Chem3410Experiment4

DeterminationoffattyacidsbyGC

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 sodium 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 sodium 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.

Chem3410Experiment4

DeterminationoffattyacidsbyGC

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 at the total flow of 75 mL/min; injector and detector temperatures set at
235 C, column temperature at programming: 125 C for 2 minutes, program to 220 C at 5
C/min, held for 5 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 1L. 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

Chem3410Experiment4

DeterminationoffattyacidsbyGC

18

Retrieve your sample data from the ChemStation; analyze them to check if integration was done
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.

Chem3410Experiment5

DeterminationofASAbyfluorometry

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.

COO-

COOH
+

2 OH -

O-C-CH 3
O
A cetyls a licylic a cid
(M W 180.16)

+ CH 3COO- + H 2O
OH
Sa licyla te ion

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
Filter paper (medium porosity)
2 - 1 L volumetric flasks
100 mL graduated cylinder

100 mL beaker
Glass funnel
9 - 100 mL volumetric flasks
hot plate or Bunsen burner

Chemicals
Aspirin tablet
Sodium hydroxide solution (4 M)

Salicylic acid (reagent grade)

mortar and pestle

buret
wash bottle

Chem3410Experiment5

DeterminationofASAbyfluorometry

20

Procedure
1.

Obtain an aspirin tablet from the instructor. Record sample number on the tablet or the
brand and/or manufacturers 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.

Chem3410Experiment5

DeterminationofASAbyfluorometry

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.

Chem3410Experiment6

DeterminationofironbyAA

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
10 mL graduated cylinder
1 mL graduated pipet
Hot plate
25 mL volumetric flask

Iron Hollow Cathode Lamp

Mortar and Pestle
4 mL pipet
5 - 13 100 mm test tube
250 mL volumetric flask

Chemicals
Acetylene
Compressed Air
Breakfast Cereal

Iron (II) Ammonium sulfate hexahydrate (reagent grade)

Nitric acid-perchloric acid solution (1:1 volume)

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.

Chem3410Experiment6

DeterminationofironbyAA

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 acidperchloric 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).

Chem3410Experiment6

DeterminationofironbyAA

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.