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Article 13

Anion-Caffeine Interactions Studied by C and H NMR and ATR-FTIR Spectroscopy 1

Nicolas O. Johnson, Taylor Patrick Light, Gina MacDonald, and Yanjie Zhang J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b12150 • Publication Date (Web): 20 Jan 2017 Downloaded from http://pubs.acs.org on January 31, 2017

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The Journal of Physical Chemistry

Anion−Caffeine Interactions Studied by

13

C and 1H NMR and ATR−FTIR

Spectroscopy

Nicolas O. Johnson, Taylor P. Light, Gina MacDonald, Yanjie Zhang* Department of Chemistry and Biochemistry, James Madison University, Harrisonburg, Virginia 22807, United States

*The author to whom correspondence should be directed. E−mail: [email protected]

1 ACS Paragon Plus Environment


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ABSTRACT This work investigates the interactions of a series of 11 anions with caffeine by utilizing 13

C and 1H NMR and attenuated total reflectance Fourier transform infrared (ATR−FTIR)

spectroscopy. The aim of this study is to elucidate the molecular mechanisms of ion interactions with caffeine and to study how these interactions affect caffeine aggregation in aqueous solution. The chemical shift changes of caffeine 13C and 1H in the presence of salts provide a measure for anions’ salting−out/salting−in abilities on individual carbon and hydrogen atoms in caffeine. The relative influences of anions on the chemical shift of individual atoms in the caffeine molecule are quantified. It is observed that strongly hydrated anions are excluded from the carbons on the six-member ring in caffeine and promote caffeine aggregation. On the other hand, weakly hydrated anions decrease caffeine aggregation by accumulating around the periphery of the caffeine molecule and binding to the ring structure. The ATR−FTIR results demonstrate that strongly hydrated anions desolvate the caffeine molecule and increase aggregation, while weakly hydrated anions have the opposite effects and salt caffeine into solution.


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INTRODUCTION The Hofmeister series ranks ions’ ability to affect the behaviors of various processes in aqueous solution.1-6 Protein crystallization,7 enzyme activity, 8-11 protein folding, 12 polymer aggregation,13-16 and micelle formation17 are a few examples among many others that have been found to follow the Hofmeister series. The typical order for the anion series is as follows: 3,4 CO32− > SO42− > S2O32− > H2PO4− > F− > Cl− > Br− ~ NO3−> I− > ClO4− > SCN− Anions on the left are strongly hydrated and usually decrease protein solubility and precipitate proteins out of solution. Anions on the right are weakly hydrated and salt proteins into solution. Chloride is considered as the dividing line between the two groups of anions. In recent years, tremendous efforts have focused on understanding how the presence of Hofmeister ions influences chemical and biological systems;18-35 However, spectroscopic evidence is needed to reveal molecular level interactions of ions with proteins, polymers, surfactants, and other analytes. Recent 1H NMR studies have enhanced the understanding of ion interactions with peptides, polymers, and small organic molecules.36-46 Weakly hydrated anions such as SCN− can bind directly to peptide or polymer backbone including the amide nitrogen and adjacent α−carbon.36,39 Strongly hydrated anions such as SO42− are repelled from peptide backbone and sidechains.36 Proteins and peptides are complex molecules containing a large variety of functional groups and ion-molecule interactions. Caffeine consists of two fused aromatic rings, two carbonyl groups, and four nitrogens, three of which have methyl substituents (Figure 1a). Caffeine has a good mix of functional groups to study multiple types of ion−molecule interactions without the complexity associated with proteins. Caffeine has limited solubility in water at room temperature due to the presence of three hydrophobic methyl groups and hydrophobic flat plane structure.47-52 Caffeine has a typical concentration dependent self-



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association in water.48,53 FTIR and 1H NMR have previously been used to study the aggregation of caffeine in the absence of cosolutes.54,55 However, spectroscopic studies on how salts influence caffeine aggregation have not be performed. The combination of 13C and 1H NMR spectroscopy provides a more complete molecular picture of ion-molecule interactions. Moreover, ATR−FTIR spectroscopy supplies information about ions’ influence on solvation and aggregation of proteins and peptides in aqueous solution.56-58 In this work, we conduct 13C and 1H NMR and ATR−FTIR experiments to explore the important anion−caffeine interactions that alter caffeine aggregation. We found different anion−caffeine interactions for strongly hydrated and weakly hydrated anions. Strongly hydrated anions are excluded from the carbon atoms on the ring that have no bounded hydrogen, namely C2, C4, C5, C6, resulting in a salting−out effect on caffeine. Weakly hydrated anions bind to these carbons to aid in salting caffeine into solution. The binding affinity of weakly hydrated anions to caffeine is evaluated. On the other hand, weakly hydrated anions accumulate around C1, C3, C7, and C8, while strongly hydrated anions do not significantly interact with these carbon atoms. To the best of our knowledge, these are the first studies employing 13C NMR to probe anion-molecule interactions. ATR−FTIR studies complement the NMR results to show that strongly hydrated anions desolvate caffeine molecules and promote aggregation while weakly hydrated anions solvate caffeine and decrease aggregation. Our recent studies have shown that anion effects on the thermodynamics of caffeine partitioning between aqueous and organic phases follow the Hofmeister series.59 Namely, strongly hydrated anions promote caffeine transfer from the aqueous to organic phase while weakly hydrated anions make the transfer process more difficult.59 The in−depth spectroscopic analysis presented herein provides a molecular level explanation for the mechanisms proposed from the thermodynamic studies.


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EXPERIMENTAL Caffeine and all of the sodium salts used in this study, including NaClO4, NaSCN, NaI, NaNO3, NaBr, NaCl, NaF, NaH2PO4, Na2S2O3, Na2SO4, and Na2CO3, were purchased from Fisher Scientific Inc. D2O for NMR and ATR−FTIR experiments was obtained from Cambridge Isotope Laboratories with 99.9% purity. 13C and 1H NMR spectra of caffeine were collected at different caffeine concentrations ranging from 1 to 100 mM in D2O. Anion effects on the chemical shift of carbon atoms and protons in caffeine were studied with 50 mM caffeine solutions containing various salts at concentrations of 0 to 2.0 M in D2O. 13C and 1H NMR analysis were performed on a Bruker TopSpin 400 MHz NMR spectrometer with an Avance III system at 25 °C. The system carried out locking, tuning, shimming, and gaining automatically before every spectrum. The chemical shift of the locking solvent (D2O) served as an internal reference for other nuclei.46 This is the default method in Bruker software.60 All the collected spectra were analyzed using Bruker TopSpin software (version 3.2). Caffeine solutions at concentrations ranging from 20 to 140 mM and 50 mM caffeine solution containing various salts at concentrations ranging from 0 to 1.3 M were prepared in D2O for infrared experiments. IR spectra were obtained on a Bruker Vertex 70 ATR-IR with a Harrick BioATR cell II (silicon crystal) equipped with a KBr beam, MIR source, and LN-MCT detector. Solution caffeine experiments were collected by co-adding 500 scans at 1 cm−1 resolution with a scanning velocity of 20 kHz, a Happ-Ganzel apodization, a phase resolution of 8, a Mertz phase correction, and a 2 mm aperture. Temperature was held constant at 25 °C using a Thermo Scientific HAAKE DC30-K10 circulating bath. IR spectra were analyzed with OPUS software (version 7.2). Salt-D2O absorbance spectra were subtracted from the respective caffeine-salt-D2O by eliminating the ~2480 cm−1 OD stretch. All infrared experiments were repeated in duplicate.



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RESULTS AND DISCUSSION The chemical structure and the numbering of carbon atoms in caffeine are shown in the inset of Figure 1a. Caffeine has eight 13C signals (three methyl carbons and five ring carbons) and four 1H signals (one attached to the ring and three methyl groups). Complete assignments of the 13C and 1H spectra for 50 mM caffeine in D2O are presented in Figure 1a and 1b, respectively. These assignments are well documented in the literature.61,62 13C and 1H spectra of caffeine were collected at caffeine concentrations ranging from 1 mM to 100 mM in D2O and the results are listed in Table 1. 1H peak positions do not shift significantly with increasing caffeine concentration in the low concentration range (1 to 10 mM). This result indicates that caffeine exists primarily in a monomeric form.62 13C has little signal at low caffeine concentrations (1 to 10 mM) and the 13C peaks for all the carbon atoms in caffeine can be observed at about 25 mM. Caffeine tends to self-associate in aqueous solution, especially when the concentration approaches the solubility limit of caffeine in water (~0.1 M at room temperature).47-50 At caffeine concentrations of 25 mM and above, we observe larger decrease in chemical shift of all the carbon atoms and protons as caffeine concentration is increased. The upfield shifts of the 13C and 1

H peaks with increasing caffeine concentration suggest that caffeine molecules form dimers and

higher order of aggregates by stacking of the molecules.55 13

C and 1H NMR studies were carried out with 50 mM caffeine solutions in D2O in the

presence of salts, including Na2CO3, NaH2PO4, Na2SO4, Na2S2O3, NaF, NaCl, NaBr, NaI, NaNO3, NaClO4, and NaSCN at varied salt concentrations. A concentration of 50 mM caffeine was chosen to conduct the experiments in order to consistently observe all peaks in the carbon spectra. The chemical shifts of individual carbon atoms and protons are uniquely altered in response to the added salts in solution (Figures 2, 3, and 4). The chemical shift change, Δδ,


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Figure 1.

13

C (a) and 1H (b) NMR spectra of 50 mM caffeine in D2O at 298 K with peak

assignments. The Chemical structure and atomic numbering of the protons and carbon atoms in caffeine molecule are shown in the inset.

Table 1. Chemical Shifts (ppm) of Proton and Carbon in Caffeine Molecule at Concentrations from 1 to 100 mM Conc. (mM) 1

C1

C3

C7

C8

C2

C4

C5

C6

H1

H3

H7

H8

-

-

-

-

-

-

-

-

3.27

3.45

3.87

7.81

5

-

-

-

-

-

-

-

-

3.25

3.43

3.86

7.80

10

27.89

29.81

-

143.51

-

-

-

-

3.24

3.42

3.85

7.80

25

27.87

29.79

33.40

143.47

152.50

148.25

107.67

156.16

3.21

3.38

3.83

7.79

50

27.85

29.76

33.39

143.43

152.29

148.09

107.45

155.89

3.17

3.34

3.80

7.78

100

27.82

29.73

33.37

143.38

152.07

147.92

107.22

155.59

3.12

3.29

3.77

7.76



0.1

0.1

(a)

(b)

0.0

Δδ (ppm)

Δδ (ppm)

0.0 -0.1 -0.2 -0.3

-0.1 -0.2 -0.3

-0.4 0.0

0.2

0.4

0.6

0.8

1.0

-0.4

1.2

0.0

Na2CO3 Conc. (M)

0.4

0.6

0.8

1.0

0.3

(c)

0.1

1.2

(d)

0.2

Δδ (ppm)

Δδ (ppm)

0.2

NaH2PO4 Conc. (M)

0.2

0.0 -0.1 -0.2

0.1 0.0 -0.1

-0.3

-0.2

0.0

0.2

0.4

0.6

0.8

1.0

1.2

0.0

Na2SO4 Conc. (M)

(e)

-0.1 -0.2 -0.3 -0.4 0.2

0.4

0.6

0.4

0.6

0.8

1.0

1.2

C1 C3 C7 C8 C2 C4 C5 C6

0.0

0.0

0.2

Na2S2O3 Conc. (M)

0.1

Δδ (ppm)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0.8

1.0

NaF Conc. (M)

Figure 2. 13C NMR chemical shift change as a function of salt concentration for caffeine in the presence of strongly hydrated anions: (a) Na2CO3, (b) NaH2PO4, (c) Na2SO4, (d) Na2S2O3, (e) NaF. Atomic numbering of caffeine is shown in the inset of Figure 1a.

in these figures represents the change in chemical shift of carbon atoms and protons in caffeine molecule in the presence of salt with respect to salt−free solutions. Figure 2 shows the effects of strongly hydrated anions on the chemical shifts of 13C in caffeine molecule. It can be seen that the chemical shifts change linearly as a function of salt concentration for all strongly hydrated anions investigated. All the linear trends in Figure 2 can be fitted by a simple equation: 8 ACS Paragon Plus Environment

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∆ = [ ]

(1)

where c is the line slope with units of ppm per molar concentration and [M] is the salt concentration. The fitting parameters by eq 1 for all the anions are compiled in Table S1 (Supporting Information). The effects of anions on individual carbons can be readily observed in Figure 2. The trends in chemical shift changes for the carbon atoms can be divided into two distinct groups. The first group includes the carbon atoms to which hydrogen(s) are covalently bonded (C1, C3, C7, and C8). These four carbons behave very similarly in the presence of strongly hydrated anions. For CO32−, H2PO4− and F−, the chemical shifts of these carbons varied very little with increasing salt concentration, suggesting that CO32−, H2PO4− and F− do not significantly interact with the methyl group carbon atoms or the ring C8. On the other hand, these carbons show increased chemical shift in the presence of SO42− and S2O32−, suggesting that SO42− and S2O32− induce a deshielding effect to the electron cloud of carbon atoms. It is surprising that SO42− and S2O32− displayed some salting−in characters on these carbons indicated by the downfield shifts of the 13C peaks. The second group of carbon atoms in Figure 2 includes the four carbons on the ring (C2, C4, C5, and C6) to which no hydrogen atoms are bonded. Remarkably different behaviors are observed in this group of carbons compared to the first group. All the strongly hydrated anions investigated herein showed salting−out effects on these carbons with negative slopes on the chemical shift vs. salt concentration graphs. The negative slopes for these anions suggest that the strongly hydrated anions are excluded from the ring surface of caffeine.36,39 It has been reported previously that the volume exclusion of strongly hydrated anion is an entropic effect.36 When weakly hydrated anions are introduced into caffeine solution the chemical shifts for the two groups of carbons mentioned above start merging together as shown in Figure 3. Still,



the chemical shift changes for C1, C3, C7, and C8 displayed a linear dependence on salt concentration for all the weakly hydrated anions. The chemical shifts for all of these carbon atoms increases (downfield shifts) with increasing salt concentration, suggesting a salting−in effect on these carbon atoms. 36,39,45 The carbons in the three methyl groups, namely C1, C3, and C7, show very similar slopes for most the anions presented in Figure 3, while C8 has a shallower slope compared to C1, C3, and C7 (NO3− is an exception). As for C2, C4, C5, and C6, the 0.5

0.8

(a)

(b)

0.6

Δδ (ppm)

Δδ (ppm)

0.4 0.3 0.2 0.1 0.0

0.4

0.2

0.0 0.0

0.5

1.0

1.5

2.0

0.0

NaCl Conc. (M) 1.4

1.0

1.5

2.0

NaBr Conc. (M)

(d)

0.4

1.0

Δδ (ppm)

Δδ (ppm)

0.5

0.5

(c)

1.2

0.8 0.6 0.4

0.3 0.2 0.1

0.2 0.0

0.0 0.0

0.5

1.0

1.5

0.0

2.0

1.0

0.5

1.0

1.5

2.0

NaNO3 Conc. (M)

NaI Conc. (M) 1.0

(e)

(f)

0.8

Δδ (ppm)

0.8

Δδ (ppm)

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0.6 0.4 0.2

0.6 0.4 0.2

0.0

0.0 0.0

0.5

1.0

1.5

2.0

0.0

NaClO4 Conc. (M)

0.5

1.0

1.5

C1 C3 C7 C8 C2 C4 C5 C6

2.0

NaSCN Conc. (M)

Figure 3. 13C NMR chemical shift change as a function of salt concentration for caffeine in the presence of weakly hydrated anions: (a) NaCl, (b) NaBr, (c) NaI, (d) NaNO3, (e) NaClO4, (f) NaSCN. 10 ACS Paragon Plus Environment

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Table 2. Fitted Values of Bmax and Kd for Weakly Hydrated Anions Binding to C2, C4, C5, and C6 in Caffeine Molecule anion

C2

C4

C5

C6

Bmax (ppm)

Kd (M)

Bmax (ppm)

Kd (M)

Bmax (ppm)

Kd (M)

Bmax (ppm)

Kd (M)

I-

1.928

4.59

2.188

7.01

3.371

3.86

2.553

4.70

NO3-

0.862

3.74

1.077

5.64

1.038

4.11

0.999

4.25

ClO4-

1.554

2.69

1.502

3.43

1.772

2.26

1.808

2.63

SCN-

1.179

2.16

1.226

2.74

1.937

2.19

1.441

2.27

chemical shifts increase linearly with increasing salt concentration in NaCl and NaBr solutions. However, it is interesting to note that the chemical shifts for C2, C4, C5, and C6 display a non−linear behavior in the presence of I−, NO3−, ClO4−, and SCN−, indicating ion binding to these sites.36,38,39 The non-linear curves for C2, C4, C5, and C6 in the presence of I−, NO3−, ClO4−, and SCN− can be fitted using eq 2: ∆ =

[ ]

(2)

[ ]

where Bmax is the maximum chemical shift change at binding saturation, and Kd represents the apparent dissociation constant in molar concentration (M). The higher the Kd value, the weaker binding. The abstracted fitting parameters for anion binding to C2, C4, C5, and C6 are listed in Table 2. It can be seen in Figure 3 that Cl−, Br−, and I− have stronger salting-in effect on the methyl carbons (C1, C3, and C7) than the carbons on the ring structure. On the other hand, NO3−, ClO4−, and SCN− show stronger salting-in effect on carbons on the ring structure compared to the methyl groups with C5 being the strongest followed by C6, C2, and C4. The chemical shifts for the hydrogen atoms attached to C1, C3, C7, and C8 follow the same trend as that in the corresponding carbons as shown in Figure 4. CO32−, H2PO4−, and F− show salting−out effects on all of protons, namely H1, H3, H7, and H8. SO42− and S2O32− have 11 ACS Paragon Plus Environment


none or slightly salting-in effects on these protons. All the weakly hydrated anions, from Cl− to SCN−, have salting−in effects on hydrogens with positive slopes on the chemical shift change vs. salt concentration graphs. All the data in Figure 4 are fitted by eq 1 and the abstracted slopes are (a)

-0.02

-0.04

-0.04

-0.06 -0.08 -0.10 -0.12

-0.06 -0.08 -0.10

0.2

0.4

0.6

0.8

1.0

1.2

0.06 0.04

0.00

0.0

Na2CO3 Conc. (M)

0.2

0.4

0.6

0.8

1.0

0.0

1.2

(d)

0.12

(e)

0.00

0.2

0.4

0.6

0.8

1.0

1.2

Na2SO4 Conc. (M)

NaH2PO4 Conc. (M)

0.14

(f)

0.4

-0.02

0.08 0.06 0.04 0.02

-0.04

Δδ (ppm)

0.10

Δδ (ppm)

Δδ (ppm)

0.08

-0.14 0.0

-0.06 -0.08 -0.10

0.3 0.2 0.1

-0.12

0.00 0.0

0.2

0.4

0.6

0.8

1.0

1.2

0.0

-0.14 0.0

0.2

Na2S2O3 Conc. (M)

0.4

0.6

0.8

0.1

0.3 0.2

1.0

1.5

2.0

0.0

0.5

NaBr Conc. (M)

1.0

1.5

2.0

0.2 0.1

(k)

0.4

Δδ (ppm)

0.3

(i)

0.3 0.2

0.0

0.5

1.0

1.5

2.0

NaNO3 Conc. (M)

NaI Conc. (M)

(j)

0.4

2.0

0.0

0.0 0.5

1.5

0.1

0.1

0.0

1.0

0.4

Δδ (ppm)

0.2

0.5

NaCl Conc. (M)

(h)

0.4

Δδ (ppm)

0.3

0.0

0.0

1.0

NaF Conc. (M)

(g)

0.4

Δδ (ppm)

0.10

0.02

-0.12

-0.14

(c)

0.12

Δδ (ppm)

-0.02

0.14

(b)

0.00

Δδ (ppm)

Δδ (ppm)

0.00

Δδ (ppm)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0.3 0.2

H1 H3 H7 H8

0.1

0.0

0.0 0.0

0.5

1.0

1.5

NaClO4 Conc. (M)

2.0

0.0

0.5

1.0

1.5

2.0

NaSCN Conc. (M)

Figure 4. 1H NMR chemical shift change as a function of salt concentration for (a) Na2CO3, (b) NaH2PO4, (c) Na2SO4, (d) Na2S2O3, (e) NaF, (f) NaCl, (g) NaBr, (h) NaI, (i) NaNO3, (j) NaClO4, (k) NaSCN. 12 ACS Paragon Plus Environment

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provided in Table S1 in Supporting Information together with the c values for the 13C chemical shift changes. The fitting parameters from the data presented in Figures 2-4 (Table S1) allow us to quantify the ion interactions with individual carbon atoms and protons in caffeine molecule. Figure 5 summarizes the c values, the slopes on the chemical shift change vs. salt concentration graphs as a function of anion identity for individual carbon and hydrogen atoms in caffeine. The relative influences of anions on the chemical shift of individual atoms can be easily seen from these plots. The positive slopes indicate a salting−in effect that increases caffeine’s solubility and decreases aggregation. These changes are consistent with the downfield shift of the carbon and hydrogen peaks as the caffeine concentration decreases (Table 1). On the other hand, negative slopes correspond to a salting−out effect, decreasing caffeine solubility and increasing aggregation. The upfield shift due to the salting−out effect is consistent with the changes in the carbon and hydrogen peaks as the concentration increases. The three methyl groups in caffeine respond similarly to the added salts. I− shows the strongest salting−in effect on C1, C3, and C7, followed by Br−, SCN−, ClO4−, Cl− and NO3−. SO42−and S2O32− also show moderate salting-in effect on C1, C3, and C7. On the other hand, CO32−, H2PO4−, and F− have very little influence on the chemical shift of these three carbons. C8 behaves similarly to the three methyl carbons in salt solutions but the difference between anions is less pronounced. The responses of the hydrogen atoms bound to these carbons follows the same trends as their corresponding carbons with much smaller slopes in general. CO32−, H2PO4−, and F− actually have a salting−out effect on these hydrogen atoms. It is interesting to note that the rest of the carbons in the caffeine molecule (C2, C4, C5, and C6) exhibit different behaviors from the methyl groups and C8. All of the strongly hydrated anions salt these carbons out with CO32− being the strongest salting−out anion followed by H2PO4−, SO42−, F−, and S2O32−. Cl− and Br− showed salting−in



0.3

2-

CO3

2-

2-

SO4

S2O3

H2PO4-

-

F-

Cl

Br-

2-

CO3

2-

SO4

S2O3

2-

H2PO4-

F-

Cl

Br-

-

2-

CO3

2-

SO4

-

2-

S2O3

H2PO4

F-

-

-

Cl

Br

2-

CO3

2-

SO4

-

2-

S2O3

H2PO4

F-

-

-

2-

CO3

2-

-

-

2-

S2O3

H2PO4

SO4

2-

CO3

2-

2-

2-

SO4

CO3

-

2-

S2O3

H2PO4

-

F-

Cl

-

-

Br

-

I

-0.4

NO3

-0.3

-0.4 SCN -

-

-0.2

-0.3

SO4

-0.2

0.0 -0.1

-

-0.1

0.1

2-

0.0

C6

S2O3

0.1

C6

(l)

0.2

H2PO4

c (ppm/M)

0.2

F-

C5

F-

SCN

0.4

C5

(k)

-

-

2-

2-

SO4

CO3

-

2-

S2O3

H2PO4

-

F-

Cl

-

Br

-

-

I

NO3

SCN

-

-0.4

-

-0.3

-0.4

0.3

-

-0.2

-0.3

0.4

NO3

0.0 -0.1

Cl

-0.2

0.1

Br

-0.1

0.2

-

0.0

C4

-

0.1

C4

(j)

Cl

c (ppm/M)

0.2

0.3

Br

C2

Cl

SCN

0.4

C2

(i)

Br

-

2-

2-

SO4

CO3

2-

S2O3

H2PO4-

-

F-

Cl

Br-

-

-

I

NO3

SCN

-

-0.4

ClO4-

-0.3

-0.1

0.3

-

-0.2

0.0

0.4

I

0.0 -0.1

-

0.1

0.1

NO3

0.2

0.2

-

0.3

H8

H8

NO3

0.4

(h)

-

c (ppm/M)

0.5

0.3

-

SCN

0.4

C8

C8

NO3

-

2-

2-

SO4

CO3

2-

S2O3

H2PO4-

-

F-

Cl

Br-

-

-

I

NO3

(g)

NO3

0.6

ClO4-

-0.4

SCN -

-0.3

-0.1

-

-0.2

0.0

0.7

-

0.0 -0.1

I

0.1

0.1

-

0.2

0.2

I

0.3

H7

-

0.4

H7

(f)

I

c (ppm/M)

0.5

0.3

NO3

-

SCN

SO4

2-

2-

0.4

C7

C7

-

(e)

CO3

-

2-

S2O3

H2PO4

-

F-

Cl

-

-

-

I

NO3

Br

-

-

ClO4

-0.4

SCN

-0.3

-0.1

-

-0.2

0.0

0.6

I

-

SCN

0.0 -0.1

I

0.1

0.1

-

0.2

0.2

ClO4

0.3

H3

H3

-

0.4

(d)

ClO4

c (ppm/M)

0.5

0.3

-

SO4

2-

2-

0.4

C3

C3

ClO4

(c)

CO3

-

2-

S2O3

H2PO4

-

F-

Cl

-

-

Br

-

I

NO3

SCN

-

-0.4

ClO4-

-0.3 ClO4

-0.2

-0.1

ClO4

c (ppm/M)

0.0 -0.1

0.0

0.7

c (ppm/M)

0.1

-

0.1

0.2

ClO4

0.2

H1

H1

-

0.3

(b)

-

0.4

0.6

c (ppm/M)

0.3

c (ppm/M)

0.5

0.7

c (ppm/M)

0.4

C1

C1

ClO4

(a)

ClO4-

c (ppm/M)

0.6

SCN

0.7

c (ppm/M)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 31

Figure 5. Fitted c values for the individual carbon and hydrogen atoms in caffeine as a function of salt identity. (a) C1; (b) H1; (c) C3; (d) H3; (e) C7; (f) H7; (g) C8; (h) H8; (i) C2; (j) C4; (k) C5; (l) C6. 14 ACS Paragon Plus Environment

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effects on these carbons but not as strong as those effects on the three methyl groups and C8. I−, NO3−, ClO4−, and SCN− exhibit a non-linear dependence of the chemical shift changes of C2, C4, C5, and C6 on salt concentration. Therefore, no slopes are available for I−, NO3−, ClO4−, and SCN− in Figure 5(i)−(l). The binding of I−, NO3−, ClO4−, and SCN− to C2, C4, C5, and C6 are modeled by eq 2, a Langmuir−shaped binding isotherm (Figure 3). The apparent dissociation constant, Kd, measures the relative strength of anion binding to these carbons. A clear trend can be observed for Kd among these anions. Namely, SCN− is the strongest binder followed by ClO4−, NO3−, and I−. On the other hand, Bmax, the maximum chemical shift change due to anion binding at saturation, characterizes the strength of salting−in effect. The order of Bmax follows I− > ClO4− > SCN− > NO3−. The trends in Kd and Bmax do not correlate. This result seems surprising as one might expect stronger ion binding (lower Kd) to produce stronger salting−in effect (larger Bmax). However, this result can be understood by employing a multiple binding site model previously used to describe anion binding to poly(N−isopropylacrylamide).14,63 Assume a caffeine solution having n apparently independent binding sites, the average binding number, v, must be between 0 and n. The v value can be related to Kd, the apparent dissociation constant, by eq 3:14,63 =

[ ]

(3)

[ ]

where [M] is the molar concentration of anion. In this binding model, v and n are correlated to the chemical shift change (Δδ) and the maximum chemical shift change at binding saturation (Bmax), respectively, in eq 2. Accordingly, a solution with higher number of binding sites, n, available would have a larger value of Bmax. In other words, Bmax characterizes the binding capacity of a caffeine solution in the presence of a particular salt. The binding capacity of a caffeine solution increases with decreasing aggregation as salting−in anions are introduced. For 15 ACS Paragon Plus Environment


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example, I− showed highest Bmax for C2, C4, C5, and C6 among the anions, which suggests that these carbons are more accessible for binding in the presence of I− than other anions. This result is consistent with the fact that I− showed the strongest salting−in effects on the three methyl groups and C8 to increase caffeine solubility and decrease aggregation as indicated by the c values for C1, C3, C3, and C8 (Figure 5). NO3− has the least salting−in effect on the three methyl groups and C8, which makes C2, C4, C5, and C6 less available to NO3− binding. On the other hand, eq 3 can be rearranged to obtain the fraction of occupied sites, Y, the ratio between v and n: =

=

[ ] [ ]

(4)

It can be seen from eq 4 that stronger binding (lower Kd value) is related to a higher fraction of binding sites in caffeine occupied by weakly hydrated anions. The NMR studies discussed above isolate the relative contributions of individual atoms in caffeine molecule to the overall caffeine−anion interactions. These spectroscopic evidence is consistent with Shimizu’s recent theoretical work on salt effects on caffeine dimerization.64 Shimizu’s calculations suggested that the exclusion of additives from caffeine enhances caffeine dimerization while the binding of additives on caffeine weakens the dimerization.64 Also, Graziano and co-worker reported in the coil-to-globule collapse transition of poly(N-isopropylacrylamide) that strongly hydrated anions such as sulfate stabilized the globular conformations of the polymer by increasing the magnitude of the solvent-excluded volume effect.65 On the other hand, weakly hydrated anions such as thiocyanate stabilized the coil conformations of the polymer by preferential binding to the polymer surface.65 The same theoretical approach by Graziano et al. has been applied to explaining the co-solvent effects of urea and tetramethylurea on coil-to-globule collapse transition of poly(N-isopropyl-acrylamide).66


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The interactions between caffeine and anions were further investigated using ATR-FTIR spectroscopy. Figure 6a shows the infrared spectra obtained on caffeine samples at concentrations ranging from 20 mM to 140 mM. The spectrum of caffeine consists of two major vibrations within the 1620−1720 cm−1 window. Previous infrared studies on caffeine have assigned the peak at ~1700 cm−1 to the stretching of the isolated carbonyl C=O(2) and the peak at ~1640 cm−1 to the stretching of conjugated carbonyl C=O(6).54,67-71 Some of these work pointed out that the conjugated carbonyl vibration is probably coupled with the ring C4=C5 stretching.68,71 Also, other infrared studies on the guanine ring in nucleic acids suggest that the conjugated carbonyl peak may be coupled with the ring stretching modes from C4=C5 and C5−C6. 72,73 As shown in Figure 6a, the intensity of both infrared bands increases with increasing caffeine concentration as expected. The increase in intensity is also accompanied by changes in the frequency of both peaks. As the caffeine concentration is increased, the frequency of both peaks increases. An increase in the frequency in carbonyl vibrations (Amide I in proteins) has been assigned to desolvation of peptides and proteins.56-58 In addition, a shoulder at ~1655 cm−1 increases intensity with increasing caffeine concentration in the ~1640 cm−1 band. Previous studies have shown increasing dimer formation as caffeine concentration increased. 49,69,74 The shoulder at 1655 cm−1 may therefore reflect caffeine dimer formation that allows hydrophobic ring stacking between caffeine molecules due to fewer water molecules being associated with caffeine at higher concentrations. For comparison, the spectra of caffeine at different concentrations were normalized using the intensity of the ~1700 cm−1 vibration as shown in the inset for Figure 6a. Both vibrations shift to higher frequency and this shift is also accompanied by band broadening of the ~1640 cm−1 vibration as caffeine concentration is increased. In order to directly compare to NMR results, 50 mM caffeine solution was used for all



additional infrared experiments with Hofmeister salts. Figure 6b−6f shows the spectra of 50 mM caffeine in the presence of selected Hofmeister salts at various concentrations. The five salts selected, Na2SO4, NaH2PO4, NaCl, NaNO3, and NaClO4, represent the spectrum of Hofmeister

0.15

3.0

(a)

0.02 M 0.05 M 0.07 M 0.10 M 0.14 M

Absorbance (A.U.)

Absorbance (A.U.)

0.20

0.10

0.05

0.00 1720

1700

1680

1660

1640

2.0 1.5 1.0

0.0

Absorbance (A.U.)

Absorbance (A.U.)

2.0 1.5 1.0

0M 0.2 M 0.5 M 0.7 M 1.0 M 1.3 M

0.5 0.0 -0.5 1720

1700

1680

1660

1640

1620

1.0

0M 0.2 M 0.5 M 0.7 M 1.0 M 1.3 M

0.0 -0.5 1720

1700

1680

1660

1640

0M 0.2 M 0.5 M 0.7 M 1.0 M 1.3 M

0.5 0.0 1700

3.0

(e)

1.5

0.5

(d)

NaCl

1680

1660

1640

1620

Wavenumber (cm-1)

2.0

1.0

1620

1.5

-0.5 1720

Absorbance (A.U.)

2.5

1640

2.0

Wavenumber (cm ) NaNO3

1660

2.5

-1

3.0

1680

Wavenumber (cm ) 3.0

(c)

NaH2PO4

2.5

1700

-1

Wavenumber (cm ) 3.0

0M 0.2 M 0.5 M 0.7 M 1.0 M 1.3 M

0.5

-0.5 1720

1620

(b)

Na2SO4

2.5

-1

Absorbance (A.U.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 31

1620

-1

(f)

NaClO4

2.5 2.0 1.5 1.0

0M 0.2 M 0.5 M 0.7 M 1.0M 1.3M

0.5 0.0 -0.5 1720

1700

1680

1660

1640

1620

-1

Wavenumber (cm )

Wavenumber (cm )

Figure 6. (a) ATR−FTIR spectra of caffeine in D2O at varied caffeine concentrations. Inset, normalized ATR−FTIR spectra of caffeine in D2O at varied caffeine concentrations. (b)−(f) Normalized ATR−FTIR spectra of 0.05 M caffeine D2O solutions in the presence of Na2SO4 (b), NaH2PO4 (c), NaCl (d), NaNO3 (e) and NaClO4 (f) at varied salt concentrations. The arrows indicate the direction of the IR peak shifts. 18 ACS Paragon Plus Environment

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anions. These salts were added to caffeine at concentrations ranging from 0 to 1.3 M. We observed large changes in the infrared spectrum of caffeine in the presence of strongly hydrated SO42− and weakly hydrated ClO4−. However, only very small changes were observed in the presence of H2PO4−, Cl−, and NO3−. The infrared frequency of both caffeine vibrations increases with increasing concentration of Na2SO4 and decreases with increasing concentration of NaClO4 (Figure 6b,f). These changes in frequency suggest differences in solvation in the presence of various salts where sulfate is desolvating and perchlorate is solvating. In addition, we observed shape changes in the ~1640 cm−1 vibration with increasing concentrations of Na2SO4 or NaClO4. High concentration of Na2SO4 causes increase in frequency and band broadening of the ~1640 cm−1 vibration while high concentrations of NaClO4 cause decrease in frequency and band narrowing of the ~1640 cm−1 vibration. The increase in frequency and band broadening of the ~1640 cm−1 vibration observed in the presence of high concentrations of Na2SO4 is similar to that observed at high concentrations of caffeine. These changes suggest that Na2SO4 weakens the solvent hydrogen bonding to the caffeine carbonyls, which desolvates caffeine and promotes caffeine dimerization. The decrease in frequency and peak narrowing of the ~1640 cm−1 vibration observed in the presence of NaClO4 is similar to that observed at low concentration of caffeine, suggesting that caffeine favors the monomer in the presence of perchlorate. Overall, we observe that solvating salts promote caffeine monomers in solution while desolvating salts promote caffeine aggregation. Figure 7a shows the infrared spectra of 50 mM caffeine in the absence and presence of Hofmeister anions at 1 M concentration. These spectra show changes in the frequency of both peaks and shape differences in the ~1640 cm−1 vibration in the presence of different salts (Figure



7a). These changes are similar to the changes seen in Figure 6. Strongly hydrated anions cause increased frequency in both peaks and band broadening of the ~1640 cm−1 vibration while weakly hydrated anions cause decreased frequency and band narrowing of the ~1640 cm−1 vibration. The trend in the observed changes follows the Hofmeister series where the strongly hydrated anions upshift from the control while the weakly hydrated anions downshift from the control. These changes in the frequency of the vibrations reflect the anion induced differences in caffeine solvation. The frequency of both caffeine vibrations was plotted for each salt in the order of the Hofmeister series for anions (Figure 7b). The ATR−FTIR results are consistent with the 13C NMR studies. Strongly hydrated anions are excluded from these moieties to desolvate caffeine and promote aggregation while weakly hydrated anions bind to these sites to solvate caffeine and decrease aggregation.

1680

1660

1640

1620

-1

Wavenumber (cm )

NaSCN

1640

1700

NaI

1642 NaClO4

NaSCN

1644

NaNO3

0.0 -0.5 1720

1646

NaCl

NaI NaClO4

1694

NaBr

0.5

1696

NaF

1.0

1698

NO Salt

NaF NO salt NaCl NaBr NaNO3

1.5

C = O (2) C = O (6)

NaH2PO4

NaH2PO4

Na2SO4

Na2S2O3

2.0

(b)

1700

Na2S2O3

Na2SO4

1702

Na2CO3

Na2CO3

-1

2.5

(a)

Wavenumber (cm )

3.0

Absorbance (A.U.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 31

Salt

Figure 7. (a) ATR−FTIR spectra of 50 mM caffeine D2O solutions in the presence of 1 M salts; (b) Peak position shown in (a) as a function of salt identity.

CONCLUSION A combination of 13C and 1H NMR and ATR−FTIR were used to explore anion−caffeine interactions at the molecular level. It was found that strongly hydrated and weakly hydrated anions influenced caffeine aggregation in solution through different mechanisms. The 20 ACS Paragon Plus Environment

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salting−out effects by strongly hydrated anions are due to the exclusion of these anions from caffeine surface. On the other hand, weakly hydrated anions associate with caffeine molecule to exert a salting−in effect on caffeine. These studies clearly showed that strongly hydrated anions do not bind to caffeine like weakly hydrated anions. This work was able to quantify the interactions of anions with individual carbon and hydrogen atoms in caffeine molecule and correlate with the changes in caffeine solvation. The results from 13C and 1H NMR and ATR−FTIR studies support the mechanisms proposed from the thermodynamics of caffeine partitioning between aqueous and cyclohexane phases.59 Strongly hydrated anions are excluded from the caffeine surface and promote the transfer from aqueous to cyclohexane phase; while weakly hydrated anions associate with caffeine molecule to make the transfer more costly in free energy. The combination of spectroscopically observed changes in ion distribution around caffeine molecule and caffeine solvation provides unique insights into understanding the mechanisms of Hofmeister anions.

AUTHOR INFORMATION Corresponding Author E−mail: [email protected] Notes The authors declare no competing financial interest.

SUPPORTING INFORMATION AVAILABLE: Fitted c Values for the Carbon Atoms and Protons in Caffeine from NMR Chemical Shift Measurements. This information is available free of charge via the Internet at http://pubs.acs.org.



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ACKNOWLEDGMENTS We thank the donors of the Petroleum Research Fund, administered by the American Chemical Society (51008−UNI4, Y.Z.), and the National Science Foundation grants NSF−RUI 0814716 (G.M.) and NSF−REU CHE−1461175 for funding.


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