Low Buffer Concentrations Research Articles

Characterization of reversed-phase liquid chromatographic columns containing positively charged functionality

To date, the most commonly used column characterization databases do not determine the relative positive charge associated with new generation RP columns, or they fail to successfully discriminate between RP columns of purportedly low level positive and neutral characters. This paper rectifies this in that it describes a convenient and robust chromatographic procedure for the assessment of the low levels of positive charge on a range of RP columns. The low degree of positive charge was determined by their electrostatic attraction towards the negatively charged 4-n-octylbenzene sulfonic acid (4-OBSA) relative to their retention of the hydrophobic marker toluene (Tol).The new parameter (α4-OBSA/Tol) was determined for 15 commercially available RP-LC columns. When this was combined with existing Tanaka parameters it was possible to guide the chromatographer towards similar columns as “backup / equivalent phases” or dissimilar columns for exploitation in method development strategies. It should be noted that under certain chromatographic conditions the retention mechanism(s) may be too complex to allow direct location of a “backup / equivalent” column(s).The α4-OBSA/Tol results indicate that even the new generation neutral alkyl phases may exhibit a small degree of positive charge at low buffer concentrations. Mobile phases containing low % MeCN were demonstrated to promote mixed mode (anionic exchange / hydrophobic) retention whereas at high % MeCN anionic exchange retention dominated.The measure of electrostatic repulsion between positively charged columns and positively charged bases was assessed by evaluating the relative retention of a range of bases and neutral analytes. The greatest electrostatic repulsion was observed with hydrophilic bases. While there was no correlation between the positive charge associated with the phases assessed by electrostatic attraction or repulsion, the columns could be broadly divided into three subsets (i.e., significant positive character, medium to low positive character and insignificant positive character).Finally, the results were used to highlight the usefulness of the column characterization database containing the new anionic exchange retention parameter (α4-OBSA/Tol) for the selection of an equivalent column possessing a low level of positive character in the analysis of a real-life biopharmaceutical application.

Spectroscopic characterization of the interactions between poly(2-(trimethylamino)ethyl methacrylate) chloride and the xanthene dyes, 2′, 7′-difluorofluorescein and 2, 4, 5, 7-tetraiodofluorescein

Intermolecular interactions in buffered aqueous solution between the polycation, poly(2-(trimethylamino)ethyl methacrylate) chloride (pTMAEMC) and two anionic xanthene dyes, 2′, 7′-difluorofluorescein (Oregon Green 488) and 2, 4, 5, 7-tetraiodofluorescein (Erythrosin B), are characterized using multiple optical spectroscopic methods. Visible absorption spectroscopy indicates the formation of ground-state pTMAEMC-dye complexes. Benesi-Hildebrand binding isotherm analysis of visible absorption spectra for pTMAEMC-dye mixtures quantifies the strength of binding interactions producing the complexes. For both Oregon Green 488 (OG) and Erythrosin B (EB) in mixtures with pTMAEMC, the concentration of the solution’s sodium acetate buffer at a fixed pH alters the binding constants, Kb, suggesting that ionic strength plays a key role in determining the binding affinity of pTMAEMC for the dyes. Comparison of Kb, for the dyes indicates stronger binding of EB under all solution conditions. Steady-state fluorescence emission spectroscopy, fluorescence quenching, excited-state fluorescence lifetime measurements and fluorescence correlation spectroscopy provide complementary data for the interactions between pTMAEMC and the dyes. Mixtures of pTMAEMC with the dyes produce fluorescence enhancements and fluorescence quenching which exhibit a dependence on the buffer concentration used in the mixture. Excited-state lifetime analysis indicates that OG interacts with pTMAEMC through ground-state interactions while EB exhibits both ground-state and excited-state interactions with pTMAEMC. The spectroscopic measurements suggest that a polyelectrolyte effect for pTMAEMC due to ionic strength variation produced by the buffer concentration affects the dye binding profile of the polycation. This conclusion is supported by fluorescence correlation spectroscopy (FCS) analyses of the hydrodynamic diameter changes in pTMAEMC-OG binding in low buffer concentration (low ionic strength) solution. FCS analyses of pTMAEMC-OG mixtures also reveal diversity in the complexes formed in low ionic strength solution suggesting that other xanthene dyes will exhibit similar binding behaviors in mixtures with pTMAEMC as a function of solution ionic strength.

Mechanistic Studies and Formulation Mitigations of Adeno-associated Virus Capsid Rupture During Freezing and Thawing: Mechanisms of Freeze/Thaw Induced AAV Rupture

Gene therapies delivered using adeno-associated virus (AAV) vectors are showing promise for many diseases. Frozen AAV drug products are exposed to freeze-thaw (F/T) cycles during manufacturing, storage, and distribution. In this work we studied the mechanisms of AAV capsid rupture during F/T. We found that exposure to interfaces, exacerbated by F/T, and the mechanical force of excipient devitrification correlated with AAV capsid rupture during F/T. There was no impact of pH shifts, cryo-concentration, or cold-denaturation. Results were similar for AAV8 and AAV9. With these mechanistic insights we identified three formulation mitigation approaches. Addition of ≥0.0005% w/v poloxamer 188 (P188) eliminated substantial recovery losses (up to ∼60% without P188) and minimized rupture to ≤1% per F/T cycle. Elimination of exothermic devitrification events during rewarming, either by formulating with a low buffer concentration, or by adding a cryoprotectant further reduced rupture during F/T. Rupture of AAV9 was <0.2% per F/T cycle in a formulation with 1 mM phosphate, 4.4 mM dextrose, electrolytes, and 0.001% P188 at pH 7.2. Rupture of AAV8 was not detected when formulated with 4% sucrose, 100 mM salt, and 0.001% P188 at pH 7.4. These results provide insights into effective strategies for stabilizing AAVs against rupture during F/T.

Effect of ion-pairing reagent hydrophobicity on liquid chromatography and mass spectrometry analysis of oligonucleotides

We performed a systematic study of thirteen alkylamines used as ion-pairing reagents for ion-pair reversed-phase liquid chromatography (IP RP LC) separations of oligonucleotides on a C18 column. We proposed a method to classify the hydrophobicity of alkylamines by their retention in RP LC. The IP reagent hydrophobicity correlated with the retention and resolution of oligonucleotides in the corresponding IP mobile phases. The baseline resolution was achieved up to 30 mer for hydrophilic, or up to 50 mer for hydrophobic IP reagents. Hydrophobic alkylamines permitted useful oligonucleotide separations at relatively low buffer concentrations, such as 5–10 mM alkylamine-acetate IP systems. These buffers were compatible with mass spectrometry detection, however, replacement of acetic acid with hexafluoroisopropanol in the mobile phase improved the MS signal by 2–3 orders of magnitude. Experiments with native and chemically modified oligonucleotides highlighted the mixed-mode nature of IP RP LC. When using hydrophobic IP reagents, the ionic retention mechanism of oligonucleotides is enhanced while hydrophobic retention is diminished.

Electropolymerised pH Insensitive Salicylic Acid Reference Systems: Utilization in a Novel pH Sensor for Food and Environmental Monitoring.

This work summarizes the electrochemical response of a salicylic acid-based carbon electrode for use as a novel solid-state reference electrode in a redox-based pH sensor. This novel reference produces a pH insensitive response over a range of pH 3–10 in solutions with low buffer concentrations, different compositions, conductivities, and ionic strengths is produced. The pH of the local environment is shown to be determined by the chemistry and the electrochemical response of the redox active species on the surface of the electrode; the local pH can be controlled by the electropolymerized salicylic acid moieties due to the acid concentration on the surface, avoiding any perturbation in environmental pH and leading to a stable novel reference system. Sensitivities of −7.1 mV/pH unit, −2.4 mV/pH unit, −0.2 mV/pH unit, and 2.5 mV/pH units were obtained for different food medias, hydroponic solution, seawater, and cell-culture media, respectively, confirming its ability to control the local pH of the electrode. This reference system is paired with a new pH sensing element based on electropolymerized flavanone to provide a calibration free, pH sensitive sensor to effectively and accurately measure the pH of various media with high viscosity, low conductivity, low/high buffer concentration or cell-culture environment, presenting a maximum error of +/−0.03 pH units.

Impact of Time Out of Intended Storage and Freeze-thaw Rates on the Stability of Adeno-associated Virus 8 and 9

There are an increasing number of clinical studies evaluating different adeno-associated virus (AAV) serotypes as vectors for gene therapy. Long-term frozen storage can maximize the stability of AAV. Freeze-thaw (F/T) cycles and exposures to room temperature (RT) and refrigerated conditions occur during manufacturing, labeling, and clinical use. In this work we exposed AAV8 and AAV9 at low and high concentrations to five F/T cycles compounded with RT and refrigerated holds in a ‘daisy chain’ time out of intended storage (TOIS) stability study, which may be a best practice in early development. We also evaluated the impact of 5 F/T cycles for multiple permutations of fast and slow cooling and rewarming rates. The quality attributes of AAV8 and AAV9 remained within acceptable ranges after the daisy chain TOIS and F/T rate studies. Potency and concentration were unchanged within method variability. There was a minor increase in non-encapsidated (‘free’) DNA released from AAV8 after F/T in a phosphate-buffered saline formulation. DNA release during F/T was minimized in a formulation with a low buffer concentration and was not detected in a formulation containing sucrose. We conclude that AAV8 and AAV9 have stability profiles that are suitable for manufacturing and clinical development.

Buffer concentration dramatically affects the stability of S-nitrosothiols in aqueous solutions

S-nitrosothiols (RSNOs) are an important group of nitric oxide (NO)-donating compounds with low toxicity and wide biomedical applications. In this paper, we, for the first time, demonstrate that the concentration of buffer remarkably affects the stability of RSNOs including naturally occurring S-nitrosoglutathione (GSNO) and synthetic S-nitroso-N-acetylpenicillamine (SNAP). For a solution with a high concentration of GSNO (e.g., 50 mM) and an initial near-neutral pH, the optimal buffer concentration is close to the GSNO concentration under our experimental conditions. A lower buffer concentration does not have adequate buffer capacity to resist the pH drop caused by GSNO decomposition. The decreased solution pH further accelerates GSNO decomposition because GSNO is most stable at near-neutral pH according to our density functional theory (DFT) calculations. A higher-than-optimal buffer concentration also reduces the GSNO stability because buffer ingredients including phosphate, Tris base, and HEPES consume NO/N2O3. In contrast to GSNO, the highest SNAP stability is obtained when the starting solution at a neutral pH does not contain buffer species, and the stability decreases as the buffer concentration increases. This is because SNAP is more stable at mildly acidic pH and the SNAP decomposition-induced pH drop stabilizes the donor. When the RSNO concentration is low (e.g., 1 mM), the buffer concentration also matters because any excess buffer accelerates the donor decomposition. Since the effect of buffer concentration was previously overlooked and suboptimal buffer concentrations were often used, this paper will aid in the formulation of RSNO solutions to obtain the maximum stability for prolonged storage and sustained NO release.

Interplay of induced charge electroosmosis and electrothermal flow in insulator-based dielectrophoresis

A depth-averaged numerical model is developed to understand the experimentally observed interplay of induced charge electroosmosis and electrothermal flow in an insulator-based dielectrophoresis microdevice. The experimentally measured nonlinear fluid velocity matches asymptotically the predicted velocity of the electroosmotic flow in a low-concentration buffer and that of electrothermal flow in a high-concentration buffer. This agreement is consistent with a scaling analysis.

Role of configurational flexibility on the adsorption kinetics of bivalent bispecific antibodies on porous cation exchange resins

The adsorption kinetics of a monoclonal antibody (mAb) used as a reference and of bivalent bispecific antibodies (BiSAb) on a macroporous cation exchanger is studied experimentally by examining the transient patterns of bound protein within the particles using confocal microscopy for a range of protein concentrations, buffer concentrations and pH, and temperatures. The mAb adsorption kinetics is controlled by pore diffusion and conforms to the classical shrinking core model. While the mAb adsorption rate increases with temperature, the ratio of effective and free solution diffusivity, De /D0, remains constant and has a value of 0.20. The BiSAb's structure is comprised of scFv domains that are genetically fused to a framework IgG through flexible peptide linkers which results in conformational flexibility leading to multiple binding forms with varying affinity for the adsorbent surface. As a result, adsorption of the BiSAbs shows complex patterns of total bound protein within the particles. These BiSAb adsorption patterns are influenced by buffer ionic strength, pH, and temperature in unique ways. Sharper intraparticle profiles are observed for conditions where the binding strength is greater (lower buffer concentration and/or pH) or when the protein is chemically crosslinked to restrict configurational flexibility. Temperature affects the BiSAb pore diffusivity as well as the interconversion kinetics. While the effects of temperature on BiSAb transport are also described by a constant De /D0 = 0.15, the temperature also affects the rate of interconversion between binding forms leading to faster equilibration at higher temperatures. A phenomenological model indicates that the interplay of pore diffusion and adsorption with the kinetically limited interconversion between binding forms is responsible for the experimental trends.

Buffering effects on the solution behavior and hydrolytic degradation of poly(β‐amino ester)s

AbstractWith a vast, synthetically accessible compositional space and highly tunable hydrolysis rates, poly(β‐amino ester)s (PBAEs) are an attractive degradable polymer platform. Leveraging PBAEs in a wide range of applications hinges on the ability to program degradation, which, thus far, has been frustrated by multiple confounding phenomena contributing to the degradation of these charged polyesters. Basic conditions accelerate hydrolysis, yet reduce solubility, limiting water access to amines and esters. Further, the high buffering capacity of PBAEs can render buffers ineffective at controlling solution pH. To unify understanding of PBAE degradation and solution properties, this study examines PBAE hydrolysis as a function of pH and buffer concentration as well as polymer hydrophobicity. At low buffer concentrations, the PBAE amines and the acid produced during hydrolysis control solution pH. Meanwhile, at high buffer concentrations that afford relatively constant pH, hydrolysis rate increases with pH, despite the reduced PBAE solubility. Increasing the hydrophobic content of PBAEs eventually hinders the capacity of the polymer to accept protons from solution, limiting the pH increase and slowing hydrolysis. These studies showcase the role of buffering on the pH‐dependent degradation and solution properties of PBAEs, providing guidance for programming degradation in applications ranging from drug delivery to thermosets.

Hydrogen production during direct cellulose fermentation by mixed bacterial culture: The relationship between the key process parameters using response surface methodology

Dark fermentation is a promising method to produce hydrogen from lignocellulosic biomass. This study assessed the influence of temperature, phosphate buffer concentration and substrate concentration on direct hydrogen production form cellulose using response surface methodology. Mixed bacterial culture was successfully enriched on cellulose and used as an inoculum for hydrogen production. The model indicated that the highest cumulative hydrogen production (CHP) of 2.14 L H2/Lmedium could be obtained at 13.5 gcellulose/L, 79.5 mM buffer and 32.6 °C. However, hydrogen yield is then only 0.58 mol H2/molhexose due to low substrate conversion efficiency (SCE). Simultaneous optimization of CHP and SCE with desirability function approach resulted in the H2 yield of 2.71 ± 0.1 mol H2/molhexose and 93.8 ± 1.8% SCE at 3.35 gcellulose/L, 69 mM buffer and 32.9 °C. Phosphate concentration above 80 mM decreased H2 production, but had positive effect on cellulose consumption. The bacterial community analysis showed that Ruminiclostridium papyrosolvens was responsible for cellulose hydrolysis. Lachnoclostridium sp. was positively correlated with ethanol production at high phosphate buffer concentration, while Caproiciproducens sp. with caproate production at low buffer concentration. The obtained results opens the possibility of simultaneous hydrogen and caproate production from cellulosic substrates.

Mitigating the negative impact of soluble and insoluble lignin in biorefineries

The presence of inhibitors is still an economic bottleneck that needs to be resolved in order to make the biorefineries feasible, requiring the development of technologies capable of improving their competitiveness in the biofuel marketplace. Soluble and insoluble lignin can impair the enzymatic hydrolysis process by inhibition, deactivation, and unproductive adsorption of enzymes. Washing the pretreated biomass or using lignin-blocking additives during saccharification could mitigate these negative effects in future biorefineries. Here, an investigation was performed of the combined mitigation processes, in terms of their technical and economic feasibility in an integrated first and second generation (1G2G) sugarcane biorefinery. Evaluation was made of the impacts of biomass washing and soybean protein addition, separately or in combination, on glucose yields for enzymatic hydrolysis in the presence of high (liquor) and low (buffer) concentrations of soluble inhibitors/deactivators. Combining washing and soybean protein addition provided the highest glucose yields, with an increase of up to 50%. The effect of the mitigation processes could be explained by a combination of catalytic mechanisms acting on both soluble and insoluble lignin. In an industrial context, biomass washing (90 °C, 15% (w/w) solids, 3 steps) followed by soybean protein addition (12% (w/v) solids) provided a cost-competitive methodology for bioethanol production, with an estimated net present value of US$ 9.16 × 107, optimizing hydrolysis process in the 1G2G sugarcane biorefinery.

Multifactorial analysis of ion concentration polarization for microfluidic preconcentrating applications using response surface method

Ion concentration polarization (ICP) in a microfluidic device requires a precise balance of forces on charged molecules to achieve high concentrating efficiency. It is, thus, of considerable interest to study the impact of all governing parameters on ICP performance. Experimental study of the ICP multifactorial phenomenon seems impractical and costly. We report a systematic approach to understand the impacts of governing parameters on the ICP phenomenon using a robust numerical model established in COMSOL Multiphysics®. We varied the buffer concentration, applied voltage, and microchannel length to study their impacts on the ICP phenomenon. Then, we developed a statistical model via the response surface method (RSM) for the numerical results to study the direct and interactive effects of the mentioned parameters on ICP optimization. It was found that the buffer concentration (Cbuffer) plays a key role in the enrichment factor (EF); however, simultaneous impacts of the applied voltage and channel length must be considered as well to enhance EF. For low buffer concentrations, Cbuffer &amp;lt; 0.1 mM, the ionic conductivity was found to be independent of Cbuffer, while for high buffer concentrations, Cbuffer &amp;gt; 1 mM, the ionic conductivity was directly linked to Cbuffer. In addition, the RSM-based model prediction for a certain buffer concentration (∼1 mM) highlighted that an electric field of 20 V/cm–30 V/cm is suitable for the initial design of experiments in ICP microdevices.

Discrete Hf18 Metal-oxo Cluster as a Heterogeneous Nanozyme for Site-Specific Proteolysis.

The selective hydrolysis of proteins by non-enzymatic catalysis is difficult to achieve, yet it is crucial for applications in biotechnology and proteomics. Herein, we report that discrete hafnium metal-oxo cluster [Hf18 O10 (OH)26 (SO4 )13 ⋅(H2 O)33 ] (Hf18 ), which is centred by the same hexamer motif found in many MOFs, acts as a heterogeneous catalyst for the efficient hydrolysis of horse heart myoglobin (HHM) in low buffer concentrations. Among 154 amino acids present in the sequence of HHM, strictly selective cleavage at only 6 solvent accessible aspartate residues was observed. Mechanistic experiments suggest that the hydrolytic activity is likely derived from the actuation of HfIV Lewis acidic sites and the Brønsted acidic surface of Hf18 . X-ray scattering and ESI-MS revealed that Hf18 is completely insoluble in these conditions, confirming the HHM hydrolysis is caused by a heterogeneous reaction of the solid Hf18 cluster, and not from smaller, soluble Hf species that could leach into solution.

Discrete Hf18 Metal‐oxo Cluster as a Heterogeneous Nanozyme for Site‐Specific Proteolysis

AbstractThe selective hydrolysis of proteins by non‐enzymatic catalysis is difficult to achieve, yet it is crucial for applications in biotechnology and proteomics. Herein, we report that discrete hafnium metal‐oxo cluster [Hf18O10(OH)26(SO4)13⋅(H2O)33] (Hf18), which is centred by the same hexamer motif found in many MOFs, acts as a heterogeneous catalyst for the efficient hydrolysis of horse heart myoglobin (HHM) in low buffer concentrations. Among 154 amino acids present in the sequence of HHM, strictly selective cleavage at only 6 solvent accessible aspartate residues was observed. Mechanistic experiments suggest that the hydrolytic activity is likely derived from the actuation of HfIV Lewis acidic sites and the Brønsted acidic surface of Hf18. X‐ray scattering and ESI‐MS revealed that Hf18 is completely insoluble in these conditions, confirming the HHM hydrolysis is caused by a heterogeneous reaction of the solid Hf18 cluster, and not from smaller, soluble Hf species that could leach into solution.

Three-Dimensional Finite Element Model to Study Calcium Distribution in Astrocytes in Presence of VGCC and Excess Buffer

The role of astrocytes in physiological processes is always a matter of interest for biologists, mathematicians and computer scientists. Similar to neurons, astrocytes propagate Ca2+ over long distances in response to stimulation and release gliotransmitters in a Ca2+-dependent manner to modulate various important brain functions. There are various processes and parameters that affect the cytoplasmic calcium concentration level of astrocytes like calcium buffering, influx via calcium channels, etc. Buffers bind with calcium ion (Ca2+) and makes calcium bound buffers. Thus, it decreases the calcium concentration [Ca2+] level. Ca2+ enters into the cytosol through voltage gated calcium channel (VGCC) and thus it increases the concentration level. In view of above, a three-dimensional mathematical model is developed for combined study of the effect of buffer and VGCC on cytosolic calcium concentration in astrocytes. Finite element method is applied to find the solution using hexagonal elements. A computer programme is developed for entire problem to simulate the results. The obtained results show that high affinity buffer reveals the effect of VGCC and at low buffer concentration VGCC effects more significantly.

Is oxidative stress an issue in peritoneal dialysis?

During the last two decades, oxidative stress (OS) has emerged as a novel risk factor for a variety of adverse events, including atherosclerosis and mortality in chronic kidney disease (CKD) patients. Increased OS occurs even in early stages of the disease, progresses with deterioration of renal function and is further aggravated by hemodialysis (HD), due to the bioincompatibility of the method. Compared to HD, peritoneal dialysis (PD) is a more biocompatible dialysis modality, characterized by a significantly reduced, but still high, OS status. The culprit for OS in PD is mainly the composition of PD solutions (low pH, lactate buffer, increased osmolarity and high glucose concentration). After heat sterilization of PD solutions, formation of glucose degradation products (GDPs) and advanced glycation end-products (AGEs) trigger inflammation and enhance OS. Chronic exposure of the peritoneum to this toxic, hyperglycemic environment leads to OS-derived morphologic damage of peritoneal cells, loss of ultrafiltration capacity and decreased technique survival. Moreover, OS is linked with peritonitis, loss of residual renal function, inflammation, atherosclerosis, cardiovascular (CV) disease, and increased mortality. To ameliorate OS status in PD, a multitargeted approach is necessary that includes use of neutral pH, low GDP, low lactate and iso-ismolar PD solutions, strict glycemic control, optimal volume management and, probably supplementation with antioxidants, N-acetylcysteine being the most promising among them.

Molecular anchoring to oxide surfaces in ultrahigh vacuum and in aqueous electrolytes: phosphonic acids on atomically-defined cobalt oxide.

In this work, we investigated the interaction of phenylphosphonic acid (PPA, C6H5PO3H2) with atomically-defined Co3O4(111) thin films, grown on Ir(100), under ultrahigh vacuum (UHV) conditions and in the electrochemical environment. In the first step, we employed infrared reflection absorption spectroscopy (IRAS) and followed the formation of a saturated monolayer (380 K) in UHV. We observed that the binding motif changes from a chelating tridentate in the sub-monolayer regime to a chelating bidentate at full monolayer coverages. In the electrochemical environment, we analyzed the interaction of PPA with the same Co3O4(111) surface by electrochemical infrared reflection absorption spectroscopy (EC-IRRAS) (0.3 VRHE-1.3 VRHE). When adsorbed at pH 10 from an ammonia buffered aqueous solution, PPA binds to the surface in form of a fully deprotonated chelating bidentate. With increasing electrode potential, we observed two fully reversible processes. At low buffer concentration, protons are released upon oxidation of surface Co2+ ions and lead to protonation of the anchored phosphonates. At high buffer concentration, most of the protons released are accepted by NH3. Simultaneously, the surface phosphonate changes its adsorption motif from bidentate to tridentate while adopting a more upright geometry.

Direct Observation of Hydrangea Blue-Complex Composed of 3-O-Glucosyldelphinidin, Al3+ and 5-O-Acylquinic Acid by ESI-Mass Spectrometry.

The blue sepal color of hydrangea is due to a metal complex anthocyanin composed of 3-O-glucosyldelphinidin (1) and an aluminum ion with the co-pigments 5-O-caffeoylquinic acid (2) and/or 5-O-p-coumaroylquinic acid (3). The three components, namely anthocyanin, Al3+ and 5-O-acylquinic acids, are essential for blue color development, but the complex is unstable and only exists in an aqueous solution. Furthermore, the complex did not give analyzable NMR spectra or crystals. Therefore, many trials to determine the detailed chemical structure of the hydrangea-blue complex have not been successful to date. Instead, via experiments mixing 1, Al3+ and 2 or 3 in a buffered solution at pH 4.0, we obtained the same blue solution derived from the sepals. However, the ratio was not stoichiometric but fluctuated. To determine the composition of the complex, we tried direct observation of the molecular ion of the complex using electrospray-ionization mass spectrometry. In a very low-concentration buffer solution (2.0 mM) at pH 4.0, we reproduced the hydrangea-blue color by mixing 1, 2 and Al3+ in ratios of 1:1:1, 1:2:1 and 1:3:1. All solution gave the same molecular ion peak at m/z = 843, indicating that the blue solution has a ratio of 1:1:1 for the complex. By using 3, the observed mass number was m/z = 827 and the ratio of 1, 3 and Al3+ was also 1:1:1. A mixture of 1, 3-O-caffeoylquinic acid (4) and Al3+ did not give any blue color but instead was purple, and the intensity of the molecular ion peak at m/z = 843 was very low. These results strongly indicate that the hydrangea blue-complex is composed of a ratio of 1:1:1 for 1, Al3+ and 2 or 3.

Electrokinetic transport properties of deoxynucleotide monophosphates (dNMPs) through thermoplastic nanochannels

The electrokinetic behavior of molecules in nanochannels (<100 nm in length) have generated interest due to the unique transport properties observed that are not seen in microscale channels. These nanoscale dependent transport properties include transverse electromigration arising from partial electrical double layer overlap, enhanced solute/wall interactions due to the small channel diameter, and field-dependent intermittent motion produced by surface roughness. In this study, the electrokinetic transport properties of deoxynucleotide monophosphates (dNMPs) were investigated, including the effects of electric field strength, surface effects, and composition of the carrier electrolyte (ionic concentration and pH). The dNMPs were labeled with a fluorescent reporter (ATTO 532) to allow tracking of the electrokinetic transport of the dNMPs through a thermoplastic nanochannel fabricated via nanoimprinting (110 nm × 110 nm, width × depth, and 100 μm in length). We discovered that the transport properties in plastic nanochannels of the dye-labeled dNMPs produced differences in their apparent mobilities that were not seen using microscale columns. We built histograms for each dNMP from their apparent mobilities under different operating conditions and fit the histograms to Gaussian functions from which the separation resolution could be deduced as a metric to gage the ability to identify the molecule based on their apparent mobility. We found that the resolution ranged from 0.73 to 2.13 at pH = 8.3. Changing the carrier electrolyte pH > 10 significantly improved separation resolution (0.80–4.84) and reduced the standard deviation in the Gaussian fit to the apparent mobilities. At low buffer concentrations, decreases in separation resolution and increased standard deviations in Gaussian fits to the apparent mobilities of dNMPs were observed due to the increased thickness of the electric double layer leading to a partial parabolic flow profile. The results secured for the dNMPs in thermoplastic nanochannels revealed a high identification efficiency (>99%) in most cases for the dNMPs due to differences in their apparent mobilities when using nanochannels, which could not be achieved using microscale columns.