CRYSTAL VIOLET RATE LAW

Investigation 11

JANUARY 17, 2018

ANTHONY HOWER, JULIA KIM, GLORIA YOO
(Procedure and Formatting), (Pre-Lab and Conclusion), (Data Collection and Computation,
Argumentation and Documentation)
Table of Contents
Pre-Lab Questions ......................................................................................................................................... 1
Materials ....................................................................................................................................................... 4
Procedure...................................................................................................................................................... 5
Data Collection and Computation................................................................................................................. 5
Argumentation and Documentation............................................................................................................. 8
Conclusion ..................................................................................................................................................... 8
[Appendix A] ................................................................................................................................................. 8
Sources ........................................................................................................................................................ 10

Pre-Lab Questions
1. Answer the following questions about the selection of a wavelength for your experiment.
a. Based on the absorption spectrum of 25 μM crystal violet in Figure 1 and taking into
account the considerations that follow, what wavelength should you use for the
Beer’s law calibration curve and subsequent reaction of CV with NaOH? Please
explain your answer.

We should use a wavelength of approximately 530nm. The instrument is not sensitive

enough to measure much over 1.0 absorbance, but we want the maximum possible
range. According to the visible spectrum, the wavelength when the absorbance is 1.0
is approximately 530nm.
.
b. Simulate the instrument readings you will get in Part 1 of the experiment by doing
the following: Trace Figure 1 onto your own paper. Draw a vertical line at the
wavelength you have chosen, intersecting the absorbance curve at that wavelength.
Where your vertical line intersects the absorbance curve is the absorbance value your
instrument should read for the stock 25 μM CV solution. Keeping in mind Beer’s law
from Equation 1, and being mindful that the wavelength and path length are fixed,
draw X’s on your vertical line where you expect the absorbance values will be for the
diluted solutions you prepare in Question 2. Use appropriate ratios of concentrations
to determine where on the vertical line to make your marks.
 Figure 1. Visible Spectrum of 25µM CV Solution

2. A calibration curve requires the preparation of a set of known concentrations of CV,

which are usually prepared by diluting a stock solution whose concentration is known.
Describe how to prepare 10. mL of a 5-, 10-, 15-, and 20- μM CV solution using a 25 μM
CV stock solution.

Use equation C1V1=C2V2 for the calculation.

For 5Mp 25(V)=5(100) V=2mL
Measure 2mL of 25Mp CV solution and put it in a measuring cylinder. Then dilute the
solution by filling up with the water up to the 10mL line.
Repeat this for the different concentrations. Each time changing the mL of 25Mp CV
solution needed.
To make the rest of the concentration 10, 15, 20Mp CV solution, the volume of 25Mp CV
solution needed is respectively 6, 4, 2mL with rest filled with water up to 10mL line.

3. During the reaction of CV with NaOH, do you expect the colorimeter’s (or
spectrophotometer’s) absorbance reading to change? How do you expect it to change if
such a change is anticipated (i.e., increase, decrease, or no change) as the reaction
proceeds? Explain your reasoning.

Because the CV molecules that are absorbing the light being transmitted degenerate and
become colorless, I expect the absorbance reading to decrease as the reaction proceeds
as the CV molecules that have reacted no longer absorb the light.

4. Answer the following questions for a reaction of CV with NaOH in these two scenarios: a
solution with a 1:1 NaOH:CV mole ratio and a solution similar to what you will be using
with a 1000:1 NaOH:CV mole ratio.
a. Using your prior knowledge of reaction stoichiometry, what is the final
percentage of each reactant remaining if each reaction went to completion? Show
work and reasoning to justify your answer.
 For the 1:1 NaOH:CV mole ratio reaction, there will be no reactant left (0%)
because the reactants react at a 1:1 ratio. For the 1000:1 NaOH:CV mole ratio
reaction, there will be 999 moles of NaOH left after the reaction is complete.
Therefore, most (99.9%) of the NaOH will be left after the reaction goes to
completion.

b. Based on this result, describe how one gets Equations 3 and 4 from Equation 2 in
the Explanation section.

In the second reaction, OH is used a thousand times as much as it is needed.

Since 99.9% of OH will remain unreacted, we can assume that the concentration
of OH does not change. Therefore, OH will have an order of reaction of zero.
After Equation 4 is simplified, the rate law will look like Equation 3.

5. Using the kinetics chapter in your textbook and websites like “Chemical Kinetics –
Integrated rate laws”
http://www.chm.davidson.edu/vce/kinetics/IntegratedRateLaws.html, describe the
graphical analysis that can be done to determine the order (considering only 0th, 1st, or
2nd order) and the value of the pseudo-rate constant, k*, of a chemical reaction from
concentration data collected through time.

The order of a reaction can be determined through graphical analysis by carrying out an
experiment where the concentration of one substance can be doubled as compared to the
other, and the power of increase of the reaction rate determines the power of the doubled
chemical, and the same thing can be done for the other chemical. Next, based on the
determined order of the chemicals and one of the measured reaction rates, you can solve
for k after plugging the rest into its corresponding equation
.
6. Based on your answer to Questions 3–5, design an experiment for the reaction of CV
with NaOH and describe the subsequent data analysis to accomplish the Central
Challenge, the determination of the value of (i) w, the order with respect to CV and (ii)
k*, the pseudo-rate constant found in the rate law in Equation 3. For simplicity, use 10.
mL for the combined volume of CV and NaOH because it is a bit more than enough to
fill cuvettes appropriately.

Measure out 8 gram of CV and 2 grams of NaOH using burettes into a beaker, then stir
and pour into cuvette and record the absorbance value periodically (every 10 seconds)
until the reaction is complete. Using the calibration curve, get the relational expression
between the absorbance and solution concentration and convert the absorbance data into
concentrations. Use excel to graph the ln[CV] and 1/[CV] value and determine w, the
order of reaction of CV. Plugging in the numbers into the second order integrated rate
law equation, solve for k.
 7. As an optional extension, design an experiment to determine from Equations 2 and 4 (i)
z, the order with respect to NaOH and (ii) k, the rate constant.

8. Answer the following questions after examining Figure 3 to address the issue of when to
stop collecting data.
a. For early parts of the three different reactions in Figure 3, all three curves seem
relatively linear with different slopes. But as the reactions progress through time,
at roughly what concentration level would you say some graphs start to look
nonlinear?

1st order reaction: about 4M

2nd order reaction: about 3.5M

b. Given that you don’t yet know the order of the reaction of CV with NaOH, how
might Figure 3 help you to decide when to stop collecting data? Hint: Think in
terms of percent completion instead of concentration.

When about 90% of the reaction is complete (when only 10% of the reactant
concentration is left), the reaction curves for both the first and second order
reactions evens out and becomes nearly flat. Therefore, stop recording data when
the reaction is 90% complete.

Materials
The materials that we used for this lab were 25.0 µM crystal violet (CV) solution, 0.200 M
sodium hydroxide (NaOH) solution, Distilled Water, Cuvettes, a spectrophotometer, Excel, 10
mL pipettes, pipette bulb, and a stopwatch.

Figure 2. Spectrophotometer
Procedure
First, we had to determine what wavelength that we would use for our experiment. Based on the
absorption spectrum of 25 μM crystal violet, we determined that the wavelength we would use
was approximately 530 nm (see pre-lab 1).
Next, we created a Beer’s law calibration curve by testing the absorption of different
concentrations of known crystal violet solutions, ranging from 0% to 100% 25 μM CV solution,
with 20% increments, so that we could convert the spectrophotometer’s raw absorbance readings
into the molar concentration of CV.
Next, we measured 8 mL of 25 μM CV and 1 mL of NaOH, after calibrating the
spectrophotometer with distilled water, we mixed the two solutions quickly, then poured it into a
cuvette and put it into the spectrophotometer. We wrote down our initial reading, then continued
to record the reading every 15 seconds until the absorbance reached 10% of the original.
However, due to time constraints, we had to stop after 15 minutes.
After we collected all the data we could within that time period, we entered the data points into
excel and determined the rate law (see Data Collection and Computation as well as
Argumentation and Documentation).

Figure 3. Diagram of CV Reacting with OH-

Data Collection and Computation

Percent solution Concentration (M) Absorbance

0% 0 0
20% 4.84×10-6 0.273
40% 9.68×10-6 0.400
50% 1.21×10-5 0.553
60% 1.45×10-5 0.602
80% 1.94×10-5 0.760
100% 2.42×10-5 1.134
Figure 4: Data Collected from 2.42×10-5M CV solution
 Absorbance vs Concentration (M)
3.00E-05
y = 2.2695E-05x + 1E-07
2.50E-05 R² = 0.9758

2.00E-05

1.50E-05

1.00E-05

5.00E-06

0.00E+00
0 0.2 0.4 0.6 0.8 1 1.2

Figure 5: Absorbance Readings Comprising our Calibration Curve

Concentration = 2.2695×10-5 × Absorbance

For figures 6-8, refer to raw data in [Appendix A].

[CV] vs Time
0.000025

0.00002

0.000015

0.00001
y = -2E-07x + 2E-05
R² = 0.9678
0.000005

0
0 45 90 135 180 225 270 315 360 405 450 495 540 585 630 675 720 765 810 855 900

Figure 6: Zero Order Reaction Graph

 ln[CV] vs Time
-10.2

450

690
0
30
60
90
120
150
180
210
240
270
300
330
360
390
420

480
510
540
570
600
630
660

720
750
780
810
840
870
900
-10.4

-10.6

-10.8
y = -0.0147x - 10.791
R² = 0.9963
-11

-11.2

-11.4

-11.6

-11.8

Figure 7: First Order Reaction Graph

1/[CV] vs Time
140000
y = 1135.5x + 44015
120000 R² = 0.9946

100000

80000

60000

40000

20000

0
210

360

510
0
30
60
90
120
150
180

240
270
300
330

390
420
450
480

540
570
600
630
660
690
720
750
780
810
840
870
900

Figure 8: First Order Reaction Graph

Rate = k[CV+][OH-]
Rate = k*[CV+]
k* = slope = -0.0147s-1
Argumentation and Documentation
The central challenge for our experiment was to determine the rate law for the reaction of crystal
violet (CV) and sodium hydroxide (NaOH). Since rate laws are calculated using concentration
and time, we chose to use concentration for our experiment as well as data analysis overall. For
the calibration curve, we had 7 data points, each of different concentrations, going up at a
constant rate (20% interval). 7 data points were used instead of 3 or 5 to increase the accuracy of
our graph. For the experiment regarding the reaction of CV and NaOH, we had lot more data
points. This was because we recorded the absorptivity changes every 15 seconds for 15 minutes
to decrease any affect that outliers might have if our data was short. Through our observation, we
could see that the absorptivity decreased over time. This was because concentration of CV
decreased as it reacted with NaOH. As CV reacts with NaOH, it also decolorizes, allowing more
light to pass through the solution, resulting in decrease of absorbance. Although we measured the
absorptivity change for 15 minutes, we failed to observe the full reaction, up to 90% decrease, as
the reaction was extremely slow.
Through graphing, we could conclude that the reaction was first order, as the line seemed most
linear with the graph ln[CV] vs time, with R2 value of 0.9963. Therefore, Rate = k*[CV+] with
k* = -0.0147s-1.

Conclusion
In conclusion, the rate law of the reaction between CV and OH is Rate = k*[CV+]. It is a first
order reaction with respect to CV, and k* = -0.0147s-1. Some sources of error that we would
improve on if the experiment was done again are the dilution process and the duration of the
reaction that we recorded data from. The sample that our group made was the outlier in the
calibration curve. Our data was higher than what it should have been, and decreased the R2-
value and the accuracy of the relational expression between the concentration and the
absorbance. Furthermore, we could only record data for 15 minutes, which made it hard for our
group to determine the end behavior of the graphs. The R2-values of the two graphs were both
fairly high because we could not record the data until the reaction was complete.

[Appendix A]
Raw Data

Time A [CV] ln[CV] 1/[CV]

0 0.944 2.14241E-05 -10.751 46676.45
15 0.919 2.08567E-05 -10.7778 47946.21
30 0.897 2.03574E-05 -10.8021 49122.15
45 0.878 1.99262E-05 -10.8235 50185.16
60 0.862 1.95631E-05 -10.8419 51116.67
75 0.845 1.91773E-05 -10.8618 52145.05
90 0.828 1.87915E-05 -10.8821 53215.66
105 0.812 1.84283E-05 -10.9016 54264.25
120 0.797 1.80879E-05 -10.9203 55285.53
135 0.782 1.77475E-05 -10.9393 56346
150 0.769 1.74525E-05 -10.956 57298.53
165 0.755 1.71347E-05 -10.9744 58361.02
180 0.742 1.68397E-05 -10.9918 59383.52
195 0.73 1.65674E-05 -11.0081 60359.68
210 0.718 1.6295E-05 -11.0247 61368.48
225 0.706 1.60227E-05 -11.0415 62411.57
240 0.696 1.57957E-05 -11.0558 63308.29
255 0.685 1.55461E-05 -11.0717 64324.92
270 0.674 1.52964E-05 -11.0879 65374.73
285 0.664 1.50695E-05 -11.1028 66359.29
300 0.654 1.48425E-05 -11.118 67373.96
315 0.645 1.46383E-05 -11.1319 68314.06
330 0.636 1.4434E-05 -11.1459 69280.77
345 0.627 1.42298E-05 -11.1602 70275.23
360 0.618 1.40255E-05 -11.1746 71298.66
375 0.609 1.38213E-05 -11.1893 72352.33
390 0.6 0.000013617 -11.2042 73437.61
405 0.591 1.34127E-05 -11.2193 74555.95
420 0.582 1.32085E-05 -11.2347 75708.88
435 0.574 1.30269E-05 -11.2485 76764.06
450 0.565 1.28227E-05 -11.2643 77986.85
465 0.557 1.26411E-05 -11.2786 79106.95
480 0.551 1.25049E-05 -11.2894 79968.36
495 0.545 1.23688E-05 -11.3003 80848.75
510 0.537 1.21872E-05 -11.3151 82053.2
525 0.53 1.20284E-05 -11.3282 83136.92
540 0.529 1.20057E-05 -11.3301 83294.08
555 0.515 1.16879E-05 -11.357 85558.39
570 0.509 1.15518E-05 -11.3687 86566.93
585 0.502 1.13929E-05 -11.3825 87774.04
600 0.495 1.1234E-05 -11.3966 89015.29
 615 0.488 1.10752E-05 -11.4108 90292.15
630 0.481 1.09163E-05 -11.4253 91606.17
645 0.475 1.07801E-05 -11.4378 92763.3
660 0.469 1.0644E-05 -11.4505 93950.04
675 0.462 1.04851E-05 -11.4656 95373.53
690 0.456 1.03489E-05 -11.4786 96628.44
705 0.45 1.02128E-05 -11.4919 97916.82
720 0.444 1.00766E-05 -11.5053 99240.02
735 0.438 9.94041E-06 -11.5189 100599.5
750 0.432 9.80424E-06 -11.5327 101996.7
765 0.426 9.66807E-06 -11.5467 103433.3
780 0.42 9.5319E-06 -11.5609 104910.9
795 0.415 9.41843E-06 -11.5728 106174.9
810 0.409 9.28226E-06 -11.5874 107732.4
825 0.403 9.14609E-06 -11.6022 109336.4
840 0.397 9.00992E-06 -11.6172 110988.8
855 0.392 8.89644E-06 -11.6299 112404.5
870 0.386 8.76027E-06 -11.6453 114151.7
885 0.381 8.6468E-06 -11.6583 115649.8
900 0.375 8.51063E-06 -11.6742 117500.2

Sources
Title picture: picture from http://2015.igem.org/Team:Oxford/Protocols
Figure 2: picture from http://www.made-in-china.com/showroom/5795517/product-
detailyelQOPvclqYo/China-UV-Visible-Spectrophotometer.html
All other figures are original.