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As I write this, I'm also grading the final exam from my "Chromatography and Separations" course. We spent approximately the first 75% of the course covering column chromatography, capillary electrophoresis (CE), and field-flow fractionation (FFF). The remainder of the course focused on chromatographic sample preparation. The final exam was based on this last section of the course. Some questions make a good review of the fundamentals of various extraction techniques. The basis for these questions may have been discussed in previous iterations of this column, in analytical chemistry textbooks, or in books on chemical separations. I generally give the final exam in my graduate courses as a take-home exam, so you are free to use whatever resources you have available to you. Here's your opportunity to test yourself, as the answers are provided. [ABSTRACT FROM AUTHOR]

Highlights • A theoretical model for separation of ellipsoidal particles in flow field-flow fractionation. • Steric-entropic mode is considered based on a recent study on rod distribution. • Shape effect on the retention ratio was investigated. Abstract A theoretical model is proposed to analyze the shape effect on the retention behaviors of rod-like particles in field-flow fractionation. This model is improved from a previous model for slender-body rods by Park and Mittal [Chromatography (2015) 2: 472–487]: The model can predict the retention behaviors of the rods, of which shape is assumed as a prolate ellipsoid, with low and high aspect ratios in various flow conditions of the flow-field flow fractionation. The effects of rod aspect ratio on the retention behaviors of the rods with the same volume are investigated in each operation mode. In normal mode, the retention ratio decreases with increasing aspect ratio. In steric-entropic mode, where we substantially improved the model to evaluate the rod orientation and the cross-sectional concentration distribution more rigorously based on our recent studies [Nanomaterials (2018) 8:130; Chem. Eng. Sci. (2018) 189:396-400], the retention ratio increases with the increasing aspect ratio. In steric mode, the retention ratio decreases with increasing aspect ratio again. Those results are discussed based on how the cross-sectional concentration distributions are affected by the aspect ratio. The new criteria for the prediction of each mode are also discussed and suggested Comparison with the experimental data shows the qualitative agreement. [ABSTRACT FROM AUTHOR]

For the purpose of developing techniques for separating biological macromolecules, the present study reports a magnetic chromatography system employing high performance liquid chromatography and superconducting magnets of 14 and 5 T. We observed chromatograms of catalase and albumin, which were eluded from columns that were exposed to magnetic fields of up to 14 T with a maximum gradient of 90 T/m. Without the magnetic fields, the chromatograms of the macromolecules showed a clear peak, while the chromatograms changed to have separated peaks for the same molecules after exposure to gradient magnetic fields. When the chromatocolumn was placed so the magnetic forces were opposite to the direction of flow, the albumin molecules separated into two groups. In addition, the chromatograms of catalase exposed to the magnetic fields indicated that the retention times of the two kinds of magnetically separated catalase were relatively changed if the column-field configuration was changed. Probably, the balance of paramagnetism in the heme and diamagnetism in the protein controlled the transport velocity under the influence of the gradient magnetic fields. In addition, the transport velocity of DNA molecules in the flow with a high gradient magnetic field was observed using a time-resolved spectrophotometric system. [ABSTRACT FROM AUTHOR]

This tutorial review focuses on the use of ICP-MS based techniques for the analysis of metal-containing nanoparticles and colloids. Within the first part the capabilities of “stand alone” ICP-MS for the analysis of total metal contents and the suitability of stable isotopes for nanoparticle tracking (stable isotope labelling and naturally occurring variation in isotope ratios) are introduced (Chapter 3). Special focus was given on single particle ICP-MS (sp-ICP-MS) mode (Chapter 4). Upon a brief introduction into the theoretical concept, critical aspects such as calibration strategies, dwell time as well as ionic background were discussed and practical advice is given. References to current data assessment sheets are provided. Furthermore, a brief chapter on general sample preparation aspects is included within the first part (Chapter 2). The second part is dedicated to fractionation/separation systems, such as field-flow fractionation (FFF), hydrodynamic chromatography (HDC), high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) coupled on-line with ICP-MS detection for metal-based nanoparticle and colloid analysis (Chapter 5). Each section starts with an introduction into the theoretical concept of the respective fractionation/separation system, followed by practical hints regarding method development (e.g. selection of appropriate carrier/mobile phase, membrane/stationary phase) as well as critical aspects and limitations. Particular attention is payed to laser ablation ICP-MS (LA-ICP-MS) for spatially resolved nanoparticle analysis. Each section concludes with selected application examples of the respective analytical technique from the most relevant fields of nanoparticle use or exposure (consumer products, food, medicine and environment), highlighting the performance of each technique in metal-based nanoparticle analysis. Chapter 6 is dedicated to aspects of quality assurance. Various critical points regarding method development and validation, mass balance, size calibration and quantification from the previous sections are revisited, discussed and practical advice is given. Finally, the authors provide some concluding remarks and future perspectives (Chapter 7). Furthermore, a flow-chart is included as a “hands-on” overview on all ICP-MS based techniques discussed within this tutorial review intended as a “method-decision tool” for users. [ABSTRACT FROM AUTHOR]

Paraquat (PQ) has caused countless deaths throughout the world. There remains no effective treatment for PQ poisoning. Here we study the blood metabolome of PQ-poisoned patients using ultraperformance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF MS). Patients were divided into groups according to blood PQ concentration. Healthy subjects served as controls. Metabolic features were statistically analyzed using multivariate pattern-recognition techniques to identify the most important metabolites. Selected metabolites were further compared with a series of clinical indexes to assess the prognostic value. PQ-poisoned patients showed substantial differences compared with healthy subjects. Based on variable of importance in the project (VIP) values and statistical analysis, 17 metabolites were selected and identified. These metabolites well-classified low PQ-poisoned patients, high PQ-poisoned patients, and healthy subjects, which was better than that of a complete blood count (CBC). Among the 17 metabolites, 20:3/18:1-PC (PC), LPA (0:0/16:0) (LPA), 19-oxo-deoxycorticosterone (19-oxo-DOC), and eicosapentaenoic acid (EPA) had prognostic value. In particular, EPA was the most sensitive one. Besides, the levels of EPA was correlated with LPA and 19-oxo-DOC. If EPA was excessively consumed, then prognosis was poor. In conclusion, the serum metabolome is substantially perturbed by PQ poisoning. EPA is the most important biomarker in early PQ poisoning. [ABSTRACT FROM AUTHOR]

Data on the selectivity in the case of infinite dilution obtained using gas–liquid chromatography have been analyzed for seven industrial separating agents. Selective agents for the extractive distillation of binary systems of various types have been recommended. [ABSTRACT FROM AUTHOR]

Two analytical separation methods-size-exclusion chromatography and asymmetrical flow field-flow fractionation-were implemented to evaluate the integrity of the colloidal composition of Chardonnay white wine and the impact of pressing and fermentations on the final macromolecular composition. Wine chromophoric colloidal matter, representing UV-visible-absorbing wine macromolecules, was evaluated by optical and structural measurements combined with the description of elution profiles obtained by both separative techniques. The objective of this study was to apply these two types of fractionation on a typical Chardonnay white wine produced in Burgundy and to evaluate how each of them impacted the determination of the macromolecular chromophoric content of wine. UV-visible and fluorescence measurements of collected fractions were successfully applied. An additional proteomic study revealed that grape and microorganism proteins largely impacted the composition of chromophoric colloidal matter of Chardonnay wines. Asymmetrical flow field-flow fractionation appeared to be more reliable and less invasive with respect to the native chemical environment of chromophoric wine macromolecules, and hence is recommended as a tool to fractionate chromophoric colloidal matter in white wines. [ABSTRACT FROM AUTHOR]

Detailed biochemical and biophysical characterization of viruses requires viral preparations of high quantity and purity. The optimization of virus production and purification is an essential, but laborious and time-consuming process. Asymmetric flow field flow fractionation (AF4) is an attractive alternative method for virus purification because it is a rapid and gentle separation method that should preserve viral infectivity. Here we optimized the AF4 conditions to be used for purification of a model virus, bacteriophage PRD1, from various types of starting materials. Our results show that AF4 is well suited for PRD1 purification as monitored by virus recovery and specific infectivity. Short analysis time and high sample loads enabled us to use AF4 for preparative scale purification of PRD1. Furthermore, we show that AF4 enables the rapid real-time analysis of progeny virus production in infected cells. [ABSTRACT FROM AUTHOR]

We developed a new technique for precise measurements of ruthenium (Ru) stable isotope compositions by multiple-collector inductively coupled plasma mass spectrometry (MC-ICPMS). Instrumental mass bias and potential isotope fractionation induced during the chemical purification of Ru were corrected using a 98Ru–101Ru double spike added prior to sample digestion. The separation and purification of Ru from natural samples is achieved by cation exchange chromatography followed by distillation of Ru as volatile oxides. A series of analytical tests demonstrates that this method results in very pure Ru cuts and accurate and reproducible Ru isotope measurements. The Ru isotope results are expressed relative to an Alfa Aesar™ Ru standard solution as the permil deviations of the 102Ru/99Ru ratio (δ102/99Ru). The external reproducibility (2 s.d.) of the entire analytical procedure (including sample digestion, chemical purification and isotope measurement) that has been determined using several doped terrestrial rock standards is 0.05‰ for δ102/99Ru. We obtained Ru stable isotope data for three different Ru standard solutions and five chromitite samples from two different localities in the Shetland Ophiolite Complex and from the Bushveld complex. All three standard solutions are isotopically distinct with δ102/99Ru values up to ca. 0.9‰. The different chromitite samples also show different Ru stable isotope compositions with δ102/99Ru values between ca. 0.2‰ and ca. 0.7‰. These natural Ru stable isotope variations can be readily resolved using the 98Ru–101Ru double spike method presented here. Overall, these data show that Ru stable isotopes hold promise as a tracer of a wide range of geochemical and cosmochemical processes. [ABSTRACT FROM AUTHOR]

A lack of reliable analytical methods has hindered the quantification of submicron protein aggregates and a detailed understanding of their formation kinetics. In this study, a simple asymmetrical flow field-flow fractionation (AF4) method with good size selectivity (>0.5) is used to investigate nanometer (<0.1 μm) and submicron (0.1-1 μm) aggregates of heat-stressed anti-streptavidin (anti-SA) IgG1. The Lumry–Eyring nucleated polymerization (LENP) model for non-native protein aggregation is fit to the AF4 data, and kinetic analysis shows that aggregates are formed via slow nucleation and aggregate condensation at long stress times. Comparison of centrifuged and uncentrifuged heat-stressed anti-SA IgG1 AF4 results show the removal of high molar mass submicron aggregates and large material (>20 μm) and suggests that centrifugation may influence the aggregation kinetics. Furthermore, qualitative LENP model analysis of centrifuged and uncentrifuged samples suggests that significant aggregate–aggregate condensation occurs even at early stress times and highlights the potential of AF4 to determine aggregation kinetics for species greater than 1 μm. [ABSTRACT FROM AUTHOR]

Dialdehyde cellulose (DAC) has gained increasing interest because its (masked) aldehyde functionalities promise a rich follow-up chemistry and further conversion into different derivatives with varying properties. So far, the question of degradation upon DAC production and the resulting molar mass changes have remained unanswered since the problem of molar mass determination of DACs had not been solved. In this study, we present a comparison of two methods based on aqueous eluants, size exclusion chromatography and asymmetric flow field-flow fractionation (AsFlFFF), to determine the molar mass distribution of highly oxidized and thus water-soluble DACs, and to elucidate the impact of oxidation on the chain length of different cellulose substrates. Molar masses were determined by multi-angle laser light scattering in the case of both separation systems. The measurements were complemented by dynamic light scattering. AsFlFFF demonstrated an overall better separation efficiency, but at the expense of increased time consumption for method development. The results of molar mass analysis strongly depended on the eluant. Hemiacetal crosslinks are stable in near-neutral, low ionic strength eluants, while they have a tendency to be cleaved in acidic eluants or in solvents of higher ionic strength. By comparison of different dissolution strategies the optimal conditions for DAC analysis are proposed for both separation techniques. It is concluded that the extent of degradation upon dissolution can be significantly minimized by controlling the dissolution parameters, but degradation cannot be avoided completely. [ABSTRACT FROM AUTHOR]

Magnetic nanoparticles (MNPs) exhibit unique magnetic properties making them ideally suited for a variety of biomedical applications. Depending on the desired magnetic effect, MNPs must meet special magnetic requirements which are mainly determined by their structural properties (e.g., size distribution). The hyphenation of chromatographic separation techniques with complementary detectors is capable of providing multidimensional information of submicron particles. Although various methods have already been combined for this approach, so far, no detector for the online magnetic analysis was used. Magnetic particle spectroscopy (MPS) has been proven a straightforward technique for specific quantification and characterization of MNPs. It combines high sensitivity with high temporal resolution; both of these are prerequisites for a successful hyphenation with chromatographic separation. We demonstrate the capability of MPS to specifically detect and characterize MNPs under usually applied asymmetric flow field-flow fractionation (A4F) conditions (flow rates, MNP concentration, different MNP types). To this end MPS has been successfully integrated into an A4F multidetector platform including dynamic ligth scattering (DLS), multi-angle light scattering (MALS) and ultraviolet (UV) detection. Our system allows for rapid and comprehensive characterization of typical MNP samples for the systematic investigation of structure-dependent magnetic properties. This has been demonstrated by magnetic analysis of the commercial magnetic resonance imaging (MRI) contrast agent Ferucarbotran (FER) during hydrodynamic A4F fractionation. [ABSTRACT FROM AUTHOR]

It is demonstrated that field-flow fractionation using a rotating coiled column (RCC) can be used for the separation and purification of the polydisperse sorbents based on polystyrene-divinylbenzene. The behavior of the sorbent at different operating parameters of RCC was studied. The mobile phase composition, column rotation speed, and the initial flow rate of the mobile phase were selected, optimized for the isolation of a monodisperse fraction with the particle size 4.5 µm and the separation of particles 0.5-2 µm in size, particle fragments, and residues of sorbent synthesis. It is shown that a 15-mL analytical RCC ensures the efficient fractionation of 100 mg of the sorbent in one experiment taking less than one hour with the yield of the desired fraction greater than 95%. The perspectives for scaling up the fractionation in RCC are evaluated; the possibility of using the method for the preparative separation of particles of different nature is discussed. [ABSTRACT FROM AUTHOR]

The article analyzes various ways wherein chromatographers and chromatography data-handling systems locate and measure peaks in liquid chromatography. Topics discussed include the development of computerized data handling, processing peaks and natural peak detection. Also mentioned is the process of using the chromatographic signal and its derivatives.

Highlights: [•] An ideal retention theory for HC and FFF in 3D rectangular channels is developed. [•] FFF devices need not be designed with large aspect ratios. [•] Even square channels are well-approximated by 2D normal-mode FFF retention theory. [•] In HC peak spacing actually improves with decreased aspect ratio. [•] Parabolic retention ratios accurately describe HC retention in rectangular channels. [ABSTRACT FROM AUTHOR]

The rapid development of protein-based pharmaceuticals highlights the need for robust analytical methods to ensure their quality and stability. Among proteins used in pharmaceutical applications, an important and ever increasing role is represented by monoclonal antibodies and large proteins, which are often modified to enhance their activity or stability when used as drugs. The bioactivity and the stability of those proteins are closely related to the maintenance of their complex structure, which however are influenced by many external factors that can cause degradation and/or aggregation. The presence of aggregates in these drugs could reduce their bioactivity and bioavailability, and induce immunogenicity. The choice of the proper analytical method for the analysis of aggregates is fundamental to understand their (size) dimensional range, their amount, and if they are present in the sample as generated by an aggregation or as an artifact due to the method itself. Size exclusion chromatography is one of the most important techniques for the quality control of pharmaceutical proteins; however, its application is limited to relatively low molar mass aggregates. Among the techniques for the size characterization of proteins, field-flow fractionation (FFF) represents a competitive choice because of its soft mechanism due to the absence of a stationary phase and application in a broader size range, from nanometer- to micrometer-sized analytes. In this paper, the microcolumn variant of FFF, the hollow-fiber flow FFF, was online coupled with multi-angle light scattering, and a method for the characterization of aggregates with high reproducibility and low limit of detection was demonstrated employing an avidin derivate as sample model. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

The article focuses on ways to improve terminology for sample analyte retention in field-flow fractionation (FFF) for chromatography. Topics discussed include the study "A new form of chromatogram employing two liquid phases I. A theory of chromatography 2. Application to the micro-determination of the higher monoaminoacids in proteins" by A.J.P. Martin and R.L.M. Synge on the subject, description of experimental determination of the retention factor, and quantification of the retention level.

Highlights: [•] Evolution of SdFFFF channel: insertion of an outlet stream splitter and downscaling. [•] Respect of channel flow path and “Hyperlayer” elution mode for cell. [•] Splitter cell recovery (>90–95%) increased with external field. [•] Improved cancer stem cells sorting: 10 fold enrichment. [Copyright &y& Elsevier]

Highlights: [•] A general plate height equation for gradient elution is proposed. [•] This equation is suited for any type of solvent strength model. [•] This equation is suited for any type of gradient profile. [•] This equation is suited for varied plate height with mobile phase composition. [•] This equation takes the effects of dwelling time into consideration. [Copyright &y& Elsevier]

Abstract: Natural organic matter (NOM) and iron colloids can coexist in surface water. These colloids might exhibit different affinities to metals and metalloids. Previously it has been shown, that organic and inorganic colloids in the low nanometer range can be fractionated using Flow Field-Flow Fractionation analyzes (FlowFFF), but it is not yet understood how the presence of inorganic colloids influences results obtained by High Performance Size Exclusion Chromatography (HPSEC). Studies that compare the use of these size-separation techniques for the analyzes of organic and inorganic colloids and associated elements are needed in order to interpret results obtained by either of these methods. Therefore, associations between colloids from a small stream draining a wetland area and a selected range of elements (Fe, Al, Ti, Pb, Cu, Ni, As, U, and Rare Earth Elements (REE)) have been investigated. FlowFFF analyzes and HPSEC analyzes were combined with ultrafiltration, functional group titration and arsenic speciation analysis. NOM and, in a sample with a pH > 5.2, slightly larger iron organo-mineral colloids, were present in the <0.2 μm fraction in the surface water. Both exhibited notably different affinities for trace elements. Cu, Ni, Al, and the REE all showed similar modes (i.e. peak maxima) and size distributions to the NOM, while Pb and As showed a preferential association with iron organo-mineral colloids. It was not possible to differentiate between NOM and iron-organo mineral colloids with HPSEC. The differences in the results regarding the apparent molecular mass distributions obtained by FlowFFF and HPSEC are discussed. [Copyright &y& Elsevier]

Abstract: The separation of the organic sulfur compounds (OSC) of petroleum or its heavy fractions is a critical step and is essential for the correct characterization of these compounds, especially due to similar physical and chemical properties of polycyclic aromatic sulfur heterocycles (PASH) and polycyclic aromatic hydrocarbons (PAH). This similarity results in coelutions among PAH and PASH and for this reason former steps of fractionation are required before gas chromatographic analysis. The objective of this study was to evaluate the potential of GC×GC for the separation and identification of OSC in a heavy gas oil sample without fractionation, after pre-fractionation in an alumina column and also after fractionation process. This last one was performed with a modified stationary phase manufactured and characterized in the laboratory, called PdII-MPSG, where palladium is chemically linked to silica through mercaptopropyl groups. The fractions obtained from both procedures were analyzed by GC×GC/TOFMS, which was effective to separate and identify various classes of OSC. A hundred and thirty-five compounds were tentatively identified in the sample that was only pre-fractionated. However, when the fractionation was also performed with the PdII-MPSG phase, a larger number of sulfur compounds were found (317). Results have shown that the analysis of a pre-fractionated sample by GC×GC/TOFMS is suitable when the goal is a general characterization of classes of compounds in the sample, while a more detailed analysis of PASH can be performed, using also the fractionation PdII-MPSG phase. GC×GC/TOFMS played a major role in the comparison of samples obtained from pre-fractionation and fractionation steps due to its high peak capacity, selectivity, organized distribution of chromatographic peaks and resolution. [Copyright &y& Elsevier]

Abstract: Interaction forces between all objects are either of repulsive or attractive nature. Concerning attractive interactions, the determination of dispersion forces are of special interest since they appear in all colloidal systems and have a crucial influence on the properties and processes in these systems. One possibility to link theory and experiment is the description of the London–Van der Waals forces in terms of the Hamaker constant, which leads to the challenging problem of calculating the van der Waals interaction energies between colloidal particles. Hence, the determination of a Hamaker constant for a given material is needed when interfacial phenomena such as adhesion are discussed in terms of the total potential energy between particles and substrates. In this work, the asymmetrical flow field-flow fractionation (AF-FFF) in combination with a Newton algorithm based iteration process was used for the determination of Hamaker constants of different nanoparticles in toluene. [Copyright &y& Elsevier]

The article discusses the miniaturization of liquid chromatography (LC), particularly noting the importance of LC systems to the fields of proteomics and metabolomics. It addresses topics including the ability of miniaturized LC systems to combine high efficiency with low flow rates, the distinction between packed and open-tubular columns, and monolithic capillary columns.

The article discusses the study which analyses the capabilities of field-flow fractionation (FFF) coupled with inductively coupled plasma-mass spectrometry (ICP-MS).The study uses several analytical methods in characterizing nanoparticles such as electron microscopy, chromatography, and nanometrology. The study reveals that the coupling of ICP-MS and FFF has lowered the detection capability which leads to critical environmental risk assessment studies of engineered nanoparticles.

Abstract: One way to potentially modify the performance of field-flow fractionation (FFF) would be to move the position of the maximum flow velocity away from the mid-point of the channel, for example by using walls with non-zero slip lengths. In this short communication, we extend the ideal theory of FFF to include the effects of two slip walls. Our calculations demonstrate that while the hydrodynamic chromatography limit of FFF (weak fields) is not improved by engineering devices with slip-walls, the performance of Normal-Mode FFF can be enhanced by having slip at the depletion wall in moderate fields. We also introduce a new regime, which we call Slip-Mode FFF, where a large external field (typical of Normal-Mode FFF) and a large slip at the accumulation wall lead to sharp separations characterized by an elution order that is similar to that of hydrodynamic chromatography. [Copyright &y& Elsevier]

In this study we present detailed characterization of a protein-PEG conjugate using two separation techniques, that is, asymmetrical-flow field-flow fractionation (AF4) and size-exclusion chromatography (SEC), which were online coupled to a series of successively connected detectors: an ultraviolet, a multiangle light-scattering, a quasi-elastic light-scattering, and a refractive-index detector (UV-MALS(QELS)-RI). Matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used as a complementary characterization technique. The results of AF4 as well as SEC on two columns connected in series, with both separation techniques coupled to a multidetection system, indicate the uniform molar mass and chemical composition of the conjugate, that is, the molar ratio of protein to PEG is 1/1, the presence of minute amounts of residual unreacted protein and the aggregates with the same chemical composition as that of the conjugate. Since the portion of aggregated species is smaller in the acetate buffer solution containing 5% sorbitol than in the acetate buffer solution with 200-mM sodium chloride, the former buffer solution is more suitable for conjugate storage. The separation using only one SEC column results in poorly resolved peaks of the PEGylated protein conjugate and the aggregates, whereas MALDI-TOF MS analysis reveal the presence of the residual protein, but not the aggregates. [ABSTRACT FROM AUTHOR]

Robust methods to detect and characterize engineered nanoparticles (ENPs) in environmental samples are an urgent need, particularly given the increasing use of ENPs in consumer products. To be successful, methods should enable differentiation of ENPs from background nanoparticulates and other system components. The element specificity of inductively coupled plasma mass spectrometry (ICP-MS) can, to some degree, satisfy this requirement. Given the polydisperse nature of particles in natural systems, combining ICP-MS with a size separation method holds particular promise. This paper compares hydrodynamic chromatography (HDC) and asymmetrical flow field flow fractionation (AF4), both coupled with ICP-MS, in their capacity to detect, quantify, and characterize nanoparticles. The detection limits, resolution, and recoveries for both techniques were determined using gold nanoparticle standards. AF4 is capable of separating mixtures of 5, 20, 50 and 100 nm gold ENPs with significantly greater resolution than HDC, with these resolution differences being most pronounced in the smaller size range. However, HDC recoveries ranged from 77 to 96%, while recovery during AF4 ranged from 4 to 89%. The low AF4 recoveries generally occurred for the largest ENPs at the lowest concentrations examined. The limits of detection for both techniques were found to be approximately 5 μg L−1, however different experimental conditions could lower this value. HDC provides an additional benefit over AF4 by proving capable of separating a dissolved signal from a NP sample. [ABSTRACT FROM AUTHOR]

Abstract: Hollow fiber flow field flow fractionation (HF5) columns can be built with minimized cost and instrumental skills by incorporation of commercial hollow fibers into holders made of classical chromatographic tubing and connectors. The proposed design leads to differential elution of human cells of different origin. Suspensions of red blood cells (RBC) and adherent human colorectal cancer (CRC) cell lines were used. These CRC nucleated population have been linked to cell-to-cell and cell to instrument interactions that are limiting factors in terms of recovery and viability. These interactions can be limited depending on injection/elution conditions. By using RBC we observed that the focalization/relaxation step played a major role in the elution process. However, HF5 opens a large potential, which completes the diversity shown by SdFFF in cell sorting methodologies and technologies. [Copyright &y& Elsevier]

Type 2 Maturity Onset Diabetes of the Young (MODY2) is a monogenic autosomal disease characterized by a primary defect in insulin secretion and hyperglycemia. It results from GCK gene mutations that impair enzyme activity. Between 2006 and 2010, we investigated GCK mutations in 66 diabetic children from southern Italy with suspected MODY2. Denaturing High Performance Liquid Chromatography (DHPLC) and sequence analysis revealed 19 GCK mutations in 28 children, six of which were novel: p.Glu40Asp, p.Val154Leu, p.Arg447Glyfs, p.Lys458_Cys461del, p.Glu395_Arg397del and c.580-2A>T. We evaluated the effect of these 19 mutations using bioinformatic tools such as Polymorphism Phenotyping (Polyphen), Sorting Intolerant From Tolerant (SIFT) and in silico modelling. We also conducted a functional study to evaluate the pathogenic significance of seven mutations that are among the most severe mutations found in our population, and have never been characterized: p.Glu70Asp, p.His137Asp, p.Phe150Tyr, p.Val154Leu, p.Gly162Asp, p.Arg303Trp and p.Arg392Ser. These seven mutations, by altering one or more kinetic parameters, reduced enzyme catalytic activity by >40%. All mutations except p.Glu70Asp displayed thermal-instability, indeed >50% of enzyme activity was lost at 50°C/30 min. Thus, these seven mutations play a pathogenic role in MODY2 insurgence. In conclusion, this report revealed six novel GCK mutations and sheds some light on the structure-function relationship of human GCK mutations and MODY2. [ABSTRACT FROM AUTHOR]

Abstract: The rapidly developing interest in nanofluidics, which is used to examine liquids on an order that ranges from an attoliter to a femtoliter, correlates with the recent interest in decreased sample amounts, such as in the field of single-cell analysis. In this paper, we have succeeded in the normal phase separation of a 101 fL sample with a high number of theoretical plates (103 plates), and a fast separation (4s), using a pressure-driven flow in extended nanochannels. A meander separation channel 1μm wide was introduced and showed no band broadening at the turns, unlike microchannels (101–102 μm wide), which is an important aspect in order to lengthen the channel in a space-limited micro/nanofluidic chip. Our device enables the fast separation of an ultra-small volume of sample with a high number of theoretical plates, and will serve as a general platform for the separation of aL to fL volumes of samples. [Copyright &y& Elsevier]

Abstract: Through a careful consideration of the retention ratio for field-flow fractionation (FFF), we show that a single unified ideal retention theory can predict a wide range of separation behaviours including hydrodynamic chromatography, normal-mode FFF and steric-mode FFF by introducing the concept of a device retention parameter. We determine the critical device retention parameter above which normal-mode does not exist and there is no clear distinction between hydrodynamic chromatography and steric-mode FFF. Numerical analysis of the elution order as a function of particle size quantitatively predicts the transitions between these regimes. The resulting map of the operational-modes shows each of the regions and their connectivity, and so may guide future device design. By extending this analysis to account for the variation of stress over particle surfaces, a hitherto unreported regime called Faxén-mode FFF is predicted, which has the same elution order as normal-mode FFF. This mode arises when particle sizes approach the channel height, as can occur when microfluidic devices are utilized for FFF. The transition from steric-mode to Faxén-mode FFF is numerically mapped and approximations for each transition are presented. [Copyright &y& Elsevier]

Submicron and micron particles present in liquid environmental, biological, and technological samples differ in their dimensions, shape, mass, chemical composition, and charge. Their properties cannot be reliably studied unless the particles are fractionated. Synthetic particles applied as components of analytical systems may also need preliminary fractionation and investigation. The review is focused on the methods for fractionation and characterization of nanoparticles and microparticles in liquid media, the most representative examples of their application, and the trends in developing novel approaches to the separation and investigation of particles. Among the separation techniques, the main attention is devoted to membrane filtration, field-flow fractionation, chromatographic, and capillary electrokinetic methods. Microfluidic systems employing the above-mentioned and other separation principles and providing a basis for the fabrication of lab-on-chip devices are also examined. Laser light scattering methods and other physical techniques for the characterization of particles are considered. Special attention is given to 'hyphenated' techniques which enable the separation and characterization of particles to be performed in online modes. [ABSTRACT FROM AUTHOR]

The article offers information about ghost peaks can be referred as artifact peaks, system peaks and spurious peaks in the U.S. It states that the peaks may arise from unknown impurities or artifacts within the liquid chromatography system. It mentions that the occurrence of ghost peaks is seen frequently in the gradient mode.

Starch consists of a mixture of two α-glucans built mainly upon α-(1,4) linkages: amylose, an essentially linear polymer, and amylopectin, a branched polymer containing 5-6% of α-(1,6) linkages. The aim of the present work was to analyze the structural properties of native starches displaying different amylose-to-amylopectin ratios and arising from different botanical sources, using asymmetrical flow field flow fractionation (A4F) and a combination of hydrodynamic and size-exclusion chromatography (HDC-SEC) coupled with multiangle laser light scattering, online quasi-elastic light scattering, and differential refractive index techniques. The procedure, based upon dimethyl sulfoxide pretreatment and then solubilization in water, generates a representative injected sample without altering the initial degree of polymerization. The amylopectin weight-average molar masses and radii of gyration were around 1.0 × 10-4.8 × 10 g mol and 110-267 nm, respectively. For each starch sample, the hydrodynamic radius ( R) distributions and the molar mass distributions obtained from the two fractionation systems coupled with light scattering techniques were analyzed. The size determination scales were extended by means of R calibration curves. HDC-SEC and A4F data could be matched. However, A4F enabled a better separation of amylopectins and therefore an enhanced structural characterization of the starches. The two advantages of this experimental approach are (1) it can directly obtain distributions as a function of both molar mass and size, while taking account of sample heterogeneity, and (2) it is possible to compare the results obtained using the different techniques through the direct application of R distributions. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

ymmetric flow field-flow fractionation (AsFlFFF) and high-performance size-exclusion chromatography (HPSEC) are techniques for separating and characterizing macromolecules; until now the latter is more utilized for analyzing polysaccharides. The demand for characterizing complex, high-molar-mass polysaccharides has raised interest in the use of AsFlFFF in analyzing polymeric carbohydrates in addition to HPSEC. In this paper, we compare the behavior of arabinoxylan aggregates present in aqueous solution in AsFlFFF and HPSEC and their effect on the obtained molecular characteristics (molar mass averages and size). Although the amount of aggregates in aqueous arabinoxylan solutions may be low, their role needs to be understood to avoid erroneous interpretations of AsFlFFF and HPSEC data. When these two separation systems were compared, AsFlFFF seemed to possess more separation power for the differentiation of aggregates from individual chains than HPSEC. To our knowledge, this is the first report on the characterization of xylans with AsFlFFF. [ABSTRACT FROM AUTHOR]

A simple, precise, shorter runtime and stability indicating reversephase high performance liquid chromatographic method has been developed and validated for the quantification of benzalkonium chloride (BKC) preservative in pharmaceutical formulation of sparfloxacin eye drop. The method was successfully applied for determination of benzalkonium chloride in various ophthalmic formulations like latanoprost, timolol, dexametasone, gatifloxacin, norfloxacin, combination of moxifloxacin and dexamethasone, combination of nepthazoline HCl, zinc sulphate and chlorpheniramine maleate, combination of tobaramycin and dexamethasone, combination of phenylephrine HCl, naphazoline HCl, menthol and camphor. The RP-LC separation was achieved on an Purospher Star RP-18e 75 mm × 4.0 mm, 3.0 μ in the isocratic mode using buffer: acetonitrile (35: 65, v/v), as the mobile phase at a flow rate of 1.8 mL/min. The methods were performed at 215 nm; in LC method, quantification was achieved with PDA detection over the concentration range of 50 to 150 μg/mL. The method is effective to separate four homologs with good resolution in presence of excipients, sparfloxacin and degradable compound due to sparfloxacin and BKC within five minutes. The method was validated and the results were compared statistically. They were found to be simple, accurate, precise and specific. The proposed method was validated in terms of specificity, precision, recovery, solution stability, linearity and range. All the validation parameters were within the acceptance range and concordant to ICH guidelines. [ABSTRACT FROM AUTHOR]

The article presents a study on accelerating liquid chromatography (LC) separations with the use of smaller particles, higher flow rates, and changes in column dimensions. It employs several experiments with the application of ultrahigh-pressure liquid chromatography (UHPLC). Further, it determines the proper change in the gradient conditions to obtain an equivalent separation upon the changes on flow rate, column size and other gradient conditions.

Methodological features of hydrodynamic chromatography (HDC) both in narrow and wide-bores are discussed in this review. HDC in narrow-bore allows separation of particles or macromolecules having relatively large dimensions; whereas, wide-bore HDC (W-HDC) allows separation of even small molecules only if they have one-order different intrinsic diffusivity. Main focus is on the latter method. This method relies on the diffusion of analytes and laminar flow. Since a stable and well-defined laminar flow is established in a capillary, it is a suitable platform of W-HDC. We discuss the basic principles followed by some applications. In addition, the strategy for rapid separation is discussed on the basis of a convenient parameter derived from the advection-diffusion equation. Separation in the sub second range is possible with W-HDC by appropriate reduction of the system dimension. [ABSTRACT FROM AUTHOR]

The author discusses the functions of liquid chromatography (LC). The author mentions the LC's historical background, where its was issued by M. Twsett in 1906. The author cites the relevance of the mobile phase components to LC's separation processes. Moreover, the author also notes the increasing applications for the LC.

Asymmetrical flow field-flow fractionation (AF4) can be used on a routine basis to separate high-molecular-weight compounds as an alternative to size-exclusion chromatography (SEC), but the principle of separation is very different and a different approach is required for optimization. This article illustrates how to choose the best experimental parameters. Two optimization parameters are proposed: the time required (TREQ) to separate two compounds that differ by a factor of two in molecular weight and the dilution factor (DILF), which describes the ratio of the actual analyte concentrations during the separation process and after elution from the channel. The TREQ parameter should be minimized to obtain fast separations and the DILF parameter should be minimized when detectability is an issue and the overloading of the separation system is a limiting factor. It is shown that in practice a compromise between these two optimization objectives has to be found. [ABSTRACT FROM AUTHOR]

Abstract: A method based on Asymmetric Flow Field-Flow Fractionation (AF4) was developed to separate different types of lipoproteins from human serum. The emphasis in the method optimization was on the possibilities to characterize the largest lipoprotein fractions (LDL and VLDL), which is usually not possible with the size-exclusion chromatography methods applied in routine analysis. Different channel geometries and flow programs were tested and compared. The use of a short fractionation channel was shown to give less sample dilution at the same fractionation power compared to a conventional, long channel. Different size selectivities were obtained with an exponential decay and a linear cross flow program. The ratio of the UV absorption signal to the light scattering signal was used to validate the relation between retention time and size of the fractionated particles. An experimental setup was developed for the simultaneous determination of the cholesterol and triglycerides distribution over the lipoprotein fractions, based on enzymatic reactions followed by UV detection at 500nm. Coiled and knitted PTFE tubing reactors were compared. An improved peak sharpness and sensitivity were observed with the knitted tubing reactor. After optimization of the experimental conditions a satisfactory linearity and precision (2–3% rsd for cholesterol and 5–6% rsd for triglycerides) were obtained. Finally, serum samples, a pooled sample from healthy volunteers and samples of sepsis patients, were analyzed with the method developed. Lipoprotein fractionation and cholesterol and triglyceride distributions could be correlated with the clinical background of the samples. [Copyright &y& Elsevier]

The retention behaviour of four active compounds from Sophora Flavescens Ait using three ionic liquids as mobile phase modifiers was examined. The effect of the pH and the amount of ionic liquid modifier on the retention of these compounds was determined in methanol/water (v/v) as the mobile phase containing different ionic liquids ranging in concentration from 0.1 mmol/L to 3.0 mmol/L. The ionic liquids showed promise as additives in high-performance liquid chromatography. [ABSTRACT FROM AUTHOR]

A broad-range, sensitive and reliable multiresidue analytical method is presented for identifying and inspecting 176 pesticide residues, including organophosphate, organochlorine, synthetic pyrethroid, organonitrogen and carbamate, in pre-harvested fruits and vegetables. Pesticide residues were extracted from samples with acetone, followed by liquid-liquid partition and solid-phase extraction. Seventy one and 83 pesticides were determined, respectively, by gas chromatography with a flame photometric detector and an electron capture detector. Nineteen carbamate pesticides and several metabolites were measured by high-performance liquid chromatography (HPLC) with a post-column derivation system and a fluorescence detector; and three additional pesticides, i.e., carbendazim, thiabendazole, and imidacloprid, were detected by HPLC using an ultraviolet detector at 280 nm. The proposed method was validated for 14 fruits and 8 vegetables using samples spiked with pesticide standards (0.1- 5.0 μg/g) in triplicate. Good sensitivity and repeatability were obtained with detection limits of 0.001- 0.03 μg/g, and all limits of detection were lower than one-fifth of their specific maximum residue levels. The recovery rates for most pesticides in various fruits and vegetables were 60-120% with relative standard deviations < 20%. With more than 4,300 samples collected from the field examined to assess the performance, it is proposed that this method be applied for routine monitoring, legislation implementation and farmer education programs. [ABSTRACT FROM AUTHOR]

Abstract: Field-flow fractionation (FFF) is a family of analytical techniques developed specifically for separating and characterizing macromolecules, supramolecular assemblies, colloids and particles. It combines the effects of a laminar flow profile with an exponential concentration profile of analyte components caused by their interactions with a physical field applied perpendicular to the flow of a carrier liquid. FFF is undergoing increasingly widespread use as researchers learn of its potential and versatility. This overview underlines the basic principle and theory behind FFF and reviews recent research efforts incorporating flow and thermal FFF methods to characterize natural, biological, and synthetic polymers. These FFF techniques will be discussed in terms of theory and practice. Selected applications of FFF and their coupling capability with other chromatographic techniques or spectrometric detection for the separation and characterization of polymers in organic and aqueous media are presented. [Copyright &y& Elsevier]

Abstract: In this article the performances of different bed structures, i.e., conventional adsorbent beads (with different diameters) and monolithic columns, in the separation of chiral species by the simulated moving bed (SMB) technology are compared. In order to assess these different morphologies the equivalent particle size for monolithic structure was calculated based on equivalent permeabilities. The performance of the SMB units was compared for the conditions quantified by maximum productivity. The configuration of the SMB unit and the total system volume were not varied. The maximum operating pressure drop of 20bar and the purity in both product streams over 99% were set as constraints in all computations. The true moving bed model was used as an analogy to simulated moving bed in order to reduce computation time. The productivity of the SMB unit using particle diameter of 27μm can be slightly higher than of the SMB with particle diameter ≥50μm but at the cost of higher eluent consumption. Moreover, there is no need to operate an SMB unit at its maximum pressure drop to get the best performance values, since in the presence of mass-transfer resistances, the contact time becomes the unit major limitation. [Copyright &y& Elsevier]

Abstract: A novel qualitative analytical method for peak tracking in impurity profiling control by the correlation of spectra was established. Two-dimensional (2D) standard spectrochromatographic data produced by high-performance liquid chromatography with diode array detection (HPLC-DAD) were compared with sample data to develop two-dimensional chromatographic spectral correlative maps. Taking full advantage of separation efficiency of HPLC and spectral specificity of the analytes, the method was successfully used to recognize impurities in quinolone antibacterials, when in combination with relative retention times (RRTs). For the comparison of spectra was expanded to three-dimensional space, simultaneous identification of the chromatographic peaks can be obtained rapidly without preparation and injection of a reference solution, even when the mobile phase changed or the peaks of multi-component samples overlapped. [Copyright &y& Elsevier]

Abstract: This work reports the application of restricted access media (RAM) column, in a multidimensional configuration, for simultaneous analysis of sulfamethoxazole (SMX) and trimethoprim (TMP) in whole eggs with ultraviolet detection. The proteins were partially precipitated by adding 0.5mL of acetonitrile into 1.0mL of blended egg followed by centrifugation. The supernatant was injected (250μL) directly into the muldimensional system. At the first dimension, a restricted access medium (RAM) bovine serum albumin (BSA) octadecyl column (100mm×46mm I.D., Luna® silica, 10μm particle size and 100Å pore size), was used for extraction and concentration of the analytes and at second dimension, an octadecyl column (150mm×46mm I.D., Luna® silica, 10μm particle size and 100Å pore size), for analysis. The developed method showed good selectivity, accuracy and precision for quantification of these different compounds in eggs, and the limits of quantification were 80ng/mL, for both compounds. The validated method is reliable and sensitive for monitoring residues in whole eggs samples and thus, to determine withdraw period for laying hens using veterinary medicine having SMX–TMP combination. [Copyright &y& Elsevier]

Abstract: The design of a new interface for comprehensive two-dimensional liquid chromatography (LC×LC) is described. To the conventionally used LC×LC system with the loop-type interface consisting of a two-position/ten-port switching valve equipped with two loops, an extra two-position/ten-port switching valve, a detector, a pump and a second column placed in parallel with the column in the second dimension, are added. The features of the interface are that the separation space in the second dimension is significantly enlarged and that the number of fractions transferred from the first to the second dimension can be increased, reducing the risk to lose resolution of the primary dimension. The potential of the system in NPLC×2RPLC is illustrated with the analysis of a standard mixture and a lemon oil extract. For the lemon oil analysis, the effective peak capacity was increased from 437 using a conventional interface to 1095 with the new interface. RPLC×2RPLC in combination with reduced modulation times was applied to the analysis of steroids and to the detection of impurities at the 0.05% relative concentration level in a sulfonamide drug sample. [Copyright &y& Elsevier]

The article illustrates some general concepts of physical chemistry and separation science through gravitational field-flow fractionation (FFF). It introduces the emerging technique of FFF. The architecture of an FFF instrument is described. Separation efficiency in FFF is monitored using the plate height. The plate height is a measure of how narrow the peak is maintained during elution. Some FFF theory which can be incorporated in lectures are outlined. The theory is useful for reinforcing some important aspects of physical chemistry such as settling and fluid flow.