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Determination of Iron in Egg Yolk


									                               Determination of Iron in Egg Yolk

Prelab Assignment – Please turn your answers individually via GaView.
Skoog et. al. calibration curves: p. 192-195 Lehninger, endopeptidases: p.95-96.

Individual assignments are due upon entering the lab. No late assignments will be accepted.

   1. Why do you add -chymotrypsin to your egg yolk? What types of bonds does this
      enzyme cleave?

   2. Calculate the concentration of Fe(II) in M in each of your 5 standards.

   3. How many blanks will you need to run what will it/they consist of?

   4. Why does the iron in the egg yolk need to be reduced from ferric iron to ferrous iron?
                              Determination of Iron in Egg Yolk

Learning Outcomes
    Acquire laboratory skill in chemical extraction.
    Practical use of visible spectroscopy for quantification.
    Acquire laboratory skills in standards preparation with micropipettors.
    Acquire laboratory skills in data analysis with calibration curves.

The iron content of an egg is typically determined by ashing the egg, dissolving the ash in acid,
and carrying out measurements using Atomic Absorption spectrometry or inductively coupled
argon plasma emission spectrometry. While this is an accurate method, it does not illustrate the
biochemical bonding of Fe to the proteins in the egg. In this lab we will isolate the iron in an egg
yolk and form an iron complex by using biochemical and chemical reactions. The quantitative
determination will be carried out by generating a calibration curve by collecting visible
absorption spectra of standard samples, and measuring the absorbance of the sample solution at
the wavelength of maximum absorption.

There are two kinds of iron-binding proteins in egg: ovotransferrin in the albumin (white) and
phosphovitin in the yolk. While practically all of the Fe in egg is bound as Fe3+ to phosphovitin
in the yolk, the ovotransferrin is deficient in Fe, serving instead as a protective mechanism for a
developing embryo in the egg. It acts by depriving invading bacteria of their iron and thereby
killing them. Thus, we will concentrate our analytical studies on the egg yolk. We will take a
weighed portion of the egg yolk, break up the protein using a 10% solution of the enzyme -
chymotrypsin, reduce the Fe3+ to Fe2+ using hydroxylamine hydrochloride, and complex the
ferrous ion with 1,10-phenanthroline in an appropriate buffer. The presence of iron will be
indicated by a orange–red complex after the addition of phenanthroline. Then we will precipitate
the proteins using trichloroacetic acid followed by centrifugation. The supernatant, which
contains the iron complex, will be used in spectrometric analysis.

1,10-phenanthroline, hydroxylamine HCl, trichloroacetic acid, ferrous ammonium sulfate, glacial
acetic acid, sodium acetate trihydrate are all irritants. Inhalation and contact with skin and eyes
should be avoided.

Experimental Procedure
Part I: Preparation of Egg Yolk Sample
The exercise will be carried out in teams of two students each. The following procedure will be
done for an egg sample and a blank. Your egg blank for spectrometric analysis should go
through the same process as the sample except that it will not contain the 1,10-phenanthroline.

1. Separate the egg yolk from the white and place the yolk in a small, tared beaker. Weigh the
beaker and record the mass of the egg yolk on your report sheet and in your laboratory notebook.
2. Set the beaker on the magnetic stirrer, add the stir bar to the beaker and start the stirrer until
the yolk is homogenized.

3. Pipette 1.00 mL of the yolk into a weighed polypropylene centrifuge tube and weigh again –
the plastic tube can lie flat on the analytical balance as the viscous egg yolk will not flow
appreciably. Record the mass on your report sheet and in your lab notebook.

4. Add 1 mL of the 1mg/mL -chymotrypsin solution to the tube, gently swirl, and allow to it to
stand for 20 minutes. Next, vortex the tube vigorously. Add 1 mL 5% hydroxylamine
hydrochloride solution and vortex again. In order to efficiently use your time, prepare the
standards (Part II below) when you have periods of inactivity during sample preparation such as
waiting for enzyme activity, color development, and centrifuging.

5. Weigh between 0.03 and 0.05 g of solid 1,10-phenanthroline, add it to the sample and vortex
again. You should see a pink color develop. Reminder: Do not add the phenanthroline to the
tube containing the blank.

6. Add 2 mL of the pH-5 buffer and 2 mL absolute alcohol to the sample and vortex again until
the pink color does not deepen further.

7. Add 1.00 mL of 30% trichloroacetic acid. The protein should immediately precipitate.
Centrifuge in the refrigerated centrifuge at 12,000 rpm for 15 minutes at 4°C.

8. Decant the supernatant into a graduated cylinder.

9. If the protein pellet in the centrifuge tube still has a pink color, add 1mL H2O, 2 mL 5%
hydroxylamine hydrochloride, 2 mL pH-5 buffer, and 2 mL absolute alcohol to the centrifuge

10. Break up the pellet with a glass rod, vortex well and centrifuge again for 15 minutes at
12,000 rpm at 4°C.

11. Combine the supernatants. If there are any fat globules, carefully siphon the fat using a
Pasteur pipette and discard it. Alternatively, you can add 1 ml of petroleum ether and, after the
fat dissolves in the ether layer, siphon off the ether layer. Dispose of the ether fraction in the red
organic waste container.

12. Transfer the de-fatted supernatants to a 50-mL volumetric flask and fill up to the mark with
distilled water.

Part II: Preparation of Standards
1. Collect six 50-mL volumetric flasks and insure that they are clean by rinsing each with
distilled water. Using a micropipettor, pipette, in order, 0, 50, 100, 150, 200, and 250 μL of the
0.0100 M Fe(II) stock solution into the clean volumetric flasks.
2. Pipette 2.00 mL 5% hydroxylamine hydrochloride solution, 5.00 mL pH 5 buffer, and 3.00
mL of 0.1% 1,10-phenanthroline solution into each of the flasks and fill up to the mark with
distilled water.

Part III: Spectrophotometric Measurements
1. Using the Shimadzu UV-visible spectrophotometer, record and print-out the absorption
spectrum (from 400-800nm) of your most concentrated standard and one egg yolk sample.
There are two different blanks to be used here: For your egg samples, the egg blank should be
used. For the Fe(II) standards, the standard that you made with 0 Fe(II) in it should be used.
Remember the blank solution goes in the back of the spectrophotometer. Determine the
wavelength of maximum absorption from your standard.

2. Record the absorbencies at the wavelength of maximum absorption for the rest of your
standards and samples in your laboratory notebook and the lab report sheet.

3. Construct a calibration curve (Concentration of Fe(II) (M) vs. Absorbance @ max) of your
standards using the Excel software spreadsheet. You should have a R2 value of 0.95 or greater or
a loss of performance points will result.

4. Determine the concentration of Fe(II) in molarity in your egg sample solutions.

5. Finally, calculate the Fe content of the egg yolk, in mg.

Trichloroacetic acid and 1,10-phenanthroline are environmental toxins and can not go down the

Trichloroacetic acid should be disposed of in a halogenated solvent waster container.
1,10-phenanthroline should be disposed of as organic waste (red jug).

Adapted from: Kevin M. Maloney, Emmanuel M. Quiazon, Ramee Indralingam, Measurement
of Iron in Egg Yolk: An Instrumental Analysis Experiment Using Biochemical Principles,
Journal of Chemical Education 2008 85 (3), 399.
                           Determination of Fe in an Egg Yolk
                                   Lab Report Sheet
This lab report will be graded one per pair of students. It consists of the Lab Report Sheet,
Standard curves generated for the standards, and copies of your spectra of one standard and one
sample. This lab is due one week from when it is performed.
Weight of the egg yolk:                     ___________________g
Weight of 1.00 mL of egg yolk:              ___________________g

max determined from spectra                ___________________nm

                  Standards       Abs @ max
                  Fe(II) M

                  Egg Yolk        Abs @ max     Calc’d        Calc’d
                  Samples                        [Fe(II)] M    Fe in mg

Provide a sample calculation of [Fe(II)] in M and Fe in mg below:

Provide the average amount of Fe in your egg yolk sample with standard deviation below:
Prep Notes for Determination of Iron in Egg Yolk

Chemicals needed:
1. Hydroxylamine hydrochloride, NH2OH_HCl, [5470–11–1]
2. Ferrous ammonium sulfate hexahydrate, Fe(NH4)2(SO4)2*6H2O, [7783–85–9]
3. 1,10-phenanthroline [66–71–7]
4. Sodium acetate trihydrate, CH3COONa*3H2O, [6131–90–4]
5. Glacial acetic acid, CH3COOH, [64–19–7]
6. -chymotrypsin, [9004–07–3]
7. Trichloroacetic acid, CCl3COOH, [76–03–9]
8. Sodium dihydrogen phosphate monohydrate, NaH2PO4*H2O, [10049–21–5]
9. Disodium hydrogen phosphate dodecahydrate, Na2HPO4*12H2O, [10039–32–4]

Mix solutions in the HOOD. Several of the chemicals are irritants if ingested or come into
contact with skin or eyes.
Any waste should be collected, trichloroacetic (halogenated waste) and phenanthroline
(organic waste-red jug) must be disposed of appropriately as they are environmental

Stock Solutions
Enough for 12 pairs of students

1. 5.0% Hydroxylamine hydrochloride: Weigh out 12.5 g and dissolve in deionized water to
250 mL.

2. 0.0100M Fe(II) stock solution: Accurately weigh about 0.196 g Fe(NH4)2(SO4)2* 6H2O and
transfer to a 50.00-mL volumetric flask. Add 5.00 mL of the hydroxylamine hydrochloride
solution before making up to the mark with deionized water. Portion this out into 1mL aliquots
into Eppendorf tubes

3. 0.1% 1,10-phenanthroline: Weigh about 0.25 g 1,10-phenanthroline and dissolve in about 15
mL ethanol. Make up to a final volume of about 250 mL with deionized water.

4. 0.1M pH 5 buffer: Weigh 4.5g sodium acetate trihydrate and dissolve in a solution of 0.75
mL glacial acetic acid in deionized water to make a final volume of 500 mL.

5. 30% Trichloroacetic acid: Weigh 15g and dissolve in deionized water to make a final volume
of 50 mL.

6. 0.05M Phosphate buffer (pH~7.5). Need ~ 100 mLs.

7. 1mg/mL -Chymotrypsin: Weigh 40 mg and dissolve in 4 mL deionized water. Make up to 40
mL with 0.05M phosphate buffer.
Equipment and Chemicals that should be placed out in the lab

Square plastic cuvettes                 4 / pair of students
50mL Volumetric flasks                  6 / pair of students
Centrifuge tubes                        2 / pair of students
Eggs                                    1 / pair of students
Stir bars                               1 / pair of students
Absolute Ethanol                        5 ml/pair of students
1,10 phenanthroline

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