UCSD Physiology: Spectrophotometry of Hemoglobin Calculations • first graph using excel develop a graph clearly showing the absorption spectra of oxyhemoglobin , deoxyhemoglobin , and methemoglobin . -compare the ferric and ferrous states of iron . • second graph develop a standard curve for cyanmethemoglobin . Use standard curve and Beer’s law to estimate the concentration of an Unknown .Also turn in the calculations using beer’s law for the unknown, show work. 580

570

590

600

Absorbance (%) vs. Wavelength (nm) for Various Hemoglobin Samples

560

550

540

530

510

520

Wavelength 500

Oxy Hb

Cyanmet

1.77.73 1.811.95/1.08/1.07/1100/088/174.57 no

.99.5.12

130/127/137.37/145.90 1.631.28

30.34 136 143.621.66.52.97 1.85:20.19

Hb

Deoxy Hb

Transfer these data to an Excel spreadsheet and construct a line graph with wave

length as the independent variable and absorbance as the dependent variable. Be sure

your name is on the graph, and that the graph is on the same sheet at the data. All of

the lines should be on the same graph, so they can be easily compared. This graph

will be due at the beginning of the next lab session.

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Part II. Determination of Line of Best Fit (Linear Regression and Beer’s Law):

A. Beer’s Law: Beer’s Law states that the absorbance of a solution at a

particular wavelength of light is proportional to the concentration of the

absorbing solute molecule.

Aknown

(CA) (A Cu

Ak

Beer’s Law: A is proportional to C where A = absorbance and C = concentration

(Cx) (Au)=(A«) (C) = Aunknown

Cknown

Cunknown

From the above relationship, we conclude that it is possible to prepare solutions of various

cyanmethemoglobin concentrations, (our “known” values) determine their absorbances at a

fixed wavelength, and finally to construct a line graph by plotting the absorbance “A” vs.

concentration “C.” Theoretically, such a graph will show a linear relationship, with “A”

dependent on “C.” This line is known as a calibration curve. The calibration curve allows

us to roughly determine the absorbance, again at “peak” wavelength, of a solution of

unknown cyanmethemoglobin concentration, locate that absorbance on our curve, and

predict the actual solute concentration by interpolation.

Sample graph A: Calibration Curve

Abs.

Conc.

37

it should therefore, be possible to measure the concentration of hemoglobin in a sample

of blood by measuring the intensity of its color. In clinical practice the blood hemoglobin is

usually measured by this method. Colorimetric methods are complicated by the fact that

red blood cells contain different types of hemoglobin, and each different type absorbs light

maximally in a slightly different region of the visible spectrum. A more accurate answer for

unknown concentration can be determined by using your given data, and constructing

a linear regression equation, using the instructions below.

your

B. Linear Regression Equation and Determination of Unknown

Cyanmethemoglobin Concentration. The algebraic equation for a straight line is:

y = mx + b

where

Y-dependent variable

X = independent variable

b=y-intercept

m= slope of the line

Procedure:

You have already determined the “peak” wavelength at which cyanmethemoglobin absorbs

light. Locate this value. It may be expected to be close to 540 nm.

Determine the absorbance of the unknown and the four tubes of known

cyanmethemoglobin concentration, at 540 nm wavelength. Use the cuvette with 0 g/di

concentration (diluent only) as your blank for this determination. The known

concentrations are as follows:

0 g/dl, 5 g/dl, 10 g/dl, 15 g/dl, 20 g/dl (values in grams

cyanmethemoglobin per deciliter (100 ml) of solution.

Enter these data in the table below:

x values

(concentration)

(g/dl)

y values

(absorbance)

(%)

0

5

0

32

154

182

10

15

1:06

20

Enter these data into an Excel spreadsheet and generate a graph. Notice that the points do not

form an exact straight line. Generate a linear regression line to obtain the equation:

Go to the front table and select any one tube of “unknown concentration” from the tubes

provided. Take the absorbance (Y value) and record here:

(Y)=_0.52

-=10

Do not empty any of the cuvettes. Leave them in the test tube rack.

Now that you have determined your equation, you can plug in the Y value and solve for X,

which is the concentration of the unknown substance. Hand-write your calculations on your

printed graph. Be sure to show your work. Use the space below to practice. Use Beer’s Law to

check your answer.

X= ___g/dl

The hemoglobin determination graph as well as the linear regression graph is due at the

beginning of the next lab session.

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