CH-317

ANALYTICAL INSTRUMENTATION

FOR BIOTECHNOLOGY

LABORATORY MANUAL

Fall 2020 EDITION

BY

ANDREW BAER, AKM RUHUL BAKSHA, GILLIAN GORING,

SINTHUJA SENATHIRAJAH, KRISHNAKANT (KEN) SHAH, and TAN
THICH

CENTENNIAL COLLEGE OF APPLIED ARTS & TECHNOLOGY

TORONTO, ONTARIO
 TABLE OF CONTENTS
Page

Laboratory safety guidelines 3

Instructions for Chemical Laboratory Report 5

Laboratory Schedule 8

Exp #

1. Determination of Caffeine and Vitamin B6 in 9

Energy Drinks by HPLC.

2. Determination of chlorophyll in pond, 13

river and/or lake samples by spectrophotometry

3. Determination of Percentage Ethanol in Beverages by GC 18

4. Determination of Fluoride Ion Concentration in Drinking Water 21

5. Determination of Concentration of Nitrate in 24

Water by UV-VIS spectroscopy

6. Sample Preparation and Analysis in Infrared Spectroscopy 28

7. Sample Preparation and Analysis in NMR Spectroscopy 31

Appendix 33

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 Laboratory Safety Guidelines - CH 317

The following safety information is provided to the student in order to ensure that all students
and college staff working in the laboratory are aware of common industrial laboratory safety
practices. Before performing any experiments in the lab you will be required to write and pass a
laboratory safety quiz on eCentennial that will test your knowledge of these rules. In order to enter
the building, you will have to have complete the mandatory training course on campus re-entry
guidelines and protocols and bring your certificate of completion, your student card and have done
the pre-screening assessment that day.

Laboratory Personal Protective Equipment (PPE):

Each student is required to have the following safety items with them during each laboratory
period:

1. Lab Coat. 2. Safety glasses or goggles. 3. Mask

Proper Laboratory Clothing:

Each student is required to wear appropriate clothing to the lab:

1. Long pants or a skirt that covers your knees when sitting.

2. Proper footwear (footwear must totally enclose your foot).

Laboratory Safety Rules:

Each student will be aware of, and abide by the following minimum laboratory safety rules as
follows:

1. Each student and teacher must wear safety glasses, lab coat and appropriate clothing in the
laboratory.

2. Do not wear contact lenses in the laboratory.

3. Do not eat, drink or smoke in the laboratory.

4. Bags and backpacks are not allowed in the laboratory or outside the laboratory door. They must
be stored in a locker.

5. Know the location, use and operation of the eye-wash fountain, emergency shower, emergency
phone and laboratory exits.

6. In case of an accident or cut, immediately notify instructor. Do not leave the laboratory to seek
help alone.

7. Clean up all spills at your work area immediately. If a chemical spills on your skin, immediately
wash the exposed area under cold water for a minimum of ten minutes. If you have been exposed
to a large body spill, quickly proceed to the emergency shower. Remove all affected clothing to
ensure the chemical spill may be washed from your skin.

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8. Always sweep up broken glassware using a brush and pan and dispose of the broken glassware
in the separate container provided.

9. Assume all chemicals are hazardous unless instructed otherwise. Never taste a chemical!

10. Do not rub your eyes, mouth or nose in the lab unless you know your hands are clean.

11. Corrosive chemicals and chemicals with unpleasant odours are to be kept in the fume hood.

12. In case of fire, notify all laboratory staff and students immediately by shouting “FIRE”. Turn
off all open flames and electrical equipment. Calmly and quickly leave the laboratory.
DO NOT FIGHT THE FIRE!

13. If someone is on fire, extinguish the fire using the emergency shower, or use the “drop and
roll” technique.

14. Hot glass and hot “hot-plates” look the same when they are hot or cold. Always approach these
items with caution. Place your hand near them to assess whether they are hot before picking them
up.

15. Never work alone in a laboratory.

Handling Chemicals and Glassware:

1. Always check the label on the bottle before you use a chemical.

2. To prevent contamination of reagents and chemicals, never insert pipettes or scoopulas into
stock bottles. The proper technique is to remove a portion of the stock bottle contents into another
piece of clean glassware from which you can obtain your sample. Take only what you need. Do
not return any unused chemicals into a stock bottle.

3. Label all glassware containing chemicals with the chemical name, date and your name.

4. Do not pipette a liquid by mouth!

5. Always wash your hands before leaving a laboratory.

6. Do not weigh chemicals directly on a balance. Always use a weighing boat, weighing paper or
container.

7. Do not pour any chemicals down the sink unless given permission by the instructor. All
hazardous chemicals and organic solvents are to be disposed of in their proper disposal containers.

8. Wear gloves when handling strong acids or bases or when instructed to do so by your instructor.

9. Do not assume that any glassware provided is clean. Always wash glassware prior to an
experiment and following the experiment.

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10. Clean and return all glassware to its proper location before the end of the lab. Do not leave
glassware near a sink, fumehood or bench.

Preparation for Laboratory Work:

1. Read the experiment and complete a pre-lab before you come to the laboratory.

2. Arrive to the laboratory on time.

CH 317 LABORATORY REPORT GUIDELINES

General Guidelines:
The laboratory component of CH 317 makes up 44 % of your final mark and consists of seven
labs. Of the seven lab reports six will be informal reports worth 5.5 % each (Experiments 2 – 7),
and one will be formal reports worth 11.0% (Experiment 1). Informal lab reports are due one
week after completion of the experiment in the online dropbox before your scheduled lab
begins. The formal lab report must be submitted by Wednesday October 21st 2020. There will be
a late penalty issued to reports not submitted at this time of 10% per day. Any labs missed will be
given a grade of “0" unless accompanied by documented reasons for absences.

The lab reports should be written in the following format.

The Informal Lab Reports will consist of the following:

A Pre-Lab Report (2 out of 10) (to be submitted to the dropbox online in week 1 or as
scheduled):

1. Your name.

2. Date Experiment was performed.

3. Experiment Number and Title.

4. Objective or Purpose:
This is a statement of the purpose or objective of the experiment. It should state what the
experiment is about and why the experiment was done. (Sentences, point form or bullets).

5. Equipment and Chemicals:

This is a list of the equipment and materials that is specifically used in this experiment.

6. Procedure:
This section is to provide the readers with a step-by-step description of the experiment to
be performed. It must contain enough detail that someone unfamiliar with the experiment
could repeat it using only this procedure. It must be written in your own words.

7. Safety Precautions:
This is a description of the precautions that must be carried out in order to perform the
experiment safely.
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8. Pre-lab Questions:
Answer any pre-lab questions and attach to your pre-lab.

Post-lab Report: (8 out of 10)

9. Observations/Results/Calculations/Discussion/Summary or Conclusions: (5 out of 10)

This section is to include all the results obtained in the experiment. Where possible they
should be summarized in neat data tables. Graphs and samples of detailed calculations
should also be included in this section. Make sure graphs have labels and titles which
explain what they show. Include the signed data sheet you filled out in class in this section.
This section should also provide the readers a commentary of the events that had taken
place during the experiment such as colour and heat changes, time requirements,
formations of new substances etc. A short summary of the findings of the lab should appear
in a 5 to 10 sentence discussion or summary.

10. Post lab Questions: (3 out of 10)

Answer the postlab questions in the appropriate quiz on eCentennial.

The Formal Lab Report will consist of the following:

A Pre-Lab Report (4 out of 20): (to be submitted to the dropbox online in week 1).
(2 marks for Pre-Lab + 2 marks for Pre-Lab Questions)

1. Your name.

2. Date Experiment was performed.

3. Experiment Number and Title.

4. Objective or Purpose:
This is a statement of the purpose or objective of the experiment. It should state what the
experiment is about and why the experiment was done. (Sentences, point form or bullets).

5. Equipment and Chemicals:

This is a list of the equipment and materials specifically used in this experiment.

6. Procedure:
This section is to provide the readers with a step-by-step description of the experiment to
be performed. It must contain enough detail that someone unfamiliar with the experiment
could repeat it using only this procedure. It must be written in your own words.

7. Safety Precautions:
This is a description of the precautions that must be carried out in order to perform the
experiment safely.

8. Pre-lab Questions:
Answer any pre-lab questions and attach to your pre-lab.
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Post-lab Report: (16 out of 20)

9. Introduction/Theory: (3 out of 20)

The purpose of an introduction is to provide your readers with any general information
they must have to understand the details that follow in the report. It also introduces them
to the circumstances leading up to the experiment. The introduction should include the
background and scope of the experiment, as well as an explanation of the scientific
principles employed by the experiment. This section is also to provide the readers
descriptions and illustrations of any special apparatus used.

10. Observations/Results/Calculations: (5 out of 20)

This section is to include all the results obtained in the experiment. Where possible they
should be summarized in neat data tables. Graphs and samples of detailed calculations
should also be included in this section. Make sure graphs have labels and titles which
explain what they show. Include the signed data sheet you filled out in class in this section.
This section should also provide the readers with a commentary of the events that had taken
place during the experiment such as colour and heat changes, time requirements,
formations of new substances etc.

11. Discussion and Conclusion: (3 out of 20)

Discussion:
This section is to provide the readers with a detailed analysis and explanation of the results
or findings of the experiment, the sources of error, the accuracy of the results with respect
to possible errors, a comparison of the results with literature values and other students'
results, the implications of the results, what can be learned or interpreted from them, and
any recommendation derived from the experiment. In writing your discussion do not use
any personal pronouns such as I, we, my, our etc. because of the impersonal nature of a
scientific report. For example, instead of saying "I believe my result is correct", you can
rephrase this to "The result of this work is believed to be correct."

Conclusion:
This is a brief statement describing how the tests, findings, and resulting analysis have met
the objective stated above. No new points should be covered here.

12. Post lab Questions: (4 out of 20)

You are to answer all of the questions following each experiment. These post lab questions
can be answered in the discussion (but make it obvious to the lab instructor that they are
part of the post lab question answers) or in the appendix where you place the answers to
the post lab questions.

13. References: (1 out of 20)

All references used in the write-up should be included here. Each formal report should
include at least three references one of which will be the lab manual. Do not use Wikipedia
as a reference!

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 CH 317 LABORATORY SCHEDULE
Fall 2020

Week Experiment/Activity
Review Lab Safety / Complete online safety quiz
1
Submit Prelabs for Expts 1 and 2 in the appropriate lab dropboxes.
Review of Lab Safety / Introduction (all students)
Review Stats Video
2
Submit Prelabs for Expts 3 and 4 in the appropriate lab dropboxes.
Lab 1 (all students) / Lab 2 (all students)
Submit Lab 2 to the appropriate dropbox
3 Submit Prelabs for Expts 5, 6 and 7 in the appropriate lab dropboxes.
Lab 4 (all students) / Lab 5 (all student)
Submit Labs 3 and 4 to the appropriate dropbox
4
Labs 5, 6 and 7 as assigned
Submit at least one of labs 5, 6 or 7 to the appropriate dropboxes
5
Labs 5, 6 and 7 as assigned
Submit remaining informal labs (5, 6 or 7) to the appropriate dropboxes
6
Submit the formal report to the appropriate dropbox

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 EXPERIMENT #1

DETERMINATION OF CAFFEINE AND VITAMIN B6

IN ENERGY DRINKS BY HPLC

REFERENCE:

This procedure is adapted from Leacock, R. E., Stankus, J. J., and Davis, J. M., "Simultaneous
Determination of Caffeine and Vitamin B6 in Energy Drinks by High-Performance Liquid
Chromatography (HPLC)”, Journal of Chemical Education, 2011, 88, 232 - 234.

INTRODUCTION:

Caffeine is found in many popular beverages (e.g. coffee, soft drinks and tea). Energy drinks
are another more recently introduced type of caffeinated beverage (e.g. Red Bull, Monster etc.).
Another common additive in energy drinks is vitamin B6 a water soluble vitamin added to many
energy drinks in the form of pyridoxine hydrochloride. The concentration of both caffeine and
vitamin B6 can be determined simultaneously by using reverse phase HPLC (High Performance
Liquid Chromatography).

Reverse phase HPLC uses a non-polar stationary phase and a polar mobile phase. This is the
reverse of traditional open column chromatography, but has become standard in HPLC. The
stationary phase is a non-polar hydrocarbon chain chemically bonded to very fine particles of silica
and the particles are tightly packed in a steel column. The mobile phase (a phosphate buffer-
methanol mixture in this experiment) is pumped through under high pressure. Various components
in a sample are separated based on their polarities. Usually, the more polar molecules will be
eluded first since they are least attracted to the non-polar hydrocarbon chain in the stationary phase.

Since columns are made up of very fine particles, trace amounts of insoluble materials in a
sample can clog a column. Therefore, it is essential that all glassware be very clean and all solvents
used are properly filtered. Glassware should be conditioned with filtered solvent. Also, dissolved
gases can damage the column, so solutions must be degassed.

PRELAB QUESTIONS:

1. Draw the molecular structure of caffeine (by hand).

2. Draw the molecular structure of vitamin B6 (by hand).

3. Calculate the expected concentrations of vitamin B6 and caffeine in the stock solution in ppm
assuming exactly 0.0506 g of pyridoxine hydrochloride (vitamin B6) and 0.1253 g of caffeine are
dissolved in a 100 mL volumetric flask.

PRECAUTIONS:

Wear safety glasses and lab coats at all times in the lab.

9
PROCEDURE:

Preparation of Stock Solutions:

Phosphate buffer (50 mM, pH 3.0):
1. In a clean 800 mL beaker dissolve 3.525 g of Na2HPO4 in 400 mL of HPLC grade water.
2. Adjust the pH of the resulting solution to 3.0 by the dropwise addition of 3 M HCl.
3. Transfer the solution to a 500 mL volumetric flask and dilute to the mark with HPLC grade
water. Mix the solution thoroughly and label the flask.

60:40 (v/v) Phosphate buffer/Methanol Solution:

1. Mix 200 mL of HPLC grade methanol and 300 mL of the above phosphate buffer.
2. Filter the mixture by using millepore filtering flask and AQUEOUS 0.45 µm millepore filter
(see filtration setup below). Use a trap to avoid backflow into the filtered solvent.
3. After filtering, degas by stirring for 10 minutes under reduced pressure.

Filtration setup:

Ask your instructor to check your set-up before proceeding with the filtration.

Caffeine/Vitamin B6 Stock Solution:

1. Weigh out exactly 0.0506 g of pyridoxine hydrochloride (vitamin B6) and 0.1253 g of caffeine
using an analytical balance and transfer into a 100 mL volumetric flask. Dilute with the phosphate
buffer/methanol mixture to the mark.

Preparation of Samples:
1. Using a 5 mL volumetric pipette add 5.0 mL of the provided Energy Drink to five clean 50 mL
volumetric flasks.
2. Spike each of the flasks with either 0.00 mL, 0.50 mL, 1.00 mL, 1.50 mL or 2.00 mL of the
caffeine/vitamin B6 stock solution. Label each flask with the amount spiked.
3. Dilute each flask to the mark with the 60:40 phosphate buffer/methanol solution.
4. Mix each flask thoroughly.
5. Obtain 5 little HPLC vials and make sure the lids fit.
6. Condition the respective vial with the appropriate solution and label with your section number,
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bench number and solution concentration. Example of labeling: 101 – b1 – 0 ppm

HPLC Testing:

1. Submit the labeled and prepared HPLC vials to your lab instructor.

2. Watch the video on eCentennial on “setting up the HPLC”.

3. Obtain your data the following week.

4. Review the lecture on setup on a standard addition curve posted in eCentennial.

POST LAB QUESTIONS / CALCULATIONS:

1. Calculate the concentrations of caffeine and vitamin B6 stock solution added to each solution.

2. Create a standard addition curve (see class notes) from the HPLC data graphing peak area of
caffeine (y-axis) versus concentration of added caffeine (x-axis).

3. Create a standard addition curve (see class notes) from the HPLC data graphing peak area of
vitamin B6 (y-axis) versus concentration of added vitamin B6 (x-axis).

4. Using the standard addition curves calculate the concentrations of caffeine and vitamin B6 in
the original energy drink.

11
 Experiment #1 Data

Name of Energy Drink: ______________________

Mass of Caffeine in Stock Solution: ____________

Mass of Pyridoxine Hydrochloride (Vitamin B6) in stock solution: _________________

HPLC data:

Caffeine Data:
Stock Solution Added Peak Area
0 mL

0.5 mL

1.0 mL

1.5 mL

2.0 mL

Vitamin B6 Data:
Sample Peak Area
0 mL

0.5 mL

1.0 mL

1.5 mL

2.0 mL

12
 EXPERIMENT #2

DETERMINATION OF CHLOROPHYLL IN POND, RIVER and/or

LAKE SAMPLES BY SPECTROPHOTOMETRY

REFERENCE:

This procedure is adapted from Aminot, A., Rey, F., "Determination of chlorophyll a by
spectroscopic methods”, ICES Techniques in Marine Environmental Sciences, 2000.

THEORY:

Chlorophyll is essential for photosynthetic processes, and consequently is found in all green
plants. Measurement of chlorophyll a and b in natural bodies of water can be used to estimate the
stock of algae and to assess the trophic status of lakes and streams.

In this procedure, chlorophyll a and b, and acidified a called pheophyton a are determined
by extraction of pheopigments into an acetone-water solvent, followed by absorbance (optical
density) measurements at a series of wavelengths both before and after acidification. The measured
absorbance at the given wavelength is the sum of the absorbance values due to the individual
chlorophyll species and the solvent. By means of mathematical equations, the concentrations (in
μg/L) of individual chlorophyll species in a water sample can be calculated. A correction for
residual turbidity in the solvent extract has been incorporated into these equations.

Suspended algal material in aqueous samples is removed by filtration through a prescribed

glass fibre or nylon filter, followed by grinding of the filter and extraction of the algal pigments
with acetone-water solvent.

PRELAB CALCULATIONS:

Given the following data set, please calculate the results for Chlorophyll c (see equation at the end
of the lab).

 (nm) A750 A664 A647 A630 A750Acid A665Acid

readings 0.0189 0.1366 0.0647 0.0847 0.0139 0.098

PRECAUTIONS:

Wear safety glasses and lab coats at all times in the lab.

INTERFERENCES:

Low pH of samples may cause chlorophyll a molecules to lose a magnesium atom and to form
pheophyton a, resulting in a shift of the wavelength of maximum absorption. This interference is
overcome by the addition of 1 mL of 0.5 % magnesium carbonate solution to each litre of water
sample.
13
 Bright light can cause fading of the chlorophyll in the acetone extract. A cool, dark place is
recommended to minimize this effect if storage is required. Evaporation of the extract will
concentrate the pigments and the result will be biased high.

REAGENTS and EQUIPMENT:

1. 90 % acetone in water solution

2. Magnesium Carbonate Suspension: 1.0 g finely powdered MgCO3 in 100 mL of distilled

water

3. Microfibre filter paper (7 cm (500 mL) or 9 cm (1000 mL))

4. Funnel (500 mL or 1000 mL) and Filter Flask

5. Tissue grinder

6. Tweezers, spatula and glass rod.

8. 1% HCl

9. Aluminum foil

10. Spectrophotometer

PROCEDURE:

1. Measure out 500 mL of the provided water sample using a graduated cylinder. Filter about
400 mL of the sample through filter paper in a funnel. Then add 0.5 mL of 1 % MgCO3 suspension
(shake the suspension before measuring it in a small graduated cylinder) to the remaining sample.
Shake to mix and then filter the remaining sample.

2. Obtain 10 mL of 90% acetone/water solution in a graduated cylinder. In reduced light (cover

the grinder with aluminum foil), grind the filter paper in a tissue grinder with 5 mL of the 90%
acetone/water solution to make a slurry in an ice bath.

3. Transfer the slurry to a clean dry centrifuge tube.

4. Add the remaining 90% acetone/water mixture to the tissue grinder and grind. Transfer the
slurry to the same centrifuge tube.

5. Centrifuge it for 20 min. at 1000 G.

6. Decant the supernatant into a 10 mL graduated cylinder and bring up to 10 mL in volume with
the 90% acetone/water solution mixing well.
14
7. Set up the Cary60 UV-VIS spectrometer to record the absorbance at 750, 664, 647 and 630 nm.

8. Zero the spectrometer at the above wavelengths using the 90% acetone/water solution in a quartz
cuvette (1 cm pathlength) as a blank.

9. Transfer a portion of the supernatant into a matched quartz cuvette, and read record the
absorbance at 750 nm (A750), 664 nm (A664), 647 nm (A647), and 630 nm (A630).

10. Add 4 drops of 1% v/v hydrochloric acid to each cuvette and mix well. Wait 2-5 min, and
reread the blank and the absorbance of the chlorophyll solution at 750 nm (A750) and 665 nm (A665).
This will be used for the calculation of the concentration of corrected chlorophyll a, henceforth
abbreviated Corr Chl a. This method corrects for turbidity and the overlap of peaks from
chlorophyll a and b and pheophyton a. Pheophyton a is a degradation product of chlorophyll a
whose peak overlaps chlorophyll a. Acidification converts all chlorophyll to pheophyton and
allows correction for possible overestimation.

POST LAB CALCULATIONS:

The concentration of chlorophyll a, b and c can be calculated according to the following equations:

Chlorophyll a = (11.85(A664-A750) – 1.54(A647-A750) - 0.08(A630-A750))*10/(L*Vf)

Chlorophyll b = (-5.43(A664-A750) + 21.03(A647-A750) - 2.66(A630-A750))*10/(L*Vf)

Chlorophyll c = (-1.67(A664-A750) – 7.60(A647-A750) + 24.52(A630-A750))*10/(L*Vf)

Where: L = pathlength of cuvette in cm.

Vf = Total filtered water volume in L.

The corrected concentration of chlorophyll a is:

Corr Chl a = 27.7*((A664 – A750) – (A665a – A750a))*10/(L*Vf)

Where: L = pathlength of cuvette in cm.

Vf = Total filtered water volume in L.

Calculated chlorophyll concentrations in all equations have the unit mg/m3.

Review the lecture posted in eCentennial concerning the statistical calculations used in Expt. 2.

1. Calculate values for the concentration of chlorophyll a, b and c as well as a value for the
corrected concentration of chlorophyll a (Corr Chl a) from your group’s data.

15
2. Using the class’s data calculate values for the corrected concentration of chlorophyll a for each
group and use these to calculate a mean, standard deviation and 95% confidence interval for the
corrected concentration of chlorophyll a.

3. Use the Grubbs test (video) on both the maximum and minimum corrected concentration of
chlorophyll a found in the class’s data to determine if either point should be rejected as an outlier.

4. Complete the Postlab Quiz for Experiment #2 on eCentennial.

16
 Experiment #2 Data

Volume of filtered water (Vf): _______ Pathlength of cuvette (L): _______

Your group’s data:
Absorbance without acid Absorbance with acid
750 nm 664 nm 647 nm 630 nm 750 nm 665 nm

Class data:
Absorbance without acid Absorbance with acid
Group 750 nm 664 nm 647 nm 630 nm 750 nm 665 nm

17
 EXPERIMENT # 3

DETERMINATION OF PERCENTAGE ETHANOL

IN BEVERAGES BY GC

THEORY:

When a complex mixture of volatile components is injected into a gas chromatograph, the
various components exit the device at different times. If the appropriate column, column
temperature, flow rate, detector etc. are selected, each component will produce a "peak" on the
final chromatogram.

The Varian Gas Chromatograph is a sophisticated instrument, capable of detecting very low
levels of organic materials. A small sample (typically 1 μL) is injected into the injection port on
top of the machine, and separated on a 1/8" diameter stainless steel column packed with a
stationary phase which has some affinity for the mixture components. As the various substances
leave the column they are mixed with air and hydrogen and burned. The resulting flame is analyzed
for the presence of ions that formed during the combustion process. Since water is ignored it has
no effect on the chromatogram.

The signal is sent to an integrator/recorder which plots the peak intensity every 1/10 second,
and labels each peak with its retention time. When the operator determines the run is complete, a
printout is requested that indicates the area of each peak. With proper calibration (often using an
internal standard), the % composition of a mixture can be quickly calculated by the integrator, or
done by hand. In the internal standard method, the sample and standard are spiked with an equal
amount of a solute whose retention time is near that of the analyte. The ratio of the area of the
standard or analyte is used to determine the unknown concentration.

PRELAB QUESTIONS:

1. You are analyzing a spirit sample. You prepared the unknown spirit sample by taking 2.0 mL
of the spirit and making up the sample volume to 10.0 mL with distilled water in a test tube. Then
your lab partner pipeted 1.0 mL of glacial acetic acid to the same test tube. The peak areas that
you found for your standard run are 2332.3 V.Min for acetic acid, 2206.4 V.Min for ethanol
and for your sample run the peaks are 5592.9 V.Min for ethanol and 2381.5 V.Min for the acetic
acid. (Use the equation listed later in this experiment to solve this question).

PRECAUTIONS:

Wear safety glasses and lab coats at all times in the lab.

PROCEDURE:

Your instructor will also provide you with alcoholic samples for analysis. Since alcohol
percentages between 1 and 10% are measured with greatest accuracy, samples of beer or wine are
easiest. If a hard liquor is used, it must be diluted first.

18
SAMPLE PREPARATION:

Prepare your two alcoholic beverages (1 known + 1 unknown) as follows:

Filter your sample through a medium fluted filter paper into a beaker. Remove all bubbles
present by shaking the beverage or by gently warming the sample. If you warm your sample, make
sure you do not overheat the sample because you would drive off some of the alcohol, and make
sure you cool it back down to room temperature before proceeding. Transfer a 10 mL aliquot of
your sample to a large dry test tube by using a clean 10 mL pipette.

Add and mix exactly 1 mL of glacial acetic acid to your 10 mL aliquot by using a 1 mL
volumetric pipette. The acetic acid acts as an internal standard in the sample. Label this test tube.

STANDARD PREPARATION:

Pipette 5.0 mL of denatured ethanol into a 100 mL volumetric flask and make up to volume
with distilled water. The resulting stock solution will contain 5% alcohol by volume.

Pipette 10 mL of the 5% alcohol stock solution into a dry large test tube. Then by using a
1.0 mL volumetric pipette add and mix exactly 1.0 mL of glacial acetic acid to the test tube as
well. Label this test tube.

GAS CHROMATOGRAPHY:

GC Instrument Settings:
Injector Settings: Oven Power: ON, Temperature: 200 ºC
Column Oven Settings: Oven Power: ON, Temperature: 120 ºC
FID Detector Settings: Oven Power: ON, Temperature: 200 ºC, Range: 9

See appendix for details on setting up and using the GC.

CALCULATION:
𝑒𝑡ℎ𝑎𝑛𝑜𝑙 𝑝𝑒𝑎𝑘
(𝑎𝑟𝑒𝑎 𝑜𝑓 𝑓𝑜𝑟 𝑡ℎ𝑒 𝑠𝑎𝑚𝑝𝑙𝑒)
𝑎𝑐𝑒𝑡𝑖𝑐 𝑎𝑐𝑖𝑑 𝑝𝑒𝑎𝑘
% alcohol = 𝑒𝑡ℎ𝑎𝑛𝑜𝑙 𝑝𝑒𝑎𝑘 x 5%
(𝑎𝑟𝑒𝑎 𝑜𝑓 𝑓𝑜𝑟 𝑡ℎ𝑒 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑)
𝑎𝑐𝑒𝑡𝑖𝑐 𝑎𝑐𝑖𝑑 𝑝𝑒𝑎𝑘

POST LAB QUESTIONS:

1. Calculate the percentage ethanol content of your unknown and known samples (Show all
calculations). Compare the calculated ethanol content of your known sample to the value given
on its bottle. You will want to note down the alcoholic percentage on the known bottle.

2. Answer the postlab quiz for Experiment #3 on eCentennial.

19
 Experiment #3 Data

Known Sample (Name): ______________________

Known Sample (Alcohol % on label): ____________

Unknown #: ____________________________

GC Manufacturer: _______________________

GC Model #: ___________________________

GC data:

Area of peaks
Sample Alcohol peak Acetic acid peak

Standard Run 1

Known Sample
Run 1

Unknown Run 1

Known calculation

Unknown
calculation

20
 EXPERIMENT #4

DETERMINATION OF FLUORIDE ION CONCENTRATION IN DRINKING WATER

Fluoride occurs naturally in some ground waters and of course in public drinking water where
a 1 mg/L level of concentration is normally maintained for the prevention of dental cares.
Excessive amounts of fluoride causes an objectionable discolourization of tooth enamel which is
referred to as mottling. For this reason, fluoride levels must be monitored carefully in accordance
with the Safe Drinking Water Act.
The fluoride concentration is determined potentiometrically using an ion-selective electrode
and a pH meter having an expanded millivolt scale. This method is applicable to the measurement
of fluoride in drinking, surface, and saline waters, domestic and industrial wastes. Concentration
of fluoride from 0.1 up to 1000 mg/L may be measured. The fluoride electrode consists of a
lanthanum fluoride crystal across which a potential is developed by fluoride ions.
Extremes of pH interfere with the electrical potential obtained from the electrode. Therefore,
pH should be between 5 and 9. Polyvalent cations of Si4+, Fe3+, and Al3+ interfere by forming
complexes with fluoride with the degree of interference depending on the concentration of the
complexing cations, the concentration of fluoride ion and the pH of the sample.
The addition of a pH buffer or total ion strength adjustment buffer (TISAB) is used to complex
the interfering ions and eliminate the pH problem. Further, it can be said that the TISAB adjusts
all unknowns and standards to essentially the same ionic strength; when this is done, the
concentration of fluoride, rather than its activity, is measured. "Activity" refers to the "effective"
concentration of the ions rather than their theoretical concentration. The pH of the buffer is about
5, a level at which F- is the predominant fluorine-containing species.

PRELAB QUESTIONS:

1. Show your calculations for the preparation of the 100 ppm stock solution.

2. Show your calculations for how you will prepare the solutions listed in step 3 of the procedure.

3. Determine the log of the concentration for each of the concentrations listed in step 3 of the
procedure.

OBJECTIVES
To determine the fluoride concentration in two tap water samples.

PROCEDURE:
1. Prepare sufficient TISAB (total ion strength adjustment buffer) for the experiment by mixing
with stirring 28 mL of glacial acetic acid, 29.0 g of NaCl, 2 g of cyclohexylamine-
dinitrilotetraacetic acid (also named as cyclohexane diamine tetraacetic acid) and 250 mL of
distilled water in a 500 mL beaker. Cool the contents in a water or ice bath, and carefully add 6 M
NaOH until a pH of 5.0 to 5.5 is reached. Dilute to 500 mL with distilled water. For long term
storage TISAB needs to be stored in a plastic bottle.

2. Weigh to the nearest milligram, about 0.22 g of pre-dried NaF into a 1-L volumetric flask.
(CAUTION: NaF is highly toxic. Wear gloves and immediately wash any skin touched by
NaF with copious quantities of water.) Dissolve in water, dilute to mark, mix well and store in
21
a plastic bottle. Calculate the exact concentration of fluoride ion in ppm by using the following
equation:

Concentration of F- in ppm = wt of NaF in mg x Atomic Weight of F

M.W. of NaF
-
The concentration of F should be about 100 ppm.

3. Prepare 100 mL each of five additional standards that contain 10 ppm, 5 ppm, 1 ppm, 0.5
ppm and 0.1 ppm F (Hint: Prepare the 10 ppm and 5 ppm solution using the 100 ppm solution
and the 1 ppm, 0.5 ppm and 0.1 ppm solutions from the 10 ppm solution). Use distilled water,
volumetric glassware and mix thoroughly each time.
Type text here
4. To prepare the standard solutions pipette 25 mL of each standard solution (0.1, 0.5, 1.0, 5,
10 and 100 ppm) into six separate clean, dry and labeled 100 mL beakers. To each of these beakers
pipette in 25 mL of the TISAB buffer (each beaker will now contain 50 mL total solution) and mix
well.

5. To prepare the tap water samples pipette 25 mL of each tap water sample into separate clean,
dry 100 mL beakers. Then add 25 mL of the TISAB buffer to each beaker (the beaker will now
contain 50 mL total solution) and mix well.

6. Obtain the fluoride ion electrode and open the air hole near the top of the electrode. Make
sure there is enough filling solution in the electrode. (If the level of the filling solution is low,
please inform your instructor.) Rinse the electrode with distilled water and dab dry it. Then
immerse the tip of the fluoride ion electrode into the beaker with the 100 ppm standard prepared
in step 5 for 15 minutes and measure the potential with the millivolt function on a pH meter.

7. Measure the potentials of the rest of the standard solutions (from highest to lowest
concentration) followed by the potentials of the two water samples. After you are done with all the
measurements, close the air hole and rinse the electrode with distilled water.

POST LAB QUESTIONS:

1. Construct a calibration curve by graphing “E” (mV) vs log [F-] (ppm) using the standard
solutions.

2. Use the calibration curve to obtain the fluorine concentration in both the tap water samples.
Note: each solution was diluted in half using the TISAB buffer, however, the calibration curve can
be constructed using the undiluted standard concentrations as each solution received the same
treatment.

3. Answer the postlab quiz for Experiment #4 on eCentennial.

22
 Experiment #4 Data

Mass of NaF for 100 ppm stock: ____________

pH of TISAB buffer: _____________

Water Sample 1 ID: ___________________

Water Sample 2 ID: ___________________

Your group’s data:

[F-] (ppm) Log [F-] E (mV)

0.10

0.50

1.0

5.0

10

100

Water Sample 1

Water Sample 2

23
 EXPERIMENT #5

DETERMINATION OF CONCENTRATION OF NITRATE in WATER by UV-VIS

SPECTROSCOPY

REFERENCE:
This procedure is adapted from American Public Health Association, American Water Works
Association, and Water Pollution Control Federation, Standard Methods for the Examination
of Water and Wastewater (21st Ed.), Part 4500-NO3- B and Part 4500-P E.

INTRODUCTION:
Significant nitrate pollution of surface water and ground water often results from the excessive
use of fertilizers and can result in significant environmental impact. Manure from livestock, food
lots, and poultry operations can also contribute to nitrate pollution of surface and ground water.
The human body is capable of reducing nitrates to nitrites in the digestive system. There are two
distinct threats to human health from nitrites. First, nitrites can oxidize the haemoglobin
(containing Fe2+), to methemoglobins (containing Fe3+), which is incapable of transporting oxygen
in the blood stream. This illness, known as methaemoglobinaemia or blue baby disease, is
especially harmful to infants since they are particularly susceptible to asphyxiation by
methaemoglobinaemia. Second, nitrites can combine with various amides in the gastrointestinal
tract to form nitrosamines, many of which are carcinogenic.
Nitrates in surface water can cause excessive eutrophication of lakes and rivers. Increased
levels of nitrates lead to excessive growth of algae and other aquatic plants. When the algae and
plants die they settle to the bottom where they decay and deplete oxygen from the water. As a
result, determining the concentration of nitrates in water is often a very important first step in
controlling eutrophication. In this experiment you will determine the amount of nitrate present in
a natural water sample.

PRECAUTIONS:
Wear safety glasses and lab coats at all times in the lab.

PRELAB QUESTIONS:

1. Calculate the concentration of nitrogen (N) in the initial nitrate stock solution (in ppm) if exactly
0.361 g of KNO3 is dissolved in a 1 L volumetric flask with distilled water. This is known as the
nitrate nitrogen (NO3-N) concentration. Remember to use a gravimetric factor correction in this
calculation.

2. Calculate the dilutions required to make the other nitrogen (N) standard solutions used in this
experiment assuming the nitrate stock solution from prelab question 1 was used. These will be the
solutions that you are preparing in step 2 of the Nitrate procedure.

24
PREPARATION OF UNKNOWN SOLUTION:

1. Obtain a water sample from your demonstrator and record down the information provided on
its sampling location and the date it was collected on your data sheets.

2. Measure 150 mL of your water sample using a graduated cylinder. Filter the sample to remove
any suspended particles using a glass fiber filter.

NITRATE PROCEDURE:
1. Prepare a nitrate stock solution by weighing 0.361 g of dry KNO3 into a 1 L volumetric flask
and diluting to the mark with distilled water. Shake to dissolve.

2. Using the nitrate stock prepare standards that are 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 ppm nitrogen
(N) in 50 mL volumetric flasks by diluting the stock with distilled water. After diluting to the
mark, pipet 1.0 mL of 1 M HCl acid into each flask and mix thoroughly.

3. Prepare a water sample by adding 1 mL of 1 M HCl to 50 mL of the prepared unknown water

sample. This can be done by filling 50 mL volumetric flasks with the samples to the 50 mL mark
and then by pipetting 1.0 mL of the HCl acid into each of these flasks. Shake well afterwards.

4. Make a second trial of the unknown sample by repeat step 3 one more time.

5. Prepare a blank using distilled water and 1 M HCl in the same ratio as the others.

6. Use the blank to set zero at 220 nm and 275 nm on the UV spectrophotometer (UV Cary 50
Bio). Then measure the absorbance of all solutions at 220 nm and 275 nm. For absorbance at these
wavelengths quartz cuvettes must to be used. See the appendix for details on the operation of the
UV-VIS spectrometer.

7. If the absorbance of a water sample is too high, dilute the sample and repeat step 6.

POST LAB QUESTIONS:

NITRATE CALCULATIONS:

Since dissolved organic matter may also absorb at 220 nm, it is necessary to correct for
interference. Subtracting two times the absorbance at 275 nm is a sufficient correction procedure
for this interference, i.e.

abscorr = abs220 - (2 x abs275)

where abscorr is the corrected absorbance for the nitrates.

Plot abscorr (y-axis) vs concentration of nitrogen nitrate (x-axis) and obtain the sample
concentration from the calibration graph. If any water samples are diluted prior to the
measurement, apply the dilution factor before reporting the results.

25
Review the lecture on Least Squares analysis in eCentennial.

1. Calculate the uncertainty in the calculated concentration of nitrate nitrogen (NO3-N) in your
unknown sample. To determine the uncertainty use the formula for the propagation of uncertainty
with a calibration curve (see class notes). An excel file set up to do this calculation is available
for download from eCentennial in the “lab” folder. Please remember that your unknown values
are not part of the calibration curve. A print screen image of your excel work with your own
annotations on the screen should be included in your lab report that is placed in the appropriate
dropbox.

2. Calculate the 95% confidence interval for the concentrations of nitrate nitrogen (NO3-N) in your
unknown using the uncertainties calculated in question 1. Remember that degrees of freedom for
a calibration curve is equal to n – 2. Please remember that your unknown values are not part of the
number of data points that are in calibration curve. Also note the mean and standard deviation
used in this question are the values you obtained in question 1.

3. Complete the Postlab Quiz for Experiment #5 on eCentennial.

26
 Experiment #5 Nitrogen Data

HC
Sample Code (from bottle): _______________________________________________________

Mass of KNO3: 0.368g

____________

Spectroscopic Data:

Absorbance at wavelength
Sample 220 nm 275 nm
0.0 ppm NO3-N
0.0001 0.0000
0.5 ppm NO3-N
0.1413 -0.0021
1.0 ppm NO3-N
0.2963 0.0070
1.5 ppm NO3-N
0.4170 -0.0073
2.0 ppm NO3-N
0.5505 -0.0088
2.5 ppm NO3-N
0.6731 -0.0084
3.0 ppm NO3-N 0.8155 -0.0071
Unknown Trial 1 0.2157 0.0832
Unknown Trial 2 0.1957 0.0895

0.117843 ~ 0.1
Average Nitrogen Nitrate Concentration of Sample: ____________________________________

0.028975 ~ 0.0
Standard Deviation of Average Nitrogen Nitrate Concentration: ___________________________

27
 EXPERIMENT #6
SAMPLE PREPARATION and ANALYSIS in INFRARED SPECTROSCOPY

REFERENCE:
This procedure is adapted from Sawyer, Donald. T., et al., Chemistry Experiments for Instrumental
Methods, John Wiley & Sons, 1984, 227 - 235.

INTRODUCTION:
Photons in the ultraviolet or visible region of the electromagnetic spectrum can excite an
electron from its grounds state to an excited state. Photons in the infrared region of the spectrum
do not contain enough energy to excite electrons. Instead photons in this region cause vibrational
excitation. Vibrational excitation results in a change in the vibrational or rotational motion of a
molecule. In their vibrations, the covalent bonds in molecules act like springs that can be stretched
and bent. The frequency at which these vibrations occur determines the frequency of IR radiation
absorbed. Particular functional groups in a molecule will appear at characteristic frequencies in
the IR spectrum. Given a library of known IR spectra, it is often possible to identify unknown
compounds. As a result, unlike UV-VIS spectroscopy IR spectroscopy is not generally used for
quantitative analysis but mainly in qualitative analysis.
The purpose of this experiment is to introduce the student to a cross section of the sampling
techniques used in IR spectroscopy. IR spectra obtained will then be analyzed to identify the
characteristic functional groups of a series of known compounds. Finally an unknown sample will
be analyzed and identified through comparison to a reference library of spectra.

PRELAB QUESTIONS:

1. Please watch the video entitled “Infrared Spectroscopy” (at least up to the end of data
analysis ~ 6 minutes of video). The link is posted to the lab folder on eCentennial.

2. Please locate the table entitled “IR data tables” in the lab folder in eCentennial. Draw out
example structures of an alcohol, a ketone, a carboxylic acid, an aromatic and an alkyne.
What are the main functional groups in each of the compounds listed above? What are the
frequency ranges of the IR peaks relevant to those functional groups?
For example an alkyl chloride (CH3CH2CH2Cl) will have CH stretches (alkane) ~ 2800 cm-1 to
3000 cm-1; CH bends (alkane) ~ 1470 cm-1 to 1450 cm-1; CH rocks (alkane) ~ 1370 cm-1 to 1350
cm-1; CH-X (alkyl halide wag) ~ 1300 cm-1 to 1150 cm-1 and 850 cm-1 to 550 cm-1 alkyl halide for
C-Cl stretch.

PRECAUTIONS:
Wear safety glasses and lab coats at all times in the lab.

PROCEDURE:

Sample Preparation / IR Spectroscopy:

28
Liquid Sampling:

(a) Each lab member must obtain one unique (in terms of functional groups) liquid organic sample
from your instructor (an alcohol, an aromatic hydrocarbon, an alkyne, a ketone or a carboxylic
acid).

(b) Inset the ATR cell into the IR spectrometer (ask your instructor for assistance).

(c) Obtain a new background scan.

(d) Place a small amount of the liquid to be analyzed with a Pasteur pipette.

(e) Obtain the IR spectrum of the liquid. (Each member of the lab group should have only 1 liquid
IR paper spectrum).

(f) Using a kimwipe and acetone, clean the ATR crystal.

(g) Repeat steps (d) to (f) for the remaining liquid samples.

(h) Place a small amount of the polymer unknown onto the ATR crystal (the small hole on the top
of the plate on the ATR attachment). Screw down the anvil above the hole (finger tight) to
compress the sample onto the ATR crystal.

(i) Obtain an IR spectrum of the polymer unknown. Each lab member needs a copy of the unknown
solid IR spectrum.

(j) Using a kimwipe and acetone, clean the ATR crystal.

KBr pellet technique:

(k) Insert the standard sample module for the IR (for solid sample analysis).

(l) Obtain a powdered polymer unknown from your demonstrator.

(m) Obtain a KBr pellet press.

(n) Weight out 1 mg of your polymer unknown and mix with 100 mg of KBr. Using a mortar and
pestle crush the polymer and KBr together.

(o) Insert one bolt into the KBr press and add enough of the above mixture to cover the bolt head.
Insert the opposite bolt and tighten the bolts using two wrenches. Leave the KBr press under
pressure for 1 – 2 minutes.

(p) Loosen and remove the bolts. If the pellet is properly formed it should remain in the press and
be clear or translucent.

(q) Place the press on a sample holder in the IR spectrometer and obtain an IR spectrum of the
polymer unknown. Each member of the lab group needs a copy of unknown with KBr IR spectrum.
29
POST LAB QUESTIONS:

1. Identify the relevant peaks for known liquid sample that you obtained in step (e). (an IR
correlation table is available for download in the lab folder on eCentennial). Label these peaks on
the paper IR spectrum.

2. Identify the diagnostic peaks on the IR spectrum for your polymer unknown. Label these peaks
on the paper IR spectra. There are two spectra that need to be analyzed: the ATR cell unknown
solid and KBR pellet technique.

3. Compare the IR spectrum of the polymer obtained from the KBR pellet technique and the ATR
cell. Which technique gave a better result? Justify your answer.

4. Using the library of polymer IR spectra in the lab folder on eCentennial attempt to identify your
polymer sample. Explain your choice.

5. Make tables of all the diagnostic IR peaks for each sample including frequency and functional
group.

6. Complete the online quiz for Experiment #6 on eCentennial.

Experiment #6 Data

Name of alcohol: ________________________________

Name of aromatic hydrocarbon: _____________________

Name of alkyne: ___________________________________

Name of ketone: __________________________________

Name of carboxylic acid: ___________________________

Unknown ID #: ___________________________________

30
 EXPERIMENT #7
SAMPLE PREPARATION and ANALYSIS in NMR SPECTROSCOPY

REFERENCE:
This procedure is adapted from Sawyer, Donald. T., et al., Chemistry Experiments for Instrumental
Methods, John Wiley & Sons, 1984, 293 - 295.

INTRODUCTION:
NMR spectroscopy involves transitions between the spin states of nuclei with non-zero nuclear
magnetic moments. Nuclei with an odd number of protons have both a charge and a spin and as a
result have a magnetic moment. Normally in a collection of atoms, these spins are randomly
arranged. However, when placed in a magnetic field, the spins will arrange themselves with
respect to the magnetic field. For a hydrogen nucleus, two orientations are possible. Transitions
between these two orientations can be induced by applying a radio frequency pulse. The frequency
of this pulse is dependent on the environment surrounding individual hydrogen nuclei.
In NMR spectroscopy, peak locations are usually measured relative to a standard compound
(usually TMS, tetramethylsilane) as a chemical shift (δ). The location of these peaks correspond
to particular chemical environments (e.g. methyl protons, aromatic protons, etc.). In addition, the
area of these peaks can be used to determine the number of protons present in each environment.
Splitting patterns of individual peaks can be used to determine the number of neighboring protons.
As a result, NMR spectroscopy is a very powerful tool for chemical structural determination.

PRELAB QUESTIONS:

1. Read the NMR operations manual in the appendix and watch the videos posted in the lab folder
dealing with how to use the NMR instrument.

2. Research the term “magnetic anisotropy”.

PRECAUTIONS:
Wear safety glasses and lab coats at all times in the lab.

PROCEDURE:

Quantitative Determination of a mixture of toluene and ethanol:

(a) Obtain the following solutions from your lab demonstrator:

(i) denatured ethanol
(ii) toluene
(iii) An unknown mixture of ethanol and toluene

(b) Inject 200 – 300 µL of each sample into the NMR spectrometer and obtain proton NMR spectra
for each of the three solutions in (a).

(c) Carefully process and integrate each spectrum (chemical shift of ethanol (CH3) = 1.25 ppm,
chemical shift of toluene (aromatic H) = 7.20).

31
POST LAB QUESTIONS:

1. Use the chemical shift table found on eCentennial and the integrals to identify and label the
major peaks in each spectrum.

2. Determine the percent of toluene and ethanol in the unknown given the following formula:

(𝑎𝑟𝑒𝑎 𝑜𝑓𝑒𝑡ℎ𝑎𝑛𝑜𝑙 𝐶𝐻3 𝑝𝑒𝑎𝑘)

% 𝑒𝑡ℎ𝑎𝑛𝑜𝑙 = × 100
3
(𝑎𝑟𝑒𝑎 𝑜𝑓 𝑡𝑜𝑙𝑢𝑒𝑛𝑒 𝑎𝑟𝑜𝑚𝑎𝑡𝑖𝑐 𝑝𝑒𝑎𝑘) + (𝑎𝑟𝑒𝑎 𝑜𝑓 𝑒𝑡ℎ𝑎𝑛𝑜𝑙 𝐶𝐻3 𝑝𝑒𝑎𝑘)
5

3. Why does the peak corresponding to the aromatic protons in toluene appear at 7.2 ppm while
the peak corresponding to the methyl protons in ethanol appears at 1.25 ppm?

4. Include a table for each sample run listing the chemical shift, splitting and identity of each peak.

5. Complete the online quiz on eCentennial for Experiment #7.

32
APPENDIX

33
Instructions (UV Cary 60):

1. Open the folder “Cary WinUV” on the desktop.

2. Open the “Simple Reads” application.
3. Once opened click the setup button or select setup from the menu.
4. Select the Cary tab if not already displayed.
5. In the “Read at wavelength” field, enter the wavelengths you require using a semi-colon
between each wavelength:
630; 647; 664; 750
6. Select Abs (Absorbance) as the Y-mode and click ok to exit the Setup mode.
7. Place the blank in the cuvette and insert the cuvette into the holder.
8. Click on “Zero” button.
9. Clean the cuvette and replace the blank with the sample to be measured and then click on the
“Read” button.
10. Clean cuvette and replace solution with the next sample to be measured and then click on the
“Read” button.
11. Repeat step 10 until all samples have been run.
12. When runs have been completed select “Print” from the File menu to print the report.

Instructions for GC (Varian 3800):

1. Ensure that the H2 and Air are turned on.
2. Under the Method section select the “Edit” button on the GC instrument.
3. Select “Method 2”, Press Enter
2. Under the Method section select the “Activate” button.
3. Select “Method 2”.
4. Press Activate Now (Middle blue button).
5. Select “Detector”
6. Select “Middle FID”
7. Select “Ignite” (left blue button).
8. Check exhaust to ensure ignition.
9. Method 2 Instrument Settings:
Injector Settings:
Oven Power: ON
Temperature: 200 ºC
Column Settings:
Oven Power: ON
Temperature: 120 ºC
FID Detector:
Oven Power: ON
Temperature: 200 ºC
Range: 9

34
Computer Settings:
10. Turn on computer, monitor, and printer
11. Load “Galaxie”
12. Select “Ok”
13. Select the system tab.
14. Confirm that the 3800GC is selected (a green check mark should be displayed by the
3800GC in the left hand menu).
15. Select “Quick Start Run” on the action bar or press F8
16. In the pop menu check that the system name reads “3800GC” and the method name reads
“Alcohol.METH”.
17. Select “Ok”.
18. Wait until the bottom of the screen reads “Waiting for Injection”
19. Inject Sample (Data collection will start automatically).

Printing Data:
20. To print select the data tab.
21. Open the file pull down menu and select “open chromatogram”
22. From the chromatograms listed select the chromatogram you wish to load into memory.
23. Data is saved using the format “CH315_Alcohol_month_day_year_time”.
24. Once loaded into memory a report on the chromatogram may be printed by opening the file
pull down menu and selecting print.

Turning off GC:

1. On the GC under Methods select “Edit”
2. Load Method 8.
3. Under Methods select “Activate”
4. Load Method 8
5. Press Activate Now (middle blue button).
6. Turn of the H2 and Air gasses (leave N2 on).
7. Turn off computer, monitor and printer.

35
Instructions for new ACCELA HPLC:

1. Turn on all three modules of the ACCELA HPLC.

2. Turn on computer, monitor and printer.
3. Double-click on ChromQuest5.0 to launch HPLC program.
4. Select Accela 1250.
5. All lights should be green except the run light on the pump which should be orange.
6. Select File from the top menu.
7. Select Method → Open
8. Open Method File window should launch
9. Select Caffeine.met, Click Open
10. Select File from the top menu.
11 Select Sequence → Open
12. Chose sequence “Caffeine315F11.seq”, Click Open
13. In the run 1 row enter a name under filename.
14. Right click on the name you have entered.
15. Select Fill down from the pull down menu.
16. Click on the arrow to the right of “Data file” under “Increment Data File”
17. Select date and time.
18. Click OK
19. Open door to Autosampler and remove the A tray.
20. Place the vials containing your samples in slots 1-6 in the order (5 ppm, 10 ppm, 15 ppm, 20
ppm, Extraction run #1, Extraction run #2).
21. Place the tray back in the autosampler and close the door.
22. To start the sequence open the Control menu and select Sequence Run.
23. Done

Printing results from new HPLC:

1. Select File from the top menu.
2. Select Data → Open
3. Select the name of the data file to be processed.
4. Select Reports from the top menul
5. Select Print → Area %
6. Repeat steps 1 – 6 for each data file to be printed.
7. Done

Turning off new HPLC:

1. Close the Accela 1250 window (do not save the current sequence).
2. Close ChromQuest 5.0
3. Shutdown Computer and turn off monitor and printer.
4. Turn off power to all four HPLC modules beginning from the top and working down.

36
Running the PicoSpin 45
Injecting a Sample:

1. Remove the PEEK plugs from the inlet and outlet fittings on the front panel.
2. Screw the Drain tube assembly (see above) into the outlet fitting on the picoSpin 45.
3. Position the drain tube over a small flask to catch any excess solution.
4. Screw the inlet filter (see above) into the inlet fitting on the picoSpin 45.
5. Screw the syringe port into the inlet filter but do not tighten.
6. Attach a blunt-tip needle onto the syringe and draw a 200-300 µL of sample into the syringe.
7. Eject any air from the syringe.
8. Insert the needle into the syringe port and tighten.

9. Have your instructor check your setup.

10. Gently inject the sample into the picoSpin 45.
Warning!!
Do not force the syringe if you feel resistance. Too much pressure (> 100 psi) could damage the
instrument!
11. Continue injecting sample until sample is exiting the drain tube and no air bubbles are visible (in the drain
tube).
12. Remove the syringe port and drain tube.
13. Plug the inlet and outlet with the two PEEK plugs.

37
Running a Sample:
1. Open Firefox and select the picoSpin 45 under Bookmarks.
2. Go to the “Files” page and select the “TOLUENE-ETHANOL” saved run.
3. Click on “use these settings with run”.
4. Under “Run Name” enter a name for your sample.
5. Set scans to 1 and click “Start Run”.
6. Call your instructor over to inspect your spectrum.
7. If the spectrum is correct set scans to 24 and click “Start Run”.
8. The spectrum will take several minutes to collect.

Transferring NMR Data to the Processing software:

1. In the NMR Program in Firefox select the “Files” tab.
2. Open the appropriate NMR file containing the spectrum to be processed and select the run data
“fid-avg.jdx”.
3. Select Open with “ACD/NMR Processor Academia Edition”.
4. The FID should now be displayed in the window.

Processing NMR Data:

1. Select “Zero Filling” → Set Points Count = 32768 → Exit the “Zero Filing” menu by selecting “OK”.
2. Select “Apodization” → Set LD = 0.3 → Exit the “Apodization” menu by selecting ✓.
3. Select “Process” → “Fourier Transform” → “Complex Transform”
4. Select “Process” → “Reverse Spectrum”
5. Use the Zoom function to zoom in on the peaks.
6. Select “Phase” → “Auto Simple” → Exit the “Phase” menu by selecting ✓.
7. Select “Baseline” → “Auto” → Exit the “Baseline” menu by selecting ✓.
8. Select “Reference” → Select center of peak to be reference using the mouse.
9. Left click on the peak to be referenced.
10. Change “Shift (ppm)” to the correct chemical shift (For ethanol the CH3 peak should have a chemical
shift of 1.25 ppm while the aromatic protons in toluene should have a chemical shift of 7.2 ppm).
11. Exit the “Reference” menu by selecting OK and then ✓.
12. Select “Peakpicking” → “Auto” → Exit the “Peakpicking” menu by selecting ✓.
13. Select “Integration” → “Auto” → Exit the “Integration” menu by selecting ✓.

Printing Processed Data:

1. Select “File” → “Print” → “Standard” → OK

38