In Situ Size Exclusion Chromatographic NMR of Sunset Yellow FCF in

Article pubs.acs.org/JPCC

In Situ Size Exclusion Chromatographic NMR of Sunset Yellow FCF in Solution Rebecca E. Joyce and Iain J. Day* School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QJ, United Kingdom ABSTRACT: Size-exclusion chromatographic NMR is the application of size-exclusion chromatographic media to modify the observed diffusion properties of analyte molecules, as measured by diffusion ordered spectroscopy. This Article demonstrates the application of this method to isotropic solutions of sunset yellow, which are known to be formed of large, noncovalent aggregates. The results show a partitioning effect of the aggregates between inpore and free solution environments, which is explained by sizeexclusion behavior. The influence of pore size on this partition is also noted.

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INTRODUCTION The noncovalent self-assembly of small molecules in solution is important in a number of areas, including drug delivery,1,2 dyestuffs,3,4 liquid crystals,5 and nanoscale engineering such as the formation of metal−organic framework materials.6 The azo dye sunset yellow FCF (sodium (E)-6-hydroxy-5-((4-sulfonatophenyl) diazenyl) naphthalene-2-sulfonate) provides an ideal system with which to investigate noncovalent aggregation phenomena in solution, as it is known to form lyotropic liquid crystals as a function of sample composition.7 This aggregation behavior has been well studied using a number of physical methods, which probe interactions occurring on different length scales and hence provide complementary information. These techniques include X-ray diffraction8−10 and optical spectroscopy.8 Nuclear magnetic resonance (NMR) spectroscopy has been successfully applied to probe the behavior of sunset yellow in isotropic solution using both concentrationdependent changes in the observed chemical shifts7,11 and changes in diffusion properties12 to monitor the assemblies formed. Generally, these investigations have either looked at structure and packing in liquid crystalline phases9,10 or the association behavior in isotropic solution.7,12 The use of diffusion NMR spectroscopy to investigate aggregation and assembly in solution is well developed, including applications to protein−protein interactions,13,14 determination of the solution aggregation state of organometallic complexes,13,14 and monitoring the formation of amyloid fibrils.15 The DOSY presentation of the technique also has the ability to afford the pseudoseparation of mixtures via the different diffusion properties of the various components.16 Recently, the development of “chromatographic NMR” or matrix-assisted DOSY, in which the solute diffusion properties are modulated by some additive to the solvent system, has demonstrated the potential to improve the observed separation in the diffusion dimension and/or provide information about solute−modifier interactions. Various © 2013 American Chemical Society

modifiers have been presented, including silica chromatography stationary phases,17−19 polymer additives,20,21 micelles,22,23 and cyclodextrins,24 depending on the system under investigation. We have demonstrated that similar effects are achievable using size-exclusion stationary phases such as Sephadex and Superdex.25 In this case, changes in the diffusion properties of a series of polymer molecular weight reference standards were observed upon addition of the size-exclusion stationary phase. The magnitude of the change in diffusion coefficient correlated with the molecular weight of the polymer and the fractionation range of the stationary phase, and can be understood using a simple description of size-exclusion phenomena.25 In this Article, we apply this SEC-DOSY methodology to aggregating solutions of sunset yellow and demonstrate that there is a partitioning between species that enter the pores of the stationary phase and the free solution surrounding the particles. We explain this behavior in terms of simple sizeexclusion behavior and demonstrate that this partitioning can be modulated by the fractionation range, that is, size of the pores, of the stationary phase.

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MATERIALS AND METHODS All chemicals were purchased from Sigma Aldrich (Dorset, UK) and used as obtained, with the exception of sunset yellow, which was purified using an ethanol precipitation as described.8,10 Solutions were prepared by diluting aliquots taken from an 833 mM stock solution of sunset yellow dissolved in deuterium oxide. Sephadex size-exclusion chromatography media were added as a dry powder to a final concentration of 60 mg mL−1. The media were allowed to swell at room temperature for at least 3 h before being transferred to standard 5 mm NMR tubes and allowed to settle for at least 30 Received: February 22, 2013 Revised: July 22, 2013 Published: August 4, 2013 17503

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shows a single species in solution. This is consistent with the fast exchange of sunset yellow monomers with the aggregates and between aggregated assemblies.7,11,12 This means that the observed chemical shifts are population-weighted averages of the chemical shifts for the monomer and the aggregates of various sizes.7,11 On addition of a size exclusion stationary phase, shown in Figure 1b−d, there is an immediate deterioration in the quality of the spectrum, with a loss of resolution arising from susceptibility broadening effects due to the presence of an inhomogeneous sample within the RF coil region.28 This effect has been observed previously with both the addition of silica17,18 and size-exclusion stationary phases.25 In addition to the reduction in spectral quality, the addition of the stationary phase results in the observation of additional resonances in the spectrum, with those new signals appearing typically to higher chemical shifts than the corresponding sunset yellow resonances in the absence of a stationary phase. Resonances that correspond to those of free sunset yellow are highlighted across Figure 1. The observed chemical shifts for sunset yellow are well-known to be concentration dependent, with increasing concentration resulting in a decrease in the observed chemical shift.7,11 This observation is typically explained by an increase in shielding upon assembly resulting from π−π stacking of the aromatic core of the sunset yellow molecules. The appearance of signals with larger chemical shifts on addition of the stationary phase therefore suggests that this causes the formation of a second pool of smaller aggregates in a slow exchange with those in free solution. The three Sephadex stationary phases used are comprised of cross-linked Dextrans with narrow pore size distributions29 and differ solely in their fractionation ranges, that is, in their pore sizes, with typical characteristic parameters for these phases given in Table 1. It is

min prior to NMR measurement. The NMR tubes were positioned such that the settled stationary phase completely filled the RF coil region.25 Diffusion ordered NMR data were obtained using the Oneshot experiment26 implemented on a Varian VNMRS 600 spectrometer (Agilent Technologies, Yarnton, UK) equipped with an X{1H} probe, capable of producing a pulsed field gradient along the z-axis of up to 0.7 T m−1. Typical acquisition parameters used diffusion encoding gradients of 4 ms duration with strengths, equally spaced in g2, between 0.0045 and 0.5625 T m−1. The diffusion encoding delay Δ was 200 ms. All data were processed using DOSY Toolbox,27 with the Stejskal− Tanner equation suitably modified for the Oneshot experiment.26 HR-MAS experiments were performed on a 4 mm magic angle spinning probe equipped with a magic angle gradient coil. Samples were transferred as 60 μL aliquots into a 4 mm glass rotor with 40 μL active volume and allowed to settle for at least 20 min. The supernatant solution (approximately 20 μL) was removed using a syringe and the rotor closed. This process ensured there was sufficient stationary phase within the rotor at a similar packing density to the standard 5 mm NMR tubes. The samples were then spun under computer control at speeds of up to 2.5 kHz about the magic angle with respect to the static magnetic field. Exchange spectroscopy (EXSY) experiments were acquired using a standard NOESY pulse sequence with a mixing time of 700 ms. The data were acquired with 1201 × 200 complex points spanning 8012 Hz in F1 and F2. The resulting spectra were processed with Gaussian apodization in both dimensions.

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RESULTS AND DISCUSSION As an initial investigation into the influence of a stationary phase on the diffusion properties of an aggregating system, three grades of Sephadex, differing in their pore sizes and hence fractionation ranges, were added to samples comprising 50 mM sunset yellow. At this concentration, previous diffusion NMR studies indicate that the aggregates are comprised, on average, of 30 molecules per aggregate.12 Figure 1a shows the aromatic region of the 1H NMR spectrum of 50 mM sunset yellow in isotropic solution. The spectrum is well resolved and clearly

Table 1. Properties of the Sephadex Stationary Phases37 fractionation range (kDa) stationary phase Sephadex G-50 Sephadex G-75 Sephadex G-100

particle size (μm)

dry bed volume (mL g−1)

globular proteins

dextrans

20−50

9−11

1.5−30

0.5−10

20−50

12−15

3−70

1−50

20−50

15−20

4−100

1−100

notable that the difference in chemical shift between the new signals and the signals at the chemical shift in the absence of the stationary phase correlates with the inverse pore size; that is, the difference is larger for the stationary phase with the smallest pores, that is, Sephadex G-50. This effect is most noticeable for the isolated doublet at 6.2 ppm, but apparent for all resonances in the spectra. This suggests that it is likely that these smaller aggregates are formed in the void space within the stationary phase pores. To confirm the assignment of these additional resonances, HR-MAS was used to attempt to remove the susceptibility broadening28 by rapid rotation of the sample about the magic angle with respect to the applied magnetic field direction. Figure 2 shows the comparison between the HR-MAS spectrum and that obtained in a standard 5 mm NMR tube for the Sephadex G-50 stationary phase. For comparison, the corresponding spectrum in the absence of stationary phase is also included. There is clearly a decrease in line width and improvement in resolution observable for some, but notably

Figure 1. NMR spectra of 50 mM sunset yellow showing the aromatic region in (a) the absence of stationary phase, and in the presence of (b) Sephadex G-50, (c) Sephadex G-75, and (d) Sephadex G-100. Dashed lines indicate the positions of the resonances, which correlate with those in the absence of any stationary phase. 17504

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Figure 2. The aromatic region of the 1H NMR spectrum of 50 mM sunset yellow in the absence and presence of Sephadex G-50. Part (a) is acquired using a 5 mm X{1H} solution state NMR probe in the absence, part (b) in the presence of stationary phase, and part (c) using a 4 mm 1H{X} HR-MAS probe. The spectrum in (c) has been vertically scaled by a factor of 2 due to sensitivity differences between the two probes. Dashed lines indicate the positions of the resonances, which correlate with those in the absence of any stationary phase.

Figure 3. Exchange spectrum (EXSY) of a sample of 200 mM sunset yellow in the presence of Sephadex G-50 stationary phase. The presence of cross peaks indicates that there is exchange occurring between the free and in-pore components.

the pore during the period Δ. This is in contrast to the observations reported previously with poly(styrene sulfonate) and SEC stationary phases.25 In that case, only minor broadening was observed on the addition of the stationary phase as a result of the polymer spectrum being broad initially due to the molecular weight. The echo attenuation profiles were well described by a single diffusion coefficient;25 however, it is known that it is extremely challenging to distinguish multiexponential behavior if the diffusion coefficients are similar.35 The sunset yellow system investigated here has the additional complication that there is a fast exchange process between monomer and aggregate, and within the aggregates,7,11,12 occurring in both the free solution, and within the pores of the stationary phase, alongside the much slower exchange between the in-pore and free species pools. The ability to differentiate sunset yellow molecules within the pores of the stationary phase and those in free solution allows the partitioning of the aggregates between the stationary phase and the surrounding free solution to be quantified. The fraction of in-pore species f p can be defined as: Ip fp = If + Ip (1)

not all, of the resonances in the spectrum. The signals that show improvement under HR-MAS conditions align with those from the sunset yellow in the absence of the stationary phase and hence are assigned to be in the free solution surrounding the stationary phase particles. The remaining broad signals arise from the smaller sunset yellow aggregates located within the pores. These resonances remain broad due to the relatively slow spinning rate (υr = 2.5 kHz) employed, which is sufficient to remove the susceptibility broadening associated with the addition of the stationary phase for the species in the free solution; however, in the pores, the motion of the aggregates is coupled to the motion of the stationary phase particles, and hence the MAS is not fast enough to fully remove the anisotropic interactions.30,31 Similar results were obtained for the other stationary phases investigated (data not shown). Diffusion measurements were not attempted under MAS conditions due to the complicating effects of rapid sample spinning.32−34 Size exclusion chromatography is essentially an entropically driven process, unlike other forms of chromatography that rely on enthalpic interactions such as adsorption to the stationary phase to affect the separation. As such, while it is possible that the sunset yellow molecules in the pores are in exchange with molecules adhered to the surface of the pores, this is unlikely. Transverse relaxation measurements for the free and in-pore components reveal similar relaxation times, which indicates that the broadening observed for the in-pore fraction is the result of diffusion in an inhomogeneous field within the pore, rather than binding to the surface of the pore. If this latter situation were the case, then shorter transverse relaxation times would be expected for the bound species due to the slower overall correlation time of the SEC particle. The observation of two distinct sets of resonances for the free and in-pore components means that the system is in slow exchange on the time scale of the diffusion measurement. This was confirmed using exchange spectroscopy, as shown in Figure 3, where cross-peaks are clearly visible between the free and inpore fractions, indicating there is exchange between these two pools. Typical displacements during the diffusion labeling period Δ will be on the order of 2−5 μm, meaning that only a fraction of in-pore species will have any probability of leaving

where If and Ip are the integrals of a particular resonance for the free and in-pore species, respectively. The partitioning as a function of sample concentration is shown in Figure 4 for the three Sephadex phases investigated. While there is a large amount of scatter in the initial data points (i.e., those below approximately 0.1 M), resulting from low signal-to-noise in the corresponding NMR spectra and therefore greater uncertainty in the determined peak areas, there is clearly a trend across the concentrations and over the stationary phases used. The data shown in Figure 4 clearly show two components for each of the stationary phases, with the change over occurring at around 0.1 M. This change over is less pronounced for the Sephadex G100 stationary phase, which has the largest pore diameter of the stationary phases investigated. These data were modeled with the straight line given in eq 2, applied independently above and below 0.1 M, with the extracted parameters given in Table 2.

fp = m[SSY] + c 17505

(2)

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Table 3. Approximate Maximum Number of Sunset Yellow Monomers in Aggregates Able To Access the Stationary Phase Pore Spaces

Table 2. Parameters Obtained from Fitting the In-Pore Fraction Data as a Function of Concentration [SSY] ≤ 100 mM

[SSY] ≥ 100 mM

m (M−1)

c

R2

m (M−1)

c

R2

Sephadex G-50 Sephadex G-75 Sephadex G-100

−1.48 −0.92 −0.64

0.64 0.58 0.59

0.85 0.75 0.67

−0.18 −0.11 −0.06

0.53 0.52 0.54

0.87 0.93 0.95

In both regimes (i.e., above and below 0.1 M), the trend in the fitted gradients correlates with the fractionation range (i.e., pore size) of the Sephadex phase (Table 1), indicating that this variation observed between the stationary phases is most likely the result of size-exclusion effects. The change over observed at around 0.1 M may be the result of a reorganization and increase in compaction of the sunset yellow aggregates, as noted by Jones et al. in an analysis of the concentration dependence of the chemical shifts and the failure of the isodesmic aggregation model to adequately model these changes.11 It is possible to estimate the maximum number of sunset yellow monomers in an aggregate, which may enter the pores of the SEC stationary phase. Using published molecular weights M and Stokes radii Rs (in nm) of a series of macromolecules,36 the approximate radius corresponding to the quoted fractionation limit for a given stationary phase37 (Table 1) can be calculated using the following relationship obtained from linear regression: R s = 3.004 log M − 10.939

exclusion limit (kDa)

calculated pore radius (nm)

number of monomers

Sephadex G50 Sephadex G75 Sephadex G100

30

2.52

∼16

70

3.62

∼22

100

4.09

∼25

DOSY spectra for a 400 mM sample of sunset yellow in the absence (Figure 5a) and presence of the various Sephadex stationary phases (Figure 5b−d). The spectrum of sunset yellow without any stationary phase appears to show differing diffusion coefficients for each component of each multiplet. This is the result of a small amount of phase modulation as a function of gradient amplitude creeping into the spectra. This is most likely the result of a combination of gradient inhomogeneity38 and a small amount of sample convection.16 While not ideal, the presence of these experimental artifacts is not detrimental to the discussion of the spectra obtained in the presence of the Sephadex stationary phases. In these DOSY spectra (Figure 5b−d), there are clearly two components present, with the signals from the sunset yellow in free solution having similar diffusion coefficients, independent of the Sephadex fractionation range. The signals for the in-pore component show a diffusion coefficient that varies with the fractionation range of the Sephadex used; that is, it is a function of the pore size. A smaller diffusion coefficient is apparent for the Sephadex G-50 than for the G-75 or G-100 stationary phases. Where resonances from the free and in-pore species overlap, the resulting diffusion peak is found part way between the free and in-pore values. This is a result of the well-known “overlap problem” in DOSY NMR and arises from the fact that the echo attenuation for the overlapping signals is no longer well described by a single exponential function.16,35 Other processing methods such as (S)CORE may have some utility in this context.39−41 Extending this investigation across a series of concentrations, for each of the stationary phases, results in the data plotted in Figure 6. For each of the three stationary phases investigated, the observed diffusion coefficient for the free component, that is, the sunset yellow aggregates that are not in the pores of the stationary phase, is similar, albeit slightly reduced from that seen in the absence of the stationary phase. This indicates that the presence of the stationary phase itself, ignoring the influence of its porosity, has little effect beyond slightly increasing the effective viscosity of the sample. The observed diffusion coefficients for the in-pore component do show a variation depending on which stationary phase is used with Sephadex G-50 having the largest effect, while the G-75 and G100 phases show broadly similar results. The observed diffusion coefficient for the species within the pores should be governed principally by the size of the pores, because the combination of the pore size and the diffusion labeling period (Δ = 200 ms) used is in the so-called long-time limit.42 An empirical relationship between the diffusion coefficients in free solution and in the pore with the porosity of the bead ϕ has been determined to be Df = Dp/√ϕ.43,44 This expression assumes a spherical pore geometry.44 Interpreting the data in Figure 6 in terms of this relationship, the smallest in-pore diffusion

Figure 4. In-pore fraction as a function of sunset yellow concentration, in the presence of three different Sephadex size exclusion stationary phases, with differing pore sizes. In-pore fraction is defined in eq 1. The resonance chosen for use in the analysis was the free and in-pore doublet at ∼6.2 ppm. The dashed lines are fits to straight lines as described in the main text.

stationary phase

stationary phase

(3)

This can then be converted into the number of sunset yellow monomers using an intermonomer spacing of 3.32 Å, as determined by Joshi et al. using X-ray diffraction studies.9 These approximate maximum aggregate sizes are given in Table 3 and indicate that only fairly small assemblies are capable of entering the pores of the stationary phase; therefore, even at low concentrations of sunset yellow, where aggregates are comprised of tens of molecules,12 there will be a partitioning between the in-pore and free component. In addition to the changes in the in-pore fraction, the effect of the stationary phases on the diffusion properties of the aggregates was also investigated. Figure 5 shows representative 17506

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Figure 5. DOSY representations of a 400 mM sample of sunset yellow in (a) the absence of stationary phase, and in the presence of (b) Sephadex G50, (c) Sephadex G-75, and (d) Sephadex G-100. In the spectra with Sephadex, the free and in-pore components are highlighted.

The general overall trend of the diffusion coefficients as a function of concentration is similar to that observed previously for sunset yellow in isotropic solution.12 This indicates that the presence of the stationary phase does not significantly alter the aggregation behavior of the sunset yellow in the solution, beyond minor alterations in solution viscosity. Further investigation of the exchange behavior between the in-pore and free components is the subject of ongoing studies.

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CONCLUSIONS The addition of size-exclusion chromatography stationary phases has previously been shown to modify the diffusion profile of polymers, with the change in diffusion coefficient related to the molecular weight of the polymer as well as the pore size of the stationary phase.25 In this Article, we have extended the in situ size exclusion chromatographic NMR method to aggregating systems, specifically solutions of sunset yellow in their isotropic phase, and demonstrated that the method yields information regarding the distribution of aggregate species, by partitioning small aggregates within the pores of the stationary phase, without greatly altering the aggregation behavior of the dye.7,8,10,12 This approach is complementary to other NMR studies of isotropic solutions of sunset yellow through analysis of chemical shifts and diffusion coefficients and has potential application to the understanding

Figure 6. Diffusion coefficients as a function of sunset yellow concentration. The filled symbols are the free components, while the open symbols represent the in-pore component associated with the stationary phases.

coefficient for Sephadex G-50 correlates with the fact that this stationary phase has the smallest pores, and hence lower porosity than the other stationary phases. This simplistic description ignores the effects of exchange between the pores and the free solution, coupled with the interaggregate exchange of sunset yellow monomers. 17507

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of aggregation in a range of molecules in solution. Further investigation of the behavior of the stationary phases is underway.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +44 1273 876622. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS R.E.J. thanks the EPSRC for a DTA award. This work was supported by the University of Sussex and the EPSRC (EP/ H025367/1).

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