Ynze Mengerink

Use of an evaporative light scattering detector in reversed-phase high-performance liquid chromatography of oligomeric surfactants

Abstract The sensitivity of the evaporative light scattering detector was found to be dependent on the analyte concentration and the organic modifier concentration: under reversed-phase gradient conditions calibration is necessary for each analyte. Detection limits for alcohol and carboxylic acid homologues were determined. The advantages of helium over carbon dioxide as a nebulization gas for both volatile and non-volatile or thermolabile analytes are a >30°C lower operating temperature and a five-fold better signal-to-noise ratio. High-resolution separations of polyether alkyl alcohols were effected by reversed-phase gradient high-performance liquid chromatography with ultraviolet transparent mobile phases. Evaporative light scattering detection was compared with refractive index and ultraviolet absorbance detection at 190 nm and found to be superior in signal-to-noise ratio as well as baseline drift. A column-switching system was designed to allow complete compositional analysis of technical samples of ethoxylated alkyl alcohols and their carboxylic acid derivatives.

Advances in the evaluation of the stability and characteristics of the amino acid and amine derivatives obtained with the o-phthaldialdehyde/3-mercaptopropionic acid and o-phthaldialdehyde/N-acetyl-L-cysteine reagents: High-performance liquid chromatography-mass spectrometry study

The composition of the amino acid and amine derivatives obtained with the o-phthaldialdehyde (OPA)/3-mercaptopropionic acid (MPA) and with the OPA/N-acetyl-L-cysteine (NAC) reagents was investigated by on-line HPLC-electrospray ionization MS. The initially formed derivatives proved to be, as expected, the corresponding isoindoles while their transformed species contained one additional OPA molecule. Based on the MS spectra of all transformed OPA derivatives a reaction pathway is suggested. This reaction mechanism was supported both by the molecular ions of the endproducts and by the presence of several selective fragment ions that served as an explanation to the structure of the believed to be less stable OPA derivatives. It has been shown that more than one OPA derivative forms in all those cases when the compound to be derivatized does contain the NH2-CH2-R moiety. Thus, amino acids like e.g. glycine, histidine, beta-alanine, gamma-aminobutyric acid, epsilon-aminocaproic acid, ornithine, and also several aliphatic mono- and diamines provide more than one OPA derivative. Analytical consequences of this experience were utilized by altering the reagents composition. Reagents containing mole ratios of [OPA]/[MPA] or [OPA]/[NAC]=1/50 resulted in two benefits, simultaneously: (i) in a decrease of the transformation rate of the initially formed derivative, and, (ii) in an increase of the overall stability of the total of derivatives.

Separation and quantification of the linear and cyclic structures of polyamide-6 at the critical point of adsorption.

The linear and cyclic structures of polyamide-6 were separated by liquid chromatography at critical conditions (LCCC) and identified with different mass spectrometric (MS) techniques and quantitated by LCCC with evaporative light-scattering detection (ELSD). Electrospray ionization MS was not suitable to identify the higher cyclic structures. For this purpose, matrix-assisted laser desorption ionization time-of-flight MS performed better and cyclic and linear structures were oligomerically resolved and separately identified in the mass spectrometer. The highest cyclic structure present and detected was the cyclic pentacontamer. It could be demonstrated that cyclic and linear oligomers follow different ionization and fragmentation routes/patterns. Quantification with ELSD of the components separated by LCCC using a universal calibration curve or an iterative procedure was developed. An area correction to account for different peak widths of coeluting components improves precision and accuracy of the calibration curve and improves quantitation accuracy for the samples analyzed. With these corrected values, no molecular mass dependency was observed for the cyclic and linear structures. Under critical conditions, the linear and cyclic structures of polyamide-6 were separated, identified and quantified.

Endgroup-based separation and quantitation of polyamide-6,6 by means of critical chromatography

Polyamide-6,6 is a polycondensation product from the two monomers adipic acid and 1,6-hexamethylenediamine. Depending on the reacted amount of these monomers, different ratios of amine and carboxylic acid endgroups can be formed. Besides linear chains, cyclic polyamides will also be formed. Using critical chromatography, polyamide-6,6 can be separated independently of molar mass. Retention is based solely on endgroup functionality. It is demonstrated that high-molecular-mass polyamide-6,6 (Mw approximately 20,000-30,000) can be separated using this approach. The separation was optimized by using different parameters, such as percentage modifier, temperature and pressure. The concentration of phosphoric acid was used for selective retention of the different endgroup functionalities. Using this property, a new method called critical gradient chromatography was performed where the mobile phase changes from a weak to a strong solvent with respect to the endgroup functionality, while retaining the critical conditions of the backbone unit. Quantification using UV detection is discussed.

Analysis of linear and cyclic oligomers in polyamide-6 without sample preparation by liquid chromatography using the sandwich injection method: I. Injection procedure and column stability

We report a method for reliable routine polymer sample introduction with minimal bias, a separation method of the first six linear and cyclic oligomers by liquid chromatography, quantification using group equivalents and long term method performance. Injecting a polymer sample in a mobile phase containing an aqueous non-solvent often results in blocked systems as the polymer precipitates in the connecting capillaries. In this first part we focus on a new injection technique, in which the dissolved polyamide is placed between two zones of formic acid, preventing the polymer to precipitate before it reaches the column. Development of this sandwich injection method makes direct injection of the polymer into an aqueous acetonitrile gradient feasible. The oligomeric polyamide recovery of this technique, extraction, dissolution/precipitation and direct injection on a hexafluoro-isopropanol (HFIP) gradient were compared. With the sandwich injection method the polymer remains on the column, slowly changing the stationary phase. The influence of this on resolution and retention was studied. Column stability allows sixty injections before cleaning or replacing the column is necessary.

Compositional analysis of nitrile terminated poly(propylene imine) dendrimers by high-performance liquid chromatography combined with electrospray mass spectrometry

Abstract Separation methods for nitrile terminated poly(propylene imine) dendrimers were developed to monitor and optimize their large scale production. Detailed analyses of defects within a dendrimer generation were performed by HPLC at alkaline pH (sodium hydroxide) on a polymer-based column or at neutral pH (ammonium acetate) on a silica-based column combined with electrospray mass spectrometry. Feasibility of operation of HPLC–electrospray MS with a mobile phase containing non-volatile components is demonstrated.

Analysis of linear and cyclic oligomers in polyamide-6 without sample preparation by liquid chromatography using the sandwich injection method: II. Methods of detection and quantification and overall long-term performance

By separating the first six linear and cyclic oligomers of polyamide-6 on a reversed-phase high-performance liquid chromatographic system after sandwich injection, quantitative determination of these oligomers becomes feasible. Low-wavelength UV detection of the different oligomers and selective post-column reaction detection of the linear oligomers with o-phthalic dicarboxaldehyde (OPA) and 3-mercaptopropionic acid (3-MPA) are discussed. A general methodology for quantification of oligomers in polymers was developed. It is demonstrated that the empirically determined group-equivalent absorption coefficients and quench factors are a convenient way of quantifying linear and cyclic oligomers of nylon-6. The overall long-term performance of the method was studied by monitoring a reference sample and the calibration factors of the linear and cyclic oligomers.

Analysis of higher polyamide-6 oligomers on a silica-based reversed-phase column with a gradient of formic acid as compared with hexafluoroisopropanol

The analysis of polyamide-6 oligomers and polymer is usually performed with expensive fluorinated alcohols like 2,2,2-trifluoroethanol (TFE) or 1,1,1,3,3,3-hexafluoroisopropanol (HFIP). Formic acid is well known as a mobile phase additive to adjust pH in reversed-phase high-performance liquid chromatography. However, formic acid is seldom used as a modifier to perform gradient elution chromatography on octadecyl-modified silica-based columns. Here we demonstrate the determination of cyclic and linear polyamide-6 oligomers using formic acid as a modifier on an octadecyl-modified silica-based column. This column was shown to be stable for more than 5000 column volumes, even when a mobile phase of 65-95% formic acid in water at a flow of 1 ml/min is applied. With formic acid under the conditions used (65-95% formic acid in water) the oligomers are retained on the column, while the polymer does not precipitate. In comparison, during adsorption and separation with a HFIP gradient, precipitation of the polymer occurs. The implications of the different separation mechanisms, i.e., adsorption vs. precipitation chromatography are discussed. Loadability is shown to be much better with the formic acid system. However, with formic acid as a modifier UV detection below 250 nm is not feasible. The less sensitive evaporative light scattering detector is used to detect the polyamide oligomers in the formic acid phase. In addition it is shown that capillary zone electrophoresis (CZE) with UV-absorbance detection using HFIP is an attractive combination as HFIP is UV-transparent and CZE allows low modifier consumption.

Low-molecular-weight model study of peroxide cross-linking of ethylene-propylene-diene rubber using gas chromatography and mass spectrometry II. Addition and combination reactions.

The dicumyl-peroxide-initiated addition and combination reactions of mixtures of alkanes (n-octane, n-decane) and alkenes [5,6-dihydrodicyclopentadiene (DCPDH), 5-ethylidene-2-norbornane (ENBH) and 5-vinylidene-2-norbornane (VNBH)] were studied to mimic the peroxide cross-linking reactions of terpolymerised ethylene, propylene and a diene monomer (EPDM). The reaction products of the mixtures were separated by both gas chromatography (GC) and comprehensive two-dimensional gas chromatography (GCxGC). The separated compounds were identified from their mass spectra and their GC and GCxGC elution pattern. Quantification of the various alkyl/alkyl, alkyl/allyl and allyl/allyl combination products shows that allylic-radicals comprise approximately 60% of the substrate radicals formed. The total concentration of the products formed by combination is found to be independent of the concentration and the type of alkene. The total concentration of the products formed by addition to the alkene increases with increasing concentration of alkene. In addition, the total concentration of the formed addition products depends strongly on the type of the alkene used, viz. VNBH>ENBH approximately DCPDH, which is a consequence of differences in steric hindrance of the unsaturation. The peroxide curing efficiency, defined as the number of moles of cross-linked products formed per mol of peroxide, is 173% using 9% (w/w) 5-vinylidene-2-norbornane (VNBH). This indicates that the addition reaction is recurrent. All these findings are consistent with experimental studies on peroxide curing of EPDM rubber. In addition, the present results provide more-detailed structural information, increasing the understanding of the mechanism of peroxide curing of EPDM. The described approach to use low-molecular-weight model compounds followed by GC-mass spectrometry (MS) and GCxGC-MS analysis is proven to be a very powerful tool to study the cross-linking of EPDM.

Quantitative analysis of morphine in dried blood spots by using morphine-d3 pre-impregnated dried blood spot cards

Two different internal standard dried blood spot (DBS) pre-impregnation procedures (prior to blood spotting) were investigated. In the first procedure DBS pre-impregnation is performed by immersing the DBS card fully into an internal standard solution. In the second procedure pre-impregnation is performed by pipetting a certain volume of an internal standard solution onto the DBS card. Morphine-d3 was used as the model compound for all experiments. The pre-impregnation procedure by immersing was further investigated with respect to homogeneity of impregnation, influence of different blood spotting techniques and the influence of spotting different blood volumes on the internal standard distribution, calibration and stability of pre-impregnated cards. Finally, the immersing procedure was used for the analysis of morphine in dried blood spots and the results were compared to the conventional procedure in which the internal standard morphine-d3 was added to the extraction solvent. The new pre-impregnated cards couple simplicity of operation and convenient use in the field to results equivalent to the conventional procedure.