05
0.05
0.05
Dilution B
7
5
5
6
6
6
4
0.05
0.05
0.05
0.05
0.05
0.05
0.05
Dilution C
5
5
5
8
5
5
5

sample #

absorba
nces

concentrati
ons
wavelegnth
s

Red and Blue

A
B

Red

0.0027

Blue
430
495
630

0.0028
0.07
0.087
0.119

C
0.001
1ml
4
0.001
1ml
4
0.068
0.07
0.081 0.089
0.13 0.117

red and yellow

A
B

sample #

absorbances

concentrati
ons

Red

0.0027 1ml

Yellow

0.0028 1ml
0.09
0.1
8
0.08
0.087
8
0.05
0.054
4

430
wavelegnth
s

495
630

sample #

absorbance
s

concentrati
ons

yellow and blue

A
B

Yellow

0.0028 1 ml

Blue

0.0028 1ml
0.09
0.097
3
0.06
0.061
1
0.112
0.12

430

wavelegnth
s

495
630

absorba
nces

concentration
s

red, blue and

yellow
sample # A

con

wavelegn
ths

C
0.001
4
0.001
4
0.175
0.086
0.055

C
0.001
4
0.001
4
0.092
0.061
0.121

red

0.0054

0.5

blue

0.0056

0.5

yellow
430
495
630

0.0055
0.113
0.1
0.14

0.5
0.114
0.102
0.141

red, blue and yellow

sample
#
A
B
C
red
0.010
0.1 0.001
9
0

C
0.001
8
0.001
9
0.001
8
0.16
0.103
0.141

centratio
ns
absorbances

wavelegn
ths

ANOVA results:

blue
yellow

0.011
1
0.011
1

430

0.18

495

0.112

630

0.266

0.5
0.5
0.18
4
0.10
7
0.27
7

0.005
0
0.005
0
0.178
0.109
0.276

One way ANOVA of absorbance and group member

430 nm
Source
Person
Error
Total

DF
2
60
62

SS
0.0000
0.8215
0.8215

MS
0.0000
0.0137

F
0.00

P
0.999

Individual 95% CIs For Mean Based on

Pooled StDev
Level
N
Mean
StDev ------+---------+---------+---------+--A
21 0.1221 0.1163 (----------------*----------------)
B
21 0.1222 0.1165 (----------------*----------------)
C
21 0.1233 0.1182 (----------------*----------------)
------+---------+---------+---------+---

0.090

0.120

0.150

0.180

Pooled StDev = 0.1170

495 nm
Source
Person
Error
Total

DF
2
60
62

S = 0.1101

Level
A
B
C

N
21
21
21

SS
0.0000
0.7275
0.7275

MS
0.0000
0.0121

R-Sq = 0.00%

Mean
0.1064
0.1065
0.1072

StDev
0.1097
0.1097
0.1109

F
0.00

P
1.000

R-Sq(adj) = 0.00%
Individual 95% CIs For Mean Based on
Pooled StDev
-------+---------+---------+---------+-(-------------------*------------------)
(-------------------*------------------)
(------------------*------------------)
-------+---------+---------+---------+-0.075
0.100
0.125
0.150

Pooled StDev = 0.1101

630 nm
Source
Person
Error
Total

DF
2
60
62

S = 0.2444

Level
A
B
C

N
21
21
21

SS
0.0000
3.5835
3.5835

MS
0.0000
0.0597

R-Sq = 0.00%

Mean
0.1514
0.1517
0.1531

StDev
0.2426
0.2428
0.2477

Pooled StDev = 0.2444

F
0.00

P
1.000

R-Sq(adj) = 0.00%
Individual 95% CIs For Mean Based on
Pooled StDev
---+---------+---------+---------+-----(-----------------*-----------------)
(----------------*-----------------)
(-----------------*----------------)
---+---------+---------+---------+-----0.060
0.120
0.180
0.240

One-way ANOVA of Absorbance and Sample

430 nm
Source
Sample
Error
Total

DF
22
40
62

SS
0.8214247
0.0000890
0.8215137

S = 0.001492

Level
Blue 1
Blue 1
Blue 2
Blue 3
Blue 4
Blue 5
Blue 6
Blue 7
Red 1
Red 2
Red 3
Red 4
Red 4
Red 5
Red 6
Red 7
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow

1
2
3
4
5
6
7

N
2
1
3
3
3
3
3
3
3
3
3
2
1
3
3
3
3
3
3
3
3
3
3

MS
0.0373375
0.0000022

R-Sq = 99.99%

Mean
0.08650
0.08700
0.07300
0.06533
0.06233
0.06033
0.05967
0.05933
0.55200
0.31333
0.18633
0.12450
0.12200
0.09100
0.07467
0.06633
0.20467
0.13067
0.09533
0.07667
0.06733
0.06333
0.06100

Pooled StDev = 0.00149

StDev
0.00071
*
0.00000
0.00058
0.00058
0.00058
0.00058
0.00058
0.00436
0.00231
0.00231
0.00354
*
0.00000
0.00058
0.00058
0.00115
0.00153
0.00058
0.00115
0.00058
0.00058
0.00000

F
16780.89

P
0.000

R-Sq(adj) = 99.98%
Individual 95% CIs For Mean Based on
Pooled StDev
------+---------+---------+---------+--*
*
*
*
*
*
*
*
*
*
*)
*
*
*
*
*)
*
*
*
*
*)
*
*
------+---------+---------+---------+--0.15
0.30
0.45
0.60

495 nm
Source
Sample
Error
Total

DF
22
40
62

SS
0.7273834
0.0001630
0.7275464

S = 0.002019

Level
Blue 1
Blue 1
Blue 2
Blue 3
Blue 4
Blue 5
Blue 6
Blue 7
Red 1
Red 2
Red 3
Red 4
Red 4
Red 5
Red 6
Red 7
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow

1
2
3
4
5
6
7

N
2
1
3
3
3
3
3
3
3
3
3
2
1
3
3
3
3
3
3
3
3
3
3

MS
0.0330629
0.0000041

R-Sq = 99.98%

Mean
0.07250
0.07200
0.06467
0.06000
0.05833
0.05700
0.05700
0.05700
0.10067
0.07733
0.06800
0.06350
0.06300
0.06000
0.05833
0.05700
0.52233
0.29333
0.17833
0.11533
0.08600
0.07100
0.06400

Pooled StDev = 0.00202

StDev
0.00071
*
0.00058
0.00000
0.00058
0.00000
0.00000
0.00000
0.00058
0.00252
0.00000
0.00071
*
0.00100
0.00058
0.00000
0.00577
0.00462
0.00153
0.00321
0.00173
0.00100
0.00100

F
8113.59

P
0.000

R-Sq(adj) = 99.97%
Individual 95% CIs For Mean Based on
Pooled StDev
-----+---------+---------+---------+---*
*
*)
*
*
*
*
*
*)
*)
(*
*)
*)
*
*
*
(*
*)
*
(*
*
*
*)
-----+---------+---------+---------+---0.12
0.24
0.36
0.48

630 nm
Source
Sample
Error
Total

DF
22
40
62

SS
3.583197
0.000329
3.583526

S = 0.002866

Level
Blue 1
Blue 1
Blue 2
Blue 3
Blue 4
Blue 5
Blue 6
Blue 7
Red 1
Red 2
Red 3
Red 4
Red 4
Red 5
Red 6
Red 7
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow

1
2
3
4
5
6
7

N
2
1
3
3
3
3
3
3
3
3
3
2
1
3
3
3
3
3
3
3
3
3
3

MS
0.162873
0.000008

R-Sq = 99.99%

Mean
1.07150
1.05900
0.58433
0.31200
0.18167
0.11767
0.08567
0.07000
0.05667
0.05567
0.05500
0.05700
0.05600
0.05567
0.05533
0.05433
0.05733
0.05500
0.05467
0.05467
0.05467
0.05467
0.05433

Pooled StDev = 0.00287

StDev
0.01626
*
0.00153
0.00436
0.00115
0.00058
0.00058
0.00000
0.00153
0.00115
0.00000
0.00141
*
0.00058
0.00058
0.00058
0.00115
0.00000
0.00058
0.00058
0.00058
0.00058
0.00058

F
19832.28

P
0.000

R-Sq(adj) = 99.99%
Individual 95% CIs For Mean Based on
Pooled StDev
--------+---------+---------+---------+*
*
*)
*)
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
--------+---------+---------+---------+0.30
0.60
0.90
1.20