Final Project:
The Determination of
the Iron Content of
Hemoglobin
Group R3
Noel Ang
Lisa Romito
Karim Sadak
Ofer Sagiv
4/ 30/ 98
Abstract
The objective of this experiment is to determine the actual weight percent of iron
(Fe) in a bovine hemoglobin molecule. Through the use of Atomic Absorption
Spectrophotometry (AAS), the weight percent of Fe was calculated to be about 0.331 
0.021%. This value can be compared to the literature value of Fe weight percent of
0.333%, which is based on a 67,000 grams/mole molecular weight of hemoglobin. This
presents an approximate error of 0.6%. Furthermore, the actual number of Fe atoms
within a molecule of hemoglobin protein was obtained; this value was 3.97  0.27 Fe
atoms per hemoglobin molecule, a 0.75% error when compared to the actual presence of
4 Fe atoms per hemoglobin molecule. To determine the Fe content in hemoglobin, the
hemoglobin molecule was kept intact. To prove that the rest of the protein did not
interfere with the absorption readings measured by the AAS, the method of additions
was utilized in which iron was added to hemoglobin in increasing amounts. This method
showed that the rest of the hemoglobin protein surrounding the iron atom did not interfere
with the absorbance readings, which therefore validates the above results.
1
Table of Contents
I. Abstract
1
II. Background
Hemoglobin
Atomic Absorption Spectrophotometry
Biomedical Relevance
3
3
4
5
III. Apparatus and Materials
6
IV. Procedure
Create the Calibration Curve for Iron (Fe)
Determine the Effect of Hemoglobin on AAS
Calculate the Weight Percent of Iron (Fe) in Hemoglobin
Determine the Number of Iron (Fe) Atoms
per Hemoglobin Molecule
7
7
7
9
V. Results
Create the Calibration Curve for Iron (Fe)
Determine the Effect of Hemoglobin on AAS
Calculate the Weight Percent of Iron (Fe) in Hemoglobin
Determine the Number of Iron (Fe) Atoms
per Hemoglobin Molecule
10
10
11
13
VI. Discussion
Suggestions
15
16
VII. Error Analysis
Error in Hemoglobin Concentrations
Error in Iron (Fe) Concentrations
Error in the Weight Percent of Iron (Fe)
Error in the Number of Iron (Fe) Atoms
17
17
17
18
20
VIII. References
21
IX. Appendix
22
9
13
2
Background
Hemoglobin
The average human body contains about four to six liters of blood. In this blood,
there are 25 trillion tiny erythrocytes, or red blood cells. Each red blood cell contains
about 250 million molecules of hemoglobin. (5) This translates into the astonishing fact
that there are 6.25 x 1021 molecules of hemoglobin in the human body. Due to its
impressive quantities alone, it is clear that hemoglobin is a primary player in the overall
function of the human body. More significantly, hemoglobin’s main function is to bind
oxygen in organs of all vertebrates and some invertebrates. It also serves to pick up
carbon dioxide that has been secreted from organs.
Hemoglobin is a globular protein with four polypeptide chains, each of which
contains a non-polypeptide component called a heme. A heme is a nitrogen containing,
disc-shaped organic molecule that is centered around an iron (Fe) molecule. The Fe is in
a ferrous state (Fe2+).
Figure 1. A hemoglobin molecule consists of four hemoglobin hemes, one shown here. As
can be seen from the figure, iron (Fe) is the centralized atom of this protein component. (6)
Hemoglobin is probably most significant when associated to the function of
respiratory organs. When blood passes through the alveoli in the lungs, oxygen is
chemically bound to the protein. This bond is due to the sharing of electrons between
iron and oxygen. The product of this reaction is oxyhemoglobin.
Hb + O2  HbO2
(Eq. 1)
Equation 1. Chemically bound blood which contains hemoglobin (Hb) and oxygen results in
oxyhemglobin.
3
The oxygen is carried to the entire body via the circulatory system. When the
oxyhemoglobin passes through cells with excess CO2, which at times has been produced
via glycolysis, the O2 is encouraged to release into the subsequent organs. At this point
the oxyhemoglobin becomes deoxyhemoglobin.
HbO2  Hb + O2
(Eq. 2)
Equation 2. This equation states the return of hemoglobin from oxyhemoglobin, the reverse of Equation 1.
The iron remains reduced and consequently, the CO2 attaches itself to the hemoglobin for
transportation back to the lungs. The reverse happens in the lungs, where the oxygen
facilitates the release of CO2. (7)
Atomic Absorption Spectrophotometry
The principal instrument used in this experiment was the Perkin-Elmer Model AA
4000 Atomic Absorption Spectrophotometer. AAS is an analytical method used to
determine the amount of substance in a sample through radiation emission and
absorbance. In this process, an electron of the element to be tested enters an excited
state. This is brought about by the change in its energy, and it consequently emits a
wavelength characteristic of that specific electron. This wavelength of light is passed
through a flame containing the same element, causing the electron to gain energy through
absorption and subsequently return to its initial state. Since more light is absorbed by
substances in which more atoms are present (or in which the concentration is higher), one
can use the data obtained from a spectrophotometer to determine the concentration of
ions of a specific element in a substance. (1)
In effect, absorbance is related to concentration through the concept of
transmittance. Transmittance relates the energy of the light beam before and after
passing through the sample. This relationship is shown by the Beer-Lambert law:
I = I0 e(-k C b)
(Eq. 3)
Equation 3. This equation relates inlet (I) and outlet (I0) intensities through variables of the proportionality
constant, molar concentration of absorbed atom and light path length.
ln T = ln (I/I0) = -k C b
(Eq. 4)
Equation 4. Through Equation 3, the log of the transmittance T, can be calculated.
I and I0 represent the intensity of the light beam before and after it goes through the
sample of gas. T is the fraction transmitted or transmittance, k is a proportionality
constant which depends on the wavelength of the initial light beam, . The length of the
light path is b and C is the molar concentration of absorbing atoms. This shows that
absorbance is related to concentration through the concept of transmittance. Using base
ten instead of base e logarithms, the relationship becomes Equation 5:
A = - log T =  C b
(Eq. 5)
Equation 5. The Beer-Lambert Law: in which A is the absorbance, T is the transmittance, is the molar absorption
coefficient, C is the concentration in moles, and b is the thickness of the light path. From this equation, one can see
that the relationship between absorption and concentration is directly proportional. (1)
4
This relationship between concentration and absorbance will play a central role in this
project as seen in the analysis of the resulting data.
Biomedical Relevance
The AAS is frequently used in the biomedical field in forensic, toxicological, and
clinical analysis. It is often necessary to determine the concentration of a certain element
in a living system to conclude whether the amount present is too toxic, or conversely, if it
is not concentrated enough. With this method, one can quickly and efficiently determine
concentrations of common or toxic substances in the body. With the aid of AAS, doctors
can more easily and accurately make a diagnosis, monitor a patient’s progress, and
determine a proper treatment.
It is also important to note that the iron content in hemoglobin has great
biomedical relevance. Those who have an iron deficiency in the erythrocytes of their
blood face a grave danger. This condition, which causes anemia, is the most common
nutritional deficiency in the world. Anemia only occurs towards the end of a process of
decreasing iron in hemoglobin, which in some cases may have been in progress for many
years. In terms of clinical medicine, it is of great importance to be able to determine how
much iron is present in blood. A person may have vague symptoms like lethargy and
dizziness; thus there must be a quick and efficient way to detect Fe amounts in blood.
Once the iron depletion is diagnosed, therapeutic intervention such as dietary
manipulation can be implemented in the life of an afflicted patient. This way the amount
of iron in their blood can be raised to a healthy and productive level.
5
Apparatus and Materials
The following materials will be found in the BE laboratory in order to conduct this
experiment:












Hemoglobin (H2500) Bovine (Lyophilized powder form)
Perkin-Elmer Model AA 4000 Atomic Absorption Spectrophotometer
Standard solution of Fe (at 1000 ppm) to make the calibration curve
Iron nitrate (Manufacturer: Fisher. Product number: I110-100 CAS number: 778261-8)
Several disposable 50mL test tubes with blue caps to hold dilutions
Various beakers to hold the deionized water (including 250mL, 300mL, and 1000mL)
Automatic pipettes (only 20 – 200 L, 100 – 1000 L, and 2 – 20 L pipettes in order
to strengthen accuracy)
Analytic balance (accuracy of 0.0005 gram)
Magnetic stirrer
Metal spatula to obtain the hemoglobin
Test tube racks
Funnel
6
Procedure
Create the Calibration Curve for Iron (Fe)
For each of the three weeks of experimentation, a calibration curve for the Fe
stock solution was constructed. In Week 1, the 1000 ppm stock of iron nitrate (FeNO3)
was diluted with deionized water to make 13 solutions. The table below illustrates the
calibration solutions made for the construction of the first calibration curve.
Table 1. This table shows the calculations to determine the correct amounts of iron stock and water needed to
create the calibration solutions. As can be seen from the above table, calibration solutions were made according
to the predicted absorbances so that a range of concentrations of low and high aborbances would be present.
When creating the diluted solutions, micro-pipettes were used to add the desired
volumes of stock and some of the water (the greater volumes of water were added with
the air suction pipettes). However, the analytic balance was employed to measure exact
amounts of the Fe stock and water. Table 1 was subsequently adjusted with these precise
weight measurements, and absorbance readings of the above solutions were then
recorded. The calibration curve was then plotted using Microsoft Excel. This same
procedure was performed for the two other calibration curves for Weeks 2 and 3. The
calibration curve of Week 2 was constructed over absorbances that ranged from 0.005 to
0.02, and the calibration curve for Week 3 was constructed over absorbances that ranged
from 0.01 to 0.2.
Determine the Effect of the Hemoglobin on the AAS
Next, the effect of hemoglobin protein on the AAS was determined by using the
method of additions (see Appendix). Three trials were conducted in which iron was
added to solutions containing constant hemoglobin concentrations. In order to achieve
this constant hemoglobin concentration, a stock hemoglobin solution was made for each
trial.
7
Table 2. The above shows how the hemoglobin stock was made for each trial. Also shown is how much
of the stock was added to each tube in each trial. As shown in the chart, less than the total available
hemoglobin stock solution was used. The amount of hemoglobin added to each solution was calculated
using the ratio of Volume added to each solution to Total volume of stock. ([C] of Hemo is based on the
final volume of 180 mL.)
Leaving one solution as the control, an increasing amount of Fe was added to
each of the remaining solutions. In trial 1 for example, Fe was added to the remaining
solutions according to Table 3.
Table 3. The table shows how much Fe was added to each of the four solutions containing a constant
amount of hemoglobin in trial 1. The desired absorbances do not include the absorbances of the
hemoglobin, so the resulted absorption values would be higher. After the hemoglobin was added from
the stock as described before, each solution was added to a volume of 180mL of water.
As can be seen, the solutions were made according to their predicted absorbances so that
a linear plot of equally spaced data points would result.
The iron from the Fe stock, hemoglobin from the hemoglobin stock, and water
were added in that precise order for each of the trials. All additions were made on the
analytic balance for greater accuracy. The amount of Fe stock was added first using the
micro-pipettes, and the concentrations were adjusted accordingly as done with the
calibration curve. The amount of hemoglobin stock added in trial 1 was 23 mL. In trials
2 and 3, 60 mL of hemoglobin stock were added to each tube as seen in Table 2. These
amounts were added using the air suction pipettes and micro-pipettes as to achieve almost
constant hemoglobin in all solutions (note: the balance changes it’s accuracy at 60 mL
from 0.0005 to 0.005 mL or mg). The water was added through a funnel and a beaker.
When the total volume of the solution approached about 175 mL, micro-pipettes were
employed to achieve a constant total volume of approximately 180 mL. This prevented
accidental over-pouring when using just the funnel and beaker. Finally, all solutions
were mixed using the magnetic stirrer and the absorbances were taken. This was
determined to be the best method in making the solutions for the method of additions.
8
Using Microsoft Excel, a linear relationship of only the Fe concentration vs. the
absorbances of the solutions was plotted. The absorbance of the control and the yintercept of that line were then compared.
Calculate the Weight Percent of Iron (Fe) in Hemoglobin
To determine the weight percent of iron, 16 solution of varying hemoglobin
concentrations were made over the 3 weeks. To make these solutions, each beaker was
placed on the analytic balance and the balance was tarred. Hemoglobin was added and
the measured amount was recorded. The amount of hemoglobin in each of the solutions
made was approximately 35 mg in 180 mL of water. A concentration of 200 ppm of
hemoglobin was not exceeded to ensure that the AAS was not damaged. Because the
balance readings fluctuated, the glass walls were put up to eliminate any interference
from the surrounding air. After the solutions were mixed thoroughly with the magnetic
stirrer, four absorbance readings were taken and then averaged. The corresponding
calibration curves for each week were used to determine the Fe concentration in the
solutions from the recorded absorbances. The ratio between the concentration of the iron
and the concentration of the hemoglobin (both in ppm’s) was calculated, giving the
appropriate weight percent of Fe in hemoglobin.
Determine the Number of Iron (Fe) Atoms per Hemoglobin Molecule
Using this experimental weight percent, various calculations (see Results) were
carried out to find the number of Fe atoms per hemoglobin molecule. All values were
average and analyzed for error.
9
Results
Create the Calibration Curve for Iron (Fe)
Figure 2 is one of the three calibration curves that were made for each of the three
weeks of experimentation.
Absorbtion
Calibration curve of pure Iron Solution
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
y = 0.0302x
R2 = 0.9963
0
50
y = -1E-08x4 + 4E-06x3 - 0.0005x2 +
0.0339x
R2 = 0.9999
100
150
Concentration(ppm)
Figure 2. The above figure shows the calibration curve of Fe for the first week
of experimentation. It can be seen that the curve become non-linear at
absorbency values of approximately 0.3A.
As seen in the figure above, the calibration curve becomes non-linear at
absorbency values of approximately 0.3A. For this reason only the first five data points
were used to find a linear relationship between the concentration of iron and the
absorbance of the Fe solutions. In order to convert absorption units (A), y, to
concentrations (ppm), x, for the hemoglobin solution of Week 1, Equation 6a below was
used. The relationship for Week 2 and Week 3 is represented by Equation 6b and
Equation 6c, respectively. These relationships were used to find the iron concentrations
in the hemoglobin solutions made in Week 2 and Week 3.
y = 0.0302 x
(Eq. 6a)
y = 0.0366 x
(Eq. 6b)
y = 0.0285 x
(Eq. 6c)
Equations 6a, 6b, and 6c. Absorption units (y) can be directly related to concentrations (x) through a single
coefficient. These three equations were used to determine the iron concentrations in the 16 hemoglobin
solutions that were made. These values were used to determine the weight percent of Fe in hemoglobin.
10
Determine the Effect of Hemoglobin on AAS
As seen in Table 2 (see Procedure), three trials were conducted in which iron was
added to solutions containing constant hemoglobin concentrations. In each case the yintercept of the best-fit line relating the Fe concentrations with the absorbances was
compared to the absorbance of the control in which no Fe was added.
In trial 2 for example, seven mixtures of Fe and constant hemoglobin were made
and their absorbances were taken. The control contained only hemoglobin and absorbed
at 0.022 absorbance units (A). It can be seen from Table 4 that the measured absorbance
values for these solutions deviated significantly from the predicted absorption starting at
mixtures of greater absorbance than 0.1A (the predicted absorbance values were
calculated using the calibration curve for Week 2).
Table 4. The above chart shows the predicted absorption of the Fe and hemoglobin mixture of trial 2
based on the calibration curve of Week 2. It can be seen that the predicted absorbance deviated from the
measured absorbance with mixtures of A > 0.1.
The graphs, which illustrate the method of additions, are shown for trial 2 below.
Figure 3 relates the added Fe concentrations of the solutions and their measured
absorbances. It can be seen that the y-intercept is 0.0271A. However, the control
absorbed at 0.022A. This value contains approximately 23% error. Excluding the
mixtures which absorb at values higher than 0.1A and subsequently fitting a line through
those points below 0.1A results in a y-intercept of 0.0217 which has approximately 1.3%
error from the control (see Figure 4).
11
y = 0.0213x + 0.0271
2
R = 0.997
Absorbency Vs. [C] of Added Pure Iron
0.25
Absorbency
0.2
0.15
0.1
0.05
0
0
2
4
6
8
10
12
Added Iron Concentration (PPM)
Figure 3. The above graph shows the relationship between the added Fe concentrations and the measured
absorbance for the mixtures made in trial 2. The control, whose absorbance was 0.022A, should have been
close to the y-intercept, 0.0271A, of this linear plot. Instead a 23% error is evident.
y = 0.0243x + 0.0217
R2 = 0.9937
Absorbency Vs. [C] of Added Pure Iron
(solution considerably above .1 abs excluded)
Absorbency
0.12
0.1
0.08
0.06
0.04
0.02
0
0
0.5
1
1.5
2
2.5
3
3.5
4
Added Iron Concentration (PPM)
Figure 4. Excluding all mixtures which absorb considerably above 0.1A results in a y-intercept of 0.0217A,
which is similar to the control whose absorbance was 0.022A. This y-intercept contains only 1.3% error
The same trend can be seen in trials 1 and 3. In trial 1, the control absorbed at
0.024A and the y-intercept of the corresponding curve to Figure 3 was 0.0258A. Because
only few mixtures which absorb above 0.1A where made, an error of 7% was seen when
all the data was included. Excluding the two mixtures which did have an absorbance
above 0.1A resulted in a y-intercept of 0.0245A, an error of only 2%. In trial 3, including
all solutions in the linear plot gave an intercept with an error of 31%; again, excluding all
solutions which absorb above 0.1A resulted in an intercept with an error of 3%. Table 5
summarizes the results for all three trials.
12
Table 5. A summary of the results from the method of additions.
Calculate the Weight Percent of Iron (Fe) in Hemoglobin
As said previously, 16 hemoglobin solutions were made and using their
absorbencies, their iron concentrations were calculated using equations 6a, 6b, and 6c
(see Table 6). The weight percent of the iron in hemoglobin could then be found through
the ratio of the iron concentration of each solution to their respective Hemoglobin
Concentration. This can be seen through the following ratio:
Equation 7. The weight percent of iron, Wt%Fe, was found through the ratio of iron concentration, [C]Fe,
to the hemoglobin concentration, [C]hemo, which were both in ppm’s.
Table 6. The above table illustrates the absorbencies of the 5 hemoglobin solutions made in week 2.
Their iron concentrations(calculated from Eq.6b) are shown. Also shown is the Hemoglobin
concentration and the corresponding weight percent.
After averaging the ratios for all 16 solutions the resulting value for the weight %
in Iron was 0.331%  0.021. This value can be compared to the literature value of Fe
weight percent of 0.333%, which is based on a 67,000 grams/mole molecular weight of
hemoglobin. This presents an approximate error of 0.6%.
Determine the Number of Iron (Fe) Atoms in a Hemoglobin Molecule
With the weight percentages known for all the 16 solutions, the actual number of
iron atoms in a hemoglobin molecule can then be calculated. This can be fulfilled
through a simple series of equations. The calculations are illustrated in Equations 8a to
8c:
13
Equation 8a. The grams of hemoglobin in each sample, which was carefully measured on the balance, can
be multiplied by the experimental weight percent of hemoglobin found previously to achieve the mass of
iron.
Equation 8b. The moles of iron can be found by dividing the mass of the iron by the molecular weight of
iron, which is 55.85 grams/mole.
Equation 8c. Avogadro’s Law is then employed that basically states that a mole is a mole, no matter the
material. Therefore, the moles of iron over the moles of measured hemoglobin will give a molar ratio,
specifically the number of iron atoms per hemoglobin molecule.
This group of calculations yielded a value of 3.97  0.27 Fe atoms per
hemoglobin molecule when taken averaging the values derived for all 16 solutions. The
percent error, when compared to the actual number of 4 atoms per molecule, was 0.75%.
(see Table 7 for derivation of this average).
Table 7. The table above shows how the number of Iron atoms per
molecule of hemoglobin was calculated for each one of the 16 solutions.
The average yielded 3.97 Iron atoms per hemoglobin molecule
14
Discussion
As seen in Figure 2, the curve at lower concentrations exhibits a linear behavior,
but as the concentration increases, the slope of this calibration curve decreases and
approaches zero. This behavior is analogous to enzyme activity; at some maximum point
of substrate concentration, the reactions of the enzymes with the substrate will reach
saturation; any following increase in substrate concentration will not increase the rate of
the reaction. Light transmitted by the spectrophotometer is analogous to this enzyme
activity. The reason for the nonlinearity at higher concentrations (above A > 0.3) of the
pure Fe calibration curve is due to the effect called coincidence. As more iron atoms are
added to the solution and the concentration increases, there is only so much light that can
be absorbed, therefore the curve tapers off to the nonlinear region. This nonlinear region
differs for each element. For example, sodium’s (Na) calibration curve becomes
nonlinear around absorbances of 0.6A. (4) The reason for this difference may be due to
the size of the atom. Fe is larger then Na and therefore absorbs more light with the same
increase in concentration.
In carrying out the method of additions it is important to note a few key points
regarding minimization of error. Instead of weighing a constant solid hemoglobin and
placing it in each of the beakers in the three trials, a hemoglobin stock solution was made.
This eliminated the large error that would result in the weighing of such small solid
quantities in the order of milligrams; instead, hemoglobin was added by volume in
milliliters. As seen in Table 2 (see Procedure), enough water was added to the
hemoglobin so the stock would not run out when adding a constant amount of
hemoglobin to each tube. The exact amount of hemoglobin that was added to each tube
within each trial was not important; what is important, however, is that the hemoglobin
concentration is constant within each trial. In addition, as can also be seen from Table 2,
the hemoglobin concentrations were made so the maximum concentration would not
exceed 220 ppm. Higher hemoglobin concentration than this limit might damage the
AAS and produce inaccurate readings.
To see a broader picture of the phenomenon showed in the method of additions
(see Results), a comparison between the calibration curve of pure Fe and the curve of
total iron concentration of all mixtures made in this experiment (with their absorbances)
is shown as Figure 4. It is seen from the above figure that the mixture line deviates from
the calibration curve at absorptions above 0.1A. In addition, the mixture line changes
linearity much faster than the calibration curve. If any of the hemoglobin protein affected
the AAS readings, a distinct shift along the x-axis by the mixture line would be observed;
in turn, the two curves would not converge. The fact that these two lines do converge at
their respective regions of linearity shows that the deviation is a result of the physical
presence of the protein, which increases the viscosity of the solutions, and not because
the hemoglobin interfered with the Fe absorptions. In this case, the calibration is affected
by only coincidence while the mixture line is effected by both coincidence and the
viscosity. Hence, the mixture line becomes nonlinear at a lower absorbance of 0.1A
compared to 0.3A for the calibration curve.
15
Effect of Hemoglobin on AAS
0.6
Pure Iron
Calibration
line
Absorbency
0.5
0.4
0.3
Mixture line
0.2
Concentrate on
this area to find
%Wt. Of Fe
0.1
0
0
5
10
15
20
25
Total [C] of Iron in Solution (PPM)
Figure 5. A clear deviation of the mixture line from the pure iron calibration line is seen. This
graph relates the approximate concentrations of all mixtures and their absorbances.
The specific aim of this project focuses on finding the weight percent of iron in a
hemoglobin molecule. To minimize error in determining this quantity several key points
should be noted. Different calibration curves were made each week in order to account
for the intrinsic drift within the AAS. Each corresponding calibration curve was then
used to find the iron concentration in the 16 solutions over the three weeks of
experimentation. Because it was shown that the viscosity of the iron and hemoglobin
mixtures affected the absorption readings at A > 0.1, the 16 hemoglobin solutions were
diluted to absorptions of approximately 0.01 < A < 0.02. This was carried out in order to
ensure that the viscosity of the solutions did not interfere with the AAS readings.
Because of the high dilutions, error was a major factor in these solutions. To
minimize this error, the volume of the solutions was 180 mL in each case, which reduces
the error in the water. However, there was still a considerable error in the amount of
hemoglobin put into the solutions due to the balance. (see Error Analysis)
Suggestions
A great value to this experiment that would minimize most of the error in the
amount of solid hemoglobin would be the use of a more accurate balance. The balance
currently present in the BE lab has an accuracy of 0.0005 g (and only reads to 0.001 g).
Other materials such as actual human hemoglobin, rather than bovine hemoglobin, would
more greatly signify the importance of this study.
16
Error Analysis
As in the results, there are two components that determine the weight percent of
iron in hemoglobin. Those are the iron (Fe) and hemoglobin concentrations. The
following shows how the error in these two components was determined. Furthermore,
the analysis illustrates the determination of the error in the weight percent of Fe in
hemoglobin as well as the number of Fe atoms in each molecule of protein.
Error in Hemoglobin Concentrations
The uncertainty of the hemoglobin concentrations throughout this experiment can
be attributed to error in the weighing of the hemoglobin and the error in the addition of
water to make the hemoglobin solutions. The analytic balance, which had an error of
approximately 0.0005 g, or 0.5 mg, was the only factor which caused error in these
values. As said in the procedure, the balance was relied on to measure all volumes and
weights. It should be noted though, that error in the 180 mL of water used was negligible
because it was such a large volume in comparison to the hemoglobin used, therefore it
was discounted. The following equations resulted in order to find the upper and lower
limits for hemoglobin concentration:
Equation 9a. The upper limit is found by dividing the maximum error in the mass of the hemoglobin by
the total volume. In this case, the maximum error is approximately 1 mg, which is the accuracy of the
scale.
Equation 9b. The lower limit is the minimum value divided by the total volume. These two equations
account for the 0.001 g uncertainty of the analytic balance.
Error in Iron (Fe) Concentrations
The error in the Fe concentrations was due to the AAS display readings, which
gave values to the nearest 0.001A (absorbance units). This error was accounted for and
further determined graphically using the corresponding calibration curve of pure Fe for
each solution as seen in Figure 6. The upper and lower limits were found by analyzing
the upper and lower 95% confidence limits of the slope of the calibration curve and
finding the maximum deviations.
17
y = 31.684x + 0.0301
R2 = 1
caibration curve day 1
(zoomed to .02<abs<.024)
0.8
Iron [C] ppm
0.78
Upper line
Eq.
y = 33.051x
R2 = 0.9966
Lower
line Eq
y = 34.417x - 0.0301
R2 = 1
Mid-line Eq
0.76
Sample's
iron [C]=.727
Abs=.022
0.74
0.72
0.7
0.68
0.02
0.0205
0.021
0.0215
0.022
Absorption
0.0225
0.023
0.0235
0.024
Figure 6. This graphical method was used in which the upper and lower 95% confidence limits of the
slope of the calibration curve was found. Because each hemoglobin solution had a certain absorption and
corresponding concentration, two other lines could be found intersecting the point indicated by the yellow
dot on the graph. Since the error in the AAS is known to be 0.001A, four more concentration values can
be found, two from each new line. The highest and lowest values of these four will determine the upper
and lower limit of the Fe concentration, respectively.
Error in Weight Percent of Iron (Fe)
Using the previously described upper and lower limits of Fe concentration and the
upper and lower limit of the hemoglobin concentrations, the upper and lower limits of the
weight percent of iron in hemoglobin could be determined. Equations 10a and 10b show
these calculations. Table 8 below shows the derived range in weight % from all the 16
solutions.
Equation 10a. The upper limit of the weight percent of Fe can be found by dividing the upper limit of the
Fe concentration by the lower limit of the hemoglobin concentration.
18
Equation 10b. Conversely, the lower limit can be found by dividing the lower limit of the iron
concentration by the upper limit of the hemoglobin concentration.
Weight Percent of Iron (*based on a molecular weight of 67000 grams for hemoglobin)
measured weight%
lower weight% of iron =
(low iron [C])/(high hemo [C])
upper weight% of iron =
(high iron [C])/(low hemo [C])
average lower limit
average higher limit
average
0.331
0.343
0.362
0.342
0.337
0.311
0.324
0.300
0.274
0.279
0.320
0.297
0.299
0.284
0.282
0.299
0.288
0.385
0.405
0.386
0.385
0.364
0.383
0.343
0.317
0.319
0.360
0.336
0.341
0.330
0.329
0.339
0.332
0.364
0.383
0.364
0.361
0.337
0.353
0.321
0.295
0.299
0.340
0.316
0.320
0.307
0.305
0.319
0.310
0.353
0.309
Table 8. This table shows the weight percent of iron in hemoglobin as well as the upper and lower
limits for all 16 solutions created in this experiment. The averages were calculated and the resulting
weight percent was 0.331%  0.021% of iron content in a hemoglobin molecule.
19
Error in the Number of Iron (Fe) Atoms
Error in the number of Iron atoms per hemoglobin molecule was calculated using
the upper and lower limits of the weight percent for all 16 solutions. Equations 8a, 8b,
and 8c were utilized once again. Table 9 shows the resulting error.
# of Iron atoms / Hemoglobin Molecule
measured Upper limit
Lower limet
4.36
4.61
4.11
4.60
4.85
4.34
4.36
4.63
4.10
4.33
4.62
4.04
4.04
4.36
3.73
4.23
4.59
3.88
3.85
4.12
3.60
3.54
3.81
3.28
3.59
3.83
3.35
4.08
4.32
3.84
3.79
4.03
3.56
3.84
4.09
3.59
3.68
3.96
3.41
3.66
3.95
3.39
3.83
4.07
3.59
3.71
3.98
3.46
averages
3.97
4.24
3.71
Table 9. This table displays the arrived value for the number of Fe atoms within a hemoglobin molecule
as well as the upper and lower limits. Again, all 16 solutions were taken into consideration and the
resultant average was 3.97  0.27 Fe atoms per hemoglobin molecule.
20
References
1.) BE210 Bioengineering Laboratory II Laboratory Manual. Spring 1998;
Experiment 3.
2.) Beaty, Richard D. “Concepts, Instrumentation and Techniques in Atomic Absorption
Spectrophotometry.” Perkin-Elmer; 1982.
3.) www.perkin-elmer.com
4.) Ang, Noel, et. al. (Group R3) “Atomic Absorption Spectrophotometry.” Spring 1998.
5.) Campbell, Neil A. Biology; Fourth Edition. Menlo Park, California: Benjamin /
Cummings Publishing Company, Inc., 1996.
6.) Bell, George Wall, et. al. “Absorption Spectra of Equine Hemoglobin.” Spring 1997.
7.) Lemberg, & Legge. Hematin Compounds and Bile Pigments. New York. 1949.
21
Appendix A
Method of Additions
The Method of Additions was the procedure used to determine the effect of the
Hemoglobin protein on the AAS. This method involves one control solution that
contains a constant amount of substance A (the hemoglobin for this experiment). All
other solutions contain the same constant amount of substance “A” as well as equally
increasing increments of substance “B” (the iron). A graph of the concentration of the
increasing amount of substance “B” vs. the corresponding absorbency values is then
analyzed, and the y-intercept will correspond to the absorbency of a solution with only
substance “A” and none of substance “B”. In this experiment, if the protein has no effect
on the absorbency readings, then this intercept absorbency value and that of the control
should be the same.
22