CHEM 312 Lab Manual_2023-7
pdf
School
Concordia University *
*We aren’t endorsed by this school
Course
312
Subject
Chemistry
Date
Oct 30, 2023
Type
Pages
72
Uploaded by AgentMoon9736
Intermediate Analytical Chemistry
CHEM 312 Laboratory Manual
Brigitte Desharnais, Cameron D. Skinner
Summer 2023 revision
Adapted from the 2008 revised version of the laboratory manual. Revised Summer 2023.
2
Table of Contents
Prelabs
............................................................................................................................................
3
Lab Report Instructions
...................................................................................................................
4
Statistics and Data Processing
......................................................................................................
11
Determination of Trace Amounts of Copper and Zinc in an Aqueous Solution by Atomic
Absorption Spectroscopy
..............................................................................................................
14
Determination of Riboflavin by Fluorescence Spectrophotometry
................................................
21
Determination of Aspartame, Caffeine and Benzoic Acid by High Performance Liquid
Chromatography (HPLC)
...............................................................................................................
26
Gas Chromatographic Determination of Isopropanol in a Mixture
................................................
32
Determination of Ascorbic Acid by Differential Pulse Voltammetry
..............................................
38
Coulometric Determination of Arsenate
........................................................................................
47
Quantitative Analysis of Citrus Essential Oils by Gas Chromatography
–
Mass Spectrometry
....
52
3
Prelabs:
You must be prepared to enter the lab. To that end, you will prepare a prelab document
for each experiment to be carried-out. It will have the following sections:
•
Your name & ID
•
Name of the experiment
•
Objectives
One paragraph explaining the experimental objectives and general procedure.
Underline 5 keywords/ideas relevant to carrying-out the lab.
•
Keywords
Prepare one sentence explanations for each of the keywords/ideas
•
Procedures
Prepare a step-by-step overview of the procedure(s). e.g.
Step 1: obtain sample (record #, dry, weigh 3.0g, record weight)
Step 2: prepare stock standard (weigh/record 1.0 g of Zinc, dissolve in 500 mls
10% HNO3, dilute to 1000 ml in volumetric)
Step 3:
etc…
•
Hazards
List known/expected hazards both chemical and physical
Prelabs (
as Word documents
) need to be emailed to
Chem312@concordia.ca
before
9 PM the night before the lab. Put “PRELAB: Experiment #” in the subject, replace the
“#” with your experiment #.
Only one prelab per email. Failure to do this will bar you
from entering the lab.
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
4
Lab Report Instructions
Late submissions will be penalized 10% per day late.
For your all your lab reports, you must use the Word template available on Moodle.
All lab reports (
as Word documents
) need to be emailed to
Chem312@concordia.ca
.
Name your electronic files/documents as:
Last name_ID number_experiment number
(e.g. Skinner_12345678_1)
.
Notice the following:
•
In the title, each “major” word is capitalized but not the minor words (e.g. “Gas
Chromatographic Determination of Isopropanol in a Mixture”). Follow the capitalization
found in the table of contents of this lab manual if you have any hesitation;
•
Pages should be numbered;
•
Section titles should be capitalized and bolded.
Abstract
This should be a concise
1 paragraph summary
of your report, approximately 100 words. It
should describe the goal(s) of the experiment, the techniques used in the analysis (instrumental
and calibration) and the significant findings (LOD, LOQ, number of the unknown and its calculated
concentration with confidence interval).
Example:
In this experiment, a gas chromatograph coupled with a flame ionization detector
was used to determine the concentration of ethanol in an unknown aqueous
sample. An internal standard calibration was performed using four samples of
known concentration. The LOD and LOQ of the method, set at 3 and 10 σ
blank
respectively, were found to be 0.001 and 0.003 mg/mL. The method was
calibrated using a linear regression analysis and the concentration of unknown
#1045 was determined to be (0.700 ± 0.005) mg/mL at the 95% confidence level.
5
Introduction
The introduction should explain the theory behind the experiment. 1-2 pages should be sufficient
(2 pages max)
. This includes a description of the theoretical principles behind the
instrumentation used and how the instrument itself works. This should show clearly how
quantification of the analyte was accomplished. You should also include details on the calibration
method used (external standard, internal standard, standard addition) and the appropriateness of
choosing this method (its advantage(s)).
The introduction should contain no specifics on the experiment itself (e.g. column used, solvent
used, analyte treated or results obtained).
NEW FOR 2023
There will be no Introduction section to any of the lab reports EXCEPT the
“oral” lab report and the electrochemistry lab report (presentation)
.
For the
Introduction section just write “Introduction: Not required”
Experimental Procedure
This section is intended to briefly describe the experimental procedure that was used to obtain
the data. However, since you usually make no changes to the procedure laid out in the lab manual,
you can simply cite it and note any changes from the established procedure.
Example:
As per lab manual (1).
Changes to the established procedure:
The standards concentrations were changed to 0, 0.2, 0.4, 0.6, 0.8 and 1.0 ppm.
Note that the number in parenthesis in the example above refers to the reference number. The
full reference of the lab manual must appear in the "Reference" section of your report, under (1).
Results
This section must include the following:
1)
Original data, in a table
2)
Treated data, in a table (data after calculations/transformations, if applicable)
3)
Calibration graph (including error bars, if possible)
4)
Calibration data, in a table (slope and intercept value, with their associated error)
5)
Unknown concentration and its associated error (confidence interval)
6)
LOD and LOQ
6
Everywhere a calculation is done (e.g. for the treated data, the unknown concentration, its error,
the LOD and the LOQ),
a sample calculation is required
. Building equations in Word documents
is quite easy now (Insert/Equation) also, many of the basic equations can be copy/pasted from
the formula sheet (Word version available on Moodle
is likely to work best, reference…)
So ALL sample calculations must be typewritten (no hand-written equations/calculations,
yes, you can skip over basic steps in a multi-step calculation. The purpose here is to make
sure you’re using the correct equation and variables/values)
Tables and graphs should be titled and numbered. Notice, the convention is to put the caption
above tables and below for figures.
Example:
Table 1: Calibration Data for Isopropanol
Concentration (
%)
Peak Area (mV*min)
Ratio
Methanol
Isopropanol
20
490248
387957
0.791348
25
411197
411094
0.999750
30
411651
480145
1.16639
35
382722
650770
1.70037
Figure 1: Calibration Curve for Isopropanol
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
7
Do not forget to put proper labels on your graph axes, and to include units (inside parentheses).
Your graph title should not be along the lines of "x vs y". This is chemistry not mathematics.
The raw data in the lab book should be photocopied and attached at the end of the lab report.
When reporting raw data (e.g. peak area) or treated data (e.g. ratio), think about how many
significant digits it is appropriate to show. The precision of the measurement can often
help you decide on the appropriate number of significant figures to use. Be aware that
thoughtless reporting of data may result in points being deducted.
Discussion
This section
(2-3 pages)
should explain the purpose and principles of the experiment, the
principles of the method and the treatment of the data as you do it (e.g. Why lithium nitrate was
used as a background support electrolyte? Why was a C18 column rather than a C8 column
used? Why was the solution sonicated? Why the solutions were filtered using ash-free paper?).
Run through the steps and try to explain or justify what needs to be. You should describe how the
data was examined (e.g. for outliers) and processed (e.g. transformations and calculations
performed). Describe the results of the experiment and compare them to the expected results (if
applicable, compare to the theory). You should also discuss the significance of the results.
This section should demonstrate your understanding of the experiment and the theory
surrounding it. Take the appropriate time to get a good understanding of the experiment before
writing this section. Be careful to correctly reference your sources of information.
References
If you are using a source of information to help you prepare your report, even though you are not
quoting this source directly, you must reference it in your lab report. Thus, your textbook and this
laboratory manual should be included in the references of all of your lab reports. If you use the
same words as the source, the text must be in quotes and referenced. Note that the goal of your
lab report is to demonstrate what you have understood, so try to use a limited number of quotes.
Paraphrasing of the source demonstrates your understanding better. In either case, you need to
put a reference number (inside parentheses, brackets, or superscripted) beside the reported
information. Any images, figures, drawings etc. that you have not created also must be
referenced.
8
Example:
High performance liquid chromatography is widely used because of its "sensitivity,
its ready adaptability to accurate quantitative determinations, its ease of
automation, its suitability for separating nonvolatile species or thermally fragile
ones, and above all, its widespread applicability" (1). It is typically coupled with an
ultraviolet detector to allow quantification of most organic analytes (2).
You then need to build a
“References” section at the end of your lab report, to describe what
sources these numbers refer to. Many referencing styles are available and adequate in chemistry.
The goal of a reference is to allow the reader to easily find, for example at the library, the document
you are referring to. It must therefore contain the information required to do so. Here we chose to
demonstrate how to quote using the American Medical Association (AMA) style. If you wish to
use another referencing style, you are welcome to do it, but all the necessary information must
be present.
In general, references for the following require
Books:
Author 1, Author 2.
Title
. Edition. Place of edition: Editor; year: pages.
Example:
James N. Miller, Jane C. Miller.
Statistics and Chemometrics for Analytical
Chemistry
. 6
th
edition. Essex: Pearson Education; 2010: 39-45.
Chapter of a Book:
Author 1, Author 2. Chapter Title. In:
Book Title
. Edition. Place of edition: Editor, year: pages.
Example:
Douglas A. Skoog, F. James Holler, Stanley R. Crouch. Atomic Absorption and Atomic
Fluorescence Spectrometry. In:
Principles of Instrumental Analysis
. 6th ed. Belmont,
California: Thomson Brooks/Cole; 2007:230-253.
9
Scientific papers:
Author 1, Author 2. Paper Title.
Journal Title
. Year; Volume (Issue or number, if available): pages.
Example:
Zhi-Min Zhang, Yi-Zeng Liang, Hong-Mei Lu, Bin-Bin Tan, Xiao-Na Xu, Miguel
Ferro. Multiscale peak alignment for chromatographic datasets.
Journal of
Chromatography A
. 2012; 1223: 93-106.
Website:
Author1, Author 2. Title of the web page. Available at: http://webaddress. Retrieved Month Day,
Year.
Example:
Department of Animal Science of Mc Gill University. Mastitits in Dairy Cows.
Available at: http://animsci.agrenv.mcgill.ca/courses/450/topics/13.pdf. Retrieved
April 24, 2012.
Note that the author can also be an organization (department of a university, company, etc.).
This lab manual:
Brigitte Desharnais, Cameron D. Skinner.
Intermediate Analytical Chemistry
–
CHEM 312
Laboratory Manual
, Concordia University: Montréal, 2012.
Statistics Introduction Document
Cameron D. Skinner.
Statistics and Data Analysis
, Concordia University: Montréal, 2012.
If one element is non-available (e.g. the author of a website), simply leave that field out.
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
10
There is software to allow you to do nearly automatic reference generation. RefWorks is available
to all Concordia Students (see http://library.concordia.ca/help/howto/refworks.php). Zotero is a
free Mozilla Firefox plugin (comes with an accompanying Word plugin), allowing you to do easy
reference storage and quotation. Word also has an included tool for referencing
(References/Insert Citation), but it is a bit longer to use than the others, because you need to
enter the source information yourself. Obviously, you can do your referencing manually, but if you
plan on continuing your studies in science at the graduate level, this will soon become
unmanageable.
Please note that
lab reports will be examined for evidence of plagiarism including using
software.
E
XPERIMENT
1
Statistics and Data Processing
Purpose:
The goal of this experiment is to familiarize yourself with the practice of calibrations, limit of
detection, limit of quantification and measurement uncertainty calculation through error
propagation. The methods you learn in this lab are to be used for all other Chem 312 labs.
Theory:
All of the experiments you will be doing in this course will require some form of data analysis.
Measurements performed in the laboratory always contain a certain amount of uncertainty (noise
or random error, systematic error). When you are calculating the concentration of an unknown
based on measurements (calibration curve), you need to acknowledge the existence of this
uncertainty and its impact on your prediction of the
unknown’s
concentration. Any values that you
report without the appropriate error treatment will be considered worthless in this course.
The theory you need to know to write your lab report (see “Calculations” Section) is available in
the “
Statistics and Data Analysis” package
available from the Moodle site. This material should
also have been covered, in part or in whole, in your class. Your textbook has an extensive chapter
on statistics, and introductory statistics are nicely presented in Harris’ textbook (see References
for details).
NOTE: Check-
out the worked example in the “
Statistics and Data Analysis” package
available
from the Moodle site. You should reproduce this worked example to test to see if you are doing
your calculations correctly then move onto the data provided for this lab!
Materials, chemicals and instruments:
•
Set of data
•
Spreadsheet program (Excel or other)
Procedure:
Solution(s) preparation:
N/A
E
XPERIMENT
1: Statistics and Data Processing
12
Measurements:
Your dataset will be provided on Moodle. This data represents typical measurements that you
will encounter in many of the labs in 312. If you develop your spreadsheet carefully you will be
able to reuse it for all of the other experiments!
Calculations: (before beginning these, READ THE STATS MANUAL)
Use a 95% confidence level for all tests performed, unless otherwise specified.
1) Plot your calibration data (signal vs concentration). Do not forget to title your graph and
your axes.
Be sure to include units. Be generous with the plot since it will help you spot
outliers in your calibration data!
2)
Test for outliers in the replicate data (standards and all samples) using the G test.
3) Eliminate the outliers from your calculations (and plots as needed). Be sure to report
which values were eliminated and justify their elimination.
4) Perform a linear regression on the calibration data to obtain the calibration coefficients
(slope, intercept, and error
–
Excel will provide many parameters, report only those you
use in the calculations below, trim reported values according to significant figures).
5) Calculate the detection and the two quantification limits (see book/lectures for
definitions). Do not forget to include units in your calculations and trim reported results.
6)
Begin your verification of the calibration method by validating that the calibration check’s
average concentration falls within the established acceptance criteria (see dataset for
expected concentration and criteria).
Failure of the calibration check would invalidate all
results in this dataset.
7) Next, verify that the method is producing acceptable results for the average
concentrations for the QClow, QChigh.
Failure of these samples indicates a problem in
sample preparation or the calibration and would invalidate the results in the dataset.
8) Next, determine if there is a significant difference between the established value of the
Certified Reference Material (CRM) and your determination.
A significant difference
indicates a biased analysis in the method and would invalidate the results.
9) Next, calculate the concentrations of the unknown samples, including appropriately
trimmed confidence intervals. (This is your best estimate of where the “true” value sits.
See the example in the stats manual.
This is more than just the value +/- the std. dev.)
Lab report:
Abstract, introduction (2023: Not required), results, discussion and references sections
are required for this lab report.
For the “Experimental” section of your lab report, simply write
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
E
XPERIMENT
1: Statistics and Data Processing
13
that it does not apply here.
Hand in your original data in your report. Send your spreadsheet(s) used for calculations to your
TA.
Clearly explain the purpose of each test you have carried and which conclusion(s) you have
reached after performing each of them.
References:
1.
Harris DC. Experimental Error. In:
Quantitative Chemical Analysis
. 7th ed. New York: W. H.
Freeman; 2007:39
–
52.
2.
Harris DC. Statistics. In:
Quantitative Chemical Analysis
. 7th ed. New York: W. H. Freeman;
2007:53
–
77.
3.
Cameron D. Skinner.
Statistics and Data Analysis
, Concordia University: Montréal, 2012
Exhaust
Waste Drain
Inlet Tube
Flame
E
XPERIMENT
2
Determination of Trace Amounts of Copper and Zinc in an Aqueous Solution by Atomic
Absorption Spectroscopy
Purpose:
The goal of this experiment is to determine the concentration of zinc and copper in an unknown
solution by using atomic absorption spectroscopy and the standard addition method.
Theory:
The atomic absorption spectroscopy instrument measures the absorption of radiation by atoms in
order to infer the concentration of these atoms in solution. Beer-
Lambert’s law states that the
absorbance varies linearly with the concentration of the analyte in solution.
Figure 1: Atomic Absorption Instrument
E
XPERIMENT
2: Determination of Copper and Zinc by Atomic Absorption Spectroscopy
15
The analyte first needs to be atomized, which means it must be converted to atoms. To do so, it
is first converted to a form suitable for atomization. The analyte is initially presented in a liquid
solution, typically aqueous. The solution is then aspirated through the inlet tube (see Figure 1).
The solution is then broken up into a spray, a process called nebulization. The instrument you will
be using uses pneumatic nebulization as a means of sample introduction, although several other
types exist (ultrasonic, electrothermal vaporization, hydride generation, laser ablation, etc.). In a
pneumatic nebulizer, the gas stream flowing around the tip of the tube will aspirate the solution
(Bernoulli effect) and break it up into small droplets. The gas containing the spray will then go
through a chamber containing several baffles, which will remove big droplets and leave the
smaller ones suspended in the gas. The droplets that impact the baffles will collect at the bottom
of the chamber and be drained to waste (see Figure 1). Most pneumatic nebulizers are extremely
inefficient and >90% of the sample will flow to waste.
The spray is then introduced into the atomizer, in this experiment a flame atomizer, although other
atomization methods such as electrothermal or glow-discharge exist. When a flame atomizer is
used, the gas flowing through the nebulizer to form the aerosol is the oxidant (air or oxygen). The
aerosol is mixed with fuel (natural gas, hydrogen, acetylene) before passing the baffle system. In
the flame, desolvation, volatilization and dissociation will occur. During desolvation, the solvent
molecules will evaporate, leaving behind a solid or a gas aerosol (depending on the analyte).
Solid aerosols will then volatilize to become gaseous molecules. Finally, molecules will dissociate
to atoms. The concentration of free atoms in the flame is proportional to the concentration of the
analyte in solution.
A radiation source produces a light beam at the right wavelength for the targeted analyte. This
beam passes through the atomizer (in this case, the flame), where atoms absorb the light in
proportion to their concentration. Your atomic absorption instrument uses a hollow-cathode lamp.
Other radiation sources, such as electrodeless discharge lamps, also exist. A diagram for a
hollow-cathode lamp is shown in Figure 2.
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
E
XPERIMENT
2: Determination of Copper and Zinc by Atomic Absorption Spectroscopy
16
Figure 2: Scheme of a Hollow-Cathode Lamp
(Wikipedia contributors. Hollow-cathode lamp.
Wikipedia, the free encyclopedia
. 2012. Available at:
http://en.wikipedia.org/w/index.php?title=Hollow-cathode_lamp&oldid=464953962. Retrieved May 20,
2012.)
The cathode and anode of this lamp are enclosed by glass in a near vacuum (1 to 5 torr). The
small amount of gas present is usually neon or argon. The anode is typically made of tungsten,
and the cathode is made out of the atom or atoms that you want to analyze (e.g. zinc, copper,
magnesium, etc.). A voltage of 300V is then applied to the electrodes, and ionization of the buffer
gas starts to occur. This creates a small current of 5 to 15 mA between the electrodes. If the
voltage is increased, ions can acquire enough kinetic energy to eject the atoms of the hollow
cathode. This process, called sputtering, produces a cloud of atoms. Some of the atoms ejected
will be excited by the current and will relax to ground state by emitting radiation in a characteristic
spectrum. This radiation obviously has the exact energy needed for the transition of an electron
in the analyte atoms and can be absorbed by the analyte atoms in the flame.
The attenuated light beam that passed through the flame is directed through a monochromator.
This allows isolation of a narrow wavelength band, therefore eliminating interferences such as the
light emitted by the flame. The monochromator uses curved mirrors to focus and a grating to
diffract light from the entrance slit to an exit slit. At the exit slit only the selected wavelength (over
a narrow bandwidth) exits to the detector. The detector used is usually a photomultiplier tube.
Atomic absorption spectroscopy is particularly used for single element determination but recent
instruments can automatically, and quickly, switch lamps and detection wavelengths for multi-
element measurements.
In order to calculate the concentration of your unknown, you will be using the standard addition
method. In this method, a known amount of the analyte is added to a given quantity of the
unknown, and the amount of analyte added is varied from one standard to the other. Therefore,
“standard
0”,
the one with no added analyte, contains only the amount provided by the unknown.
The remaining standards have a concentration of “unk+x”, that is the amount provided by the
unknown (unk) plus the known amount added (x). The signal measured (in this experiment, the
E
XPERIMENT
2: Determination of Copper and Zinc by Atomic Absorption Spectroscopy
17
absorbance) is plotted against the added analyte concentration. This yields a graph similar to
Figure 3.
Figure 3: Example of a Standard Addition Calibration Curve
The calibration graph has a non-zero intercept, since standard 0 does contain some analyte. The
unknown analyte concentration can be found simply by using the absolute value of the x intercept
of this graph.
Materials, chemicals and instruments:
•
14 volumetric flasks of 100 mL
•
2 sets of glass pipettes (10, 8, 6, 4, 3 and 2 mL)
•
Micropipette (100
–
1000 μL)
•
Micropipette plastic tips
•
1000 ppm copper solution
•
1000 ppm zinc solution
•
Distilled water
•
Unknown solution
•
Atomic absorption spectrophotometer
E
XPERIMENT
2: Determination of Copper and Zinc by Atomic Absorption Spectroscopy
18
Procedure:
Solution(s) preparation:
Using the 1000 ppm stock solutions, prepare a 100 ppm solution for copper and a 10 ppm
solution for zinc in two 100 mL volumetric flasks. For this operation, you will need to use the
micropipette. The solvent used is 1% HNO
3
solution.
Each student needs to prepare their own series of 6 volumetric flasks (100 mL). To each of the 6
flasks, add 3 mL of the unknown solution. Then, add sufficient 100 ppm copper solution to make
up solutions of 0, 1, 2, 3, 4 and 5 ppm solutions. Do the same thing with the 10 ppm zinc solution
to make up solutions of 0, 0.1, 0.2, 0.3, 0.4 and 0.5 ppm solutions. Thus, in all of the flasks but
the “Standard 0”, you should have three solutions mixed: your unknown, the zinc solution and
the copper solution. The “Standard 0” flask will contain only 3
mL of the unknown solution. Once
the three solutions are added in each flask, use 1% HNO
3
solution dilute. Mix well each solution,
and be sure to identify them clearly.
Measurements:
To select the element you wish to analyze, click on the “Lamp” tab, then click on the “Element”
box (see Figure 4). A
“Select
Element”
window will appear: select zinc or copper and click ok. The
instrument will go through a series of optimizations (setting slit width and wavelength, peaking
turret and wavelength, etc.). Wait until this is finished and the screen is no longer displaying an
operation. Do not forget to note the wavelength used, as it will be needed for the discussion in
your lab report.
Figure 4: Lamp Selection
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
E
XPERIMENT
2: Determination of Copper and Zinc by Atomic Absorption Spectroscopy
19
If this is not already done, put the sample inlet tube in a bottle of distilled water and allow the
blank to rinse the system for 15 seconds.
To set zero, click on the “Analyze” tab, then on “Analyze Blank” (see
Figure 5). The instrument
will take three absorbance readings for distilled water, and set this absorbance level as zero.
Figure 5: Sample Analysis
Remove the sample inlet tube from distilled water and wipe it clean using a Kimwipe tissue. Place
it in the next solution you wish to analyze. Click on “Analyze Sample”. When the absorbance
readings are done, take down in your lab notebook the three replicate measurements displayed
on the screen (see Figure 5).
When all samples have been analyzed for the chosen element, return to the
“Lamp”
tab to change
the element to be analyzed. Set zero again using distilled water, and analyze all the samples as
specified above.
Calculations:
Plot the absorbance against the added concentration of copper (0, 0.2, 0.4, 0.6, 0.8 and 1.0 ppm).
Using the linear regression equation, determine the true amount of copper in the unknown
solution. Do not forget to take into account the dilution of the unknown solution performed during
the preparation.
Perform a similar analysis for zinc.
Do not forget to include LOD, LOQ and uncertainty values.
E
XPERIMENT
2: Determination of Copper and Zinc by Atomic Absorption Spectroscopy
20
Lab report:
The following questions should be addressed in your lab report. The answers should be included
in the body of the text, not as separate sections.
•
Discuss the value of the wavelength used for copper and zinc. To what physical process
does this correspond?
•
For nearly every element there are multiple wavelengths that can be used. Discuss what
factors go into selecting the wavelength to use experimentally.
•
Different flame structures exist in atomic absorption instruments. The instrument you were
using has a long thin flame. What is the advantage of this shape over, say, a round flame
(similar to a Bunsen burner)?
•
The flame is used to atomize the analyte. This requires a high temperature flame. Why, in
general,
don’t
we always use the hottest flame type available (an oxygen-acetylene flame
is about 500-900 degrees hotter than the air-acetylene you used)?
•
What are the advantages and disadvantages of standard addition calibration in general,
and in the context of atomic absorption spectroscopy in particular?
•
Are there advantages, or disadvantages, to using more than three replicate
measurements?
References:
1.
Douglas A. Skoog, F. James Holler, Stanley R. Crouch. Atomic Absorption and Atomic
Fluorescence Spectrometry. In:
Principles of Instrumental Analysis
. 6th ed. Belmont, California:
Thomson Brooks/Cole; 2007:230-253.
2.
Harris DC. Quality Assurance and Calibration Methods. In:
Quantitative Chemical Analysis
.
7th ed. New York: W. H. Freeman; 2007:78-95.
3.
Harris DC. Spectrophotometers. In:
Quantitative Chemical Analysis
. 7th ed. New York: W. H.
Freeman; 2007:424-452.
4.
Harris DC. Atomic Spectroscopy. In:
Quantitative Chemical Analysis
. 7th ed. New York: W. H.
Freeman; 2007:453-473.
5.
James D. Ingle, Stanley R. Crouch. Atomic Absorption Spectrophotometry. In:
Spectrochemical Analysis
. Upper Saddle River, New Jersey: Prentice Hall; 1988:273-306.
6.
Kahn HL. Instrumentation for atomic absorption
—
Part two.
J. Chem. Educ.
1966;43(2):A103.
7.
Lajunen LHJ.
Spectrochemical analysis by atomic absorption and emission
. Cambridge:
Royal Society of Chemistry; 1992.
E
XPERIMENT
3
Determination of Riboflavin by Fluorescence Spectrophotometry
Purpose:
The goal of this experiment is to determine the concentration of riboflavin in an unknown solution
using fluorescence spectrophotometry.
Theory:
Fluorescence
and
phosphorescence
are
two
categories
of
photoluminescence.
In
photoluminescence, a molecule is excited by absorption of photons. The molecule then returns
to its electronic ground state by emitting photons. In fluorescence, this emission occurs very
shortly after the excitation (<10
-8
s), whereas it is longer for phosphorescence (10
-4
s to minutes).
In both cases, the wavelength of emission is longer than the wavelength of excitation. This shift
in wavelength is due to loss of energy through collisions with solvent or analyte molecules.
Fluorescence is typically observed for molecules with a
π system,
although other molecules can
also produce fluorescence. Solvent, temperature and pH can have a strong effect on the intensity
of fluorescence and the wavelength of emission.
The emission spectrum of riboflavin excited at 372 nm can be found in Figure 1.
Figure 1: Emission Spectrum of Riboflavin Excited at 372 nm
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
E
XPERIMENT
3: Determination of Riboflavin by Fluorescence Spectrophotometry
22
The peak observed around 375 nm is due to
scattered excitation light that the instrument hasn’t
blocked successfully. The fluorescence occurs around 525 nm.
At low absorbance of the excitation beam (A<0.05), the fluorescence emission intensity will
increase linearly with analyte concentration. A detailed explanation of why this is the case can be
found in your textbook (Reference 1, section 15A-4).
The instrument used to measure fluorescence is called a spectrofluorometer. An excitation
source emits light across the full spectrum, which is then filtered by a monochromator to select
the excitation wavelength that is transmitted to the sample. The source is usually a low-pressure
mercury vapor lamp or a high- pressure xenon arc lamp. The excited molecules emit light in all
directions. A second monochromator placed at 90° from the excitation source isolates the desired
emission wavelength and transmits it to a transducer. The photomultiplier is the most commonly
used transducer, but charge-transfer devices (e.g. CCDs) are also used.
Materials, chemicals and instruments:
•
Riboflavin
•
Glacial acetic acid
•
Unknown solution
•
1 volumetric flask of 1000 mL
•
7 volumetric flasks of 100 mL
•
1 set of glass pipettes (10, 8, 6, 4 and 2 mL)
•
2 glass pipettes of 10 mL
•
Erlenmeyer flask of 2 L
•
Waste beaker
•
Weighing paper
•
Balance
•
Quartz cell
•
Spectrofluorophotometer (Shimadzu RF-5301)
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
E
XPERIMENT
3: Determination of Riboflavin by Fluorescence Spectrophotometry
23
Procedure:
Solution(s) preparation:
Prepare 2 L of 5% (v/v) acetic acid in the Erlenmeyer flask. A certain amount of this solution might
remain from preceding groups, in which case you can use what is left.
Prepare a 10 ppm riboflavin solution by weighing 0.010 g of riboflavin and dissolving it in 1000 mL
of 5% acetic acid. Use the volumetric flask for this operation. Be careful, riboflavin takes some
time and shaking to dissolve properly. Make sure all of the riboflavin is dissolved before bringing
the volume to 1000 mL. Also, make sure to record the exact mass of riboflavin used.
Using this 10 ppm riboflavin solution, prepare 100 mL solutions of 0.2, 0.4, 0.6, 0.8 and 1.0 ppm.
Use the volumetric flasks for this operation and 5% acetic acid for dilution. The same set of
standards is used for both students.
Dilute a 10 mL aliquot of your unknown solution to 100 mL with 5% acetic acid.
Measurements:
Be careful, quartz cuvettes must be handled with care! They are expensive and fragile.
The spectrophotofluorometer can be found in room 332.01.
Pour the 5% acetic acid blank into the quartz cuvette. Open the instrument cover and place it in
the cuvette holder. Make sure to note in which position your cuvette is (e.g. brand name on one
side of the cuvette facing you). This position should be used for all of your measurements, in order
to reduce variability. Close the cover.
On the top right corner of the computer screen, you should see the icons shown in Figure 2.
Figure 2: Buttons for Panorama Software
Click on the “Go to WL” icon. A “Wavelength Selection” window should appear (see Figure 3).
Make sure that the excitation wavelength is 372 nm and the emission wavelength is 523 nm. Click
on “Shutter” to open the shutter and let the light pass through. The “Shutter” icon should now
show an open shutter (yellow circle in its center).
Click on
“Autozero”
to zero the emission level with the blank.
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
E
XPERIMENT
3: Determination of Riboflavin by Fluorescence Spectrophotometry
24
Remove the cuvette containing the 5% acetic acid blank from the instrument. Throw the contents
in a waste beaker, rinse and dry the cuvette thoroughly.
Pour your first standard into the cuvette, and place it in the holder in the instrument.
Click on the “Go to WL” icon. Then click on “Read”. The intensity of the fluorescence emission
will appear in the small box above the
“Read” button. Note this intensity in your lab book.
Figure 3: Wavelength Selection Window
Read two more times the fluorescence intensity for this first standard, for a total of three intensity
measurements.
Throw the contents of the cuvette in the waste, rinse and dry your cuvette and repeat the
measurement process for all of your standards and unknowns.
Calculations:
Plot the intensity of fluorescence versus the concentration of riboflavin. Make sure you use the
real concentration of riboflavin in your standards, that is, the one calculated from the actual mass
of riboflavin you weighed. This should be clearly shown in your lab report.
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
E
XPERIMENT
3: Determination of Riboflavin by Fluorescence Spectrophotometry
25
Lab report:
The following questions should be addressed in your lab report. The answers should be included
in the body of the text, not as separate sections.
•
Why do you need to use quartz cuvettes and not plastic ones?
•
Why do, in general, the fluorescence intensities decline with replicate number?
•
Explain the geometry of the instrument (detector vs. excitation source).
•
Why do you need to place the cuvette in the same position for every measurement you
take?
•
Why is fluorescence so susceptible to solvent effects (e.g. pH, salts, chlorides etc.)
•
Why is fluorescence more sensitive than absorption (lower detection limits)?
References:
1.
Douglas A. Skoog, F. James Holler, Stanley R. Crouch. Molecular Luminescence
Spectrometry. In:
Principles of Instrumental Analysis
. 6th ed. Belmont, California: Thomson
Brooks/Cole; 2007:399-429.
2.
Harris DC. Spectrophotometers. In:
Quantitative Chemical Analysis
. 7th ed. New York: W. H.
Freeman; 2007:424-452.
3.
James D. Ingle, Stanley R. Crouch. Molecular Luminescence Spectrometry. In:
Spectrochemical Analysis
. Upper Saddle River, New Jersey: Prentice Hall; 1988:164-188.
4.
Utecht RE. Identification and quantitation of riboflavin in vitamin tablets by total luminescence
spectroscopy.
J. Chem. Educ.
1993;70(8):673.
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
E
XPERIMENT
4
Determination of Aspartame, Caffeine and Benzoic Acid by High Performance Liquid
Chromatography (HPLC)
NB: 2023
–
this experiment does not require a complete lab report.
It will be evaluated in two
parts: 1- the Results 2- An oral examination that also includes the GC experiment.
Purpose:
The goal of this experiment is to determine the concentration of aspartame, caffeine and benzoic
acid in an unknown solution using high performance liquid chromatography (HPLC) coupled to a
UV-Vis detector.
Theory:
The purpose of all chromatographic techniques is to separate different species that compose a
mixture. In all chromatography techniques, a mobile phase and a stationary phase are used. The
mobile phase travels through the stationary phase and carries the analytes with it. The more
strongly an analyte interacts with the stationary phase, the longer it will take to elute (exit) from
the column. Since each analyte interacts differently with the stationary and mobile phases, they
will elute at different times. The time elapsed between the injection of an analyte and its exit from
the column is called the retention time.
In liquid chromatography, the mobile phase is a liquid. In this experiment, it is a mixture of
acetonitrile and aqueous acetate buffer (20:80 v:v). The composition of the mobile phase can be
fixed (isocratic elution, as in this experiment), or it can be varied through the chromatographic run
(gradient elution). The liquid mobile phase is pushed through a column packed with stationary
phase using a high pressure pump (Figure 1). A liquid chromatography column is typically 10 to
30 cm long (Figure 2) and filled with derivatized silica particles. The silanol groups at the surface
of these particles are reacted with other groups to provide the surface that interacts with the
mobile phase and the analytes dissolved in it. This thin surface layer is the stationary phase. In
this experiment, a C18 stationary phase is used, which means that octadecylsilane has been used
to derivatize silanol groups on the surface of silica particles to provide R
3
Si-O-Si(OH)
2
-C
18
H
37
.
The hydrocarbon chain covers the surface to such an extent that the analytes can usually be
assumed to interact only with this (and not the underlying silica). C18 is probably the most
common stationary phase used in LC, but other phases such as C8, CN and phenol are also
used: a wide variety of columns exist. Liquid chromatography performed on a column packed with
a hydrophobic stationary phase (such as C18) is called reversed phase. This is in opposed to
normal phase chromatography, where a column with a hydrophilic stationary phase (such as non-
derivatized silica) is used. In reverse phase liquid chromatography, the most hydrophobic
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
E
XPERIMENT
4: Determination of Aspartame, Caffeine and Benzoic Acid by HPLC
27
UV Detector
Column Compartment
Autosampler
Quaternary Pump
Degasser
Mobile Phases
molecules will interact the stationary phase most strongly, and will therefore have a higher
retention time.
Figure 1: HPLC Instrument
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
E
XPERIMENT
4: Determination of Aspartame, Caffeine and Benzoic Acid by HPLC
28
Figure 2: Analytical Column
(Hi Tech Trader.com. Agilent Zorbax Eclipse XDB-C18 HPLC Column - Product Details. Available at:
http://www.hitechtrader.com/detail.cfm?autonumber=84831. Retrieved May 30th, 2012.)
When the analyte peak exits the column, it is dissolved in mobile phase. This mixture flows
through a UV detector cell, where absorbance is measured. A plot of absorbance vs. time elapsed
since injection yields a chromatogram (Figure 3). The area under the peaks is calculated using
an integration software. Peak area can then be plotted against concentration, and this calibration
curve can be used to quantitate an analyte.
Figure 3: Chromatogram
(Modified from: Statistical Designs. How to Develop Validated HPLC Methods: Rational Design with
Practical Statistics and Troubleshooting Short Courses. 2012. Available at:
http://www.statisticaldesigns.com/sc_hplc.htm. Retrieved May 30
th
, 2012.)
Materials, chemicals and instruments:
•
Aspartame
•
Caffeine
•
Benzoic acid
•
Acetonitrile
•
Aqueous acetate buffer (0.5% (w/v) ammonium acetate and 2% (v/v) glacial acetic acid)
•
Unknown solution, in a 25 mL volumetric flask
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
E
XPERIMENT
4: Determination of Aspartame, Caffeine and Benzoic Acid by HPLC
29
•
1 volumetric flask of 100 mL
•
4 volumetric flasks of 50 mL
•
1 volumetric flask of 25 mL
•
1 volumetric flask of 1000 mL
•
4 glass pipettes of 25 mL
•
7 beakers of 10 mL
•
7 luer-lock syringes (3 mL)
•
7 nylon membrane filters (Progene, 0.45
μ
m pores)
•
7 HPLC vials (2 mL) and their caps
•
HPLC instrument, including a quaternary pump, a UV-visible detector and an autosampler
(Agilent 1200 Series)
Procedure:
Solution(s) preparation:
In a volumetric flask, prepare 1 L of the mobile phase, consisting of 20% (v/v) acetonitrile and
80% (v/v) of the aqueous acetate buffer.
To a 100 mL volumetric flask, add 40 mg of caffeine, 40 mg of benzoic acid and 200 mg of
aspartame. Make sure to note the exact weight added to the flask in order to properly calculate
your concentrations. To dissolve all of the analyte, you will need to shake the solution thoroughly.
Make sure everything is dissolved before proceeding to dilution to full volume using the mobile
phase. This is your most concentrated mixed standard solution.
In 50 mL volumetric flasks, prepare 4 serial dilutions (1:1). Thus the first dilution is prepared by
aliquoting 25 mL of the most concentrated solution and completing to 50 mL with mobile phase.
The next dilution is prepared by aliquoting 25 mL of the first dilution and completing to 50 mL with
mobile phase, and so on for the next two dilutions. This should yield five different standard
solutions. Identify them clearly (make sure you know which one is the most concentrated and
which one is the most dilute).
The 25 mL volumetric flask you received contains 5 mL of an unknown solution. Complete to
25 mL using mobile phase.
In a 10 mL beaker, pour some of your first standard solution. Using a plastic syringe, draw in
about 2 mL of this solution. Screw on a nylon membrane filter, and push the syringe plunger to
deliver about 1.5 mL of this solution in a HPLC vial. Close the vial with a cap, and identify it.
Repeat this operation for all of your standards, a blank and unknown solutions.
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
E
XPERIMENT
4: Determination of Aspartame, Caffeine and Benzoic Acid by HPLC
30
Measurements:
The following HPLC parameters are used for the production of chromatograms:
Injection volume: 25
μ
L
Flow rate: 1.0 mL/min
Elution: Isocratic
Solvents: 20% B (acetonitrille), 80% A (acetate buffer)
Column: Zorbax Eclipse XDB-C18 (5
μ
m, 4.6 x 150 mm) (Agilent)
Column thermostat: deactivated
Run time: 8.00 min
Detection wavelength: 254 nm
HPLC runs will be performed in duplicate using the autosampler. The instrument set up will be
performed by the lab technician or your TA, but you will assist to them. Once the run is started,
there is no need for you to stay: the injection sequence will be performed automatically by the
instrument and will last around 2 hours. You will receive your chromatograms the following week.
Calculations:
Calculate the concentrations of caffeine, benzoic acid and aspartame in your five standards (in
μ
g/mL). Make sure you use the real concentration of caffeine, benzoic acid and aspartame in
your standards, that is, the one calculated from the actual mass you weighed. This should be
clearly described in your lab report.
Plot the intensity of absorption versus the concentration of caffeine, benzoic acid and aspartame.
You should therefore have three different graphs and calibration curves (one for each analyte).
From the calibration data, calculate the concentrations of caffeine, benzoic acid and aspartame
of your unknown. Make sure you take into account the dilution you performed.
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
E
XPERIMENT
4: Determination of Aspartame, Caffeine and Benzoic Acid by HPLC
31
Lab report:
Part 1: This will be an abbreviated lab report. It is due on the normal due date. Complete the
following sections only: title, abstract, results and calculations and references (as appropriate) in
the lab report template as per usual.
All other sections
of the report should be denoted as “Not
required”
Part 2: Prepare yourself for an oral lab report (see separate instructions on Moodle) but
essentially you will be asked general questions on HPLC as well as specific questions about this
experiment. The oral report will also cover the GC-TCD experiment (Expt. 5). You will need to
schedule a report date with your TA once BOTH experiments are complete.
Give some thought to the following questions when preparing for the report (there may be more
in the Moodle instructions)
•
Why is it necessary to filter the prepared solutions before injecting them on the HPLC?
•
Why do you use mobile phase to dissolve your samples instead of water or acetonitrile
(what would be the consequences on the separation and peak shapes)?
•
What is the order of elution of the analytes and why is this the case? Make reference to
the type of interaction with the stationary phase and the molecular structure of the
analytes.
•
Calculate plate number and plate height, and discuss the result.
•
Discuss the different
chromatograms’
features (baseline, peak resolution, peak symmetry
and shape, etc.). Specify average retention times for all analytes.
•
Why would you use column thermostatting?
•
Why is 254 nm used as the detection wavelength? Give some consideration to the mobile
phase too.
•
Why would you use gradient elution instead of isocratic elution?
•
In the past, students needed to degas their mobile phase in an ultrasonic bath for 20
minutes. Why is this not necessary anymore?
References:
1.
Delaney MF, Pasko KM, Mauro DM, et al. Determination of aspartame, caffeine, saccharin,
and benzoic acid in beverages by high performance liquid chromatography: An undergraduate
analytical chemistry experiment.
J. Chem. Educ.
1985;62(7):618.
2.
Douglas A. Skoog, F. James Holler, Stanley R. Crouch. An Introduction to Chromatographic
Separations. In:
Principles of Instrumental Analysis
. 6th ed. Belmont, California: Thomson
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
E
XPERIMENT
4: Determination of Aspartame, Caffeine and Benzoic Acid by HPLC
32
Brooks/Cole; 2007:762-787.
3.
Douglas A. Skoog, F. James Holler, Stanley R. Crouch. Liquid Chromatography. In:
Principles of Instrumental Analysis
. 6th ed. Belmont, California: Thomson Brooks/Cole;
2007:816-855.
4.
Harris DC. High-Performance Liquid Chromatography. In:
Quantitative Chemical Analysis
. 7th
ed. New York: W. H. Freeman; 2007:556-587.
5.
Pavia DL, Lampman GM, Kriz GS, Vyvyan JR. Ultraviolet Spectroscopy. In:
Introduction to
Spectroscopy
. 4th ed. Brooks Cole; 2009:381-417.
6.
Snyder LR, Kirkland JJ, Glajch JL.
Practical HPLC Method Development, 2nd Edition
. 2nd
ed. Wiley-Interscience; 1997.
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
Oven
Column
Keyboard
Injection Port
TCD Detector
E
XPERIMENT
5
Gas Chromatographic Determination of Isopropanol in a Mixture
NB: 2023
–
this experiment does not require a complete lab report.
It will be evaluated in two
parts: 1- the Results 2- An oral examination that also includes the HPLC experiment.
Purpose:
The goal of this experiment is to determine the percentage of isopropanol (v/v) present in an
unknown solution using a gas chromatography with thermal conductivity detection and internal
standards calibration.
Theory:
In gas chromatography, the liquid sample to be analyzed is introduced into the column through a
heated injection port (Figure 1) that flash vaporizes it. The temperature of both the injection port
and the column must be at least 50°C above the boiling point of the least volatile constituent of
the mixture. Thus, thermolabile molecules cannot be analyzed by gas chromatography, because
they will degrade before reaching the detector. Once vaporized, the sample goes into the column,
carried by the flow of mobile phase. In gas chromatography, an inert gas is used as the mobile
phase (usually helium, although hydrogen is gaining in popularity). The column, which is typically
around 30 m long, is placed inside of an oven that keeps the temperature stable. The oven
temperature must be high enough for the sample to stay in the gas phase. If the temperature is
too low, some of the analytes can condense onto the column, ruining the chromatogram and in
some cases the column.
Figure 1: Gas Chromatograph
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
E
XPERIMENT
5: Gas Chromatographic Determination of Isopropanol in a Mixture
33
A variety of columns are available for gas chromatography. Almost all GC columns used are fused
silica wall-coated open tubular (FSOT). This means that the column is an open capillary made up
of fused silica, and that the inside wall of this capillary, made of silica, have been derivatized using
the stationary phase. Usually, non-polar molecules are used as the stationary phase, but other
options are available as well.
When the sample exits the column, it goes through a detector. Several GC detectors are available:
electron capture, nitrogen-phosphorus, flame ionization, mass spectrometer, etc. In this
experiment, a thermal conductivity detector is used (TCD; see Figure 2).
Figure 2: Thermal Conductivity Detector (TCD)
(Wikipedia contributors. Thermal conductivity detector.
Wikipedia, the free encyclopedia
. 2007. Available
at: https://en.wikipedia.org/wiki/Thermal_conductivity_detector. Retrieved May 30th, 2012.)
The TCD detector box contains a series of fine metallic wire elements. Current is passed through
this fine wire, whose resistance depends on the temperature. The temperature, in turn, depends
on the heat capacity of the gas exiting the column which surrounds the wire. If species with a high
heat capacity exit the column, they will lower the temperature of the wire. By lowering the
temperature of the wire, its resistance will also change. By placing a voltmeter across the wire,
one can associate changes in voltage to the passage of an analyte through the detector. The
sensitivity of the measurement can be increased by measuring the difference in voltage between
a reference flow, which provides a reference signal, and the column flow as shown in the above
figure. A further enhancement in sensitivity is possible by arranging a pair of these
sample/reference elements in what is called a Wheatstone bridge configuration as shown in the
above figure. The voltage change from the bridge detector will be proportional to the concentration
of analyte passing through the detector. Software is used to integrate the peaks and determine
the area under each peak.
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
E
XPERIMENT
5: Gas Chromatographic Determination of Isopropanol in a Mixture
34
In order to calculate the concentration of your unknown, you will be using the internal standard
method. In this method, a constant amount of a species closely related to your analyte is added
to all standards and unknowns, before any sample treatment is performed. This species is known
as the internal standard. The signal of both the analyte and the internal standard are then
measured. In order to build a calibration, the ratio of the analyte signal to the internal standard
signal is plotted against the concentration. Table 1 and Graph 1 below illustrate this calibration
technique. Notice that the internal standard signal varies even though it should be constant if a
constant volume of sample is injected. This can be due, for example, to variations in the manual
injection. However, both the analyte and the internal standard signals vary together with injection
volume so that the ratio reflects the correct concentration signal as can be seen in the “Ratio”
column. From this calibration curve, the concentration of the unknown can be determined.
Table 1: Data Illustrating the Internal Standard Calibration
Concentration
(ng/mL)
Analyte
Signal (mV)
Internal
Standard
Signal (mV)
Ratio
1
1.25
5
0.25
2
1.50
3
0.50
3
3.00
4
0.75
4
3.00
3
1.00
5
10.00
8
1.25
Figure 3: Calibration Curve Illustrating the Internal Standard Calibration
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
E
XPERIMENT
5: Gas Chromatographic Determination of Isopropanol in a Mixture
35
Materials, chemicals and instruments:
•
Isopropanol
•
Methanol
•
Ethanol
•
Unknown solution
•
6 volumetric flasks of 10 mL
•
10
μ
L glass syringe
•
Volumetric pipette (1000
–
5000
μ
L) and plastic tips
•
Gas chromatograph (Shimadzu GC-17A)
Procedure:
Solution(s) preparation:
In the 10 mL volumetric flasks, prepare 5 calibration standards of 0% (blank), 20%, 25%, 30%
and 35% isopropanol. All standards should also contain 20% methanol. You will need to use a
micropipette for this operation. Once the isopropanol and the methanol are added to the
volumetric flasks, perform dilution using ethanol.
Measurements:
The following GC-TCD parameters are used for the production of chromatograms:
Oven temperature: 150°C
Injector: 220°C
Column: Alltech ECONO-CAP
TM
EC
TM
-5 (30 m x 0.25 m ID x 0.25 μm)
Flow rate: 10 mL/min
TCD current: 50 mA
The instrument should be started and ready to use. In the GC Software toolbar (Figure 4), click
on the blue arrow button (first of the two arrows). The “Single Run Acquisition” window should
open up (Figure 5). Fill out the
“Sample
ID”
field (e.g. 20% Isopropanol, Run 1) and the
“Data
file”
field (e.g. 2012-05-17-
01, each injection must have a different data file name). Click on “Start”.
Figure 4: GC Software Menu
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
E
XPERIMENT
5: Gas Chromatographic Determination of Isopropanol in a Mixture
36
Figure 5:
“Single
Run
Acquisition”
Window
The instrument will equilibrate, and eventually a “Waiting for trigger” message will appear at the
bottom of the software window. When this message appears, the instrument is ready for you to
inject your sample.
Figure 6: GC Keyboard
Be careful, the glass syringe needle and plunger are fragile and must
be handled with care to prevent bending of the plunger or the needle.
Rinse the syringe three times with the first standard solution. Draw in a
volume of 1
μ
L. Make sure no bubbles are present. Insert the needle
in the injection port carefully, while keeping your hand over the syringe
plunger (if you don’t “guard” the plunger, the pressure in the inlet will
push it out). Softly push the plunger to release the 1
μ
L inside the inlet
and simultaneously press start on the GC keyboard (Figure 6).
The chromatographic run will last 2 minutes and a report will be printed
automatically at the end. You can then start another run of the same
sample, using the same procedure described above. Try to be as
consistent as possible in your manner of injecting samples. While one
sample is running, take the preceding data report and calculate the
area ratio of isopropanol to methanol (area of isopropanol / area of
methanol). The difference between the highest and lowest area
ratios for different replicate measurements of a sample should be smaller than 5%. Once you get
three chromatograms for your first standard solution fitting this criterion, you can start injecting
the next standard.
Once all your standards are analyzed, inject your unknowns. They already contain the internal
standard (20% methanol), which is why you can simply draw the solution from the bottle and inject
it as is. Again, the maximum area ratio difference should be 5% or less.
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
E
XPERIMENT
5: Gas Chromatographic Determination of Isopropanol in a Mixture
37
Calculations:
Do not include your blank as a standard in the calibration since it is impossible to integrate the
analyte peaks. Instead, use it to verify that the chromatogram is devoid of peaks at the retention
time of the analytes i.e. there is no peak when there is no analyte. Plot the area ratio (area of
isopropanol / area of methanol) versus the percent of isopropanol (v/v).
From the calibration data, calculate your
unknown’s
content in isopropanol (in %) and its error.
Lab report:
Part 1: This will be an abbreviated lab report. It is due on the normal due date. Complete the
following sections only: title, abstract, results and calculations and references (as appropriate) in
the lab report template as per usual.
All other sections
of the report should be denoted as “Not
required”
Part 2: Prepare yourself for an oral lab report (see separate instructions on Moodle) but
essentially you will be asked general questions on GC-TCD as well as specific questions about
this experiment. The oral report will also cover the HPLC experiment (Expt. 4). You will need to
schedule a report date with your TA once BOTH experiments are complete.
Give some thought to the following questions when preparing for the report (there may be more
in the Moodle instructions)
•
What are the advantages and disadvantages of internal standard calibration? In which
context is it usually used?
•
Would it make a difference in the quantification if a volume of unknown other than 1 μL
was injected? Consider a small difference, say 0.8 μL and a large difference, say 5 μL.
•
Calculate plate number and plate height, and discuss the result. How does it compare to
other, similar, columns?
•
Discuss the different
chromatograms’
features (baseline, peak resolution, peak symmetry
and shape, etc.). Specify average retention time for all analytes.
•
What advantages are gained by using a temperature gradient during a chromatographic
run (instead of keeping it constant, as you did in the lab)?
•
What are the advantages and disadvantages of a TCD detector? Would another detector
be better suited for the analytes quantified in this experiment?
References:
1.
Douglas A. Skoog, F. James Holler, Stanley R. Crouch. An Introduction to Chromatographic
Separations. In:
Principles of Instrumental Analysis
. 6th ed. Belmont, California: Thomson
Brooks/Cole; 2007:762-787.
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
E
XPERIMENT
5: Gas Chromatographic Determination of Isopropanol in a Mixture
38
2.
Douglas A. Skoog, F. James Holler, Stanley R. Crouch. Gas Chromatography. In:
Principles
of Instrumental Analysis
. 6th ed. Belmont, California: Thomson Brooks/Cole; 2007:788-815.
3.
Harris DC. Quality Assurance and Calibration Methods. In:
Quantitative Chemical Analysis
.
7th ed. New York: W. H. Freeman; 2007:78-95.
4.
Harris DC. Gas Chromatography. In:
Quantitative Chemical Analysis
. 7th ed. New York: W.
H. Freeman; 2007:528-555.
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
E
XPERIMENT
6
Determination of Ascorbic Acid by Differential Pulse Voltammetry
NB: 2023
–
this experiment does not require a traditional lab report.
Instead you will prepare
your results and a Powerpoint presentation with voice-over as a lab report/presentation for
both electrochemistry experiments.
Specific instructions are on Moodle (lab section)
Purpose:
The goal of this experiment is to determine the concentration of ascorbic acid in an unknown
solution using differential pulse voltammetry.
Theory:
Ascorbic acid can be oxidized to dehydroascorbic acid (Figure 1), with a cell potential (E°) of
0.29 V (vs Ag/AgCl) at pH 4.0.
Figure 1: Oxidation of Ascorbic Acid
Voltammetric techniques are a group of electrochemical methods which measure the current as
a function of the potential applied on an electrode. A reference electrode (such as Ag/AgCl), an
auxiliary electrode (e.g. platinum) and a working electrode (e.g. glassy carbon) are used to
perform these types of experiments. When a potential is applied to an electrode, the
electrochemical reaction occurs only at the surface layer of the electrode, that is, the thin layer of
solution immediately adjacent to the electrode. The concentrations of oxidized and reduced
species in this layer will governed by the Nernst equation:
where E is the potential applied to the electrode, E°' is the standard cell potential, R is the gas
constant, T is the temperature (in
°
K), n is the mole of electrons exchanged in the reaction and
F is Faraday's constant. Following Ohm's law, the current measured at the electrode should be
I=E/R,
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
E
XPERIMENT
6: Determination of Ascorbic Acid by Differential Pulse Voltammetry
39
and the current should increase linearly with the potential applied to the electrode. However,
polarization effects modify this relationship at the extremes of the curve. A graph of the current
plotted against the potential, also called a voltammogram, therefore shows a sigmoidal curve
(Figure 2). Figure 2 shows that as voltage is increased, current reaches a plateau. This is caused
by concentration polarization, which means that at these voltages, the rate of transfer of reduced
species from the bulk solution to surface layer is not fast enough to allow the current to increase
further. Since the rate of transfer of species is proportional to the concentration, the intensity of
the current reached by each plateau increases (is more negative) with concentration of reduced
species in solution (see Figure 2).
(Image: Harris DC.
Quantitative Chemical Analysis
. 8th ed. W. H. Freeman; 2010, p. 376)
Figure 2: Linear Sweep Voltammogram
In differential pulse voltammetry, a pulse is superimposed on a linear scan (see Figure 3) of
applied potential.
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
E
XPERIMENT
6: Determination of Ascorbic Acid by Differential Pulse Voltammetry
40
(Skoog DA, Holler FJ, Crouch SR.
Principles of Instrumental Analysis
. 6th ed. Brooks Cole; 2006, p.743)
Figure 3: Waveform of Differential Pulse Voltammetry
The values used analytically are the difference in the currents measured when the potential E is
applied to the electrode (S1 in the figure) and the current measured when the potential E + step
height (usually 50 mV) is applied to the electrode (S2 in the figure). Notice that this is basically
the derivative of the linear sweep voltammogram and is called a differential pulse voltammogram
(Figure 4) where Δi is
plotted against the initial voltage applied (before the pulse).
(Skoog DA, Holler FJ, Crouch SR.
Principles of Instrumental Analysis
. 6th ed. Brooks Cole; 2006, p.743)
Figure 4: Differential Pulse Voltammogram
As you can see on the linear sweep voltammogram (Figure 2), the slope of the linear portion
increases with increasing reduced agent concentration. In the differential pulse voltammogram
(Figure 4), this translates to an increased peak height. Therefore, Δi (peak current) is correlated
linearly with analyte concentration (look at the relation of [Fe(II)] to the slope on Figure 2).
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
E
XPERIMENT
6: Determination of Ascorbic Acid by Differential Pulse Voltammetry
41
Materials, chemicals and instruments:
•
1 volumetric flask of 50 mL
•
1 glass pipette of 10 mL
•
1 micropipettor
•
1 beaker
•
L-Ascorbic acid
•
0.1 M Acetate buffer (pH = 4.0)
•
Unknown ascorbic acid solution (in a 50 mL volumetric flask)
•
Polishing Alumina Solution
•
Glassy Carbon Electrode
•
Ag/AgCl reference electrode
•
Platinum wire auxiliary electrode
•
Polarographic cell (15 mL)
•
Polarographic cell cap with 4 holes
•
Magnetic stir bar
•
EmStat4S Potentiostat
Procedure:
Solution(s) preparation:
Prepare 50 mL of 3 mM ascorbic acid solution in a volumetric flask. Use acetate buffer to dilute
your ascorbic acid. Make sure to note the exact weight of ascorbic acid used.
You will receive 2 mL of unknown solution in a volumetric flask. Dilute this solution to 50 mL with
acetate buffer.
Measurements:
This experiment contains many important small steps. Make sure you follow the instructions
carefully, because it is easy to go off track. Read these instructions carefully before you go into
the lab, so that you will have a clear picture of what you will do.
Using the glass pipette, transfer 10 mL of acetate buffer solution into the polarographic cell. Add
the magnetic stir bar. Open the Faraday Cage and pivot the stir motor to the right. Bring the cell
up from underneath, around the electrodes, and seat on the cell top. Pivot stir motor back under
the cell.
Reference electrode, auxiliary electrode and gas inlet should already be placed in the
appropriate holes in the cap as indicated in Figure 5.
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
E
XPERIMENT
6: Determination of Ascorbic Acid by Differential Pulse Voltammetry
42
Magnetic Stir Motor
Magnetic Stir Bar
Polarographic Cell
Cell Cap (4 Holes)
Gas Inlet
Auxiliary Electrode
Glassy Carbon Electrode
Ag/AgCl Reference Electrode
Figure 5: Polarographic Cell Setup
Using a micropipettor, add 0.4 mL of your ascorbic acid standard solution in the polarographic cell
through the front hole.
Toggle the “Purge” and “Stir” switches to ON position. Stir
and degas this solution for 5
minutes. Once the degassing step is finished, switch off the purge gas and stir on the cell stand.
***Make sure purge and stir are OFF during measurements***
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
E
XPERIMENT
6: Determination of Ascorbic Acid by Differential Pulse Voltammetry
43
Shake the polishing alumina solution and put a few drops on the polishing pad placed on the
benchtop. Polish the glassy carbon electrode by making light, circular motions on the wet pad
for about 60 seconds and rotate the electrode 90º every 15 seconds to prevent uneven wear of
the electrode. Rinse well the carbon electrode with distilled water and carefully blot dry using a
Kimwipe. Insert the glassy carbon electrode in its hole and connect the electrode to the
potentiostat using the alligator clip. Make sure that the bottom of the electrode is in the solution,
that no bubbles are present on the electrode surface and that the electrodes do not touch each
other. Close the Faraday Cage.
INSTRUCTIONS FOR USING PSTrace Software
1.
On the Desktop open the PSTrace software.
2.
Click “Connect” button to connect
to the instrument.
Stir Switch
Purge Switch
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
E
XPERIMENT
6: Determination of Ascorbic Acid by Differential Pulse Voltammetry
44
3.
Click
“Load method” and select “Experiment 6”
4.
Click on the
GREEN ARROW
in the toolbar
***The experiment will start***
5.
When the run is finish, in the Plot area click on Autodetect Peaks command
at the top of the plot.
6.
The peak height will be visible.
Record the height value
listed on the top of the peak.
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
E
XPERIMENT
6: Determination of Ascorbic Acid by Differential Pulse Voltammetry
45
In order to begin another run you just click again on the green arrow.
Perform a duplicate run on the same solution. To do so, degas and stir the solution for 2 minutes.
When it is done, turn off the gas flow, turn off the stirring, and perform a run as described above.
Once the run is complete, find the current peak value, using the same data treatment steps as
described above.
Perform analysis of a second standard solution. The second standard solution is prepared by
adding 0.4 mL of the 3 mM ascorbic acid solution to the polarographic cell. Degas and stir for 2
minutes, then start a run. Perform duplicate analysis, as specified above. Repeat this procedure
3 other times (for a total of 5 standards).
To analyze the unknown solution, throw your last standard solution in the waste. Rinse and dry
the polarographic cell. Pour the diluted unknown solution (10 mL) in the empty polarographic cell.
Degas and stir for 5 minutes, then start a run. Perform a duplicate analysis. Repeat this procedure
for the second unknown (second student).
Calculations:
Plot the intensity of the peak current versus the concentration of ascorbic acid. Make sure you
use the real concentration of ascorbic acid in your standards, that is, the one calculated from the
actual mass of ascorbic acid you weighed. This should be clearly shown in your lab report.
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
E
XPERIMENT
6: Determination of Ascorbic Acid by Differential Pulse Voltammetry
46
Additionally, you need to take into account the dilution factor of each standard analyzed. The first
standard is 0.4 mL of your 3 mM standard in 10 mL of buffer, for a total volume of 10.4 mL; the
second standard is 0.8 mL of the 3 mM standard in a total volume of 10.8 mL, and so on. From
the calibration curve built with your standards, you can calculate the concentration of your
unknown. Do not forget to take into account the dilution of your unknown solution.
Lab report:
Part 1: This will be an abbreviated lab report. It is due on the normal due date. Complete the
following sections only: title, abstract, results and calculations and references (as appropriate) in
the lab report template as per usual.
All other sections
of the report should be denoted as “Not
required”
Part 2: You will prepare a PowerPoint presentation covering both this experiment and the
Coulometry experiment together.
There are two specific PowerPoints on Moodle to help you
understand how to structure a Scientific presentation (similar to what is given at a Scientific
conference) and how to prepare a PowerPoint with voice-over.
They should be found in the
“Lab” section of Moodle.
The presentation is due one week after the submission of the second electrochemistry
abbreviated report (the second Part 1)
References:
1.
Harris DC. Electroanalytical Techniques. In:
Quantitative Chemical Analysis
. 7th ed. New
York: W. H. Freeman; 2007:348
–
377.
2.
Osteryoung J. Pulse voltammetry.
J. Chem. Educ.
1983;60(4):296.
3.
Mirceski V, Komorsky-Lovric S, Lovric M.
Square-Wave Voltammetry Theory and Application
.
Available at: http://0-www.springerlink.com.mercury.concordia.ca/content/j45550/?MUD=MP.
Accessed June 20, 2012.
4.
Douglas A. Skoog, F. James Holler, Stanley R. Crouch. Voltammetry. In:
Principles of
Instrumental Analysis
. 6th ed. Belmont, California: Thomson Brooks/Cole; 2007:716
–
760.
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
3
E
XPERIMENT
7
Coulometric Determination of Arsenate
NB: 2023
–
this experiment does not require a traditional lab report.
Instead you will prepare
your results and a Powerpoint presentation with voice-over as a lab report/presentation for
both electrochemistry experiments.
Specific instructions are on Moodle (lab section)
Purpose:
The goal of this experiment is to determine the concentration of arsenate in an unknown solution
using coulometry.
Theory:
Coulometric analysis relies on using the quantity of electricity needed to complete an electrolytic
reaction to calculate the quantity of analyte present. The completion of the electrolysis reaction
can be evaluated through electrochemistry, but in this experiment we use colorimetric detection.
In this experiment, a series of electrochemical reactions are used to quantify arsenate.
Iodide (I
-
) is first oxidized at the anode to produce the active species iodine:
triiodide (I
-
) (an iodine molecule complexed with one iodide ion) then acts as an oxidant for
arsenate (conversion of As(III) to As(V)):
The triiodide is thus converted back to iodide. However, once all of the arsenate is converted,
excess triiodide (I
3
-
) starts to build up. Since starch and iodine (present in the excess triiodide)
form an intense blue-purple coloured complex, colouration of the solution indicates completion of
the electrolysis reaction.
As with all electrochemical cell reactions oxidation (of iodide) occurs at the anode and the
accompanying reduction (of H
+
) occurs at the cathode. Both electrodes are immersed in
conductive phosphate buffer. The anode is immersed in a solution containing iodide, starch and
arsenate. Charge balance is maintained by movement of ions through the frit present at the
bottom of the beaker containing the anode (see Figure 2). Since this is an electrolytic cell, it must
be activated by a power source. The circuit used for this experiment (Figure 1) also includes
resistances and a voltmeter. The circuit can be closed by a switch.
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
E
XPERIMENT
7 Coulometric Determination of Arsenate
50
e
-
Figure 1: Electrical Circuit
The quantity of electrons needed to complete the electrolytic reaction can be calculated by using
the time elapsed during the reaction and the current:
where Q is the charge (in Coulombs, C), I is the current (A) and t is the time (s).
And the current passing through the circuit can be calculated using its characteristics:
where I is the current (A), U is the potential (V) and R is the resistance
(Ω).
The quantity of analyte oxidized or reduced can then be calculated using
Faraday’s
law:
where n
A
is the number of moles of analyte reduced or oxidized, Q is the charge (C), n is the
number of moles of electrons lost or gained per oxidation or reduction and F is
Faraday’s
constant
(96 485.3365 C/mol of e
-
).
-
+
3300
Ω
100
Ω
390
Ω
Anode
(Outer Electrode)
Cathode
(Inner Electrode)
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
E
XPERIMENT
7 Coulometric Determination of Arsenate
51
Materials, chemicals and instruments:
•
1 beaker of 250 mL
•
Frit bottomed beaker
•
3 beakers of 100 mL
•
Rubber stopper with holes
•
2 electrodes
•
Electrical circuit, including wiring and resistances
•
Voltmeter
•
Stopwatch
•
2 glass pipettes (2 mL)
•
Pasteur pipettes
•
Magnetic stir plate
•
Heating plate
•
Starch
•
Anhydrous sodium carbonate
•
Sodium iodide
•
Phosphate buffer (pH 8.0)
•
Arsenate unknown
Procedure:
Solution(s) preparation:
In a 100 mL beaker, add 0.3 g of starch and approximately 10 mL of water. Use a heating plate
to bring the solution to ebullition. The solution should be opalescent after stirring.
To the 250 mL beaker (outer beaker), add approximately 150 mL of phosphate buffer, 9 g of KI,
0.3 g of Na
2
CO
3
, 2 mL of arsenate unknown, 1 mL of starch (20 drops using the Pasteur pipette)
and the magnetic stir bar.
Fill the frit bottomed beaker (inner beaker) to the 3/4 mark with phosphate buffer solution. Insert
the small green rubber stopper in this beaker, and insert this assembly in the big black rubber
stopper.
Insert the black rubber stopper (with the green rubber stopper and the inner beaker attached to
it) in the outer beaker. The level of solution in the outer beaker should be slightly above the bottom
frit of the inner beaker (see Figure 2).
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
E
XPERIMENT
7 Coulometric Determination of Arsenate
52
Figure 2: Picture and Sketch of Coulometry Setup
Measurements:
The circuit should already be built for you, but make sure it is properly done using the diagram
placed on the bench top. Using the alligator clip, connect the red wire of the switch to the outer
electrode, and the black wire from the power supply to the inner electrode.
Start stirring the solution. Stirring should be brisk without creating a “vortex” in the beaker. The
solution should be stirred during the whole experiment. Switch the power supply on. Switch the
voltmeter on by placing the wheel at
“2”
in the
“DCV”
section (see Figure 3). Switch the circuit on
using the switch. The meter should show a reading of approximately 1 V. Notice that the outer
electrode is producing a purple colouration in the solution, which disappears as the solution is
stirred. Switch off the circuit when the solution has just entirely turned purple. Remove the black
rubber stopper, and pour a small quantity of the purple solution in a 100 mL beaker. This will be
used as your colour control.
Magnetic Stir Plate
Outer Beaker
Rubber
Stopper
Magnetic Stir Bar
Inner (Frit Bottomed)
Beaker
Outer Electrode
Inner Electrode
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
E
XPERIMENT
7 Coulometric Determination of Arsenate
53
Figure 3: Voltmeter Configuration
Add exactly 2 mL of arsenate unknown. Switch on the circuit and start the stop watch
simultaneously. Note down the voltage displayed on the voltmeter. Place your colour control
beaker right beside the outer beaker, and when the colouration in the outer beaker becomes the
same as your colour control, stop the stopwatch and turn off the circuit simultaneously. Note the
time elapsed during the completion of the electrolysis reaction.
Add again 2 mL of arsenate unknown. Repeat the electrolysis procedure, monitoring again the
voltage and the time elapsed.
Repeat this procedure until you have at least three time data where the difference between the
highest and the lowest values is less than 5%.
Calculations:
Using the equations presented in the theory above, calculate the quantity of arsenate reduced
after each unknown addition (in mol). From there, you can easily calculate the concentration of
arsenate in the unknown. Be careful while choosing the value of the resistance you use for this
calculation (think about over which resistance the voltage is measured).
Lab report:
Part 1: This will be an abbreviated lab report. It is due on the normal due date. Complete the
following sections only: title, abstract, results and calculations and references (as appropriate) in
the lab report template as per usual.
All other sections
of the report should be denoted as “Not
required”
Part 2: You will prepare a PowerPoint presentation covering both this experiment and the DPV
experiment together.
There are two specific PowerPoints on Moodle to help you understand
how to structure a Scientific presentation (similar to what is given at a Scientific conference) and
how to prepare a PowerPoint with voice-
over.
They should be found in the “Lab” section of
Moodle.
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
E
XPERIMENT
7 Coulometric Determination of Arsenate
54
The presentation is due one week after the submission of the second electrochemistry
abbreviated report (the second Part 1)
References:
1.
Douglas A. Skoog, F. James Holler, Stanley R. Crouch. Coulometry. In:
Principles of
Instrumental Analysis
. 6th ed. Belmont, California: Thomson Brooks/Cole; 2007:697
–
715.
2.
Harris DC. Electroanalytical Techniques. In:
Quantitative Chemical Analysis
. 7th ed. New
York: W. H. Freeman; 2007:348
–
377.
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
E
XPERIMENT
8
Quantitative Analysis of Citrus Essential Oils by Gas Chromatography
–
Mass Spectrometry
Purpose:
The primary purpose of this experiment is to identify and quantitate several components of
citrus essential oils
Theory:
Essential oils are the generic term applied to the chemical components extracted from a
natural source, usually from fruits, flowers, leafs or nuts. They typically have strong flavours and/or
fragrances characteristic of their source. These essential oils find a wide variety of uses, especially in
cooking (vanilla, orange, almond, peppermint, etc.), the fragrance and aroma therapy industry (rose,
citrus, bergamot etc.) and other industries (c
itronella, peppermint, lemon/orange, clove, etc.).
Given their use in fragrance/aroma therapy, and their economic importance, it is critical to have a viable
method of measuring the key components of essential oils.
The fact that, by definition, essential oils are fragrant implies that many of the components are volatile at
relatively low temperatures. As a natural product, it is also probable that they will be a complex mixture of
different species with many of them being closely related compounds. Taken together, these two facts
suggest that the analytical technique must be capable of measuring many different, but similar, species.
Gas chromatography is an ideal technique for separating complex mixtures into its components due to its
high resolving power (each component appears in the chromatogram as a separate peak) as demonstrated
in the following separation of 30 different species.
GC multi-residue pesticide standard with 30 components. Image taken from:
http://www.restek.com/chromatogram/view/GC_FS0605/prod::41958
, September 2017
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
E
XPERIMENT
8 Citrus oil by GC-MS
55
However, achieving a complete separation is not sufficient (see
“Purpose”)
since it can be extremely difficult
to identify the components from the chromatogram alone, especially in a complex natural product. There
can also be overlapping (unresolved) peaks, even with high resolution separations. To provide
unambiguous identification of the components of a mixture it is also necessary to obtain molecular
information. This is best done using mass spectrometry
–
as explained more completely below. For these
reasons, this experiment will use GC-MS.
Basics of Gas Chromatography
In successful gas chromatography (GC) the analytes MUST be vaporized. This means they need to be
reasonably volatile (bp less than 250-300 C usually) and stable at the elevated temperature of the oven.
In chromatography the analytes partition between the mobile phase (MP) and the stationary phase (SP)
(i.e. there is an equilibrium process where the analyte moves into/out of the MP and into/out of the SP).
Therefore, both the MP and the SP must be reasonably good solvents for the analyte(s).
While the analyte is in the MP it is carried/swept down the column towards the detector, when the analyte is
in the SP it is, effectively, stationary. Depending on the type of MP, type of SP and the available energy
(temperature in GC) different types of molecules will have differing equilibrium points and therefore spend
different amounts of time in the MP and SP.
The analytes that have high energies AND/OR low affinity for the SP spend little time in the SP and
consequently progress down the column quickly. Analytes with low energy (high bp) AND/OR high affinities
for the SP will spend a large fraction of their time in the SP and will progress down the column slowly.
Therefore, the order of emergence (and detection) from the column depends on the
analyte’s
interactions
with the MP, SP and its energetic requirements (thermodynamic properties).
In GC we intentionally use an inert gas as the MP so the (chemical) interaction with the MP is uniform for all
species.
Therefore, it is only the analyte’s interaction with the SP and the energetic (thermodynamic)
properties that give rise to the separation.
When choosing a GC column it is critical that the analyte(s) be capable of interacting with the SP
(otherwise…).
To that end, there are a wide variety of commercially available columns with different
SP’s.
The most general property to select from is the polarity of the SP which may run from extremely non-polar
(hydrocarbon/waxes) through to highly polar (glycols, amines, acids).
Once the column (and therefore the SP) is chosen the only remaining variable to control the separation is
the energy available to alter the equilibrium point of the analyte(s). In GC this is controlled via the
temperature that may be altered during a separation to optimize the separation (a lower temperature
maximizes the interactions with the SP and therefore increases retention and increases separation of
closely related species, increasing the T reduces interaction and therefore reduces retention and improves
overall separation time
–
do you really want a 14 hour long separation?). A well designed separation
method (a relatively long column, a carefully chosen SP and precise temperature ramp program) achieves
good separation of analytes from each other and non-analyte components while still being relatively fast.
Chromatography is a powerful tool for separating complex mixtures (hopefully) into individual components
that can be measured and quantitated BUT unless there is some particular insight, or foreknowledge, there
is no way of knowing the identity of all of the peaks in a chromatogram. In some instances it is possible to
identify species by adding (spiking) a small amount of the purified analyte into a sample and observing
which chromatographic peak increases but even this
isn’t
a guarantee of identification since several species
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
E
XPERIMENT
8 Citrus oil by GC-MS
56
can elute from the column with the same retention time. This co-elution problem is more prevalent in
complex samples
–
such as natural products. What is needed is a detector that can provide molecular
information
–
mass spectrometry.
Basics of Mass Spectrometry
The analyte molecule(s) are neutral while undergoing chromatography however, they must be charged
(ionized) to be successfully analysed in a mass spectrometer. There are a wide variety of ionization
methods available but in GC-MS the most common is by electron impact. As the gas, containing the
analytes, emerges from the column it passes through a relatively high energy electron beam (usually about
70 eV). The majority of molecules are not impacted by the electrons and are therefore undetected,
however, some fraction are struck by an electron which causes ionization via extraction of one of the
molecule’s
electrons. This results in a molecular ion (cation) with the
molecule’s
original mass (the electron
has such a small mass it is negligible). This molecule is called the parent ion. In addition to the formation
of the parent ion, some electron impacts occur such that chemical bonds can be broken (most chemical
bonds require about 4-8 eV and the ionizing electron has 70 eV) resulting in ions that undergo
rearrangement to produce fragment ions. During these rearrangements, thermodynamically favored (i.e.
reasonably stable) structures are favored, as opposed to just any old structure.
These fragments will have
a mass that is lower than the parent ion and more importantly they will be characteristic of the parent ion’s
structure. Thus analysis of the fragmentation data, both the masses AND the relative intensities, reveals
significant information about the parent ion and can be used to unambiguously identify the parent ion
(molecule). For example, below is the GC-MS mass spectrum of two nearly identical species
–
the only
difference is the ring closure bond. The parent ions have, as expected, identical masses but, the
fragmentation data differs since the ring alters the types of fragment ions that were produced (243, 258 vs
246 Da) and the intensities (at 271, 299 Da etc).
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
E
XPERIMENT
8 Citrus oil by GC-MS
57
O H
O
Mass spectra of delta-9-tetrahydrocannabinol and cannabidiol. Mass spectra are usually scaled to
the maximum ion intensity and the x-axis is in mass/charge units (Da). All the ions produced in
electron impact ionization carry a +1 charge.
Given that the parent ion appears at the molecular mass and that the highly structure dependant
fragmentation pattern (masses and intensities) is reproducible it is possible to build a database/library of
mass spectra that can be used to identify species in samples. In some instances the identification will be
unambiguous but, in many cases the database search will provide a list of potential species and the analyst
will need to provide the final identification by spiking the sample with the pure analyte.
Basics of the method of Internal Standards
A common problem faced in analytical chemistry is the potential for uncontrolled losses/gains of analyte
during sample preparation and changes of instrument sensitivity during analysis. These problems will result
in signals (measurements) that do not accurately reflect the actual concentration of the analyte(s) in the
original sample.
These problems can be largely addressed by including a known amount of internal standard (IS) in the
sample (and standards too). The IS needs to match the analyte(s) physio-chemical behaviour as closely as
possible in order to also experience the same losses/gains throughout the preparation/measurement
processes. If MS detection is employed, deuterated analogs of the analyte(s) make the perfect IS since
delta-9-Tetrahydrocannabinol
100
299
231
314
271
50
41
243
91
55
67
81
115
174
258
147
193
217
0
60
90
120
150
180
210
240
270
300
330
(swgdrug) delta-9-Tetrahydrocannabinol
H
O
O
H
Cannabidiol
100
231
50
0
43
55 67 77
60
91
90
107
121
120
174
147
150
193
207
246
180
210
240
271 285
270
299
314
300
330
(swgdrug) Cannabidiol
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
E
XPERIMENT
8 Citrus oil by GC-MS
58
they have identical physio-chemical behaviour as the
“light”
version of the analyte but can be readily
distinguished from the analyte based on the additional mass from the deuterium.
In practice, the same amount of IS is added to all samples, blanks and standards and provides a constant
reference signal/measurement. To determine the concentration of the analyte(s) the ratio of analyte signal
to IS signal is used for all calibration and concentration calculations (i.e. take the ratio of signals then treat
this ratio as your “new signal” and proceed as if doing the usual external standards calibration)
In the event of any losses/gains both the analyte(s) and IS are changed by the same proportion which does
not affect the ratio and therefore the quantity determined (neat huh?)
Calibrating from scratch
In this lab you will prepare your standards from scratch
–
i.e. you will need to determine the range of
concentrations needed to calibrate. The general rule is that, ideally, the unknown should fall into the center
of the calibration standards. However, without knowing the concentration of the analyte(s) a priori it is difficult
to prepare the correct standards. To address this it is necessary to perform one measurement (separation)
of the unknown and one measurement of a standard at a known concentration (usually at the best guess of
the concentration of the analyte). From the standard measurement it is possible to establish a ROUGH value
for the sensitivity (signal/unit of concentration). Use this value and the measured signal from the sample to
estimate the concentration in the sample. Round this value to a convenient value to facilitate preparation of
the standards (i.e. if the [ ] = 62 ppm round it to 50 and make your standards in multiples of 50 ppm). Prepare
standards that will surround the sample
–
usually aim for ≈ ¼, ½, 1, 2, 4 X the sample concentration.
For example. If your chromatograms looks like:
Pure standard at 350 ppm
Rough concentration in sample:
[sample]
≈
Signal
sample
/ sensitivity = 3500/13000 x 350 ppm = 94.23
≈
100 ppm
Prepare your middle standard at 100 ppm
Sample at XX ppm
Signal
sample
≈
3500
Signal
std
≈
13000
sensitivity
≈
13000/350 (ions/s)/ppm
Analyte
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
E
XPERIMENT
8 Citrus oil by GC-MS
59
Materials, chemicals and instruments:
Procedure:
Overview: you will be provided a sample of lime peel. Add a known amount of deuterated IS and extract
the essential oils using methanol. Separate this sample by GC-MS in an initial separation. Your TA will
assign you an analyte to quantitate and provide you with chromatographic information necessary to find the
analyte in the sample chromatogram. Verify the identity of the analyte, estimate its concentration, prepare
calibration standards (including IS) and calibrate the instrument. Measure the sample and determine the
concentration of the analyte in your sample.
Glassware and Chemicals
50 mL beaker
10 mL graduated cylinder
Methanol
7 cm Whatman filter paper
Dichloromethane
Micropipette (2
–
20 μL)
Anhydrous sodium sulfate
Micropipette (20
–
200 μL)
Diluted
D
-linalool (Internal standard)
Micropipette (200
–
1000 μL)
Vial
≈
0.2 g of grated whole lime flavedo
Filtration funnel
Short Pasteur pipette
GC vials
Analytical balance
GC-MS
Procedure
Part 0: Already done for you
•
The lime will have already been washed with soap to dewax the peel and just the flavedo (the
exterior, oil rich part, none of the white pith) will have been grated, alliquotted (≈0.2 g) and frozen
at -80 C.
Removal of the flavedo by grating. Note no pith should be included.
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
E
XPERIMENT
8 Citrus oil by GC-MS
60
Part I: pipetting exercise with DCM
You will use methanol and dichloromethane (DCM) throughout this experiment. They are very
low viscosity and high volatility solvents so that solvent losses, and losses during transferring
operations (pipetting) are likely to constitute the major source of error.
•
Use a 100 μL pipette and draw in 100 μL of water and hold the pipette vertically for 10-15
seconds. Note if any of the water is expelled/lost from the pipette.
•
Repeat the above with Methanol and DCM
Consider these results when transferring and pipetting during the remainder of the experiment.
Preparation of DCM solvent
•
Prepare the DCM solvent by dissolving 100 μL of the internal standard into 50 ml of DCM using
a volumetric flask.
Extraction of essential oils
•
Record the exact mass of the glass vial containing the grated whole lime flavedo including the cap.
•
Add 7 mL of methanol and 100 μL of diluted
D
-linalool (internal standard), then securely close the cap
of the vial. Shake the glass vial gently a few times before opening the cap to release any pressure
buildup. Continue with this process for 5 minutes.
•
Add approximately one gram of anhydrous sodium sulfate (kept in a desicooler) to the glass vial and
allow to dry for 5 min to remove any water.
•
Filter the solid-liquid mixture by gravity filtration and collect it in a 50 mL beaker. Rinse the vial with 3
mL methanol and use it to rinse the filter paper.
•
Record the accurate mass of the cleaned and dried glass vial with cap to determine the mass of the
grated flavedo used for extraction.
•
Prepare 2 samples for injection on the GC-MS by diluting the methanol extract. To dilute, transfer 10
μL of the methanol extract into each GC vial and add 990 μL of dichloromethane in each. SEAL THE
VIALS CAREFULLY
–
the solvents are volatile!
Measurement of sample signal (initial run)
•
Place your sample vials in the auto-sampler, verify the position numbers
•
On the computer screen double click
“Chromeleon
7”
to open the main page of the software.
•
When the page opens, click on
“Data”
shown on the lower left side of the screen.
•
Double-click CHEM 312 and select
“Experiment
#8”
(or any number you would like to give to the
experiment)
•
In this window, click on “Click here to add a new injection”. The software will introduce a new line at the
bottom. Enter a new
“Name”
for your sample, the
“Position”
of ONE of your vials in the autosampler and
leave the rest unchanged.
•
Press on “Save” and then “Resume” to start the run. It might take 5 to 10 minutes for the instrument to
adjust itself to the proper temperature and flow rate of the carrier gas. When ready, the LED light
“RUN”
on
the GC instrument will turn blue and the sample will be injected.
•
The run takes 15 min.
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
E
XPERIMENT
8 Citrus oil by GC-MS
61
Preparing your calibration standards
–
IMPORTANT ASK YOUR TA FOR ANY MODIFICATIONS TO THIS SECTION
•
Your TA will assign an analyte for quantitation, collect a chromatogram from this standard, overlay the initial
sample separation and the standard chromatogram to identify the analyte in the sample chromatogram.
•
Use the MS data to verify the identity of your analyte (compare the MS from
both…).
•
Use the standard chromatogram data and its concentration to establish the (approximate) sensitivity.
•
Use the results of your initial run and the sensitivity to estimate the analyte concentration in the sample.
•
Using the known concentration of the provided stock (usually in the ballpark of 10X the most concentrated
calibration standard) transfer appropriate volumes to produce calibration standards that are: 0 (a blank), 4, 2,
1, 0.5, 0.25 times the estimated sample concentration in 6 volumetric (10 ml) flasks. (should be about 4000,
2000, 1000, 500 and 250 μL transferred, set the pipette to 1000 μL for the more concentrated standards and
250 μL for the last two
–
think about why…).
•
Dilute the blank and standards to the 10 ml mark with DCM containing the IS.
•
Prepare the standards for injection by transferring 1 mL to each of the GC vials. BE SURE THE VIALS ARE
SEALED
Collecting calibration and sample data
•
Your two sample vials should already be in the auto-sampler, add the 6 standards and note their positions in
the rack.
•
Run the samples and standards (see above for instructions).
Calculations:
Recall that this is the method of internal standards. Use linear regression to determine the calibration
equation.
Determine the true concentration and confidence interval of the analyte (μg/g) in the peel.
Determine the LOD, LLOQ and ULOQ
Lab report:
The following questions/points should be addressed in your lab report. The answers should be included in
the body of the text, not as separate sections.
•
Why is it necessary to dry the extract?
•
Why is it best to have the calibration standards bracket the unknown?
•
Why is the blank included in the calibration set (what do you expect in a blank, what did you actually observe)?
•
There are many options for
IS’s
(see the book), why are deuterated
IS’s
preferred over analogs of the analyte?
•
What determined the order of elution? Does the MS affect the separation?
•
Demonstrate that you have correctly identified the targeted analyte using the information available to you.
•
Identify the major peaks in the chromatogram based on their mass spectra.
Compounds known to be found in citrus essential oils (mostly lemon but many should also be
found in lime), in no particular order.
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
E
XPERIMENT
8 Citrus oil by GC-MS
62
Myrcene
α
- Thujene
Octanal
α
–
Pinene,
β
- Pinene
α
-Phellandrene,
β
-Phellandrene
Sabinene
3-Carene
Heptanal
Nerol
Citronellal
p-Cymene
Terpinen-4-ol
δ
-Limonene
γ
-Terpinene,
α
-Terpinene
Terpinolene
Linalool
Nonanal
α
-Terpineol
Decanal
cis-3,7-Dimethyl-2,6-octadien-1-ol
Neral
Geraniol
Geranial
Nonyl acetate
Citronellyl acetate
Neryl acetate
Geranyl acetate
Lauric aldehyde
trans-
α
-Bergamotene
β
-Bisabolene
Camphene
References:
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
E
XPERIMENT
8 Citrus oil by GC-MS
63
Appendix: Software
To open the software and check the instrument parameters
1)
On the desktop, double-click on the
“Chromeleon
7”
Software. The software main window will appear.
2)
On the lower left corner of the screen, click on
“Data”.
3)
In the list under
“Data”,
double-click on the folder
“Summer
2017”.
Select
“orange
oil”.
4)
Place your oil vial sample into the auto-injector. Look at what slot position it is.
5)
On the lower left corner of the screen, click on
“Instrument”.
A window with options and parameters to the
machine and the conditions of the machine are displayed.
6)
Under the
“Filter”
tab, select both boxes: TIC and +c EI Full ms[50.00-300.00]
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
E
XPERIMENT
8 Citrus oil by GC-MS
64
7)
Under
“ISQ1601519”
tab, make sure the parameters of the instrument are as followed:
-
MS Transfer Line temp: 260
℃
-
Ion Source Temp: 275
℃
-
Electron Energy: 70.0 [eV]
8)
Under
“Sampler”
tab, make sure the parameters of the injection volume is 0.1μL. For the position number,
select the number where your sample is placed.
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
E
XPERIMENT
8 Citrus oil by GC-MS
65
To create a file for injection
1)
On the main window, click on
“Click
here to add a new
injection”.
A row will appear.
2)
Fill in the slots as followed:
a.
Title: Give a name for your essential oil sample (Example: [YourInitials] Lime Oil);
b.
Type: Unknown;
c.
Volume: 0.1;
d.
Position: the slot number where your sample is placed (1 to 8);
e.
Other parameters are unchanged.
Repeat steps 5 and 6 for other injections (make sure to select the right vial positon number if more than one
vial is being injected and analysed). Save your new injection file by clicking on
“Save”,
at the top of the main
window.
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
E
XPERIMENT
8 Citrus oil by GC-MS
66
3)
Click on “Resume” to start the run. It may take some time for the instrument to adjust itself to the proper
temperature and flow rate to start the run. On the GC, the LED light on the
“Run”
sign will turn blue, and the
auto-injector will start moving and injecting.
To analyse the chromatogram
1)
When the sample is running, click on the
“Data”
tab, then double click on the small chromatogram picture of
your sample run.
2)
The tab “Data Processing” is selected in a new
window (at the lower left corner). This is where you will see
your chromatogram. At first you may not see it, because the instrument is still preparing for separation (you
may see a red writing on the “Data Processing” screen). When the peaks appear, click on the F5 button to
refresh the chromatogram every 2-3 minutes. The run will take 14 minutes, and then will see your
chromatogram like so:
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
E
XPERIMENT
8 Citrus oil by GC-MS
67
3)
To zoom in on your peaks, select a portion of the chromatogram by holding the left-button and dragging the
mouse to form a blue rectangle. The selected blue portion will zoom in.
*To zoom out, bring your mouse to the lower left corner of the chromatogram screen and a button will
appear. Click on that button to zoom out.
Print the chromatogram by holding the buttons Ctrl + P, choose the printer Samsung Driver and click OK.
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
E
XPERIMENT
8 Citrus oil by GC-MS
68
4)
On the
“Layout”
tab at the top of the software window, select on
“Time
Spectra”
at the top right.
5)
Chose a peak you want to analyse and identify, and select a time frame of the peak by left-clicking and
dragging to mouse inside the peak. A mass spectra will appear under your chromatogram.
6)
Right-click on the mass spectra, select
“Spectrum
with
NIST”.
A new window with the mass spectra,
molecular structure and list of possible compounds will appear.
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
E
XPERIMENT
8 Citrus oil by GC-MS
69
7)
Identify the peak by mass fragmentation and choose the compound that is most likely present in the
essential oil (use reference list for help). Close the window by clicking
“X”
→
This is how you can identify
the compounds manually.
*To identify the peaks using the software, click on the chromatogram. Under the “Data Processing Home”
tab, click on “Interactive Results”. A new small window under the chromatogram will appear. Click on
“Library
Search”
under the new window, and you will see three lists of high percentage possible compounds
for each peak on your chromatogram. Chose the compounds that you think represents best the peak. You
may print this table by clicking on the table and hold Ctrl + P and print (with the Samsung printer).
Identify each peak.
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
70
E
XPERIMENT
8 Citrus oil by GC-MS
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
8)
On the Layout tab, click on
“Delimiters”.
Double-click on a peak until it turns pink. Right-click on that peak,
and select “Peak Properties”.
9)
A small window with the properties of the peak will appear. Write down the retention time, the name of the
peak and its area.
Your preview ends here
Eager to read complete document? Join bartleby learn and gain access to the full version
- Access to all documents
- Unlimited textbook solutions
- 24/7 expert homework help
Related Documents
Related Questions
1. Solid Lead react with Copper (II) nitrate
Please write
Complete reaction /
Ionic reaction
Net ionic reaction
Oxidized half
Reduced half
Find oxidation agent/
Find reduction agent/
................................................................................
Balan2. Balance the following redox reaction using the half-reaction method under basic conditions.
MnO4- + 2CHCl3 ® 3Cl2 + 2CO2 + Mn2+
arrow_forward
[15 minutes] Predict the chemical formula for the ionic compound that will form
when the elements listed below combine as ions, and then name the
compound.
A. Al and S
B. Kand N
C. Sr and O
D. Al and Cl
E. Na and F
6/24/24
WILBERTH A. NARVAEZ | ELEMENTARY CHEMISTRY
19
19
arrow_forward
Hydrochloric acid (aq) reacts with the mineral calcite (s) to produce carbon dioxide (g), water (l), and calcium chloride (aq). Based on what you have learned in activities 1, 2, and 3, what are three things you could do to make the reaction occur more quickly?
.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,..,.,,.,
arrow_forward
Preview
File
Edit
View
Go
Tools
Window
Help
Thu 25 Feb 5:13 PM
Tutorial Rate Law.pdf
Search
1 page
Tutorial Rate Law.pdf
OMY AND
GENETICS THINKING SKILLS
COMPUTER
IOLOGY
SCIENCE
reacts with iodine according to the following equation: Â ( C...
time
Tutorial
lude all
Rate Law
writing, or
he rate of the ...
Diethylhydrazine reacts with iodine according to the following equation:
|) 2 ( …..
(C2H5)2(NH)2 (1) + l2 (aq)
» (C2H5)2N2 (1) + 2HI (aq)
bjects!
I we want
ed time ask
ubjects as
ey don't
Table 1 shows the data at a certain temperature.
and more?
[(C2H5)2(NH)2] /
Experiment
[l2] /M
Rate /Mh1
count!
M
bject experts
1.08 х 104
1.56 x 104
2.30 х 104
3.44 х 104
0.150
0.250
uestions are
fast as 30
2
0.150
0.362
Political
Science
0.200
0.400
4
0.300
0.400
Table 1
d time:
Calculate the order of reactions for each of the reactants and write the rate law for the
ubject and
reaction.
esponse time
Privacy - Terms
nger for new
OTO
28
FEB
CC
25
étv
280
PDF
arrow_forward
4
arrow_forward
The gas phase reaction between hydrogen and iodine is proposed to occur as follows:.....step 1.....fast:......I2 2 I.....step 2.....slow:....H2 + 2 I 2 HI(1) What is the equation for the overall reaction? Use the smallest integer coefficients possible. If a box is not needed, leave it blank.
+
+
(2)
Enter the formula of any species that acts as a reaction intermediate? If none leave box blank:
(3) Complete the rate law for the overall reaction that is consistent with this mechanism.Use the form k[A]m[B]n... , where '1' is understood (so don't write it if it's a '1') for m, n etc.
Rate =
arrow_forward
I#12 please. C would be The equation for a second order with 1/A.
woykd not the answe mr for A v T be reverse and answet B . This is not a graded question as it is a practice question . I am 60 years old and helping my son prepare for the AP exam in a few months. We do questions at the back of the textbook by Zumdahl and Zumdahl
arrow_forward
Can you please complete and check if the answers are correct!!! Thank you in advance!!
arrow_forward
1) In the laboratory, a general chemistry student measured the pH of a 0.554 M aqueous solution of nitrous acid to be 1.787.
Use this information she obtained to determine the Ka for this acid?
Ka .......................... ?
2) How many grams of solid potassium hydroxide ( molar mass= 56.1 g/mol) are needed to react with 26.7 mL of a 1.75 M hydroiodic acid solution?
KOH + HI......> kI + H2o
................ grams
arrow_forward
The rate constant for the decomposition of H2O2 was determined to be 2.89×10¬2 s1. How long will it take for 98.7% of the H2O2
to decompose?
arrow_forward
LUTCA
Chapter 3 Homework: Allie Burt
← Chapter 3 Homework
Question 13 of 14
View Policies
Current Attempt in Progress
r
Save for Later
L
WP NWP Assessment Player Ul Application
education.wiley.com
g of malachite were originally used.
Chemistry I (Science Majors) (CHEM-141...
Ć
Malachite is a green copper mineral with the formula Cu₂CO3(OH)2. It can be converted into copper metal in a number of ways. One
method extracts the metal by reacting the malachite with sulfuric acid to form CuSO4, and then adding iron to the CuSO4 solution
which, after they react, forms FeSO4 and copper metal. The balanced chemical reactions are:
Cu₂CO3(OH)2 (s) + 2 H₂SO4 (aq) →2 CuSO4 (aq) + 3 H₂O (1) + CO₂ (g)
CuSO4 (aq) + Fe (s) →FeSO4 (aq) + Cu (s)
If the final solution is dried, and 3.28 g of iron sulfate is collected, how many grams of malachite were originally used?
W Wiley Course Resources
-/1 = :
Attempts: 0 of 3 used
Submit Answer
Quest
Multipar
Quest
Multiple
Ques
Numer
Ques
Numer
Units
Que
Multip
Que…
arrow_forward
Q4.
lodide ions are oxidised to iodine by hydrogen peroxide in acidic conditions.
H₂O₂(aq) + 2H+(aq) + 21-(aq) → 1₂(aq) + 2H₂O(1)
The rate equation for this reaction can be written as
rate = k [H₂O₂] [1] [H*]c
In an experiment to determine the order with respect to H*(aq), a reaction mixture is made
containing H*(aq) with a concentration of 0.500 mol dm-³
A large excess of both H₂O2 and 1 is used in this reaction mixture so that the rate
equation can be simplified to
rate = k₁ [H*]c
(a)
Explain why the use of a large excess of H₂O2 and I means that the rate of reaction
at a fixed temperature depends only on the concentration of H+(aq).
(b)
Samples of the reaction mixture are removed at timed intervals and titrated with
alkali to determine the concentration of H*(aq).
State and explain what must be done to each sample before it is titrated with alkali.
arrow_forward
I need help! Pls
arrow_forward
Bb Take Test: QUI
A Ims.uhb.edu.sa/webapps/assessment/take/take.jsp?cours...
لا تعيد السمكات إلى ا. . . الاثاد
M Gmail
O YouTube
Human Biology Ch...
A Apps
Remaining Time: 15 minutes, 04 seconds.
* Question Completion Status:
0.5 points
Saved
Question 6
Find the major product of following dehydration.
OH CH3
CH,-CH-CH-CH2-CH3
+ H20
CH3
OA CH2=CH-ĊH-CH2-CH3
OCH3
O B. CH3-C=c-CH2-CH3
OC. CH3-C-CH,-
-CH2-CH2-CH3
O CH3
OD. CH3-C-CH-CH2-CH3
CH3
CH3-CH=C-CH2-CH3
» A Moving to another question will save this response.
Question 6 of 10>
.w.
MacE
arrow_forward
A learning.mheducation.com
Types of Reactions assignment and quiz F.
Question Mode: Multiple Select Question
Explain the different types of chemical react...
https://moodle.coastal.edu/pluginfile.php/106...
https://moodle.coastal.edu/pluginfile.php/106...
Exit Assignment X
Mc
Graw
Hill
13 of 36 Concepts completed
i
Multiple Select Question
Select all that apply
LI
Which of the following reactions are expected to occur as written, with
Ba
reference to the activity series for metals (shown)? Select all that apply.
Ca
O Cu (s) + 2HCI (aq) → CuCl2 (aq) + H2 (g)
Na
Mg
Mg (s) + Co(NO3)2 (aq) → Co (s) + Mg(NO3)2 (aq)
Al
Mn
O Pb (s) + 2HCI (aq) → PbCl2 (s) + H2 (g)
Zn
Cr
O 3Ag (s) + Fe(NO3)3 (aq) → 3ÄGNO3 (aq) + Fe (s)
Fe
NI
Sn
Pb
H2
Cu
Hg
Ag
Au
Copyright O McGraw-Hll
Education
EText Alternative
Need help? Review these concept resources.
E Reading
Rate your confidence to submit your answer.
High
Medium
Low
O2021 McGraw-Hill Education. All Rights Reserved.
Privacy
Terms of Use
80
888
DII
DD
F10…
arrow_forward
A
??
OMe
B
OMe
arrow_forward
Is it correct
arrow_forward
18)
08
Veek 6 Homework # 6
Week 6 Homework
Topic: Week 6 Recorded X
Marc Foster's Personal M X +
stuniversity.edu/courses/6957/assignments/165042
Maps
EReading
5 of 16
MISSED THIS? Watch KCV 11.9; Read Section 11.9. You can click on the Review link to access the section in your e Text.
The hydrogen gas formed in a chemical reaction is collected over water at 30 °C at a total pressure of 732 mm Hg . The vapor pressure
of water at 30 °C is 31.8 mm Hg.
deos
Part A
What is the partial pressure of the hydrogen gas collected in this way?
Express your answer in millimeters of mercury to three significant figures.
tions
PH,
mm Hg
Next
- Previous
2:14 PM
85°F Sunny
10/3/2021
ASUS VivoBook
delete
insert
prt sc
home
F12 Pgdn
f10 end
因
94
61
f5
backspace
&
5
6
24
4.
9.
3.
arrow_forward
SEE MORE QUESTIONS
Recommended textbooks for you
Physical Chemistry
Chemistry
ISBN:9781133958437
Author:Ball, David W. (david Warren), BAER, Tomas
Publisher:Wadsworth Cengage Learning,
Related Questions
- 1. Solid Lead react with Copper (II) nitrate Please write Complete reaction / Ionic reaction Net ionic reaction Oxidized half Reduced half Find oxidation agent/ Find reduction agent/ ................................................................................ Balan2. Balance the following redox reaction using the half-reaction method under basic conditions. MnO4- + 2CHCl3 ® 3Cl2 + 2CO2 + Mn2+arrow_forward[15 minutes] Predict the chemical formula for the ionic compound that will form when the elements listed below combine as ions, and then name the compound. A. Al and S B. Kand N C. Sr and O D. Al and Cl E. Na and F 6/24/24 WILBERTH A. NARVAEZ | ELEMENTARY CHEMISTRY 19 19arrow_forwardHydrochloric acid (aq) reacts with the mineral calcite (s) to produce carbon dioxide (g), water (l), and calcium chloride (aq). Based on what you have learned in activities 1, 2, and 3, what are three things you could do to make the reaction occur more quickly? .,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,..,.,,.,arrow_forward
- Preview File Edit View Go Tools Window Help Thu 25 Feb 5:13 PM Tutorial Rate Law.pdf Search 1 page Tutorial Rate Law.pdf OMY AND GENETICS THINKING SKILLS COMPUTER IOLOGY SCIENCE reacts with iodine according to the following equation: Â ( C... time Tutorial lude all Rate Law writing, or he rate of the ... Diethylhydrazine reacts with iodine according to the following equation: |) 2 ( ….. (C2H5)2(NH)2 (1) + l2 (aq) » (C2H5)2N2 (1) + 2HI (aq) bjects! I we want ed time ask ubjects as ey don't Table 1 shows the data at a certain temperature. and more? [(C2H5)2(NH)2] / Experiment [l2] /M Rate /Mh1 count! M bject experts 1.08 х 104 1.56 x 104 2.30 х 104 3.44 х 104 0.150 0.250 uestions are fast as 30 2 0.150 0.362 Political Science 0.200 0.400 4 0.300 0.400 Table 1 d time: Calculate the order of reactions for each of the reactants and write the rate law for the ubject and reaction. esponse time Privacy - Terms nger for new OTO 28 FEB CC 25 étv 280 PDFarrow_forward4arrow_forwardThe gas phase reaction between hydrogen and iodine is proposed to occur as follows:.....step 1.....fast:......I2 2 I.....step 2.....slow:....H2 + 2 I 2 HI(1) What is the equation for the overall reaction? Use the smallest integer coefficients possible. If a box is not needed, leave it blank. + + (2) Enter the formula of any species that acts as a reaction intermediate? If none leave box blank: (3) Complete the rate law for the overall reaction that is consistent with this mechanism.Use the form k[A]m[B]n... , where '1' is understood (so don't write it if it's a '1') for m, n etc. Rate =arrow_forward
- I#12 please. C would be The equation for a second order with 1/A. woykd not the answe mr for A v T be reverse and answet B . This is not a graded question as it is a practice question . I am 60 years old and helping my son prepare for the AP exam in a few months. We do questions at the back of the textbook by Zumdahl and Zumdahlarrow_forwardCan you please complete and check if the answers are correct!!! Thank you in advance!!arrow_forward1) In the laboratory, a general chemistry student measured the pH of a 0.554 M aqueous solution of nitrous acid to be 1.787. Use this information she obtained to determine the Ka for this acid? Ka .......................... ? 2) How many grams of solid potassium hydroxide ( molar mass= 56.1 g/mol) are needed to react with 26.7 mL of a 1.75 M hydroiodic acid solution? KOH + HI......> kI + H2o ................ gramsarrow_forward
- The rate constant for the decomposition of H2O2 was determined to be 2.89×10¬2 s1. How long will it take for 98.7% of the H2O2 to decompose?arrow_forwardLUTCA Chapter 3 Homework: Allie Burt ← Chapter 3 Homework Question 13 of 14 View Policies Current Attempt in Progress r Save for Later L WP NWP Assessment Player Ul Application education.wiley.com g of malachite were originally used. Chemistry I (Science Majors) (CHEM-141... Ć Malachite is a green copper mineral with the formula Cu₂CO3(OH)2. It can be converted into copper metal in a number of ways. One method extracts the metal by reacting the malachite with sulfuric acid to form CuSO4, and then adding iron to the CuSO4 solution which, after they react, forms FeSO4 and copper metal. The balanced chemical reactions are: Cu₂CO3(OH)2 (s) + 2 H₂SO4 (aq) →2 CuSO4 (aq) + 3 H₂O (1) + CO₂ (g) CuSO4 (aq) + Fe (s) →FeSO4 (aq) + Cu (s) If the final solution is dried, and 3.28 g of iron sulfate is collected, how many grams of malachite were originally used? W Wiley Course Resources -/1 = : Attempts: 0 of 3 used Submit Answer Quest Multipar Quest Multiple Ques Numer Ques Numer Units Que Multip Que…arrow_forwardQ4. lodide ions are oxidised to iodine by hydrogen peroxide in acidic conditions. H₂O₂(aq) + 2H+(aq) + 21-(aq) → 1₂(aq) + 2H₂O(1) The rate equation for this reaction can be written as rate = k [H₂O₂] [1] [H*]c In an experiment to determine the order with respect to H*(aq), a reaction mixture is made containing H*(aq) with a concentration of 0.500 mol dm-³ A large excess of both H₂O2 and 1 is used in this reaction mixture so that the rate equation can be simplified to rate = k₁ [H*]c (a) Explain why the use of a large excess of H₂O2 and I means that the rate of reaction at a fixed temperature depends only on the concentration of H+(aq). (b) Samples of the reaction mixture are removed at timed intervals and titrated with alkali to determine the concentration of H*(aq). State and explain what must be done to each sample before it is titrated with alkali.arrow_forward
arrow_back_ios
SEE MORE QUESTIONS
arrow_forward_ios
Recommended textbooks for you
- Physical ChemistryChemistryISBN:9781133958437Author:Ball, David W. (david Warren), BAER, TomasPublisher:Wadsworth Cengage Learning,
Physical Chemistry
Chemistry
ISBN:9781133958437
Author:Ball, David W. (david Warren), BAER, Tomas
Publisher:Wadsworth Cengage Learning,
Browse Popular Homework Q&A
Q: The freezing point of benzene, C6H6, is 5.500 °C at 1 atmosphere. Kf(benzene) = 5.12 °C/m
In a…
Q: Which of the following interactions would be seen between the R-groups of His and Gln at pH 8?…
Q: True or False
compared with NAD+, electrons transferred to FAD lead to less proton translocation in…
Q: A livestock cooperative reports that the mean weight of yearling Angus steers is 1144 pounds.…
Q: n=1
(-1) + 1n
30
X ≤S≤
X
Q: to access important values if needed for this question.
The boiling point of ethanol, CH3 CH₂OH, Iis…
Q: sin x
5. (a) Use the Squeeze Theorem to evaluate lim
(b) Graph f(x) = (sin x)/x. How many times does…
Q: Let T (n) be the number of moves in our solution to the n-disc.
•move the top n −1 discs from the…
Q: The Parent consolidated group reports the following results for the tax year.
Entity Income or Loss…
Q: protein.
O
H
H "
H₂N-CH-C-N-CH-C-O
O
||
I
CH3
Ala Arg Asn Asp
Cys Glu
Gln Gly His lle Leu Lys
Q: A proton has a speed of 2.0 x 105 m/s at a point where the electrical potential is 1000 V. It moves…
Q: The position of a mass oscillating on a spring is given by x=(7.4cm)cos[2πt/(0.71s)]
Part A
What…
Q: When a work’s author is designated as “Anonymous,” which of the following is the correct APA 7.0…
Q: Some soluble compounds are listed in the table below.
Complete the table by filling in the name or…
Q: Which statement describes tumor cells?
A. Tumor cells acquire a drive to proliferate that does not…
Q: what measure of variability is to be computed with a median
Q: Like new express
had
machines in Stock at
Inventory records for
Q: 1. Balance the following redox reaction under acidic conditions.
S406²
S203² + 12
Q: Based on the thermodynamic properties provided for water, determine the amount of energy needed for…
Q: The time to complete an exam is approximately Normal with a mean of 45 minutes and a standard…
Q: Q6. Fill the following options given below into blanks. Please note the options given below can be…
Q: In the figure, the vehicle has a mass of 2000 kg, and is traveling at a speed of v = 50 m/s. If the…
Q: compressed
shoot 1.80 kg packages
into a truck, as the figure(Figure 1) shows. The spring constant…
Q: The letters A, B, and C are marked on the histogram. Describe the shape of the data.
Then determine…
Q: In step C, which mechanistic step(s) is(are) taking place? Choose all that apply
Nucleophilic attack…