CHEM 3410 – INSTRUMENTAL METHODS OF ANALYSIS

LAB MANUAL
 1

Chemistry 3410 Index - 2007

Experiment # Title Page

Index of 3410 experiments 1

Laboratory safety rules 2

Safety agreement (note to be completed & submitted) 4

Equipment list 5

1 Quantitative determination of caffeine in nonalcoholic

beverages by UV spectrophotometry. 7

2 UV spectrophotometry: Simultaneous determination

of caffeine and acetylsalicylic acid in an analgesic. 9

3 Flame photometry: Quantitative determination

of sodium in fruit juices. 14

4 Gas chromatography: Quantitative determination

of fatty acid composition in oils and fats. 16

5 Fluorometric determination of acetylsalicylic

acid in aspirin tablet. 19

6 Determination of iron in a cereal by atomic 22

absorption spectrophotometry.

7 Analytical method development- Seminar presentation. 25

Chem 3410     Lab Safety Rules  2

CHEMISTRY LABORATORY RULES

AND SAFETY PRECAUTIONS

1. Never work alone in the laboratory.

2. Smoking is not permitted.

3. Unauthorized experiments are prohibited.

4. Know the location and use of the fire extinguisher, safety showers and first aid kit.

5. It is required that you wear prescription glasses or safety glasses at all times in the
laboratory for your own protection. Contact lenses are particularly dangerous and they
must not be worn in the laboratory.

6. Report all injuries to your instructor at once.

7. Never taste chemicals or solutions.

8. Use the fume hoods for all poisonous reactions or any reactions which produce noxious
gases.

9. When diluting concentrated acid or base always add the concentrated acid or base to
water (never the reverse), while stirring the solution. Be very careful with sulfuric acid.

10. Keep an orderly, clean laboratory desk. Return glassware to the lab drawer when finished
using it to keep the work area from becoming cluttered.

11. Place unneeded books, etc. on the shelves at the side of the laboratory.

12. Waste crocks are provided for the disposal of all solid chemicals and paper, etc.

13. Some stock reagent bottles and specialized glassware are placed with specific experiment
kits please return them to their respective kits when finished.

14. Always read the label twice before taking any chemical from a bottle. If you are not sure
if you have the right chemical, ask!

15. When pouring reagents, hold the bottle so the label points upwards facing the palm of the
hand. The accumulation of reagent on bottle lip may be removed by touching the bottle lip
to the rim of the receiving vessel.
Chem 3410     Lab Safety Rules  3

16. Avoid using an excess of reagent. If you happen to have measured out too much, see if
someone else can use the excess.

17. Due to possible contamination of the contents of a whole stock bottle, never return unused
chemical to the stock bottle.

18. Always check your glassware before you use it. If it is broken or cracked, exchange it for
a new one.

19. There is one crock reserved for broken glass. All broken glassware should be placed in
this crock and no other.

20. If corrosive chemicals or liquids come in contact with the skin or clothing, flood with
copious amounts of water for an extended period of time.

21. Spilled chemicals should be wiped up immediately; spilled acid or base should be rinsed
with plenty of water and wiped up with a sponge and the sponge rinsed after.

22. Inserting glass tubing or thermometers through a rubber stopper - first lubricate the tube
and stopper with glycerol or water, then holding the tube near the end to be inserted insert
slowly while rotating the tube. BE VERY CAREFUL!

24. When you are ready to leave the laboratory, your bench area should be rinsed off with a
wet sponge and the water, gas, and air valves shut off.

25. The chemistry store room is out of bounds to students. If you require apparatus, ask your
instructor.
Chem 3410  Safety Agreement  4

This form must be completed, signed, and submitted to the laboratory 
instructor before any laboratory work is begun. 

********

I have read and understood the safety rules that appear on preceding pages of this
manual, recognize that it is my responsibility to observe them, and agree to abide
by them throughout this course.

Name (please print) ________________________________

Date ______________ Signature ____________________________________

Chem 3410    Equipment list  5                

Glassware and Equipment List

In Week 1 of the laboratory course you will be assigned a locker of glassware and laboratory
equipment. It is your responsibility to see that all elements of your kit are present and that it is
clean and ready for use. Any equipment on the list below which is not in your locker should be
reported to the instructor.

Name of Apparatus Number in Locker Check In Check Out

Beakers 50 mL 2
150 mL 1
250 mL 1
400 mL 2
600 mL 1

Bottles, Plastic 1L 1
Wash 1

Bottles, Weighing 34 × 12mm 1

29 × 12mm 1

Brush, Sample Transfer 1

Brushes, Test tube large 1

small 1

Drying Glass 1

Flasks, conical 50 mL 1
250 mL 4
500 mL 4

Flasks, volumetric 50 mL 4
100 mL 2
250 mL 2
500 mL 1

Funnels, buret 1
ordinary 1
powder 1

Measuring Cyl. 10 mL 1
100 mL 1

Medicine Dropper 1
Chem 3410    Equipment list  6                

Pipets, Transfer 5 mL 1
10 mL 1
20 mL 1

Pipet, Measuring 10 mL 1

Rods, Stirring Glass 3

Rubber Bulb 1

Stoppers, glass 2
plastic 2

Spatula 1

Test tubes, pyrex 13 × 100 mm 9

Thermometer -10 – 260 °C 1

Vial, glass 1 dr. 1

3 dr. 1

Watch glasses, 65 mm 1
100 mm 1

At the end of the lab course all glassware should be washed and cleaned. Re-check the contents
of your locker and report any missing items, together with your locker number to the instructor.

The following items will be provided for you in Week 1 of the laboratory course. You should
attach a label to them containing your name and locker number. Burets should be filled with
distilled water and stoppered between sessions. Ask your instructor about their storage.

Apparatus Number Check In Check Out

Buret 50 mL 1
stop cock 1

Volumetric Flask 1 L 1

Glass Bottle 1L 1
Chem 3410 ‐ Experiment 1    Caffeine in nonalcoholic beverages  7
       

Quantitative determination of caffeine in nonalcoholic beverages

Introduction

Caffeine, a nervous system stimulant, is naturally present in coffee and is incorporated

into many non-alcoholic beverages. The stimulant caffeine is incorporated into many non-
alcoholic beverages. The method described below for the quantitative determination of caffeine
is recommended by the Association of Official Analytical Chemists as an official method. In
this experiment, we will compare the amount of caffeine present in Coca-cola and Pepsi. The
method described below uses the instrumental technique of UV/Visible spectrophotometry.
(Suggested reading: Chapters 18 – 20 in “Quantitative Chemical Analysis” by Harris and
Chapters 13 & 14 in “Principles of Instrumental Analysis” by Skoog, Holler and Neiman)

Apparatus

2 – Spectrophotometer Cuvet 200-1000 µL pipet 50 mL beaker

6 – 50 mL Volumetric Flasks 50-200 µL pipet 100 mL grad cylinder

4 – 100 mL Volumetric Flasks Filter Paper 125 mL vacuum flask

125 mL separator funnel 2 – 250 mL beaker magnetic stir bar

Chemicals

Potassium Permanganate Sodium Hydroxide

Recrystallized Caffeine Chloroform
Sodium Sulphite Phosphoric acid
Potassium Thiocyanate

A. Reagents

(a) Reducing solution: Prepare 100 mL of aqueous solution containing 5 wt % Na2SO3

and 5 wt % KSCN.
(b) Dilute phosphoric acid solution - Prepare 100 mL of 15 vol % H3PO4.
(c) Sodium hydroxide solution - Dissolve 25 g NaOH in 75 mL of water.
(d) Caffeine stock solution - Prepare 50 mL of solution containing 1mg caffeine /mL of
CHCl3
Chem 3410 ‐ Experiment 1    Caffeine in nonalcoholic beverages  8
       

B. Preparation of Standard Curve

Prepare diluted standard solutions containing 0.05, 0.125, 0.25, 0.50, 0.75, and 1.00 mg
caffeine/50 mL CHCl3. Determine wavelength of maximum absorbance for caffeine by
measuring its absorption spectrum from 190 nm to 400 nm using a 0.50 mg/50 mL solution and
determine the absorbance of all solutions at this wavelength (see Note 1). Prepare a calibration
curve.

C. Determination of Caffeine in Samples

Degas the beverage samples by using a vacuum aspirator (Take care not to get the tap
water into your sample). Pipette 10 mL degassed sample into a 125 mL separator funnel; add 5
mL of 1.5% w/v KMnO4 solution and mix. After exactly 5 minutes, add 10 mL of reducing
solution and mix. Add 1 mL of the dilute H3PO4 solution, mix, add 1 mL of NaOH solution,
mix, extract with 50 mL of CHCl3 for 1 minute. After separation, drain the lower layer through
7 cm filter paper into a 100 mL ground stoppered volumetric flask. Add 2-3 mL CHCl3 to the
separator and drain through paper to rinse the separator stem. Wash the paper with 2-3 mL of
CHCl3. Re-extract the solution with 40 mL CHCl3, and wash stem, and paper as before. Dilute
to volume with CHCl3. Determine the absorbance of this solution at the wavelength of
maximum absorbance against CHCl3 with matched cells. Determine the amount of caffeine in
your samples from the standard curve. Report the caffeine content as mg caffeine/100 mL of the
beverage.

Notes
(1) You will be using only one UV cell for the absorbance determinations. Therefore it will be
necessary to obtain a baseline correction run of CHCl3 against a reference of air.
Chem 3410 ‐ Experiment 2    Caffeine and Acetylsalicylic acid by UV‐Vis  9

Simultaneous Determination of Caffeine and Acetylsalicylic Acid

in an Analgesic by Ultraviolet Spectrophotometry

Introduction

Whenever possible it is advantageous to analyze a mixture for its components without

performing a prior separation of the components. In certain circumstances, spectrophotometry
can be used for the simultaneous analysis of various analytes in a mixture. In an ideal case, each
component of a mixture exclusively absorbs radiation at a particular wavelength and does not
absorb radiation at a wavelength of any other component. In that case, Beer's law can be used at
the wavelength which is characteristic of each component to determine its concentration.
Since ultraviolet-visible absorption bands of polyatomic species are usually broad, it is
rarely possible to find a wavelength for each component at which no other component absorbs
radiation. If a wavelength is chosen for an analysis at which more than one analyte absorbs
radiation, then ideally the absorbance of the mixture at that wavelength is the sum of the
absorbance of its components. For a sample which contains two absorbing components
(analytes), the absorbance at a wavelength λ1 is given by the equation

Aλ1, sample = Aλ1,1 + Aλ1,2

where Aλ1,1 is the absorbance at wavelength 1 of component 1 and Aλ1,2 is the absorbance of
component 2. Substitution from Beer's law into the equation yields

Aλ1,sample = ελ1,1bC1 + ελ1,2bC2

The values of ελ1,1, ελ1,2 and b can be independently measured, and Aλ1,sample is obtained from
the absorption measurement. Substitution of these values into the equation results in a single
equation with two unknown terms (C1 and C2). Consequently for a two component mixture,
absorbance measurements must also be made at a second wavelength λ2 for which the following
equation can be written.

Aλ2,sample = ελ2,1bC1 + ελ2,2bC2

The two equations can be simultaneously solved for the concentrations of the two analytes.
Chem 3410 ‐ Experiment 2    Caffeine and Acetylsalicylic acid by UV‐Vis  10

Although it is theoretically possible to use any two wavelengths for the absorbance
measurements, in practice, the accuracy limitations of the measuring instrument make it
desirable to choose two wavelengths at which ε1, and ε2 significantly differ; i.e., two
wavelengths are chosen such that at one wavelength component 1 absorbs strongly and
component 2 weakly, and at the other wavelength component 2 absorbs strongly and component
1 weakly. In addition, the two wavelengths must be chosen such that Beer's law is obeyed and
their absorbances are additive.
In this experiment, an analgesic capsule or tablet is simultaneously analyzed for
acetylsalicylic acid (an analgesic) and caffeine (a stimulant).
(Suggested reading: Chapters 18 – 20 in “Quantitative Chemical Analysis” by Harris and
Chapters 13 & 14 in “Principles of Instrumental Analysis” by Skoog, Holler and Neiman)

Apparatus
11, 50mL Volumetric Flask Mortar and Pestle
3, 250mL Volumetric Flask
10mL Graduated Cylinder
200-1000uL Eppendorf pipet

Chemicals
Acetylsalicylic Acid Caffeine (sublimed)
Anacin or other analgesic tablet Methanol

Procedure

(1) Weigh 0.121 g of caffeine to the nearest 0.1 mg. Quantitatively transfer to a 250 mL
volumetric flask using the solvent methanol. Fill the flask to the mark with methanol (conc.
caffeine is approx 2.50 x 10-3 M)

(2) Prepare 5 different concentrations (typically between 25 µM to 125 µM) of standard

solutions using 50 mL volumetric flasks for constructing the calibration curve. Dilute each flask
to the mark with methanol.

(3) Weigh 0.113 g of acetylsalicylic acid to the nearest 0.1 mg. Quantitatively transfer the
acid to a labeled 250 mL volumetric flask using methanol as the solvent. Make solution up to
the mark with methanol (conc. is approx 2.50 x 10-3 M).
Chem 3410 ‐ Experiment 2    Caffeine and Acetylsalicylic acid by UV‐Vis  11

(4) Prepare 5 different concentrations (typically between 25 µM to 125 µM) of standard

solutions using 50 mL volumetric flasks for constructing the calibration curve. Dilute each flask
to the mark with methanol.

(5) Use Eppendorf pipet to deliver 0.5 mL of the caffeine stock solution and 1 mL of the acid
stock solution to a 50 mL volumetric flask. Fill the flask to the mark with methanol and mix
well.

(6) Weigh to the nearest 0.1 mg the sample analgesic tablet provided. Transfer the weighed
solid to a labeled 250-mL volumetric flask. Add about 200 mL of methanol to dissolve the
sample and then fill the flask to the mark with methanol.

(7) Use an Eppendorf pipet to transfer accurately 0.5 mL of the sample solution into each of
three 50 mL volumetric flasks. Dilute each flask to the mark with methanol.

(8) Obtain a baseline spectrum of the methanol solvent referenced against air from a λmin of
220 nm to λmax of 320 nm.

(9) Obtain a baseline corrected spectra, between the same wavelength limits, of the solution
prepared in step (5), and of the three sample solutions which were prepared in step (7). Label
each spectrum.

(10) Obtain spectra for the five caffeine solutions which were prepared in step (2) over the
same limits.

(11) Obtain spectra for the five acetylsalicylic acid solutions which were prepared in step (4)
over the same limits.

Calculations

(1) Calculate the concentrations of the caffeine and acetylsalicylic acid which are in the
stock and standard solutions.

(2) From the caffeine spectra choose a wavelength (about 270 nm) on a maximum of the
absorbance spectra.
Chem 3410 ‐ Experiment 2    Caffeine and Acetylsalicylic acid by UV‐Vis  12

(3) Similarly choose a second wavelength (about 225 nm) from the acetylsalicylic acid
spectra.

(4) Tabulate the absorbance of each of the solutions at the two chosen wavelengths.

(5) Prepare two working curves by plotting the absorbance of each of the standard caffeine
solutions as a function of concentration at each of the two wavelengths.

(6) Similarly prepare two working curves for the standard acid solutions.

(7) If the curves are linear and go through the origin, Beer's law is obeyed for each
component at each wavelength. If the curves are not linear and/or do not go through the origin,
choose a different pair of wavelengths for the analysis. If Beer's law is obeyed use the slope of
the working curves to calculate the molar absorptivity of each component at each wavelength.

(8) Use the molar absorptivities to calculate the expected, correct absorbance at each
wavelength for the solution which contains a known concentration of both acid and caffeine
(prepared in step (5)). If the calculated absorbance agrees with the observed absorbance with in
5-10%, the absorbance of the two components are additive at the two chosen wavelengths. If
there is disagreement, a new pair of wavelengths must be chosen.

(9) Write an equation of the following form at each of the two wavelengths

A λ,sample = ελ,caffeine bCcaffeine + ελ,ASA bCASA

Make the proper substitutions into the two equations and solve for Ccaffeine and CASA in each of
the three sample solutions.

(10) Determine the mean and standard deviation of the percent ASA and caffeine in each
analgesic tablet.
Chem 3410 ‐ Experiment 3 Determination of sodium by flame photometry 14

Flame Photometry:
Quantitative determination of sodium in fruit juices

Introduction

Flame photometry (aka flame atomic emission spectrometry) is an atomic emission

spectroscopic method. It is routinely used for the detection of alkali and alkaline earth metals
such as Na, K, Li, Ca and Ba. The experimental procedure described below for the determination
of sodium has been adapted from an official procedure reported by the Association of Official
Analytical Chemists and may be used to obtain the sodium contents of many fruits and their
juices.
(Suggested reading: Chapter 21 in “Quantitative Chemical Analysis” by Harris and
Chapter 10, section 10C-1, in “Principles of Instrumental Analysis” by Skoog, Holler and
Neiman)

Apparatus

7 - 100mL Volumetric Flasks Large beaker for waste Glass Funnel

8 - stoppers 50 mL beaker Filter Paper
250 mL Erlenmeyer flask 10 mL beaker
1 L Volumetric Flasks 5 mL and 10 mL pipet

Chemicals

Dried Sodium Chloride

Preparation of Standard Solutions

Dry reagent grade sodium chloride at 100 °C overnight. Allow sample to come to room
temperature in a desiccator. Prepare 100 mL of stock solution containing about 1000 ppm of
sodium. Dilute this to prepare a ~100 ppm working standard. From this, prepare series of
standard solutions of concentration ~1, 2, 4, 6, 8, and 10 ppm in Na respectively. Ensure all
solutions are tightly stoppered. Calculate the exact concentration of the stock solution.
Chem 3410 ‐ Experiment 3 Determination of sodium by flame photometry 15

Preparation of Juice samples

Mix containers of juice thoroughly by shaking. This will ensure uniform sampling.
Filter the juice through absorbent cotton or rapid filter paper and collect the filtrate (take care to
remove all solids).

Flame Photometry Measurements

Open the valve on the propane regulator. Turning on the power switch to the instrument
will automatically ignite the burner. Open the viewing port to ensure that the flame is burning
evenly and does not flicker.
Aspirate your reference solution from a 10 mL beaker. Zero the digital scale using the
"zero" control for sodium (n.b. the instrument scale should be placed in reduced range mode by
depressing the Na "Range" button). Replace the reference solution with the most concentrated
standard solution and adjust the scale to approximately 50 using the "Calib" control.
Aspirate the other standard solutions from 10 mL beakers and record the resultant
relative intensities, which if possible should read 300 or as high as is possible. Allow readings to
stabilize before recording. Remember to re-zero the instrument with the reference solution
between each reading and repeat each measurement twice. Construct a working curve of relative
intensity versus concentration from your results.
Dilute juice samples if necessary to produce relative intensity readings which are
bracketed by the standards.

Calculations

Using the working curve constructed from your standard solutions, the relative intensities
of your juice samples and any relevant dilution factors to calculate the mean and standard
deviation of the concentration of sodium in each juice sample.
Chem 3410 ‐ Experiment 4 Determination of fatty acids by GC 16

Gas Chromatography: Determination

of Fatty Acid composition in Fats and Oils

Introduction

The following procedure for the determination of fatty acid composition is an official
method of the American Oil Chemists' Society. The composition of fatty acids in different fat/oil
samples (personal samples are recommended), will be determined.
Gas chromatography is a technique for carrying out the separation and measurement of
mixtures of materials that can be volatilized. These materials may be gases, liquids, or solids
that have appreciable vapor pressures at temperatures up to a few hundred degrees. In capillary
gas chromatography a stationary phase, generally a stable non-volatile liquid, is spread in a thin
film on a wall of column. A carrier gas acts as an inert moving phase to transport the sample
components from an injection port at the head of the column through the column to a detector.
Sample injection is an arrangement by which a solid, liquid, or gaseous sample is transmitted as
a short pulse into the carrier-gas stream before it enters the column. The sample should be
vaporized and carried to the leading end of the column in negligible time. The detector,
commonly flame ionization, monitors the composition of the carrier-gas stream as it leaves the
column. Simple, sensitive, and stable, the flame ionization detector has contributed in a major
way to the explosive growth of gas chromatography. A significant advantage is that it provides a
recorder response proportional to concentration of substance in the effluent from a column.
(Suggested Reading: Chapters 23 & 24 in “Quantitative Chemical Analysis” by Harris and
Chapter 27 in “Principles of Instrumental Analysis” by Skoog, Holler and Neiman)

Preparation of reagents and apparatus

(a) Prepare 100 mL of an esterification reagent - 0.50 M potassium methoxide solution
(check as this maybe prepared for you by your instructor).

(b) Prepare 100 mL of saturated salt solution in water.

(c) Prepare 35 mg sample of lipids (fat or oil) in threaded test tube. Ensure that the top edge
of the tube is not broken. Add 1 mL of iso-octane and dissolve the sample in it. Add 10 mL of
esterification solution (0.50 M potassium methoxide), close the tube, vortex for 2 minutes and
place in a hot heating block for 10 minutes and remove the tubes and allow the contents to cool
undisturbed for 30 minutes.
Chem 3410 ‐ Experiment 4 Determination of fatty acids by GC 17

(d) Add 6 mL of iso-octane and top with ~5 mL of the salt solution. Close tube and mix by
inverting only, vigorous mixing causes emulsion formation and the 2 phases will not separate.
Place this tube in a test tube stand and wait until phases separate and is clear. A centrifuge can be
applied to speed up separation and clarification. Note: Always place an equal counterweight in
the centrifuge.

(e) Transfer the upper clear phase into an auto sampler vial up to neck of the vial, using a
Pasteur pipet.

(f) Set carrier gas H2 at the total flow of 75 mL/min; injector and detector temperatures set
at 225 °C, column temperature at programming: 125 °C for 2 minutes, program to 215 °C at 5
°C/min, held for 2 minutes. Program the running sequence on ChemStation, program controlling
GC to do injection and data acquisition.

(g) Place vials into autosampler carousel, set injection to 1µL. With the samples, add at least
two standard mixtures to verify elution order of components. Standard samples have to be placed
at the beginning and close to end of set of samples. When all parameters are set start your run
with the ChemStation and not on the GC.

Figure 4-1. Schematic drawing of a gas chromatograph

Data presentation

Retrieve your sample data from the ChemStation; analyze them to check if integration was done
Chem 3410 ‐ Experiment 4 Determination of fatty acids by GC 18

properly. Check how the baseline was drawn and correct it if not as expected by changing
integration parameters.

Identify peaks on chromatogram base on retention data from analyzed standard samples.

Print report and chromatogram for each sample analyzed. Prepare a report which should have
printed identification of peaks and data on the composition of fatty acids in your samples.
Calculate the weight % of trans fatty acids and weight % of saturated fatty acid in your sample.
Chem 3410 ‐ Experiment 5 Determination of ASA by fluorometry 19

The Fluorometric Determination of

Acetylsalicylic Acid in an Aspirin Tablet

Introduction

Acetylsalicylic acid is an analgesic (pain reliever) which is found in aspirin tablets. In

addition to acetylsalicylic acid, aspirin tablets contain other ingredients such as binders and
buffering agents. In this experiment a portion of an aspirin tablet is dissolved in water and
converted to salicylate ion by the addition of sodium hydroxide.

COOH COO-
+ 2 OH - + CH 3COO- + H 2O
O-C-CH 3 OH
O
A cetyls a licylic a cid Sa licyla te ion
(M W 180.16)

The salicylate ion strongly fluoresces at about 400 nm after it has been excited at about
310 nm. A series of standard solutions of salicylate ion are prepared; the fluorescence of the
standards and the samples are measured; and the working curve method is used to determine the
concentrations of salicylate ion in the sample solutions. The concentration is used to calculate
the percentage of acetylsalicylic acid in the aspirin tablet.
(Suggested reading: Chapters 18 – 20 in “Quantitative Chemical Analysis” by Harris and
Chapter 15 in “Principles of Instrumental Analysis” by Skoog, Holler and Neiman)

Apparatus

2 L beaker 100 mL beaker mortar and pestle

Filter paper (medium porosity) Glass funnel buret
2 - 1 L volumetric flasks 9 - 100 mL volumetric flasks wash bottle
100 mL graduated cylinder hot plate or Bunsen burner

Chemicals

Aspirin tablet Salicylic acid (reagent grade)

Sodium hydroxide solution (4 M)
Chem 3410 ‐ Experiment 5 Determination of ASA by fluorometry 20

Procedure

1. Obtain an aspirin tablet from the instructor. Record sample number on the tablet or the
brand and/or manufacturer’s name if it is available.

2. Place the tablet in a clean, dry mortar. Use a clean pestle to grind the tablet into a powder.
Weigh 0.1 g of the powder to the nearest 0.1 mg into a 100 mL beaker.

3. Place about 1 L of distilled or deionized water in a 2 L beaker. Heat the water to just
below boiling.

4. Fold a piece of filter paper and place it in a glass funnel. Place the funnel in the top of a 1
liter volumetric flask. Use a spray of distilled or deionized water from a wash bottle to
rinse the powder in the 100 ml beaker into the funnel. Allow the solution which flows
through the funnel to drain into the volumetric flask.

5. Slowly pour the hot water which is in the 2-L beaker over the solid and through the funnel.
The acetylsalicylic acid, which is in powder form, slowly dissolves in water and drains into
the funnel. Some tablets contain binders which will not dissolve. The insoluble binders
are separated from the acetylsalicylic acid during this step. After the solid has completely
dissolved, or after no further solid appears to dissolve, allow the solution in the flask to
cool to room temperature. Dilute the solution to the mark with room-temperature water.
Pour at least 500 mL of the hot water through the funnel before concluding that the solid
will not dissolve further.

6. Weigh 0.077 g of salicylic acid to the nearest 0.1 mg. Place the weighed acid in a labeled,
1-L volumetric flask. Add about 500 ml of distilled and deionized water and swirl the
contents of the flask until the solid has dissolved. Dilute the solution to the mark with
water. The result is a stock solution of salicylic acid.

7. Respectively label nine, 100 mL volumetric flasks with B, U1, U2, U3, 1,2,3,4, and 5. Use
a pipet to deliver 2 mL of 4 M sodium hydroxide solution to each of the nine flasks. Use a
buret to add 2, 4, 6, 8, and 10 mL, respectively, of the salicylic acid stock solution to the
100 ml volumetric flasks which are labeled 1, 2, 3, 4, and 5. Use a pipet to place 10 mL of
the 1-L solution of the tablet into each of the flasks which are labeled U1, U2, and U3. Fill
each flask to the mark with distilled or deionized water.
Chem 3410 ‐ Experiment 5 Determination of ASA by fluorometry 21

8. Measure the excitation and emission spectrum of salicylic acid using standard ‘5’. Get the
exact excitation and emission wavelengths and adjust the monochromator which controls
the excitation wavelength and emission wavelength appropriately.
9. Fill a cuvet with the well-stirred solution from one of the nine 100 mL volumetric flasks.
Place the cuvet in the fluorometer. Measure and record the relative fluorescence of the
solution. The instructor will provide the operating instructions for the fluorometer.
10. Similarly measure and record the relative fluorescence of each of the remaining eight
solutions.

Calculations

1. Use the mass of the salicylic acid (MW 138.13) to calculate the concentration of salicylic
acid in the stock solution.

2. Use the volumes of the stock solution which were added to the 100 mL volumetric flasks to
calculate the concentrations of salicylate ion which are in flasks 1, 2, 3, 4, and 5.

3. Prepare a working curve

4. From the working curve, determine the concentration of salicylate ion which is in flasks
U1, U2, and U3.

5. Use the dilution factor to calculate three values for the concentration of acetylsalicylic acid
in the 1-L solution.

6. Use the three acetylsalicylic acid concentrations and the mass of the tablet which was used
to prepare the solution to calculate three values of the percentage of acetylsalicylic acid in
the tablet.

7. Determine the mean and standard deviation of the results.

Chem 3410 ‐ Experiment 6    Determination of iron by AA  22

Determination of Iron in Cereal by Atomic Absorption

Spectrophotometry

Introduction

The nutritional value of trace amount of certain metals, such as iron and manganese, is
well known. In this experiment, the amount of iron present in a dry, breakfast cereal is
determined. The powdered cereal is digested with a nitric acid-perchloric acid solution. Atomic
absorption spectrophotometry is used to determine the concentration of iron in the resulting
solution. The effect of potentially interfering substances in the cereal is minimized by use of the
standard-addition technique.
(Suggested reading: Chapters 5 (5-3) & 21 in “Quantitative Chemical Analysis” by Harris and
Chapter 9 in “Principles of Instrumental Analysis” by Skoog, Holler and Neiman)

Apparatus

50 mL beaker Iron Hollow Cathode Lamp

10 mL graduated cylinder Mortar and Pestle
1 mL graduated pipet 4 mL pipet
Hot plate 5 - 13 × 100 mm test tube
25 mL volumetric flask 250 mL volumetric flask

Chemicals

Acetylene Iron (II) Ammonium sulfate hexahydrate (reagent grade)

Compressed Air Nitric acid-perchloric acid solution (1:1 volume)
Breakfast Cereal

Procedure

1. Weigh appropriate amount of iron(II) ammonium sulfate hexahydrate to the nearest 0.1 mg
to prepare 100 mL of ~100 mg/L aqueous iron standard solution.

2. Grind about 2 g of the cereal using a mortar and pestle into a powder. Weigh to the nearest
0.1 mg about 0.5 g of the powdered cereal and transfer it into a 50 mL beaker.
Chem 3410 ‐ Experiment 6    Determination of iron by AA  23

Caution: The remainder of the experiment could be hazardous. The use of safety glasses
is required at all times.

3. Place the beaker on a hot plate in the hood. Cautiously add 10 mL of the nitric acid-
perchloric acid solution. Gently warm the beaker until the sample is colorless. The acid
helps to dissolve iron present in the sample during this step.

4. After digestion is complete, transfer the solution to a 25 mL volumetric flask. Rinse the
beaker twice with 5 mL portions of distilled or deionized water. Pour the rinsing into the
volumetric flask. Dilute the solution to the mark with distilled or deionized water.

6. Label five 13 × 100 mm test tubes as S, 1, 2, 3, and 4. Use a pipet to add 4 mL of the 25
mL of the sample solution to each of the five test tubes. Use the 1 mL graduated pipet to
transfer 0.20 mL of the 100 mg/L iron solution to tube 1, 0.30 mL to tube 2, 0.40 mL to
tube 3, and 0.60 mL to tube 4.

7. Use a graduated 1 mL pipet to add 1.00 mL of distilled or deionized water to tube S, 0.80
mL to tube 1, 0.70 mL to tube 2, 0.60 mL to tube 3, and 0.40 mL to tube 4. Each tube
should contain a total of 5.00 mL of solution.

8. Insert the iron hollow cathode lamp into the atomic absorption spectrophotometer. Refer to
the supplied instructions and optimize the signal and prepare for the analysis.

9. Successively aspirate the solutions in the five test tubes into the flame. Record the
instrumental reading from each solution.

10. Close the acetylene quick-shut-off valve and allow the flame to extinguish. After the flame
has extinguished, close the valve on the acetylene tank, open the quick-shut-off valve and
allow the acetylene to drain from the acetylene line. Shut off the air supply and turn off the
instrument. Turn off the hood.

Calculations

1. Calculate the exact concentration (mg/L) of iron (AW 55.847) in the standard solution
using the mass of the iron(II) ammonium sulfate hexahydrate (MW 392.14) and the volume
of the solution (100.0 mL).
Chem 3410 ‐ Experiment 6    Determination of iron by AA  24

2. Use the concentration of the standard solution, the volume of the solution which was added
to each of test tubes 1 through 4, and the final solution volumes (5.00 mL) to calculate the
concentration (mg/L) of iron which was in each of the tubes.

3. Prepare a plot of absorbance (or instrumental reading) (y axis) as a function of the added
iron concentration for the solutions that are in tubes S, 1, 2, 3, and 4. Draw a straight line
through the data points and extrapolate it to intersection with the concentration axis. The
distance on the concentration axis between the origin and the intersection with the
extrapolated line corresponds to the concentration of iron which is in tube S. (Refer to
section 5-3 in your textbook on constructing a calibration curve for standard addition
experiments and obtaining the concentration of the analyte in the unknown sample).

4. Use the dilution factor and the iron concentration that is in tube S to calculate the
concentration in the undiluted sample solution. Use that concentration and the total
sample-solution volume (25.0 mL) to calculate the mass (mg) of iron in the sample.
Express your result as mg of iron/g of cereal.
Chem 3410 ‐ Experiment 7 Laboratory Development  25

Analytical Method Development

This section of the 3410 laboratory course will require at least 3 weeks to complete.
Together with a partner you are required to design and implement a quantitative analytical
chemistry experiment which utilizes any of the instrumentation available in the instrumental lab.
You may select any sample that interests you as the source for your analysis. Please note limits
will be imposed on the number of similar samples.

Proposed Method of Completion

(1) Week 1
Library work + initial experimental investigations.

(2) Week 2
Continued experimental work + fine tuning of experimental approach.

(3) Week 3
Continued experimental work + start preparation of a 10 minute seminar on your research
topic to be presented to the 3410 group. A final project report which details your investigations
is due at the conclusion of your seminar on the due date.