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).     

Separation and determination of polyethylene glycol fatty acid esters in cosmetics by a reversed-phase HPLC/ELSD

A comprehensive analytical method was established for the separation of polyethylene glycol (PEG) stearates according to the distribution of ethylene oxide (EO) and subsequent determination of the surfactants in cosmetic samples by using a high-performance liquid chromatography–evaporative light scattering detection. Separation of the PEG stearates comprising approximately up to 82 EO adducts was performed on a reversed-phase YMC-Pack C₈ column using water–acetonitrile gradient elution. The PEG oligomers were separated in order of the increasing number of EO adducts. Quantitation of the PEG fatty acid esters, which was separated as single peak per each component, was performed by chromatography on a reversed-phase Wakosil 10 C₁₈ column using water–methanol gradient elution. The standard curve to quantify the PEG stearates was constructed by the log–log plot, which showed good linearity with the correlation coefficients (R²) 0.998 and more. Working range, repeatability, limit of detection and recovery were acceptable for analysis of the surfactants in cosmetic products. The analytical methods were applied to characterize the PEG stearates according to the EO distributions, then to quantify the surfactants in cosmetic products.     

Carbon dioxide modified subcritical water chromatography

A novel method of increasing the elution strength in subcritical water chromatography (SWC) by adding CO2 to the water mobile phase is presented. Since the polarity of water reduces dramatically with increasing temperature, this property is used in SWC to create an isocratic mobile phase with tunable elutropic strength in reversed-phase separations. Unfortunately, thermal stability of the stationary phase dictates the upper temperature limit and therefore also the minimum available mobile phase polarity. As a result SWC is often not very effective at eluting non-polar analytes. However, when CO2 is blended into subcritical water, a considerable reduction in mobile phase polarity results and improves such separations. For example, in conventional SWC 1-octanol is not observed to elute from a PRP-1 column after several hours at the maximum column temperature of 200 degrees C. In contrast to this, when CO2 is present at 180atm (1atm=101325Pa) in the mobile phase, 1-octanol elutes with good peak shape in less than 4min at only 100 degrees C. The technique is applied to the separation of a variety of analytes which have previously been challenging or even not possible to analyze by conventional SWC. Further, the ability to use temperature and composition programming with the blended CO2/water mobile phase in SWC is also presented and discussed. Overall, the developed method considerably extends the range of non-polar analytes amenable to SWC analysis, while maintaining the beneficial conventional SWC features of flame ionization detection and environmental compatibility.     

Rapid column heating method for subcritical water chromatography

A novel resistive heating method is presented for subcritical water chromatography (SWC) that provides higher column heating rates than those conventionally obtained from temperature-programmed gas chromatography (GC) convection ovens. Since the polarity of water reduces dramatically with increasing temperature, SWC employs column heating to achieve gradient elution. As such, the rate at which the mobile phase is heated directly impacts the magnitude of such gradients applied in SWC. Data from the current study demonstrate that the maximum column heating rate attainable in a typical SWC apparatus (i.e. using a GC convection oven) is around 10 degrees C/min, even at instrument oven settings of over three times this value. Conversely, by wrapping the separation column with ceramic insulation and a resistively heated wire, the column heating rates are increased five-fold. As a result, elution times can be greatly decreased in SWC employing gradients. Separations of standard alcohol test mixtures demonstrate that the retention time of the latest eluting component decreases by 35 to 50% using the prototype method. Additionally, solute retention times in this mode deviate by less than 1% RSD over several trials, which compares very well to those obtained using a conventional GC convection oven. Results suggest that the developed method can be a useful alternative heating technique in SWC.     

Separation of nucleotide oligomers by unitary anion-exchange.

This paper is the first report on the retention behavior of synthetic oligonucleotides and nucleotide oligomers on a continuous-bedmatrix, strong-anion-exchange column. The separation mechanism is predominantly an anion-exchange process, but hydrophobic interaction plays a role as well. The separation is based on the chain length of the oligonucleotide. Both the addition of organic mobile phase modifiers and changes in column temperature affect the retention of oligomers significantly. A volatile buffer system (e.g., triethylamine acetate) could be employed to purify oligonucleotides, and no desalting procedure would be required after the column separation step. The recoveries from the separation are 70% or higher. The maximum loading capacity of an analytical column (35 x 7-mm i.d.) was found to be more than 366 micrograms.     

Determination of poly(ethylene glycol)s by both normal-phase and reversed-phase modes of high-performance liquid chromatography

A normal-phase HPLC system using an amino column has been developed to characterise oligomers of poly(ethylene glycol)s (PEGs) of average M_r 400 to 2000 with derivatisation by dinitrobenzoate. Normal-phase HPLC with gradient elution using ternary solvents of hexane, dichloromethane and methanol has produced a baseline resolution for oligomers of PEG 400, 600 and 1000, while PEG 1000 and 2000 were analysed by using binary solvents of acetonitrile and water. Mixtures of PEGs have been determined by these HPLC systems. PEG 400 in a textile finish has also been determined with satisfactory recovery. It has been found that the hydroxyl group of solvents in normal-phase HPLC plays an important role in resolution and retention of PEG oligomers. Derivatisation efficiency for PEGs by dinitrobenzoyl chloride and quantitative determination of derivatised PEGs by HPLC have been studied. A reversed-phase (RP) mode of HPLC was examined for determination of PEG 400 oligomers. The normal-phase system provided greater resolution for oligomers of PEGs.     

Two-dimensional and serial column reversed-phase separation of phenolic antioxidants on octadecyl-, polyethyleneglycol-, and pentafluorophenylpropyl-silica columns

The separation selectivity of octadecyl-silica (C18) and of bonded pentafluorophenylpropyl-silica (F5) and PEG-silica columns was compared for natural phenolic antioxidants. The separation selectivities for phenolic antioxidants on C18 and F5 columns are strongly correlated, but low selectivity correlation indicating strong differences in the retention mechanism was observed between the C18 and PEG columns. Hence, the combination of a C18 and a PEG column is useful for separation of phenolic antioxidants that are not fully separated on single columns. Two-dimensional comprehensive liquid chromatography using a short PEG-silica column in the first dimension and a conventional C18-silica in the second dimension has the advantage of on-column focusing of the fractions transferred onto the C18 column in the second dimension, as a weaker mobile phase is used in the first dimension than in the second dimension. However, a stop-flow set-up in the first dimension system is necessary after the transfer of each fraction to the second dimension. Peak capacity is considerably larger but the separation time is much longer than with serially coupled PEG and C18 columns, which were employed for separation of beer and hop extract samples in connection with coulometric detection.     

On-column amperometric detection in capillary electrophoresis with an improved high-voltage electric field decoupler

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.     

Base sequence- and <Emphasis Type="Italic">T</Emphasis><Subscript>m</Subscript>-dependent DNA oligomer separation by open tubular capillary columns carrying complementary DNA oligomers as probes

DNA chips prepared on a flat glass surface have unavoidable drawbacks when used for quantitative analysis. In an attempt to overcome this problem, we constructed an HPLC-type system suitable for quantitative analysis that enables base sequence- and T _m-dependent DNA oligomer separation in a flow system. A small open tubular capillary column (300-mm × 100-μm I.D.) was used. The DNA oligomers used as probes had an amino group at the 5′-end and were immobilized on the inner silica surface of the capillary column which had been sequentially treated with 3-aminopropyltriethoxysilane, butyltrimethoxysilane, and disuccinimidylglutarate. Using the combination of probe-immobilized column placed in a column oven equipped with temperature gradient function, a nano-flow-controllable pump, a small sample-loading injector, and a capillary-fitted UV detector, we succeeded in separating complementary and non-complementary DNA oligomers in specific and quantitative modes. We also designed a temperature gradient strategy for efficient separation of target DNA oligomers in DNA mixture samples. Using a column carrying two different probes with similar T _m values, their complementary target DNA oligomers were also separated and detected. The developed DNA open tubular capillary column system investigated in the present study could be further improved as an alternative tool to DNA chips to be used for the quantitative analysis of DNA or mRNA samples. Kamakshaiah Charyulu Devarayapalli and Seung Pil Pack contributed equally to this paper.     

Determination of polyethylene glycol in low-density polyethylene by large volume injection temperature gradient packed capillary liquid chromatography

Polyethylene glycol (PEG) 20000 in low-density polyethylene has been determined using column switching and inverse temperature programming in reversed-phase packed capillary liquid chromatography with evaporative light scattering detection. PEG 20000 was extracted into water from the polyethylene dissolved in toluene and PEG 35000 was added as an internal standard (I.S.). The samples in aliquots of 100 microl were reconcentrated on the enrichment column using a loading mobile phase of acetonitrile-water (3:97, v/v) at a flow-rate of 75 microl/min for 3 min, then back-flushed and separated on the analytical column with acetonitrile-THF-water (40:5:55, v/v) as mobile phase. The column temperature was reduced from 68 to 55 degrees C with a ramp of -1.5 degrees C/min, held constant for 3 min and then reduced further to 45 degrees C with a -1.5 degrees C/min ramp and kept constant for 1 min. The analysis runtime was 20 min. The recovery of PEG 20 000 was determined to 65.1% with 2.8% RSD and the mass limit of detection of PEG 20 000 was 1.25 microg. The within-assay and between day precision of the retention times of both PEG 20000 and PEG 35000 displayed RSD of less than 1.1% (n = 9), while the overall area ratio RSD of 100 microg/ml PEG 20000 over PEG 35000 was 1.3% (n = 9). The method was linear within the investigated concentration range 25-125 microg/ml (R2 = 0.9983). In addition, a mixture of PEG 4000, 8000, 10000, 20000 and 35000 was analysed on the system to demonstrate the possibility of analysing several PEGs in a sample with the use of temperature gradient elution.     

Dual Fiber-In-capillary Annular Column with Ternary Stationary Phase for Gas Chromatographic Separation

In the present work a novel strategy for improving and/or tuning the selectivity of gas chromatographic (GC) separation by combining three different stationary phases (SPs) without premixing was introduced. A fused silica fiber coated with polydimethylsiloxane (SE30) and another coated with cyanopropylphenylmethylpolysiloxane (OV1701) were serially inserted into an 8-m polyethylene glycol 20 M (PEG20M) capillary column to form a GC annular column with ternary SP, abbreviating as SE30-OV1701-CF/PEG20M-CC. The separation capability of this ternary SP annular column was compared with a SE30-coated fiber-in-PEG20M-coated capillary annular column and a PEG20M-coated open tubular column by a test mixture of 19 organic compounds. Among these three columns, SE30-OV1701-CF/PEG20M-CC produced the best separation when the SE30-coated fiber and OV1701-coated fiber was 3 and 5 m, respectively. Selectivity can be easily tuned by changing the length of the SP-coated fibers in the ternary SP annular column. The proposed ternary SP annular column shows additional tunability, thus making it a promising tool for separation of organic solvents that are often used in the manufacturing process of pharmaceutical formulations and lacquer thinners.     

Analysis of linear and cyclic oligomers in polyamide-6 without sample preparation by liquid chromatography using the sandwich injection method. III. Separation mechanism and gradient optimization

The first six linear and cyclic oligomers of polyamide-6 can be quantitatively determined in the polymer using HPLC with the sandwich injection method and an aqueous acetonitrile gradient. In this final part of the triptych concerning the determination of the oligomers in polyamide-6, the irregular elution behavior of the cyclic monomer compared to the cyclic oligomers was investigated. We also optimized the separation of the involved polyamide oligomers, with respect to gradient steepness, stationary phase, column temperature and mobile phase pH. The irregular elution behavior of the cyclic monomer could be caused by its relatively large exposed/accessible hydrophobic surface, which permits relatively easy penetration into the hydrophobic stationary phase giving extra retention. The dipole moment of the different oligomers was used as a measure for this exposed/accessible hydrophobic area to correlate the retention factors using quantitative structure-retention relationships. We also studied the retention behavior of the polyamide, which is injected each run directly onto the column and modifies the stationary phase. Using a 250-microl post gradient injection zone of formic acid on a 250x3 mm Zorbax SB-C18 column, the polyamide could be effectively removed from the stationary phase after each separation. The linear solvent strength (LSS) model was used to optimize the separation of the first six linear and cyclic oligomers. As the LSS model assumes a linear correlation between the modifier concentration and the logarithm of the retention factor and the cyclic monomer and dimer show extreme curvation of this relation in the eluting region, we investigated different models to predict gradient elution from isocratic data. A direct translation of the isocratic data to gradient retention times did not yield adequate retention times using the LSS model. It was found that the LSS model worked acceptably if gradient retention times were used as input data. Even for fast non-linearly eluting components, an average error of 0.4 resolution units of 4sigma was obtained. Using the LSS model in combination with different column temperatures and mobile phase pH values, a separation of the first six linear and cyclic oligomers was accomplished.     

Unified capillary chromatography

The idea of unified capillary chromatography is described. Different-mode separations could be carried out by changing the column temperature and the pressure by using a single system. Gas chromatographic separation of aromatic hydrocarbons followed by supercritical fluid chromatographic separation of styrene oligomers was carried out in series in a single chromatographic run.     

The use of shift reagents in ion mobility-mass spectrometry: Studies on the complexation of an active pharmaceutical ingredient with polyethylene glycol excipients

Gas-phase ion mobility studies of mixtures containing polyethylene glycols (PEG) and an active pharmaceutical ingredient (API), lamivudine, have been carried out using electrospray ionization-ion mobility spectrometry-quadrupole-time-of-flight mass spectrometry (ESI-IMS-Q-TOF). In addition to protonated and cationized PEG oligomers, a series of high molecular weight ions were observed and identified as noncovalent complexes formed between lamivudine and PEG oligomers. The noncovalent complex ions were dissociated using collision induced dissociation (CID) after separation in the ion mobility drift tube to recover the protonated lamivudine free from interfering matrix ions and with a drift time associated with the precursor complex. The potential of PEG excipients to act as “shift reagents,” which enhance selectivity by moving the mass/mobility locus to an area of the spectrum away from interferences, is demonstrated for the analysis of lamivudine in a Combivir formulation containing PEG and lamivudine.     

Preparation of nanocrystalline porous titania films on titanium substrates by a sol–gel method with polyethylene glycol as a template

Porous nanocrystalline TiO₂ films have been prepared on cp-Ti substrates for biomedical usage by a sol–gel process from the solutions containing polyethylene glycol (PEG) as a template. Variations of the crystal structure with heat-treatment temperature determined by XRD are different for TiO₂ films and powders, due to the effect of titanium substrate. The surface texture of porous TiO₂ films is analyzed by means of SEM and found to greatly depend on the concentration and molecular weight of PEG. The pore formation mechanism is discussed in relation to the self-assembly of PEG and phase separation between PEG adsorbed on TiO₂ oligomers and ethanol.     

Multidimensional LC x LC analysis of phenolic and flavone natural antioxidants with UV-electrochemical coulometric and MS detection

A comprehensive 2-D LC x LC system was developed for the separation of phenolic and flavone antioxidants, using a PEG-silica column in the first dimension and a C(18) column with porous-shell particles or a monolithic column in the second dimension. Combination of PEG and C18 or C8 stationary phase chemistries provide low selectivity correlations between the first dimension and the second dimension separation systems. This was evidenced by large differences in structural contributions to the retention by -COOH, -OH and other substituents on the basic phenol or flavone structure. Superficially porous columns with fused core particles or monolithic columns improve the resolution and speed of second dimension separation in comparison to a fully porous particle C(18) column. Increased peak capacity and high orthogonality in different 2-D setups was achieved by using gradients with matching profiles running in parallel in the two dimensions over the whole 2-D separation time range. Multi-dimensional set-up combining the LC x LC separation on-line with UV and multi-channel coulometric detection and off-line with MS/MS technique allowed positive peak identification. The Coularray software compensates for the effects of the baseline drift during the gradient elution and is compatible with parallel gradient comprehensive LC x LC technique. Furthermore, it provides significant improvement in the sensitivity and selectivity of detection in comparison to both UV and MS detection. The utility of these systems has been demonstrated in the analysis of beer samples.     

HPLC-electrospray mass spectrometry for the analysis of temoporfin-poly(ethylene glycol) conjugates

The photodynamic therapeutic agent temoporfin, 5,10,15,20-tetra(m-hydroxyphenyl)chlorin (m-THPC) conjugated with poly(ethylene glycol) 2000 (PEG), has been analysed by high performance liquid chromatography (HPLC), linked to electrospray ionisation mass spectrometry (ESI-MS). Sufficient separation of m-THPC-PEG 2000 oligomers was achieved, enabling determination of molecular mass. The use of ESI-MS alone could not achieve this, because of too great a complexity in the mass spectrum, resulting from the presence of four PEG 2000 side chains with a wide molecular mass distribution. The technique is applicable to similar PEG conjugated compounds.     

硅胶整体柱的制备及制备条件对整体柱的影响

研究了硅胶整体柱的制备工艺及温度、pH对凝胶化过程的影响,探讨了不同的四甲氧基硅烷／聚乙二醇比例、不同制也剂浓度对柱结构的影响,以及不同焙烧温度和酸活化处理方法对柱体Si0H含量和线性收缩率的影响,由此确定最佳制备条件。结果表明,采用改进的溶胶-凝胶工艺制备的硅胶整体柱,无开裂变形现象;不同制备条件,可以产生具有不同微米级鞍形骨架、微米级大孔和纳米级中孔的整体柱,可适合不同物质的分离和分析。     

Quantitative comparison of a corona-charged aerosol detector and an evaporative light-scattering detector for the analysis of a synthetic polymer by supercritical fluid chromatography

A corona-charged aerosol detector (CAD) was developed to improve the sensitivity, reproducibility and quantitativeness of detection as compared to evaporative light-scattering detector (ELSD) for liquid chromatography. Our laboratory used the corona CAD as a detector for supercritical fluid chromatography (SFC) and evaluated its performance compared to the ELSD by using a certified reference material of poly(ethylene glycol) (PEG) and a well-defined equimass mixture of uniform PEG oligomers. The corona CAD was able to detect a 10 times more dilute solution of uniform oligomers compared to the ELSD. Although the original data of molecular mass by ELSD was 4.6% smaller than the certified value of PEG 1000, molecular mass distribution obtained by corona CAD was virtually almost the same as the certified value without any calibrations.     

Characterization and utilization of a novel triflate ionic liquid stationary phase for use in comprehensive two-dimensional gas chromatography

A novel triflate (trifluoromethylsulfonate) ionic liquid (IL) thin film (0.08 microm) stationary phase was implemented for use within the second column of a comprehensive GC x GC configuration. The first column in the configuration had a 5% phenyl/95% dimethyl polysiloxane (DMPS) stationary phase with a 0.4 microm film. The DMPS x IL column configuration was used to separate a mixture of 32 compounds of various chemical functional classes. The GC x GC results for the IL column were compared with a commercially available polar column (with a 0.1 microm PEG stationary phase film) used as the second column instead. Additional studies focused on the rapid and selective separation of four phosphorous-oxygen (P-O) containing compounds from the 32-compound matrix: dimethyl methylphosphonate (DMMP), diethyl methylphosphonate (DEMP), diisopropyl methylphosphonate (DIMP), and triethyl phosphate (TEP). van't Hoff plots (plots of ln k vs. 1/T) demonstrated the difference in retention between the P-O containing compounds (with DMMP reported in detail) and other classes of compounds (i. e., 2-pentanol and n-dodecane as representative) using either the IL column or the commercial PEG column. The selectivity (alpha) of the triflate IL column and the commercially available PEG column were also compared. The IL column provided significantly larger selectivities between DMMP and the other two compounds (2-pentanol and n-dodecane) than the commercial PEG column. The alpha for DMMP relative to n-dodecane was 3.0-fold greater for the triflate IL column, and the alpha for DMMP relative to 2-pentanol was 1.7-fold greater for the triflate IL column than for the PEG column.