ANALYTICAL

CHEMISTRY
Laboratory Guide

This module provides guidelines for safety, Laboratory

Report and note book. It’s also providing students with
Laboratory Manual for subject of Analytical Chemistry
(CLD 10402).

3rd EDITION
SEPT 2015
 Laboratory Information

Before each lab session, you should prepare by reading the lab manual, reference book and
summarized it in a jotter book. We expect you to have a good understanding of the purpose,
details of the procedure, the use of all chemicals and any significant hazards, and the underlying
science of the experiment when you come to lab.

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 CONTENTS

Preface

Laboratory safety Guidelines

Safety Declaration Form

Chemistry Laboratory Report Guidelines

Laboratory Notes Books Guidelines

Experiment

1 Analysis of Food Colour by UV-Vis Spectrophotometer

2 Using FT-IR as an Analytical Buddy: To Compare and Identify

3 Determination of Copper (Cu) in Tap Water by Atomic Absorption

Spectroscopy (AAS)

4 Determination of Caffeine in Soft Drink by HPLC

5 Separation of an Alcohol Mixture by Gas Chromatography

REFERENCES

APPENDICES

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 PREFACE

This manual provides laboratory guidelines, safety declaration form, Chemistry Lab
Report guidelines and Laboratory manual for subject of Analytical Chemistry (CLD
10402).

The primary purpose of this manual is to compile all necessary information regarding
laboratory component in one manual.

The manual contains four parts. Part 1 provides a description of laboratory guidelines
and safety declaration form. It is compulsory for student to understand all those
guidelines and submit safety declaration for recording purposes. Part 2 is laboratory
report guidelines containing all requirements such as format and arrangement in order to
produce good quality of laboratory report. Part 3 is guidelines for preparation of
laboratory notes book. Part 4 is compilation of laboratory manual that will provide
student practical guidelines in Analytical Chemistry.

There may be shortcomings which we had overlooked but hopefully these should not
hinder the process of enhancing laboratory skill.

DR MOHD ZULKHAIRI BIN ABDUL RAHIM

DR NORZAHIR BIN SAPAWE
DR NOR NADIAH BT MOHAMAD YUSOF
Ms NORHAYATI BT MOHD IDRUS

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 PART 1

LABORATORY SAFETY GUIDELINES

General Guidelines

1. Conduct yourself in a responsible manner at all times in the laboratory.

2. Be familiar with your lab assignment before you come to the lab. Follow all
written and verbal instructions carefully. If you do not understand a direction or
part of a procedure, ask the instructor before proceeding.
3. No student may work in laboratory alone. The lab instructor or co-coordinator
grant exceptions on a case by case basis.
4. When first entering a laboratory, do not touch any equipment, chemicals or other
materials in the laboratory area until you are instructed to do so.
5. Do not eat, drink beverages or chew gum in the laboratory. Do not use laboratory
glassware as containers for food or beverages.
6. Smoking is not allowed in any indoor area.
7. No music allowed in the laboratory. Radio (including walkman) and other
entertainment devices are not permitted.
8. No cellular phone is allowed in this laboratory.
9. Perform only those experiments authorized by the instructor. Never do anything
in the laboratory that is not called for the laboratory procedures or by your
instructor. Carefully follow all instructions, both written and oral. Unauthorized
experiments are prohibited.
10. Observe good housekeeping practices. Work areas should be kept clean and tidy
at all times.
11. Horseplay, practical jokes, and pranks are dangerous and prohibited.
12. Always work in a well-ventilated area.
13. Bring only your laboratory instructions, worksheets and report to the work area.
Other materials (books, purses, backpacks, etc) should be stored in the cabinet.
14. Know the locations and operation procedures of all safety equipment including
the first aid kit, eyewash station, safety shower, spill kit and fire extinguisher.
15. Be alert and proceed with caution at all times in the laboratory. Notify the
instructor immediately of any unsafe condition you observe.
16. Label and equipment instructions must be read carefully before use. Set up and
use the prescribed apparatus as directed in the laboratory instructions provided by
your instructor.
17. Experiments must be personally monitored at all times. You will be assigned a
laboratory station at which to work. Do not wander around the room, distract
other students or interfere with laboratory experiments or others.

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18. Write your name and equipment use every time you come in to the laboratory in
the log book.
19. Defeating safety devices or using equipment in a manner other than that which is
intended will be grounds for dismissal from the lab.

Clothing
1. Safety goggles and safety jacket must be worn whenever you work in lab.
2. Gloves should be worn whenever you use chemicals that cause skin irritations or
need to handle hot equipment.
3. Mask should be worn every time you prepare the chemicals.
4. Safety shoes and hard hat should be worn at all times while in the laboratory.
5. Contact lenses should not be worn in the laboratory unless you have permission
from your instructor.
6. Dress properly during a laboratory activity.
7. Long hair, dangling jewelry and loose or baggy clothing are a hazard in the
laboratory. Long hair must be tied back and dangling jewelry and loose or baggy
clothing must be secured.
8. Sandal, open-toed shoes, high heels or shoes with holes in the sols will not be
worn in the lab.
9. Short and skirts are not permitted.
10. Instructor and laboratory assistant have a right dismiss to you from the laboratory
if they found that you are not wearing proper safety clothing.

Handling Chemicals
1. Treat chemicals with respect and understand the chemicals you are using with
Material Safety Data Sheet (MSDS). The MSDS are available in the analytical
room.
2. All chemicals in the laboratory are to be considered dangerous. Do not touch,
taste or smell any chemical unless specifically instructed to do so.
3. Check the label on chemical bottles before removing any of the contents. Take
only much chemical are you need. Smaller amounts often work better than larger
amounts.
4. Label all containers and massing papers holding dry chemicals.
5. Never return unused chemicals to their original containers.
6. Never use mouth suction to fill a pipette. Use pipette bulb or pipette filler.
7. Acids must be handled with extreme care. Always add acids slowly to water, with
slow stirring and swirling, being careful of the heat produced, particularly with
sulfuric acid.
8. Handle flammable hazardous liquid over a pan to contain spills. Never dispense
flammable liquids anywhere near a flame or source of heat.
9. Never take chemicals or other materials from the laboratory area.
10. Take good care when transferring acids and other chemicals from one part of the
laboratory to another. Hold them securely and in the method demonstrated by the
instructor as you walk.

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11. All wastes generated during the course of an experiment must be disposed of
according to the lab instructor’s directions.
12. Never mix chemicals in sink drains.
13. Sinks are to be used only for water and those solutions designated by the
instructor.
14. Solid chemicals, metals, matches, filter paper, and all other insoluble materials are
to be disposed of in the proper waste containers, not in the sink.
15. Checks the label of all waste containers twice before adding your chemicals waste
to the container.
16. Cracked or broken glass should be placed in the special container for “broken
glass”.
17. Keep hands away from your face, eyes, mouth and body while using chemicals.
Wash your hands with soap and water after performing all experiments.

Personal Hygiene
1. Wash hands before leaving the lab and before eating.
2. Gloves should be removed before leaving the lab, using telephones, or entering
common areas

Accidents and Injuries

1. Report any accidents (spill, breakage, etc) or injury (cut, burn, etc) to the
instructor immediately, no matter how trivial it may appear.
2. If you or your lab partners are hurt, immediately tell to the instructor.
3. If a chemical should splash in your eye(s), immediately flush with running water
from the eyewash station for at least 20 minutes. Notify the instructor
immediately.
4. Spills should be cleaned up immediately.

Handling Glassware and Equipment

1. Inserting and removing glass tubing from rubber stopper can be dangerous.
Always lubricate glassware (tubing, thistle tubes, thermometer, etc) before
attempting to insert it in a stopper. Always protect your hands with tower or
cotton gloves when inserting glass tubing into, or removing it from a rubber
stopper.
2. When removing an electrical plug from its socket, grasp the plug, not the
electrical cord.
3. Hands must be completely dry before touching an electrical switch, plug or outlet.
4. Examine glassware before each use. Never use chipped or cracked glassware.
5. Never use dirty glassware.
6. Do not immerse hot glassware in cold water; it may shatter.
7. Report damaged electrical equipment immediately. Look for things such as frayed
cords, exposed wires and loose connections. Do not use damaged electrical
equipment.

7
8. If you do not understand how to use a piece of equipment, ask the instructor for
help.
9. Be careful when lifting heavy objects. Lift comfortably, avoid unnecessary
bending, twisting, reaching out, and excessive weights, lift gradually and keep in
good physical shape.
10. Do not transfer a glassware form one laboratory to another without permission
from instructor.

Heating Substances
1. Do not operate a hot plate by yourself. Take care that hair, clothing, and hands
are a safe distance from the hot plate at all times. Use of hot plate is only allowed
in the presence of the teacher.
2. Heated glassware remains very hot for a long time. They should be set aside in a
designated place to cool, and picked up with caution. Use tongs or heat protective
gloves if necessary.
3. Never look into a container that is being heated.
4. Do not place hot apparatus directly on the laboratory desk. Always use an
insulated pad. Allow plenty of time for hot apparatus to cool before touching it.
5. If leaving a lab unattended, turn off all ignition sources and lock the doors.

Ended the Experiments

1. At the end of the laboratory sessions, you should;
 Shut-off main gas outlet
 Turn-off the water inlet
 Desk top, floor area and sink are clean
 All equipment is cool, clean and arranged
2. All equipment use should be flushed using deionized water.

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 SAFETY DECLARATION FORM

Dean / Head of Campus

Universiti Kuala Lumpur
Malaysian Institute of Chemical and Bioengineering Technology (UniKL-MICET)
Lot 1988 Vendor City, Taboh Naning, 78000 Alor Gajah,
Melaka, Malaysia.

Dear Sir,

SAFETY DECLARATION

I ………………………………..………………………………………………………….
ID No ………………………. declare that I have read and understood the safety rules and
regulations in UniKL-MICET. I hereby agree to abide by all the rules and regulations
stated in the safety guidelines.

 I hereby understood the contents and will disciplinary action will be taken
against me, if I do not abide by the stated rules.
 I am fully responsible for all my actions during laboratory sessions.

Thank you.

Yours faithfully,

……………………………….
Name:
Student ID No:
Group:
Subject:
Date:

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 PART 2

CHEMISTRY LAB REPORT FORMAT

You should type your lab report. Make sure that you check your document for any spelling
errors. Each lab report is worth 100 points. You should also read the student handbook on the
subject of plagiarism. Your data and observations will be similar, but your interpretations
should not be written identically. You may not copy another student’s lab report in part or in its
entirety. If you are found guilty of this infraction, you and the person from whom you copied
will both lose points or shared total marks. In extreme cases or repeated offenses, both students
may receive a zero for the lab.

Result & Discussion

Analyze all data qualitative and quantitative. Then transfer finding into Table, Graph,
Histogram and Pie chart if necessary. This includes any observations. Make sure that your
graphs have titles, labeled axes with units, and legends. You should include the proper units
with any numbers, as well as use the proper number of significant figures based upon the lab
equipment used. DO NOT place any calculations or data analysis in this section. It may be a
good idea to reproduce here any data tables that you completed during the lab. Base on above
point, discuss on your findings and relate to your theory and objective of experiment.

Example:
Table 1: X versus Y

Samples X (unit) Y (unit)

A
B
C
D

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 50
45 y = 4.4557x + 0.9714
40 R2 = 0.9893

parameter X (unit)
35
30
25
20
15
10
5
0
0 2 4 6 8 10 12
parameter Y (unit)

Figure 1: Relationship between X and Y

Conclusions

This is the most important section. Please include the summary of the results and relate in brief
the findings / results with the theory. Answer the questions, “What did you learn?”, “Did I
accomplish the purpose?”, “How would I improve the experiment next time?”.
Recommendation is optional. The conclusion should be one paragraph of 5 – 7 sentences.

References

Write down any sources such as your textbook, the Internet, electronic
encyclopedia, books, etc. that you used.

 A list of lab manuals, books, reports, journal, world wide web (www) etc.
 Arrangement (year, alphabetical order)
 Author, title, publisher, year, chapter or page number

Example:
Smith J.M and Van Hess H.C., Introduction to Chemical Engineering Thermodynamics,
McGraw-Hill, New York, 2001, p229.
Rahim M.Z.A., Sapawe N., Yusof N.N.M. and Idrus N.M., Introduction to Chemical
Engineering Thermodynamics, Journal of Chemical Sciences, 2001, 20-30.

Appendix

Here is where you attach any material that you think is pertinent to the lab report such as
summary of calculation involved. Also answer any questions here that are in the lab report.
You do not have to re-write the questions, but label and number them appropriately.

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 PART 3

LABORATORY NOTEBOOKS

You are required to use a bound notebook in CLD 10004 Lab to record all primary data and
observations. You should prepare your notebook before coming to a lab by writing the
title of the experiment on a new numbered page, summarizing relevant information from
the lab manual, and starting calculations involving molar masses, etc. Take note of
theoretical ideas and special instructions given by your instructor at the start of each
experiment. Your notebook should be a complete record of your work in lab. Notes should be
able to understand in the future, not just during the current experiment. Good note taking in a
lab is a valuable skill that you can learn with a little effort and practice.

Guidelines to be followed:
1. Always bring your notebook with you to lab. You will be graded on the completeness of
your previous note taking and your preparation for the current experiment. You may use
your notebook during a lab quiz.
2. Number the pages sequentially and reserve space at the beginning for a table of contents.
3. Take your notebook during laboratory hours and record all values directly in it – not on
loose scraps of paper.
4. Specify each measured quantity by name and include the units.
5. If you make a mistake in your notebook, simply draw a solid line through the error and write
the correction nearby.
6. Tables greatly simplify data entry; they should be set up before coming to lab.
7. Write down all observations if necessary – don’t rely on your memory.
8. Save time by doing trial calculations in your notebook before filling out any report sheets.
9. Save time by making preliminary sketches of graphs (flow Chart) on the ruled lines in your
notebook.

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 EXPERIMENT 1

ANALYSIS OF FOOD COLOUR

OBJECTIVE

(1) To determine max of Colourant (wavelength scan)

(2) To prepare a serial dilution and generate a standard calibration graph for sample
quantitation (photometric scan)

INTRODUCTION

Food coloring (colouring) is any substance that is added to food or drink to change its color.
Synthetic Food Colours, also known as Artificial Food Colours, are manufactured chemically
and are the most commonly used dyes in the food, pharmaceutical and cosmetic industries.
Besides that, a growing number of natural food dyes are being commercially produced, partly
due to consumer concerns surrounding synthetic dyes. Some examples include Caramel
coloring (E150), made from caramelized sugar, used in cola products and also in
cosmetics.Annatto (E160b), a reddish-orange dye made from the seed of the Achiote A green
dye made from chlorella algae (chlorophyll, E140) and etc.

All those colourant can be analyze by using an ultraviolet/visible spectrophotometer. Figure 1

below gives a schematic diagram of a double beam spectrophotometer. Instruments for
measuring the absorption of U.V or visible radiation are made up of the following components;
Sources (UV and visible), Wavelength selector (monochromator), Sample containers, Detector,
Signal processor and readout.

Figure 1: Schematic diagram of double beam UV – Vis spectrophotometer

13
When a beam of parallel radiation passes through a layer of solution of thickness, b (cm) and a
concentration, C (moles per liter) of an absorbing species, absorption of radiation occurs. The
transmittance (T) of the solution is the fraction of incident radiation transmitted by the solution.
Transmittance is often expressed as a percentage. The absorbance (A) of a solution is defined
as the negative log of the transmittance (T) of the solution. The absorbance is directly
proportional to the path length of the radiation through the solution and the concentration of the
absorbing species.

A=bc

Where; A = absorbance (no unit)

 = molar absorptivity coefficient (M-1 cm-1)

b = pathlength (cm)

c = concentration (M or mol/L)

This relationship between absorbance, A and bc is known as Beer’s Law. Beer’s Law is
successful in describing the absorption behavior of dilute solutions only. At high
concentrations, absorbance of the solution does not obey Beer’s Law and is no longer
proportional to C.

In this experiment you are going to conduct two main applications which are wavelength
scanning and photometric scanning.

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 MATERIALS AND METHODS

Instrument:

Perkin – Elmer UV/Vis Spectrophotometer Lambda EZ210

Chemicals:

100 ppm colorant stock (100 ml), Unknown (two samples), Distilled water

Apparatus:

Sample cuvettes, path length 1 cm, volumetric flask 50 ml (five) , pipette 5 ml, 10 ml and
25 ml (one each), rubber bulb (three), beaker 100 ml (one), graduated cylinder 50 ml
(one), dropper (one), labeling sticker, tissue paper.

Methods:

1. Prepare serial dilutions (5 ppm, 15 ppm, 25 ppm, 35 ppm and 45 ppm) in 50 ml

volumetric flask from the 100 ppm carmoisine stock.
2. Calculate the volume needed, V1 from the 100 ppm food dye stock for all dilutions

by using dilution formula (M1V1=M2V2). *Show your calculation.

Where;
M1 = Initial concentration in ppm
V1 = Initial volume in ml (volume of solution required)
M2 = Final concentration in ppm
V2 = Final volume in ml (volume of volumetric flask used)

3. In order to prepare a dilution, draw an exact volume of V1 from the food dye stock

and pour into a 50 ml volumetric flask. Then add distilled water up to the mark
level of the volumetric flask. Then, shake the volumetric flask properly.
4. Repeat procedure No. 3 for all the dilutions.
5. After preparing the serial dilutions, your instructor will brief on the standard
operating procedure of Perkin – Elmer UV/Vis Spectrophotometer Lambda EZ210.

15
 6. Fill a cuvette with 45 ppm dilution and another cuvette with blank solution; insert
them in the sample compartment. Wipe clean the sides of the cuvettes and
remember not to touch on the clear surface. Do the wavelengths scan and obtain
the max. Record your data.
7. For photometric scan, fill the cuvette as step NO. 6 but use the serial dilution
prepared and scan one by one. Record the absorbance readings and look at the
standard calibration graph produced.
8. Determine the concentrations of Unknown #1 and Unknown #2.
9. Record all data completely and clean your work station properly before leave.

Analysis of data:

1. Use the spectrum obtained from the wavelength scan to identify the max for
Carmoisine and then record.

2. Record different concentration of standards absorbance and construct a standard

calibration curve (concentration vs absorbance).

3. Measure the unknown 1 and unknown 2 concentration peak in the soda sample
chromatograph, and Use standard calibration curve (concentration vs absorbance) to
determine the concentration of unknown 1 and unknown 2.

Note: All raw data must be record in table form.

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 FLOW CHART [10 Marks]

(Hints: Draw a flow chart of the experiment in this page, simplify the procedure & include the
calculation of dilution)

17
 RESULTS [20 Marks]
Part A: Wavelength Scan

(i) Instrument Parameters

Starting Wavelength = _________________ nm

Ending Wavelength = _________________ nm
Path length = _________________ mm

(ii) Result for Wavelength Scan

Sample used = _________________

Concentration of the sample = _________________ ppm

max obtained = _________________ nm

Part B: Photometric Scan

(i) Instrument Parameters

Wavelength, max = _________________ nm

Path length = __________________ mm

(i) Results for Photometric Scan (Creating a Standard Calibration Curve)

Record the absorbance readings and the unknown concentrations in the table below:

Table 1: Data
No. Sample Carmoisine Absorbance (nm),
Concentration (ppm), y-axis
x-axis
1 Std 1
2 Std 2
3 Std 3
4 Std 4
5 Std 5
6 Unknown 1
7 Unknown 2

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 GRAPH

(Hints: Plot your graph from the data obtained in Table 1 using EXCEL. Attach the graph in
this page)

19
 DISCUSSION [30 Marks]

(Hints: Discuss your findings and relate to your theory and objective of experiment)

20
 CONCLUSION [10 Marks]

(Hints: Conclusion should contain summary of the results and relate in brief the findings /
results with the theory)

21
 RECOMMENDATION [10 Marks]

(Hints: Suggest improvements in apparatus or measurement procedure, or experimental

procedures for future)

22
 TUTORIALS [10 Marks]

1. State the Beer’s Lambert law.

2. What is the volume needed to prepare a 50 ppm of carmoisine from a 100 ppm of
carmoisine in 100 ml volumetric flask?

3. Why we need to wipe the sides of the cuvette clear surface?

4. Describe the function of wavelength scanning and photometric scanning.

23
 REFERENCES [5 Marks]

(Hints: Write down any sources such as your textbook, the Internet, electronic encyclopedia,
books, etc. that you used)

24
 APPENDICES [5 Marks]

(Hints: Attach all the printed data from this experiment and jotter notes at this page)

25
 EXPERIMENT 2

USING FTIR AS AN ANALYTICAL BUDDY: TO COMPARE AND IDENTIFY

OBJECTIVE

(1) To conduct background scanning

(2) To conduct sample scanning
(3) To compare sample's peak
(4) To identify impurities from peak comparison

INTRODUCTION

The most common purpose of analytical method is either to indentify, to compare, or to quantify
which will lead to more exciting methods in product developing, quality controlling and new
inventions. FTIR has become one of the most favourite equipment in analytical method because it
is the most less tedious and easy to conduct. The basic operation is easy to understand and the
method is easy to apply. This experiment will let you experience on how to use FTIR to compare
and identify chemicals used daily in our life. The industries usually will use this technique to
determine the Quality of their product and then as the development of materials grow, we can
identify the add-in substance or impurities.

MATERIALS AND METHODS

Instrument:
Fourier Transform Infra Red Spectrometers

Sample:
Sample 1, Sample 2

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Method:

1. Choose the right sample compartment that is suitable for the sample you are going to
analyzed. Since we are going to analyze imulsions, using the ATD is best recommended.
2. Insert carefully the ATD and make sure the source light is turned on.
3. Scan Banckground
Instrument Scan Background
4. Scan Sample 1 and Sample 2.
Instrument Scan Sample
5. Print graph
6. To compare
Process Compare
7. Print graph
8. To Identify
Compare plotted graphs with a set of compiled data to identify the impurities. See Graph A,
Graph B and Graph C.

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 FLOW CHART [10 Marks]

(Hints: Draw a flow chart of the experiment in this page, simplify the procedure)

28
 RESULTS [20 Marks]

1. Spectral Analysis

Sample 1: _______________________

Table 1: Data
Peaks Bond Compound Frequency range, cm-1

Note: The number of peak is depend on your sample

Sample 2: _______________________

Table 2: Data
Peaks Bond Compound Frequency range, cm-1

Note: The number of peak is depend on your sample

29
 DISCUSSION [30 Marks]

(Hints: Discuss your findings and relate to your theory and objective of experiment)

30
 CONCLUSION [10 Marks]
(Hints: Conclusion should contain summary of the results and relate in brief the findings /
results with the theory)

31
 RECOMMENDATION [10 Marks]
(Hints: Suggest improvements in apparatus or measurement procedure, or experimental
procedures for future)

32
 TUTORIALS [10 Marks]

1. What is the purpose of correlation charts?

2. What is the range of the wave number of infra red region?

3. State an example of IR spectroscopy application in industries.

33
 REFERENCES [5 Marks]

(Hints: Write down any sources such as your textbook, the Internet, electronic
encyclopedia, books, etc. that you used.)

3
 APPENDICES [5 Marks]

(Hints: Attach all the printed data from this experiment and jotter notes at this page)

4
 EXPERIMENT 3

DETERMINATION OF COPPER (Cu) IN TAP WATER BY USING

ATOMIC ABSORPTION SPECTROSCOPY (AAS)

OBJECTIVE

(1) To optimize the burner system by flame atomic absorption spectroscopy using
standard solution
(2) To check the performance (sensitivity) of the atomic absorption spectroscopy
using standard solution
(3) To identify the present of Copper in drinking water sample
(4) To determine amount of Copper in drinking water sample

INTRODUCTION

Atomic absorption spectroscopy is the elemental analysis that widely used in analyzing
samples from environment, metal, food, pharmaceutical and chemical industries. For
measurement, sample must be converted into free atom.

Figure below is the schematic diagram of atomic absorption spectrometer. Hollow

cathode lamp will emit the line spectrum of the element to be analyzed. Samples are then
atomized in the flame. Selection of wavelength of interest done by the monochromator.

Figure 1: Schematic diagram of Atomic Absorption Spectrometer

5
The sample must be introduced in the aerosol form. When the samples enter the flames,
the metal ions inside the samples are then converting into free atoms and evaporate all the
solvent. The common flames used are air-acetylene and nitrous oxide-acetylene.

A premix burner is commonly used with the flame. Sample aspirated through the
capillary tube, sprayed into the spray chamber in a fine mist by a nebulizer and mixed
with oxidant-air mixture. Samples are then introduced into the flame. Large droplets of
sample flow to waste and only a small amount of samples with the smallest droplets
remained.

Optimization of atomic absorption spectroscopy requires the several conditions such as to

make sure the unit is operating correctly, flame conditions is correct for the element,
alignment of the burner system and proper integration time. In order to achieve
optimization, we need to check few items in the system; lamp, nebulizer, gas flows and
burner position condition. Setup element is needed to assess instrument performance
which should have the following characteristic;

 Should not be sensitive to slight changes in flame conditions

 Wavelength should be greater than 250 nm (minimizes potential interferences).

 Standard solution should be easy to prepare.

 Standard solution should produce a signal of about 0.200 absorbance.

In this experiment, the ideal setup element used is copper which have a wavelength at
324.8 nm and aspiration of solution of 4.0 mg/L should produce of 0.200 absorbance.
Characteristic concentration is the concentration of analyte that gives 1% absorption or
0.0044 absorbance (as shown in checking the Performance part). Characteristic
concentration is very useful in assessing instrument performance by showing a low
characteristic concentration value which indicates a higher sensitivity.

6
 MATERIAL AND METHODS

Instrument:
Perkin Elmer Atomic Absorption Spectrometer model 100 AAnalyst, Copper
hollow cathode lamp

Apparatus:
Eight 100 ml volumetric flasks, Beakers, Pipette (2ml and 5 ml)

Chemicals
1000ppm Copper (Cu) standard solution, Concentrated nitric acid (69%),
Deionized water
Caution: Dispose all the chemicals in the proper waste container!

Method

Part A: Preparation of Copper standard solution

1. Pour about 10 mL of 100 ppm Copper standard solution into a small beaker and
prepare 100 ppm copper standard solution in 100 ml volumetric flask using the
following equation;

M1V1 = M2V2

Where;
M1 = Initial concentration in ppm
V1 = Initial volume in ml (volume of solution required)
M2 = final concentration in ppm
V2 = final volume in ml (volume of volumetric flask used)

2. Add 1% (v/v) of concentrated nitric acid (1% = 1 ml) into the 100 ml volumetric flask
and mark up to the volume using deionized water.

WARNING! Concentrated nitric acid is highly corrosive! Please use a glove for
protection when you are dealing with the corrosive chemicals!

7
 3. By using the freshly prepared 100 ppm copper standard solution above , prepare
2.0 ppm and 4.0 ppm of copper standard solution using 100ml volumetric flask
(calculate the volume of solution required using the above equation).
4. Prepare the 0.5ppm, 1.0ppm, 1.5ppm, 2.0ppm and 2.5ppm copper standard
solution using 100ppm copper standard solution. Add 1% v/v of concentrated
nitric acid to each of the standard solution and mark up to the volume.
5. The standards solution prepared are now ready to be analyzed.

Part B: Operating the instrument

Please refer to the Appendix. Step 1 until 5 and then do the following order as below;

1. Optimizing the Burner System.

a. Prepare a blank solution.

b. Aspirate the known standard using 2ppm and 4ppm.

c. Adjust the burner position using the horizontal adjustment knob and the
nebulizer adjustment nut until a maximum absorbance is displayed on the
screen.

d. Check the absorbance of blank solution again. It should be zero.

2. Checking the Performance

a. In the Analyses menu, click on Characteristic Concentration.

b. Enter the sample concentration and instrument reading (the maximum

absorbance).
c. Press the Tab key.
d. The measured Characteristic Concentration should be within 20% of the
Comparison Characteristic Concentration value.
e. Close the Characteristic Concentration window. The characteristic
concentration value can also be calculated using the following equation;

0.0044 × known conc. used

Characteristic Conc. Value = _____________________________ (Equation 1)
Absorbance for known conc. used

8
3. Creating a Calibration Curve and Analyzing Samples.
a. Creating a calibration curve.
i. Immerse the nebulizer tube into blank solution. In the Manual Analysis
Control window, click on Analyze Blank.

ii. Select the calibration standard you want to analyze from the drop-down
list. The software completes the concentration entry according to the
value entered in the method.

iii. Click on Analyze Standard. Repeat this step for every calibration
solution; 0.5ppm, 1.0ppm, 1.5ppm, 2.0ppm and 2.5ppm.

iv. Check the calibration curve. If any of the standards appear to be off the
calibration curve, then you may wish to edit the calibration curve.

b. Analyzing the samples.

i. Immerse the nebulizer tube into sample solution.
ii. Click on Analyze Samples.

4. Shut down the instrument by following step 7 in the Appendix.

9
 FLOW CHART [10 Marks]

(Hints: Draw a flow chart of the experiment in this page, simplify the procedure &
include the calculation of dilution)

10
 RESULTS [20 Marks]

1. Checking performance of the instrument

Calculate characteristic concentration for Standard solution 2 ppm & 4 ppm using
equation below:

0.0044 × known conc. used

Characteristic Conc. Value = _____________________________
Absorbance for known conc. used

2. Creating a Standard Calibration Curve

Record the absorbance readings and the unknown concentrations in the table below:

Table 3: Data
No. Sample Copper Concentration Absorbance (nm),
(ppm), x-axis y-axis
1 Std 1
2 Std 2
3 Std 3
4 Std 4
5 Std 5
6 Sample 1
7 Sample 2

11
 GRAPH

(Hints: Plot your graph from the data obtained in Table 1 using EXCEL. Attach the
graph in this page)

12
 DISCUSSION [30 Marks]

(Hints: Discuss your findings and relate to your theory and objective of experiment)

13
 CONCLUSION [10 Marks]

(Hints: Conclusion should contain summary of the results and relate in brief the findings
/ results with the theory)

14
 RECOMMENDATION [10 Marks]
(Hints: Suggest improvements in apparatus or measurement procedure, or experimental
procedures for future)

15
 TUTORIALS [10 Marks]

1. What is the function of monochromator?

2. You are given a 100 ppm mercury stock solution. What is the volume needed in mL to
prepare a 15 ppm standard stock solution in 50 mL volumetric flask?

3. How would you determine the performance of the atomic absorption spectrometer?

4. Calculate the Characteristic Concentration Value for 2 ppm copper standard that has the
absorbance value of 0.194 absorbance.

5. Label all the components below with the correct component name.

16
 REFERENCES [5 Marks]

(Hints: Write down any sources such as your textbook, the Internet, electronic
encyclopedia, books, etc. that you used)

17
 APPENDICES [5 Marks]

(Hints: Attach all the printed data from this experiment and jotter notes at this page)

18
 EXPERIMENT 4

DETERMINATION OF CAFFEIN IN SOFT DRINK

OBJECTIVE

(1) To identify the present of caffeine in soft drink sample.

(2) To determine amount of caffeine in soft drink sample.

INTRODUCTION

High performance liquid chromatography (HPLC) is a type of chromatography which is very

similar to liquid chromatography. In HPLC, however, the stationary phase column is more tightly
packed than in other types of liquid chromatography. In this lab, the column is packed with C18

particles that are less than 10 μm in diameter. The small diameter of the particles allows
unprecedented resolution and high efficiency. Since the particles in the column are so small, it is
necessary to pump the mobile phase through the column at a very high pressure. The pump keeps a
precise flow rate so that the positions of the peaks in time can be used to identify the species in a
sample. This is done by comparing the chromatographs of prepared standards of the particular
species to be determined. The common peak is an indication of the standard.

Figure 1: Schematic Diagram of High Performance Liquid Chromatography

19
 MATERIALS AND METHODS

Instrument:

Isocratic HPLC system with UV detector, C18 column,

Chemicals

Caffeine 1000 ppm standard (stock solution), Methanol (HPLC grade), Double
distilled water (filtered with 0.45 μm filter paper), Soft drink sample

Apparatus:

Vacuum, Funnel, 0.45 μm filter paper, 0.45 μm filter syringe,100 μL syringe, 60

mL syringe,Volumetric flask

Methods:

Part A: Preparation of Caffeine standards

1. Prepare standard caffeine samples of 20 ppm, 40 ppm, 60 ppm, 80 ppm and 100
ppm by diluting portions of the 1000 ppm solution with distilled water. Calculate
the volume needed, V1 from the 1000 ppm Caffein stock for all dilutions by using

dilution formula (M1V1=M2V2). *Show your calculation.

Where;
M1 = Initial concentration in ppm
V1 = Initial volume in ml (volume of solution required)
M2 = final concentration in ppm
V2 = final volume in ml (volume of volumetric flask used)

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Part B: Preparation of soda samples

1. Obtain a soft drink sample. *Bring your own soda sample.

2. Degas the sample by placing it in a vacuum flask and connecting the flask to a
vacuum pump or water aspirator. Leave it under vacuum until no more bubbles
appear in the soda sample. (If no vacuum is available, allow the soda to stand
open overnight.)

3. Filter the degassed soda through #42 filter paper.

4. After preparing the serial dilution and sample, your instructor will brief on
standard operating procedure of HPLC.

21
 FLOW CHART [10 Marks]

(Hints: Draw a flow chart of the experiment in this page, simplify the procedure &
include the calculation of dilution)

22
 RESULTS [20 Marks]

(i) Instrument Parameters

Mobile phase = ___________________________

Ratio of mobile phase = ___________________________
Flow rate = ___________________________

(ii) Creating a Standard Calibration Curve

Record the retention time, peak area and the Caffein concentrations in the table below:

Table 1: Data
No. Sample Caffeine Retention Peak Area,
Concentration (ppm), Time, (min) y-axis
x-axis
1 Std 1
2 Std 2
3 Std 3
4 Std 4
5 Std 5
6 Soft drink 1
(______________)
7 Soft drink 2
(______________)

23
 GRAPH

(Hints: Plot your graph from the data obtained in Table 1 using EXCEL. Attach the
graph in this page)

24
 DISCUSSION [30 Marks]

(Hints: Discuss your findings and relate to your theory and objective of experiment)

25
 CONCLUSION [10 Marks]

(Hints: Conclusion should contain summary of the results and relate in brief the findings
/ results with the theory)

26
 RECOMMENDATION [10 Marks]

(Hints: Suggest improvements in apparatus or measurement procedure, or experimental

procedures for future)

27
 TUTORIALS [10 Marks]

1. Briefly explain how HPLC is used as a separation technique.

2. What is the purpose of the mobile phase and the stationary phase?

3. What is the purpose of the caffeine standards?

4. Why does the syringe have to be carefully rinsed before each use?

5. Retention of caffeine in standards. How could you be identify a peak in the soft drink
was caffeine and not another substance by using retention time?

28
 REFERENCES [5 Marks]

(Hints: Write down any sources such as your textbook, the Internet, electronic
encyclopedia, books, etc. that you used)

29
 APPENDICES [5 Marks]

(Hints: Attach all the printed data from this experiment and jotter notes at this page)

30
 EXPERIMENT 5

SEPARATION OF AN ALCOHOL MIXTURE BY GAS CHROMATOGRAPHY

OBJECTIVE

(i) To identify the component of an unknown alcohol mixture

(ii) To determine the composition of an unknown alcohol mixture

INTRODUCTION

Many samples of interest to chemists are complex mixtures of many individual

components. Analysis of a particular substance in the mixture is complicated by the
presence of other substances. Therefore complex mixtures must often be separated into
the individual components prior to analysis.

One technique used for separating and analyzing the components of a solution is gas
chromatography (GC). This technique is useful for gases or volatile compounds that
vaporize quickly. It is based on the partition, or distribution, of the sample components
between a gaseous mobile phase and a liquid phase that is coated on the surface of an
inert solid (the GC column).

A sample is injected onto the head of a column (see Figure 1). The injector and column
are heated to a temperature that is high enough to vaporize the sample. -A gas such as
helium flows through the column; this is the mobile phase or carrier gas and it serves to
carry the sample from the injector, through the column and into the detector. The
column (inert solid chemically coated with a liquid) is called stationary-phase; the
stationary phase selectively interacts with the components in the sample and
accomplishes the separation. A competition is established between the mobile and
stationary phase for the individual compounds passing through the column. The rate at
which the sample component passes through the column depends on the degree of
interaction between the sample and the column and on the boiling point of the sample.
If the component does not interact with the column, it moves through the column, swept
by the mobile phase, in a relatively short time. If the component interacts with the
column, a longer time is needed for the sample to travel through the column.

31
A component's identity can be determined by its retention time. Retention time is the time
required for the component to appear at the detector. Components that move through the
column quickly have short retention times while components that interact with the
stationary phase and linger on the coloring have longer retention time. In this experiment,
the composition of an unknown alcohol mixture is determined by using gas
chromatography (Figure 2).

Figure 1 Figure 2

Instrument:
GC system, Perkin Elmer AutoSystem XL Gas Chromatograph — Thermal
conductivity detector.

Apparatus:
1. Column — V4-in. (o.d.) x 0.5- m Pgropack column.
2. Micro syringe (10μL)

Chemicals:
1. Methanol.
2. Ethanol
3. Unknown mixture of alcohols
CAUTION: Dispose of all organics in the proper waste container.

Safety:
Handle the syringes carefully. The syringes are very dangerous, especially when
filled with hazardous chemicals. They also break easily. Do not allow the syringe
to roll off of the lab bench. When injecting the sample, push the plunger straight so
that it does not bend. The gas chromatograph is like an oven, so the port may be
hot. Use caution.

32
Methods:

1. Turn on the GC.

2. Set the following conditions:

Column temperature 100 °C
Detector temperature 130 °C
Injection port temperature 120 °C
Flow rate 60 mL min-1

3. Obtain a micro liter syringe and a vial of methanol.

4. Clean the syringe by rinsing it at least 6 times with methanol. With the plunger fully
depressed, place the needle into the methanol. Slowly draw up the plunger to obtain a
sample of methanol in the syringe. Remove the syringe from the methanol sample.
Discharge this sample into a waste container. Depress the plunger, and put the syringe
needle back into the methanol. Draw up the sample and discharge it into a waste
container. Repeat.

5. Place the syringe needle back into the methanol sample, and obtain 1 microliter of
the methanol. Wipe the needle with a tissue.

6. Insert the needle carefully into the injection port of the GC until the needle stops.

7. Inject the sample, and start the data collection simultaneously by depressing the
syringe plunger and pressing the space bar on the computer at the same time.

8. After the peak is obtained, record data or print the graph as instructed.

9. Repeat steps 5-8 for the other 2 known alcohols and the unknown alcohol mixture.
When running an unknown sample, be certain to let the data collect long enough to get
all possible peaks.

10. Calculate the percentage composition of each alcohol in the mixture.

Percentage X = Peak area of X × 100

Total peak area of all peaks

33
 FLOW CHART [10 MARKS]

(Hints: Draw a flow chart of the experiment in this page, simplify the procedure)

34
 RESULTS [20 Marks]

(i) Instrument Parameters:

Injector temperature = ___________________________

Column temperature = ___________________________
Detector temperature = ___________________________

(ii) Chromatogram data:

A. Alcohols standard

Standard Retention Time

Methanol
Ethanol
Isopropanol

B. Identification of unknown sample

Unknown Sample Retention Time Peak Area

(min) (V.s)
Peak 1
Peak 2
Peak 3

C. Percentage Composition (%)

By using formula determine the percentage composition of each alcohol present in the
unknown alcohol mixture. Show the calculation.

35
 DISCUSSION [30 Marks]

(Hints: Discuss your findings and relate to your theory and objective of experiment)

36
 CONCLUSION [10 Marks]

(Hints: Conclusion should contain summary of the results and relate in brief the findings
/ results with the theory)

37
 RECOMMENDATION [10 Marks]

(Hints: Suggest improvements in apparatus or measurement procedure, or experimental

procedures for future)

38
 TUTORIALS [10 Marks]

1. What is Gas Chromatography (GC)?

2. Explain the separation column in GC?

3. Why we need to make sure there is no bubble in syringe during injection of sample
into GC.

39
 REFERENCES [5 Marks]

(Hints: Write down any sources such as your textbook, the Internet, electronic
encyclopedia, books, etc. that you used)

40
 APPENDICES [5 Marks]

(Hints: Attach all the printed data from this experiment and jotter notes at this page)

41
 APPENDICES

Mass Force
1 lbm = 0.453592 kg 1 lbf = 4.448222 N
1 ton = 2000 lbm 1N = 0.224809 lbf = 1 kg.m/s2
1 kg = 2.20462 lbm

Volume Density
1 ft3 = 0.028317 m3 1kg /m3 = 0.062428 lbm/ ft3
1 L = 0.001 m3
1 m3 = 35.32 ft3
1 cm3 = 0.06102 in3
1 gal =0.0037854 m3
1 gal/min = 6.31 x 10‾5 m3/s

Pressure

1 pascal (Pa) = 1 N/m2

1 atm = 760 mmHg = 760 torr
1 atm = 101.325 KPa

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 Table 1: Characteristic Infrared Absorption frequencies

Bond Compound Type Frequency range, cm-1

2960-2850(s) stretch
Alkanes
1470-1350(v) scissoring and bending
C-H
1380(m-w) - Doublet - isopropyl, t-
CH3 Umbrella Deformation
butyl
3080-3020(m) stretch
C-H Alkenes
1000-675(s) bend
Aromatic Rings 3100-3000(m) stretch
C-H Phenyl Ring Substitution Bands 870-675(s) bend
Phenyl Ring Substitution Overtones 2000-1600(w) - fingerprint region
3333-3267(s) stretch
C-H Alkynes
700-610(b) bend
C=C Alkenes 1680-1640(m,w)) stretch
C C Alkynes 2260-2100(w,sh) stretch
C=C Aromatic Rings 1600, 1500(w) stretch
C-O Alcohols, Ethers, Carboxylic acids, Esters 1260-1000(s) stretch
Aldehydes, Ketones, Carboxylic acids,
C=O 1760-1670(s) stretch
Esters
Monomeric -- Alcohols, Phenols 3640-3160(s,br) stretch
O-H Hydrogen-bonded -- Alcohols, Phenols 3600-3200(b) stretch
Carboxylic acids 3000-2500(b) stretch
3500-3300(m) stretch
N-H Amines
1650-1580 (m) bend
C-N Amines 1340-1020(m) stretch
Nitriles 2260-2220(v) stretch
1660-1500(s) asymmetrical stretch
NO2 Nitro Compounds
1390-1260(s) symmetrical stretch

v - variable, m - medium, s - strong, br - broad, w - weak

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 THIS IS THE END OF THE LABORATORY MANUAL ~

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