4 Results
4.3 Light induces chelation of copper
The initial rate was now so high that it could not be measured with several simultaneous experiments after titrating them to the same pH. Moreover, two samples could be measured a day, so it was decided to skip pH titration and optimize one sample at a time instead, keeping it in the spectrophotometer, where it previously had been discarded after measurement.
4.3.1 First evidence of light induced chelation
The absorption of the first sample, measured without leaving the spectrophotometer, showed an increase with a curve having sharp bends and almost linear shape between these bends (figure 4.14). When analysed for reaction speed (the slope), it was revealed that there were steep drops in reaction speed when the duration between the measurements increased. This indicated that measuring the sample increased reaction speed significantly, even though a measurement only took 0.5 second and the light from the spectrophotometer is all but intense to avoid photochemical reactions in the samples.
Coordination of 0.2 mM Cu2+ to 0.4 mM [35]adz
R2 = 0,9977
R2 = 0,9994
R2 = 0,998
R2 = 0,9977
R2 = 0,9994
R2 = 0,998
0 0,2 0,4 0,6 0,8 1
0 20 40 60 80 100
Minutes
Absorption at 289 nm
Reaction speed
0 0,01 0,02 0,03 0,04
0 20 40 60 80
Minutes
ΔAbs./min.
Figure 4.14: CuSO4 was mixed with (H4[35]adz)Br4 to a concentration of 0.2 mM Cu2+ and 0.4 mM [35]adz in 1 x PBS (without Cl-) and a pH of 7.4. The first 5 minutes the sample spectrum was measured every minute (blue). Then the spectrum was measured every second minute until 15 minutes had passed (dark green) and every 5 minutes until 35 minutes had passed (light green). The final 3 spectra were measured after 43, 60 and 85 minutes (orange). To the left coordination, measured as the absorption at 289 nm, is shown and to the right the reaction speed, measured as the increase in absorption at 289 nm per minute, is shown. The red line is an attempt to fit the data to an exponential decrease in rate.
To test this unexpected hypothesis, a sample of Cu2+ and [35]adz in PBS was mixed and half of it transferred to a cuvette and measured every 5 minutes (figure 4.15 a). That was compared to an identical sample, which was mixed, transferred and measured every minute, so the only difference was the measuring frequency.
Measurement was stopped when the absorption at 289 nm had surpassed 1.15 as this was calculated to be 100 % coordination of Cu2+. Then the second half of the samples, which had been kept in the dark, was measured. The results decisively confirmed the hypothesis that measuring enhances the rate of coordination.
It took 64 minutes to reach absorption of 1.15 at 289 nm for the sample measured every minute, while it took 104 minutes for the sample measured every fifth minute. The samples kept in the dark and only measured once were only about 2/3 of the way to 1.15 after 112 minutes. As there is no other influence on the samples from the spectrophotometer than the light beam passing through, the hypothesis that irradiation of the samples increased the rate of coordination of copper was confirmed.
The samples were measured in a 500-µL cuvette. Since it is only a part of the sample, which is irradiated in such a cuvette, it seems reasonable to expect a two phase reaction, where there is a fast reaction initially where the irradiated part reacts, followed by a slow reaction limited by diffusion. This is in fact, exactly what is seen when analysing for reaction speed. The sample, which was irradiated every minute, had a steep drop in reaction speed the first 7 minutes. Then the rate dropped a bit slower the next 10 minutes, followed by another period with a step drop, which flattens off as it approached zero. The sample, which was irradiated every fifth minute, had a much wider plateau. The UV spectra of the sample measured every fifth minute have a distinct isosbestic point at 258 nm confirming that only one product, the chelate, is formed (figure 4.15 d). In order to minimize the diffusion factor, the 500-µL cuvette was afterwards replaced with a 150-µL cuvette where most of the sample was irradiated. In order to obtain reproducible results the cuvette had to be filled beyond the window, as curvature due to surface tension otherwise results in a large background.
Coordination of 0.20 mM Cu2+ to 0.40 mM [35]adz in phosphate buffer
0 1.15
0 50 100 150
Minutes
Absoption at 289 nm Once
every minute Once every 5 minutes
Spectra of sample measured every 5th minute
0 0.2 0.4 0.6 0.8 1 1.2 1.4
200 250 300 350 400
nm
Absorption
5 10
15 20
25 30
35 40
45 50
55 60
65 70
75 80
85 90
95 100 105 110
Figure 4.15: Chelation of Cu2+ from by [35]adz, same solution as in figure 14. a) Chelation measured by absorption at 289 nm. The theoretical maximum of 1.15 corresponding to chelation of 100 % of the copper is marked by a dashed line. The encircled dots show the absorption of the second half of the two samples being measured for the first time after the first halves have reached an absorption of 1.15. b) The UV spectra of the sample measured every 5th minute.
4.3.2 A comparison between coordination of copper to [35]adz and to [24.31]adz
After replacement of the cuvette with one where almost all of the sample could be irradiated simultaneously, continuous irradiation resulted in a 20-fold increase in reaction speed. This enabled a large increase in experiments per day, and they indicated an adverse effect on chelation rate of other Cu(II) binding
molecules. Another 20-fold increase in reaction speed resulted from removal of phosphate from the sample solution; chelation became so fast that it was almost finished before measurement could begin and completed after only 13 seconds (figure 4.16 a). This rate encourage us to give the more rigid [24.31]adz a try.
a) b)
Chelation of Cu2+ by [35]adz
0 0.5 1 1.5
0 10 20 30
Seconds
Absorption
239 nm 256 nm 289 nm
Chelation of Cu2+ by [24.31]adz
0 0.2 0.4 0.6
0 1000 2000 3000 4000 Seconds
Absorption
230 nm 285 nm 600 nm 20*600 nm 285 nm -20*600 nm
Figure 4.16: 0.20 mM CuSO4 and 1/3 mM adamanzane measured continuously in a diode array spectrophotometer. a) Chelation of Cu2+ by [35]adz. b) Chelation of Cu2+ by [24.31]adz.
In the first attempt to induce coordination of Cu2+ to [24.31]adz with UV light, the reaction did not finish within 4000 seconds (figure 4.16 b). This was measured by absorption at 285 nm and at 600 nm. As the absorption peak at 600 nm is 20 fold lower than the peak at 285 nm, they can be compared by multiplication.
The figure shows that the increase in absorption is exactly 20 fold higher at 285 nm than at 600 nm as the two curves are parallel except at 3300 seconds, where a spectrophotometrical error occurred. This makes an increase in background during the experiment unlikely. After 2400 seconds the absorption at 230 nm began to rise again, which indicate a secondary process after prolonged irradiation.
The products were analysed by MS (app. B), which confirmed the formation of [Cu([35]adz)]2+ and [Cu([24.31]adz)]2+ with different anions as 5th ligands.
As [Cu[24.31]adz]2+ is more robust than [Cu[35]adz]2+ and [24.31]adz is easier to functionalize with side groups, it was decided to switch focus to [24.31]adz.
4.3.3 Measurement of light induction
In a chelation assay of Cu2+ to [24.31]adz where the contents of all the samples were identical and the only parameter chanced was the time the samples were exposed to light from the spectrophotometer, the chelation rate is shown to be correlated to light quantity (figure 4.17). As measuring required irradiation of the
samples, it was not possible to obtain information of the reaction speed with no irradiation, and at 0.3 % of full spectrophotometrical irradiation, not until after 900 seconds. As expected, the reaction speed decreases over time - due to a reduction of reactant concentrations, UV light absorption by the product or both. It is also evident that irradiation by the spectrophotometer increases the reaction speed. E.g.: Increasing the irradiation of the spectrophotometer from 20 % to 100 % of the time, effectively doubles the reaction speed measured after 30 seconds. Interestingly, the reaction speed drops very fast initially at low irradiation, whereas there is a steadier decline at high irradiation. This could indicate that the product absorbs some of
a) b)
the photons otherwise destined to induce chelation. At high irradiation, more photons compensates for this if high irradiation causes some saturation. This seems to be the case, as it takes a 5-fold increase in irradiation to double the reaction speed. If no secondary processes takes place, the rate in abs./min can be converted to mM Cu2+/min, which also is shown on the figure. The rate is not high, but almost 5 % per minute after 2 minutes is acceptable at 25 °C and pH 7.5, where no chelation would take place without UV light.
Chelation rate of 200 µM Cu2+ from by 300 µM [24.31]adz at 25 °C
0 0.005 0.01 0.015 0.02 0.025
0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
Irradiation
ΔAbs./min. at 285 nm
0 2 4 6 8 10 12
µM Cu2+ /min.
30 s 60 s 120 s 240 s 360 s 900 s 2700 s
Figure 4.17: Chelation rate of Cu2+ by [24.31]adz as a function of irradiation at pH 7.5. A solution of Cu(ClO4)2 and a solution of [24.31]adz were mixed to concentrations of 200 µM and 300 µM respectively. pH was adjusted by adding NaOH to the adamanzane solution so that the pH after mixing with the copper solution was 7.5. Full spectra were measured at regular intervals in a manner so the shutter of the diode array spectrophotometer was open approximately the stated percentage of time. The rate was measured as the change in absorption at 285 nm at certain time intervals.
The rate of µM copper chelated per minute is shown on the second y-axis. The x-axis show the percentage of the time the shutter of the spectrophotometer was open. Supplementing figures are shown in appendix C.
All samples had their full time spectra measured after addition of HClO4 to remove any pH effects on the spectra (figure 4.18). The adjusted full time spectra confirm that chelation is affected by irradiation but they also show that the most irradiated samples have an absorption peak at a lower wavelength than the lesser
irradiated samples. The addition of HClO4 ensured that this could not be a result of hydroxide as 5th ligand, and since the lowering of the peak was proportional to irradiation, it could be a side effect of UV irradiation.
UV spectra after 1 hour's incubation and HClO4
0 0.1 0.2 0.3 0.4 0.5 0.6
200 250 300 350 400
nm
Absorption
100%
75%
50%
20%
3%
0.3 % water
HO-Figure 4.18: Final UV absorption spectra of the samples (120 µL) from figure 4.17 after addition of 30 µL 10 mM HClO4 resulting in final concentrations of: ctotal copper = 160 µM; ctotal adz = 240 µM and volume of 150 µL. The water spectrum is the calculated spectrum of 160 µM [Cu([24.31]adz)H2O]2+. The HO- spectrum is 160 µM
[Cu([24.31]adz)HO]+.
4.3.4 Blocking UV-light with λ < 220 nm prevents the secondary process
To investigate the secondary process, a solution of [Cu([24.31]adz)]2+ was irradiated in a spectrophotometer for en hour, with or without a UV filter with a cut-off at 220 nm (figure 4.19). When the filter is in place nothing happens, but without the filter, the spectrum peak is shifted to a lower wavelength and increased.
4.3.5 Only UV light induces chelation of Cu2+ to [24.31]adz
To distinguish the effect of irradiation by its wavelength, light filters were placed between the light source and the samples. An object glass with a cut off at about 300 nm and the side of a 4 mL disposable cuvette from Ratiolab with a cut off at about 280 nm, were used as UV block filters. One hour with irradiation through the object glass resulted in a 6 % higher chelation, and the plastic cuvette side in a 14 % higher chelation than a control kept in darkness. A sample kept in an Eppendorff tube for one hour had a chelation of 96 % of the control, indicating that Cu2+ might stick to the sides of Eppendorff tubes. Since almost no chelation is expected to take place at room temperature without light induction, the absorption of the control should be the calculated value unless the concentration of the (identical) samples were slightly higher than intended or unless the 0.5 second irradiation during measurement is sufficient to explain the deviance.
Effect of 1 h irradiation with shortwaved UV-light
0 0.1 0.2 0.3 0.4 0.5 0.6
200 250 300 350 400
nm
Absorption
F 10 s F 1800 s F 3600 s 10 s 1800 s 3600 s
Figure 4.19: Final UV absorption spectra of 200 µM [Cu([24.31]adz)]2+ irradiated by a diode array spectrophotometer.
The orange lines show the samples irradiated with a 2 mm 50 mM NaBr UV filter, cut-off at 220 nm, and the blue lines show the samples irradiated with unfiltered light.
Absorption at 285 nm after 1 hour at room temp.
0 0.07
Object glas filter
Plastic filter Epp. tube dark
Cuvette dark
Absorption
Calculated absorption
before incubation and
measuring
Figure 4.20: Absorption of 0.10 mM Cu2+ and 0.15 mM [24.31]adz at pH 7 after 1 hour incubation, either in darkness or irradiated through a UV filter. Filter cut-off: object glass - 300 nm; plastic cuvette side - 280 nm.