20Control
7.2. Effect of nitrogen substitution of aggregation
Chapter 7 109
110 Facile synthesis of spindles, rods and niddles from HPBI analogues: AIEE from ESIPT
benzene ring also brought changes in the responses of dye towards environmental factors like temperature [284], ions [288], micro-heterogeneous medium like β-CD [218], micelle, BSA [102] etc. With rise in temperature, the population of cis-enol of HPBI decreases and the nonradiative decay of tautomer increases [284]. Nitrogen substitution in the benzene ring increases the nonradiative deactivation to the ground state. Nitrogen substitut io n changes the response of the fluorophores towards Cu2+ ion. The 140 folds increase in intensity ratio of HPBI decreased to 2-3 folds upon pyridyl nitrogen substitution [288]. The pyridyl nitrogen substituted molecules showed more affinity towards β-CD compared to HPBI [218]. All the three molecules donate the proton to DMSO to form monoanion. The presence of extra nitrogen stabilizes the monoanion of HPIP-b and HPIP-c and makes them more sensitive to environment than HPBI. HPBI and HPIP-c bind in the hydrophobic site of BSA [102]. On the other hand, HPIP-b binds at the hydrophilic site of BSA. Though both HPBI and HPIP-c bind at hydrophobic sites, their binding pockets and interaction with BSA are different. Therefore, the aggregation of HPIP-b and HPIP-c is investigated. The biocompatibility and utility of all the fluorophores as aggregates within cells are discussed in this chapter.
7.2.1. Absorption characteristics:
Figure 7.2.1: Absorption spectra of HPIP-b in water (green line) and of reprecipitate mixture (dotted line) after 13 days. Inset shows the expanded spectra of HPBI in water and aggregated mixture in the 350 nm-450 nm region.
0 0.2 0.4 0.6 0.8 1
290 340 390 440 490
A bs or ban ce
Wavelength (nm)
0 0.01 0.02 0.03 0.04
390 410 430 450 470
Absorbance
Wavelength (nm) Aggregate
In water
0 days 13 days
Chapter 7 111
Alike HPBI, the pyridoimidazoles also experienced increase in the absorbance for the re-precipitation mixture initially [Figure 7.2.1 and 7.2.2]. The instant increase in absorbance was due to presence of methanol rich environment as explained in earlier section. With increase in time, it started decreasing with a bathochromic shift. The long tailing due to Mie scattering was also observed. The absorption spectra of HPIP-b and HPIP- c are red shifted by 16 nm and 2 nm, respectively. The absorption spectra of the aggregated structures became less structured. The decreased absorbance, less structured absorption band and scattering in the longer wavelength indicated the formation of particles through aggregation. But from the absorbance point of view, the decrease in absorbance for HPIP-c with time is 1.3 fold whereas for HPIP-b, the absorbance decreases by 2 fold. Compared to these nitrogen substituted analogous, HPBI undergoes more decrease (3 fold) in absorbance.
This gives clear indication that N substitution of HPBI has effect on the aggregate formatio n.
Figure 7.2.2: Absorption spectra of HPIP-c in water (Red line) and of reprecipitate mixture (dotted line) after 13 days. Inset shows the expanded spectra of HPBI in water and aggregated mixture in the 400 nm-450 nm region.
0 0.1 0.2 0.3 0.4 0.5 0.6
260 310 360 410 460
Int ens it y (a .u. )
Wavelength (nm)
0 days
13 days
0 0.01 0.02 0.03 0.04
390 400 410 420 430
Absorbance
Wavelength (nm) In water Aggregate
112 Facile synthesis of spindles, rods and niddles from HPBI analogues: AIEE from ESIPT
7.2.2. Fluorescence characteristics:
Both HPIP-b and HPIP-c have weak emission in aqueous medium. Upon aggregation, the fluorescence of both fluorophores have impressive enhancement. From the Figures 7.2.3 and 7.2.4, it is obvious that aggregation of the fluorophores led to the enhancement in the emission intensity. The enhancement varies depending on the position of the substitution. The ratio of the tautomer emission to normal emission of HPIP-b was 2.5 and 1.04 in the monomeric and aggregated states. The same of HPIP-c is 5.0 and 3.33 in the monomeric and aggregated states. In contrast, ratio of the tautomer emission to normal emission increases upon aggregation of HPBI (2.6 to 3.1). Alike to absorbance, this shows the effect of pyridyl nitrogen substitution.
Figure 7.2.3: Fluorescence spectra of HPIP-b in (a) water and (b) 2% v/v water methano l re-precipitation mixture. (λexc = 320 nm)
0.0E+0 8.0E+4 1.6E+5 2.4E+5 3.2E+5 4.0E+5
340 400 460 520 580 640
Int ensi ty (a. u. )
Wavelength (nm) (a)
(b)
Chapter 7 113
Figure 7.2.4: Fluorescence spectra of HPIP-c in (a) water and (b) 2% v/v water methano l re-precipitation mixture. (λexc = 310 nm)
7.2.3. Lifetime analysis:
Figure 7.2.5: Fluorescence decay of normal emission of HPIP-b (b) in water, (c) in reprecipitate solution and HPIP-c (d) in water, (e) reprecipitate solution, (a) is the instruments response function with residuals.
0.0E+0 4.0E+4 8.0E+4 1.2E+5 1.6E+5 2.0E+5
320 380 440 500 560
Int ensi ty (a. u. )
Wavelength (nm) (b)
(a)
5.0E+0 5.0E+1 5.0E+2
0 5 10 15 20
Counts
Time (ns)
HPIP-c
1.0E+0 1.0E+1 1.0E+2 1.0E+3
0 5 10 15 20 25
Counts
Time (ns)
HPIP-b
(a) (b) (a)
(c)
(d) (e)
-6 -3 0 3 6
0 200 400 600 800 1000 1200
Time (ns) -8
-4 0 4 8
0 100 200 300 400 500 600 700 800
Time (ns)
114 Facile synthesis of spindles, rods and niddles from HPBI analogues: AIEE from ESIPT
Table 7.2.1. Fluorescence lifetime of the monomer and the aggregates of HPIP-b and HPIP- c.
Fluorophore
τ
Nτ
Tχ
2Relative Enhancement in lifetime with respect to
monomer
τ
Nτ
THPIP-b Aggregate 0.92 (36.24) 2.51 (63.76) 1.0
51 % 4 %
Monomer 0.61 (42.46) 2.41 (57.54) 1.0 HPIP-c Aggregate 0.62 (27.83) 3.06 (72.17) 1.1
5 % 23 %
Monomer 0.59 (26.6) 2.48 (73.4) 1.0
The lifetimes of monomer and the aggregates of HPIP-b and HPIP-c are summar ized in the Table 7.2.1. Unlike that of HPBI, the fluorescence lifetime of both the emissio ns increases upon aggregation [Figure 7.2.5]. Hence, not only the cis enols but the Trans enols are also present in the aggregates. But the relative enhancement in lifetime is much higher in the normal emission of HPIP-b whereas that of tautomer is little. The reverse is true for HPIP-c. It is clear that like in TICT here too the presence and the position of the pyridyl nitrogen plays a significant role.
7.2.4. Morphology:
Fluorescence microscopic images showed that the aggregates emit strong fluorescence upon excitation with 350 nm light [Figure 7.2.6]. HPIP-b aggregated to form long needle type structures which showed green fluorescence. These needles are appeared to be originating from one center. Unlike to HPIP-b for another nitrogenous analogue HPIP- c, small rods were formed and emits green color.
Chapter 7 115
Figure 7.2.6: Fluorescence microscopy images of HPIP-b and HPIP-c after 10 min of re- precipitation observed under white light [right] and excited with UV light (λex=350 nm) [left].
Figure 7.2.7: FESEM images of the samples made from aqueous suspensions of HPIP-b (left side) and HPIP-c (right side) after re-precipitation in water with 2% v/v methanol after 24 hours.