33 research outputs found
Super-resolution imaging reveals resistance to mass transfer in functionalized stationary phases
Chemical separations are costly in terms of energy, time, and money.
Separation methods are optimized with inefficient trial-and-error approaches
that lack insight into the molecular dynamics that lead to the success or
failure of a separation and, hence, ways to improve the process. We perform
super-resolution imaging of fluorescent analytes in four different commercial
liquid chromatography materials. Surprisingly, we observe that chemical
functionalization can block over fifty percent of the porous interior of the
material, rendering it inaccessible to small molecule analytes. Only in situ
imaging unveils the inaccessibility when compared to the industry-accepted ex
situ characterization methods. Selectively removing some of the
functionalization with solvent restores pore access without significantly
altering the single-molecule kinetics that underlie the separation and agree
with bulk chromatography measurements. Our molecular results determine that
commercial stationary phases, marketed as fully porous, are over-functionalized
and provide a new avenue to characterize and direct separation material design
from the bottom-up
Unified superresolution experiments and stochastic theory provide mechanistic insight into protein ion-exchange adsorptive separations
Chromatographic protein separations, immunoassays, and biosensing all typically involve the adsorption of proteins to surfaces decorated with charged, hydrophobic, or affinity ligands. Despite increasingly widespread use throughout the pharmaceutical industry, mechanistic detail about the interactions of proteins with individual chromatographic adsorbent sites is available only via inference from ensemble measurements such as binding isotherms, calorimetry, and chromatography. In this work, we present the direct superresolution mapping and kinetic characterization of functional sites on ion-exchange ligands based on agarose, a support matrix routinely used in protein chromatography. By quantifying the interactions of single proteins with individual charged ligands, we demonstrate that clusters of charges are necessary to create detectable adsorption sites and that even chemically identical ligands create adsorption sites of varying kinetic properties that depend on steric availability at the interface. Additionally, we relate experimental results to the stochastic theory of chromatography. Simulated elution profiles calculated from the molecular-scale data suggest that, if it were possible to engineer uniform optimal interactions into ion-exchange systems, separation efficiencies could be improved by as much as a factor of five by deliberately exploiting clustered interactions that currently dominate the ion-exchange process only accidentally
Single-Molecule Imaging in Commercial Stationary Phase Particles Using Highly Inclined and Laminated Optical Sheet Microscopy
We resolve the three-dimensional, nanoscale locations
of single-molecule
analytes within commercial stationary phase materials using highly
inclined and laminated optical sheet (HILO) microscopy. Single-molecule
fluorescence microscopy of chromatography can reveal the molecular
heterogeneities that lead to peak broadening, but past work has focused
on surfaces designed to mimic stationary phases, which have different
physical and chemical properties than the three-dimensional materials
used in real columns and membranes. To extend single-molecule measurements
to commercial stationary phases, we immobilize individual stationary
phase particles and modify our microscope for imaging at further depths
with HILO, a method which was originally developed to resolve single
molecules in cells of comparable size to column packing materials
(∼5–10 μm). We describe and characterize how to
change the angle of incidence to achieve HILO so that other researchers
can easily incorporate this method onto their existing epi- or total
internal reflection fluorescence microscopes. We show improvements
up to a 32% in signal-to-background ratio and 118% in the number of
single molecules detected within stationary phase particles when using
HILO compared to epifluorescence. By controlling the objective position
relative to the sample, we produce three-dimensional maps of molecule
locations throughout entire stationary phase particles at nanoscale
lateral and axial resolutions. The number of localized molecules remains
constant axially throughout isolated stationary phase particles and
between different particles, indicating that heterogeneity in a separation
would not be caused by such affinity differences at microscales but
instead kinetic differences at nanoscales on identifiable and distinct
adsorption sites
Single-Molecule Imaging in Commercial Stationary Phase Particles Using Highly Inclined and Laminated Optical Sheet Microscopy
We resolve the three-dimensional, nanoscale locations
of single-molecule
analytes within commercial stationary phase materials using highly
inclined and laminated optical sheet (HILO) microscopy. Single-molecule
fluorescence microscopy of chromatography can reveal the molecular
heterogeneities that lead to peak broadening, but past work has focused
on surfaces designed to mimic stationary phases, which have different
physical and chemical properties than the three-dimensional materials
used in real columns and membranes. To extend single-molecule measurements
to commercial stationary phases, we immobilize individual stationary
phase particles and modify our microscope for imaging at further depths
with HILO, a method which was originally developed to resolve single
molecules in cells of comparable size to column packing materials
(∼5–10 μm). We describe and characterize how to
change the angle of incidence to achieve HILO so that other researchers
can easily incorporate this method onto their existing epi- or total
internal reflection fluorescence microscopes. We show improvements
up to a 32% in signal-to-background ratio and 118% in the number of
single molecules detected within stationary phase particles when using
HILO compared to epifluorescence. By controlling the objective position
relative to the sample, we produce three-dimensional maps of molecule
locations throughout entire stationary phase particles at nanoscale
lateral and axial resolutions. The number of localized molecules remains
constant axially throughout isolated stationary phase particles and
between different particles, indicating that heterogeneity in a separation
would not be caused by such affinity differences at microscales but
instead kinetic differences at nanoscales on identifiable and distinct
adsorption sites
Extending single molecule fluorescence observation time by amplitude-modulated excitation
We present a hardware-based method that can improve single molecule fluorophore observation time by up to 1500% and super-localization by 47% for the experimental conditions used. The excitation was modulated using an acousto-optic modulator (AOM) synchronized to the data acquisition and inherent data conversion time of the detector. The observation time and precision in super-localization of four commonly used fluorophores were compared under modulated and traditional continuous excitation, including direct total internal reflectance excitation of Alexa 555 and Cy3, non-radiative Förster resonance energy transfer (FRET) excited Cy5, and direct epi-fluorescence wide field excitation of Rhodamine 6G. The proposed amplitude-modulated excitation does not perturb the chemical makeup of the system or sacrifice signal and is compatible with multiple types of fluorophores. Amplitude-modulated excitation has practical applications for any fluorescent study utilizing an instrumental setup with time-delayed detectors