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Taylor Dispersion Analysis of polysaccharides using backscattering interferometry Phoonthawee Saetear, Joseph Chamieh, Michael N. Kammer, Thomas J. Manuel, Jean-Philippe Biron, Darryl J. Bornhop, and Hervé Cottet Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 22 May 2017 Downloaded from http://pubs.acs.org on May 25, 2017

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Analytical Chemistry

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Taylor Dispersion Analysis of polysaccharides using backscattering interferometry

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Phoonthawee Saetear1,*, Joseph Chamieh1, Michael N. Kammer2,3, Thomas J. Manuel4,

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Jean-Philippe Biron1, Darryl J. Bornhop2,3, Hervé Cottet1,*

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Institut des Biomolécules Max Mousseron (UMR 5247 CNRS - Université de Montpellier – Ecole Nationale Supérieure de Chimie de Montpellier), place Eugène Bataillon CC 1706, F-34095 Montpellier Cedex 5, France. 2 Department of Chemistry, Vanderbilt University, Nashville, TN 37235, USA. 3 The Vanderbilt Institute for Chemical Biology, Vanderbilt University, Nashville, TN 37235, USA. 4 Department of Agricultural and Biological Engineering, Mississippi State University, MS 39762, USA. ABSTRACT: Taylor Dispersion Analysis (TDA) allows the determination of the molecular diffusion coefficient (D) or the hydrodynamic radius (Rh) of a solute from the peak broadening of a plug of solute in a laminar Poiseuille flow. The main limitation plaguing the broader applicability of TDA is the lack of a sensitive detection modality. UV absorption is typically used with TDA, but is only suitable for UV-absorbing or derivatized compounds. In this work, we present a development of TDA method for nonUV absorbing compounds by using a universal detector based on refractive index (RI) sensing with backscattering interferometry (BSI). BSI was interfaced to a capillary electrophoresis-UV instrument using a polyimide coated fused silica capillary and an inhouse designed flow-cell assembly. Polysaccharides were selected to demonstrate the application of TDA-BSI for size characterization. Under the conditions of validity of TDA, D and Rh average values and the entire Rh distributions were obtained from the (poly)saccharide taylorgrams, including non-UV absorbing polymers.

49 1. INTRODUCTION Taylor Dispersion Analysis (TDA) is a simple and abso- 50 lute method for the determination of diffusion coefficient (D), 51 and thus hydrodynamic radius (Rh), of solutes of virtually any 52 size from angstroms to submicrons. It is based on the analysis 53 of the peak broadening of a solute plug in a laminar Poiseuille 54 flow, originated from the work of Taylor1,2 and Aris3. TDA 55 was first employed to determine diffusion coefficient of ana- 56 lytes in gaseous phase4 and lately in liquid phase5-7. In liquid 57 phase, TDA can be implemented with high performance liquid 58 chromatography (HPLC) equipment using either UV5-7 or RI8- 59 10 detections. Limitations of TDA with HPLC equipment are 60 large injection volume (about 10 – 20 µL) and quite long 61 analysis time (about an hour) to fulfill the conditions of validi- 62 ty for TDA. As pointed out by Bello et al in 199411, the capil- 63 lary electrophoresis (CE) instrumentation and the commercial- 64 ly available narrow bore fused-silica capillaries are well suited 65 for doing TDA within a few minutes and low sample con- 66 sumption (a few nL). TDA has been performed on a CE-UV 67 apparatus, for a broad range of applications, including size 68 characterization of small molecules12,13, proteins11,14-19, pep- 69 tides20, polymers21-23, pharmaceuticals24 and nanoparticles25,26. 70 However, many solutes exhibit little or no UV absorbance, 71 72 limiting the applicability of TDA by CE-UV. There are several approaches available for detecting non- 73 UV absorbing samples by CE. One common approach is to 74 perform chemical derivatization of the species using a UV- 75 absorbing reagent. This approach does have limitations 76 though; it requires the presence of a reactive group on the 77 solute and can be time-consuming. Another approach is based 78

on the use of capacitively coupled contactless conductivity detector (C4D) which can only be used for the detection of charged (ionic) solutes27. Mass spectrometry is also a very attractive alternative detection mode for TDA28. However, MS is restricted to volatile electrolytes, it is relatively expensive and requires a liquid-gas interface increasing instrument complexity. In the case of polymer analysis, ion suppression with MS detector is also an issue for quantitative analysis. In the specific case of saccharide analysis, photooxidation reaction of mono- and disaccharide was used for direct UV detection in capillary electrophoresis at 270 nm29. High pH (~12-13) of background electrolyte was required to produce some UV-absorbing intermediate species by photooxidation. However, this approach was only reported of monoand disaccharides, not for polysaccharides. The refractive index (RI) detector is also a potential alternative detector, suitable for quantifying compounds which show no absorption in the UV region. In general, RI detectors are mass sensitive, bulk property and nondestructive sensors. RI detectors are reasonably sensitive, providing a signal for essentially all analytes, as long as the difference in RI or refractive index increment of the solute and its matrix is not zero. RI detection coupled to TDA has been reported with HPLC system8-10. However, the detection volume of most commercially available liquid chromatography (LC)-RI detectors is ~10 µL. This value is two-three orders of magnitude larger than what is suitable for TDA, when performed on 25100 µm i.d. capillaries using CE equipment. Surface plasmon resonance (SPR)-based detector coupling with Taylor Dispersion injection (TDi) was also reported to generate the continu-

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ous analytes titration for both steady-state and kinetic anal- 63 yses30. SPR is capable of detecting changes of RI occurring 64 near the surface of a sensor chip. This work used a commer- 65 cially available SensíQ Pioneer to determine D for biomole- 66 cules. However, this system was coupled to HPLC system and 67 required about 20 µL injection volume. In addition, the SPR 68 signal is interrogating the liquid at about 100 – 200 nm from 69 the surface which cannot be considered as a true bulk meas- 70 71 urement. There have been many attempts to develop RI detectors 72 with volumes in the nanoliters regime31-36, with the most suc- 73 cessful involving some form of beam interference and direct 74 interrogation of the capillary. The backscattering interferome- 75 try (BSI), approach has been shown to enable the determina- 76 tion of ∆RI changes at the 10-7 RIU level in a probe volume of 77 250 pL. Furthermore, BSI has been successfully used in CE37- 78 39 . The BSI detector works by interrogating the interference pattern produced from the laser-capillary interaction, viewed in the backscatter configuration33,40-42. The interference pattern is a set of periodic dim and bright spots (fringes) whose positions are related to the RI of the fluid in the capillary tubing. By measuring phase changes after Fourier-Transform of these patterns of light in the backscatter configuration, it has been shown that BSI can serve as a very sensitive RI detector. While BSI has been used for numerous applications, some of the physical parameters affecting the sensitivity of the measurement in the capillary format has been studied43. Given that BSI has been successfully used in CE detection for the analysis of some organic dyes and carbohydrates37, cations38 and caffeine in beverages39, it represents a good candidate for use in TDA. Here we report on our efforts to incorporate BSI detection into TDA framework, by interfacing BSI to a CE-UV instrument using an in-house designed flow cell and polyimide coated capillary. Polysaccharides and their monomers were selected to demonstrate this application, as they constitute an 79 important class of (macro)molecules that generally do not 80 absorb in UV. To our knowledge, this is the first time that 81 TDA-BSI has been used for sizing non-UV absorbing species. 82

83 2. EXPERIMENTAL 84 2.1. Chemicals 85 All saccharide compounds used in this work can be cate- 86 gorized in 3 groups based on the degree of polymerization: 87 monomers, oligomers and polymers (see Figure SI-1 in the 88 Supporting Information for the chemical structures). Mono- 89 saccharides are D-(+)-glucose monohydrate (Alfa Aesar 90 GmbH & Co KG, Germany) and D-(+)-galacturonic acid 91 monohydrate (Fluka, Slovakia). Oligosaccharides are D-(+)- 92 maltose monohydrate (Sigma Aldrich, Japan) and malto- 93 tetraose, Dp4 (C24H42O11, product no. 47877, lot no. 94 LC13397V, Supelco, USA). Polysaccharides are dextran 95 sulfate (product no. D-6001, lot no. 052K2615, Sigma Al- 96 drich, Switzerland), dextran T500 (lot no. 276838, Amersham 97 Pharmacie, France), pullulan from Aureobasidium pullulans 98 (P416, lot no. SLBH6388V, Sigma Aldrich, Japan), glycogen 99 from oyster-Type II (product no. G8751, lot no. SLBN2162V,100 Sigma Aldrich, Japan), sodium hyaluronate (product no.101 F002205, lot no. 0910001, Bioiberica, Spain) and pectin from102 citrus peel (product no. P9135, lot no. SLVN9007V, Sigma103 Aldrich, Denmark). Pectin sample has a galacturonic acid104 content ≥75% and methoxy content ≥6.7%. 105

106

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Following chemicals were used for the preparation of buffers. Sodium phosphate monobasic, sodium phosphate dibasic and 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS) were purchased from Sigma Aldrich, France. 4-(2Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) was from BDH chemicals (VWR, Paris, France). Tris(hydroxymethyl)aminomethane was from Riedel-De Haën AG Seelze-Hannover (Germany). Sodium tetraborate decahydrate was from Merck (Germany). The pH of buffer solutions was adjusted with 1 M NaOH and 1 M HCl. All saccharide solutions were prepared by dissolving the appropriate amount in phosphate buffer (ionic strength 160 mM; pH 7.4; 1.09 × 10-3 Pa⋅s viscosity at 25 °C) that was prepared by dissolving 15.7 mM Na2HPO4, 3.2 mM NaH2PO4 and 110 mM NaCl in ultra-pure water (18 MΩ cm) purified on a Milli-Q system from Millipore (Molsheim, France).

Figure 1. Schematic diagram of instrumentation setup for Taylor Dispersion Analysis of saccharide compounds showing (a) a capillary electrophoresis-UV instrumentation interfaced to backscattering interferometry (BSI) and (b) an in-house designed flow cell assembly for BSI. 2.2. BSI interface with the CE-UV equipment for TDA A commercial CE system (P/ACETM MDQ, Beckman, USA) was used to operate TDA with modification of the CE cartridge using two external detector adapters (EDA part n°149852, Beckman, USA) to allow simultaneous detection for BSI and UV (200 nm), shown as in Figure 1. The picture of instrumentation setup is shown in Figure SI-2 in the Supporting Information. The BSI setup and detection was performed outside the MDQ system. Bare fused-silica capillary (Polymicro technologies, USA) of 100 µm i.d. and 200 µm o.d. with a total length of 155.9 cm was used and inserted in the Beckman cassette. In Figure 1a, the coolant tubing conducts the capillary from the inlet to the outlet of CE cartridge passing through detection windows of BSI and UV at 80.5 am and 145.9 cm, respectively. The cooling of the capillary was ensured by the MDQ system using the EDA parts. Detection by BSI is able to work without removing the polyimide coating, whereas detection by UV requires the removal of polyimide coating to allow UV transparency. The principle of the BSI setup is shown on the right hand side of the block diagram in Figure 1a and has been explained


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in detail elsewhere40,41,44. When applying BSI, the fused-silica 64 capillary serves as the optics and laser light impinges on a 65 fused-silica capillary containing the fluid of interest. As the 66 light passes through the different capillary interfaces 67 (air/glass/fluid), it is reflected and refracted at each interface 68 creating a series of high contrast fringes. A CCD camera is 69 used to record the backscattered interference fringe pattern. 70 By measuring the positional shift of the fringes, the changes in 71 RI can be quantified. 72 All components and flow cell assembly are rigidly 73 mounted on a square optical breadboard (60 cm × 60 cm, 74 Newport Corp., USA). Manual micrometer-driven-translation 75 stages (Newport Corp., USA) are used to provide reproducible 76 motion of the CCD array detector, flow cell assembly and all 77 components. A linearly polarized 4 mW Helium-Neon (He- 78 Ne) laser (632.8 nm, 25 LHP-121-249, Melles Griot, USA) 79 coupling with optical fiber lens (600 µm spot size, Oz optics, 80 Canada) was used as the light source. Capillary on the flow 81 cell assembly was located 10 cm from head of the fiber cou- 82 pler lens. The CCD array (CCD-53600-D, ALPHALAS 83 GmbH, Germany) located 22 cm from the capillary was used 84 85 to detect the backscattered fringes. The in-house designed flow cell assembly shown in Figure 1b consists of a fused-silica capillary mounted on a massive, black anodized aluminum block. The aluminum block/capillary assembly was temperature stabilized with a Peltier thermoelectric cooler (TE-127-1.0-1.3, TE Technology, Inc., USA) controlled by a digital temperature controller (LFI3751, Wavelength Electronics, USA) set at 25°C and wired in feedback from a calibrated thermocouple (OMEGA Engineering, Inc., USA). By-pass coolant tubing was connected to make diversion of coolant, allowing the laser beam to impinge only on the capillary at the detection point of BSI which measures the bulk properties of the flowing liquid in the capillary. For ease in positioning, the flow cell assembly was mounted on two stacked transition stages.

86 2.3. Taylor Dispersion Analysis TDA was performed using the previously described 87 Beckman P/ACETM MDQ instrumentation interfaced with the 88 BSI detection (located on the optical breadboard) and UV (200 89 nm, from CE equipment) at detection windows of 80.5 cm and 90 145.9 cm from the inlet side, respectively (see Figure SI-3 in 91 the Supporting Information). New capillaries were conditioned 92 with the following flushes at 20 psi: 1M NaOH for 60 min; 93 water for 60 min and buffer for 60 min. TDA experiments 94 were carried out at 25°C (cooling was ensured by the MDQ 95 system using the external detector adapter). Samples were 96 injected hydrodynamically on the inlet side of the capillary 97 (0.3 psi, 20 s, 59.2 nL injected volume) for online detection by 98 BSI (Vi/Vd = 0.94%) and by UV (Vi/Vd = 0.52%), where Vi/Vd 99 is the ratio of finite volume of the injected sample volume to100 the volume of capillary from inlet to the detector. Experiments101 were performed using a mobilization pressure of 1, 2 or 5 psi102 depending on the samples and the operation (see Figure cap-103 tions). Between each run, the capillary was rinsed with water104 105 (5 min) and buffer (5 min) at 20 psi. The taylorgrams were recorded in sequence by BSI and106 UV (see Figure SI-3 in the Supporting Information). For BSI107 detection, data was recorded and stored on a computer via108 USB cable of the CCD array camera with an in-house software109 written using LabVIEW 2015 (National Instruments, USA).110 The phase (signal from BSI) of the recorded fringe pattern is111

extracted by using a Fourier-algorithm40,41. In our BSI configuration the CCD camera orientation is upside-down. In other words, we obtain negative going phase values for increasing RI signals (see Figure SI-3 in the Supporting Information). Therefore, to meet with convention (increasing RI gives an increasing signal (phase value), we have taken the absolute value (multiplied by -1) of the phase shift values in our data set. This approach allows positive ∆RI as increasing phase values. For UV detection, the absorbance detector was used and operated by Beckman Coulter's 32 Karat™ Software 8.0. All data obtained from BSI and UV were exported to Microsoft Excel for subsequent data processing using Microcal Origin 6.0. The peak variance was determined by Gaussian fitting and by integration method described elsewhere45 to obtain the average values of weight-average Rh. Deconvolutions of the signal by fitting with a sum of two or three Gaussian contributions46, and by mathematical approach based on Constrained Regularized Linear Inversion (CRLI)47 were also applied to obtain the size distribution of the species in the sample. All samples were analyzed 5 times and the average D (or Rh) values, as well as the Rh distribution were reported.

Figure 2. Taylorgrams obtained for (a) D-(+)-glucose monohydrate and (b) D-(+)-galacturonic acid monohydrate in frontal mode by BSI and UV at 200 nm. Experimental condition: fused silica capillary 160 cm total length × 100 µm i.d. (200 µm o.d.); detection window from inlet: 80.5 cm (BSI) and 150 cm (UV). Eluent: phosphate buffer, 15.7 mM Na2HPO4, 3.2 mM NaH2PO4 and 110 mM NaCl (160 mM ionic strength, pH 7.4, 1.10 × 10-3 Pa⋅s at 25°C). Mobilization pressure: 5 psi. Samples were prepared in the eluent at the concentration mentioned on the graph. 3. RESULTS AND DISCUSSION 3.1. BSI detection on narrow bore capillaries and determination of LOD/LOQ in frontal mode Here we set out to demonstrate that BSI can be used for the detection and quantification of both non-UV and UV absorbing species in TDA using narrow bore fused silica capillaries. To do so, the signal was first recorded in frontal mode (continuous injection of the sample, see Figure SI-3a in the Supporting Information) to allow a precise determination of the limit of detection (LOD) and the limit of quantification (LOQ) without dilution of the sample zone. D-(+)-glucose monohydrate (non-UV absorbing species) and of D-(+)galacturonic acid monohydrate (UV absorbing species) were selected as model compounds. Each TDA experiment allowed



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simultaneous BSI and UV detection (200 nm) at different 48 detection times, noted t1 and t2 respectively in Figure SI-3b. 49 Figure 2 shows the elution profiles for various concentrations 50 of glucose and galacturonic acid, keeping the same elution 51 time window from 0 to 6 min. As expected in Figure 2a, glu- 52 cose response was only reported by BSI detection and not in 53 UV absorption. On the other hand, Figure 2b shows the signal 54 changes in both BSI and UV for galacturonic acid solution. As 55 mentioned in experimental section, the phase values in our 56 data sets on elution profiles were multiplied by -1 to allow 57 positive ∆RI to be displayed by increasing phase values. Elu- 58 tion profiles of glucose and galacturonic acid obtained from 59 BSI show a positive phase shift since the RI of the glucose and 60 galacturonic acid solutions have higher values than that of the 61 phosphate buffer eluent. This positive shift makes sense be- 62 cause BSI is reporting a relative measurement of changes in RI 63 between the eluent and solute in the eluent. Higher concentra- 64 tions of solute (with a higher RI) will lead to larger changes in 65 RI thus to larger positive phase shift due to the increase in the 66 measured RI. This observation maybe non-intuitive and is in 67 contrast to a conventional RI detector, which typically em- 68 ploys a cell which contains two equal volume sides, one con- 69 taining the mobile phase the other the column eluent. Elution 70 profile of galacturonic acid obtained by UV shows positive 71 absorbance since the solute contains a carboxylic group (mo- 72 lecular structure shown in Figure SI-1 in the Supporting In- 73 74 formation) which can absorb the UV light at 200 nm.

75 Table 1. Sensitivity, limit of detection (LOD) and quantifica- 76 tion (LOQ) of saccharide compounds obtained from BSI and 77 UV detections in frontal mode on 100-µm i.d./200-µm o.d. fused-silica capillary. Phosphate buffer pH 7.4 was used as eluent. Mobilization pressure was 5 psi.

Compound

Glucose Galacturonic acid Maltose Maltotetraose Dextran sulfate Dextran T500 Pullulan Glycogen Sodium hyaluronate Pectin

34 35 36 37 38 39 40 41 42 43 44 45 46 47

Slopea (mrad L g-1) 182.8 164.5

BSI LODb (mg L-1) 35 39

LOQc (mg L-1) 116 129

UV at 200 nm Slopea LODb LOQc (mAU (mg (mg L g-1) L-1) L-1) ND ND ND 9.6 9.4 31

78 79 80 81 171.5 59 197 ND ND ND 82 165.7 53 176 ND ND ND 83 180.4 50 166 15.0 10 33 84 137.7 52 173 24.4 3.7 13 85 86 168.7 56 188 23.6 5.1 16 87 a From linear calibration curves. 88 b Defined as 3σ/slope. 89 c Defined as 10σ/slope. ND: not detected. 90 91 Calibration curves were obtained by plotting the absolute 92 values of phase shift (|∆φ|, in radian unit) for BSI or the sig93 nal height (∆AU) for UV, according to the solute concentra94 tion. Therefore, slope of the calibration curves are always 95 positive values. The LOD and LOQ were determined based on 96 3σ/slope and 10σ/slope, respectively. The sigma (σ) is ob- 97 tained from the standard deviation of the signal response (400 98 - 500 data points) and the slope is obtained from the linear 99 regression of the calibration line. Figures of merit relative to100 182.0 177.3 130.5

21 37 82

69 124 273

ND ND ND

ND ND ND

ND ND ND

the calibration curves are gathered in Table 1. Even though

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lower LOD and LOQ were obtained by UV for galacturonic acid; glucose could be only detected by BSI with LOD and LOQ in the order of 35 mg L-1 and 116 mg L-1, respectively. It is worth noting that the LOD and LOQ values obtained in frontal mode are lower than those that would be obtained in plug mode due to dilution factor (e.g. about 7-fold for glucose and galacturonic acid). Clearly frontal mode provides better LOD and LOQ than plug mode. 3.2. Effects of fused silica capillary dimensions and eluent composition Several types of buffers were investigated: phosphate buffer (ionic strength 160 mM; pH 7.4), HEPES-Na (20 mM; pH 7.4), TRIS-HCl (20 mM; pH 7.4), borate-Na (20 mM; pH 9.2) and borate-CAPS-Na (33 mM borate and 13.3 mM CAPS; pH 9.2). The figures of merit derived from the calibration curves are provided in Table SI-1 in the Supporting Information. Results showed that all buffers gave comparable detection sensitivity (slopes of the linear regression were between 80-90 mrad L g-1), except for TRIS-HCl eluent which gave lower sensitivity (61 mrad L g-1). The corresponding LOD and LOQ vary between 54 to 71 mg L-1 and 179 to 237 mg L-1, respectively, and were significantly higher for TRISHCl (98 mg L-1and 326 mg L-1). At that point, phosphate buffer pH 7.4 was selected as eluent. Table 2. Effect of capillary dimension on the LOD and LOQ for analysis of D-(+)-maltose monohydrate measured by BSI in frontal mode. Phosphate buffer pH 7.4 was used as eluent. Mobilization pressure was 5 psi. Thickness of polyimide coating (µm)a 18.1 – 18.8

Slopec (mrad L g-1)

LODd (mg L-1)

LOQe (mg L-1)

50 / 360

Wall thick ness (µm)b 155

41.7

152

506

75 / 360

142.5

18.5 – 19.4

79.7

71

237

100 / 360

130

20.7 – 21.1

118.6

32

106

Dimension (i.d./o.d., µm)a

100 / 200 50 12.6 – 12.8 182.0 21 69 a Indicated by manufacturer. b(o.d.–i.d.)/2. cFrom linear calibration curves. dDefined as 3σ/slope. eDefined as 10σ/slope. i.d.: internal diameter; o.d.: outer diameter.

For UV detection, there is well-known and established relationship between path length and sensitivity (BeerLambert Law). When working in the linear range, even with capillaries (not a square cuvette) it is normally found that the sensitivity is directly proportional to the capillary inner diameter. In the case of BSI detection, our earlier observations43 appeared to indicated that there was little influence on sensitivity as a function of capillary inner diameter because the capillary acts as an optical resonator-like cavity allowing the beam to make numerous passes through the sample43,48 and because of the inherent difficulty to thermostat the solution in the capillary. As with all preliminary scientific observations, we have since found the situation to be more complicated. As our ability to drive the noise floor down, decreasing dn/dT contributions49 and to refine our modeling efforts50 we have come to understand that, with more effective temperature control49, the sensitivity of BSI depends on both the capillary i.d. and the wall thickness. Thus, the theoretical relationship is more complicated than just defining the capillary i.d. Here


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were performed a series of experiments to determine the sensi- 63 tivity of our TDA-BSI system. These empirical sensitivity data 64 given in Table 2, show that when the capillary i.d. was varied, 65 while keeping the capillary o.d. constant at 360 µm we obtained a quantifiable change in response or ∆RI sensitivity. 66 Using these experimental values we were able to optimize the 67 detection scheme, for both S/N and separation efficiency con- 68 siderations. As shown in Table 2, the capillary of 100 µm 69 i.d./200 µm o.d with approximate 13-µm thickness of polyi- 70 mide coating gave the best sensitivity of detection (slope of 71 the calibration curve) and the best LOD and LOQ of 21 mg L-1 72 and 69 mg L-1. We chose to use this capillary for further ex- 73 periments described here. 74

75

3.3. Application to the Taylor Dispersion Analysis of 76 (poly)saccharides 77 TDA experiments were performed in plug injection mode 78 (Figure SI-3b in the Supporting Information) using the buffer 79 and capillary dimensions selected from section 3.2. Figure 3 80 shows the taylorgrams obtained for mono- and oligosaccharides; whereas Figure 4 displays those for polysaccharides. 81 The corresponding figures of merit are given in Table 1. As 82 expected, oligosaccharides (maltose and maltotetraose), which 83 consist of a number of the monomeric units (repeating units) of glucose gave a response only with BSI, not in UV (see the insets in Figures 3c and 3d). The taylorgrams were then fitted with Gaussian peaks (see red and black lines in Figure 3) for the mono- and oligosaccharides since they are monodisperse samples45, allowing the corresponding D and Rh values to be determined (Table 3). It was found that the D and Rh values are comparable for the two detection modes. The polysaccharides used in this work consist of a number of glucose or glucose-like monomeric units (see Figure SI1 in the Supporting Information for chemical structures of dextran sulfate, dextran T500, pullulan and glycogen), and glucose-like structure containing carboxylic acids or ester moieties (sodium hyaluronate and pectin). The four first samples only constituted of glucose/glucose sulfate should have no UV absorbance. Our TDA-BSI experiments (Figures 4a to 4c), 84 shows this to be the case for this class of species, except for 85 glycogen (see Figure 4d). According to the product specifica- 86 tion, glycogen from oyster, used in this work contains ≥ 75% 87 of glycogen. It was reported in the literature that the product 88 contains not only glycogen but also fat, protein and salts51. 89 Proteins are UV-absorbing at 200 nm and may be the cause of 90 the UV response for the glycogen sample (Figure 4d). Sodium 91 hyaluronate contains D-glucuronic acid and D-N- 92 acetylglucosamine (see Figure SI-1), both of which are UV 93 absorbing species. Pectin is a complex ester-based polysaccha- 94 ride derived from galacturonic acid and methanol which has 95 some absorbance at 200 nm. The galacturonic acid units con- 96 tained within pectin can be either partially methyl-esterified, 97 acetylated or both. 98 The taylorgrams for the polysaccharides displayed in 99 Figure 4 are non-Gaussian due to the polydisperse nature of100 the samples. Residual monomers or salts (small molecules)101 also contributes to this non-Gaussian shape. Here we observed102 peak shape typical of bimodal mixture, with a thin peak prin-103 cipally due to the small molecule or monomer, displaying on104 the top of a broader peak due to the polymer52. First, the105 weight-average hydrodynamic radius (Rh) was calculated over106 all the solutes for these taylorgrams. This calculation is per-107

108

formed by integration of the taylorgram and using the following equations:

η

(1)

where kB is the Boltzmann constant, T is the temperature (in K), RC is the capillary radius, η is the viscosity of the eluent and t0 is average elution time. is the temporal variance of the elution profile that can be calculated using eq. (2)23,45:

∑

∑

(2)

where h(t) is the detector response, ti is elution time for a given point i of the taylorgram, and n and m are the starting and ending points that are considered for the integration of the Taylorgram. The integration of signal is generally performed on the left part of the signal45 (t – t0 ≤ 0) in order to avoid any error due to possible adsorption of the solutes onto the capillary wall. In all measurements, the conditions met the requirement for TDA, with the numerical values of the dimensionless residence time τ ≥ 1.25 (τ = Dt0/Rc2) and the Péclet number Pe ≥ 40 (Pe = Rcu/D)53. Here, both conditions were verified for all samples, the values varied between 1.7 to 574 for τ and 51 to 1.7×104 for Pe.

Figure 3. Taylorgrams obtained by BSI and UV (200 nm, insets) detections of (a) D-(+)-glucose monohydrate, (b) D(+)-galacturonic acid monohydrate, (c) D-(+)-maltose monohydrate and (d) maltotetraose, Dp4. Experimental condition: fused silica capillary, 155.9 cm total length × 100 µm i.d. (200 µm o.d.); detection window from inlet: 80.5 cm (BSI) and 145.9 cm (UV). Eluent: phosphate buffer, 15.7 mM Na2HPO4, 3.2 mM NaH2PO4 and 110 mM NaCl (pH 7.4; ionic strength 160 mM, 1.10 × 10-3 Pa⋅s viscosity at 25°C). Mobilization pressure: 2 psi. Injection at 0.3 psi for 20 s (59.2 nL injected volume, Vi/Vd ratio are 0.94% for BSI and 0.52% for UV). All solutions were at 10 g L-1 in the eluent. Gaussian curve fitting are displayed in red; experimental data in black. The D and Rh values were found to correlate well with the molar mass given in Table 3 (i.e., higher molar mass (or higher degree of polymerization (DP)) leads to larger Rh (or lower D)). The standard deviation for D and Rh values are given in Table 3 for five repetitions. The average relative standard deviation on D calculated on all samples given in Table 3 is 2%. While similar average Rh values, , were reported by UV and BSI, for glycogen and hyaluronate, this was not the case for pectin. UV detector leads to higher average Rh compared to the RI detector (see comparison of values in Table 3).



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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 61 62

Pectin is known to be a polydisperse polysaccharide having different responses between RI and UV detectors due to the polymer heterogeneity in composition. As reported earlier, the macromolecules with higher molar masses have fewer UV absorbing molecules associated with them than their lower molar mass counterparts54,55. A second approach applicable to the processing of the taylorgrams from the polysaccharide samples (Figure 4) is to curve fitting with two (or three) Gaussian functions52. This approach allows the size and mass proportion of each two/three populations to be determined. Rigorously, the proportion of signal coming from the fraction of peak area of the two populations is similar to the mass proportion, only if the two populations have the same response coefficients. In the present case, there is no reason that these response coefficients would be similar since the chemical structures of both populations are different. The curve fitting results are displayed in red in Figure 4 and the corresponding quantitative values of D and Rh obtained from both BSI and UV detection methods are gathered in Table 3. The Rh of the population of smallest size (population 1) ranges between 0.14 and 3.10 nm, which may correspond to small ions (salt) and oligosaccharides, respectively. 63 The proportion of population 1 expressed in terms of peak area varies between 5% and 41%. Rh of population 2, which is 64 more representative of the polymer size compared to the aver- 65 age Rh value, varies between 6.49 nm and 47.14 nm, depend- 66 67 ing on molar mass and on the structure of the polymers. To demonstrate that the two/three Gaussian curve fitting 68 69 approach to analyzing the taylorgrams can effectively provide 70 the population size of each of the polymers, dextran T500 and 71 glycogen from oyster were dialyzed against the phosphate 72 buffer used as eluent prior TDA analysis. This purification 73 step aids to significantly remove the small molecules from the mixture which appear as thin peaks for both polymers in BSI and UV taylorgrams (Figure SI-4, Supporting Information). Interestingly, this dialysis step was found to be unnecessary. Here the Rh of polymers obtained before and after dialysis are comparable (see the values in Figure SI-4 in the Supporting Information), as far as the small molecule contribution was subtracted from the taylorgrams for the non-dialyzed samples. This observation represents an advantage over other particle sizing methods, making it quite straightforward to vanish the small molecule contribution. A third approach based on the deconvolution of the taylorgrams using constrained regularized linear inversion (CRLI)47 has also been applied to all polymeric samples presented in Figure 4. This approach has the advantage of providing the entire Rh distribution without any assumption on the number of populations contained in the sample. Figure 5 shows the whole Rh distribution as obtained by CRLI from BSI (Rh distribution from UV detection is shown in the Figure SI-5). The average size of each population and their percentage in mass proportion extracted from the distribution are also tabulated in Table 3. In the whole, the results obtained by the CRLI approach are consistent with the second approach (see values in Table 3), even if the CRLI provides a more detailed view of the whole distribution and therefore, more information on the size dispersion of the sample (see Figures 5 and SI-5 in 74 the Supporting Information). Using CRLI, dextran T500, 75 pectin and sodium hyaluronate appear as the most polydis- 76 perse samples in the collection of the studied polysaccharides. 77

78

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Figure 4. Taylorgrams obtained from BSI and UV (200 nm, insets) of (a) dextran sulfate, (b) dextran T500, (c) pullulan from Aureobasidium pullulans, (d) glycogen from oyster, (e) sodium hyaluronate and (f) pectin from citrus peel. Experimental condition as in Figure 3. Mobilization pressure: 1 psi for sodium hyaluronate and pectin, and 2 psi for others. Injection at 0.3 psi for 20 sec (59.2 nL injected volume, Vi/Vd ratio are 0.94% for BSI and 0.52% for UV). All solutions were at 10 g L-1 in the eluent. Curve fitting with two Gaussian peaks are displayed in red; experimental data in black.

Figure 5. Hydrodynamic radius distribution as obtained by CRLI with BSI for (a) dextran sulfate, (b) dextran T500, (c) pullulan from Aureobasidium pullulans, (d) glycogen from oyster, (e) sodium hyaluronate and (f) pectin from citrus peel.


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4. CONCLUSIONS Here we present, for the first time, the application of TDA to the size-characterization of polysaccharides using backscattering interferometry (BSI). We accomplished these polymer characterization measurements by building a TDA-RI instrument with nanoliter detection volume by coupling a BSI optical set-up to a commercial CE-UV instrument. The universal detection capability of BSI allowed us to circumvent one of the main limitation plaguing the use of TDA, which is that only a small class of candidate molecules exhibit significant UV absorption. Under validated TDA operating conditions, it was shown that BSI can be used to determine the molecular diffusion coefficient (D) and the corresponding hydrodynamic radius (Rh) of (poly)saccharide compounds. In addition, both average Rh values and the entire Rh distributions were derived from results obtained by TDA-BSI, important size characterization parameters for non-UV absorbing species, including polydisperse polymer samples. Due to the simplicity of the TDA approach, the absence of stationary phase (that can generate abnormal elution as in SEC), the low injection volume, the universal character of BSI, the TDA-BSI method represents a potentially powerful technique for the sizecharacterization of a large variety of samples.

26 ASSOCIATED CONTENT 27 Supporting Information 28 The Supporting Information is available free of charge on the 29 ACS Publications website. 30 31 Figure SI-1, structure of saccharide compounds; Figure SI-2, 32 picture of instrumentation setup; Figure SI-3, schematic represen33 tation of the mobilization; Figure SI-4, taylorgrams from dialysis 34 experiment; Figure SI-5, Rh distribution obtained by CRLI with 35 UV detection; Table SI-1, investigation of type of buffer (PDF) 36 37 AUTHOR INFORMATION 38 *Corresponding Authors 39 HC: Tel.: +33 4 67 14 34 27; Fax: +33 4 67 63 10 46. 40 E-mail: [email protected] 41 PS: E-mail: [email protected] 42 Author Contributions 43 PS performed the experiments under the guidance of JC, DJB and 44 HC. MNK and TJM wrote the LabVIEW code for recording the 45 BSI signal. JPB analyzed data using CRLI. PS and HC wrote the 46 manuscript. 47 ACKNOWLEDGMENTS 48 HC thanks the support from the Institut Universitaire de France 49 (IUF junior member, 2011-2016). The authors are grateful to the 50 postdoctoral scholarship from the Development and Promotion of 51 Science and Technology Talents Project (DPST) under Thai 52 Royal Government given to PS. DJB, MNK and TJM 53 acknowledge support of the National Science Foundation (NSF) 54 through Grant CHE-1307899. 55 56



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Table 3. Quantitative data on molecular diffusion coefficient (D) and hydrodynamic radius (Rh) of saccharide compounds obtained from TDA with BSI and UV detectiona. Compound

Molar mass (g mol-1)

BSI

UV at 200 nm

Glucose Galacturonic acid Maltose Maltotetraose, Dp4 Dextran sulfate

198.16b 212.15b 360.31b 666.58b 5×105 b

D × 10-11 (m2 s-1)c 69.7 ± 0.79d 71.6 ± 0.27d 52.6 ± 0.19d 38.8 ± 0.67d 2.59 ± 0.06e

Dextran T500

5×105

2.96 ± 0.07e

6.17 ± 0.16e

Pullulan

3.34 -6.14 × 105 [56]

3.00 ± 0.05e

6.62 ± 0.12e

Glycogen

5×106 b

1.97 ± 0.07e

10.07 ± 0.36e

Sodium hyaluronate

3.0×104 – 2.0×106 b

1.72 ± 0.02e

10.55 ± 0.15e

9.99 ± 0.37f 11.29 ± 0.41g (88%) 0.37 ± 0.06g (12%)

Pectin

2.0×104 – 2.0×106 [57]

2.89 ± 0.14e

6.87 ± 0.34e

7.53 ± 0.51f 11.74 ± 0.84h (54%) 2.81 ± 0.13h (41%) 0.16 ± 0.01h(5%)

Rh (nm) 0.28 ± 0.003d 0.28 ± 0.002d 0.38 ± 0.001d 0.51 ± 0.01d 7.66 ± 0.17e

Two/Three-Gaussian ND ND ND ND 6.97 ± 0.16f 7.95 ± 0.18g (87%) 0.20 ± 0.01g(13%) 6.17 ± 0.01f 6.49 ± 0.01g (95%) 0.19 ± 0.001g (5%) 6.42 ± 0.06f 7.42 ± 0.07g (84%) 1.12 ± 0.004g(16%) 10.34 ±0.35f 11.17 ± 0.38g (92%) 0.30 ± 0.06g (8%)

CRLI ND ND ND ND 6.64i 7.65j (99.50%) 0.23j (0.50%) 6.41i 6.80j (99.64%) 0.62j (0.31%) 0.10j (0.05%) 6.80i 7.97j (95.50%) 1.65j (4.50%) 10.23i 11.22j (99.70%) 0.35j (0.30%) 10.22i 14.74j (78.10%) 6.59j (21.40%) 0.54j (0.30%) 0.26j (0.20%) 7.77i 12.94j (83.95%) 2.77j (15.95%) 0.16j (0.10%)

a

D × 10-11 (m2 s-1)c ND 62.6 ± 0.16d ND ND ND ND

Rh (nm) ND 0.32 ± 0.001d ND ND ND ND

Two/Three-Gaussian ND ND ND ND ND ND

CRLI ND ND ND ND ND ND

ND ND ND

ND ND ND

ND ND ND

ND ND ND

ND ND

ND ND

ND ND

ND ND

2.04 ± 0.09c

9.75 ± 0.41e

9.77 ± 0.30f 15.90 ± 0.50g (60%) 0.39 ± 0.004g (40%)

1.39 ± 0.01c

14.28 ± 0.14e

14.18 ± 0.37f 14.79 ± 0.36g (95%) 1.79 ± 0.58g (5%)

0.75 ± 0.03c

26.48 ± 0.88e

21.99 ± 0.15f 47.14 ± 0.33h (45%) 3.10 ± 0.01h (23%) 0.14 ± 0.001h (32%)

8.08i 13.68 j (97.87%) 1.64 j (0.19%) 0.38 j (1.94%) 14.74i 17.61j (85.88%) 7.95j (13.90%) 2.34j (0.20%) 0.22j (0.02%) 23.10i 64.47j (91.29%) 7.31j (7.93%) 1.55j (0.41%) 0.84j (0.20%) 0.13j (0.17%)

Experimental condition as described in Figures 3 and 4. Indicated by manufacturer. c Corresponding to the d From Gaussian fitting (n=5). e By left-part integration of the taylorgram using eq. (1) and (2), (n=5). f values calculated from the mass proportion of each population by two/three Gaussian functions. g By curve fitting of the left part of the taylorgram with two Gaussian functions. The mass proportion (or peak area proportion) of each population is given in parenthesis (n=5). h By curve fitting of the left part of the taylorgram with three Gaussian functions. The mass proportion (or peak area proportion) of each population is given in parenthesis (n=5). i values obtained from the integration of whole Rh distribution (n=1). j By deconvolution of the taylorgram using constrained regularized linear inversion (CRLI) approach. The mass proportion (or peak area proportion) of each population is given in parenthesis (n=1). ND: not detected. b


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