MEASUREMENTS AND DILUTIONS
Materials:
Pipetmen, glass pipettes and pipette helpers
50 µM solution of phenol red in boric acid buffer (Molecular weight = 354 g/mol)
Test tubes
Scanning Spectrophotometers- Cary 50’s
Disposable cuvettes
Solution of unknown concentration of phenol red
Safety:
Disposal:
Wear safety glasses and nitrile gloves
All solutions can be rinsed down the sink.
Disposable test tubes placed in glass waste receptacles.
Procedures:
A.
Spectrum
560
1. The figure on the side shows absorption
spectrum of 50 µM phenol red solution. At
which wavelength, does it absorb maximum
light?
2. Phenol red solution looks red, but it does
not absorb significantly in the red wavelength
range. Why?
3. If you want to monitor phenol red
concentration, at which wavelength will you
set the spectrophotometer to measure
absorbance?
B.
1.
Preparation of a standard curve for Phenol Red
Calculate the volumes of the phenol red solution and water need for each dilution in table
below. Have your calculations checked by the Instructor before making the dilutions.
Set-up the assay, in duplicate, as indicated in the table below. Prepare the dilutions
using glass measuring and automatic pipets, which you have performance verified, to
deliver the determined volumes into disposable cuvettes. Check the volume level for
uniformity and repeat any suspect preparations. Label the cuvettes on the upper edge
making sure to avoid the light path window.
Wavelength (nm)
1
Cf
Cuvette
No.
1 a, b
2 a, b
3 a, b
4 a, b
5 a, b
6 a, b
Vf
Ci
Vi
Final
Conc. P.
Red
(M)
Volume
(1.5
mL)
Stock(given)
Conc. P. Red
(M)
Volume
Phenol
Red
(1.5mL)
Water
(mL)
0
2
3
6
9
12
1.5
1.5
1.5
1.5
1.5
1.5
50
50
50
50
50
50
0
1.5
Absorbance
a
b
Calculation: Cf Vf = Ci Vi
Remember, the final volume is always 1.5 mL. Calculate the volume of 5 M phenol red
stock and add water to bring final volume to 1.5 mL.
2. Use the contents of assay #1 to zero the instrument following the directions for a
single wavelength read in the photometric mode. Proceed to measure and record the
absorbance of each dilution.
Do not discard the solutions, just in case you will need
to take another reading of a sample.
3. Using graph paper provided, plot the Concentration (x) versus Absorbance (y). Repeat
any readings or prepare new dilutions if the data does not fall in a linear fashion.
Outside of class, use Excel® (or comparable graphics program) to make a computer
plot and obtain a best-fit line and equation.
4. Obtain a test tube containing a solution of phenol red with an unknown concentration
and record the unknown code. Transfer the solution to a clean cuvette and record the
absorbance ______________. From the equation obtained from the standard curve
graph determine the concentration of the unknown.
Record your unknown code: _____________
Unknown concentration: _________M
Molecular weight of Phenol Red is 354 g/mol.
.
C. Calculations:
1 1.
If you want to make 100 mL of 0.1% phenol red solution (wt/vol), how many grams of
phenol red do you need to dissolve in water to make 100 mL volume?
2
2.
How will you make the following solutions of phenol red?
a. 12 mL of 2 M solution using solid phenol red
b. 10 mL of 10 mM solution using 1 M stock solution
c. 5 mL of 1mM using solid phenol red
d. 10 mL of 60 µM solution using:
1) 1 M
stock solution
2) 1 mM stock solution
e. 1 mL of 150M solution using solid phenol red
Check calculations in class.
3