CH 149: Chemical Principles. Fall KP1019 Module TA Manual

CH 149: Chemical Principles Fall 2012 KP1019 Module TA Manual

2 Table of Contents KP1019 Synthesis 3 ORP Titrations...10 Electrochemistry 15 ph Kinetics 19 Temperature/NaCl Kinetics...24

3 Equipment (per lab group): Hot Plate Stir Bar 25mL Graduated Cylinder 50mL Erlenmeyer Flask Thermometer Frits Filtration Flask 100mL Graduated Cylinder Tub of ice (to store chemicals) o Ethanol o Diethyl Ether 150mL Beaker Glass Pipettes Pipette Bulb Clamp Equipment (Lab): Gloves 12M HCl Indazole Weigh boats Balance KP1019 Synthesis Set-Up Preparing Ru(III) Solution (ALSO SEE DIAGRAMS BELOW): Equipment: o 100mL round-bottom flask o Weigh boat o RuCl 3 3H 2 O o Condenser o Mineral Oil Bath o Paper Clip for Mineral Oil Bath o Hot Plate o Stir bar o Rotary Evaporator o Cable ties (to hold tubing in place) o Worked in gaseous Nitrogen with H 2 O Protocol:

4 o In a 100mL round-bottom flask, add 1g RuCl 3 3H 2 O to 20mL 12M HCl and 20mL ethanol (rinse the weigh boat with ethanol to get all of Ru solid, if needed continue with HCl; also be sure to add a stir bar to the round bottom) o Wrap ground-glass joint of condenser with Teflon tape o Place condenser into round-bottom flask making sure that the joints are sealed o In a mineral oil bath, reflux solution by heating on a hot plate and stirring Immerse flask halfway in bath Ensure stir bar is in round-bottom Add paper clip to oil bath At this point, turn nitrogen and H 2 O taps in hood on low (make sure that the water tube is going into the drain, use cable ties to hold the tubing) flow should be almost a drizzle Set hot plate to approximately a third Watch for condensation in flask as this indicates proper temperature Make sure oil is not boiling o Reflux 1 hour (start time at the first spot of condensation) o Cool to room temperature o Place on rotary evaporator for approximately one hour (half of the solution should be gone when the ethanol is removed) Check clips on rotary evaporator to make sure they aren t broken (if slightly pulled apart cracks should become visible) Turn on water and check that it is going down the drain Turn on vacuum Begin rotating the round bottom (do not rotate too quickly) Adjust heating of water bath and vacuum pressure to prevent solution bubbling into rotary evaporator Ensure fume tubing is in the hood Attach flask in a bucket of ice underneath the condenser to collect evaporated solution When ethanol and Ru solution are separated, the solution will turn from dark brown to greenish black (also looks thicker) When finished, make sure to turn off vacuum and water Add HCl to ruthenium solution to a final volume of 40mL. Divide into four parts (10mL for each lab group).

5 Ensure stopcock is open Nitrogen Bubbler Nitrogen Tubing Condenser Teflon Tape Mineral Oil Bath Water Tubing Hot Plate

6 Round Bottom Flask Containing Ru Solution Ensure to close stopcock before starting Knob to control rotation Handle (controls elevation) Vacuum Pump Collection Flask on Ice Hot Water Bath

7 Condenser Clip Round Bottom Flask Sample Data: 2.5 UV-Vis Spectrum 0.5mM KP1019 in Water (Shay) 2 Absorbance 1.5 1 0.5 0 350 450 550 650 750 850 Wavelength (nm) Max peak at 420 nm: 0.638 Max peak at 360 nm: 2.0848

8 UV-Vis Spectrum 0.5mM KP1019 in Water (Sydney) 2.5 2 Absorbance 1.5 1 0.5 0 350 450 550 650 750 850 Wavelength (nm) Max peak at 420 nm: 0.6389 Max peak at 360 nm: 2.0426 2.5 UV-Vis Spectrum 0.5mM KP1019 in Water (Allison) 2 Absorbance 1.5 1 0.5 0 350 450 550 650 750 850 Wavelength (nm) Max peak at 420 nm: 0.5986 Max peak at 360 nm: 1.9534

9 UV-Vis Spectrum 0.5mM KP1019 in Water (old) 2.5 2 Absorbance 1.5 1 0.5 0 350 450 550 650 750 850 Wavlength (nm) Max peak at 420 nm: 0.6613 Max peak at 360 nm: 1.9953 Above: UV-Vis spectra taken of 0.5mM KP1019 in water and run from 360-800 nm. The first three samples (Shay, Sydney, and Allison s samples), were produced using the modified synthesis protocol for the students in August 2012, the last sample (Old sample), was produced in June 2012 by halving the original protocol and was from the stock KP1019 that had been used throughout the summer.

10 Glassware for each group: 100mL Beaker 250mL Beaker 10mL Graduated Cylinder 100 ml Graduated Cylinder 25mL Volumetric Flask 2x50mL Beakers Other Equipment for each group: Small Stir Bar Drop Counter ORP Sensor LabQuest Small Stir Plate Clamp Ring Stand Plastic Pipettes Small Weigh Boats Kimwipes Spatula Balance? Sonicator? Parafilm Solutions: 0.5M Phosphate Buffers: ph 7.2 ph 6.2 ph 5.4 ph 4.4 0.2mM KMnO 4 0.2mM Ascorbic Acid ORP Titration Lab Set-Up Preparation of Solutions(pH Buffers do not need to be exact): 1M KH 2 PO 4 Stock: dissolve 136.086g KH 2 PO 4 in 1L dh 2 O 1M KHP Stock: dissolve 204.23g in 1L dh 2 O (may begin to add NaOH to help in dissolving the KHP; however, be sure not to go over the ph of the desired buffer (ph 5.4 and ph

11 4.4)) if you begin to add NaOH, once the KHP has dissolved, half the solution into two 1L beakers and then add additional NaOH as needed ph 7.2 Buffer: Add 500mL of 1M KH 2 PO 4 stock to 2000mL beaker on a stir plate. Add stir bar. Add approximately 300mL 1M NaOH solution while monitoring the ph using a ph sensor making sure not to go over desired ph Monitor ph and add more NaOH dropwise as needed until desired ph is reached Add to stock bottle and label. Dilute to the shoulder of the bottle with dh 2 O (1L) ph 6.2 Buffer: Add 500mL of 1M KH 2 PO 4 stock to 2000mL beaker on a stir plate. Add stir bar. Add approximately 100mL 1M NaOH solution while monitoring the ph using a ph sensor making sure not to go over desired ph Monitor ph and add more NaOH dropwise as needed until desired ph is reached Add to stock bottle and label Dilute to the shoulder of the bottle with dh 2 O (1L) ph 5.4 Buffer: Add 500mL of 1M KHP stock to 2000mL beaker on a stir plate. Add stir bar. Add approximately 300mL 1M NaOH solution while monitoring the ph using a ph sensor making sure not to go over desired ph Monitor ph and add more NaOH dropwise as needed until desired ph is reached Add to stock bottle and label Dilute to the shoulder of the bottle with dh 2 O (1L) ph 4.4 Buffer: Add 500mL of 1M KHP stock to 2000mL beaker on a stir plate. Add stir bar. Add approximately 70mL 1M NaOH solution while monitoring the ph using a ph sensor making sure not to go over desired ph Monitor ph and add more NaOH dropwise as needed until desired ph is reached Add to stock bottle and label Dilute to the shoulder of the bottle with dh 2 O(1L) 0.2mM KMnO 4 Stock: Add 0.0316g KMnO 4 to 500mL dh 2 O Add 10mL 4.5M H 2 SO 4 Add 250mL 18M H 2 SO 4 to 750mL dh 2 O (make sure to add acid to water) Add to stock bottle and label

12 Dilute to shoulder of the bottle with dh 2 O (1L) 0.2mM Ascorbic Acid Stock: Add 0.017613g Ascorbic Acid to 1L dh 2 O Add to stock bottle and label Tips: Students should use 15.4mg KP1019 to make 25mL 1mM stock Placement of buret o If drop counter isn t reading the buret is too close to the Drop Counter or too far from the Drop Counter, so readjust It takes approximately 30mL of titrant for potential to stabilize Caution the students about getting all the KP1019 into solution o Rinsing the weigh boat with buffer o Rinsing beaker with buffer o Pipetting undissolved solid into flask o Sonicating well Caution the students to check power on LabQuests Caution the students on opening stopcock on buret tips too quickly Wear gloves working with KP1019 If students spill storage solution for ORP sensor, replace with 3M KCl solution Sample Data: Below are samples of the oxidation and reduction titration curves seen when KP1019 is titrated with KMnO 4 and ascorbic acid as well as sample reduction potentials calculated from the midpoints of these curves for each ph. The overall trends in the graphs should be similar with variance in ph (i.e. the oxidation curves should rapidly increase and the reduction curves should rapidly decrease), but the reduction potentials should be affected by ph changes.

13 0.2mM KP1019 in 0.5M ph 6.26 Phosphate Buffer-0.2mM KMnO 4 650 600 Potential (mv) 550 500 450 400 350 0 5 10 15 20 25 30 35 40 45 50 Volume KMnO 4 Titrated (ml) Midpoint: 516.15mV 380 360 340 0.2mM KP1019 in 0.5M ph 4.47 Phosphate Buffer-0.2mM Ascorbic Acid Potential (mv) 320 300 280 260 240 220 0 5 10 15 20 25 30 35 Volume Ascorbic Acid Titrated (ml) Midpoint: 302.75mV

14 Potentials for Oxidation of 0.2mM KP1019 with 0.2mM KMnO 4 ph of Phosphate Buffer (0.5M) Starting Potential (mv) Midpoint of Titration Curve (mv) 7.24 232.0 398.5 238.0 414.55 6.26 390.6 502.4 408.0 516.15 5.42 463.0 577.75 483.4 577.6 4.47 522.0 632.6 553.6 651.65 Potentials for Reduction of 0.2mM KP1019 with 0.2mM Ascorbic Acid ph of Phosphate Buffer (0.5M) Starting Potential (mv) Midpoint of Titration Curve (mv) 7.24 287.0 226.4 280.0 216.15 6.26 303.0 249.55 284.8 235.15 5.42 310.0 256.5 310.1 256.85 4.47 328.1 279.65 354.0 302.75

15 Electrochemistry Lab Set-up Equipment (per lab group): 25mL volumetric flask 2x50mL beakers Small spatula (for KP1019) Equipment (Lab): Balance Weigh boats Various ph buffers (4, 5, 6, and 7) Transfer pipettes Sonicator Gloves Kimwipes Solutions for Student Use: Preparation of Solutions(pH Buffers do not need to be exact): o 1M KH 2 PO 4 Stock: dissolve 136.086g KH 2 PO 4 in 1L dh 2 O o 1M KHP Stock: dissolve 204.23g in 1L dh 2 O (may begin to add NaOH to help in dissolving the KHP; however, be sure not to go over the ph of the desired buffer (ph 5.4 and ph 4.4)) if you begin to add NaOH, once the KHP has dissolved, half the solution into two 1L beakers and then add additional NaOH as needed o ph 7.2 Buffer: Add 500mL of 1M KH 2 PO 4 stock to 2000mL beaker on a stir plate. Add stir bar. Add approximately 300mL 1M NaOH solution while monitoring the ph using a ph sensor making sure not to go over desired ph Monitor ph and add more NaOH dropwise as needed until desired ph is reached Add to stock bottle and label. Dilute to the shoulder of the bottle with dh 2 O (1L) o ph 6.2 Buffer:

16 Add 500mL of 1M KH 2 PO 4 stock to 2000mL beaker on a stir plate. Add stir bar. Add approximately 100mL 1M NaOH solution while monitoring the ph using a ph sensor making sure not to go over desired ph Monitor ph and add more NaOH dropwise as needed until desired ph is reached Add to stock bottle and label Dilute to the shoulder of the bottle with dh 2 O (1L) o ph 5.4 Buffer: Add 500mL of 1M KHP stock to 2000mL beaker on a stir plate. Add stir bar. Add approximately 300mL 1M NaOH solution while monitoring the ph using a ph sensor making sure not to go over desired ph Monitor ph and add more NaOH dropwise as needed until desired ph is reached Add to stock bottle and label Dilute to the shoulder of the bottle with dh 2 O (1L) o ph 4.4 Buffer: Add 500mL of 1M KHP stock to 2000mL beaker on a stir plate. Add stir bar. Add approximately 70mL 1M NaOH solution while monitoring the ph using a ph sensor making sure not to go over desired ph Monitor ph and add more NaOH dropwise as needed until desired ph is reached Add to stock bottle and label Dilute to the shoulder of the bottle with dh 2 O(1L) Cyclic Voltameter Setup Make sure you have the following equipment: o Carbon electrode Ensure that to polish it on a felt pad Add silica to the pad Wet with dh 2 O Hold the electrode vertically to the felt pad while applying a small amount of pressure and gently polish the electrode in a circular motion (make sure to move your arm not your wrist) After a few seconds of polishing in one direction, continue to polish in the opposite direction (counterclockwise vs. clockwise) and occasionally rotate the electrode in your hand

Rinse off with dh 2 O and gently blot off with a Kimwipe o Reference electrode o Counter electrode (platinum wire) o Bubbler Connect the electrodes to the wiring inside the cyclic voltameter o Each electrode should have a wire that fits it perfectly: Carbon electrode to black wire Reference electrode to white wire Counter electrode attached with a connecter to red wire Bubbler connected to tubing with test sample label Turn on the nitrogen by opening the valves on the tank (gas should come through the bubbler) Turn on the cyclic voltameter: o This consists of two boxes Long rectangular device with a switch on the back Cube containing the electrode wiring with a switch on the back Open the CV50W program on the computer desktop Set the machine to automatically stir and purge o File > Setup Options o Make sure that Stir and Purge are checked o Click OK De-gas a blank solution ( 25-40mL ph buffer students will be using for KP1019 solutions in a 50mL beaker with a stir bar) by bubbling for approximately five minutes o At this time ensure that nitrogen gas is issuing from the bubbler and that the solution is stirring, otherwise, return to Setup Options to check these options (see previous bullet) If solution is not purging and the Purge option is selected, check nitrogen tank Measure rest potential: o Control > Measure Rest Potential o Test twice to make sure it is consistent Set parameters: o Method > General Parameters o Initial: 800 o High: 800 o Low: -1000 o Scan Rate: 150 o The students will do this as well. Start run: o Control > Start Run 17

18 Make sure that the blank looks normal (not too noisy, no random peaks) o Noise?? Check to see if there is a bubble on the Carbon electrode in the solution o Random peaks?? Polish Carbon electrode and run again Polish the electrode after each lab group has run their experiment Run a blank for each lab group depending on the ph they are assigned (run the blank in the correct ph solution) Sample Data: 1.00E-05 8.00E-06 6.00E-06 4.00E-06 Potential of KP1019 in Phosphate Buffer Current (A) 2.00E-06 0.00E+00-2.00E-06-4.00E-06 ph 4.46 ph 5.42 ph 6.26 ph 7.24-6.00E-06-8.00E-06-1.00E-05 450-50 -550-1050 Potential (mv) Above: An overlay of cyclic voltameter data for all four ph conditions that KP1019 was observed under. Note how the oxidation peaks shift to the right (the potential becomes more negative) as ph increases. Also note the overall shape of the graph for KP1019 in phosphate buffer and the lack of some reversible peaks.

19 KP1019 ph Kinetics Set-Up Equipment (per lab group): Laptop computer with Logger Pro Program Vernier Spectrometer 2x Plastic Cuvettes 2x Cuvette Caps 2x Small Glass Pipettes Pipette Bulb 2x 50mL Beakers 25mL Volumetric Flask Phosphate Buffer Solution (ph 4, 5, 6, or 7) General Equipment Balance Weigh Boats Plastic Pipettes Parafilm Kimwipes Spatulas Sonicator Preparation of Solutions (ph Buffers do not need to be exact): 1M KH 2 PO 4 Stock (used for ph 6.2 and ph 7.2 buffers): -dissolve 136.086g KH 2 PO 4 in 1L dh 2 O 1M KHP Stock (used for ph 4.4 and ph 5.4 buffers): -dissolve 204.23g in 1L dh 2 O (may begin to add NaOH to help in dissolving the KHP; however, be sure not to go over the ph of the desired buffer (ph 5.4 and ph 4.4)) -if you begin to add NaOH: once the KHP has dissolved, halve the solution into two 1L beakers and then add additional NaOH as needed ph 7.2 Buffer: -Add 500mL of 1M KH 2 PO 4 stock to 2000mL beaker on a stir plate. -Add stir bar. -Add approximately 300mL 1M NaOH solution while monitoring the ph with a ph sensor, making sure not to go over desired ph

20 -Monitor ph and add NaOH dropwise as needed until desired ph is reached -Add to stock bottle and label. -Dilute to the shoulder of the bottle with dh 2 O (1L) ph 6.2 Buffer: -Add 500mL of 1M KH 2 PO 4 stock to 2000mL beaker on a stir plate. -Add stir bar. -Add approximately 100mL 1M NaOH solution while monitoring the ph with a ph sensor, making sure not to go over desired ph -Monitor ph and add NaOH dropwise as needed until desired ph is reached -Add to stock bottle and label -Dilute to the shoulder of the bottle with dh 2 O (1L) ph 5.4 Buffer: -Add 500mL of 1M KHP stock to 2000mL beaker on a stir plate. -Add stir bar. -Add approximately 300mL 1M NaOH solution while monitoring the ph with a ph sensor, making sure not to go over desired ph -Monitor ph and add NaOH dropwise as needed until desired ph is reached -Add to stock bottle and label -Dilute to the shoulder of the bottle with dh 2 O (1L) ph 4.4 Buffer: -Add 500mL of 1M KHP stock to 2000mL beaker on a stir plate. -Add stir bar. -Add approximately 70mL 1M NaOH solution while monitoring the ph with a ph sensor, making sure not to go over desired ph -Monitor ph and add NaOH dropwise as needed until desired ph is reached -Add to stock bottle and label -Dilute to the shoulder of the bottle with dh 2 O(1L) Tips: Students should use 7.7mg KP1019 to make 25mL of 0.5mM solution Caution the students about getting all of the KP1019 into solution: o Rinsing the weigh boat with buffer o Rinsing beaker with buffer o Pipetting undissolved solid into flask o Sonicating well Caution the students to check power on LabQuests

21 Wear gloves working with KP1019 Caution students against bumping/moving spectrometers while running the kinetics experiments as this will disturb proper data collection Ensure students wipe cuvettes with Kimwipes prior to reading with the spectrometers Sample Data: Absorbance 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0.5mM KP1019 in 0.05M ph 4.4 KHP/NaOH Buffer 0 50 100 150 Time (min) 410.0 nm 420.0 nm 430.0 nm 492.0 nm Absorbance 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0.5mM KP1019 in 0.5M ph 5.42 KHP/NaOH Buffer 0 20 40 60 80 100 120 Time (min) 410.0 nm 420.0 nm 430.0 nm 492.0 nm

22 0.5mM KP1019 in 0.5M ph 6.24 KH2PO4/NaOH Buffer Absorbance 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 410.0 nm 420.0 nm 430.0 nm 492.0 nm 0 0 20 40 60 80 100 120 Time (min) 0.5mM KP1019 in 0.5M ph 7.24 KH2PO4/NaOH Buffer Absorbance 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 410.0 nm 420.0 nm 430.0 nm 492.0 nm 0 0 20 40 60 80 100 120 Time (min) Above: Note how in ph 4.4 and ph 5.42 buffer solutions the kinetics studies at the selected wavelengths show very little change in the drug. However, at ph 6.24 a significant change in absorbance over time becomes noticeable, reflecting some change in the drug. At ph 7.24 an even greater change in absorbance over time is seen: the initial decrease in absorbance followed by an increase in absorbance suggests that a two step reaction is observed during this time frame. These changes in drug conformation can be visually seen at these phs: the KP1019 solution in the cuvettes will change from an amber, golden color to a dark, blue-green color.

A 0.5mM KP1019 solution in ph 4.4 buffer (left) and in ph 7.22 buffer (right) after a 2.5 hour kinetics run. 23

24 KP1019 and Temperature/NaCl Kinetics Set-Up Equipment (per lab group): Laptop computer with Logger Pro Program Vernier Spectrometer 2x Plastic Cuvettes 2x Cuvette Caps 2x Small Glass Pipettes Pipette Bulb 2x 50mL Beakers 25mL Volumetric Flask Phosphate Buffer Solution (ph 7 for temperature) Phosphate Buffer solution with 0.5M NaCl (ph 6 or 7) General Equipment Balance Weigh Boats Plastic Pipettes Parafilm Kimwipes Spatulas Sonicator 4x Water Baths (on ice, and at approximately 25 C, 30 C, 37 C) o 4x Ringstands o 4x Temperature Probes o 4x Labquests o 4x Cylindrical Dishes o Styrofoam Bucket o Ice o 3x Hotplates o 4x Clamps Plug in temperature probe to Labquest and set in clamp on ringstand Fill cylindrical dish ½ full with water and place on hotplate or in styrofoam bucket with ice Lower clamped temperature probe into water Adjust hotplate temperature to get water bath to desired temperature (ice bath should be around 2.0-2.5 C) Preparation of Solutions (ph Buffers do not need to be exact):

25 1M KH 2 PO 4 Stock (used for ph 6.2 and ph 7.2 buffers): -dissolve 136.086g KH 2 PO 4 in 1L dh 2 O ph 7.2 Buffer: -Add 500mL of 1M KH 2 PO 4 stock to 2000mL beaker on a stir plate. -Add stir bar. -Add approximately 300mL 1M NaOH solution while monitoring the ph with a ph sensor, making sure not to go over desired ph -Monitor ph and add NaOH dropwise as needed until desired ph is reached -Add to stock bottle and label. -Dilute to the shoulder of the bottle with dh 2 O (1L) ph 6.2 Buffer: -Add 500mL of 1M KH 2 PO 4 stock to 2000mL beaker on a stir plate. -Add stir bar. -Add approximately 100mL 1M NaOH solution while monitoring the ph with a ph sensor, making sure not to go over desired ph -Monitor ph and add NaOH dropwise as needed until desired ph is reached -Add to stock bottle and label -Dilute to the shoulder of the bottle with dh 2 O (1L) ph 7.2 buffer with 0.5M NaCl - dissolve 2.922g NaCl in 100mL ph 7.2 phosphate buffer ph 6.2 buffer with 0.5M NaCl -dissolve 2.922g NaCl in 100mL ph 6.2 phosphate buffer Tips: Students should use 7.7mg KP1019 to make 25mL of 0.5mM solution Caution the students about getting all of the KP1019 into solution: o Rinsing the weigh boat with buffer o Rinsing beaker with buffer o Pipetting undissolved solid into flask o Sonicating well Caution the students to check power on LabQuests Wear gloves working with KP1019 Caution students against bumping/moving spectrometers while running the kinetics experiments as this will disturb proper data collection

26 Ensure students wipe cuvettes with Kimwipes prior to reading with the spectrometers Ensure students using the cold water bath allow time to remove and prevent condensation on their cuvettes so that data collection will be accurate Sample Data: Absorbance 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.5mM KP1019 in 0.5 ph 7.26 Phophate Buffer in Ice Bath (2.2 C, 2 hrs elapsed time) 0 380 480 580 680 780 880 Wavelength (nm) 1.2 0.5mM KP1019 in 0.5M ph 7.26 Phosphate Buffer in Hot Water Bath (30 C, 2 hrs elapsed time) 1 Absorbance 0.8 0.6 0.4 0.2 0 380 480 580 680 780 880 Wavelength (nm)

27 Above: Temperature kinetics studies with 0.5mM KP1019 in 0.5M ph 7.26 phosphate buffer (samples were kept in water baths at the appropriate temperatures and removed from the baths and tested in a spectrometer every ten minutes for 2 hours). The ice bath showed little overall change in the absorption spectrum of the drug. However, the 30 C water bath showed rapid increase in the conformational change of the drug: the initial peak seen at 420 nm flattens within the first 40 minutes and a second broad peak at 620 nm becomes visible, likely a result of the forming blue-green precipitate. At this temperature, the second peak increases and then begins to decrease just before 2 hours have elapsed, perhaps because of the precipitate settling out of solution. 1.2 0.5mM KP1019 in 0.5M ph 7.26 Phosphate Buffer (1.5 hrs elapsed time) 1 Absorbance 0.8 0.6 0.4 0.2 0 380 480 580 680 780 880 Wavelength (nm)

28 Absorbance 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.5mM KP1019 in 0.5M ph 7.26 Phosphate Buffer/0.5M NaCl (1.5 hr elapsed time) 0 380 480 580 680 780 880 Wavelength (nm) Absorbance 0.5mM KP1019 in 0.5M ph 7.26 Phosphate Buffer/1.0M NaCl (1.5 hrs elapsed time) 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 380 480 580 680 780 880 Wavelength (nm) Above: Kinetics with 0.5mM KP1019 in 0.5M phosphate buffer with NaCl (samples were taken with a spectrometer every 10 minutes for 90 minutes). With increased concentration of NaCl, the observed change in drug conformation (as reflected by the absorption spectra) decreases.