Chemistry Practical III - Lab Manual

UNIVERSITI TEKNOLOGI PETRONAS
CHEMISTRY PRACTICAL III (YAB 2052)

LABORATORY MANUAL
SEMESTER: JAN 2020
COURSE COORDINATOR:
Dr Noraini Abd Ghani
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TABLE OF CONTENT
EXPERIMENT Topics
NO
SAFETY IN LABORATORY
CHEMICAL ASSESSMENT FORM

- To be completed for each experiment
EXPERIMENTAL FLOW CHART FORM
- To be completed for each experiment
1 Experiment 1: Potentiometric Titration of Hydrogen Peroxide
2 Experiment 2: Determination of Water Hardness by Atomic Emission
Spectrophotometry
3 Experiment 3: Quantitative analysis of aspirin tablets using UV-VIS
Absorption Spectroscopy
4 Experiment 4: Determination of functional groups using infra-red
spectroscopy
5 Experiment 5: Analysis of alcohols using gas chromatography
6 Experiment 6: Identification of Isomeric Compounds Commonly Found in

Gasoline using gas chromatography /mass spectrometer
7 Experiment 7: Determination of caffeine in beverages using liquid
chromatography
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SAFETY IN LABORATORY
1. Before entering the laboratory or doing any experiment, you must
1.1 Wear a fully covered low heels shoes, not sandals or slippers. Sport shoes are permitted, but it
is advisable not to wear canvas shoes.
Students who are not wearing proper attire will not be allowed to enter the laboratory.
1.2 Wear and button up your laboratory coat.
Fail to wear your laboratory coat, RM 10.00 will be penalized.
1.3 Wear safety glasses in laboratory when dealing with hazardous chemicals.
1.4 Students with long hair should tug in their hair inside the laboratory coat, or tie it properly.
1.5 Know the positions and the use of safety equipment and the fire extinguishers.
1.6 Know the exit of the building.
2. You are NOT PERMITTED to
2.1 Be playful in the laboratory.
2.2 Eat or drink in the laboratory.
2.3 Smoke in the laboratory.
3. While using the chemicals, you must remember that
3.1 All chemicals especially organic substances are flammable, poisonous and can endanger your
health. Avoid holding, tasting, touching, sniffing or inhaling the vapor directly. A safer method
of sniffing the chemical is as shown in Figure A.
Figure A: Safer way of sniffing chemical
3.2 Do not use your mouth for drawing liquid into a pipette, use a rubber bulb.
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3.3 Do not dispose chemicals in the sink even if the chemicals are not used. Ask your
demonstrator what should be done with the wastes.
3.4 Put the chemical waste into the labeled waste bottle as instructed.
3.5 Do not use chemicals from unlabelled bottle.
3.6 Label all test tubes that you use.
4. While using apparatus or doing experiments, you must remember
4.1 Avoid crowding in doing experiment.
4.2 Use the fume hoods as instructed.
4.3 While heating, do not direct the test tube's mouth or any apparatus used towards your
friends.
4.4 Your table must be clean and dry, free from any chemical spills, books, papers or anything
that are not being used.
4.5 Check the glassware apparatus for any defect or crack and have it replaced. Do not try to use
it as it will cause danger.
4.6 If any accident happens, no matter how small it is, the demonstrator or the laboratory staff
should be acknowledged.
a) If the chemical splash enters the eye, quickly wash it with plenty of water.
b) If it spills on any part of the body, wash with water or use the shower provided.
5. Fire
5.1 If your clothes caught fire, roll over on the floor and splash yourself with water or cover
yourself with wet cloth.
5.2 If any part of the laboratory caught fire, tell your demonstrator or laboratory staff quickly. Use
the fire extinguisher to avoid it from spreading. If the condition gets worse, try to get out of
the laboratory safely.
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COMMON LABORATORY APPARATUS
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CHEMICAL ASSESSMENT FORM
Name:___________________________________________ Student’s signature: _________________
Student ID:_______________________________________ Demonstrator’s signature: ____________
Title of experiment: _______________________________ Date: _________________
No. Chemical Approx. Assessment Hazard First aid

measures
Amount
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EXPERIMENTAL FLOW CHART
Name:___________________________________________ Student’s signature: _________________
Student ID:_______________________________________ Demonstrator’s signature: ____________
Title of experiment: _______________________________ Date: _________________
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EXPERIMENT 1
Title: The Potentiometric Titration of Hydrogen Peroxide
Objectives:
In this experiment, you will
1. Conduct the potentiometric titration of the reaction between commercially available
hydrogen peroxide and potassium permanganate.
2. Measure the potential change of the reaction.
3. Determine the concentration of the hydrogen peroxide solution.
Chemicals & Apparatus:

Chemicals Equipment/tools
3% hydrogen peroxide, H2O2, solution Lab Quest
Drop Counter
Vernier ORP Sensor
4.5 M sulfuric acid, H2SO4 solution Reagent reservoir
0.020 M potassium permanganate solution, ring stand /utility clamp
KMnO4, in H2SO4
distilled water 250mL beaker
10mL pipet and pump
50mL graduated cylinder
magnetic stirrer with stirring bar
Introduction:
One method of determining the concentration of a hydrogen peroxide, H 2O2, solution is
by titration with a solution of potassium permanganate, KMnO4, of known concentration.
The reaction is oxidation-reduction and proceeds as shown below, in net ionic form.
5H2O2 (aq) + 2MnO4– (aq) + 6H+ (aq) → 5O2 (g) + 2Mn2+ (aq) + 8H2O (l)
In this experiment, you will use an ORP (Oxidation-Reduction Potential) Sensor to
measure the potential of the reaction. Your data will look like an acid-base titration curve.
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The volume of KMnO4 titrant used at the equivalence point will be used to determine the
concentration of the H2O2 solution.
Experimental procedure:
1. Prepare an acidified and diluted hydrogen peroxide, H2O2, solution for the titration.
a) Prepare 100mL of 0.3% H2O2 from stock solution of 3% H2O2.
b) Measure out precisely 10mL of the diluted H2O2 solution into a 250mL beaker.
Add 25mL of distilled water and 10 mL of 4.5 M sulfuric acid, H2SO4, solution.
CAUTION: H2SO4 is a strong acid, and should be handled with care.
2. Place the beaker of hydrogen peroxide solution on a magnetic stirrer and add a
stirring bar.
3. Assemble the apparatus, as shown in Figure 1:
Figure 1
a) The Drop Counter has been calibrated prior experiment.
b) Insert the ORP Sensor through the large hole in the Drop Counter into the
beaker.
c) Adjust the positions of the Drop Counter and reagent reservoir so they are both
lined up with the center of the magnetic stirrer.
4. Switch on Lab Quest. Attach Drop counter and ORP Sensor to DIG 1 and Channel
1, respectively. You will see the reading for volume (mL) and potential (mV) at the
screen.
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5. Rinse and fill the reagent reservoir with 0.020M MnO4– solution. Open both valve so
that the tip is filled with the MnO4– solution, then close the top valve. CAUTION:
Handle the KMnO4 solution with care; it has been mixed with H2SO4, which can
cause painful burns if it comes in contact with the skin.
6. Switch on magnetic stirrer. Make sure the stirring bar does not hit the ORP Sensor.
7. You are now ready to begin the titration. At the equivalence point, you will see a faint
pink color of unreacted MnO4– solution.
a) Before adding MnO4– titrant, tap ► to start data collection.
b) Carefully open the top valve so that the MnO4– solution drops once for every
second. You can see the plot at the meter screen as well as the volume of
MnO4– solution added at the bottom of the screen.
c) Wait for the plot to make a huge jump. You can see the linear increase of
the plot. Allow the MnO4– solution to continue dropping for another 2mL.
Then stop the titration.
d) Tap ■ to stop data collection.
e) Examine the titration curve and estimate the volume of MnO4– solution used
to reach the equivalence point of the titration. Record this value in your data
table for Trial 1.
9. Dispose of the reaction mixture as directed. Rinse the ORP Sensor with distilled water
in preparation for the second titration.
10. When you continue for the second trial, tap the Cabinet icon next to word Run 1, the
graph for Run 1 will be stored temporarily. The screen will show new graph with name
Run 2. Repeat the titration for another two times with a new sample of H2O2 solution.
Be careful by adding the MnO4– solution drop by drop in the region near the
equivalence point, so that you can precisely identify the equivalence point of the
reaction.
11. After you have completed all trials, tap the word Run 3. You will be able to select
previous run or select all runs. The graph of the selected run will be displayed.
12. Print/ save a copy of the titration data curve for the trial that you intend to use in your
data analysis.
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WORKSHEET
EXPERIMENT 1
Title: The Potentiometric Titration of Hydrogen Peroxide
DATA
1. Instrument:
a. Type of instrument: _____________________________________
b. Brand of instrument:_____________________________________
c. Type of electrode: ______________________________________
2. Titration data:
Trial Volume of KMnO4 (V) Potential (E)
(mL) (mV)
Before jump After jump Before jump After jump
1
2
3
Average mL mL mV mV
Standard
deviation
RESULTS AND CALCULATIONS

1. Plot the potential (E) versus reagent volume (V) for each trial and indicate the
equivalence point.
2. Plot the derivative of the titration curve (∆E/∆V) for each trial and determine the
equivalence point from the point of inflection
3. Write the oxidation and reduction half-reactions for the redox reaction taking place
in this laboratory. Identify the oxidizing agent for this reaction. Justify your answer.
4. How many moles of electrons are transferred in the balanced redox reaction?
Justify your answer.
5. State the value of equivalence point obtained from the derivative plot (∆E/∆V).
Calculate the moles of MnO4– used to reach the equivalence point.
6. Use the number of MnO4– moles to calculate the moles of H2O2 in the sample of
solution.
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7. Calculate the molar concentration of the H2O2 solution.
DISCUSSIONS
1. How closed are the values of the equivalence points obtained from the two plots?
2. Describe sources of errors in this experiment
3. What are advantages of a potentiometric titration over a direct potentiometric
measurement?
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EXPERIMENT 2:
Title: Determination of Water Hardness by Atomic Emission Spectrophotometry
Objectives:
1. Determine concentration of Ca2+ and Mg2+ ions
2. Compare the hardness of different types of water

Chemicals/ Materials Equipment/tools
Stock solution Ca, 100 ppm Microwave plasma- Atomic Emission Spectrometer
(MP-AES)
Stock solution Mg, 100 ppm Volumetric flasks , 50 ml ( 7)
Deionized water Pipets (adjustable 50 ul – 1 ml)
Tap H2O
Mineral H2O
Unknown samples
Introduction
Atomic Emission Spectroscopy
Atomic emission spectroscopy (AES) is a method of chemical analysis that uses the
intensity of light emitted from a flame, plasma, arc, or spark at a particular wavelength to
determine the quantity of an element in a sample. The sample that needs to be studied
should be first converted to highly excited free atoms. The liquid samples are generally
nebulized and carried to the source of excitation by flowing gas. Solid samples are
introduced into a source by slurry or laser ablation in a stream of gas. Another way of
dealing with solid samples would be to directly vaporize the sample and excite it by using
a laser pulse or a spark between electrodes. The excitation source should desolvate the
sample, atomize and excite the atoms.
In atomic emission, a sample is subjected to a high energy, thermal environment in order

to produce excited state atoms, capable of emitting light. The energy source can be an
electrical arc, a flame, or more recently, a plasma. The emission spectrum of an element
exposed to such an energy source consists of a collection of the allowable emission
wavelengths, commonly called emission lines, because of the discrete nature of the
emitted wavelengths. This emission spectrum can be used as a unique characteristic for
qualitative identification of the element. Emission techniques can also be used to
determine how much of an element is present in a sample. For a "quantitative" analysis,
the intensity of light emitted at the wavelength of the element to be determined is
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measured. The emission intensity at this wavelength will be greater as the number of
atoms of the analyte element increases.
Figure 1: Microwave Plasma - Atomic Emission Spectroscopy
Background
Water hardness is defined as the total concentration of alkaline earth metal ions in water.
Because the concentrations of Ca2+ and Mg2+ are usually much higher than those of other
alkaline earth ions, hardness can be equated to [Ca2+] + [Mg2+]. Individual hardness refers
to the individual concentration of each alkaline earth ion. Hardness is commonly
expressed as the equivalent number of milligrams of CaCO3 per liter. Thus, if [Ca2+] +
[Mg2+] = 1 mM, we would say that the hardness is 100 mg CaCO3 per liter (because 1
mmol CaCO3 = 100 mg CaCO3).
Water whose hardness is more than 60 mg per liter is considered to be "hard".

Sometimes, hard water is considered "bad". For example, it causes the formation of scale
in a water boiler or heater, and it consumes soap that would otherwise be useful for
cleaning. However, it is not believed that "hard" water is unhealthy. Hardness can be
determined by many methods such as EDTA titration, and atomic absorption and
emission spectrophotometry.
In this experiment, you will use emission spectrophotometry (AES) to determine the
concentrations of Ca2+ and Mg2+ in tap water, mineral water, and in three unknown
samples. You can bring your tap water from home or use the stuff in the lab, bathroom,
or drinking fountain.
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Procedure
1. Prepare seven 50 mL Ca standard solutions of 0.1, 0.2, 0.5, 1, 1.5, 3 and 5 ppm by
dilution of the 100 ppm Ca stock solution by using deionized water.
(Note: 1 ppm = 1 mg/L)
2. Prepare seven 50 mL Mg standard solutions of 0.1, 0.2, 0.5, 1, 1.5, 3 and 5 ppm by
dilution of the 100 ppm Mg stock solution by using deionized water.
3. Set up the spectrophotometer as described in the operating instructions. Please refer
to Attachment 2: MP-AES
4. Measure the emission intensity of the standards, tap water, mineral water and the
unknown samples.
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WORKSHEET
EXPERIMENT 2:
Title: Determination of Water Hardness by Atomic Emission Spectrophotometry
DATA
1. Instrument:
a. Type of instrument: _____________________________________
b. Brand of instrument:_____________________________________
c. Method file name: ______________________________________
(attach the method file with the report)
d. Scanning parameters (excitation source, wavelengths, flow rate, etc)
2. Emission data:
Wavelength for Mg measurement: ____________________

Wavelength for Ca measurement: _____________________
Sample name [Mg2+] [Ca2+] Intensity Intensity

(Trial 1) (Trial 2)
mg/L or ppm mg/L or ppm
Standard 1
Standard 2
Standard 3
Standard 4
Standard 5
Standard 6
Standard 7
Tap H2O
Mineral H2O
Unknown
1. Plot Intensity versus concentration of standard solutions. Obtain the slope of the
calibration curve.
2. Calculate the concentrations of calcium and magnesium in the tap water, mineral
water and the unknown samples using the slope of the calibration curve.
3. Calculate the hardness of the water samples.
DISCUSSION
1. Classify the water samples as "soft" or "hard". Discuss the difference in hardness
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between the tap water and mineral water, if any is observed.
2. What are some of the advantages of plasma sources compared with flame sources
for emission spectrometry?
3. Discuss possible interferences encountered in this instrument.
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Attachment 2: MP-AES
ABBREVIATED INSTRUCTIONS FOR MICROWAVE PLASMA -ATOMIC EMISSION

SPECTROMETER (MP-AES)
(AGILENT MP-AES- 4200)
New instrumental technique for elemental determinations using atomic emission

• Microwave plasma excitation source
• Nitrogen based plasma: runs on air
Reduced operating costs, improved safety:
• Runs on Nitrogen– eliminates need for Acetylene, Argon, etc.
• Eliminates need for source/hollow cathode lamps
• Safe, reliable unattended multi-element overnight operation
What is the MP-AES
Atomic Emission Spectrometer
• Similar to Inductively Coupled Plasma Optical Emission systems
• Samples of interest are introduced into an excitation source
• Excited atoms decay back to ground states by emitting characteristic
wavelengths of light
• Emission Intensity/Concentration relationship is established using solutions of
known concentration (Calibration Curve)
• Sample Atomic Emissions are measured and converted into a concentrations
using the established Emission Intensity/Concentration relationship.
• IT IS NOT AN ICP-AES
MP-AES Major Components
The MP-AES consists of four major components;
(a) Sample introduction system
• Transports sample to the excitation source
(b) Plasma source
• Atomize
• Excite sample material
(c) Spectrometer
• Collects, filters and detects the light from the excitation source, according to
wavelength
(d) Detector
• Measures the intensity of the emitted light after filtering
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SAMPLE INTRODUCTION
• Purpose of the sample introduction system is to produce small particles suitable
for transport and introduction to a plasma source.
• Sample Introduction system consists of
- Peristaltic pump to deliver solution to a nebulizer
- A nebulizer to convert the solution into a fine aerosol (small droplets)
- A Spray Chamber to filter out larger droplets
THE MICROWAVE PLASMA SYSTEM

• The Plasma generation system consists of 4 components
- Plasma gas control system
- Magnetron
- produces an electro-magnetic field
- Waveguide
- a resonant cavity that “contains” and focuses the electro-magnetic
energy. The torch is positioned at the maximum magnetic field strength
position within the waveguide to produce and sustain the plasma
- High Voltage DC Supply is required to operate the magnetron
• MP-AES uses Magnetic Fields for plasma creation
- Results in a Toriodal plasma when sample is injected into the central
region
- Produces temperature (est. 5000K)
- Very similar in structure to that of an Argon ICP plasma
Figure 2. Excitation process
STANDARD OPERATING PROCEDURE FOR MP-AES 4200 INSTRUMENT

Start up procedure
1. Turn on air compressor.
2. Turn on Nitrogen generator.
3. Power on MP-AES, auto sampler and exhaust fan.
4. Turn on PC. Log in with user name : admin and password : 3000hanover.
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5. Connect the sample and drain tubing on peristaltic pump. Clamp the tubing.
6. Check that all tubing on the spray chamber, nebulizer, peristaltic pump and auto
sampler are correctly connected.
7. Make sure the waste bottle is not full, empty it if necessary.
8. To start the MP Expert software, double-click the MP Expert icon on the desktop.
9. Click Plasma in the top toolbar and select Plasma On.
10. Let the MP-AES warms up for approximately 30 minutes before analyzing any
sample.
Developing a Method
1. Create a new worksheet or create a worksheet from an existing template.
2. On the Elements page, select the element(s) to be included in the method, as well
as suitable wavelength for their measurement.
3. Click Add.
4. Click the Conditions tab to modify both common settings for the run and settings
for each element.
5. Click Standards to define the analytical calibration information for the method. Use
this page to select number of standards, edit correlation coefficient limit, enter
standards concentration and define the standard units to be used in analysis. The
parameters to fit the calibration curves can be set as well.
6. Click QC to set up the QC tests and parameters for each test to be used to monitor
the quality of analytical data. The QC tab only appears if Enable QC has been
selected on the Elements page.
7. Click Sequence to specify the end of run actions, number of samples, edit the
sample labels and rack tube locations.
8. If using a SPS 3 Sample Preparation System, click the Autosampler tab to select
the racks and probe depth (if needed). This only appears if selected Autosampler
in the Sample introduction field on the Conditions page.
9. Save as a template (if needed).
10. Click Run in the top toolbar to run the analysis.
11. It will pop up a message to request to save the worksheet before proceed with
analysis. Click OK and give a name to the worksheet.
12. When the analysis completed, interpret analysis data.
13. Click Report in the top toolbar and select Report to PDF to save the results.
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EXPERIMENT 3
Title: Quantitative analysis of aspirin tablets using UV-VIS Absorption
Spectroscopy
Objectives:
1. Prepare and measure the absorbance of salicylic acid solutions
2. Prepare a calibration curve of the standard solutions
3. Quantitatively analyze aspirin tablets using UV-VIS Absorption Spectroscopy

0.1 N NaOH UV-VIS spectrophotometer (Cary)
Salicylic acid Cuvette
Commercial aspirin tablets Volumetric flasks , 100ml, 250 ml
Deionized water Pipets (adjustable 50 ul – 1 ml)
Introduction:
UV-Visible spectroscopy is an analytical technique which involves the absorption of
electromagnetic radiation at the near ultraviolet (200nm) region and the visible region.
The ultraviolet and visible absorption is based on molecules containing π- electrons or
non-bonding electrons (n- electrons) which can absorb the energy in the form of ultraviolet
or visible light to excite these electrons to higher anti-bonding molecular orbitals. UV-
Visible spectroscopy is particularly important for the qualitative and quantitative
determination of many organic compounds especially those with a high degree of
conjugation and transition metal ions. Figure 1 shows the block diagram of a UV-VIS
spectrometer.
Figure 1. The components of UV-VIS spectrometer

The basic principle of quantitative determination by UV-Visible spectroscopy lies in
comparing the extent of absorption of a sample solution with that of a set of standards
under radiation of a selected wavelength through the application of Beer- Lambert law.
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The Beer-Lambert law (also called the Beer-Lambert-Bouguer law or simply Beer's law)
is the linear relationship between absorbance and concentration of an absorber of
electromagnetic radiation. The general Beer-Lambert law is usually written as:
A = ε· b · c
where A is the measured absorbance, ε is a wavelength-dependent absorptivity
coefficient, b is the path length, and c is the analyte concentration.
Background:
Aspirin (acetylsalicylic acid)
• Aspirin is widely used as an analgesic (pain reliever) and an antipyretic (for

reducing fever). It is also used to help prevent heart attacks, strokes, and blood
clot formation in people at risk of developing blood clots.
• Aspirin (acetylsalicylic acid) is an aromatic compound containing both a carboxylic
acid functional group and an ester functional group.
• Aspirin is a weak acid that is only slightly soluble in water.
• Aspirin can be prepared by reacting salicylic acid and acetic anhydride in the
presence of an acid catalyst.
Structure of Aspirin (acetylsalicylic acid)
Aspirin (acetylsalicylic acid) contains three groups:
carboxylic acid functional group (R-COOH)

ester functional group (R-O-CO-R')
aromatic group (benzene ring)
Properties of Aspirin (acetylsalicylic acid)
Acidity
Aspirin is a monoprotic weak acid, Ka = 2.8 x 10-4 at 25oC, so very little of the molecular
aspirin (acetylsalicylic acid) dissociates to form acetylsalicylate ions.
For the equilibrium dissociation reaction:
aspirin (acetylsalicylic acid) acetylsalicylate ion + H+
+ H+
the equilibrium position lies well to the left, favoring molecular aspirin.
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Solubility
Aspirin is only slightly soluble in water and acidic solutions such as is present in the
stomach. Aspirin contains polar functional groups which can form hydrogen bonds with
polar water molecules. Aspirin is more soluble in basic (alkaline) solutions, so it readily
dissolves in the duodenum which is the first part of the intestine. Ionic salts of aspirin,
such as sodium acetylsalicylate, are more soluble in water since they form stronger ion-
dipole interactions with water. These ionic salts of aspirin are sometimes marketed as
"soluble aspirin". When you add water to the soluble aspirin, eg, sodium acetylsalicylate,
it dissociates to form sodium ions and acetylsalicylate ions:
sodium acetylsalicylate → acetylsalicylate ions + sodium ions
→ + Na+
C9H7O4-Na+ → C9H7O4- + Na+
Reaction of aspirin
Hydrolysis: cleavage of a covalent bond in a molecule by reaction with water
acetic acid
aspirin + water → salicylic acid +
(ethanoic acid)
(acetylsalicylic acid)
+ H2O(l) → + CH3COOH(aq)
C9H8O4(s) + H2O(l) → C7H6O3(s) + C2H4O2(aq)
The amount of aspirin (acetylsalicylic acid) in commercial analgesics can be determined

once it has been hydrolyzed to salicylic acid and diluted to a concentration were Beer’s
Law is obeyed.
Experimental procedures:
Preparation of standards
1. Accurately weigh (to 0.1 mg) about 0.1g of pure salicylic acid (weight by difference
using a weighing bottle) and quantitatively transfer to a 100 ml volumetric flask.
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2. After thoroughly mixing, dilute to the mark with a NaOH solution so that the final
concentration is 0.1 N NaOH.
3. Calculate the salicylic acid concentration (mg/L) in this Stock solution.
4. Prepare five dilutions of your stock solution using 1,2,3,4, and 5 mls diluted to 100ml
with 0.1 N NaOH.
5. Calculate the salicylic acid concentration (mg/L) in your stock solution and each
standard.
Preparation of calibration curve

1. Refer to the abbreviated procedure for the UV-VIS instrument, as shown in
Attachment 3.
2. Using 3ml dilution of salicylic acid, measure the UV absorption spectra with the UV-
VIS Spectrophotometer from 250 nm – 450 nm. Determine the wavelength of
maximum absorbance.
3. Measure the absorbance of each of the standards at the wavelength of maximum
absorbance. Repeat the measurement three times.
4. Plot a calibration curve for your salicylic acid standards (average absorbance versus
concentration).
Aspirin analysis
1. Allow one commercial aspirin tablet to dissolve in 0.1 N NaOH solution contained in
a 250 ml volumetric flask.
2. Bring the volumetric flask to volume and mix thoroughly.
3. Dilute this solution 1 to 50 with 0.1 N NaOH and measure the absorbance of this
solution at the same wavelength as your standards. Repeat the measurement three
times.
4. Use the calibration curve to find the concentration of salicylic acid present in the
solutions using the scanning UV-VIS spectrometer.
Unknown analysis:
5. Measure the absorbance of unknown solution at the same wavelength as your

standards. Repeat the measurement three times.
6. Determine the concentration of salicylic acid in the unknown solution, after
appropriate dilutions if needed, using the calibration curves from the scanning UV -
VIS.
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WORKSHEET
EXPERIMENT 3
Title: Quantitative analysis of aspirin tablets using UV-VIS Absorption
Spectroscopy
DATA
1. Instrument:
a. Type of instrument: __________________________
b. Brand of instrument:___________________________
c. Method file name: ______
(attach detailed parameters in the developed method file with the report)
d. Scanning parameters (mode, light source, wavelength range, speed, slit
width, cell path length, etc)
2. Absorbance data:
Wavelength of absorbance measurements (max ): ____________________
Sample name Concentration Absorbance Absorbance Absorbance

(mg/L) (Trial 1) (Trial 2) (Trial 3)
Standard 1
Standard 2
Standard 3
Standard 4
Standard 5
Aspirin
Unknown
RESULTS & CALCULATIONS:

1. Plot absorbance versus wavelength for one of the standard salicylic solutions.
Determine the wavelength of maximum absorbance (max ) from this spectrum.
2. Construct a calibration curve by plotting average absorbance versus concentration of
standard salicylic acid solutions at max . Determine the absorptivity coefficient (ε) of
the salicylic acid.
3. Determine the concentration of salicylic acid in the aspirin and the unknown samples
using the absorptivity coefficient (ε) determined from the calibration curve.
4. Calculate the weight of acetylsalicylic acid (ASA) in the aspirin tablet applying the
appropriate stochiometric factors and considering the dilutions involved.
5. Compare your result to the content specified on the label and compute the percent
recovery.
DISCUSSION:
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1. Why did you use a 0.1 N NaOH solution as the solvent in this experiment?
2. How would your test results be affected if you left fingerprints on the sides of the
cuvette in line with the light path of the spectrometer (or colorimeter)?
3. Give possible explanations for the recovery you found when analyzing the aspirin
tablet. Are your results within expected variations allowed for over-the-counter
analgesics?
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ATTACHMENT3: UV-VIS
ABBREVIATED INSTRUCTIONS FOR UV-VIS SPECTROPHOTOMETER
(CARY100)
Absorbance (Abs) measurement for liquid sample
1.) The UV of the sample is measured in a 1-cm cuvette. Insert the liquid sample
holder into the designated space
2.) Setup the software. Choose mode to ‘Abs’ for Absorbance measurement and
enter range of scanning (Max: 500nm, Min: 200nm). Choose ‘Baseline’, then
‘Baseline Correction’.
3.) Choose ‘zero’. Do not load any samples. And click ‘OK’
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4.) Once done, choose ‘Baseline’ and insert blank sample (NaOH) into both
reference and sample compartment for calibration.
5.) After baseline correction, put the cuvette containing the sample in the sample
compartment. Press ‘Start’ and obtain the spectrum/absorbance of the sample.
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EXPERIMENT 4
Title: Determination of functional groups using infra-red spectroscopy
Objectives:
1. Learn how an infra-red spectrophotometer operates
2. Analyze the spectrum to characterize organic families of compounds

1. Carboxylic acid (Acetic acid, benzoic acid) Fourier-Transform infra-red
2. Alkane (Heptane, hexane) spectrophotometer (FT-IR)
3. Alcohol (Ethanol, propanol)
4. Ketone (Acetone)
5. Chloroform
6. Unknown
Introduction
Theory:
Infrared spectrometry is one of the most useful tools available for the analysis of organic
compounds. An infrared spectrum helps the analyst determine the functional group(s)
present in the compound as well as something about the structural environment of the
group. When an infrared analysis is conducted on a sample dissolved in a solvent, the
technique is non-destructive, which means the sample is not destroyed by the analysis.
Thus, once an IR spectrum has been taken on the unknown compound, the entire sample
may be recovered from the solvent for further analysis.
Figure 1 is a block diagram of an FTIR spectrometer. A common FTIR spectrometer
consists of a source, interferometer, sample compartment, detector, amplifier, A/D
convertor, and a computer. The source generates radiation which passes the sample
through the interferometer and reaches the detector. Then the signal is amplified and
converted to digital signal by the amplifier and analog-to-digital converter, respectively.
Eventually, the signal is transferred to a computer in which Fourier transform is carried
out.
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Figure 1. The Components of FTIR Spectrometers
What is Infrared Radiation? Like visible light, infrared radiation is electromagnetic

radiation. The difference among the various kinds of electromagnetic radiation is how
energetic the radiation is.Very high-energy radiation such as that found in cosmic rays is
at one end of the electromagnetic spectrum and very low-energy radiation such as that
found in radio waves is at the other end.
Infrared radiation is less energetic than visible light. As you know, visible light is absorbed
by certain materials. This absorption produces the complementary color in the human
eye. We cannot see infrared radiation; therefore, it is not detectable by the human eye.
We need an instrument called an infrared spectrometer to measure the amount of infrared
radiation that is absorbed by a given compound.
What is the energy of infrared radiation? Because the energy (E), frequency (ν), and
wavelength (λ) of electromagnetic radiation are related as shown by equation 1, any of
these variables may be used by an analyst to represent energy.
E = hn = hc/ λ (1)
E is the energy of any electromagnetic radiation in joules (J). Planck’s constant, h, is 6.63
x 10-34 J s, ν (nu) is the frequency of the radiation or wave in reciprocal seconds s-1, c is
the speed of light 3 x 1010 cm s-1, and λ (lambda) is the wavelength of the radiation in
cm.
As shown in equation 1, E is inversely proportional to λ. Therefore, the term wavenumber

was created. Wavenumber ¯ ν¯ (nu bar) is the reciprocal of wavelength. Thus, nu bar is
directly proportional to energy, and the greater nu bar the greater the energy of the IR
radiation.
¯ ν¯ = 1/ λ (2)
-1
The units for nu bar are reciprocal centimeters or cm .
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How are IR peaks reported?
For the past few years, chemists have attempted to use a common set of units. For IR
radiation, the standard unit for reporting the position of absorption of a given IR band is
nu bar, which is reported in reciprocal centimeters. Thus, an absorption that corresponds
to the presence of a carbonyl group in a ketone might be expressed as 1715 cm -1. This
is an energy equivalent unit. The reciprocal of 1715 cm-1 is 1/1715 = 0.000583 cm.
Clearly, it is more user friendly to describe an absorption as 1715 as compared to
0.000583. Therefore, nu bar or reciprocal centimeters is the preferred unit. An older unit
called a micrometer or micron might be encountered. A micron (μm) is one-millionth of a
meter or 1 x 10-6 m, which equals 1 x 10-4 cm. Thus, 0.000583 cm = 5.83μm. A 1715
cm-1 wave is a 5.83μm wave. In this course, we shall use wavenumbers in reciprocal
centimeters as the standard for reporting IR spectral bands.
How does IR radiation interact with molecules?
Two atoms joined by a covalent bond can be considered as two masses joined by a
spring. The vibration of two such masses joined by a spring can be described by Hooke’s
law. According to Hooke’s law, the stretching frequency of two masses joined by a spring
is directly proportional to the strength of the spring and inversely proportional to masses
joined by the spring. For two atoms, the bond strength is analogous to the strength of a
spring. Thus, bonds involving hydrogen such as C—H and O—H have higher stretching
frequencies than do C—O and C—C bonds. Double and triple bonds are stronger than
the corresponding sigma bonds holding two atoms together. Therefore, the stretching
frequencies for C=C and C=O are higher than those for C-C and C-O, respectively.
Likewise, triple bonds, C≡C or C≡N are at even higher frequency than double bonds, but
lower than C—H or O—H stretching frequencies. Thus, IR radiation causes vibrations in
molecules, particularly at the natural stretching frequencies of the bonds. Stretching is
one kind of vibration known as a mode of vibration. A mode of vibration is how the
vibration occurs. Stretching is one mode, but bending and scissoring are also modes of
vibration. The most intense stretching vibrations occur when the stretch involves a change
in the dipole moment of a bond. A carbonyl group, C=O, undergoes a large change in its
dipole moment when the double bond in the group is stretched, leading to one of the most
intense IR absorptions for a carbonyl group as compared to other IR absorptions. On the
other hand, a symmetrically substituted triple bond such as the one in 2-butyne,
CH3C≡CCH3, produces a very weak IR signal, if any, because there is no change in the
dipole moment when this kind of a symmetrical molecule is stretched at the triple bond.
How does an instrument record the data?

The infrared region of the electromagnetic spectrum is scanned from 4000 cm-1 to about
800 cm-1. Infrared radiation interacts strongest with a molecule when the IR radiation
corresponds to a mode of vibration in the molecule (i.e., stretching, bending, or scissoring,
etc.) The sample is placed in the IR beam, and the IR region scanned by the instrument.
As the region from high to low energy is swept, both the location and intensity of the
absorption is registered. Think about walking down a street with numbers on the houses.
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You might find Smith at 3500 and Jones at 1700. That is the number may be correlated
with a family. Exactly the same thing is true of IR spectra. As the spectrum is obtained,
significant absorption occurs at numbers that can be correlated with organic families. For
example, alcohols are found at 3300 - 3500cm-1 and ketones are found at 1680 -1800
cm-1. IR is most useful to us in the correlation of peaks we find in a spectrum with the
families or structural features that we know absorb at these wavenumbers.
What are sampling techniques for FTIR?

FT-IR spectroscopy allows measuring all types of samples whether they are solid, liquid
or gaseous. Yet, the preparation of the sample for a transmission measurement is a rather
complex task. Liquid samples need to be filled into a liquid cell with suitable path length;
solids typically have to be diluted with the IR-inactive KBr and pressed to the well-known
“KBr-pellet”. However, both types of measurement techniques have their drawbacks:
• Liquid cells need to be filled without air bubbles and are not easy to clean.
• KBr is hygroscopic, hence not easy to handle and to store. A good KBr pellet is
difficult to make, time consuming and requires a special tool kit including a
hydraulic press
• Too much sample in a pellet will result in total absorption.
In order to overcome these disadvantages of KBr pellets and liquid cells nowadays IR-
measurements are mainly performed in ATR (Attenuated Total Reflection) mode as this
technique is much more comfortable to use than the conventional transmission mode. All
types of samples (e.g. solids, liquids, powders, pastes, pellets, slurries, fibers etc.) are
placed undiluted on the ATR crystal. The measurement then is typically performed within
seconds.
How ATR Works?

With ATR sampling we direct the IR beam into a crystal of relatively higher refractive
index. The IR beam reflects from the internal surface of the crystal and creates an
evanescent wave, which projects orthogonally into the sample in intimate contact with the
ATR crystal (Figure 2). Some of the energy of the evanescent wave is absorbed by the
sample and the reflected radiation (some now absorbed by the sample) is returned to the
detector. This ATR phenomenon is shown graphically in the following representation of a
single reflection ATR.
Figure 2. Graphical Representation of a Single Reflection ATR.
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Experimental:
Follow the instructions for the start up of the instrument, as shown in Attachment 4.
1. Obtain four unknown samples from the instructor and record the numbers of the
unknowns in your notebook. The family names of the four compounds will be known.
2. Fill the ATR sample cell with one of the unknowns, using the instructor-demonstrated
technique for filling the cell.
3. Obtain the IR spectrum of the unknown and print it out.
4. Clean the cell with chloroform and obtain IR spectra for the other three unknowns.
5. Clean up all of the equipment and sample cells are return them to their storage
locations.
5. Compare the spectra of your unknowns with the spectra provided by the instructor.
6. Identify your four unknowns by comparing your spectra band for band with those
provided.
7. Verify the identity of the four unknowns with your instructor.
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WORKSHEET
EXPERIMENT 4
Title: Determination of functional groups using infra-red spectroscopy
DATA
1. Instrument:
d. Scanning parameters (mode, light source, wavelength range, speed, slit
width, cell path length, no. of scans, resolution, etc)
2. Display FTIR spectra for all the compounds and label the peaks obtained.
RESULTS & CALCULATIONS
Table 1: The Identification of Unknown Compounds by their IR Spectra
Family Wavenumber Shape of Complete Partial

name range of major bands (sharp, Structure structure
bands intense,
broad)
Acid
Alkane
Alcohol
Ketone
Unknown
1. A structural feature produces the observed signal. That is a major tenet of

spectroscopy. We want to learn as many structure-spectra correlations as possible. For
each family identified in Table 1, place an X in the appropriate box. For example, the
alkane contains a methylene (CH2) group; therefore, an X is placed in the box in the
Alkane row and the –CH2- column.
2. From Table 1, what partial structure is common to an acid and an alcohol?

Answer________________________
What is the shape of this band? Answer____________
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3. Compare the spectrum of your acid with that of your alcohol. Find an IR band in Table
1 that is common to only the acid and alcohol. What is the wavenumber (range)?
Answer________________________
4. From Table 1, what partial structure is common to an acid and a ketone?

Answer________________________
5. Compare the spectrum of your acid with that of your ketone. Find an IR band in Table
1 that is common to only the acid and ketone. What is the wavenumber (range)?
Answer________________________
What is the shape of this band? Answer___________
DISCUSSION
1. List other types of instrument/methods that can be used to confirm the identity of
the functional groups.
2. What are major advantageous of Fourier Transform IR instruments over dispersive
IR instruments?
3. Why run a background spectrum before every sample?
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ATTACHMENT 4: FTIR
ABBREVIATED INSTRUCTIONS FOR

FOURIER TRANSFORM INFRARED SPECTROSCOPY (FTIR) WITH [ATR]
(Nicolet 5S FTIR)
START UP
1. Double click on the OMNIC shortcut icon from the windows desktop
2. Click on the Experiment Setup from the Collect menu
3. Choose the Collect
4. Type in the desired No of scans and Resolution. Typically settings use for an
experiment is
a. No of scans: 16
b. Resolution: 4
c. Final format: % Transmittance or Absorbance
d. Tick on Save Interferograms (recommended)
e. Background Handling: Collect background before every sample
(recommended)
5. Choose Bench, check the Gain. Recommended is Autogain.
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6. Choose OK to close the Experiment Setup dialog box.
7. Choose Collect Sample from the Collect menu and then follow the instructions that
appear on the screen.
8. For choosing the recommended on Background Handling. A message will pop up
to ask you to prepare to collect the background spectrum, remove any sample
from the beam path (or install the background material) and then choose OK. If
you choose Cancel, the data collection procedure will end.
9. A message asks you to prepare to collect the sample spectrum, install your sample
in the appropriate sample holder or accessory and then choose OK. If you choose
Cancel, the data collection procedure will end.
10. Type a title for the sample spectrum and then choose OK or accept the default title
in the text box by just choosing OK.
11. A message asks whether to add the spectrum to a spectral window, choose Yes
to add the sample spectrum to the indicated window; choose No to end the
procedure without saving the spectrum; or choose Cancel to return to the Collect
Sample window.
Converting spectra to Absorbance unit
1. Select the spectra

2. Choose Absorbance from Process menu
Converting spectra to %Transmission unit
1. Select the spectra

2. Choose Transmission from Process menu
Saving a spectrum (.spa)
1. Select the spectrum that need to be saved
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2. Choose Save As from File menu
3. Type in the desired filename
4. Click OK button
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EXPERIMENT 5
Title: Analysis of alcohols using gas chromatography
Objectives:
1. learn how a gas chromatograph operates
2. analyze the data to get qualitative and quantitative results

1.Methylene Chloride Gas chromatograph with FID detector (GC/FID)
2. Acetone Carrier gas: Helium/Argon
3. Methanol Column: Blood alcohol column (7.5mx0.320 mm ID)
4.Ethanol Syringe ( 10 ul)
5.1-propanol
6.Isopropyl alcohol (2-
propanol)
7.Water (HPLC grade)
Introduction
Chromatography literally means to separate by colors, because early separations were

detectable by the colors of the various compounds that were separated. The word
chromatography as it is used today still implies a separation technique. The words that
lead in to chromatography define how the separation is accomplished. Gas
chromatography, also called vapor-phase chromatography, is the technique we will study
in this block of instruction.
Chromatographic separations involve two phases (recall that phases are solids, liquids,
or gases). In chromatography, one of the two phases is a stationary phase (i.e., it does
not move during the analysis), and the other phase is a mobile phase, which moves
through the stationary phase. The mixture of compounds to be separated, let’s say
Compound A, Compound B, and Compound C, is placed on the stationary phase and is
separated into pure A, pure B, and pure C as the mobile phase carries them from the
starting point to the ending point. The chemicals to be analyzed are called analytes. The
chemical principle that is involved in all separations of this type is that the properties of
compounds are a function of their structure. Hence, compounds that differ in structure will
differ in the rates at which they travel during the chromatographic process and will be
separated over time. If compounds A, B, and C differ sufficiently in structure for the
chromatographic technique employed, they can be separated. However, you should be
aware that a mixture of compounds, very similar in structure, might be inseparable by
standard chromatographic techniques. For example, a mixture of sterols might appear to
be a pure compound by TLC. Thus, finding the right column to separate a novel mixture
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might require some experimentation to find the parameters that work. For the alcohol
analyses, we will not be changing columns; we will use the one installed in our instrument.
Gas Chromatography (GC)
The Major Uses of Gas Chromatography

There are three major applications of gas chromatography. One use is to determine the
number of component compounds in a mixture; another use is to determine whether or
not a specific compound is present in a sample; and the third use is to determine the
specific amount of a given compound in a sample. In short, these three uses are the
separation of a mixture, a qualitative analysis, and a quantitative analysis.
Gas chromatography is accomplished with an instrument known as a gas

chromatograph. We will focus our attention on the basics or fundamentals of GC.The
following discussion is an introduction to your laboratory work and is not intended to be a
comprehensive treatment of gas chromatographic analysis.
As the name implies, a gas is the mobile phase in GC. This gas is commonly called the
carrier gas,because it carries the mixture through a stationary phase. The carrier gas
must be inert (i.e., nonreactive) to our mixture of compounds. The stationary phase is
prepared by packing a long (~10 m), small diameter (6 mm) coiled metal tube, called a
column, with a high-boiling compound that will be in the liquid phase when the separation
is carried out. Fortunately, you will not have to prepare a column, because a variety of
them are commercially available, depending upon the separation to be accomplished.
Thus, the chromatographic system is a gas-liquid system. The mobile phase is the gas,
and the stationary phase is the liquid. Helium is a commonly used carrier gas, because it
is readily available and is an inert chemical. You select a column from those available
that will best separate your mixture. A typical column contains ground up fire brick, which
serves as a support for a high-boiling liquid phase, which is a material such as Carbowax.
Carbowax is essentially non volatile, meaning that it will not boil away during its use and
will remain stationary (i.e., the stationary phase); however, it will be a liquid at the
temperature of the analysis. The separation begins by the injection of the mixture from a
syringe through a silicon septum onto the column, which is heated by an oven. The oven
can be programmed to rise in temperature during the analysis but is often set at a constant
temperature when the sample is a simple mixture of known structures.
The sample is vaporized by a heating element as it is injected. That is, the temperature
of the heating element immediately causes all components of the liquid sample to boil,
thus making certain that they are in the vapor or gaseous state (i.e., the gas phase). The
carrier gas enters the column near the injection port and sweeps the sample through the
column. The various compounds in the mixture vary in their solubility in the stationary
liquid phase. Thus, the compounds in the mixture will travel through the column according
to their solubility in the liquid phase. The compounds being separated first dissolve in the
liquid and then vaporize from it and continue this process of dissolving and vaporizing
until they emerge from the column. In the vapor form, the helium moves the compound
along. Thus, the more soluble a compound is in the stationary liquid, the longer it stays
dissolved in the liquid during each dissolution-vaporization cycle, and the longer it takes
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for it to pass through thecolumn. The time it takes for a given compound to pass through
the column from the time it is injected to the time it is detected is called the retention
time. That is, the retention time is how long the compound is retained on the column. The
greater the compound’s affinity for the column, the longer is its retention time. As a
compound reaches the end of the column, it is sensed by a detector, which sends an
electronic signal either to a strip recorder where the signal is recorded as a peak on the
strip chart or to a computer data station where the signal is captured electronically. This
kind of analysis in which the signal is proportional to the concentration of the analyte is
called a concentration technique. The strip chart of the entire sample, called a
chromatogram provides documentation of the analysis. Likewise, an electronic
chromatogram, captured by a computer data station, allows an investigator to save the
data for follow-on interpretation and analysis. When the mixture is totally separated into
its components, the chromatogram shows each compound as a separate peak. The area
under a given peak is proportional to the amount of the compound that causes the signal.
Hence, this technique can be used quantitatively to determine amounts or concentrations
as well as to separate the components of a mixture. Modern instruments perform the
integration function for the analyst.
Schematic of Gas Chromatograph Gas chromatography includes three basic

operational steps. They are injection, separation, and detection.
Schematic diagram of a GC
Figure 1. Schematic diagram of a gas chromatograph
Columns
A variety of columns are available to the analyst. Like the detector, the column selection
will depend upon the application. One column might be suitable for hydrocarbon analysis
in the absence of water, whereas another column might be suitable for alcohol analysis
in the presence of water. To pick a column, first determine what you want to analyze.
Then try to marry the requirement with an available column. You should start by checking
with the vendor whose instrument you are using.
For our analyses, we are using a proprietary column made by Agilent Technologies
especially for alcohol/water solutions. You will not be changing columns or detectors
during your analyses today. These parameters, perhaps the most important ones, will
remain constant throughout your GC studies.
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Table 2. HP 7890 series GC/FID Parameters for Alcohol in Water Analysis (A Simulated
Blood Alcohol Analysis)
Parameter Initial setting

Inlet system
Split inlet 50:1 split ratio
Volume injected 1 ul
Inlet temperature 250 C
Inlet pressure Constant pressure mode
Helium
He 6 psi @120 C, 2 mL/min ( 55 cm/sec)
Detector temperature FID temperature 300 C
Column oven temperature Program: 120 C ( 1 min)
25 C/min ( 1 min)
Flame Ionization Detector (FID)

This is the kind of detector you will use in lab. The FID is similar to the FPD. Again, the
sample is burned in a hydrogen-rich flame. The combustion of organic compounds
produces ions in a large excess of the number of ions produced by the carrier gas. A
voltage is applied across the gas stream such that the ions are attracted to a collector
near the flame. The ions produce an electrical current (i.e., ions are charged particles).
The current produced is proportional to the number of ions, and the number of ions is
proportional to the sample size. The current is sensed by an electrometer, converted into
a digital form, and sent to the data station for storage in a computer file.
A Summary of this Week’s Lab

During this lab period, you will be introduced to gas chromatography and its application
to forensic analysis. You will learn how a gas chromatograph operates, what data you
can get from it, and how to analyze the data to get qualitative and quantitative results. As
your introduction to the instrument, you will be allowed to prepare and inject three
samples. The first sample is a mixture that contains four known alcohols. You will be given
the names of the four alcohols and the order in which they appear on a chromatogram
(the retention times or relative order of elution).
The second sample is an unknown alcohol, which is one of the four possible known
alcohols. You will compare the retention time of your unknown to the retention times of
the four known alcohols and make a preliminary determination about the identity of your
unknown from the comparison.
You will complete the identification of your unknown by preparing and injecting a third
sample. You will prepare the third sample by adding a small amount (~ 1μL) of your
unknown to the known mixture of alcohols. You will inject the third sample and obtain a
chromatogram. You will identify the unknown on the basis of the peak-enhancement you
observe. You will be able to conclusively identify the unknown, because you know it to be
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one of only four possible alcohols. If you did not know that the unknown is one of four
alcohols, then you could state only that your unknown and one of the known alcohols
have identical retention times. An identification of this type, in which you identify an
unknown, is called qualitative analysis.
Procedure:
A. Preparation of the Mixture of Alcohols (Sample 1):

1. Thoroughly clean four 25-mL Erlenmeyer flasks and rinse them with acetone. Wipe
the outsides of the flasks with a paper towel and allow them to dry as much as possible.
2. Rinse each Erlenmeyer flask several times with HPLC water, which will serve as the
solvent for your four alcohols.
3. Into one of the flasks, weigh exactly 0.060 g (60.0 mg) of methanol.
4. Add approximately 10 mL of HPLC water to the flask and mix the contents
thoroughly.
5. Clean and prepare a 100-mL volumetric flask by rinsing it with acetone and then
HPLC water. It’s okay to leave a small amount of HPLC water in the flask, because you
are going to fill the volumetric flask with HPLC water momentarily.
6. Pour the contents of the Erlenmeyer flask into the clean 100-mL volumetric flask with
the aid of a short stem or burette funnel and rinse the Erlenmeyer with two or three 2-
mL portions of HPLC water. Pour each rinse solution, in turn, into the volumetric flask.
This is a quantitative transfer. We want every molecule of the alcohol to be
transferred.
7. You will now add the other three alcohols to the volumetric flask in the same way you
added methanol.
8. Starting with one of the remaining clean Erlenmeyer flasks, repeat steps 3-5 with
ethanol.
9. Starting with one of the two remaining clean Erlenmeyer flasks, repeat steps 3-5 for
1-propanol (also known as propanol or n-propanol or propyl alcohol).
10. Repeat steps 3-5 for 2-propanol (isopropyl alcohol), using the last remaining clean
Erlenmeyer flask.
11. The 100-mL volumetric flask is now more than half full.
12. Swirl the volumetric flask to ensure the contents of the flask are thoroughly mixed.
13. Add HPLC water to the flask until the mixture reaches the neck of the flask, and
then remove the funnel and add HPLC water with a medicine dropper or controllable
pipette until the concave meniscus in the neck of the flask just touches the calibration
mark on the 100-mL volumetric flask.
14. Place the lid into the volumetric flask and shake the flask vigorously to ensure that
the contents of the flask are thoroughly mixed.
15. You now have a standard solution. A standard solution is one for which the
concentration is known exactly. The concentration of your sample is 60.0 mg/100 mL
HPLC water for each alcohol. Keep your standard solution in the volumetric flask until
you are ready to inject it onto the gas chromatograph.
B. Unknown sample (Sample 2):
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1. Obtain the unknown from the instructor
C. Preparation of the enhanced mixture of Alcohols (Sample 3):

1. Carefully transfer exactly 10.00 mL of your standard stock solution to a clean dry 25-
mL Erlenmeyer flask.
2. Tare the Erlenmeyer flask (be sure to close the sliding glass window on the balance
before you tare it) on a balance that weighs to 0.0000 g.
3. Clean and rinse a 10-μL syringe with acetone. Wipe it clean and withdraw 8.0-μL of
your unknown into the syringe. If you have an ample amount of unknown sample, expel
the unknown from the syringe onto a paper towel. If not, continue.
4. Add exactly 8.0 μL of your unknown to the Erlenmeyer flask while the flask remains
on the balance. Close the glass door and record the mass of the added unknown.
5. Carefully swirl the contents of the flask to ensure even mixture and a homogeneous
concentration.
6. Inject a 1-μL sample that is enhanced with your unknown onto the GC column.
7. Compare the chromatogram of the enhanced sample with that of the four known
alcohols and determine which your unknown is.
Preparing the Sample for Injection:
1. Transfer a small amount of your standard solution from the 100-mL volumetric flask
to a clean vial fitted with a stopper. Replicate the measurement for each sample
three times. Place all vials containing samples in the autosampler of the GC.
2. Obtain chromatograms for all the samples and note the retention times for all the
compounds.
Pre-operational Check of the Gas Chromatograph:

Refer to attachment 5 for the abbreviated instructions on the GC. This method is adapted
for a HP 7890 series gas chromatograph equipped with a flame ionization detector. The
column is a proprietary HP (now Agilent) Blood Alcohol Column (7.5 m x 0.320 mm.
Before injecting your sample, call up the identified method file.
Then, verify that the parameters (in Table 2) have been entered for the method.
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WORKSHEET
EXPERIMENT 5: Analysis of alcohols using gas chromatography
DATA
1. Instrument:
d. GC conditions (column type, detector, carrier gas, flow rate, oven
temperature, ramp rate, inlet temperature, split ratio, etc)
2. Display chromatograms for all the compounds and label the GC peaks.
1. For Table 1, insert the experimentally observed retention times and peak areas for
the four alcohols.
Table 1: GC data for standard solution of alcohols (Sample 1)
Compound Concentration Retention Time Peak area Response

(mg/L) (Minute) (counts) factor
(counts/
mg/L)
Injection No 1 2 3 Average 1 2 3 Average
Methanol
Ethanol
2-Propanol
1-Propanol
2. For Table 2, insert the data you obtained from the sample you enriched with the
unknown (Sample 3). The retention times for the four alcohols in Table 1 should closely
match the data you enter into Table 2. Three of the alcohols should give peak
heights/areas close to their values in Table 1; the unknown’s peak height/area should be
significantly enhanced.
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Table 2: GC data for the sample enriched with the Unknown alcohol (sample 3)
Compound Retention Time Peak area Enhanced

(Minutes) (Counts)
Injection 1 2 3 Average 1 2 3 Average

Methanol Yes / No
Ethanol Yes / No
2-Propanol Yes/No
1-Propanol Yes/ No
3. The experimentally determined RT for the unknown alcohol is

_________________. The unknown alcohol is________________.
4. Based on data in Table 2, determine the concentration of the alcohol that has been
enhanced for sample 3.
DISCUSSION
1. List the parameters that can influence the retention time of the compounds.
2. Which parameter can be changed in order to reduce the retention time?
3. What is meant by temperature programming in gas chromatography?
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ATTACHMENT 5: GC-FID
ABBREVIATED INSTRUCTIONS FOR GAS CHROMATOGRAPH
(AGILENT 7890B GAS CHROMATOGRAPH)
General Start-up Procedures

1. Turn on the Compressed Air, Hydrogen, Helium and Nitrogen Gas flows.
2. Check gas source pressures at the marking level
3. Turn on PC power
4. Turn on the GC power. Wait for Power on successful to display.
5. Launch GC Software from OpenLAB
Liquid Sample Injection
1. From the Method and Run Control view select Method > Load Liquid Method
2. Wait for the detector(s) to stabilize before acquiring data
3. Put sample in Vial and cap it
4. Place the vial in ALS Tray
5. From the Method and Run Control menu, clickRun Control and then Sample Info
6. Fill in sample information> Run Method
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EXPERIMENT 6
Title: Identification of Isomeric Compounds Commonly Found in Gasoline using
gas chromatography /mass spectrometer
Objectives:
1. learn how a gas chromatograph/mass spectrometer operates
2. analyze the mass spectra

1. Pentane Gas chromatograph with mass spectrometer detector
2. Hexane (GC/MS)
3. Heptane Carrier gas: Helium/Argon
4. Octane Column: SLB-5 ms ( 15 m x 0.53 mm ID)
5. Decane
Introduction
NS
During this lab period, you will be introduced to a method of analysis in which two
instruments (gas chromatograph and mass spectrometer) are linked. You will inject
various compounds that might be found in gasoline into a gas chromatograph. The
samples will be both pure hydrocarbons and also mixtures of hydrocarbons. The function
of the GC is to separate the hydrocarbon mixtures into their component compounds, so
they can be analyzed as pure compounds by the mass spectrometer.
Because gasoline contains many hydrocarbons in the C6-C10 range, we will focus on
analyzing hydrocarbons whose numbers of carbon atoms fall within this range. As each
compound emerges from the GC column, it will be split into two samples. The GC will
detect one sample, and the other sample will be directed into the mass spectrometer
where it will be ionized and excited. As the excited molecular ions lose energy, they will
fragment into pieces. The positive-ion fragments will be detected by the MS’s transducer
and sent electronically to the data station. The output from each compound will be a mass
spectrum. You will analyze the spectra of pure compounds and then compare the spectra
of the pure compounds with those found in the mixture. You will determine the
composition of your mixture by comparing the mass spectra of each compound with a
reference spectrum obtained on the same instrument and under the same conditions as
you used to obtain your spectra. Once you have correlated actual structures with mass
spectra, you will also attempt to correlate the compounds with their order of emergence
from the GC.
The number of applications of GC-MS is vast and growing every day. Therefore, only a
few important forensics applications will be mentioned. The application of most interest
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to us is in the area of arson investigations. Because the technique lends itself to the
identification of small quantities of compounds in the gaseous state, it is one of the best
techniques for identifying volatile remnants from a fire. Another important application is
the identification of drugs (and their metabolites) of abuse in blood, urine, and saliva. In
addition, any information this technique offers to a general chemist might also be of
interest to a forensics chemist. For example, the determination of the structure of an
organic compound, the identity of components of a thin-layer chromatogram, and
pesticide analyses are applications that might be of forensic interest.
General Considerations of Mass Spectrometry

There are two types of mass spectrometry, atomic and molecular. We will consider only
molecular mass spectrometry. As the name implies, mass spectrometry is a technique
that measures masses. In molecular mass spectroscopy, molecules from a sample are
ionized. The ions break apart into fragments. If a fragment is a charged particle or ion, it
will have a mass to charge ratio (m/z). When the charged fragment passes through a
magnetic field, the field causes the ion’s path to bend with a radius of curvature that is a
function of m/z. The fragment impinges on a transducer that converts the m/z into an
electronic signal that is amplified, sent to a data station and plotted along with the m/z’s
of other fragments to give a spectrum of m/z’s. If z =1, then m/z = the mass of the
fragment. Hence, the technique can give us valuable information about the structure of
the molecule by the initial ionization and subsequent fragmentation. The fragmentation
pattern is somewhat like a fingerprint of the molecule’s structure. In this laboratory period
we will focus on the interpretation of mass spectra.
Important Terms
Carbon-12 is the reference standard for all atomic masses; one mole of carbon-12 has a
mass of exactly 12.0000 grams. Carbon-12 has one isotope, carbon-13. Isotopes differ
by the number of neutrons in their nuclei. Hence, isotopes are chemically the same
because their electronic structures are the same. A mass spectrometer distinguishes
fragments of differing isotopic composition by their different mass to charge ratios. A mass
spectrum reflects the natural isotopic abundance of atoms in a sample of molecules.
Since carbon-13 constitutes about 1/100th of all carbon atoms, fragments containing one
13C atom will show up one m/z unit higher than the corresponding fragment containing
only 12C atoms. The intensity of the peak for the 13C-containing fragment will be tiny
compared to the peak for the 12C-containing fragment. The intensity of a given peak
reflects the number of fragments having that m/z. High-resolution mass spectrometers
measure masses very precisely, up to many decimal places. The presence of isotopes
gives rise to several different molecular weights. The integral molecular weight is the
molecular weight of a compound comprised of the lowest-mass isotopes of all elements
in the molecule. The average or chemical molecular weight is a weighted average that
includes all isotopes, according to their natural abundance. The nominal mass is the
whole number one gets by rounding a decimal. The mass spectrum of acetone in Figure
1 will be used to describe other terms.
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Figure 1 Mass Spectrum of Acetone
Relative abundance refers to the number of ions with a given mass/charge ratio. The peak
in the spectrum of the ion with the largest relative abundance is called the base peak.
The base peak serves as the reference peak. The intensity of the base peak is arbitrarily
set at 100, and the intensity of every other peak in the spectrum is found as a percentage
of the base peak. In Figure 1, the base peak is the peak at m/e 43 (the electronic charge
may be represented by e or z). Likewise, the peak at m/e 58 has a relative abundance of
approximately 50%, because its height is approximately 50% that of the base peak. The
relative abundance is read as a percent in terms of the base peak. Thus, the peak at
m/e 15 has a relative abundance of about 15%. It is generally true that modern mass
spectrometers are designed so that only singly charged ions are recorded. When the
charge in the mass/charge ratio is one, the m/e simply becomes the mass of the fragment.
Thus, we often just refer to the fragments by their mass when we really mean
mass/charge. We can do that because the numbers are the same, and it simplifies the
discussion. With some exceptions, the largest significant mass that we find in a spectrum
is called the molecular ion. The molecular ion is the radical-cation that forms when an
electron is ejected from an intact molecule. The molecular ion for acetone is shown in
Figure 1; it has a mass of 58. For our purposes, the mass of the molecular ion is the
molecular weight (molar mass) of the molecule. The terms molecular ion and parent ion
may be used interchangeably; they both refer to the mass of the radical-cation formed
from the initial ionization of the molecule. The base peak at m/e 43 arises from the
fragmentation between one of the methyl groups and the carbonyl group in acetone.
Often, by subtracting the mass of a fragment from the mass of the molecular ion, one gets
the m/e of a significant peak in the spectrum. In the case of acetone, the subtraction of
the mass of a methyl group (15) from M+ (58) gives the base peak (43).
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Spectra of Aliphatic Compounds
Consider the mass spectrum of octane (Figure 2). Initial ionization produces a radical-
cation.
Figure 2 Mass Spectrum of Octane
The radical-cation fragments by the loss of a radical to form a set of even-electron ions.
The lost radical is often a methyl group of mass 15. A typical fragmentation is shown in
Figure 2. Significant peaks are predicted to be at m/z 43, 57 and 71. These peaks are
indeed found in Figure 2. The fragmentation pattern is explicable by the Even-Electron
Rule. Molecules contain an even number of electrons. When an electron is ejected from
an intact molecule, the ensuing radical-cation contains an odd number of electrons.
Hence, a molecular ion contains an odd number of electrons. Odd-electron ions
decompose by the loss of either a radical or a molecule.
The Instrument
As noted earlier, our mass spectrometer is linked to an Agilent 7890 gas chromatograph.
The eluents from the gas chromatograph enter a HP 5973 selective mass detector. Figure
2 shows both instruments. The mass chromatograph is on the left side of the gas
chromatograph. The system is equipped with an auto-injector, which can be seen on top
of the gas chromatograph.
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Figure 2 schematic diagram of a Gas Chromatograph-Mass Spectrometer
Fundamentals of Mass Spectroscopy

The following discussion illustrates the basic principles of the simplest of instruments. A
compound is introduced as a vapor into a system that is under a very high vacuum. See
Figure 2. In the inlet system, the molecules become widely dispersed. A portion of the
sample is channeled into a beam of electrons. The energy of the electron beam is (~70
eV = 7620 kJ/mol) is sufficient to ionize the sample (Ion Source). The ions are accelerated
and analyzed (Mass Analyzer). Inside the analyzer, the ions are accelerated in a magnetic
field H. The magnetic field deflects the ions in a circular path of radius r. The radius is
given by Equation 1, which is the fundamental equation of mass spectroscopy.
r = SQRT {2V(m/z)/ H2} Equation 1
From the form of Equation 1, we see that the radius of curvature is a function of both the
applied potential V and the field strength H. The detector converts the beam of ions into
an electrical signal that is processed (Signal Processor) and either stored as a mass
spectrum or printed out. The requirement for a high vacuum distinguishes the technique
of mass spectroscopy from most of the other commonly used techniques by chemists.
The care and maintenance of the high-vacuum system is a laboratory necessity that
comes with experience and training. Our primary focus is to learn how these instruments
can be used in chemical analysis.
Ion Sources
Some compounds are thermally stable, and some are not. Likewise, some molecules are
volatile, easily vaporized, and others are not. Accordingly, a variety of methods has been
developed to ionize various compounds, so they can be subjected to mass spectral
analysis. The two major categories of ion sources are gas-phase and desorption sources,
and examples of both types will be described below. Sources may also be described as
hard or soft. A hard source imparts more energy to a sample than does a soft source. The
more energy that is imparted, the more the sample fragments to produce daughter ions.
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Thus, soft sources produce a limited number of peaks, perhaps only a molecular ion peak;
whereas, hard sources produce many peaks. Whether to use a hard source or soft source
for an application depends upon the level of detail needed from the spectrum. If the
analyst is certain of the compound in question and only wants to confirm its presence, a
soft source might be sufficient. If the analyst is trying to prove the structure of a compound,
a hard source is required.
Gas-Phase Sources
Gas-phase sources are generally useful for compounds that boil below 500oC and are
stable up to that temperature. Three kinds of gas-phase sources, electron impact (EI),
chemical ionization (CI), and field ionization (FI) are described below.
Electron Impact (Gas-Phase Source)

The sample (is introduced into the system via a so-called molecular leak, meaning that
only a small portion of the incident ions is allowed into the chamber. Electrons are emitted
from a heated tungsten (W) or rhenium (Re) filament. They pass through a potential of
about 70V, giving them an energy of 70 eV. The electron beam and sample beam cross
paths at right angles. Radical-cations are produced when the high-energy electrons come
close enough to eject an electron from a sample molecule by electrostatic repulsion (i.e.,
this is the electron impact). Although the ionization of the analyte molecule produces a
radical-cation, it will be written as M+ in this manual and M+ will mean molecular ion (i.e.,
radical-ion). The emergent ions are accelerated into the analyzer. Because the ionizing
electrons have a small mass but high kinetic energy, they eject electrons from M and
create M+ ions that are at higher vibrational and rotational energy levels but not at a
substantially higher translational energy level. Therefore, the M+ ions lose energy by
fragmenting into lower-mass daughter ions. The presence of these daughter ions makes
electron impact sources very useful when identification of the analyte is important.
Experimental
Standard Procedure for GC-MS

Start up the instrument in accord with procedures, as shown in Attachment 6.
Hydrocarbon Samples
The instrument has been set to the standard conditions for hydrocarbons.
1. Obtain a set of hydrocarbons from the instructor. The set will contain five samples. Four
of the samples are pure unbranched hydrocarbons each of which contains from 5-10
carbon atoms (i.e., they range from pentane to decane). You will be given the identity of
the four pure hydrocarbons. The fifth sample is a mixture of hydrocarbons. The mixture
contains from three to five hydrocarbons. These five hydrocarbons might be any of the
C5-C10 unbranced hydrocarbons.
2. In turn, inject each sample and print out the output in the form of a mass spectrum. For
the mixture, you should obtain one mass spectrum for each component.
3. Analyze the spectra and fill in the boxes in the report form.
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WORKSHEET
EXPERIMENT 5: Identification of Isomeric Compounds Commonly Found in
Gasoline using gas chromatography /mass spectrometer
DATA
1. Instrument:
d. GC/MS conditions (column type, detector, carrier gas, flow rate, oven
temperature, ramp rate, inlet temperature, split ratio, etc)
2. Display the total ion chromatograms (TIC) and mass spectra for all the
compounds.
Unknown Mixture Number ________________
1. Analyze your mass spectra and complete Table 1 by filling in the missing data. For
hydrocarbons #1- #4, write in the name of the pure hydrocarbon in the sample. For
the mixture, fill in the data for each compound but do not name the compound. You
will name the unknowns after you have tabulated the data on them.
Table 1 Mass Spectra of Unbranched Hydrocarbons

Molecular Ion (M+) Base Peak (m/e) Other Significant
Compound Name Peaks
Hydrocarbon #1
Hydrocarbon #2
Hydrocarbon #3
Hydrocarbon #4
Unknown mixture
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2. Identify the components of your unknown mixture from your data in Table 1, and write
the name next to its molecular weight (M+) in Table 2.
Compound M+ Name of Hydrocarbon
3. The unknown mixture number _____________ contains the following

compounds:____________________________
DISCUSSION
What type of mass analyzer did you use in this experiment? Briefly describe its
operating principle. What are its limitations?
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ATTACHMENT 6: GC – MS
ABBREVIATED INSTRUCTIONS FOR GAS CHROMATOGRAPH- MASS

SPECTROMETER
(Agilent GC/MS)
1. Click on the icon GCMS MassHunter Acquisition Software.

2. Go to [method]>[load method] and load the desired method.
3. If you wish to edit the method or create a new method, please go to
[method]>[Edit Entire Method].
4. Tune the MSD by clicking this icon
5. Please make sure that N2 percentage in tuning report is less than 10%
6. Click on the icon below to run single run
7. Fill in information such as Operator name, data Path, data File name, sample
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name and vial number.
8. Click [OK and Run Method] to run.
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EXPERIMENT 7:
Title: Determination of caffeine in beverages using liquid chromatography
Objective:
1. To understand basic function and operation system of liquid chromatography
2. To determine the caffeine content in daily beverages

Chemicals Apparatus
Methanol HPLC with UV-VIS detector
Iso-propanol Column: C18
Coffee Vials
Decaffeinated Coffee Samples
Tea Samples
Soft Drink Beverages
Introduction:
Chromatography is a process used to separate the components of a mixture. A mixture
is injected into a chromatography column, where it lands on a substrate, also known as
the stationary phase. The stationary phase may be polar, attracting polar substances, or
nonpolar, attracting nonpolar substances. When a mixture is injected into a
chromatography column, the substances in the mixture cling to the stationary phase.
Next, a solvent is injected into the column. The solvent is called the mobile phase. As the
solvent moves along the stationary phase, it will carry the components with it. When and
how quickly the substances are carried out of the column by the solvent depends on the
polarity of the substances and their solubility in the solvent. If the solubilities and/or
polarities of the individual parts of the mixture are significantly different, the substances
in the mixture will separate from each other as the mixture travels along the substrate.
The substance that is the most strongly attracted to the solvent will be the first to leave
the chromatography column. The substrate (stationary phase) and the solvent (mobile
phase) can be in any phase, depending on the properties and concentrations of the
components in a given mixture. Therefore, there is solid, liquid, and gas chromatography.
Figure 1 shows the block diagram of a high-performance liquid chromatograph (HPLC).
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Figure 1. Schematic diagram of HPLC system
Many beverages such as soft drinks, coffee and tea contain the mild stimulant caffeine
(C8H10N4O2). The caffeine content varies widely from about 100 μg/mL (100 ppm) in
sodas to over 1000 μg/mL in certain types of coffee. In this experiment the caffeine
content of a diluted soft drink will be determined using high performance liquid
chromatography (HPLC).
The chemical structure of caffeine is shown here.
Spectrophotometry provides a sensitive method for the detection and measurement of

caffeine. The UV absorption spectrum (see figure below) of caffeine exhibits a pair of
absorption bands peaking at 205 nm and 273 nm with a characteristic absorption shoulder
between them.
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Typically, caffeine content is determined by measuring the absorbance at 275 nm. Soft
drinks contain a wide variety of substances, many of which absorb UV light at 275 nm.
Consequently, the direct measurement of the caffeine absorbance in soft drinks is not
possible and one must first separate the caffeine from other components before making
the absorbance measurement.
The caffeine separation is easily achieved using HPLC. Liquid chromatography can be
divided into several types, including normal-phase (e.g., a silica or alumina column),
reversed-phase and ion exchange. Reversed-phase partition chromatography uses a
non-polar organic coating on a silica structure for the stationary phase. The non-polar
coating is commonly formed by reacting an organochlorosilane with the OH groups on
the silica surface. With normal-phase HPLC, the most commonly used solvents are
hexane, isopropanol or THF, whereas a more polar mobile phase such as methanol/water
or acetonitrile/water is commonly used for reversed-phase partition chromatography.
In this experiment, the caffeine separation will be done using a non-polar C18 column
and a methanol/water mobile phase. A series of caffeine standards that bracket the
unknown sample concentration will be measured to construct a calibration curve. A
comparison of the caffeine peak area in the soft drink sample compared to those for the
standards permits a quantitative determination of the caffeine content.
High performance liquid chromatography (HPLC) is carried out by injecting a small

amount of liquid sample into a moving stream of liquid that passes through a column
packed with particles of a stationary phase.
Reversed-phase HPLC will be used to determine the concentration of caffeine in various
beverages such as coffee, tea, and soft drinks. The traditional method for the
determination of caffeine is via solvent extraction followed by spectrophotometric
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quantitation. HPLC allows for the rapid separation and quantitation of caffeine from the
many other substances found in these beverages, including tannic acid, caffeine and
sucrose. Retention time, tR is used as a qualitative measure and peak area as a
quantitative measure for external standard determination.
Experimental Procedure:
A. Preparation of caffeine standards
1. Accurately weigh out 10.0 mg of caffeine then transfer it to a clean 100 mL
volumetric flask.
2. Dilute to the mark with HPLC grade water.
3. Carry out a series of dilutions of the stock 0.1 g/L solutions to obtain standards of
0.01 g/L, 0.025 g/L, 0.05g/L, and 0.075 g/L. Make 10 mL of each of the dilutions.
Use HPLC grade water to make the dilutions.
4. Shake the five caffeine solutions to insure adequate mixing.
B. Sample preparation:
1. Coffee: Pipette 5 mL of coffee into a clean and dry 50 mL volumetric flask and then
dilute to the mark with HPLC grade water. Be sure to filter the sample into a clean vial.
Rinse the filter before collecting the sample by filtering the first 1-2 mL to waste.
2. Decaffeinated Coffee Samples: Pipette 25 mL of the decaffeinated coffee into a clean
and dry 50 mL volumetric flask and then dilute to the mark with HPLC grade water. Be
sure to filter the sample into a clean vial. Rinse the filter before collecting the sample by
filtering the first 1-2 mLs to waste.
3. Tea Samples: Pipette 10 mL of your tea sample into a clean and dry 50 mL volumetric
flask. Dilute to the mark with HPLC grade water. Be sure that you filter your sample into
a clean vial. Rinse the filter before collecting the sample by filtering the first 1-2 mL to
waste.
4. Soft Drink Beverages: If your beverage is carbonated, you must decarbonate it by
pouring it back and forth between two beakers until the bubbling ceases. Now pipette
25 mL of your beverage into a clean and dry 50 mL volumetric flask and dilute to the
mark with HPLC grade water. Be sure to degas this sample for 5 minutes. Also, be sure
to filter the sample into a clean vial. Rinse the filter prior to collecting the sample by
filtering the first 1-2 mL to waste.
C. Determination of the UV absorbance spectrum of caffeine using the HPLC instrument.

1. Refer to Attachment 7 for the abbreviated instruction on the HPLC instrument.
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2. The mobile phase composition is 50:50 methanol:water solution (volume %)
Flow rate: 1.0 ml/min
Detection wavelength: 276 nm
Injection volume: 20 microLiter
Cautions (!):
1. Record three chromatograms for each beverage.
2. Do not change the sensitivity of the detector. If your samples are off-scale on the
detector, you must make an additional dilution of your sample and run it again.
3. Filter each solution through the syringe filter into a clean vial. Filter the least
concentrated sample first. Wash the filter between samples by filtering 1 mL into
waste before you collect the rest. Do not use tape to label vials, it can get the vials
stuck. Use a pen.
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WORKSHEET
EXPERIMENT 7:
Title: Determination of caffeine in beverages using liquid chromatography
DATA
1. Instrument:
d. HPLC conditions (column type, detector, wavelength, mobile phase,
stationary phase, flow rate, etc).
2. Display the chromatograms for all the samples.
3. Enter data obtained for all samples in Table 1.
Sample Concentration Peak area

(g/L)
Trial 1 Trial 2 Trial 3
Caffeine
standard 1
Caffeine
standard 2
Caffeine
standard 3
Caffeine
standard 4
Coffee
Decaffeinated
coffee
Tea
Soft drinks
1. Plot the average area of the caffeine peak against the concentration in g/L for the
caffeine standards. Determine the slope of the calibration plot.
2. Calculate the concentration of caffeine in coffee, decaffeinated coffee, tea and soft
drink samples using the slope of the calibration plot.
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DISCUSSION
1. Comment on the amounts of caffeine in these samples. Discuss possible source
of errors.
2. Discuss how retention time depends on the methanol content and pH of the mobile
phase. What factors determine the choice of mobile phase composition and pH in the
present analysis?
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ATTACHMENT7- HPLC
ABBREVIATED INSTRUCTIONS FOR HPLC

(Agilent 1200 Series HPLC)
Sample preparation prior to analysis
I. Powering on and preparing the instrument

II. Creating a method
III. Creating a sequence
IV. Data retrieval and analysis
Sample preparation
1. Ensure samples are free of particulate matter. A 0.45 μm disposable filter is

recommended to filter samples.
2. Load samples into labeled HPLC vials. Each vial holds a maximum of 1.5 mL but
do not need to be completely filled; because the autosampler needle collects
sample from the bottom of the vial. The cap is then screwed onto the vial as normal.
3. Place sample vials in the autosampler tray, making note of the position of each
sample in the tray (you will need this information later).
I. Powering on and preparing the instrument
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1. Turn on the computer (the password and username can be found in the HPLC
register book).
2. Turn on the HPLC by turning on the power strip. The instrument will proceed through
its normal startup procedure. Wait for the startup procedure to finish; all lights
should be green. Orange or red lights indicate a problem.
3. Open the program “LC1200 Online” otherwise known as “ChemStation”. The

program should start up and open after initializing. If the instrument modules are
not recognized, repeat previous steps.
4. To enable instrument control, make sure “Method and Run Control” in the lower
left corner of the program is highlighted.
5. Ensure solvent bottles are filled with solvent (methanol/acetonitrile/deionized
water/ etc). If running low, fill bottles with HPLC grade solvent. Then adjust the
solvent bottle fill level in the program by clicking on the picture of the solvent bottles
and selecting “solvent bottles filling,” inputting your new volumes.
6. Ensure that the waste jug underneath the HPLC is not full, or near full, or it will
overflow during the run. Dispose of waste as necessary.
7. Install your column, making sure a secure connection is established by tightening
screw- plugs to finger-tight. NOTE: when putting away columns, ensure they are
stored in the box with endcaps in place. Columns should not be allowed to dry out.
8. If you already have a method created, load that method (Method→Load Method...,
then select your method from the list). If not, navigate to and complete section II:
creating a method. Method must be created and loaded to proceed with instrument
operation.
9. In the instrument control panel in ChemStation, hit the “on” button. The instrument
will begin to pump solvent. NOTE: if the instrument detects a leak via dripping
solvent, it will automatically turn red and shut off Depending on your column, it is
recommended to flush with organic solvent first until column appears clean (no
peaks eluting in the screen). Quickly change solvents by clicking on the arrows
above the solvent and selecting “Set up Pump...” Once clean, set the solvent levels
to match your initial chromatographic conditions to equilibrate the column. Column
should be equilibrated until pressure and UV-vis baseline has stabilized.
10. You are now ready to create a sequence to run or, alternatively, perform single
runs. Select between these options by clicking the image of three vials (vs. the
single vial) in the upper left-hand corner of the chemstation program. A sequence
may be started by pushing the “start” button.
II. Creating a method
1. Choose a method on which to base your own. Then save it as your own by going
to File→Save As→Method... whereupon you will be prompted to select a name for
your new method. NOTE: it is useful to save your file name ending in a number
(ex. Name_1) so that new versions may be saved when changes are made.
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2. Now edit your method by selecting Method→Edit Entire Method... and select the
desired method sections to edit (typically all checkboxes are checked). Select
“OK.”
3. You will now be able to fill in your method information which should include your
analyte, solvents, detection wavelengths, injection volume, flow rate, column type,
chromatographic conditions, and total runtime. This information may be edited
later. Select “OK.”
4. Set up Pump: on this screen you will set your flow rate, stop time, chromatographic
conditions, and pressure limits. Initially, ensure your solvents are labeled correctly
and the letters A, B, C, and D match the actual solvent bottles.
5. For an isocratic run, only use of the “solvents” section is required.
6. If a gradient run is required, use “timetable;“ create a timetable using the “insert”
and “append” buttons to modify your chromatographic run. Make sure to include
ample time in your run for your column to equilibrate back to initial conditions.
NOTE: information in the “timetable” section will always override information in the
“solvents” section. Select “OK.”
7. Set up Injector: here you will select your injection type and volume. Standard
injection of 10 μL is a recommended starting point. Select “OK.”
8. Ensure the “store” box is checked so that data is saved! If you require detection in
the UV region, make sure the required lamps Vis and UV are selected; if the UV
region is not needed, unselect this box. Make sure in the “time” section that
“stoptime” is set to “as Pump.” All other conditions may be changed as desired.
Select “OK.”
9. Column thermostat method: here you can select the temperature at which the
column will be maintained. If temperature is to change throughout a run, push the
“more” button and input the timetable desired using the insert and append buttons.
Select “OK.” Signal Details: Each signal that is to be stored must be added to the
method by selecting it from the drop-down menu and then pushing the “add to
method” button. In the example shown, three signals have been added and are
therefore stored simultaneously. Select “OK.”
10. Edit Integration Events: make sure “manual events” box is checked, then select
“OK.”
11. Specify Report: here you will adjust what displays on the screen, and the default
settings are generally acceptable. Select “OK.”
12. Run Time Checklist: the boxes next to data acquisition, standard data analysis,
and save method with data should be checked. Select “OK.” Your method is now
ready to use.
III. Creating a sequence
1. Access the sequence table by clicking on the image of the sample tray and
selecting “sequence table.” Alternatively, this may be accessed via
Sequence→Sequence Table.
2. Here you will program your sequence. Under “vial,” input the number only (do not
type “vial”) that corresponds to the location of the vial in the sample tray. Chose a
unique name for your sample, then click the “method name” box and select your
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desired method from the drop-down list. Then continue to fill in injections/vial
(typically 1), sample type (typically Sample), and injection volume (which should
ideally match your method). Use the “insert” and “append line” buttons to add
samples to the sequence list.
3. When sequence is created as desired, select “OK.”
4. SAVE YOUR SEQUENCE! Select Sequence→Save Sequence As... or
File→Save As→Sequence... You will then be prompted to enter a name for your
sequence. NOTE: this name is how you will locate your data later so it is
important to remember the name you select.
IV . Data Retrieval and Analysis
The data analysis screen in chemstation.
1. In the lower left-hand panel of chemstation select “Data Analysis”

2. Select the name of your sequence (or, alternatively, your single run) from the list
to the left.
3. Now you may select the individual sample you wish to analyze from the list
displayed at the top of the screen. To view all spectra collected for this sample,
show “all loaded signals”. Alternatively, if only one signal is desired, select it
instead; this is recommended for integration.
4. Record your integration results. This is your data! Congratulations.
• When finished with HPLC, shut down the system to conserve energy. This is done
by turning off the power strip behind the instrument (note: do not touch the
buttons on the instrument) and turning off the computer.
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UNIVERSITI TEKNOLOGI PETRONAS

CHEMISTRY PRACTICAL III (YAB 2052)

SEMESTER: JAN 2020

CHEMICAL ASSESSMENT FORM

6 Experiment 6: Identification of Isomeric Compounds Commonly Found in

1.2 Wear and button up your laboratory coat.

Fail to wear your laboratory coat, RM 10.00 will be penalized.

1.6 Know the exit of the building.

2. You are NOT PERMITTED to

2.1 Be playful in the laboratory.

2.2 Eat or drink in the laboratory.

2.3 Smoke in the laboratory.

3. While using the chemicals, you must remember that

Figure A: Safer way of sniffing chemical

3.5 Do not use chemicals from unlabelled bottle.

3.6 Label all test tubes that you use.

4. While using apparatus or doing experiments, you must remember

4.1 Avoid crowding in doing experiment.

4.2 Use the fume hoods as instructed.

CHEMISTRY PRACTICAL III (YAB 2052)

CHEMICAL ASSESSMENT FORM

Name:___________________________________________ Student’s signature: _________________

Student ID:_______________________________________ Demonstrator’s signature: ____________

Title of experiment: _______________________________ Date: _________________

No. Chemical Approx. Assessment Hazard First aid

CHEMISTRY PRACTICAL III (YAB 2052)

EXPERIMENTAL FLOW CHART

Name:___________________________________________ Student’s signature: _________________

Student ID:_______________________________________ Demonstrator’s signature: ____________

Title of experiment: _______________________________ Date: _________________

Chemicals & Apparatus:

RESULTS AND CALCULATIONS

Title: Determination of Water Hardness by Atomic Emission Spectrophotometry

Chemicals & Apparatus:

Atomic Emission Spectroscopy

In atomic emission, a sample is subjected to a high energy, thermal environment in order

Figure 1: Microwave Plasma - Atomic Emission Spectroscopy

Water whose hardness is more than 60 mg per liter is considered to be "hard".

Title: Determination of Water Hardness by Atomic Emission Spectrophotometry

Wavelength for Mg measurement: ____________________

Sample name [Mg2+] [Ca2+] Intensity Intensity

RESULTS AND CALCULATIONS

ABBREVIATED INSTRUCTIONS FOR MICROWAVE PLASMA -ATOMIC EMISSION

New instrumental technique for elemental determinations using atomic emission

THE MICROWAVE PLASMA SYSTEM

Figure 2. Excitation process

STANDARD OPERATING PROCEDURE FOR MP-AES 4200 INSTRUMENT

Chemicals & Apparatus:

Figure 1. The components of UV-VIS spectrometer

• Aspirin is widely used as an analgesic (pain reliever) and an antipyretic (for

Structure of Aspirin (acetylsalicylic acid)

Aspirin (acetylsalicylic acid) contains three groups:

carboxylic acid functional group (R-COOH)

Properties of Aspirin (acetylsalicylic acid)

C9H7O4-Na+ → C9H7O4- + Na+

C9H8O4(s) + H2O(l) → C7H6O3(s) + C2H4O2(aq)

The amount of aspirin (acetylsalicylic acid) in commercial analgesics can be determined

Preparation of calibration curve

5. Measure the absorbance of unknown solution at the same wavelength as your

Wavelength of absorbance measurements (max ): ____________________

Sample name Concentration Absorbance Absorbance Absorbance

RESULTS & CALCULATIONS:

Name:___________________________ Student’s signature: _

Student ID:___________________________ Demonstrator’s signature:

Title of experiment: _______________ Date: _

Name:___________________________ Student’s signature: _

Student ID:___________________________ Demonstrator’s signature:

Title of experiment: _______________ Date: _