Determination of Metals in Cereal with Flame Atomic

Absorption Spectroscopy
Ali Bazzi, Bette Kreuz, Jeffrey Fischer
Department of Natural Sciences, University of Michigan, Dearborn__________________________

Introduction and Background

Atomic absorption spectroscopy is an analytical technique used for the determination of elements and at
times compounds, based on the absorption of radiation by unexcited gaseous free atoms. In order to be
able to make measurements with atomic absorption spectroscopy, it is necessary to convert the analyte
being determined to a population of ground state gaseous atoms and pass the resonance radiation of the
element of interest through the population of atoms. To accomplish this, either a flame or a graphite
furnace is used. In flame atomic absorption technique, the sample solution is sprayed as a fine mist and
atomized with a nebulizer/burner assembly. In AAS the source of radiation is normally a hollow cathode
lamp which gives a sharp line spectrum characteristic of the desired element. The isolated resonance line,
after traversing the flame, reaches the detector where the signal is amplified and converted into an
absorbance reading.

Atomic absorption spectroscopy (AAS) is similar in principle to molecular absorption spectroscopy, in

that Beer’s Law is followed. The absorbance is directly proportional to the path length through the flame
and to the concentration of the atomic vapor in the flame. Obviously, both of these are difficult to
determine; however, the length of the path in the flame is held constant and the concentration of the free
gaseous atoms in the flame is directly proportional to the concentration of the analyte in the solution
aspirated into the flame. Thus, the absorbance is proportional to the concentration of the analyte in the
solution.

In this experiment, flame atomic absorption spectroscopy (FAAS) is used to determine calcium, zinc, and
magnesium in cereal to illustrate the use of this important technique in the analysis of food products.

First, it is necessary to destroy the organic material of cereal. This can be accomplished by wet-ashing,
digestion with concentrated acid (i.e. HNO3 or HNO3/H2SO4).

The organic material can also be destroyed by dry-ashing, heating at high temperature in a crucible
without any acid. The advantages of dry-ashing are that it is safer, simpler, and allows for the dissolution
of the cereal residue in a small volume of relatively dilute hydrochloric acid, which helps to minimize the
generation of hazardous waste.

In this experiment, the cereal sample is crushed and dry-ashed in a marked glazed silica crucible using a
high temperature muffle furnace. This is followed by dissolving the sample residue in 5 mL of 6 M
hydrochloric acid solution and using the two increment standard addition method to determine the amount
of calcium in cereal. The prepared solutions are aspirated into a air/acetylene flame and absorbance
measurements are made using the resonance line each of the analytes. The specific parameters are
described later in the handout.
Solutions and Reagents
- Full Standard: 1000 ug/mL standard solutions of calcium, zinc, and magnesium will be provided
by the TA.
- Working Standards: Using these prepared standards prepare working standards at 20 ug/mL for
each of the analytes. This can be done by pipetting 10 mL of the standard solution into a 500 mL
volumetric flask and then diluting.
- 6M HCl: Also prepared by your TA
- Cereal Sample: Prepared as described in the Sample Preparation section.

Procedure
Each group will be analyzing samples of cereal for calcium, zinc, and magnesium. You will make
4 solutions from the product of your dry ashing. These solutions will be used to prepare all the
standard spike solutions required for this experiment. They will also be analyzed for each of the
analytes, so don’t be wasteful with them.

Hazards
Flame-resistant leather or glass fiber gloves and jumbo (18'') crucible tongs must be used when inserting
and removing the silica crucibles from the muffle furnace. Care must be taken when working with the
hydrochloric acid. The instrument operates with an open flame, so take care around it to make sure you
don’t set something on fire

Sample Preparation
Crush about 5 g of the cereal to be analyzed into a fine powder using a mortar and pestle. Weigh to the
nearest 0.0001 g three 0.5 g samples of the crushed cereal into three marked glazed silica crucibles. Then
place the crucibles in a muffle furnace for two hours at 600 C. Also place an empty crucible in the
furnace to serve as a blank crucible. Remove the four crucibles from the furnace and allow to cool for 30
minutes. Add 5 mL of 6 M hydrochloric acid to each crucible to dissolve the residue (note: There is no
residue in the blank.) and transfer to four separate 100 mL volumetric flasks which are labeled A, B, C
and Blank. Make sure to rinse each crucible with 5 mL portions of distilled water three times and add the
rinse water to the respective flask. Dilute each flask to volume with distilled water and mix well by
inversion.

Pipet a 5 mL aliquot from volumetric flask A and transfer to a 50 mL volumetric flask. Dilute to volume
with distilled water and label as A1 and mix well by inversion. Pipet another 5 mL aliquot from
volumetric flask A and transfer to another 50 mL volumetric flask. Pipet a 20 mL aliquot of one of the 20
g mL–1 working standard solutions and dilute to volume with distilled water and mix well by inversion.
Label this flask (A1 + standard, with mention of which analyte it is for). Repeat this procedure for
volumetric flasks B and C. To prepare the working blank solution, pipet a 5 mL aliquot from the 100 mL
volumetric flask labeled Blank and transfer to 50 mL volumetric flask and dilute to volume with distilled
water and mix well by inversion. Label this 50 mL flask as Working Blank. Repeat this standard
solution preparation for all three analytes (Ca, Zn, Fe). When this is complete the samples are ready to be
analyzed.

Instrumentation
A Buck Scientific Model 200A atomic absorption spectrophotmeter instrument with variable (Ca, Zn, Mg)
hollow cathode lamps and air/acetylene flame are used. Absorbance measurements are made using the
calcium resonance line at l = 422.7 nm, zinc resonance line at l = 213.9, iron resonance line at l = 213.9,
0.5 nm slit width, and 5 mA lamp current. Follow the detailed instrument instructions and consult with
instructor before making the measurements.

Measurement Procedure
The instrument must be allowed to warm up for roughly 30 minutes before any samples are run. Also
before you start running you samples you will need to check the flow rate through the instrument by
timing how long it takes to draw 10 mL of water through the instrument. Your TA will tell you what the
flow rate should be for optimal performance of the instrument.

After that is done you can begin running your samples. To analyze a sample simply put the intake tube in
to the sample and allow it draw in sample until you get a consistent absorbance reading from the
instrument read out. Record this absorbance value in your notebook, it is recommended that you develop
a table ahead of time to contain the data you will be collected. Each run of a sample shouldn’t take much
more than 10 seconds, so this will go quickly when you get comfortable with it.

Remember to only analyze the samples that contain the element that can be detected by the lamp that is
currently installed on the instrument. If you try to analyze the wrong element you will not get very good
results. We will take turns on the instrument to minimize the amount of lamp switching that will be
occurring. This means that everyone will do the calcium measurement in rapid succession and then we
will move onto the other elements of interest.

Calculations
Once you have collected the absorbance data you will need to calculate the amount of each element that
was present in your sample. The method to do this is explained below:

The amount of the metals in the cereal is calculated using the following equation.
A sample C std Vs
C sample (g mL-1 ) = (1)
 
 A sample+std A sample  Vx
 

Csample and Cstd are the concentrations in mg mL–1 of the sample and standard solutions, respectively.
Asample and Asample+std are the corrected absorbance values of the unspiked and spiked solutions,
respectively. These values are obtained by subtracting the absorbance of the blank solution from their
respective absorbance measurements. Vs is the number of mL of the standard solution added and Vx is
the number of mL of the aliquot of the unknown solution. The value obtained from the above equation is
then converted to mg Ca g–1 cereal taking into account the sample preparation and the mass of the
individual cereal sample.

Results for Lab Report

Use the average value for mg metal/g cereal and the serving size provided by the instructor to calculate
the %RDA, given that for calcium, zinc, and iron the Recommended Dietary Allowance are 1200 mg, 8-
11mg, and 8-18 mg respectively for age group 19+ years.

Also compare your calculated concentration of metals to what is expected according to the Nutritional
facts on the side of the box.