PNP spectrochemistry Prelab assignment, Spring 2010 Page 1 PNP ...

PNP prelab assignment, PNP spectroscopy lab. Name ____________________
Lab day___________________
1. Using only stock solutions of 0.1 M KH2PO4 and 0.1 M K2HPO4 you wish to prepare 5.0 mL solutions
of potassium phosphate buffers with pH values of 6.5, 7.2 and 7.6. Noting that the pKa of KH2PO4 to
K2HPO4 is 6.82, use the Henderson‐Hasselbalch equation to calculate the relative amounts of the
two stocks needed to generate these pH values. Show your work in the space below the table to
get full credit.
Hndsn/Hssblch
predicted pH
6.5
7.2
7.6
Calculated ratio
Volume of
0.1 M KH2PO4
(mL)
Volume of
0.1 M K2HPO4
(mL)
Total Volume
(mL)
0.47863 3.4 1.6 5
2.399 1.5 3.5 5
6.025 0.7 4.3 5
Note that this question is similar to the table you must complete in the PNP protocol. You should
copy your answers from this assignment into that table!
2. Using a 0.1 mM PNP solution and pH 10 buffer for dilution, prepare a series of 2 mL standards with
final concentrations of PNP of 2, 4, 6, 10, 15, and 25 μM.
Sample
Name
Std. 1
Std. 2
Std. 3
Std. 4
Std. 5
Std. 6
Vol. of 0.1 mM
(100 μM) PNP
(μL)
Vol. of pH
10 buffer
(μL)
Total
Volume
(μL)
Final
Conc. of
PNP
(μM)
40 1960 2000 2
80 1920 2000 4
120 1880 2000 6
200 1800 2000 10
300 1700 2000 15
500 1500 2000 25
Note that this question is similar to the table you must complete in the PNP protocol. You should
copy your answers from this assignment into that table!
PNP spectrochemistry Prelab assignment, Spring 2010 Page 1

Sample calculation for problem 1
For pH 6.5:
6.5 6.82
0.32
0.47863
We have 2 variables, and 2 equations:
Equation 1: Equation 2:
A‐+HA = 5 mL A‐/HA = ratio (in this case 0.47863)
Start:
A‐/HA=0.47863
A‐=0.47863HA
Plug this into equation 1
0.47863HA + HA = 5
1.47863HA =5
HA = 3.4
Plug this into Equation 1:
A‐+3.4 = 5.0
A‐ = 1.6
For problem 2, use M1V1 = M2V2 where M1=100 uM, M2=value in column 5, and V2=2000 uL. Solve for
V1. Volume of pH 10 buffer is simply 2000 uL – V1.
PNP spectrochemistry Prelab assignment, Spring 2010 Page 2