ORP Protocol
KP1019 is a very unique and intriguing potential anticancer drug largely in part because
of its central ruthenium atom. As you learned last week, ruthenium has three oxidation states,
Ru(II), Ru(III), and Ru(IV). In order for ruthenium to change between these different oxidation
states, the atom must be either oxidized or reduced. When the ruthenium is oxidized, the atom
loses electrons and the oxidation number increases. Reversely, when the ruthenium is reduced,
the atom gains electrons and the oxidation number decreases.1 This is significant for KP1019
because researchers believe that the drug is activated by reduction, and this characteristic of the
drug is thought to relate to its ability to target tumor cells.2 Tumors are hypoxic: because of their
rapid growth, they cannot develop sufficient blood vessels to supply themselves with oxygen and
thus are more reliant on glycolysis, producing lactic acid and a reductive environment within the
tissue.3 The reduction of KP1019 is thought to have an effect on its ability to bind other
molecules, such as DNA.
Figure 1: The reduction of Ru(III) to Ru(II)
You will be performing redox titrations with solutions of your KP1019 at different pH
values. However, you will not focus on determining the concentration of any solution (that is
already known), rather you will analyze the titration curve produced by the reaction to determine
the reduction potential of your KP1019 solutions. The ORP (oxidation reduction potential)
sensor that you will be using will measure the potential of your KP1019 solution as you titrate
oxidizing or reducing agent. This data will be plotted against the volume of titrant added that the
1
Silberberg, M. S. Chemistry: The Molecular Nature of Matter and Change, Fifth Edition; McGraw-Hill: New
York, 2009.
2
Kapitza, S.:Pongratz, M.;Jakupec M. A.; Heffeter, P.; Berger,W.; Lackinger, L.; Keppler, B. K.; Marian, B.
“Heterocyclic complexes of ruthenium(III) induce apoptosis in colorectal tumor cells.” J. Cancer Res. Clin. Oncol.
2005, 131, 101-110.
32
Hartinger, C. G.; Jakupec, M. A.; Zorbas-Seifried, S.; Groessl, M.; Egger, A.; Berger, W.; Zorbas, H.; Dyson, P.
K.; Keppler, B. K. “KP1019, a new redox-active anticancer agent – preclinical development and results of a clinical
phase I study in tumor patients”. Chem. Biodivers. 2008, 5, 2140-2155.
drop counter will measure. The midpoint of the titration curve that is consequently produced
should reflect the reduction potential of the KP1019 solution and can be verified by the
[!"#$%&']
application of the Nernst equation where Q is [!"#$%#&%]:
!"
Ecell = E°cell – !" lnQ
At the midpoint, or equivalence point, of the titration curve, Q = 1, so lnQ = 0 and
Ecell = E°cell, or the standard reduction potential of the cell1. You will record these KP1019
reduction potentials and compare them to determine if pH has any effect on the reduction
potential of KP1019.
Protocol:
Begin by setting up the drop counter. First, place the drop counter on the ring stand.
Assemble the buret by connecting the tip to the end of the buret. Rinse the buret with deionized
water and allow it to dry. Clamp the buret to the ringstand above the drop counter, making sure
that the tip is just above the opening of the drop counter (if the tip is too close or too far from the
drop counter, the LabQuest will not count the drops correctly). Make sure that the valves of the
buret tip are in the closed position (horizontal). Add your titrant to the buret to just above 60mL.
Next, you will need to fill the tip of the buret with titrant. Place a waste beaker below the buret
and turn both valves to the open (vertical) position, allowing the titrant to run out. Close the
valves once the tip is filled. To calibrate the drop counter, connect the drop counter to the
LabQuest using the DIG1 port. At this time, also connect the ORP sensor to the CH1 port. Once
this is completed, tap on the drop counter display on the LabQuest screen. Click calibrate. A
new screen will pop up: click “Calibrate now”. Obtain a 10mL graduated cylinder and place it
under the buret. Open bottom valve, and slowly and carefully open the top valve to allow a
slow, steady drip of titrant. Make sure the LabQuest is counting the drops. Fill the graduated
cylinder in this manner to approximately 9mL. Close the top valve of the buret. Measure and
record the precise volume of titrant in the graduated cylinder. Enter this in the space provided on
the drop counter calibration screen (be sure to record the number of drops of titrant and the
volume of titrant in your lab notebook). Press OK in the bottom right corner of the screen. The
original screen should now read the volume of titrant used in the calibration step. If not, go back
to the calibration screen. Select the “Equation” section at the top of the screen. Calculate the
drops/mL used and enter this value to the nearest whole number. Click OK at the bottom of the
screen. Your drop counter should now be calibrated.
Place a stir plate under the drop counter. Prepare 25mL of a 1mM KP1019 solution in
one of the buffers you will be testing. Be sure to sonicate the solution to allow the KP1019 to
dissolve. Add 20mL of this same buffer to a 100mL beaker. Then add 5mL of the 1mM KP1019
solution to the beaker. Place a small stir bar in your solution, and then place the beaker on the
stir plate under the drop counter. Stir at a fast rate. Rinse the ORP sensor with DI water and
wipe it gently with a Kimwipe. Place the ORP sensor through the space in the drop counter and
into the solution. Click play (green play button in bottom left) on your LabQuest App. Slowly
open the top valve of the buret, and allow titrant to drop at a steady pace. There should be a
sharp increase or decrease in the potential very soon after beginning the titration. Record data
until the potential stabilizes after the oxidation or reduction peak. Close the valve, and click the
stop button (same as play button). Save the run by tapping on the file cabinet (make sure to note
in your lab notebook the identity of each run). You can discard the leftover KP1019 solution in
the sink. Repeat this procedure for the remaining pH buffers. After completing all of the runs,
find the midpoint potential for each of them.
Save all runs by clicking File at the top of the LabQuest screen, and then save. Save the
file under an appropriate name. Connect a FlashDrive to the USB port and transfer the data by
clicking File>Export. Select the flashdrive picture and then click on the location within your
flashdrive you would like to save the file. You will be required to rename the file as you save it.
After renaming the file and clicking “Ok”, the file should be saved onto your flashdrive.