Dynamic control of split flow in packed column supercritical fluid chromatography using dual resistively heated restrictors

Remote control of the vent/detector split flow ratio in packed column supercritical fluid chromatography (pSFC) with flame ionization detector (FID) is demonstrated using a dual heated restrictor method. Restrictors stemming from a Tee at the separation column outlet were, respectively, fixed into an FID and a vent port, and their individual temperatures were controlled using resistively heated wires. Subsequently, both system pressure and split flow could be manipulated. For example, for applied restrictor temperatures examined up to 600°C, corresponding vent/FID split flow ratios between 2 and 7 were observed depending on the port heated. As well, column pressures around 16–23 MPa were also achievable over the same range. Conversely, isobaric altering of the split flow ratio was possible when opposing positive and negative temperature gradients were applied at the two restrictors. Under these conditions, the system pressure varied less than 1% RSD over a 10 min period. As an application, the method was used to establish stable detector operation in the analysis of n‐alkanes under pSFC‐FID conditions that initiated flame instability. Results indicate that this technique could be a relatively simple and inexpensive means of controlling system pressure and detector split flow ratios in pSFC‐FID.     

Separation of polyethylene glycol oligomers using inverse temperature programming in packed capillary liquid chromatography

Inverse temperature programming in packed capillary liquid chromatography coupled to evaporative light-scattering detection has been used to resolve native polyethylene glycol (PEG) oligomers. The model compound, PEG 1000, was separated on a 300 mm x 0.32 mm I.D. capillary column packed with 3 microm Hypersil ODS particles with acetonitrile-water (30:70, v/v) as mobile phase. The retention of the PEG oligomers increased with increasing temperature, different from what is commonly observed in liquid chromatography. The retention times of the oligomers were approximately doubled for each 25 degrees C increment of the column temperature in the temperature range 30-80 degrees C. The oligomers were almost unretained and co-eluted at a column temperature of 30 degrees C. At 80 degrees C a baseline separation of more than 22 peaks was obtained, but the last eluting peaks were severely broadened and all oligomers did not elute. When a negatively sloped temperature ramp from 80 to 25 degrees C at -1.5 degrees C/min was applied, the peak shapes were improved, additional peaks were detected and the analysis time was reduced by 48%. In the temperature programming mode, the intra-day precision of the retention times ranged from 0.5 to 5.8% (n=5).     

Some peculiarities of thermodynamic behaviour of oligomeric mixtures with specific interactions between components

Using inverse gas chromatography, the temperature and concentration dependences of the interaction parameter χ₂₃ were estimated for mixtures of various oligomers capable of specific interaction. Mixtures of polyethylene glycol with polypropylene glycol and polyethylene glycol adipate were studied, including mixtures of polyethylene glycol fractions of different molecular weights. It was found that there is a maximum and a minimum in the temperature dependence of χ₂₃, and the bimodal character phase diagrams was established. Experimental data were explained by aggregation or association phenomena in mixtures. The comparison of experimental values of χ₂₃ with those calculated on the basis of the new Flory theory shows essential differences, especially for enthalpy and entropy contributions, this being explained by the specific interaction between components.     

Supercritical fluid chromatography with a slurry-packed capillary column

A versatile system with a slurry-packed capillary column was developed for supercritical fluid chromatography, which is capable of programming both inlet and outlet pressure independently, as well as using a restrictor to apply back pressure. This system revealed the relationships between pressure drop, flow rate, and linear velocity in pressure-programmed supercritical fluid chromatography. In the restrictor system, both the pressure drop and the flow rate increased almost linearly with inlet pressure, while under conditions of constant pressure drop characteristic behavior was observed which depended on the density-viscosity relationships of supercritical fluid. Resolution in the separation of polysiloxane oligomers was found to be increased by increasing the ratio of pressure drop to pressure-programming rate, although the sensitivity decreased due to the increase in peak volume. The system controlling both inlet and outlet pressure has distinct advantages over the restrictor system controlling both inlet and outlet pressure has distinct advantages over the restrictor system in practical in practical operations.     

Control of flow rate in supercritical fluid chromatography

Summary Supercritical Fluid Chromatograph was constructed using two pumps and two restrictors. With this system in conjunction with the fused silica column packed with relatively large particles (40 μm), the flow rate was controlled both for isobaric and pressure programmed operations. Application of this system to the determination of molecular weight distribution of polystyrene oligomers was presented.     

A model for quantitatively describing linear velocity programming in capillary SFC

Summary Linear velocity in capillary SFC is commonly controlled by restricting capillaries. In this paper, a model is described that quantitatively predicts the linear velocity of a supercritical fluid in SFC using tapered or ceramic frittype restrictors. In this model, the flow from the restricting capillary is assumed to be an isentropic expansion. The variation of the linear velocity as a function of pressure, temperature and cross-sectional area of the restricting aperture was predicted by this model. This predictive capability is important to the use of gradient programming in capillary SFC. Finally, the ideal variable restrictor for gradient programming was found to be one that could reversibly increase or decrease the linear velocity independent of the pressure, temperature, and/or density conditions used to create the gradient.     

Changes in the order of elution of some organic compounds in high-resolution gas chromatography on polar columns depending on the injection method and water content in the sample

During the GC analysis of apple aroma essence using a capillary column with polar stationary phases, i.e., bonded/crosslinked polyethylene glycol (HP-Innowax) and acid-modified polyethylene glycol (FFAP) the phenomenon of changing the elution order of some compounds was observed ("cross-over phenomenon"). The elution order within two pairs of closely eluting compounds: 2-methylbutyl acetate/pentan-3-ol (internal standard) and trans-2-hexen-1-al/2-methylbutan-1-ol differed between samples introduced onto the column with direct injection and with static headspace method. Experiments carried out on standard solutions of investigated compounds in water-acetone mixtures of gradually reduced water ratio (99:1 to 0:100) proved that changes of elution times leading first to co-elution and subsequently to reversion of elution order of compounds within both pairs depended on the amount of water in the injected sample. The same effect was observed while the amount of water was diminished by reducing the sample volume introduced into the column.     

Pressure‐Tunable Dual‐Column Ensembles for High‐Speed GC and GC/MS

A series‐coupled ensemble of two capillary GC columns of different selectivity with an adjustable pressure at the column junction point is used to obtain tunable selectivity for high‐speed GC and GC/TOFMS. An electronic pressure controller with a 0.1‐psi step size is used to obtain numerous computer‐selected unique selectivities. System configurations for conventional, atmospheric‐pressure outlet operation with flame ionization detection and for vacuum‐outlet operation with photoionization detection are described for GC‐only experiments. Polydimethylsiloxane is used as the non‐polar column and polyethylene glycol (atmospheric outlet) or triflouropropylpolysiloxane (vacuum outlet) is used as the polar column. For GC/TOFMS experiments, 5% phenyl polydimethylsiloxane was used as the non‐polar column, and polyethylene glycol was used as the polar column. The time‐of‐flight mass spectrometer can acquire up to 500 complete mass spectra per second. Since spectral continuity is achieved across the entire chromatographic peak profile, severely overlapping peaks can be spectrally deconvoluted for high‐speed characterization of completely unknown mixtures. For mixture components with significantly different fragmentation patterns, spectral deconvolution can be achieved for chromatographic peak separations of as little as 6.0 ms. This can result is very large peak capacity for time compressed (not completely resolved) chromatograms. The use of columns with tunable selectivity allows for precise peak‐position control, which can result in more efficient utilization of available peak capacity and thus further time compression of chromatograms. The limits of tunability and deconvolution are tested for near co‐elutions of different classes of hydrocarbon compounds as well as for more multi‐functional mixtures.     

Characterization of ultrafiltration membranes. Part II — Mass transport measurements for low and high molecular weight synthetic polymers in water solutions

An experimental study was carried out on the ultrafiltration of two linear synthetic polymers: polyethylene glycol (PEG) and polyvinylpyrrolidone (PVP) in a stirred batch system. Seven fractions of PEG in the molecular weight range 400 to 20,000 and three fractions of PVP (with molecular weights 23,400, 39,000 and 80,000) have been studied using IRIS 3038, IRIS 3042, and UM 10 membranes, in the pressure range 0.2 to 1.2 atm. The permeability ratio α, a dimensionless parameter which measures the deviation of the flux from the “normal” value as given by Darcy's law, was used to analyse the flux-pressure behavior. Rejection data were corrected for concentration polarization using a classical thin-film theory. For aqueous PEG solutions the observed rejection was nil and an increase in applied pressure resulted in more than a proportional increase in permeate flux, i.e. α appears to approach the value 1 asymptotically. This phenomenon was related to the “deformability” of the PEG solute. For aqueous PVP solutions, the data agree with a gel or pre-gel polarization model.     

Evaluation and prediction of the shape of gas chromatographic peaks

The evaluation and prediction of the shape of asymmetric gas chromatographic peaks is important as the knowledge of the amount of tailing permits to foresee the resolution between closely eluting peaks and to select the best analytical conditions for an efficient and rapid separation. A model function was tested in order to approximate the true peak shape obtained on non-polar column by injecting different compounds. The trend of the parameters involved in the used equation has been investigated as a function of column temperature and inlet pressure. The reproduction of the symmetrical or asymmetrical shape of gas chromatographic peaks is satisfactory and the method also permits to predict the shape of peaks obtained in different conditions of temperature and pressure.     

Evaluation of mass discrimination effects in the quantitative analysis of polydisperse polymers by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using uniform oligostyrenes

Mass discrimination effects in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) were quantitatively investigated using equiweight and equimolar mixtures of uniform polystyrene (PS) oligomers. Uniform PS oligomers were separated by preparative super-critical fluid chromatography (SFC) from commercial standard PS samples. The separated PS oligomers, with degrees of polymerization n = 2–25, have absolutely no molecular weight distributions. Equiweight and equimolar mixtures of uniform PS oligomers were accurately prepared by weighing by microbalance, and their spectra were recorded using a MALDI-TOF mass spectrometer. In the lower molecular weight region (less than about 10³) the oligomers with lower molecular weights give lower mass spectral intensities, with no correlation with laser power. In contrast, higher laser powers yield a decrease of mass spectral intensities in the higher molecular weight region. These results clearly show that mass discrimination effects occur at lower and higher molecular weights depending on the laser power, and provide quantitative information about the discrimination. Using the data on equiweight and equimolar mixtures of PS oligomers, it was possible to calibrate the MALDI-TOF mass spectral data for an analysis of molecular weight distribution of a standard monodisperse PS sample with number-averaged molecular weight of 10³, and to compare it with the molecular weight distribution measured by analytical SFC. The result from the calibrated MALDI-TOF mass spectrum, however, does not agree perfectly with that from the SFC results, because undetectable peaks in MALDI-TOF mass spectra at lower and higher molecular weights could not be included in the calibration of peak intensities. Copyright © 2001 John Wiley & Sons, Ltd.     

Comparison of Two Novel Techniques for Eliminating Liquid Chromatography Gradient Ghost Peaks

Abstract

Distilled water to organic gradients in reversed phase liquid chromatography are often plagued with UV-detectable “ghost” peaks that can obscure sample peaks and complicate interpretation of results.^3,4 These contaminants usually come from the distilled water³ although it is possible for them to originate in the organic eluent⁴. The ghost-peak problem can be eliminated by using specially prepared “LC-grade” water, although this is expensive. In addition water can be cleaned with the Milli-Q ion-exchange system, containing a carbon absorption column⁵ but not where these systems contain buffers or organic amine eluting agents such as triethylamine phosphate. Gurkin and Ripphahn have shown that a C-8 silica column can be used off-line in a low- pressure mode to clean water⁶.     

Development of a fritless packed column for capillary electrochromatography-mass spectrometry

A novel procedure was developed for the fabrication of a fritless packed column for the coupling of capillary electrochromatography (CEC) to mass spectrometry (MS). The process involved the formation of internal tapers on two separate columns. Once the internal tapers are formed and the columns are packed, the untapered ends of each column were joined together by a commercially available connector. Several advantages of the fritless columns are described. First, the design used here eventually eliminates the need for any frits thus reducing the possibility of bubble formation seen with fritted packed columns. In addition, this is the first report in which the internal tapers are formed at both the inlet and outlet column ends making the fritless CEC-MS column more robust compared to only one report with externally tapered counterparts. Second, a comparison of internally tapered single frit packed CEC-MS (previously developed in our laboratory) column versus fritless CEC-MS column reported here shows that the latter provides better efficiency, suggesting no dead volume with equally good sensitivity and chiral resolution of (±)-aminoglutethimide. The fritless column procedure is universal and was used to prepare a series of columns with a variety of commercially available packing material (mixed mode strong cation exchange, SCX; mixed mode strong anion exchange, SAX; C-18) for the separation and MS detection of short chain non-chromophoric polar amines, long chain nonchromophic anionic surfactant as well as oligomers of non-chromophoric non-ionic surfactants, respectively. The fritless columns showed good intra-day repeatability and inter-day reproducibility of retention times, chiral and achiral resolutions and peak areas. Very satisfactory column-to-column and operator-to-operator reproducibility was demonstrated.     

Separation range and separation efficiency in high-speed gel filtration on TSK-GEL SW columns

Separation ranges for gel filtration on TSK-GEL SW columns, G2000SW, G3000SW and G4000SW, were measured for globular protein, dextran and polyethylene glycol. It was possible to separate globular proteins of molecular weights from 5000 to 7,000,000, dextrans of molecular weights from 1000 to 500,000 and polyethylene glycols of molecular weights from 500 to 250,000 by using these three columns of different pore sizes. The column having the highest separation efficiency for globular proteins was also determined. The highest separation efficiency for molecular weights of less than 30,000, 30,000–500,000 and greater than 500,000 was exhibited by G2000SW, G3000SW and G4000SW, respectively.     

High-speed aqueous gel-permeation chromatography

High-speed gel-permeation chromatography (GPC) of water-soluble polymers has been investigated by using TSK-GEL, type-PW columns packed with small porous particles. Being semirigid, the column packing could be operated under high pressure and, therefore, it was possible to achieve high-speed GPC. A resolution higher than that of ordinary GPC in organic solvent system was attained when the measurement of one sample was completed within 40 min with a 6-ft column set. Samples with a wide range of molecular weights, from oligomers to polymers having a molecular weight greater than 10⁶, could be fractionated. Dextran, polyethylene glycol, polyacrylamide, poly(vinyl alcohol), and poly(vinyl pyrrolidone) were separated according to molecular size with no evidence of adsorption. Investigation of other water soluble polymers is now in progress.     

A molecular approach to flavor synthesis. I. Menthol esters of varying size and polarity

For a series of menthol esters of increasing size and polarity, “mintyness” decreased rapidly as molecular weight increased. For molecular weights above about 700, the compounds were tasteless to a significant proportion of the taste panel. As polarity increased, the “sweet minty” taste of hydrophobic menthol esters became increasingly bitter, until water-soluble menthol esters were strongly bitter. Bitterness was still apparent in quite high-molecular-weight water-soluble esters; a different receptor may be involved for bitterness than for mint. An overall hypothesis relating flavor to molecular solubility parameters is proposed.     

The physicochemical characteristics of the 1-butyl-3-methylimidazolium bromide-polyethylene glycol-water system

Mixtures of 1-butyl-3-methylimidazolium bromide (BMImBr) with polyethylene glycol (PEG) (molecular weights 1500 and 40000) containing 4–74 wt % BMImBr were studied. Thermogravimetric and differential scanning calorimetry measurements showed that the compatibility of BMImBr and PEG improved as the molecular weight of the polymer decreased. At a BMImBr: PEG₁₅₀₀ weight ratio of 1: 3, a phase-stable composite formed. The results of conductometric measurements were used to determine the diffusion coefficients D _± and conductivity f of BMImBr in mixtures. BMImBr behaved as a strong electrolyte in the mixtures under consideration. Cyclic voltammetry with a platinum electrode was used to show that, as distinct from the BMImBr-H₂O system, water in the BMImBr-PEG₁₅₀₀-H₂O system did not exhibit electrochemical activity within the “electrochemical window” of BMImBr.     

Isomorphism within the hexagonal columnar mesophase of molecular and macromolecular self- and co-assembled columns containing tapered groups

The phase behaviour of binary mixtures of self-assembled tapering molecules based on monoesters of oligooxyethylene glycol and 3,4,5-tris[4-(n-dodecan-1-yloxy)benzyloxy]benzoic acid, their corresponding polymethacrylates, and of 4'-methyl (benzo-15-crown-5)-3,4,5-tris[4-(n-dodecan-1-yloxy)benzyloxy]benzoate within their hexagonal columnar mesophase (Φ_h) is described. The binary blends between molecular tapers co-assemble into a single supramolecular column resulting in isomorphism within their Φ_h mesophase over the entire range of composition. The binary blends between polymethacrylates containing tapered side groups co-assemble into a single Φ_h phase only when the columns of the parent polymers are of similar diameters. This results in binary mixtures which are isomorphic within the Φ_h mesophase over the entire composition range. When the diameters of the columns formed by the parent polymers are dissimilar, isomorphic mixtures are obtained only over a narrow range of composition. Binary mixtures between molecular tapers and macromolecular systems containing tapered side groups co-assemble into a single column to the extent that intercalation of the molecular taper, within the column formed by the macromolecular system containing tapered side groups, is permissible. In all systems increased intracolumnar interactions can be induced by complexation of CF₃SO₃Li by the oligooxyethylenic receptors leading to isomorphism in otherwise non-isomorphic mixtures. Ternary mixtures between molecular tapers with non-specific oligooxyethylenic receptors and specific crown ether receptors and CF₃SO₃ Na as the third component are non-isomorphic within their Φ_h phase due to preferential complexation of the alkali metal cation by the column of the crown ether containing the molecular taper. This results in two columns of dissimilar diameters, which are isomorphic in the Φ_h phase only within a limited range of composition.     

Variable flow liquid chromatography-tandem mass spectrometry and the comprehensive analysis of complex protein digest mixtures

A microscale, variable flow, liquid chromatography-electrospray ionization source has been coupled to an ion trap mass spectrometer and used to analyze both simple and complex protein mixtures. By using an analytical technique described as “peak parking,” the sample analysis times for components eluting from a liquid Chromatograph can be greatly increased without sacrificing Chromatographic resolution. This was achieved by instantaneously reducing the column head pressure (and hence, flowrate) under peaks of interest to prolong their elution from the tip. With the technique, it is possible to perform manual parent ion selection, higher resolution narrow mass range scans, and multiple stages of tandem mass spectrometry (MS² and MS³) within the context of a single eluting peak. Benefits normally associated with an off-line tandem mass spectrometry analysis, such as the optimization of collision induced dissociation parameters and the analysis of more than one charge state, can now be obtained with the increased sensitivity and selectivity afforded by the chromatography. The utility of this method for the comprehensive analysis of complex mixtures was illustrated with the analysis of an in-gel protein digest mixture derived from a single spot from a two-dimensional electrophoresis gel. In addition to the expected enzyme digestion products, other peptides derived from nonspecific cleavage by the enzyme and protein impurities, as well as those having oxidized, derivatized, or deamidated amino acid residues were fully characterized.