Chem 161
Fall, 2011
Lab C: Identification of Factors Affecting Rate of Oxidation of
Ascorbic Acid in RO Water and in Orange Juice
Prelab Assignment to be done in a Group or Individually. Groups 3 and 4 will begin the
experiment on Sept. 29. Groups 1 and 2 will begin on Oct. 13.) Please turn these in by noon.
Week 1:
1. Describe how you will prepare 250 mL of 2000 ppm ascorbic acid.
2. Calculate the expected absorbance at 244 nm from 20 ppm ascorbic acid. Use Beer's Law and
the molar absorptivity given in the introduction. The cell path, b, is 1.0 cm.
3. Calculate the volume of 12 M HCl to measure to prepare 500 mL of 1M HCl.
4. Calculate the mass of NaOH required to make 100 mL of 1 M NaOH.
5. Calculate the weight of CoCl2 · 6H2O (s) needed to prepare 200 mL of 100 ppm Co
(1.7 x 10-3 M Co).
6. Calculate the weight of Cu(NO3)2 · 3H2O (s) needed to prepare 200 mL of 100 ppm Cu (1.6 x
10-3 M Cu).
7. Manager only: Identify the group member who has been notified that his or her first job will
be to gather glassware that needs to be acid soaked.
Week 2:
1. Calculate the expected concentration (ppm and molar) of ascorbic acid in orange juice as
prepared for normal drinking. Assume that the nutrition label says that a serving supplies 130%
of the recommended daily amount of ascorbic acid. Assume that a serving is 8 fluid ounces and
that 100% of the recommended daily amount is 60 mg.
2. Describe how you will prepare the 10 ppm ascorbic acid standard prepared for constructing
the calibration curve. Begin with solid ascorbic acid.
INTRODUCTION
Ascorbic acid occurs naturally in fruits and vegetables. Dietary intake of this substance
prevents scurvy. Ascorbic acid is a reducing agent, and it therefore reacts with molecular
oxygen, O2, to form dehydroascorbic acid (Figure 1, next page). At low temperature and low pH,
the rate of the reaction is slow. Cool solutions of acidic ascorbic acid are relatively stable, with
only slight reaction occurring over a period of weeks, even in the presence of 0.2 atm oxygen.
The rate increases as temperature increases, and it also increases in the presence of catalysts such
as transition metal ions and hydroxide ion.
Because ascorbic acid absorbs light strongly in the ultraviolet
(ε = 9.8 x 103 M-1 cm-1 at 244 nm, pH = 1), molecular absorbance spectrometry is a natural
method for determining its concentration in solution. Unfortunately, the non-ascorbic acid part of
orange juice (i.e., the matrix) also absorbs strongly over a wide wavelength range in the
ultraviolet, and UV molecular spectrophotometry is not highly specific. Thus, absorbance at 244
nm is related to the concentrations of ascorbic acid and many other substances.
Fortunately, there is a way to find out how much of the absorbance is due to ascorbic
acid. This is done by creating an instrumental blank that contains the matrix of orange juice with
zero ascorbic acid, i.e., an analyte-free blank. When the instrument is blanked with such a
solution, the absorbance of an orange juice sample at 244 nm is due only to ascorbic acid, at a
first approximation. This solves a major part of the matrix problem but not all of it because the
chemical composition of the matrix depends on the history and age of the orange juice. (You can
be sure that week old orange juice held at room temperature is chemically different from
refrigerated orange juice in more ways than just the ascorbic acid concentration.) The slightly
different matrix spectra detract from the information content about ascorbic acid at 244 nm.
Figure 1: Reaction of Ascorbic Acid with Dioxygen
OH
OH
O
O
OH
O
OH
O
+ H2O
+ 1/2 O2
O
OH
OH
ascorbic acid
O
dehydroascorbic acid
A second method of getting past the lack of selectivity is to use the first derivative of the
absorbance (dA/dλ) rather than the absorbance as the signal. The intensity of the derivative
signal is a linear function of the ascorbic acid concentration. The first derivative of absorbance
tends to be relatively insensitive to the baseline variations that you can expect to see in your
samples. For, example, the first derivative spectra for six different concentrations of saccharin
can be used to illustrate how this might be done.
Figure 2: Derivative UV Spectra of Saccharin at 0, 2.5, 5, 6.25, 7.5, and 10 ppm
0
d1(Abs orbanc e)
-0.01
-0.02
-0.03
5 ppm
-0.04
-0.05
-0.06
7.5 ppm
-0.07
10 ppm
-0.08
215
220
225
230
235
240
245
Wavelength (nm)
The signal at 237 nm (and at 229 nm and 223 nm) is a linear function of concentration.
A third method for enhancing reliability is to use dual wavelength spectrophotometry
(remember this from the NiEDTA experiment). A baseline wavelength is chose, where the signal
is independent of the concentration of analyte. For the derivative spectra of saccharin, the signal
at 234 nm (or at 227 nm) would be a good first choice for a baseline signal.
Thus, the combined approach of using an analyte-free blank and first derivative
spectrophotometry with baseline correction allows you to do a rapid analysis of a complex
mixture. While the results will be less reliable than with a chromatographic approach, they will
be sufficiently reliable to solve the problem at hand (see the next paragraph). The
spectrophotometric approach is at least an order of magnitude faster than the chromatographic
approach.
This experiment is an introduction to screening designs. The goal of screening is to
identify the few variables that are important, out of the many candidates that might be
hypothesized early in a study. In this study, you will be looking for very large effects of active
variables, so that high precision is unnecessary. Thus, you can use a rapid, but nonselective
method of analysis, derivative UV absorbance spectrophotometry.
You can appreciate some of the sources of difficulty when using UV spectrophotometry
by examining the spectra in Figure 3. While the absorbance spectrum of ascorbic acid in dilute
hydrochloric acid has a nearly ideal shape (curve a), the spectrum of ascorbic acid in aged,
acidified orange juice can look suspiciously non-ideal (curves b and c). While "b" is imperfect,
the spectrum is quantitatively useful. In contrast, the presence of the large absorbing band at 300
nm in "c" would make me suspicious that an ascorbic acid concentration calculated from the
absorbance at 244 nm would be biased. There was mold growing in the very dark orange juice
that gave spectrum "c". (If you needed to have highly accurate results for this experiment, you
would do high performance liquid chromatography, and you would spend at least an hour doing
the sample filtration and instrumental measurements.)
Figure 3: UV Spectra of Ascorbic Acid in Different Matrices
Legend
a. Ascorbic acid in 0.1 M
HCl
b. Week-old OJ #1
c. Week-old OJ #2 with
metabolic product present.
Ascorbic Acid in Water and OJ
a
Absorbance
0.4
c
b
0.2
0.0
205
-0.2
230
255
280
Wavelength (nm )
305
330
OVERVIEW
Important Chemical Facts:
• The reaction between ascorbic acid (vitamin C) and molecular oxygen (O2) is spontaneous,
but slow in the absence of a catalyst.
• The hydroxide ion, OH- (aq), and transition metal cations are reported to be catalysts for the
reaction.
• The UV spectrum of ascorbic acid changes with pH, but the changes are very small when the
pH is near 1 (0.1 M HCl). If all solutions taken for absorbance spectrophotometry are near
pH 1, the spectra will be similar in profile and the intensity with vary linearly with ascorbic
acid concentration.)
Overall Goals:
• Week 1--Use factorial design and UV spectrophotometry to identify factors that accelerate
the O2 oxidation of ascorbic acid in RO water.
• Week 2—Study four factors that could affect the reactivity of ascorbic acid in orange juice.
New Unit Operations Used: Centrifugation, oxidative preparation of analyte-free matrix
(blank), first derivative molecular absorbance spectrophotometry, factorial design.
Quality Control:
• You will prepare two 20 ppm ascorbic acid controls during week 1 to verify that your bench
methods were not seriously in error.
• All absorbance measurements will be made in completely random order. During week two,
you will make measurements of analytical standards in one random sequence and
measurements of orange juice samples in two separate sequences because the instrumental
blanks will be different.
Group Lab Considerations:
During week one, half of the group will be preparing orange juice samples that will be
reacting for a week. The other half of the group will be doing the solution preparation for the
catalyst experiment. The entire group will reconvene for the spectrophotometry part of the
catalyst experiment.
During week two, everyone will be involved in bench and instrumental chemistry
associated with measuring ascorbic acid concentration in orange juice. You will all prepare
individual unknowns for analysis.
The operations done in week two are many and involved. There are many possibilities for
forgetting to do a step of the planned operations. When a well prepared group does this
experiment, it takes 3 to 3.5 hours to complete the entire experiment. The major cause of delay
could be preparation of the analyte free instrument blanks. Get going on this preparation as soon
as you have samples of centrifuged “fresh orange juice”.
EXPERIMENTAL
Safety Considerations Specific to this Experiment:
12 M hydrochloric acid (“concentrated” HCl) is corrosive and a health hazard.
Highly irritating and water soluble HCl (g) is emitted from 12 M HCl. Work in the hood. Wear a
face shield and goggles. Use gloves while working with the 12 M HCl. The MSDS is available at
the J. T. Baker Web site at http://www.avantormaterials.com/search.aspx?searchtype=msds
and in the MSDS folder in Seaver North 6.
The aqueous 0.1 M HCl and 1 M HCl are corrosive and irritating. They may be used in
the laboratory as the amount of HCl (g) emitted is minimal. HCl spills may be neutralized with
NaHCO3.
Solid and aqueous sodium hydroxide (NaOH) are caustic. The MSDS for NaOH is also
available on the web and in the lab.
The hot plate and the hot water used to heat samples can cause burns.
The centrifuge must be used with the cover closed. Allow the rotor to stop spinning
before opening the cover. Opposing pairs of centrifuge tubes must be equal in weight to avoid an
unbalanced spin that could cause rotor failure. Details of centrifuge operation are in appendix 3.
Contamination Avoidance:
All glassware, except for cuvettes, that is in contact with ascorbic acid, must be treated to
avoid contamination from transition metal cations, especially Cu2+ (aq) and Fe3+ (aq). Because
solutions of transition metal cations are common in undergraduate labs, you must acid soak the
inside of your glassware to remove them by an ion exchange process where H3O+ (aq) displaces
them from the surface of the glass. To accomplish this goal, all glassware should be rinsed three
times with RO water, soaked for 20 minutes with 0.1 M HCl, and rinsed three times with RO
water.
Standards and Stock Solution Preparation:
Week 1:
Clean the inside surfaces of all your glassware with 0.1 M HCl and RO water. The
manager should assign this job in advance so that the group member can determine in advance
what glassware needs to be acid rinsed. We will supply a large volume of 0.1 M HCl to do this
job.
Prepare 500 mL of approximately 1 M HCl. This solution will be used both weeks.
Caution: Work in the hood. Wear a face shield, goggles, and gloves. 12 M HCl is very reactive,
poisonous, and volatile.
• Add approximately150 mL of water to an acid cleaned 500 mL Erlenmeyer flask.
• Measure the desired volume of 12 M HCl using an appropriately sized, acid cleaned
graduated cylinder.
• Transfer the 12 M HCl to the water in the 500 mL Erlenmeyer flask. Swirl to
homogenize.
• Add water until the final volume is approximately 500 mL. Homogenize the solution by
pouring into another acid cleaned 500 mL Erlenmeyer flask. Label the flask
appropriately.
•
Rinse the graduated cylinder used for 12 M HCl with water before you remove it from
the hood. Otherwise the HCl fumes may irritate someone in the lab.
Prepare 250 mL of a solution that contains roughly 2000 ppm ascorbic acid and 0.1M
HCl. Use solid ascorbic acid and 1 M HCl. This preparation does not have to be made to the
quality of a primary standard, as it will be used for comparative, rather than absolute,
measurements. If you weigh to the nearest 10 mg and dilute casually in an acid cleaned Class A
volumetric flask, you will get fine results and have a container with an air-tight lid. The
concentration of HCl is not critical. (If you were "off" by 20% in the HCl concentration, it would
have no observable effect on the spectra.)
You will dispose of this solution at the end of the first lab session. Rinse the flask well
and keep it handy for use during the second week when you prepare 2500 ppm primary standard
ascorbic acid in 0.1 M HCl.
Prepare 100 mL of 1 M NaOH, starting with solid NaOH. Caution: Solid NaOH is
caustic. Do not handle the pellets with your hands. Use a top-loader balance rather than an
analytical balance. The preparation can be done in an acid cleaned 125 mL Erlenmeyer flask.
This solution should not be stored with a ground glass stopper. Strong base reacts slightly with
glass, and the ground glass joint can be “cemented” shut.
Prepare 200 mL of 100 ppm cobalt (II) (1.7 x 10-3 M Co2+), starting with
CoCl2 · 6H2O (s). Use a top-loader balance rather than an analytical balance. The preparation can
be done with graduated cylinders. These graduated cylinders do not have to be acid rinsed. This
solution will be used during week 1 to determine if cobalt (II) can be used as a catalyst in the
formation of an analyte free instrumental blank.
Prepare 200 mL of 100 ppm copper (II) (1.6 x 10-3 M Cu2+), starting with
Cu(NO3) · 3H2O (s). Use a top-loader balance rather than an analytical balance. The preparation
can be done with graduated cylinders. These graduated cylinders do not have to be acid rinsed.
Week 2:
Clean your glassware, as needed, with 0.1M HCl and RO water.
Prepare 100 mL of 2500 ppm primary standard ascorbic acid in 0.1 M HCl. Use solid
ascorbic acid, 1 M HCl and RO water. The solution need not be 2500 ppm on the nose, but you
must know its concentration accurately. Weigh to 0.1 mg, dilute carefully in an acid cleaned
class A volumetric flask. The concentration of HCl is not critical.
Then dilute this solution accurately by pipetting a 4 mL aliquot into an acid cleaned 50
mL volumetric flask. Add HCl and water so the final solution is 0.1 M HCl and 200 ppm
ascorbic acid.
Finally, use the 200 ppm ascorbic acid solution to prepare six working standards in the
range of 0 to 10 ppm ascorbic acid, with all six solutions containing 0.1 M HCl. Again, the
concentration of HCl is not critical.
Sample Preparation:
Week 1: Preparations to investigate factors affecting the rate of oxidation of ascorbic
acid.
Prepare a warm water bath, large enough to contain four 125 or 250 mL Erlenmeyers.
The temperature should be between 40 and 50oC.
Label ten acid-cleaned Erlenmeyer flasks and identify them with numbers 1 through 10.
Add 79 mL of water to each flask (100 mL graduated cylinder). Pipette 1.00 mL of 2000 ppm
ascorbic acid into each flask.
Add 10 mL of 1M HCl and 10 mL of water to flasks 9 and 10. (Both flasks get the HCl
and the water.) Stopper these and set them aside to be used as 20 ppm ascorbic acid controls. The
ascorbic acid should be stable in these flasks.
Treat samples 1 through 8 according to Table 1. Please read the written instructions all
the way through to the end before you begin preparations to get these 8 solutions! (This is a
standard design for a three factor, two level factorial design experiment. The rational for two
level factorial designs is given on page V-11.) Note that each solution will have 20 ppm ascorbic
acid when ultimately diluted to 100 mL, prior to any reaction taking place. Variations of a few
percent in concentration (i.e. a few percent in final volume) are not important since you are
expecting to see large differences in ascorbic acid concentration due to the effect of any active
variables.
Table 1: Design for Solutions Made Week 1
Factor A
Factor B
Sample #a
1 M NaOHb 100 ppm Co2+ b additional waterb,c
1
2
3
4
5
6
7
8
0 mL
1 mL
0 mL
1 mL
0 mL
1 mL
0 mL
1 mL
0 mL
0 mL
5 mL
5 mL
0 mL
0 mL
5 mL
5 mL
10 mL
8 mL
5 mL
3 mL
10 mL
8 mL
5 mL
3 mL
Factor C
Temperature,
o
C
20
20
20
20
45
45
45
45
a
Samples 9 and 10 are the 20 ppm ascorbic acid controls. See section titled “Standards and
Stock Solution Preparation”
b
Add these volumes to 79 mL water plus 1 mL 2000 ppm ascorbic acid
c
The additional water is not a variable. These volumes are added to make the volume of each
solution 100 mL after the final addition of HCl.
Swirl all eight samples well to homogenize them and to ensure a high concentration of
dissolved oxygen. Place samples 5, 6, 7, and 8 into the warm water bath. Swirl all samples every
five minutes to ensure oxygenation. After 30 minutes, cool samples 5, 6, 7, and 8 to room
temperature in an ice bath.
After the 30 minute reaction time, add 10 mL of 1M HCl to flasks 1, 3, 5, and 7
(containing no NaOH). Add 11 mL of 1M HCl to flasks 2, 4, 6, and 8 (containing 1 mL 1M
NaOH). All flasks should now have a volume of approximately 100 mL and a pH of
approximately 1 (0.1 M HCl). Bring the ten samples to the spectrometer.
Week 1: Preparation of orange juice samples:
Read the label on the cans of orange juice. Note any useful facts in your notebook.
Measure eight 47.5 mL portions of Minute Maid orange juice into acid-cleaned 125 or
250 mL Erlenmeyer flasks, labeled 1-8 plus your group name. Measure eight 47.5 mL portions
of Safeway orange juice into acid-cleaned 125 or 250 mL Erlenmeyer flasks, labeled 9-16 plus
group name. Use Table 2 on the next page, which is the standard design for a four factor, two
level factorial design, to tell you how to treat each of the sixteen samples. These labeled samples
will react at either low temperature or high temperature. Half of the samples will be kept cool in
the refrigerator for a week, and the Factor B descriptor will be "refrigerator". The other half of
the samples will react for five days at room temperature and then two days in the refrigerator and
the Factor B descriptor will be "room". Half will be tightly stoppered to exclude molecular
oxygen. Half will be unstoppered.
Week 2: Individual unknown preparation:
Before you plan your strategy and do these operations, consider that your goal will be to
determine the mass percent of ascorbic acid in this mixture of solid ascorbic acid and solid NaCl.
Get an unknown from the laboratory assistant. Note the number in your notebook. This
unknown is a mixture of solid ascorbic acid and solid NaCl. Place 10 mL of 1M HCl and 40 mL
of water in an acid-cleaned 100 mL class A volumetric flask. Weigh by difference and transfer
the entire sample to the flask, swirl to dissolve, and dilute to volume with RO water. Take a 2
mL aliquot of the sample and dilute to volume in a 50 mL class A volumetric flask. Use HCl and
water to make the final solution 0.1 M in HCl. Take a 4 mL aliquot from the 50 mL flask and
transfer it to a 100 mL class A volumetric flask. Add HCl and water so that the final solution is
about 0.1 M HCl. The solution in this 100 mL flask will be taken to the spectrophotometer.
(Again, the concentration of HCl need not be adjusted exactly.)
Table 2: Design of Orange Juice Experiment
Factor A
Additional
Factor B
Sample
Volume of 100
volume of
ppm Cu2+ (mL) a
water (mL) Temperature
Factor C
Tight
Stopper
Factor D
no
no
no
no
yes
yes
yes
yes
no
no
no
no
yes
yes
yes
yes
Minute Maid
Minute Maid
Minute Maid
Minute Maid
Minute Maid
Minute Maid
Minute Maid
Minute Maid
Safeway
Safeway
Safeway
Safeway
Safeway
Safeway
Safeway
Safeway
Brand
a,b
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
0
2.5
0
2.5
0
2.5
0
2.5
0
2.5
0
2.5
0
2.5
0
2.5
2.5
0
2.5
0
2.5
0
2.5
0
2.5
0
2.5
0
2.5
0
2.5
0
refrigerator
refrigerator
room
room
refrigerator
refrigerator
room
room
refrigerator
refrigerator
room
room
refrigerator
refrigerator
room
room
a
Add these volumes to 47.5 mL of orange juice
The additional water is not a variable. These volumes are added to make the volume of each
solution 50 mL
b
Week 2: Orange juice sample centrifugation, dilution, and acidification before
absorbance measurements.
Examine the sixteen orange juice samples that you prepared last week. It is likely that
water evaporated from the unstoppered samples. Use a graduated cylinder and RO water to bring
all solutions back to a total volume of 50 mL.
Centrifuge 12.0 mL portions of each of the sixteen orange juice samples (and two
duplicated “fresh orange juice samples”—see two paragraphs down). Instructions are in
Appendix 3. The centrifugation should give a good size pellet at the bottom of the tube, although
the supernatant may still be cloudy. The spectrophotometer can handle the relatively small
amount of suspended solids, so longer centrifugation is not necessary or desirable.
Pipette 1 mL aliquots of the supernatant liquid into 100 mL volumetric flasks. The
suspended solids in the supernatant will make pipetting less accurate than you will like. In the
context of this experiment, you can assume that the pipetting errors are small. Add
approximately 50 mL of water and 10 mL of 1M HCl. Dilute to the mark with water.
Week 2: Duplicate Fresh Orange Juice Samples.
Centrifuge duplicate portions of freshly prepared Minute Maid orange juice and duplicate
portions of the freshly prepared Safeway orange juice. (Freshly prepared orange juice samples
will be available.)
Both of the duplicates will be used as samples for chemical analysis. Pipette 1 mL
aliquots of the supernatant liquid from each of the four centrifuge tubes into 100 mL volumetric
flasks. Add approximately 50 mL of water and 10 mL of 1M HCl. Dilute to the mark with water.
This will give you two samples for fresh Minute Maid and two samples for fresh Safeway orange
juice.
You will also use a 1 mL aliquot of Minute Maid and a 1 mL aliquot of Safeway orange
juice to prepare analyte-free instrumental blanks. See the next paragraph.
Week 2: Preparation of two analyte-free instrumental blanks.
Your results from last week should enable you to devise a method that will cause the
rapid oxidation of ascorbic acid in diluted orange juice. The method that you use should be the
mildest method that will do this. (If you do a harsh treatment, other solutes in the sample are
more likely to be chemically transformed, affecting the absorbance of the blank. To the
maximum extent possible, you want to react only ascorbic acid.)
For each of the orange juice types, pipette a 1 mL aliquot of the supernatant liquid of the
centrifuged fresh orange juice into a 125 mL Erlenmeyer flask. Add 70 mL of water. Then add
an appropriate amount of either NaOH or Co2+ (aq). If your results suggest that heating is
necessary, heat the two portions gently. Allow the oxidation to occur for 30 minutes, being
certain to swirl the solution frequently. Add 10 mL of 1M HCl and dilute with RO water to a
final volume of 100 mL.
Bring the six standards, the twenty orange juice samples, the four individual unknown
preparations, the two analyte-free blanks, a waste beaker, and a squeeze bottle of R.O. water to
the Cary 50 spectrometer in SN 217.
Molecular Absorbance Spectrometry:
General instructions for operating the Cary 50 UV-VIS spectrophotometers are on page
II-11. Specific instructions for derivative spectrometry (and for using the Cary 300) are in
Appendix 1. Set up to acquire data from 220 nm to 300 nm. Use quartz (not glass or plastic)
cuvettes.
Week 1 Ascorbic Acid samples:
Blank the instrument with RO water. (A blank of 0.1 M HCl would give equivalent
results. Pure water is simpler.) Obtain spectra for the ten ascorbic acid samples in random order.
Duplication is not necessary. Store these spectra on the hard drive in the 161f11/ascorbic
folder. Back up the files on an external device. Print the overlaid spectra. Export the numerical
data as a CSV file.
Use the Math function to calculate the first derivative spectra. Determine a wavelength to
use to measure the concentration of ascorbic acid. The best wavelength is the one where the
derivative signal varies most strongly with concentration of ascorbic acid. Determine a
wavelength for baseline correction in the first derivative spectra. A good wavelength for the
baseline is one where the derivative signal changes least with change in ascorbic acid
concentration. This examination of first derivative spectra will help get ready for observing first
derivative spectra of orange juice samples during week two. Export the numerical data as a CSV
file.
Week 2 Analysis of Orange Juice and Individual Unknown Samples:
NOTE!!--When you select wavelengths, make sure you understand how this will extract
the information available in the raw spectral data. See Prof. Taylor or Prof. Stolzberg for
suggestions if the strategy is not clear.
Set up the instrument to measure absorbance.
Blank the instrument with RO water. Obtain spectra for the six standards and the
individual unknowns in random order. Consideration of good experimental design suggests that
it is worth duplicating the absorbance measurements of the unknowns. Examine the data to be
certain the calibration is good and to verify the analytical wavelength. (If there is a problem, get
help.) Store these absorbance spectra in the 161f11/ascorbic folder. Print the overlaid spectra,
(or copy and paste to Word and email the file for printing later.) Export the numerical data as a
CSV file. These absorbance data will be used for analysis of the individual unknowns.
Use the Math function to calculate and display the first derivative spectra. Determine a
wavelength to use to measure the concentration of ascorbic acid. Determine a wavelength for
baseline correction in the first derivative spectra. If you export the data again as a CSV file, the
derivative data will be exported along with the absorbance data. .
Clear the spectra. Blank the instrument with the Minute Maid analyte-free blank. Obtain
absorbance spectra for the two fresh Minute Maid unknowns and the eight samples made with
Minute Maid orange juice (#1 through 8) in random order. Duplication is not necessary. Print the
overlaid spectra. (You might need to refer to these to determine if any of the spectra look like
example c in Figure 2.) Export the numerical data as a CSV file.
Use the Math function to calculate and display the first derivative spectra for Minute
Maid samples 1-8. Print the overlaid spectra. Copy and paste the spectra into a Word file. Export
the numerical data as a CSV file. The file will contain the absorbance and the first derivative
data.
Clear the spectra. Blank the instrument with the Safeway analyte-free blank. Obtain
absorbance spectra for the two fresh Safeway unknowns and the eight samples made with
Safeway orange juice (#9 through 16) in random order. Duplication is not necessary. Print the
overlaid spectra.
Use the Math function to calculate and display the first derivative spectra for Minute
Maid samples 1-8. Print the overlaid spectra. Copy and paste the spectra into a Word file. Export
the numerical data as a CSV file. The file will contain the absorbance and the first derivative
data.
Clean up the area. Wash the cuvettes with soapy water and rinse with R.O. water.
Orange juice and ascorbic acid may be disposed of in the sink.
Wash glassware that contained orange juice with soap and water.
Plastic and centrifuge tubes may be disposed of in the trash.
RAW DATA WORKUP
Ascorbic Acid Experiment--Week 1:
Examine the overlaid spectra. Examine the absorbance data. See if there are any
conditions that give very high absorbance (no reaction) or very low absorbance (complete
reaction) at 244 nm. See if you can figure out what variables affect degree of reaction.
Make three "pair-wise comparison" plots of absorbance versus level of one variable (4
cold results and 4 hot results, 4 results with NaOH and 4 results without NaOH, 4 results with
Cu2+ and 4 results without Co2+.) Because you are looking for conditions that cause nearly
complete oxidation of ascorbic acid, you should not have to split hairs. Identify which of the
three variables are active. These active variables will be used to prepare instrumental blanks
during the second week.
Individual Unknowns--Week 2:
Examine the overlaid individual unknown and standard spectra. Examine the absorbance
data.
The quantitative analysis is done with external standards of 0 to 10 ppm ascorbic acid.
Construct an absorbance Beers Law plot with the six standards, and calculate the concentration
of ascorbic acid in the final unknown solutions using cal161.xls.
Use stoichiometry to determine the mass percent of ascorbic acid in the original solid
unknown sample.
Orange Juice Experiment--Week 2:
Examine the two sets of ten overlaid derivative and two sets of ten overlaid absorbance
spectra. Critically evaluate if all of the spectra appear reliable. Examine the numerical derivative
data.
The quantitative analysis is done with external standards of 0 to 10 ppm ascorbic acid.
Construct a derivative Beers Law plot with the six standards, and calculate the concentration of
ascorbic acid in the twenty diluted orange juice samples using cal161.xls. Use stoichiometry to
calculate the concentration of ascorbic acid in the original undiluted orange juice samples.
Graphically analyze the data for the sixteen treated orange juice samples. (Order them
into groups of 8 cold vs. 8 warm, 8 stoppered vs. 8 unstoppered, etc.). If the two groups of eight
look like those in Figure 3a, you could infer that the presence of Cr had the effect of causing a
smaller “result”. That is, Cr would be an interesting experimental variable. On the other hand, if
the results looked like Figure 3b, you would not believe that Zn is a variable that affects the
result.
After you have visually examined your data, use the Design Expert 6 (DE6) software to
generate normal probability plots and to identify outliers. Normal probability plots are explained
on page V-13 to V-16. Operating instructions for the DE6 software are in Appendix 2.
Figure 3. Examples of Active (a) and Inactive (b) Variable Results
Figure 3a
10
Figure 3b
150
result
result
0
No Cr
Cr Present
0
No Zn
Zn Present
REPORT--Due 5:00 PM, Oct. 11 (groups 3 & 4) or Nov. 1 (groups 1 & 2)
You should address the eight specific questions listed below. Note that questions 2 - 6
ask you to interpret the results of the orange juice experiment. Write an appropriately detailed
Results and Discussion section, including figures, and text. Although there are eight numbered
items, the report should not look like the answer to eight individual homework problems. Try to
make links where appropriate.
1. What did you discover about the effects of NaOH, Co2+, and temperature on the reactivity of
ascorbic acid and O2 in RO water?
2. Did the Cu2+ (aq) catalyze the oxidation of ascorbic acid in orange juice?
3. How did temperature affect ascorbic acid reactivity in orange juice?
4. Did the free access to atmospheric O2 increase the amount of oxidation of ascorbic acid over
the week-long reaction time? (This is the “stopper” effect.)
5. Is the ascorbic acid equally reactive in the two brands of orange juice?
6. Is there evidence of an interaction of two variables? Which ones? Can you make chemical
sense of any observed interactions?
7. Report the measured concentration and standard deviation of ascorbic acid in the two types
of freshly-made orange juice. Is the consumer getting at least the amount promised on the label
of the can of frozen concentrate? Is the consumer getting a better deal (amount of ascorbic acid
per dollar) with Safeway or with Minute Maid?
8. For the individual unknowns, make a table with each unknown number, the analyst, the total
mass of each sample, the mass of ascorbic acid in each sample, the mass % ascorbic acid, and the
95% confidence interval of the mass % ascorbic acid in each sample.
9. Make four honest estimates of the expected accuracy of each determination. Base this on
each member's perception of how he or she did the preparations. Any group that has all members
achieve suitably accurate analyses will get five bonus points in addition to the total of 100 points
available. While this is an evaluation of individual ability, the group as a whole will benefit if
every member of the group has learned from the others. I will tell you the true values when I
return the lab reports.
For numbers 2 - 6, try these preliminary operations. Make a tabulation of the concentrations
of ascorbic acid in the sixteen samples of orange juice. Use the four pairwise comparison graphs,
normal probability plots, and effects plots from DE6 to help you answer questions 2 to 6 and to
convince the reader. (The DE6 results should be in agreement with your pairwise comparison
plots. There may be subtleties of the data or interactions of variables that are detected with DE6
that are not detected with simple pairwise comparison plots.) Attempt to put your observations in
the context of what you know about stoichiometry, kinetics, and catalysis from other Chemistry
courses and from your first weeks' experiment. If a result is as expected, note that. If a result is
surprising, tell the reader why. (Note that a saturated solution of air in water gives a molar
concentration of O2 of about 0.001 M.)
Please review pages IV-3 to IV-5 of the laboratory manual, Guidelines and Instructions
for Group Lab Reports. Pay particular attention to the "mechanical details" which begin at the
bottom of page IV-4. If the cover sheet is incomplete or if we do not have your data files, we
cannot grade the lab effectively.
Please email both faculty the following:
• easily understood, succinct spreadsheets used to make final interpretations of the data.
• the original spectral data files that were used to prepare the report.
Please name them suitably so we do not have to guess what they contain and who made them.
Use an appendix in the printed copy to show us your statistical calculations/inference and
your calculations for questions 7 and 8. Feel free to hand write the appendix if this will be easier
than doing it at the keyboard.
The printed copy of the report is due Oct. 25 (groups 3 & 4) or Nov. 3 (groups 1 & 2)
(Oct 11, 2011)
Appendix 1 for Experiment C
Acquiring and Derivatizing Spectra of Ascorbic Acid with the Cary 50
Data acquisition will be similar to that for NiEDTA, with the following changes in Setup:
• In the Cary tab, acquire data from 300 nm to 220 nm.
• In the Cary tab, set scan speed to Slowest (60 nm / min, 0.5 nm data interval, 0.5 s
average time).
To take the derivative of all of the spectra on the display,
• Click on the graph window. This should make all of the spectra “active”.
• Choose Maths on the top bar.
• Click the Focused Graph button.
• In Trace,
• Select Smooth (filter size 11, interval 1)
• Apply (look at top of Maths window as operations stack up)
• Select Deriv1 (filter size 5, interval 1)
• Enter Filter Size (5) and Interval (1 nm).
• Apply (look again)
• In the Display Options group, select New Graph.
• Click on the equals (=) button.
• You will have to repetitively (one per spectrum derivatized) answer a question about the
y-axis label. Enter D1 for the first request. Then enter the default (“?”) choice. You will
get the proper y-axis label.
• The result will be displayed in the Graphics area.
• Copy and paste the absorbance and the derivative graphs into Word and email it to
everyone.
• Export the data as a CSV file. The file will contain absorbance and derivative data sets.
(over for Cary 300)
Acquiring and Derivatizing Spectra of Ascorbic Acid with the Cary 300
Data acquisition will be similar to that for NiEDTA, with the following changes in Setup:
• In the Cary tab, acquire data from 300 nm to 220 nm.
• In the Cary tab, enter scan speed = 60 nm / min, data interval = 0.5 nm, average time =
0.5 s).
• In the Options tab, set spectral band width (SBW) = 2.0 nm, beam mode = Double,
Source = UV.
• In the Baseline tab, choose Baseline Correction
To take the derivative of all of the spectra on the display,
• Click on the graph window. This should make all of the spectra “active”.
• Choose Maths on the top bar.
• Click the Focused Graph button.
• In Trace,
• Select Smooth (filter size 11, interval 1)
• Apply (look at top of Maths window as operations stack up)
• Select Deriv1 (filter size 5, interval 1)
• Enter Filter Size (5) and Interval (1 nm).
• Apply (look again)
• In the Display Options group, select New Graph.
• Click on the equals (=) button.
• You will have to repetitively (one per spectrum derivatized) answer a question about the
y-axis label. Enter D1 for the first request. Then enter the default (“?”) choice. You will
get the proper y-axis label.
• The result will be displayed in the Graphics area.
• Copy and paste the absorbance and the derivative graphs into Word and email it to
everyone.
• Export the data as a CSV file. The file will contain absorbance and derivative data sets.
Appendix 2 for Experiment C
Using Design-Expert 6 for Interpreting the Orange Juice Factorial Design Experiment
To load the software
From the desktop, click on Start, Programs, Stat-Ease, Design-expert 6, OK.
You should see the Stat-Ease logo and an option for a new design or an existing design.
To make the design
At the tool bar, click on File, New Design.
In the Factorial tab, choose 2-Level Factorial.
Click on the design matrix at the point where your number of experiments and your
number of factors (variables) intersect. For the orange juice experiment, you have 16
experiments and four factors.
Choose 1 replicate, 1 block, and 0 center points per block.
Click on Continue. This will get you to the 2-Level Factorial Design screen.
(If you need to get back to the design matrix, press the Back button.)
To finish and print design information
Click on Continue.
Fill out the table with information about the variables. Replace A, B, C, D with good
descriptive names: Vol. Cu (mL), Temperature, Stopper, Brand. The data type can be chosen as
either numerical (volume Cu) or categorical (other three variables). Click on Continue.
Fill out the table with information about the number of responses (one) and the name and
units of the response (Conc. Asc. Acid, ppm). Click on Continue.
The design matrix should appear on the display. It will originally appear in “Run Order”,
which is the random order in which the experiments should be run. Change the design to
"Standard Order" by clicking on View, Standard Order. Verify that the design is equivalent to
that in Table 2, page VIII-9.
To get a summary of your design, move the cursor to the left-most window, and click on
Status (under Design).
Save your design using File, Save As.
Print any interesting screens with File, Print. (Do this throughout the processing of your
data.)
To Enter Responses
Click on Design in the left-most window.
Click on View and be certain that Design Layout and Standard Order have been checked.
Enter your sixteen ascorbic acid concentrations in the right-most column labeled
Response.
Save your design and results.
Analyzing the Results with a Normal Probability Plot of Effects
In the left hand frame, click on the name of your response (Conc. Asc. Acid).
Click on Transform at the top of the screen, and choose “none” as the transform.
Click on Effects. You will see a "half-normal" probability plot. Change this to a "normal"
probability plot by clicking on View, Normal Graph. If there is a warning that the model is not
hierarchical, ignore it. If there is a warning about aliases, ignore it.
Examine the normal probability plot. Identify significant factors by looking for ‘outlier”
effects that are especially large or small compared to the swarm of nonsignificant effects on a
line near zero effect size. You can toggle the effects on and off by clicking on the point. The
chosen active factors (colored symbols) will be used later in models, plots, and diagnostics.
Click on ANOVA. This will give statistical details for the linear model based on the
active factors chosen in the Effects window. Unambiguously significant effects will have
“Prob>F” of less than 0.05. In a screening experiment like this, you might manually choose
factors with “Prob>F” of less than 0.10 (or even larger), if you are in desperate need of
significant factors.
Identifying Outlier Experiments
Click on Diagnostics to hunt down outliers. The normal plot of residuals is a good
starting place. If there are no outliers, the residuals should fall on a straight line centered on zero.
A point in the far upper right or the lower left signifies an outlier experiment. The identity
(experiment number) of any point can be determined with a click of the mouse.
Other choices in the Diagnostics Group should be investigated. An Outlier is indicated by
being far from the rest of the group or by some nonrandom structure in residuals. For example, a
large (3.5 or greater) "outlier t" indicates an experiment that does not seem to be consistent with
the other experiments and the indicated model. See Prof. Stolzberg for suggestions.
If you identify an experiment that appears to be an outlier, you may go back to the Design
folder (left-most frame) and delete the suspect response. Rerun the analysis by clicking on the
name of your response (absorbance, yield, etc.) under Analysis. You will be warned about
missing data. Allow the software to make calculations with the suspect point(s) removed. Look
at the results and continue your interpretation. You should remember that you are doing
preliminary, exploratory data analysis to find some interesting variables.
Making Graphical Interpretations of the Results
Click on Model Graphs to view the effect of variables. The default choice may be all you
need. See Prof. Stolzberg for suggestions of other tools in this part of the software.
For one active variable, you will see an Effect Plot. For two active variables, a Square
Plot would be desirable, if it is available. For three active variables, a Cube Plot will be
displayed. Choose to display the actual (rather than the predicted) response in these plots.
When you have finished, save your file.
Appendix 3 for Experiment C
Instructions for Using the Allegra 6R Centrifuge to De-Pulp Orange Juice
Your goal will be to get most, but not all, of the pulp out of your 20 orange juice samples.
A 10 minute spin at 2000 rpm does the job nicely. Refrigerating the sample at 10oC is a nice
addition.
The centrifuge bucket inserts have a capacity of 14 tubes (not 6 as in the original
instructions), so this operation should not be a bottleneck. (Two inserts are used simultaneously,
so as many as 28 tubes could be centrifuged.)
When you fill centrifuge tubes, each pair* must be balanced for weight. I have been told
a couple of approaches: adjust the volume (12.0 mL of OJ) in the centrifuge tube by eye, using
the volumetric marks on the tube or adjust the weights on the two-pan balance in SN 217. I like
the “adjust carefully by eye” approach, using a Pasteur pipette to make the adjustments. Prof.
Negritto assured me that this is a fine way of balancing tubes. In addition, the total weight of the
two bucket inserts must be within 6 g of each other.
When you place the centrifuge tubes into the two bucket inserts, two criteria must be
satisfied.
• The bucket inserts must be loaded symmetrically with respect to their pivotal axis.
• The rotor should be loaded symmetrically with respect to its center of rotation.
The figure defines some of the terms and gives examples.
Here are three additional warnings gleaned from the instruction manuals:
• Don’t open the door when the rotor is still spinning.
• Don’t lean on the centrifuge or place items on the centrifuge during operation.
• Don’t come within 3 inches of the centrifuge during operation except to adjust the
controls.
*In practice, all 20 tubes should contain 12.0 mL of OJ, so physical pairing of volumes or
weights is unnecessary.
(over)
To prepare and load the centrifuge:
• Verify that the POWER switch is ON.
• Move the manual lock lever to the left (UNLOCK).
• Press DOOR to the OPEN position. Lift up the door.
• Place the two exquisitely balanced bucket inserts into the swinging bucket rotor yokes.
Have Prof. Stolzberg inspect your handiwork before proceeding.
======================================================
• Close the door. Push firmly down until you hear a clicking (latching) sound, indicating
that the door is latched.
• Move the manual lock lever to the right (LOCK).
To start a run:
• Press and hold the ACCUSET button.
• Rotate the SPEED dial to 2000 rpm.
• Select the brake position to HIGH.
• Set the temperature knob to 10oC.
• Turn the time control to 10 minutes. This will start the centrifuge aspinnin’.
• The run will end automatically, with a braking period of less than one minute.
To end a run in progress for any reason, turn the ITME control to OFF.
To liberate your centrifuged samples:
• After the rotor stops spinning, move the manual lock lever to the left (UNLOCK).
• Press the DOOR switch to OPEN.
• Visually inspect the inside of the centrifuge to verify that nothing has broken or spilled.
• Remove the bucket inserts with your centrifuge tubes.
• Remove the centrifuge tubes from the bucket inserts, replace the inserts in the centrifuge,
and close the door.
• If no one else is going to use the centrifuge, turn the POWER to OFF.
Oct 5, 2011