fluorescence switch on-off-on receptor constructed … calix[4]arene for selective recognition of...

Fluorescence switch on-off-on receptor constructed on quinoline

allied calix[4]arene for selective recognition of Cu2+

from blood

serum and F- from industrial waste water

Pinkesh G Sutariya

a, Alok Pandya

a, Anand Lodha

b and Shobhana K Menon

ab*

*aDepartment of Chemistry, *

b Department of Forensic Science, School of Sciences

Gujarat University, Navrangpura,

Ahmedabad 380009, Gujarat, India.

[email protected]

Contents

1. Materials and method

2. Absorption and Luminescence

3. Binding Study

4. Real sample preparation

5. Synthesis procedure

6. Binding constant curve for Cu2+

and F-………………………………………S1-S2

8. Absorption spectra of TDQC ligand with Cu2+

and F--……………………….S3-S4

9. Absorption spectra of TDQC ligand with Cu2+

and F- at various concentrations.S5-S6

10. pH study of TDQC ligand with Cu2+

and F- ………………………………………..S7-S8

11. Job’s plot obtained from the absorption titration of TDQC with Cu2+

and F-…S9-S10.

12. Competitive emission spectra of TDQC ligand with Cu2+

complex with other cations. S11

13. FT-IR spectra of TDQC…………………………………………………………………… S12

14. Proposed binding mechanism through hydrogen bonding and electro static interaction with

TDQC ligand by Cu2+

and F-- …………………………………………………………….. S13-S14

15 Real sample analysis table…………………………………………Table S1- S2

16. Comparison of proposed TDQC fluorescence sensor with various previously reported Cu2+

and

F- determination methods…………………………………………………….. Table S3

Electronic Supplementary Material (ESI) for AnalystThis journal is © The Royal Society of Chemistry 2013

mailto:[email protected]

1. Materials and methods

All the reagents and chemicals like DCC (N,N'-Dicyclohexylcarbodiimide), DMAP (4-

Dimethylaminopyridine) and 1-pyrenebutyric acid used were of analytical grade purchased from

sigma aldrich. Silica gel (Merck, 0.040-0.063 mm) was used for column chromatography. Metal

salts used for the titrations were their perchlorate salts (Caution: since perchlorate salts are

known to explode under certain conditions, these are to be handled carefully!) with formula,

M(ClO4)2.xH2O.Melting points were taken on Opti-Melt (Automated melting point system). The

FT-IR spectra were recorded as KBr pellet on Bruker TENSOR-27 in the range of 4000-400 cm-

1. Discover BenchMate system-240 V (CEM Corporation) microwave synthesizer was used for

synthesis. 1H NMR spectra was scanned on 400 MHz FT-NMR Bruker Avance-400 in the range

of 0.5 ppm -15 ppm and 13

C NMR spectra was recorded on a Bruker DPX-300 spectrometer

using internal standard tetramethylsilane (TMS) and deuterated DMSO as a solvent in the range

of 0.5 ppm to 250 ppm. ESI Mass spectra were taken on a Shimadzu GCMS-QP 2000A.

Emission spectrum was recorded on Horiba Jobin, Fluorolog, and Edinburgh F900. UV–Vis

absorption spectra were acquired on a Jasco V-570 UV–Vis. spectrometer. Working standard

solutions were prepared daily in deionized water.

2. Experimental

2.1 Synthesis of compound A: Microwave assisted synthesis of p-tert-butylcalix[4]arene (A)

A mixture of p-tert-butyl phenol (4.0 g, 0.33 mM), sodium hydroxide (NaOH) (1 g) and

formaldehyde(1.8 ml,0.18 mM) solution was taken in an open vessel and was irradiated with 50


W power in a microwave synthesizer Discover(CEM)by stirring for 3 min. Cooling for 10 min,

resulted in yellow solid mass. Next, 4 ml of toluene and 30 ml of diphenyl ether were added in

this yellow solid, again irradiated with microwave power of 100 W for 5 min with stirring and

obtained a dark brown solution. Further, this solution was added in to 75 ml of ethyl acetate and

kept for 2 h. Finally, white precipitate was obtained which was filtered and washed with ethyl

acetate and finally dried. Yield, 3.5 g (96%). Elemental analysis for C44H56O4

:Calcd.C,81.44;H,8.70% Found:C,81.11;H,8.56%. FT-IR (KBr) ʋ: 3230cm-1

(Ar-CH), 3450cm-

1(Ar-OH).

1HNMR: δH (CDCl3, 500 MHZ): 1.18(36H, t-butyl, s), 3. 81 (8H, ArCH2Ar, s), 7.12

(8H,s,Ar-H), 9.71(4H, Ar-OH, s). ESI-MS (m/z) 648 (M+1).

2.2 Synthesis of compound B

A mixture of p-tert-butylcalix[4]arene 1 (3.5 g. 0.80 mM), K2CO3(1.9 g, 14.0 mM) and 1-

iodomethane (4 ml, 14.0 mM) in dry acetone (150 ml) was stirred for 24 hrs . The actual reaction

time was considered by taking thin layer chromatography (TLC) at regular interval of time by

using mixture of ethylacetate:hexane (8:2). The solvent was then evaporated under vacuum and

the residue taken up with CH2Cl2 .The organic phase was washed with 0.1 M HCl up to

neutrality and dried over anhydrous Na2SO4 . After complete evaporation of the solvent, the

resulting crude product was purified by column chromatography (silica gel, hexane: ethyl acetate

1); 2.9 g, yield (81%). Elemental analysis for C46H60O4 : C,81.61; H,8.93 % Found: C,81.38;

H,8.52 %. FT-IR (KBr) ʋ: 3280cm-1

(Ar-CH), 3430cm-1

(Ar-OH). 1H NMR: δH

(CDCl3,500MHZ), 1.20 (18H, t-butyl, s) 0.96(18H, t-butyl, s), 4.28 (4H, -OCH3, t), 3.83 (4H,

ArCH2Ar ,d), 4.30(4H, ArCH2Ar, d), 6.42(4H, Ar-H, s), 6.85(4H, Ar-H, s), 9.19 (2H, OH, s),

m.p. 223-228OC. ESI MS (m/z) 677.1 (M+1).

2.3 Synthesis of compound C


In a solution of p-tert-butylcalix[4]arene (A) (2.60 g, 2 mmol) in freshly distilled acetone (80

mL), ethyl bromo acetate (1.86 g, 5 mmol) and K2CO3 (0.70 g, 2.5 mmol) were added and the

reaction mixture was heated at reflux temperature for 20 h under inert atmosphere. The solution

was then allowed to cool to room temperature and evaporated to dryness by rotary evaporation.

The residue was then triturated three times with ethanol (40 mL each time) and filtered off the

compound as white solid, which was dried overnight in high vacuum for overnight. Yield: 2.24

g, (71 %). Elemental analysis for C50H64O8: C, 75.72; H, 8.13% Found: C, 74.89; H, 7.95 %

FT-IR (KBr) ʋ: 3200cm-1

(Ar-CH), 1680cm-1

(-C=O). 1H NMR (500 MHz, CDCl3): δ = 7.23 (s,

4H, Ar-H), 6.81 (s, 4H, Ar-H), 4.52 (s, 4H, –OCH2CO), 4.32 (d, 4H,J= 13.2 Hz, ArCH2Ar), 3.85

(s,6H,–OCH3), 3.34 (d, 4H, J= 13.2 Hz, ArCH2Ar), 1.24 (s, 18H, –C(CH3)3), 0.92 (s, 18H, –

C(CH3)3) ppm. m.p. 215-217OC ESI-MS (m/z) 832.13.

2.4 Synthesis of compound D:

A solution of compound C (1 g, 0.0012 mol) and 2.5 ml of hydrazine hydrate in 15-20 ml of

absolute ethanol was refluxed for 6-8 hrs. The solvent was evaporated and crude was crystalized

with ethanol. The white product was then dried over Na2SO4. Yield 75% Elemental analysis for

C48H64N4O5 : C,74.19; H,8.30; N,7.21 % Found: C,74.11; H,8.12; N,6.90 % FT-IR (KBr):

3260cm-1

(-NH), 3350cm-1

(-CH), 1710cm-1

(-C=O). 1H NMR (400 MHz, CDCl3): δ = 7.23 (s,

4H, Ar-H), 6.81 (s, 4H, Ar-H), 4.32 (d, 4H, J= 13.2 Hz, ArCH2Ar), 3.85 (s,6H,–OCH3), 3.34 (d,

4H, J= 13.2 Hz, ArCH2Ar), 8.17 (s, 2H, –CONH), 4.31 (s, 4H, –NH2),1.24 (s, 18H, –C(CH3)3),

0.92 (s, 18H, –C(CH3)3) ppm. m.p. 198-200OC ESI-MS (m/z) 817.12 (m+1)

2.5 Synthesis of compound E:

A mixture of compound D (1 g, 0.001mol) and 3-quinoline carboxylic acid (1.48 g, 0.004 mol)

were taken into anhydrous tetrahydrofuran (20-25 ml) and stirred this solution for 15 minutes.


Then N, N'-Dicyclohexylcarbodiimide (DCC) (1.06 g, 0.004 mol) and catalytic amount of 4-

Dimethylaminopyridine (DMAP) were added into reaction mixture. This reaction mixture was

stirred at room temperature for 48 hours. The reaction progress was monitored by tlc in

chloroform:methanol (7:3). After the completion of reaction, solvent was evaporated by rotary

evaporation. The crude product was then washed with 1N HCl followed by NaHCO3 for the

removal of unreacted 3-quinoline carboxylicacid and compound D. Then product was crystalized

with dichloromethane. Yield 72% Elemental analysis for C70H78N6O8 : C,74.31; H,6.95;

N,7.43 % Found: C,74.18; H,6.68; N,7.25 % FT-IR (KBr)ʋ: 3380cm-1

, 3120 cm-1

(-NH2), 2970

cm-1

(-CH), 1700cm-1

(-C=O). 1H NMR (400 MHz, CDCl3): δ = 1.24 (s, 18H, –C(CH3)3), 0.92 (s,

18H, –C(CH3)3) , 3.81 (s,6H,–OCH3), 4.26 (d, 4H, J= 13.2 Hz, ArCH2Ar), , 3.45 (d, 4H, J= 13.2

Hz, ArCH2Ar), 7.25 (s, 4H, Ar-H), 7.82 (s, 4H, Ar-H), 8.29(s, 2H, –CONH), 8.32(s, 2H, –

CONH), 7.84-8.78 (m, 12H, Ar-H), m.p. 128-130OC ESI-MS (m/z) 1132.7 (m+1) and 1196.7

(m+Cu2+

).

3. Absorption and luminescence

Absorption spectra of lower rim substituted calix[4]arene di-amidoquinoline (TDQC) was

recorded in acetonitrile and the data are given in experimental section. This compound shows

absorption band in the region between 290-390 nm, the band at 307 nm indicates π- π* transition

of quinoline system. This compound shows a strong luminescence band at 476 nm in acetonitrile

with excitation at the absorption maxima (λmax) of the quinoline moiety , which is at 370-380 nm.

The fluorescence study has been carried out at λex= 410 nm, λem= 476 nm.

4. Ion-binding study


Stock solutions of the complex TDQC (1 x 10-9

M) and that of perchlorate salts (1 x 10-7

M) of

various metal ions (Zn2+

, Cd2+

, Fe2+

, Hg2+

, Na+, K

+, Cu

2+, Ni

2+, Mn

2+, Cr

3+, Pb

2+,Sr

2+, Ce

3+,Co

2+,

Ca2+

, Mg2+

, Ba2+

, Cs+, Rb

+, Ag

+, Be

2+, V

5+, Th

4+ and Fe

3+ ) were prepared in freshly purified

acetonitrile. Then 2 mL stock solution of the complex and 2 mL stock solution of each metal

salts were taken in a 5 mL volumetric flask, so that the effective concentration of the complex is

1 x 10-9

M and that of the metal ions are 1 x 10-7

M (100 fold). The spectra of the cation added

solutions were compared with that of the original solution to ascertain the interactions of the

metal ions with the ionophore. For emission titration study, the same stock solutions of the

complexes were used and the metal perchlorate solutions of desired concentration (1.0 – 100.0

equivalents) were prepared by proper dilution of the stock solution .The ion-binding property of

fluoroionophore TDQC was investigated with a large number of cations as their perchchlorate

salts(Zn2+

, Cd2+

, Fe2+

, Hg2+

, Na+, K

+, Cu

2+, Ni

2+, Mn

2+, Cr

3+, Pb

2+,Sr

2+, Ce

3+,Co

2+, Ca

2+, Mg

2+,

Ba2+

, Cs+, Rb

+, Ag

+, Be

2+, V

5+, Th

4+ and Fe

3+ ) and anions (F

-, Cl

-, Br

-, I

-, CH3COO

- and H2PO4

-

) as their tetrabutyl ammonium salts in acetonitrile. Then solution for Cu2+

and F- were prepared

in the concentration range of (0-100 nM) with 1×10-9

M concentration of TDQC ligand. The ion-

recognition process was monitored by luminescence, UV-Vis and ESI-Mass spectral changes.

According to this procedure, the fluorescence intensity (F) scales with the metal ion

concentration ([M]) through (F0-F)/(F-Fœ) = ([M]/Kdiss)n. The binding constant (Ks) is obtained

by plotting Log [(F0-F)/(F-Fœ)] vs. Log [M], where F0 and Fœ are the relative fluorescence

intensities without addition of guest metal ions and with maximum concentration of metal ions

(when no further change in emission intensity takes place), respectively. The value of Log [M] at

Log [(F0-F)/(F-Fœ)]= 0 gives the value of log(Kdiss), the reciprocal of which is the binding

constant (Ks).


5. Real sample preparation

For the analytical application of proposed fluorescence probe we have applied this probe for real

sample analysis in blood serum for Cu2+

and industrial waste water for F-. The blood sample was

allowed to clot and serum was obtained by centrifugation at 3000 rpm for 30 minutes. We have

used HEPES buffer for this experiment and blood serum sample was stored at -150

C. The blood

sample was diluted upto 1000 fold and analyzed by present sensor. Pipet 2.0 ml. of serum into a

small test tube, add 1.0 ml. of ascorbic acid solution, mix and allow to stand let stand for 5 min.

Add 1.0 ml. of 20% tnichioroacetic acid and 1 ml. of chloroform. Stopper and shake vigorously

for 10-45 sec. Centrifuge for 10 min. Carefully decant the supernatant into another small test

tube; leave behind a supernatant protein. The solution should be water clean; if not, it is

recentrifuged. Now, take 1 ml of TDQC + 0.5 ml. of ascorbic acid solution + 0.5 ml. of 20%

trichioroacetic acid2. This titration has been done by taking an in situ generated Cu

2+ complex of

TDQC with blood serum. Furthermore, the proposed sensor was successfully used in the

determination of fluoride in spiked water samples. The waste water samples (100 ml) were

collected from industrial water (vatava) where the amount of fluoride is much more than 2

ppm. The fluoride containing water sample was subjected for extraction procedure. Our

compound was soluble in chloroform as well as in acetonitrile but as acetonitrile is miscible

with, the TDQC was dissolved in chloroform to prepare solution for analysis. Then in separating

funnel, we took 60 ml of ligand solution and 40 ml of water sample and shake for half an hour.

The organic layer was separated and dried with anhydrous sodium sulphate. The organic extract

was made up to 100 ml and measured the fluorescence intensity to find out the concentration of

fluoride in organic layer. The results of real sample analysis are given in Table S1-S2.


Fig. S1 Binding constant plot for Cu2+

with TDQC ligand from emission titration.


Fig. S2 Binding constant plot for F- with TDQC ligand from emission titration.

Fig. S3 Absorption spectral changes of TDQC (1×10-6

M) ligand in the presence of Cu2+

(1×10-

6M).

307 nm

351 nm


Fig. S4 Absorption spectral changes of TDQC (1×10-6

M) ligand in the presence of F- (1×10

-6M).

Fig. S5 The plot demonstrates the absorption spectral changes of TDPC in the presence of

different concentrations of Cu2+

(200 nM, 175 nM, 150 nM, 125 nM and 100 nM)

307 nm 383 nm

200 nM

100 nM

L L + F-


Fig. S6 The plot demonstrates the absorption spectral changes of TDQC in the presence of

different concentrations of F- (200 nM, 175 nM, 150 nM, 125 nM and 100 nM)

Fig. S7 Shows the effect of fluorescence intensities of TDQC with Cu2+

complex varying pH.

200 nM

100 nM


Fig. S8 Shows the effect of fluorescence intensities of TDQC with F- complex varying pH

Fig.S9 Job’s plot obtained from the absorption titration of TDQC with Cu2+

.

300

800

1300

1800

2300

2800

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Rel

ati

ve

Inte

nsi

ty

pH

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0.18

0.2

0 0.2 0.4 0.6 0.8 1

Ab

s.

nM

Mole fraction; (nM)


Fig.S10 Job’s plot obtained from the absorption titration of TDQC with F-.

Fig. S11 Competitive emission spectra of TDQC (1×10-6

M) with Fe3+

in presence of other

cations (a = Ligand + Cu2+

, b = a + Zn2+

, C = a + Cd2+

, D = a + Fe2+

, E = a+ Hg2+

, F = a+ Na+,

G = a+ K+ , H = a + Fe

3+, I = a+ Ni

2+, J = a + Mn

2+, K = a + Cr

3+, L = a + Pb

2+,M = a + Sr

2+,N =

0

0.05

0.1

0.15

0.2

0.25

0.3

0 0.2 0.4 0.6 0.8 1

Ab

s.

nM

Mole fraction; (nM)

0

5000

10000

15000

20000

25000

30000

35000

40000

Lig

an

d a b c d e f g h i j k lm n o p q r s t u v w x y

Re

lati

ve

In

ten

sity


+ Ce3+

,O = a + Co2+

, P = a + ca2+

, Q = a + Mg2+

, R = a + Ba2+

,S = a + Cs+ ,T = a + Rb

+ and U=

a + Ag +

)

Fig.S12 FT-IR spectra of TDQC ligand showing absence of –OH group.

Fig. S13 Proposed binding mechanism of Cu2+

with TDQC ligand through electrostatic

interaction.


Fig. S14 Proposed binding mechanism of F- with TDQC ligand through hydrogen bonding.

Table S1: Results of the determination of Cu2+

in blood serum.

Sample Spiked

Cu2+

(nM)

Found by

AAS

(nM)

Found

by proposed

sensor

(nM)

Recovery (%)

Blood serum 1 0 7.6 7.4 ---------

Blood serum 2 10 18.1 18.2 104.58 ± 1

Blood serum 3 20 30.8 31.7 115.69 ± 2

Blood serum 4 30 40.3 39.8 106.41 ± 4

Blood serum 5 100 110.4 109.7 102.41 ± 3


. Table S2: Results of the determination of F- in industrial waste water samples.

Method Recognized

ion

Linear

range

Limit of

detection

Reference

Foating organic drop

micro extraction

Cu2+

1.0–25.0 ng

mL−1

0.4 ng mL−1

1

Colorimetric

determination

Cu2+

0.16 to 32

µM

5.0 µg g-

2

Flame atomic absorption

spectroscopy

Cu2+

0.01–15 μg

ml−1

0.004 μg ml−1

3

High-resolution

continuum

source spectrometer

F-

0.03 mg L−1

0.041± 0.002 mg

L−1

4

High-resolution

molecular absorption

spectrometry with

electrothermal

vaporization

F- 0.5 and 25

mg L−1

0.16 mg L−1

5

Fuorimetric flow

injection

F- (1.0–50.0) ×

10−6

M

1.0 × 10−6

M 6

Present method Cu2+

and F- 0-100 nM 4.16 nM and

2.15 nM

--------

Table S3: Comparison of proposed TDQC fluorescence sensor with various previously reported

Cu2+

and F- determination methods.

Sample Spiked

F-

(nM)

Found

F-

(nM)

Recovery (%)

Water sample 1 0 11.5 ---------

Water sample 2 10 22.4

106.04 ± 3

Water sample 3 20 32.7 103.80 ± 2

Water sample 4 30 44.6 107.46 ± 2

Water sample 5 100 116.4 104.39 ± 1


References:

1. C. I. Arpa., S. I.Tokgöz, Analytica Chimica Acta ., 2010, 667, 83–87.

2. L.Feng, Y.Zhang, L. Wen, Z. Shen, Y. Guan., Talanta, 2011, 84, 913–917.

3. Z. Es’haghi, R. Azmoodeh, Arabian Journal of Chemistry, 2010, 3, 21–26.

4. H. Gleisner, B. Welz, J. W. Einax, Spectrochimica Acta Part B, 2010, 65, 864–869.

5. S. Morés, G. C. Monteiro, F. da. S. Santos, E. Carasek, B. Welz., Talanta, 2011, 85,

2681–2685.

6. M. Nakaya, M. Oshima, T. Takayanagi, S. Motomizu, H. Yamashita.,Talanta, 2011,

84,1361–1365


fluorescence switch on-off-on receptor constructed … calix[4]arene for selective recognition of...

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