An apparatus for continuous separation of macromolecules by combined electrophoresis and gel filtration

Summary An apparatus for continuous separation of soluble and particulate macromolecules, and of other electrically charged particles by combined electrophoresis and gel filtration, is described. The two principles, electrophoresis and gel filtration, which operate simultaneously at right angles, enable separation between the various macromelecules, depending upon differences in electric charge and molecular wight. The sample to be fractionated is fed continuously into the apparatus while the separated macromolecules reaching the distal end of the instrument are collected at the outlet ports. Data for a continuous fractionation of human plasma proteins using the instrument are given.     

Process analytical technology for chromatography

A major United States Food and Drug Administration effort is devoted to process analytical technology (PAT), which is emerging as the likely "surprise" of the second half of the decade. PAT is an approach to monitoring, manufacturing, and other processes on a continuous rather than discrete basis. It carries the future promise of new methods of production. Building PAT into a chromatography system provides a significant cost and quality advantage to high volume multitest laboratories and provides a significant marketing advantage to the first suppliers able to implement such an approach.     

Membrane-based sample preparation coupled on-line to chromatography or electrophoresis.

A review on the use of membranes for on-line sample preparation prior to chromatographic and electrophoretic analysis is provided. The current state-of-the-art of four membrane-based techniques (dialysis, electrodialysis, filtration and membrane extraction) is described by reviewing their principles and applications. Possible future developments are discussed.     

A quantitative study of continuous flow-counterbalanced capillary electrophoresis for sample purification

A systematic evaluation of continuous flow-counterbalanced capillary electrophoresis for microscale preparative applications is presented. The success of the technique was found to depend on the specific nature of the analyte and background electrolyte and was most heavily influenced by factors such as solution conductivity and rate of buffer depletion. The performance of the technique for the system studied was ultimately limited by contamination arising from changes in analyte mobilities during extended run times. Marked improvements in both purification rate and yield were observed using the zwitterionic buffer 2-(N-cyclohexylamino)ethanesulfonic acid when compared to similar runs carried out using a borate background electrolyte. A quantitative evaluation of the technique was performed for the analytes studied and the performance of the technique has been compared to fraction collection methods. The highest recovery achieved was 5.2% of the fastest migrating component present in the original sample with a resolution of 9 to the adjacent peak and required 100 min under optimized conditions. Recovery could be further increased to 9.0% in 120 min for the same analyte using pressure-ramped flow-counterbalanced capillary electrophoresis.     

Continuous superporous agarose beds for chromatography and electrophoresis

Continuous agarose beds (monoliths) were prepared by casting agarose emulsions designed to generate superporous agarose. The gel structures obtained were transected by superpores (diameters could be varied in the range 20-200 microns) through which liquids could be pumped. The pore structure and the basic properties of the continuous gel were investigated by microscopy and size exclusion chromatography. The chromatographic behaviour was approximately the same as for beds packed with homogeneous agarose beads with a particle diameter equivalent to the distance between the superpores. In one application, the superporous continuous agarose bed was derivatized with a NAD+ analogue and used in the affinity purification of bovine lactate dehydrogenase from a crude extract. In another application, a new superporous composite gel material was prepared by adding hydroxyapatite particles to the agarose phase. The composite bed was used to separate a protein mixture by hydroxyapatite chromatography. In a third application, the continuous superporous agarose material was used as an electrophoresis gel. Here, a water-immiscible organic liquid was pumped through the superpores to dissipate the joule heat evolved, thus allowing high current densities.     

Identification of ADP-ribosylated histones by the combined use of high-performance liquid chromatography and electrophoresis

Reversed-phase high-performance liquid chromatography (HPLC) was employed for analysing mono- and oligo(ADP-ribosyl)ated histones. Under the chromatographic conditions described, the ADP-ribosylated histones showed similar retention times to the unmodified histones, although the molecular weight and the charge of the proteins are significantly altered by their modification. The simultaneous elution of unmodified and labelled modified histones was detected by two types of gel electrophoresis and by autoradiography. In addition, the HPLC fractions did not display overlapping ladders of the multiply modified histones, as is commonly seen in one-dimensional electrophoretic analyses of unfractionated material. Hence individual bands could be unambiguously assigned. After in vitro labelling of isolated rat liver nuclei, the following ADP-ribosylated and unmodified histones were identified by HPLC and gel electrophoresis: histone H1(0), four histone H1 subfractions, histone H2A.1, histone H2A.2, oxidized histone H2A.2, histone H2A.X, histone H2A.Z, histone H2B, three histone H3 variants and histone H4.     

Ion chromatography: A powerful and versatile analytical technique for environmental chemical characterization

The advanced IC instrumentation systems currently offered on the market are attractive due to their particular ability to solve a great number of analytical problems in different fields of utilization, including the environment. The most significant features of the IC system are:

• the versatility in assuming a proper configuration tailored for the actual features of the analysis;

• the possibility of preconcentrating the analytes on line and eliminating most of the interference components of the sample matrix, by using ancillary columns and devices in the same IC environment;

• the ability to separate and determine solutes which have similar chemical behavior and are present in the sample in an extreme mass ratio (f.i. 1:10⁸);

• the low detector limits (namely ppb or sub-ppb) which can be reached by changing the loop volume, or by using columns (and the associated flow paths) with a low internal diameter;

• the large dynamic range (typically four or five decades);

• the large number of analytes that can be analyzed per run (up to 10 well-separated peaks in a 20-min run), coupled to a short reequilibration time between runs;

• the large number of classes of compounds (ionic, nonionic, organic, inorganic, etc.) which can be separated and detected;

• the low coefficient of variation (typically 0.2–1% for the concentration range 0.1–50 ppm; 10% for lower concentrations).

For all the above features IC is becoming the most competitive instrumental technique in the environment for the analysis of major, minor, and trace components in natural water, snow, ice, rain, and air. Some significant results obtained with modern advanced IC instrumentation are illustrated. Instrumental precision and accuracy are also discussed.     

Multivariate curve resolution combined with gas chromatography to enhance analytical separation in complex samples: A review

This review describes the major advantages and pitfalls of iterative and non-iterative multivariate curve resolution (MCR) methods combined with gas chromatography (GC) data using literature published since 2000 and highlighting the most important combinations of GC coupled to mass spectrometry (GC–MS) and comprehensive two-dimensional gas chromatography with flame ionization detection (GC × GC-FID) and coupled to mass spectrometry (GC × GC–MS). In addition, a brief summary of some pre-processing strategies will be discussed to correct common issues in GC, such as retention time shifts and baseline/background contributions. Additionally, algorithms such as evolving factor analysis (EFA), heuristic evolving latent projection (HELP), subwindow factor analysis (SFA), multivariate curve resolution-alternating least squares (MCR-ALS), positive matrix factorization (PMF), iterative target transformation factor analysis (ITTFA) and orthogonal projection resolution (OPR) will be described in this paper. Even more, examples of applications to food chemistry, lipidomics and medicinal chemistry, as well as in essential oil research, will be shown. Lastly, a brief illustration of the MCR method hierarchy will also be presented.     

Potential of flow-counterbalanced capillary electrophoresis for analytical and micropreparative separations.

The potential of flow-counterbalanced capillary electrophoresis (FCCE) in chiral and achiral separations was investigated in this work. Unlimited increase of the separation selectivity can be achieved for binary mixtures using FCCE. This was shown for the enantioseparation of (+/-)-chlorpheniramine (CHL) with carboxymethyl-beta-cyclodextrin (CM-beta-CD) as chiral selector. The other example is the separation of alpha- and beta-isomers of a dipeptide aspartame (AS). The carrier ability of the (chiral) selector or pseudostationary phase, the electroosmotic flow (EOF), the pressure-driven flow or hydrodynamic flow can be used as a counterbalancing flow to the electrophoretic mobility of the analyte or vice versa. This mechanism can also be used for micropreparative purposes. FCCE also bears the potential for stepwise separation and fraction collection of multicomponent mixtures.     

Non-particulate (continuous bed or monolithic) acrylate-based capillary columns for reversed-phase liquid chromatography and electrochromatography

Three approaches are described to synthesize acrylic non-particulate beds (also called continuous beds or monoliths) in aqueous polymerization media for reversed-phase capillary liquid chromatography/electrochromatography. In the first, hexyl acrylate comonomer was dissolved together with water soluble polar comonomers using a non-ionic detergent. In the second, a new alkyl ammonium salt comonomer, (3-allylamino-2-hydroxypropyl)dodecyldimethylammonium chloride was used, which is water soluble and has detergent properties itself. The alkyl group of this comonomer provides hydrophobicity while the ionic groups generate electroosmosis in the non-particulate bed. In the third approach, the alkyl comonomer was used as a detergent to dissolve another hydrophobic comonomer in an aqueous polymerization medium. All three approaches were evaluated with respect to hydrophobicity, efficiency and electroosmotic properties of the beds. Hydrophobicity expressed as methylene group selectivity for the three types of the beds in 50% methanol mobile phase was 1.86, 1.16 and 1.78, electroosmotic mobility -5.14 x 10(-5), 6.89 x 10(-5) and 6.37 x 10(-5) cm2 V(-1) s(-1) and efficiency for the retained compound (methylparabene) 67,000, 93,000 and 110,000 plates m(-1) correspondingly. The columns were tested using pressure driven capillary chromatography and capillary electrochromatography. The influence of polymerization temperature on hydrodynamic permeability, separation impedance and inverse size exclusion porosimetry characteristics were used to evaluate the separation columns. The increase of the polymerization temperature resulted higher permeability of the bed, separation impedance and lower polymeric skeleton porosity. Further characterisation was provided by examining the separation efficiency observed for a series of benzoic acid esters and alkyl parabens.     

A continuous acetic acid system for polyacrylamide gel electrophoresis of gliadins and other prolamines

A polyacrylamide gel electrophoresis system buffered by acetic acid alone was developed for electrophoresis of prolamines. When applied to gliadin electrophoresis, the acetic acid system produces more bands than does a conventional aluminum lactate-lactic acid system (using 12% acrylamide gels). The acetic acid system is relatively simple, requiring a single buffer component that is universally available in high purity.     

A combined version of planar chromatography

A new version of thin-layer chromatography (TLC) was developed, in which a combination of conventional TLC versions, ascending, descending, and horizontal, were used on one plate. Such a combination decreases the time of analysis and increases the separation efficiency with respect to the conventional methods of planar chromatography. The method was used to analyze liquid kerosene pyrolysis products obtained using various catalytic systems.     

Luminol-type chemiluminescence derivatization reagents for liquid chromatography and capillary electrophoresis

The present paper provides the principles for chemiluminescence of luminol-type compounds and their wide and powerful application to the detection system in liquid chromatography and capillary electrophoresis as derivatization reagents. The reagents can be classified into two types, chemiluminescence labeling and chemiluminogenic reagents. The former reagents are highly chemiluminescent themselves and used for tagging their intense chemiluminophores to analytes, whereas the latter are weakly chemiluminescent but generate intense chemiluminescence by reaction with analytes. The liquid chromatographic methods utilizing chemiluminescence derivatizing reactions with luminol-type reagents allow the analytes to be detected at pmol–sub-fmol levels. Furthermore, the chemiluminogenic reactions show high selectivity owing to their selective reaction against the analytes permitting facile and reproducible detection.     

Ultra-high-speed liquid chromatography combined with mass spectrometry detection analytical methods for the determination of nitrosamine drug substance-related impurities

In the present study, five simple, feasible, and sensitive Ultra-high-speed liquid chromatography combined with mass spectrometry detection methods, using electrospray ionization are proposed. These methods were developed and validated for the determination of four different nitrosamine drug substance-related impurities—N-nitrosoacebutolol, N-nitrosobisoprolol, N-nitrosometoprolol, and N-nitrososotalol—in five beta blockers active pharmaceutical ingredients—acebutolol HCl, bisoprolol fumarate, metoprolol tartrate, metoprolol succinate, and sotalol HCl. The proposed methods were validated as per regulatory guidelines. Acquity HSS T3 (3.0 × 100 mm, 1.8 μm) column and formic acid 0.1% in water combined with methanol or acetonitrile were used for chromatographic separation in all methods. The limit of detection and the limit of quantification were found to be in the range of 0.02–1.2 and 2–20 parts per billion, respectively. The accuracy and precision of the five methods have been demonstrated in the working range of each one, giving values of recovery within the range of 64.1%–113.3%, and the regression coefficients (R) were found to be in the range of 0.9978–0.9999. These methods could be used for controlling nitrosamine drug substance-related impurities content for beta blockers drug substances batches manufactured at Moehs group.     

Multilayer coil planet centrifuge for analytical high-speed counter-current chromatography

A compact portable model of a high-speed counter-current chromatograph enables efficient analytical separations of microgram sample quantities within 10 min. A series of preliminary experiments was conducted to study retention of the stationary phase of various two-phase solvent systems in short coils with different helical diameters. Under the optimal experimental conditions, analytical capability of the apparatus was successfully demonstrated in separation of flavonoids from a crude sea buckthorn ethanol extract in a multilayer coil with a total capacity of 8 ml.