Ultrahigh molar mass component detected in ethylhydroxyethyl cellulose by asymmetrical flow field-flow fractionation coupled to multiangle light scattering.

Asymmetrical flow field-flow fractionation (flow FFF) was connected to multiangle light scattering (MALS) and refractive index (RI) detectors for characterization of the molar mass distribution and molecular radius of a cellulose derivative, ethylhydroxyethyl cellulose (EHEC). Experimental conditions were optimized to allow study of a wide range of molar mass including even ultrahigh molar mass (UHM) components. The weight-average molar mass was 3.1 x 10(5) g x mol(-1) representing a very broad range (of molar mass) from 4.0 x 10(4) to 10(7) g x mol(-1), which corresponds to from <20 to 200 nm rms radius. The light scattering signal showed the presence of an UHM component, possibly an aggregate of extreme size, i.e., approximately 10(8) g x mol(-1) with a hydrodynamic diameter of 0.35 microm. Careful choice of the pore size in in-line filters is necessary in order to minimize MALS detector noise without removing the UHM component. Flow FFF-MALS-RI was demonstrated to be uniquely suited to detect the presence of UHM components.

Retention behaviour of amylopectins in asymmetrical flow field-flow fractionation studied by multi-angle light scattering detection.

Asymmetrical flow field-flow fractionation (FFF) with multi-angle light scattering (MALS) detection was applied for the fractionation of amylopectins from four different sources. Samples originated from genetically modified potatoes and waxy maize. Amylopectins were dissolved in a 1 mol l(-1) sodium hydroxide solution or water. With an injected mass of 0.2 microg, well below overloading conditions, a decrease of the apparent hydrodynamic radius with increasing inlet flow-rate was observed. Moreover, a decrease of the radius of gyration with increasing elution volume was recorded by the MALS detector. Steric/hyperlayer effects are a feasible explanation for this behaviour. The observed radius of gyration at the steric inversion point was in the order of 0.3 microm, which is smaller than the theoretically calculated inversion point. Apparently, the amylopectin behave as macromolecules with a larger hydrodynamic radius than expected on basis of their radius of gyration and are subjected to significant lift forces. The results were confirmed by four fractionations with varying flow-rates but constant ratio of cross to outlet-flow. In contrast to the normal mode operation, the retention of the amylopectins depended strongly on the applied flow-rates and was close to that of a much smaller 10 kDa dextran. Apparent molar masses in the order of between 10(7) and 10(9) g mol(-1) were obtained. The results are contrasted with enzymatically degraded and oxidised starch samples that were fractionated in the normal mode.

Quantitative analysis of film coating in a fluidized bed process by in-line NIR spectrometry and multivariate batch calibration.

A method is described which enables real-time analysis of film coating on pharmaceutical pellets during an industrial manufacturing process. Measurements were conducted on the solid particulate material by near-infrared (NIR) spectrometry utilizing a diffuse reflectance fiber-optic probe positioned inside a fluidized bed process vessel. Time series of NIR spectra from 11 batches generated a three-way data matrix that was unfolded and modeled by partial least squares (PLS) in a multivariate batch calibration. The process conditions were deliberately varied according to an experimental design. This yielded good predictability of the coating thickness with a best model fit, R2 = 0.97, for one PLS-projection, and a root-mean-square error of calibration = 2.2 microm (range tested 0-50 microm). The regression vector was shown to be highly influenced by responses that are both direct (aliphatic C-H stretch overtones) and indirect (aromatic C-H stretch overtones), from film component and core material, respectively. The impact of different data pre-treatment methods on the normalization of the regression vector is reported. Justification of the process calibration approach is emphasized by good correlation between values predicted from NIR data and reference image analysis data on dissected pellets and a theoretical nonlinear coating thickness growth model. General aspects of in-line NIR on solids and multivariate batch calibration are discussed.

Analysis of film coating thickness and surface area of pharmaceutical pellets using fluorescence microscopy and image analysis.

A method is presented which enables geometrical characterisation of pharmaceutical pellets and their film coating. It provides a high level of details on the single pellet level. Image analysis was used to determine the coating thickness (h) applied on the pellets and the surface area (A) of the pellet cores. Different definitions of A and h are evaluated. Hierarchical analysis of variance was used to resolve different sources contributing to the total variance. The variance within pellets and the variance between pellets were found as significant sources of variation. Special emphasis was put on evaluation of A/h due to its influence on the release rate of an active drug substance from the pellet core. The pellet images were thus used to predict variations in the release rate using a mathematical model as a link between the image data and the release rate. General aspects of image analysis are discussed. The method would be useful in calibration of near infrared spectra to h in process analytical chemistry.

Expression of Vitreoscilla hemoglobin in Escherichia coli enhances ribosome and tRNA levels: a flow field-flow fractionation study.

Asymmetrical flow field-flow fractionation (FFF) was used to separate and quantitate 70S ribosomes, the 30S and 50S subunits, and tRNA in one single analytical procedure. The method was applied to an investigation of the effect of Vitreoscilla hemoglobin (VHb) on the translational machinery of the recombinant Escherichia coli cells. The number of active 70S ribosomes per cell increased dramatically, more than 2-fold, as did also the tRNA levels for the VHb-expressing strain relative to VHb-negative control at the end of a 30-h fed-batch cultivation. This was accompanied by a corresponding 61% increase of a cloned marker enzyme activity. The results clearly indicate that VHb promotes the level of translational components. There should be many other cases in bioengineering where it is important to relate the protein production level in a bioreactor to the ribosome and tRNA levels.

Monitoring of a film coating process for tablets using near infrared reflectance spectrometry.

A process analytical chemical method using near infrared diffuse reflectance spectrometry was developed for the determination of the amount of tablet coating on single tablets. This method is based on calibration of the spectra versus the added mass of coating solution. The tablet core was composed of two halves of different chemical composition and spectra were recorded from both sides of the tablets. The calibration was carried out using the chemometric methods principal component analysis (PCA), partial least squares (PLS), and multiplicative signal correction (MSC). The PLS-model utilised spectra obtained from both sides, pretreated with MSC, and ordered into one object. This method can be used in process analytical chemistry at-line. Additional characterisation of the measurements was obtained by calibrating the spectra versus coating thicknesses obtained from optical microscopy. Using PCA, it was possible to roughly estimate the maximum depth in the coating material that returns chemical information, the 'information depth', which was 0.1-0.2 mm.

Transferred nuclear Overhauser effect spectroscopy study of a peptide from the PapG pilus subunit bound by the Escherichia coli PapD chaperone.

Interaction of the Escherichia coli PapD chaperone with the synthetic peptide PapG308-314 (Thr-Met-Val-Leu-Ser-Phe-Pro), corresponding to the seven C-terminal residues of the PapG pilus subunit, was studied by transferred nuclear Overhauser effect (TRNOE) spectroscopy. The observation of cross-peaks corresponding to either intraresidue or sequential C(alpha)H/NH and C(beta)H/NH TRNOEs and the absence of sequential NH(i)/NH(i+1) TRNOEs indicate that the peptide binds to PapD in an extended conformation. In addition, line-broadening effects gave information of the peptide's mode of interaction with PapD. These observations were in excellent agreement with a recent crystal structure of a PapG peptide complexed with PapD.

Use of flow field-flow fractionation for the rapid quantitation of ribosome and ribosomal subunits in Escherichia coli at different protein production conditions.

Asymmetrical flow field-flow fractionation was used for rapid (8-14 min) separation of ribosomes and their subunits. The amount of ribosomes and the mass fraction of ribosomes was determined in growing Escherichia coli cells. These quantities changed significantly at different growth phases. Ribosomal composition was monitored after the insertion of a protein-encoding plasmid and after the addition of an antibiotic agent. The results suggest that the method will be useful in studies of, e.g., the relationships between the protein production capacity of cells and the ribosomal composition. The analysis time is substantially shorter than ultracentrifugation run times.

Determination of relative amounts of ribosome and subunits in Escherichia coli using asymmetrical flow field-flow fractionation.

The 30S and 50S subunits and the 70S ribosome of Escherichia coli were separated in 6 minutes by using asymmetrical flow field-flow fractionation (FFF). The total analysis time for determination of the relative amounts of ribosomes and free subunits in a preparation from a cell suspension was 8 min. The method can detect a change in the mass fraction of ribosomes if it exceeds approx, 10%. The separation is based on differences in diffusion coefficients, i.e., hydrodynamic diameters, and these can be determined from observed retention times. The hydrodynamic diameters were in good agreement with literature values obtained from electron microscopy. The mass fraction of ribosomes changed as a function of the magnesium ion concentration which confirms previous knowledge and shows the accuracy of the method. The method appears as an alternative to ultracentrifugation analysis and avoids some of its drawbacks and artefacts. An obvious application can be the optimisation of cell design in metabolic engineering in order to maximise translation and protein production.

Separation and quantitation of monoclonal antibody aggregates by asymmetrical flow field-flow fractionation and comparison to gel permeation chromatography.

During the downstream processing of monoclonal antibodies (Mab) the formation of dimers and/or oligomers may occur. Today, the most common technique for determination of aggregate content is gel permeation chromatography (GPC) but it has limited resolution and separation speed. Oligomers are particularly difficult to resolve. Asymmetrical flow field-flow fractionation (FFF) is a new analytical method for the separation of Mab aggregates. The monomer and dimer peaks were well resolved and three additional peaks, thought to be trimers, tetramers, and pentamers were partially resolved. The total time for a separation can be kept as short as 6 min. The GPC separation takes about 15 min with less resolution. A study of the reproducibility for the two techniques revealed that the precision was slightly better for GPC. Investigation of the dependence of sample concentration showed that the load limit for FFF was about 4 micrograms of Mab. The GPC technique requires prefiltered samples in order to avoid clogging of the column. For FFF it is possible to inject samples containing precipitated material without any pretreatment. The flow conditions can be adjusted so that the precipitated material elutes with the front, well separated from the monomer peak.

Improved separation speed and efficiency for proteins, nucleic acids and viruses in asymmetrical flow field flow fractionation.

The performance of the asymmetrical flow field-flow fractionation channel has been improved by the use of a much thinner (0.12-mm) channel than before and by flow programming (stepwise gradient elution). The thinner channel contributes to a decreased zone broadening which enables complete resolution in a shorter time. Three protein peaks, representing molecular weights from 12,000 to 136,000, wer completely resolved within 3 min. Flow programming speeds up the elution of the high-molecular-weight materials which occur late in a fractogram. This enabled separation of a small plasmid fragment (700 base pairs) from the large amounts of a big fragment (4600 base pairs) in 30 min and improved detection of a presumed trimer of albumin. Two viruses (1.8 10(6) and 50 10(6) daltons) were eluted as narrow peaks within 5 min.

Application of an asymmetrical flow field-flow fractionation channel to the separation and characterization of proteins, plasmids, plasmid fragments, polysaccharides and unicellular algae.

The asymmetrical flow field-flow fractionation channel has been improved by introduction of a different sample loading method, downstream central injection, which reduces sample relaxation and focusing time considerably and allows the concentrations of material in the channel, thereby enabling the loading of very large sample volumes. The performance of the channel was demonstrated by the separation of a protein from its dimer, retention of nucleic acids, i.e., plasmids and enzymatically cleaved plasmids, hyaluronate and unicellular algae.

Precolumn-venting plug technique for direct injection of untreated blood plasma samples into reversed-phase liquid chromatography systems.

A simple column switching technique for the direct injection of untreated blood plasma samples is presented for the determination of drugs and related compounds. The system consists of injector valves, a precolumn, a three port valve and a separation column. The precolumn is used for trace enrichment and sample clean-up. Aqueous plugs are introduced on both sides of the plasma sample before it enters the precolumn. This results in a stable system, because contact between plasma proteins and the organic solvent usually present in the mobile phase is prevented. The proteins are eluted to waste with the aqueous plug fluid. The stability of the chromatographic system was studied with respect to efficiency and column back-pressure. The influence of the concentration of organic solvent in the eluent, type and particle size of the packing material, precolumn filters, pH, and ionic strength was investigated for large sample volumes. Optimal stability was obtained at low concentrations of organic solvent in the eluent, with plugs of phosphate buffer (mu = 0.1) and with a spreader at the inlet and a screen (2 microns) at the outlet of the precolumn. The precolumn was packed with 10-microns particles for trace enrichment. Under favourable conditions 40-50 large-volume (0.5-ml) plasma injections can be made into a single precolumn.

The determination of lidocaine and its metabolites by a direct injection LC method using large sample volumes of untreated plasma.

Lidocaine and its N-dealkylated metabolites (glycylxylidide and monoethyl-glycylxylidide) have been determined at their therapeutic levels. The plasma samples were centrifuged and then injected directly into a liquid chromatograph containing a reversed-phase column with LiChrosorb RP-Select B as solid phase and 1-propanolaqueous buffer containing decanesulphonate as eluent. A pre-column venting plug technique was used, in which the chromatographic system consisted of two injector valves, a precolumn, a valve and a separation column. Lidocaine and its dealkylated metabolites were detected in the eluent by UV detection (210 nm) and the quantitations were performed by measurement of the peak areas of the samples and external standards. Lidocaine and its metabolites were determined in the therapeutic range with a percentage recovery close to 100% and inter-assay precision (RSD) of 1.0-2.2%.

The liquid chromatographic determination of theophylline in untreated plasma by simple direct injection.

A liquid chromatographic method for determination of theophylline by direct injection of untreated plasma samples is described. Theophylline is detected without interference from related compounds such as paraxanthine. A precolumn venting technique is used which considerably increases column life. The lifetimes of the separation columns are unaffected by plasma injections whereas the precolumn has to be changed after 140 injections (10 mul of plasma). The peak purity of theophylline is examined spectrophotometrically. Determinations are performed by external standardization with recoveries close to 100% with a precision better than 2.3% (RSD).

Procedures for direct injections of untreated blood plasma into liquid chromatographic columns with emphasis on a pre-column venting technique.

The stability of reversed-phase liquid chromatographic systems when untreated blood plasma samples are applied was studied with respect to retention, peak efficiency and column back-pressure. The influence of the content of organic solvents in the eluent, the flow-rate and the particle diameter of the support was investigated. Many of the column-deteriorating effects seem to be due to denaturation of the plasma proteins and the kinetics of this process. The stability is increased by decreasing the content of organic modifier in the eluent. The flow-rate should be moderate, as both high and low flow-rates give decreased stability. The stability increases with increasing particle size of the solid phase. A small pre-column is used (1) as a guard column and (2) to effect a pre-separation of the solute from plasma matrix components by a pre-column venting technique. The pre-column venting technique considerably increases the lifetime of the system and especially the separation column. Under optimal conditions hundreds of injections (10 microliters of plasma) can be performed without loss of stability.

Elimination of peak deformation in the liquid chromatographic separation of a strongly protein-bound drug from directly injected blood plasma samples.

Direct injection of blood plasma samples into reversed-phase columns resulted in skewed chromatographic peaks for the drug naproxen. The skew is shown to be due to strong binding of naproxen to albumin present in the blood plasma. Methods to eliminate the peak skew have been investigated. They include changes of the composition of the eluent and of the sample solution in order to decrease the degree of binding of the drug to albumin. The methods studied were dilution, addition of displacers, change of pH and change of methanol concentration. Calculations based on known binding constants indicate that the degree of peak skew was directly influenced by the degree of protein binding of the drug in the sample solution.