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Photometry
CHEM4401 Biochemistry I Laboratory
(Please see Photometry laboratory in your manual for additional background material)
Purpose
-Principles of spectrophotometry
- Determine optimal absorption wavelength for an analyte
- Determine molar extinction coefficient for an analyte
- Determine pKa for analyte from data and Henderson-Hasselbach equation
Spectrophotometry
Versatile method:
- usually nondestructive (unless photochemical rxn occurs)
- can be very specific for compound or functional group of interest
- fast
- sensitive to low concentrations
Large number of biomolecules absorb light in UV or visible spectrum
- compounds with conjugated double bonds (-C=C-C=C-)
-nucleotides (purines and pyrimidines)
-aromatic compounds (amino acids, metabolites, pigments, etc.)
- etc.
Absorption of light of a particular wavelength is related to concentration via the Lambert-Beer Law
Io
= εbc
I
Abs = absorbance
Abs = log
I = incident light (light falling on absorbing medium)
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I = transmitted light
e = molar extinction coefficient
b = path length of cell containing absorbing compound
c = concentration of absorbing compound
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Caution! - various factors can affect accuracy of measurement”
- light must be monochromatic (specific wavelength)
- side reactions in cell can change concentration of absorbing species
- pH stability of compound
- temperature stability of compound
Today - Using a spectrophotometer to follow dissociation of p-nitrophenol
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OH
O-
+ H+
NO2
HA
(p-nitrophenol)
NO2
<==>
A
+
H+
(phenolate ion, absorbing species)
Q: If [p-nitrophenol] = [phenolate ion] when pH = pKa, what will happen to the [p-nitrophenol] when
pH increases?
Blanks: blanks are used to obtain baseline readings for your instrument. Usually NOT H O. Blanks
contain all components except the compound of interest (analyte).
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Laboratory goals:
1. Determine optimal wavelength for measurement of phenolate ion (λmax)
2. Determine molar extinction coefficient (ε) for phenolate ion
3. Estimate pKa for p-nitrophenol dissociation reaction (graphically)
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CHEM4401 Biochemistry I Laboratory
Photometry
Materials & Reagents
p-nitrophenol (0.2 mM)
Tris buffer (0.5 M, pH 9.0)
NaH2PO4 (0.05 M)
Na2HPO4 (0.05 M
Tris HCl (0.05M)
Tris Base (0.05M)
13 x 100 mm glass test tubes
Spectrophotometer
(per student group)
6 ml (fresh daily, pH 9.0 Tris buffer (0.05M)
(Make a 2mM soln, then dilute 1:10 to 0.2 mM)
500 ml (for 0.05M solutions of p-nitrophenol)
50 ml
50 ml
20 ml
20 ml
12/group (6 blanks, 6 w/ p-nitrophenol)
1/group
Experimental Procedure
1. Student groups (6) will prepare 200 ml of each buffer listed in the table below.
2. Set up 12 test tubes as follows:
3. Determine the optimal wavelength (λmax) for measurement of the phenolate ion (see attached
instructions for proper operation and zeroing of spectrophotometer)
a. Set the spectrophotometer wavelength to 360 nm.
b. Zero the machine using tube 9B (a “blank”, contains NO p-nitrophenol)
c. Shake tube 9 (pH 9.0 buffer plus p-nitrophenol) well. Replace the blank with tube 9
and record the absorbance.
d. Change the spectrophotometer wavelength to 365 nm.
e. Repeat step b.
f. Repeat step c.
g. Increase the spectrophotometer wavelength by another 5 nm and repeat steps b and c.
Continue this procedure at 5 nm intervals until you reach a wavelength of 430 nm.
h. Determine the wavelength at which the maximum absorbance of tube # 9 occurs. Be
sure you obtain an absorbance value > 0.6 for your λmax reading. Otherwise your
extinction coefficient (ε) value will be too low and you will get a negative value of
[HA] for some pH values. If it is too low, repeat your absorbance readings using more
p-nitrophenol (2 vs 1 ml, etc.).
4. Set your spectrophotometer wavelength to λmax. Zero the spectrophotometer with tube 6B.
Remove the blank and record the absorbance of tube 6 (shake well). Repeat for remaining
sample tubes.
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Experimental Results (17 pts)
1. Prepare a plot (Figure 1) of absorbance (y-axis) vs. wavelength for the pH 9.0 sample using
the Microsoft Excel “Graph Wizard” (or similar program). Note the λmax on your plot. Be sure
figure has a legend, axes are labeled and units are indicated where appropriate (4 pt).
2. Plot (by hand is OK) the absorbance values for the pH 6.0, 6.5, 7.0, 7.5, and 8.0 samples on
figure 1 (1 pt).
3. Calculate the Molar Extinction Coefficient (ε) for your sample (2 pt).
According to Beer’s Law, A = ε.b.c may be rearranged as:
ε=
where:
A
bc
ε = the molar extinction coefficient
A = the absorbance of the pH 9.0 sample at λmax
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b = the path length of the spectrophotometer chamber (1 cm)
c = concentration of the phenolate ion
The concentration of the phenolate ion is directly dependent upon the concentration of pnitrophenol. Recall that 1 ml of 0.2mM p-nitrophenol was added to 4 ml of buffer, for a total
volume of 5 ml. It’s concentration in the pH 9.0 solution can be determined using the dilution
equation
C1V1 = C2V2
(0.2mM)(1 ml) = C2.(5 ml)
C2 = 0.04 mM = 0.00004M
Let’s assume that the p-nitrophenol (HA) is completely ionized (100% in the phenolate ion
(A) form) when the pH = 9.0. The extinction coefficient for phenolate ion may then be
calculated as:
ε=
Absorbance of pH 9.0 sample
(1 cm)(0.00004 M)
Final units should be in cm-1.M-1 (2 pt).
4. Prepare a tabe (No.1) as follows:
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(phenolate ion [A]) can be calculated for each sample using Beer’s law. Since the total
concentration of p-nitrophenol and the phenolate ion remains constant, [HA] can be calculated
as:
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[HA] = total conc. (0.00004M) – [A]
Show one set of example calculations for the determination of [A], [HA] and log [HA]/[A].
Note: It’s OK to get negative values for some log [A]/[HA] values, especially at lower pH.
This means that [HA] > [A], which we expect at lower pH. Table should be titled. (3 pt)
5. The pKa for any weak acid can be determined from the Henderson Hasselbach Equation
[A]
pH = pKa + log
[HA]
Notice that this equation is similar to that for a straight line:
y = mx + b
We can take advantage of this fact to find an estimate for the pKa of p-nitrophenol using data
€ if we make the following substitutions:
from our table. For example,
y = pH
m = slope of the line = 1.0 ( each increase in pH represents a directly proportional
increase in log [A]/[HA]
x = log [A]/[HA]
b = pKa
then we can estimate the pKa for p-nitrophenol from a graph of pH vs. log [A]/[HA]
Prepare a second graph (Figure 2) of pH (y-axis) vs. log [A]/[HA] (x-axis) using the values
from Table 1. Use the “scatter” plot option in Microsoft Excel or similar and produce a line of
best fit (regression) through the data points. Be sure to include a figure legend, label axes and
indicate units where appropriate. (5 pt)
6. Lab performance (2 pt)
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Spectrophotometer Instructions
(Spectronic 20+ spectrophotometer)
1) Turn the spectrophotometer on by turning the “Power Switch/Zero Control” knob (front left side
of instrument) clockwise. Allow the spectrophotometer to warm up for 15 minutes.
2) Set the filter level to the position appropriate for your desired wavelength (340-599 nm or 600-950
nm).
3) Switch ‘mode’ to “Transmittance”. Adjust the spectrophotometer to 0% T (Transmittance) with
the Power Switch/Zero Control knob. Make sure the sample compartment is empty and the cover
is closed.
• The purpose of this step is to zero the detector with the shutter closed and no light hitting
the detector.
4) Fill a clean cuvette (test tube) with appropriate volume (about 2/3 of the cuvette) of your blank
solution. Wipe the cuvette with a Kimwipe to remove liquid droplets, dusts, and fingerprints.
5) Place the cuvette in the sample compartment. Close the lid.
6) Adjust the spectrophotometer to 100 % T with the Transmittance/Absorbance Control knob (front,
right side of instrument).
7) Remove the blank cuvette from the sample compartment.
8) Fill a new cuvette (test tube) with appropriate volume (about 2/3 of the cuvette) of test sample.
Wipe with a Kimwipe.
9) Insert sample cuvette into the sample compartment and close the lid. Change mode to absorbance.
10) Read the appropriate value (% Absorbance).
11) When all measurement are completed, turn off the spectrophotometer by turning the Power
Switch/Zero Control knob counterclockwise until it clicks. Place plastic cover on instrument.
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