Probing the GelatinâAlkylammonium Salt Mixed Assemblies through

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Probing the Gelatin-Alkylammonium Salts Mixed Assemblies through Surface Tensiometry and Fluorimetry Suprava Maharana, and Pramila Kumari Misra J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b00338 • Publication Date (Web): 26 Apr 2018 Downloaded from http://pubs.acs.org on April 26, 2018

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The Journal of Physical Chemistry

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Probing the Gelatin-Alkylammonium Salts Mixed Assemblies through Surface Tensiometry and Fluorimetry

Suprava Maharana and Pramila K. Misra* Centre of Studies in Surface Science and Technology, School of Chemistry, Sambalpur University, Jyoti Vihar 768019, Odisha, India

Author Information Suprava Maharana Centre of Studies in Surface Science and Technology, School of Chemistry, Sambalpur University, Jyoti Vihar768019, Odisha, India Email: [email protected]

Dr. (Mrs.) Pramila K. Misra* (Corresponding author) Centre of Studies in Surface Science and Technology, School of Chemistry, Sambalpur University, Jyoti Vihar 768019, India Email: [email protected], [email protected] Telephone: +916632430983(R), +919938333244(M) Fax: +916632430158(O)

ACS Paragon Plus Environment


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ABSTRACT: The interactions, nature of the organization, and physicochemical properties of alkyltrimethylammonium bromides (CnTAB , n=12, 14, and 16)–gelatin mixed assemblies were investigated by UV–visible spectrometry, surface tensiometry, and fluorimetry techniques. The synergistic interaction between the surfactant and gelatin was established from the decrease in critical micellar concentration (CMC) and increase in molecular parking area of surfactants with increase in percentage of gelatin from 0 to 0.4%; e.g., CMC of C16TAB decreased from 0.93 mM in water to 0.44 mM in presence of 0.4% gelatin whereas its Amin increased from 134.98 to 325.55 Å2. The fluorescence anisotropy data and polarity parameters of pyrene indicated the progressive change in the anisotropy and micropolarity of the mixed systems media with gelatin percentage, respectively. The decrease in aggregation number with increase in gelatin concentration can be attributed to the enhanced compatibility of surfactants with the bulk microenvironment. The maximum rigidity of mixed system was also significant from the lifetime data of tyrosine. The formation of Menger micelles on gelatin segments was supported by surface tension and anisotropy data. The overall observations can be attributed to the formation of micelles via gelatin-surfactant aggregates; gelatin segments are localized within the microdomain of these aggregates.


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1. INTRODUCTION With increasing demand for food products, drugs, cosmetics, and body care products due to rampant growth of population, it is essential to develop suitable formulations of these products. This requires the engineering of a suitable medium (carrier) with desired properties and morphology that would facilitate the administration of requisite solute or suspension1-3. Certain properties such as fluidity, micropolarity, size, viscosity, and anisotropy are a few characteristic parameters of a medium that affect the ease of solubilization and flowability of solute within the formulated products4-5. Considering the site of applications and interest, several micro-heterogeneous media such as surfactant–surfactant, surfactant–polymer, and surfactant–protein assemblies have been established and successfully used for this purpose6-8. Surfactant–protein assemblies, in particular, have received considerable attention owing to the similarities of structural hierarchy of surfactant assemblies with globular proteins9-10. The analytical techniques like surface tensiometry, conductivity and viscometry11-14 and spectral techniques such as UV–visible spectrometry15, fluorimetry16, nuclear magnetic resonance17, electron spin resonance18 and calorimetric analyses12 are some important techniques that are used to efficiently determine the physicochemical properties of organized assemblies pertaining to surfactant alone or its mixed systems. The surface tensiometry technique, in particular, is considered as the main tool to analyze the assembling behavior of surfactants as it is the direct yardstick of the surface activity of a surfactant accounting for its organization characteristics13,19-22. When coupled with fluorescence spectral technique, comprehensive understanding of the properties of the medium such as micropolarity, size, rigidity and aggregation number can be achieved12, 18, 23. Gelatin, a high-molecular-weight denatured collagen, is an important constituent in different consumer items such as emulsifiers, drugs, pharmaceuticals, food products, and gelling agents. To obtain the desired formulations, the characteristic properties of gelatin, i.e.,



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the physicochemical properties of media formed from it, are significantly modulated using different additives and technologies13,17,20,24-27. The underlying mechanisms of formulations are mostly affected by both hydrophobic and electrostatic interactions between gelatin and additives 14,20,28. Gelatin is mostly used with one or more surfactants during product formulation, and hence surfactant–gelatin interactions have been extensively studied13,17,20,24,26,28. Formation of gelatin–surfactant complexes below critical micellar concentration (CMC) has been reported13. Above CMC, the resulting complex is solubilized within free micelles as evident from the estimation of transition points, counterion binding of the mixed and free micelles, enthalpy of binding interactions and energetic of micellization of mixed systems13. Studies on a mixture of gelatin and alkyl sulfate surfactant of varying alkyl chain lengths through pulsed-gradient spin-echo NMR spectroscopy17 showed that the equilibrium between gelatinbound micelles with freely diffusing unimeric surfactant depends on the extent of diffusivity of surfactants. The interaction of gelatin with surfactants of longer alkyl chains resulted in minimum diffusion due to greater hydrophobic interaction17. The interaction between a cationic gemini surfactant 1,2-ethane bis(dimethyldodecylammonium bromide) with gelatin was explored using various techniques24. The alteration in the polarity of resulting microenvironment was attributed to the enhanced electrostatic and hydrophobic interactions of twin hydrocarbon chains and charges of gemini surfactant with gelatin24. Dynamic lightscattering technique26 was used to understand the binding of both ionic and nonionic surfactants with gelatin in aqueous buffer (pH= 7.0). The size dependence of gelatin– surfactant aggregates on the nature of surfactant head groups and the necklace-bead model of interaction were confirmed in this study. However, till date, the overall studies mainly focused on the types of interactions generated within gelatin–surfactant mixed systems, but attention has been rarely paid to bulk properties such as the micropolarity and rigidity of


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mixed system with the practical utility from product formulation perspective. To the best of our knowledge, the effect of gelatin concentration on such properties at biological pH has not been reported. In our studies on protein–surfactant assemblies involving native and denatured proteins20,29, we have used several techniques to elucidate the mechanism of organization and specific interactions involved during the formation of surfactant–protein assemblies. The specific roles of native and denatured proteins in the formation of mixed organized assemblies and premicellar aggregates were determined20,24,29. To investigate the physicochemical characteristics of these mixed aggregates comprehensively, in this study, we have used strategically designed surfactant–protein mixed systems involving a series of cationic surfactants, alkyltrimethylammonium bromides (CnTAB) with varying hydrocarbon chain lengths (n= 12, 14, 16) and gelatin, with varying concentrations. The point of changeover of premicellar and micellar aggregates and specific interactions involved therein were identified. The intrinsic and extrinsic fluorescence properties were used to estimate the rigidity and micropolarity of organized assemblies. The initial detection of formation of mixed assemblies along with physicochemical parameters, thermodynamic parameters, and orientation of surfactant at interfaces was achieved by surface tensiometry. In addition, the information about the environment and restricted motion/free dangling of gelatin segments was obtained from fluorescence anisotropy, time-resolved fluorimetry, and rotational correlation time data. The generation of Menger micelle has been supported.

2. EXPERIMENTAL 2.1. Materials. The MERCK chemicals (Germany) CnTAB surfactants, i.e., cetyltrimethylammonium

bromide

(C16TAB),

tetradecyltrimethylammonium

bromide

(C14TAB) and dodecyltrimethylammonium bromide (C12TAB) were recrystallized from an



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alcohol/acetone mixture30 before use. Gelatin purchased from MERCK (Germany) was used as such. The buffer used for this study was prepared using disodium hydrogen phosphate and sodium dihydrogen phosphate (each of 99.5% purity) procured from S.D. Fine Chemicals (India). Pyrene and cetyltrimethylpyridinium chloride (CPC) were purchased from Aldrich, USA and used as received without further purification. 2.2. Preparation of Samples. A stock solution of gelatin (0.5% w/v) was prepared by soaking the gelatin powder in the buffer for ∼1h followed by heating up to 40 °C with mild stirring12,26. The phosphate buffer was used to maintain a pH of 7.0 throughout the experiments31. The samples, i.e., surfactant solutions (with and without gelatin) were aged for 24 h at 303 K to reach the equilibrium before taking each measurement32. All the solutions were prepared in Milli-Q water (resistivity 18.3MΩ-cm). The entire experimental studies were performed at ambient temperature (T =303 K). 2.3. Methods. Each of the following measurements was an average of at least three readings and the data were almost reproducible. The standard deviations are provided in the footnote of the Tables. 2.3.1. Surface Tension Measurements. The surface tensions of aqueous surfactant solutions were measured using a Nima Manual Tensiometer, Model ST 500-man (Nima Tech, England) equipped with the Wilhelmy plate at 303 K and 105 Pa. The plot of surface tension vs. concentration was used to insinuate the point of crossover among the species formed along the organization pathway. The critical concentrations such as CMCs and critical aggregation concentrations (CACs) in the presence and absence of gelatin were estimated as the points of intersections of the extrapolated straight segments as reported by Mitra et al13. The representative plot is shown in Figure S1. 2.3.2. UV–Visible Spectral Measurements. UV–visible spectra in the absence and presence

of

gelatin

were

recorded

using

a

Shimadzu


UV-2450

double-beam

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spectrophotometer (Japan) in the wavelength range 200–600 nm. All the measurements were performed using clean quartz cuvettes. 2.3.3. Steady-State Fluorescence Measurements. The intrinsic fluorescence of gelatin and extrinsic fluorescence of pyrene were investigated by recording their fluorescence spectra and intensities using Hitachi F7000 fluorescence spectrophotometer (Japan). The quartz cuvettes of 1 cm path length equipped with a thermostated cell holder were used for measurements. 2.3.3.1. Intrinsic Fluorescence of Tyrosine. The fluorescence emission spectra of gelatin

samples were monitored at a fixed excitation wavelength of 280 nm (Absorption λmax of tyrosine) in the wavelength range 300–600nm. The spectral bandwidth, scan speed and data interval during the measurements were adjusted to 2.5 nm, 1200 nm/min, and 2 nm, respectively. 2.3.3.2. Determination of CMC Using Pyrene. The concentration of pyrene was

maintained low (10−6 M) to avoid the formation of its own excimer or any perturbation to the gelatin–surfactant organized system16,33. The emission spectra of pyrene were recorded in the range 350–600 nm by exciting the solution at 335 nm (Absorption λmax of pyrene). The CMCs of the surfactants in the presence of various amounts of gelatin was measured by plotting the micropolarity of the system, i.e., I3/I1 vs. [surfactant], where I1 and I3 are the emission intensities at 374 and 384 nm, respectively. The spectral bandwidth and scan speed during the measurement were adjusted at 2.5 nm and 1200 nm/min, respectively. 2.3.3.3. Determination of Aggregation Number. The steady-state fluorescent quenching

method was used to measure micellar aggregation number34-37, which is an accurate method to determine the aggregation number of up to C12TAB, corroborating the predominance of hydrophobic interactions in CnTAB-gelatin systems.

400

Maximum Intensity / 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

350

300

250

[C16TAB]-G(0.1%)

200

[C14TAB]-G(0.1%) [C12TAB]-G(0.1%)

150 0.000

0.005

0.010

0.015

0.020

0.025

[CnTAB] / M

Figure 5. Maximum fluorescence intensity as a function of [CnTAB] in presence of 0.1% of gelatin. 3.5. Micropolarity and CMC. Steady-state fluorescence measurements were achieved by taking pyrene as the most frequently used extrinsic fluorophore, which is a suitable fluorescent probe to investigate the polarity of micro-organized systems59. The intensity ratio of the third (I384 or I3) and first (I374 or I1) vibrational peaks in the fluorescence spectrum of pyrene, i.e., I384/I374 or I3/I1 is sensitive to the polarity of microenvironment60. The ratio increases with the decrease in the polarity of the environment, and hence it is called polarity parameter24. In agreement with the literature24,61, the I3/I1 value in water was observed at 0.55. The higher values of I3/I1 in the presence of gelatin compared with water indicate that the pyrene is localized on the hydrophobic regions of gelatin segments. In our earlier study20, the I3/I1 of pyrene was found to be 1.58 in the presence of a native protein, BSA, which is much higher than that of gelatin (I3/I1 =1.17) obtained in this case. This indicates that the pyrene experiences a decreased polar environment in the presence BSA– surfactant mixed assemblies compared with that of gelatin–surfactant mixed assemblies. Because BSA is a native protein, it folds itself, so that its hydrophobic residues are buried



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within to avoid hydrophobic water interactions. A higher polarity parameter value of pyrene in the presence of gelatin, therefore, is an apparent observation. In gelatin, the unfolded hydrophobic segments are exposed relatively more to the bulk water compared with BSA on which pyrene anchors. Addition of surfactant adds to the nonpolarity of the environment in proportion to the increase in hydrocarbon chain length (Figures 6, S14, and S15). This study further supports that gelatin-coupled micelle has less polarity compared with surfactant micelle24.

1.12 1.10 1.08 1.06

PP of Pyrene

1.04 1.02 1.00 0.98 0.96 0.94

G=0% G=0.1% G=0.2% G=0.4%

0.92 0.90 0.88 0.86 -4.5

-4.0

-3.5

-3.0

-2.5

-2.0

Log[C16TAB / M]

Figure 6. Plot of polarity parameter of pyrene in presence of different Log[C16TAB] with and without gelatin. The polarity decreases with the increase in hydrocarbon chain length of CnTAB (Figure 7).

1.10

C16TAB-G(0.1%)

1.05

C14TAB-G(0.1%) C12TAB-G(0.1%)

1.00 0.95

PP of Pyrene

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

0.90 0.85 0.80 0.75 0.70 0.65 -5.0

-4.5

-4.0

-3.5

-3.0

-2.5

-2.0

-1.5

-1.0

-0.5

0.0

Log[CnTAB / M]


0.5

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Figure 7. Plot of polarity parameter of pyrene in presence of different Log[CnTAB] with gelatin (0.1%). On comparison of I3/I1 values of pyrene within the micelle in the presence and absence of gelatin (Figures 6, S14 and S15), it was observed that the I3/I1 vs. surfactant concentration curve has a sharp transition at the CMC in the absence of gelatin, whereas in the presence of gelatin, a gradual change in the I3/I1 values was observed till the plateau was obtained. The gradual increase in I3/I1 values indicates the progressive decrease in the polarity of environment experienced by pyrene as the surfactant concentration increases. Such gradual increase in I3/I1 values is attributed to the formation of micelles/mixed micelles in a step-wise process i.e. via premicellar aggregates30,55. At a low concentration, pyrene resides in water or within the microdomain of discrete gelatin–surfactant complex of diverse sizes. The complex gradually turns into a fully formed micelle within which the segments of gelatin are entrapped. The micropolarity indexes of pyrene beyond CMC in the presence of 0.1 wt % of gelatin were 1.06, 0.92, and 0.69 in the presence of C16TAB, C14TAB, and C12TAB, respectively, consistent with the decrease in carbon chain length of surfactants (Figure 7). 3.6. Determination of Aggregation Number. When a surfactant is dissolved in water, the distortion of water structure due to the unfavorable hydrocarbon tail–water contact and consequent free energy constraints enforce the surfactant molecule to aggregate, leading to the formation of a micelle29. The number of surfactant monomers constituting the micelle is known as aggregation number. The energetic benefit due to sequestering the hydrophobic side chains away from the polar environment

15-16

and the delicate interplay between the

electrostatic repulsion of charged groups and the hydrophobic attractive interactions of hydrocarbon tail of the surfactant itself determine the magnitude of aggregation number62.



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Alternatively, the more is the compatibility between surfactants and water (solvent) the less will be the propensity of surfactant to aggregate which in turn decreases aggregation number. The aggregation number of micelles in the presence of CnTAB was calculated using the steady-state fluorescence quenching method proposed by Turro and Yekta involving Pyrene-CPC ion pair as the fluorophore–quencher dyad system and considers the quenching process in micelles to be very rapid with respect to the fluorescence lifetime of the probe62-63. The Turro and Yekta plots are shown in Figures S2-S4, and the aggregation numbers are provided in Table 2 (Figure S16). Table 2. Determination of Aggregation Number Different Wt% of Gelatin C16TAB C14TAB b Gelatin CMC Nagg CMCb Nagg (Wt %) mM mM G=0% 0.93 90.53 3.52 60.63

C12TAB CMCb Nagg mM 18.30 52.05

G=0.1%

0.85

72.73

1.56

48.59

11.50

37.97

G=0.2%

0.80

70.94

1.00

40.09

10.10

30.37

0.45

56.95

0.60

33.74

9.50

26.57

G=0.4%

of CnTAB’s at

b

NB: CMC = CMC measured by Fluorimetry method, G =Gelatin. Standard deviations of the parameters: cmcb = ± 0.06; Nagg (C16TAB) = ± 0.05; Nagg (C14TAB) = ± 0.09; Nagg (C12TAB) = ± 0.04. For each surfactant micelle, the aggregation number decreases with the increase in the percentage of gelatin, thereby demonstrating that the surfactant prefers to aggregate slightly as the gelatin content in the bulk aqueous phase increases. The micropolarity of bulk phase (indicated by micropolarity index, Figures 6, S14 and S15) decreases with the increase in gelatin percentage in the bulk water phase. Hence, the surfactant hydrophobic chains prefer less to confiscate from the environment, resulting in a lower aggregation number. 3.7. Time-Resolved Fluorescence Studies. The time a molecule spends in its excited state before returning to the ground state with or without emitting radiation (photon) is known as a lifetime. The environment that does not allow a molecule to purge out its excitation


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energy through any phenomenological events leads to an increase in the lifetime of the molecule. Lifetime measurements can yield information about the microenvironment within which the probe is localized and thus help to understand the microenvironment experienced by the probe molecule within micelles. Fluorescence lifetime is more authoritative than fluorescence intensity because it is independent of the inner filter effects, static quenching, and variations in the fluorophore concentration56. These data allow us to better understand the photophysics of the system. The average lifetimes of the tyrosine fluorophore instituted on gelatin were evaluated as a function of surfactant concentrations. The changes in the average lifetime (‹τ›) with [CnTAB] are shown in Figures 8, and S17-S19 (Tables S1-S3).

3.0 2.9 2.8 2.7

Average Lifetime / ns

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

2.6 2.5 2.4 2.3 2.2

G=0.1% G=0.2% G=0.4%

2.1 2.0 1.9 -0.001 0.000

0.001

0.002

0.003

0.004

0.005

0.006

0.007

0.008

[C16TAB] / M

Figure 8. Plot of average lifetime versus [C16TAB] in presence of various wt% of gelatin. The gradual increase in the ‹τ› values with increase in [CnTAB] is due to the progressive partition of the tyrosine molecule from bulk water to the micellar phase. The ‹τ› values experience a transition at some critical concentrations of the surfactants, which coincide with the CMC values of surfactants obtained from surface tension and fluorescence measurements. This finding supports our proposition of the formation of micelles via the following sequence: surfactant monomer → discrete aggregates of gelatin–surfactant



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aggregates→ to micelle. The probe (tyrosine) has both nonpolar (due to C-C and C-H bonds in the ring) and the polar (phenolic OH) regions capable of forming H-bonds with water and hence, with proper orientation, it can cope up with both hydrophilic and hydrophobic environment. The solubilization site for this type of probe needs a rough surface as proposed by Menger, where a hydrophobic part of the substrate can suitably interact with the hydrophobic patches of the micelle64-65. In contrast to Hartley’s micellar characteristics66, where the surface is proposed as a smooth one with distinct polar surface enveloping a hydrophobic fluid like core, Menger micelle has a hydrophilic surface with fatty patches due to the protrusion of hydrophobic group of the surfactants. With kinetic evidence, we have reported the existence of 9% fatty patches at the surface of a CTAB micelle67. The earlier reports on the necklace bead model26 of surfactant-protein interaction have been explained through Hartley type model of surfactant aggregation around the protein. The present findings, however, can only be explained if tyrosine is positioned at the micellar surface of Menger micelle. Both the hydrophilic –OH group and the aromatic π-cloud are compatible with the hydrophilic head group of the surfactant, while the methylene unit and the aromatic ring can attune with the hydrophobic patches of the Menger micellar surface. In a recent report on the interaction of long chains esters of tyrosine with CTAB using a number of techniques68, the interactions of π electron clouds with the cationic headgroups have been envisaged. With increasing number of the methylene group in the surfactant tail, the hydrophobicity increases (0.5 unit /methylene unit69) leading thereby, the increase in the hydrophobic patches. As the lifetime of excited tyrosine increases with increase in nonpolar environment70, the trend in the lifetime C16 TAB > C14 TAB> C12 TAB indicates the localization of tyrosine in the fatty patches of the Menger micelle. Beyond CMC, the marginal effect on ‹τ› values is due to increasing in the number of micelles.


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Intriguingly, although the trend in lifetime vs. surfactant concentration is retained at all the concentrations of gelatin, the values of lifetimes decreased as the gelatin percentage in the surfactant solution increased. This may be due to increase in the non-radiative transitions due to the increase in hydrogen bonding, dipole-dipole interactions because of the increase in gelatin concentration in bulk phase71-72. 3.8. Determination of Fluorescence Anisotropy or Steady-State Fluorescence Measurements. Generally, a system is said to be anisotropic if some of its properties are sensitive to the direction of measurement. Fluorescence anisotropy, in particular, is the phenomenon where the light emitted by a fluorophore has unequal intensities along different axes of polarization. The fluorescence anisotropy measured by anisotropy parameter, r value, is low in an isotropic medium, and the value increases as the anisotropy of the medium increases. The r values of tyrosine fluorescence were measured as a function of [CnTAB] in the presence of various amounts of gelatin and are shown in Figures 9, S20, and S21 (Table S4).

0.20

G=0.1% G=0.2% G=0.4%

0.19 0.18

Fluorescence Anisotropy

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

cmc 0.17 0.16 0.15 0.14 0.13 0.12 0.11 0.10 0.000

0.002

0.004

0.006

0.008

[C16TAB] / M

Figure 9. Anisotropy of [C16TAB] in presence of different wt% of gelatin.



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At a fixed amount of gelatin, the r value increased with the increase in surfactant concentration till a maximum value was obtained at an intermediate surfactant concentration. The concentration at the maximum r values (Figures 9, S20, and S21) of the anisotropy data vs. surfactant concentration coincides with the CMC values obtained from the surface tension and fluorescence measurements of pyrene/tyrosine (Table 1). On further increasing the surfactant concentration, the r value decreased to a minimum value, which does not change with a further increase in surfactant concentration. This phenomenon can be explained as follows: In water (Figure 10a) or at a very low concentration of surfactant, the segmental mobility of gelatin is random; therefore, tyrosine experiences a uniform environment, resulting in a very low value of r. With the increase in surfactant concentration, the global motion of gelatin segment is gradually restricted due to the entrapment of a portion of gelatin segments within the progressively formed gelatin–surfactant discrete aggregates/micelles erratically distributed throughout the bulk phase (Figure 10b). The tyrosine molecule present in gelatin segment thus feels a disconnected environment consisting of bulk water and surfactant aggregates. The value continues to increase till the discrete aggregates are converted to full-grown micelles (Figure 10d). Beyond CMC, the r value starts decreasing with the increase in the number of micelles. The step-wise formation of micelles is also revealed from surface tension and micropolarity measurements. At higher concentrations, when the micelles are enough in number to provide a continuous domain to gelatin segments, a minimum r value is yielded that does not change with the change in surfactant concentration (Figure 10c). These findings can also be explained by the formation of Menger micelle64-65 (Figure 10d) in the vicinity of gelatin segments due to the presence of both charged and hydrophobic sites on its segments as revealed from lifetime data. The entrapment of gelatin segment within the isotropic environment of micelles allows its free movement. Thus, the global motion of gelatin segments is the critical factor for the magnitude of


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anisotropy values. The fact that for a particular gelatin concentration, the r value decreases in the

order

C16TAB-gelatin

>

C14TAB-gelatin

>

C12TAB

-gelatin

confirms

our

conjecture23,40,73. Because the micelle is formed relatively earlier with the increase in gelatin concentration, the anisotropy value decreases with the increase in gelatin percentage, further corroborating the proposed hypothesis.

Figure

10.

Gelatin

segments

entrapped

within

the

gelatin-surfactant

discrete

aggregates/micelles distributed throughout the bulk phase. These results also validate the lifetime data. However, CAC could not be recognized from the anisotropy data as there is a continuous increase in anisotropy with the increase in surfactant concentration until a large number of micelles are formed in the bulk medium, providing a similar environment to all fluorescent probes present over gelatin segment. To further authenticate our proposition concerning some of the properties of medium experienced by tyrosine during its lifetime, its apparent rotation correlation time (θ) was calculated using Eq. 5. The representative plots of θ vs. surfactant concentration are shown in Figures 11-12 (Figures S22-23). θ also follows the same trend as that of anisotropy data (Figure 9), supporting that tyrosine lifetime is mainly controlled by the segmental motion of gelatin and is independent of its rotational correlation time56.



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Apparent Rotational Correlation Time / ns

4.5

G=0.1% G=0.2% G=0.4%

4.0 3.5 3.0 2.5 2.0 1.5 1.0 -0.001 0.000

0.001

0.002

0.003

0.004

0.005

0.006

0.007

0.008

[C16TAB] / M

Figure 11. Rotational correlation time of [C16TAB] in presence of different wt% of gelatin

4.5

Apparent Rotational Correlation Time / ns

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

C16TAB-G(0.1%) C14TAB-G(0.1%)

4.0

C12TAB-G(0.1%) 3.5

3.0

2.5

2.0

1.5 -0.001 0.000 0.001 0.002 0.003 0.004 0.005 0.006 0.007 0.008 0.009 0.010

[CnTAB] / M

Figure 12. Rotational correlation time of [CnTAB] in presence of 0.1% of gelatin

4. CONCLUSION


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The ever-changing scientific scenario always projects advanced technologies that crumble/authenticate the established facts/concepts in the global scientific horizon. Consequently, a large number of technologies are being continually developed. Fluorescence spectroscopy has emerged as one of the advanced techniques that provide indubitable information on the mechanism of formation of surfactant assemblies at the molecular level as well as about their physicochemical properties, and hence it is more frequently used in such studies. In this article, surface tension coupled with fluorescence studies were used for investigating the organization characteristics and physicochemical properties of surfactant– protein mixed assemblies formed from a denatured protein and a series of cationic surfactants of variable hydrocarbon chain lengths. The synergistic interaction generated in the mixture was evident from the decrease in CMC and increase in the Amin of surfactant at air-water interface. The formation of mixed assemblies can be attributed to both electrostatic and hydrophobic interactions between the surfactant and protein. A step-wise association was proposed:

surfactant

monomer

→

discrete

aggregates

of

gelatin–surfactant

aggregates→micelle to account for the formation of mixed assemblies. The entrapment of gelatin segment within the microdomain of organized assemblies was evident from both the lifetime of tyrosine and fluorescence anisotropy data. The lifetime, micropolarity index of pyrene, and intrinsic fluorescence intensity of tyrosine increased with the increase in surfactant till a plateau was obtained beyond CMC. This can be attributed to the increase in the rigidity of medium generated in the mixture that did not allow the easy tumbling of the fluorophore. The apparent correlation time and anisotropy, in contrast, followed a different trend: the values increased to a maximum that coincided with the CMC of mixed surfactant systems. With further increase in surfactant concentration, these values decreased to a flat minimum at a higher concentration. These observations validated the proposition of



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formation of mixed micelles via the surfactant-gelatin discrete aggregates, whereby the fluorophore was transferred from a discontinued environment consisting of discrete premicellar aggregates to a large microdomain of the micelle. The entrapment of the segments within a Menger micelle could account for such conclusive results. To the best of our knowledge, such distinct outcome differentiating premicellar regions from post-micellar regions with respect to their micropolarity, rigidity, and point of transition is reported for the first time in this study. Although the toxicity and biodegradability of CnTAB were not evaluated, a literature survey reveals that cationic surfactants with alkyl and benzyl groups are less aquatic toxic and easily biodegradable74; hence, these results can be used while delivering any solutes (drugs, food supplements, etc.) into biological systems using CnTAB-gelatin as a potent carrier.

Acknowledgment The authors thank UGC, New Delhi, for providing a BSR fellowship to S.M. (F.No.25-1/2014-15

(BSR)/7-166/2007(BSR)).

Financial

support

of

the

UGC

(No.F.540/14/DRS/2013 (SAP-I)) and DST (SR/FST/CSII-021/2012(G)) to the School of Chemistry are also gratefully acknowledged. Special thanks to Prof. B. K. Mishra and Dr. H. Chakraborty of School of Chemistry, Sambalpur University for useful discussion during the preparation of this manuscript and fluorescence measurements. SUPPORTING INFORMATION The details of figures related to surface tension, fluorescence (intensity, micropolarity, aggregation number, anisotropy, lifetime) and the data on the fluorescence lifetime and anisotropy as a function surfactants and gelatin concentration studied (PDF). This material is available free of charge via the Internet at http://pubs.acs.org.


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