Attenuation Coefficients of Tubular Conduits for Liquid Phase Absorbance Measurement: Shot Noise Limited Optimum Path Length.

We trace the history of liquid core waveguides (LCWs, also called liquid core optical fibers) and the role Teflon AF (TAF) has played in their development. We show that, in any shot noise limited situation, the optimum signal-to-noise ratio (S/N) occurs at a path length of 1/αa{ln[1 + 2(αa/αb)]}, approximately 2/αb under most conditions, αa and αb being the light attenuation coefficient due to the analyte and the background, respectively. The analysis shows that LCW length should be selected depending on the applicable αb value. An overly long LCW may exhibit a lower signal-to-noise ratio. Water-filled TAF-clad fused-silica (FS) tubes show the lowest attenuation across the wavelength range. Nevertheless, except at λ ≥ 600 nm, the observed αb values far exceed those reported for pure water: it appears that both impurities in the water and waveguide losses are involved. In examining the attenuation in various water-filled tubes, we find that the transmission of air-surrounded FS tubes is second only to TAF-clad FS tubes and is better than that of TAF tubes or externally mirrored FS tubes. Surprisingly, except for a window centered at ∼250 nm, light transmission in a water-filled poly(tetrafluoroethylene) (PTFE) tube is worse than in poly(ether ether ketone) (PEEK) tubing. Light transmission in PTFE tubes improves with increasing wall thickness.

Flow-Cell-Induced Dispersion in Flow-through Absorbance Detection Systems: True Column Effluent Peak Variance.

Following a brief overview of the emergence of absorbance detection in liquid chromatography, we focus on the dispersion caused by the absorbance measurement cell and its inlet. A simple experiment is proposed wherein chromatographic flow and conditions are held constant but a variable portion of the column effluent is directed into the detector. The temporal peak variance (σt,obs2), which increases as the flow rate (F) through the detector decreases, is found to be well-described as a quadratic function of 1/F. This allows the extrapolation of the results to zero residence time in the detector and thence the determination of the true variance of the peak prior to the detector (this includes contribution of all preceding components). This general approach should be equally applicable to detection systems other than absorbance. We also experiment where the inlet/outlet system remains the same but the path length is varied. This allows one to assess the individual contributions of the cell itself and the inlet/outlet system.to the total observed peak. The dispersion in the cell itself has often been modeled as a flow-independent parameter, dependent only on the cell volume. Except for very long path/large volume cells, this paradigm is simply incorrect.

Width Based Characterization of Chromatographic Peaks: Beyond Height and Area.

The preceding paper ( Anal. Chem. 2017 , 10.1021/acs.analchem.6b04857 ) introduced width-based quantitation (WBQ). The present paper focuses on (1) situations where WBQ is effective while height/area-based linear calibrations fail, e.g., when (a) the detector is in a nonlinear response region, (b) the detector/data system is saturated, causing clipping/truncation of the signal, or (c) the detector signal is not a single-valued function of concentration, as when a fluorescence signal goes into the self-quenched domain. (2) Utilization of WBQ in postcolumn reagent addition methods where the reagent produces a significant detector background. WBQ can minimize added reagent without sacrificing the upper determination limit; a limited reagent amount truncates peaks from high analyte concentrations but does not hamper WBQ at a low height. (3) A description of peak asymmetry via leading/trailing half-widths vs relative height (fraction of maximum height) plots. (4) A holistic description of chromatographic peaks through six parameters describing the two independent generalized Gaussian distributions that underlie the WBQ chromatographic peak model. (5) Characterization of shape by widths at multiple heights and shape-based impurity detection.

Width Based Quantitation of Chromatographic Peaks: Principles and Principal Characteristics.

Height- and area-based quantitation reduce two-dimensional data to a single value. For a calibration set, there is a single height- or area-based quantitation equation. High-speed high-resolution data acquisition now permits rapid measurement of the width of a peak (Wh), at any height h (a fixed height, not a fixed fraction of the peak maximum) leading to any number of calibration curves. We propose a width-based quantitation (WBQ) paradigm complementing height or area based approaches. When the analyte response across the measurement range is not strictly linear, WBQ can offer superior overall performance (lower root-mean-square relative error over the entire range) compared to area- or height-based linear regression methods, rivaling weighted linear regression, provided that response is uniform near the height used for width measurement. To express concentration as an explicit function of width, chromatographic peaks are modeled as two different independent generalized Gaussian distribution functions, representing, respectively, the leading/trailing halves of the peak. The simple generalized equation can be expressed as Wh = p(ln h̅)q, where h̅ is hmax/h, hmax being the peak amplitude, and p and q being constants. This fits actual chromatographic peaks well, allowing explicit expressions for Wh. We consider the optimum height for quantitation. The width-concentration relationship is given as ln C = aWhn + b, where a, b, and n are constants. WBQ ultimately performs quantitation by projecting hmax from the width, provided that width is measured at a fixed height in the linear response domain. A companion paper discusses several other utilitarian attributes of width measurement.

Highly flexible UV-vis radiation sources and novel detection schemes for spectrophotometric HPLC detection.

The concept and performance of the first multiwavelength deep UV light-emitting-diode-based high-performance liquid chromatography (HPLC) absorbance detector are presented. In single-wavelength mode and with optical reference, the limit of detection (LOD) is comparable to conventional state-of-the-art HPLC absorbance detectors. In multiwavelength mode--at present up to eight wavelengths without optical reference--the LOD is about 10 times higher than in single-wavelength mode. Multiplexing and demultiplexing methods are used to separate chromatographic signals in multiwavelength mode and keeps the detector configuration simple and yet flexible. Depending on the operation mode, stray light is either totally negligible or controlled electronically and digitally.

Relation between chromatographic resolution and signal-to-noise ratio in spectrophotometric HPLC detection.

Absorption spectrophotometry has been and still is the industry standard for detection in HPLC. Limit of detection (LOD) and linear dynamic range (LDR) are the primary performance requirements and have driven continuous improvement of spectrophotometric HPLC detectors. Recent advances in HPLC column technology have led to low flow-rate HPLC such as capillary HPLC and nanoflow HPLC and put higher demands on optical HPLC signal detection. However, fundamental principles in spectrophotometric HPLC detection have not been reviewed for many years. In particular the relationship between the detector's signal-to-noise ratio (SNR) and band broadening needs to be re-evaluated. In this work, a new quantitative model is presented which allows the calculation of the trade-off made between chromatographic resolution and SNR in spectrophotometric HPLC detection. Modern optics flow cells based on total internal reflection are included and compared to conventional flow cells.

Fully automated isotopic dimethyl labeling and phosphopeptide enrichment using a microfluidic HPLC phosphochip.

Quantitative detection of phosphorylation levels is challenging and requires an expertise in both stable isotope labeling as well as enrichment of phosphorylated peptides. Recently, a microfluidic device incorporating a nanoliter flow rate reversed phase column as well as a titania (TiO(2)) enrichment column was released. This HPLC phosphochip allows excellent recovery and separation of phosphorylated peptides in a robust and reproducible manner with little user intervention. In this work, we have extended the abilities of this chip by defining the conditions required for on-chip stable isotope dimethyl labeling allowing for automated quantitation. The resulting approach will make quantitative phosphoproteomics more accessible.

Exploring the human leukocyte phosphoproteome using a microfluidic reversed-phase-TiO2-reversed-phase high-performance liquid chromatography phosphochip coupled to a quadrupole time-of-flight mass spectrometer.

The study of protein phosphorylation events is one of the most important challenges in proteome analysis. Despite the importance of phosphorylation for many regulatory processes in cells and many years of phosphoprotein and phosphopeptide research, the identification and characterization of phosphorylation by mass spectrometry is still a challenging task. Recently, we introduced an approach that facilitates the analysis of phosphopeptides by performing automated, online, TiO(2) enrichment of phosphopeptides prior to mass spectrometry (MS) analysis. The implementation of that method on a "plug-and-play" microfluidic high-performance liquid chromatography (HPLC) chip design will potentially open up efficient phosphopeptide enrichment methods enabling phosphoproteomics analyses by a broader research community. Following our initial proof of principle, whereby the device was coupled to an ion trap, we now show that this so-called phosphochip is capable of the enrichment of large numbers of phosphopeptides from complex cellular lysates, which can be more readily identified when coupled to a higher resolution quadrupole time-of-flight (Q-TOF) mass spectrometer. We use the phosphochip-Q-TOF setup to explore the phosphoproteome of nonstimulated primary human leukocytes where we identify 1012 unique phosphopeptides corresponding to 960 different phosphorylation sites providing for the first time an overview of the phosphoproteome of these important circulating white blood cells.

Improved hydrophilic interaction chromatography LC/MS of heparinoids using a chip with postcolumn makeup flow.

Heparan sulfate (HS) and heparin are linear, heterogeneous carbohydrates of the glycosaminoglycan (GAG) family that are modified by N-acetylation, N-sulfation, O-sulfation, and uronic acid epimerization. HS interacts with growth factors in the extracellular matrix, thereby modulating signaling pathways that govern cell growth, development, differentiation, proliferation, and adhesion. High-performance liquid chromatography (HPLC)-chip-based hydrophilic interaction liquid chromatography/mass spectrometry has emerged as a method for analyzing the domain structure of GAGs. However, analysis of highly sulfated GAG structures decasaccharide or larger in size has been limited by spray instability in the negative-ion mode. This report demonstrates that addition of postcolumn makeup flow to the amide-HPLC-chip configuration permits robust and reproducible analysis of extended GAG domains (up to degree of polymerization 18) from HS and heparin. This platform provides quantitative information regarding the oligosaccharide profile, degree of sulfation, and nonreducing chain termini. It is expected that this technology will enable quantitative, comparative glycomics profiling of extended GAG oligosaccharide domains of functional interest.

Impact of conduit geometry on the performance of typical particulate microchip packings.

This work investigates the impact of conduit geometry on the chromatographic performance of typical particulate microchip packings. For this purpose, high-performance liquid chromatography (HPLC)/UV-microchips with separation channels of quadratic, trapezoidal, or Gaussian cross section were fabricated by direct laser ablation and lamination of multiple polyimide layers and then slurry-packed with either 3 or 5 microm spherical porous C8-silica particles under optimized packing conditions. Experimentally determined plate height curves for the empty microchannels are compared with dispersion coefficients from theoretical calculations. Packing densities and plate height curves for the various microchip packings are presented and conclusively explained. The 3 microm packings display a high packing density irrespective of their conduit geometries, and their performance reflects the dispersion behavior of the empty channels. Dispersion in 5 microm packings correlates with the achieved packing densities, which are limited by the number and accessibility of corners in a given conduit shape.

Packing density, permeability, and separation efficiency of packed microchips at different particle-aspect ratios.

HPLC microchips are investigated experimentally with respect to packing density, pressure drop-flow rate relation, hydraulic permeability, and separation efficiency. The prototype microchips provide minimal dead volume, on-chip UV detection, and a 75 mm long separation channel with a ca. 50 microm x 75 microm trapezoidal cross-section. A custom-built stainless-steel holder allowed to adopt optimized packing conditions. Separation channels were slurry-packed with 3, 5, and 10 microm-sized spherical, porous C8-silica particles. Differences in interparticle porosity, permeability, and plate height data are analyzed and consistently explained by different microchannel-to-particle size (particle-aspect) ratios and particle size distributions.

Separation efficiency of particle-packed HPLC microchips.

We report an experimental study of separation efficiency in microchip high-performance liquid chromatography (HPLC). For this study, prototype HPLC microchips were developed that are characterized by minimal dead volume, a separation channel with trapezoidal cross section, and on-chip UV detection. A custom-built stainless steel holder enabled microchip packing under pressures of up to 400 bar and ultrasonication. Bed densities were investigated with respect to the packing conditions and consistently related to pressure drop over the packed microchannels and separation efficiency under isocratic elution conditions. The derived plate height curves show a decrease of mobile phase mass transfer resistance with increasing bed density. High bed densities are critical to separation performance in noncylindrical packed beds, because only at low bed porosities does hydrodynamic dispersion in noncylindrical packings come close to that of cylindrical packings. At higher bed porosities, the presence of fluid channels of advanced flow velocity in the corners of noncylindrical packings affects hydrodynamic dispersion strongly. We demonstrate that the separation channels of HPLC microchips can be packed as densely as the cylindrical fused-silica capillaries used in nano-HPLC and that consequently microchip-HPLC separation efficiencies comparable to those of nano-HPLC can be achieved.

G-index: A new metric to describe dynamic refractive index effects in HPLC absorbance detection.

High performance liquid chromatography (HPLC) with a solvent gradient and absorbance detection is one of the most widely used methods in analytical chemistry. The observed absorbance baseline is affected by the changes in the refractive index (RI) of the mobile phase. Near the limited of detection, this complicates peak quantitation. The general aspects of these RI-induced apparent absorbance effects are discussed. Two different detectors with fundamentally different optics and flow cell concepts, a variable-wavelength detector equipped with a conventional flow cell and a diode-array detector equipped with a liquid core waveguide flow cell, are compared with respect to their RI behavior. A simple method to separate static - partly unavoidable - RI effects from dynamic RI effects is presented. It is shown that the dynamic RI behavior of an absorbance detector can be well described using a single, relatively easy-to-determine metric called the G-index. The G-index is typically in the order of a few seconds and its sign depends on the optical flow cell concept.

Molecular absorption measurements with an optical fibre coupled array of ultra-violet light-emitting diodes.

A photometric detector based on eight different light-emitting diodes covering the ultraviolet range from 255 nm to 350 nm is described. These are coupled with fused silica optical fibres to a conventional cuvette with 1 cm optical path length or to a low volume flow through cell for detection in high-performance liquid chromatography. Photodiodes are employed for the measurement of the transmitted intensity as well as of a reference signal and the photocurrents are processed with a log-ratio amplifier to obtain a voltage proportional to absorbance values. The wavelength desired for the measurement at hand is selected by electronically switching on the requisite light-emitting diode. The detector was found to have a low noise level of 80 µAU. In batch-wise measurements as well as in detection for high-performance liquid chromatography dynamic ranges of 2-3 orders of magnitude were possible. Reproducibilities in peak areas for the latter application were better than 1%.

Chip-Based Enrichment and NanoLC-MS/MS Analysis of Phosphopeptides from Whole Lysates.

Protein phosphorylation may be the most widespread and possibly most important post-translational modification (PTM). Considering such a claim, it should be no surprise that huge efforts have been made to improve methods to allow comprehensive study of cellular phosphorylation events. Nevertheless, comprehensive identification of sites of protein phosphorylation is still a challenge, best left to experienced proteomics experts. Recent advances in HPLC chip manufacturing have created an environment to allow automation of popular techniques in the bioanalytical world. One such tool that would benefit from the increased ease and confidence brought by automated 'nanoflow' analysis is phosphopeptide enrichment. To this end, we have developed a reusable HPLC nanoflow rate chip using TiO 2 particles for selective phosphopeptide enrichment. Such a design proved robust, easy to use, and was capable of consistent performance over tens of analyses including minute amounts of complex cellular lysates.

Chip-based nano-LC-MS/MS identification of proteins in complex biological samples using a novel polymer microfluidic device.

Although the proteome of each organism is unambiguously coded in its genome, the proteome shows the real biology in action in each particular organism. New powerful tools are being developed for biochemists and biologists to analyze complex biological samples for studying the complete protein supplement of the genome, i. e., the proteome. There are several methods available for proteome analysis including 2-DE and several forms of MS. In recent years, technologies such as microfluidics and array-based systems have appeared in the field of analysis, identification, and quantification of proteins. These novel approaches might help in solving current technical challenges in proteomics. This paper presents a practical application of the first commercially available microfluidic nano-ESI device coupled with nano-LC (i. e., HPLC-chip) for the analysis of samples of some biological protein mixtures.

An experimental study of chromatographic dynamics in open and packed non-cylindrical conduits.

A systematic investigation of the influence of the perimeter shape of open and particle packed fused silica capillaries on chromatographic properties such as resistance to flow and dispersion of solutes propelled through these channels has been conducted. Verifications of these uncommon experiments with existing theoretical treatments are presented and the insights transferred to a novel polymer chip design with integrated facilities for complex separations. A comparison of the chromatographic performance of a real life proteomics sample on this chip with a capillary column of "similar" dimensions is presented.