Rapid Separation Technique Research Articles

Large scale Gravitational SPLITT Fractionation (GSF)에서의 시료 throughput에 관한 연구

SPLITT Fractionation (SF)는 콜로이드 입자 및 거대분자들을 둘 혹은 그 이상의 부분(fraction)으로 분획하는 기술이다. SF에서는 시료를 지속적으로 주입하므로 대용량 분획이 가능하다. 일반적으로, SF에서는 얇은 리본모양의 채널을 이용하는데, 채널의 입구와 출구부분에는 각각 flow stream splitter가 설치되어 있어서 채널의 입구와 출구가 위 아래로 두 개씩 존재한다. SF에는 두 가지 작동방법이 있는데, 하나는 conventional mode 이고 다른 하나는 전액 공급 모드(full feed mode, FFD)이다. FFD 모드에서의 분리도는 conventional mode 에 비해 떨어지지만, FFD 모드에는 몇 가지 독특한 장점이 있다. FFD 모드에서는 용매의 주입이 필요하지 않으므로 채널의 디자인 이나 작동이 더 간단하다. 따라서 입구 쪽에 flow stream splitter를 필요로 하지 않으며, 시료와 용매를 주입하기 위하여 두 개의 펌프가 필요한 conventional 모드와는 달리 한 개의 펌프만으로 작동이 가능하다. 또한 용매의 주입이 없으므로 시료가 희석되지 않는다. 이는 환경시료와 같이 콜로이드의 농도가 낮은 시료를 분획하고자 하는 경우 유리하다. 농도가 낮은 환경시료의 분획을 위해서는 종종 농축이 필요하다. 본 연구에서는 입구에는 물론 출구에도 splitter를 사용하지 않는 새로운 대용량 FFD 모드 SF 장치를 만들었다. Splitter가 없으므로 장치를 대형화 할 수 있어서 시료처리량(throughput, TP)을 크게 증가시킬 수 있었다. 산업용 폴리우레탄(polyurethane, PU) 라텍스 입자들을 이용하여 새로운 SF 장치를 테스트하였으며, 폴리아크릴레이트(polyacrylate, PA) 입자를 대상으로 새로운 SF 장치의 TP를 확인하기 위하여 유속 및 <TEX>$d_c$</TEX>에 따르는 TP의 변화를 조사하였다. Split-flow thin cell (SPLITT) fractionation (SF) is a rapid separation technique capable of separating colloidal particles or macromolecules into two or more fractions. SF allows fractionations in a preparative scale as sample is fed continuously. Generally SF uses a thin ribbon-like channel equipped with two flow stream splitters at the inlet and outlet of the channel. Thus there exist two flow inlets and two flow outlets at the top and bottom of the inlet and outlet of the channel, respectively. There are two operating modes in SF, conventional mode and full-feed mode (FFD). Although the resolution in the FFD mode is lower than that in the conventional mode, FFD mode has some merits. The design of the channel and operation are simpler in the FFD mode, as it does not require the feeding of the solvent. Thus there is no flow stream splitter at the channel inlet, and only one pump is needed, unlike the conventional mode, where two pumps are required for the feedings of the sample and the solvent separately. Also the sample is not diluted in the FFD mode as there is no solvent feeding, which is important for fractionation samples with low colloidal concentrations such as environmental samples. For some of environmental samples, pre-concentration is often required. In this study, a new large-scale splitter-less FFD-SF channel was implemented, where there is no splitter at the outlet as well as at the inlet of the channel. It was possible to build the channel in a much larger dimension than conventional ones, allowing much higher sample throughput (TP). The new channel was tested and optimized with polyurethane (PU) latex beads, and then applied to large-scale separation of Polyacrylate (PA).

Preparation of phytosterols and phytol from edible marine algae by microwave-assisted extraction and high-speed counter-current chromatography

A rapid method of microwave-assisted extraction coupled with high-speed counter current chromatography and UV detector was established for the preparation of phytosterols from edible marine algae. Extraction conditions, namely microwave power, liquid/solid ratio, irradiation time and extraction temperature were optimized using orthogonal array design. The microwave-assisted extraction of Undaria pinnatifida and Sargassum fusiforme were separated and purified with a non-aqueous two-phase solvent system composed of n-hexane–acetonitrile–methanol (5:5:3, v/v/v). The isolation was done in less than 220min, and a total of 13.0mg fucosterol, 1.5mg 24-methylenecholesterol and 10.7mg phytol were obtained from 15.0g U. pinnatifida; 4.6mg fucosterol, 0.3mg 24-methylenecholesterol and 3.5mg phytol were obtained from 15.0g S. fusiforme. The purities of all products were over 97% determined by high performance liquid chromatography. The results demonstrate that microwave-assisted extraction coupled with high-speed counter-current chromatography is a feasible, economical and efficient technique for rapid extraction, separation and purification of effective compounds from natural products.

Separation and purification of furanocoumarins from Toddalia asiatica (L.) Lam. using microwave‐assisted extraction coupled with high‐speed counter‐current chromatography

A method of microwave-assisted extraction coupled with high-speed counter-current chromatography was established for separation and purification of isopimpinellin, pimpinellin and phellopterin from Toddalia asiatica (L.) Lam. The conditions of MAE including the extraction solvent, size of sample, solid/liquid ratio, extraction temperature and extraction time were optimized by a mono-factor test. That is, 2.0 g dried powder of T. asiatica (L.) Lam of 0.30-0.15 mm size was extracted with 20 mL (solid/liquid ratio of 1:10, g/mL) methanol under 50 °C for 1 min. The crude extract was separated and purified by high-speed counter-current chromatography with hexane-ethyl acetate-methanol-water (5:5:5.5:4.5, v/v/v/v) solvent system. 0.85 mg/g of isopimpinellin, 2.55 mg/g of pimpinellin and 0.95 mg/g of phellopterin were obtained from original sample in one-step within 240 min, the purity determined by high performance liquid chromatography was 95.0%, 99.1% and 96.4%, respectively. Their chemical structures were further identified by mass spectroscopy and nuclear magnetic resonance spectroscopy. The results demonstrated that microwave-assisted extraction coupled with high-speed counter-current chromatography was a feasible, economical and efficient technique for rapid extraction, separation and purification of effective compounds from natural products.

Inertial lift enhanced phase partitioning for continuous microfluidic surface energy based sorting of particles

A new microfluidics technique that exploits the selectivity of phase partitioning and high-speed focusing capabilities of the inertial effects in flow was developed for continuous label-free sorting of particles and cells. Separations were accomplished by introducing particles at the interface of polyethylene glycol (PEG) and dextran (DEX) phases in rectangular high aspect-ratio microfluidic channels and allowing them to partition to energetically favorable locations within the PEG phase, DEX phase or interface at the center of the microchannel. Separation of partitioned particles was further enhanced via inertial lift forces that develop in high aspect-ratio microchannels that move particles to equilibrium positions close to the outer wall. Combining phase partitioning with inertial focusing ensures selectivity is possible using phase partitioning with sufficient throughput (at least an order of magnitude greater than phase partitioning alone) for application in the clinical and research setting. Using this system we accomplished separation of 15 μm polystyrene (PS) particles from 1-20 μm polymethylmethacrylate (PMMA) particles. Results confirm the feasibility of separation based on phase partitioning and enhancement of separation via inertial focusing. Approximately 86% of PS particles were isolated within the PEG phase whereas 78% of PMMA particles were isolated within the DEX phase. When a binary mixture of PS and PMMA was introduced within the device, ~83% of PS particles were isolated in the PEG phase and ~74% of PMMA particles were isolated in the DEX phase. These results confirm the feasibility of this technique for rapid and reliable separation of particles and potentially cells.

Assessment of soil organic carbon and total nitrogen under conservation management practices in the Central Claypan Region, Missouri, USA

Conservation management practices including upland vegetative filter strips (VFS) and no-till cultivation have the potential to enhance soil carbon sequestration and other ecosystem services in agroecosystems. A modified two-factor analysis of variance (ANOVA) with subsamples was used to compare SOC and TN on a concentration, soil volume and soil mass basis in claypan soils planted to different conservation management practices and as a function of landscape position. The three conservation management practices (no-till cultivation, grass VFS and agroforestry VFS) and four landscape positions (summit, shoulder, backslope and footslope) investigated were compared 10 years after VFS establishment in a no-till system planted to maize ( Zea mays. L.)–soybean ( Glycine max (L.) Merr.) rotation. Two soil depth increments (0–5 cm and 5–13 cm) were modeled separately to test for treatment effects. In the surface layer, mean SOC concentration was significantly greater in the VFS soils compared to no-till. On a soil volume or mass basis, no significant differences in SOC stocks were found among treatment means. Concentration and mass based TN values were significantly greater in the grass VFS relative to no-till in the surface layer. A rapid slaking stability test, developed to separate particulate, adsorbed and occluded organic carbon (PAO-C) and nitrogen (PAO-N), showed that VFS soils had significantly greater mean PAO-C and PAO-N concentrations, soil volume and soil mass based stocks than no-till. In addition, comparison of SOC:TN and PAO-C:PAO-N ratios suggest reduced decomposition and mineralization of SOC in the PAO fraction. No significant treatment effects were detected in total or PAO soil fractions in the subsurface layer or among landscape position in either depth increment. Study results emphasize the need to compare soil carbon and nitrogen stocks on a soil volume and/or soil mass basis using bulk density measurements. Additionally, the rapid PAO separation technique was found to be a good indicator of early changes in SOC and TN in the systems studied. Overall, this research indicates that grass VFS may sequester TN more rapidly than agroforestry VFS and that a greater proportion of SOC and TN may be stabilized in VFS soils compared to no-till.

Preparation of flavonoids and diarylheptanoid from Alpinia katsumadai hayata by microwave-assisted extraction and high-speed counter-current chromatography

In the present work, five flavonoids and diarylheptanoid, namely, alpinetin, pinocembrin chalcone, pinocembrin, cardamomin and 1,7-diphenyl-4,6-heptadien-3-one, were simply and rapidly prepared from Alpinia katsumadai hayata ( A. katsumadai) by microwave-assisted extraction (MAE) followed with high-speed counter-current chromatography (HSCCC). The critical conditions of MAE and HSCCC were optimized. 2.0 g crude sample was extracted with MAE, and then directly separated and purified by HSCCC with a two-phase solvent system composed of hexane–ethyl acetate–methanol–water (3:7:6:4, v/v/v/v), and 7.5 mg of alpinetin, 1.9 mg of pinocembrin chalcone, 2.6 mg of 1,7-diphenyl-4,6-heptadien-3-one with purities of 98.3%, 96.9% and 93.0% were obtained respectively in a single step within 4 h. 4.0 mg mixture of cardamomin and pinocembrin from HSCCC was further separated by prep-HPLC, 2.1 mg of 99.4% pinocembrin and 1.4 mg of 99.8% cardamomin were obtained respectively. The purities of compounds were determined by HPLC and their chemical structures were further identified by ESI-MS and 1H NMR. The results demonstrated that MAE coupled with HSCCC was a feasible, economical and efficient technique for rapid extraction, separation and purification of effective compounds in natural products.

Separation of trace amount of silver using dispersive liquid–liquid based on solidification of floating organic drop microextraction

In the present work, dispersive liquid–liquid microextraction based on solidification of floating organic drop was developed as a simple and rapid technique for separation of silver ions from aqueous samples. In this technique, 700 μL 0.02% of 5-(4′-dimethylamino benzyliden)-rhodanine (chelating agent) was added into the 10 mL analyte sample in a test tube and 30.0 μL 1-undecanol (extraction solvent) was injected shortly thereafter. The test tubes were sonicated, centrifuged and then some effective parameters on extraction and complex formation, such as type and volume of extraction and disperser solvent, pH, the amount of chelating agent and extraction time were optimized. The effect of the interfering ions on the analytes recovery was also investigated. The calibration graph was linear in the range of 0.10–10.0 ng mL −1 with detection limit of 0.056 ng mL −1 ( n = 8). The relative standard deviation (RSD) was±4.3% ( n = 8, C = 5.0 ng mL −1) and the enrichment factor was 250.0. The proposed method was applied for extraction and determination of silver in different water samples.

A rapid separation technique for overcoming suppression of triacylglycerols by phosphatidylcholine using MALDI-TOF MS

Phospholipids and triacylglycerols (TAGs) are important classes of lipids in biological systems. Rapid methods have been developed for their characterization in crude samples, including MALDI time-of-flight MS. For mixtures, MALDI often selectively shows only some components. For example, phosphatidylcholine (PC) suppresses detection of other lipids. Most rapid MS methods detect either TAGs or phospholipids but not both. Herein, we demonstrate a simple approach to rapidly screen mixtures containing multiple lipid classes. To validate this approach, reference lipids [PC, tripalmitin (PPP), and phosphatidyl-ethanolamine (PE)] and real samples (beef, egg yolk) were used. In a binary mixture with a strong suppressor (PC), PPP was greatly suppressed. After a simple separation, suppression was virtually eliminated. A mixture of nominally nonsuppressing lipids (PE and PPP) was not adversely affected by separation. Ground beef and egg yolk were used to demonstrate detection of known lipid compositions where other methods have missed one or more lipids or lipid classes. Separation was performed using solid phase extraction with a PrepSep florisil column. A 10 min separation allows rapid screening for lipids and changes in lipids. It is sufficient to clearly detect all lipids and overcome suppression effects in complex lipid mixtures.

Rapid separation of lanthanides and actinides on small particle based reverse phase supports

Abstract This paper presents the results on the use of short columns (3−5 cm long) with small particle size (1.8 μm) for high performance liquid chromatographic separation of individual lanthanides and uranium from plutonium as well as uranium from thorium to achieve rapid separations i.e. separation time as short as 3.6 min for individual lanthanides, 1 min for thorium-uranium and 4.2 min for uranium from plutonium. These advantages can be exploited to significantly reduce analysis time, liquid waste generation as well as dose to operator when radioactive samples are analysed e.g. burn-up determination. In the present work, a dynamic ion-exchange chromatographic separation technique was employed using camphor-10-sulfonic acid (CSA) as the ion-pairing reagent and α-hydroxy isobutyric acid (α-HIBA) as the complexing reagent for the isolation of individual lanthanides as well as the separation of uranium from thorium. Uranium was separated from Pu(III) as well as Pu(IV) by reverse phase HPLC technique. The reverse phase HPLC was also investigated for the isolation and quantitative determination of uranium from thorium as well as lanthanide group from uranium. The dynamic ion-exchange technique using small particle support was demonstrated for measuring the concentrations of lanthanide fission products such as La, Ce, Pr, Nd and Sm in the dissolver solution of fast reactor fuel. Similarly, the assay of uranium in the dissolver solution of fast reactor was carried out using reverse phase HPLC technique. The rapid separation technique using reverse phase HPLC was also demonstrated for separation of lanthanides as a group from uranium matrix; samples of LiCl-KCl eutectic salt containing chlorides of lanthanides in uranium matrix (typically 1:2000) were analysed.

Simultaneous concentration and separation of microorganisms: insulator-based dielectrophoretic approach

Microanalytical methods offer attractive characteristics for rapid microbial detection and concentration. There is a growing interest in the development of microscale separation techniques. Dielectrophoresis (DEP), a nondestructive electrokinetic transport mechanism, is a technique with great potential for microbe manipulation, since it can achieve concentration and separation in a single step. DEP is the movement of particles due to polarization effects in nonuniform electric fields. The majority of the work on dielectrophoretic manipulation of microbes has employed alternating current fields in arrays of microelectrodes, an approach with some disadvantages. An alternative is to employ insulator-based DEP (iDEP), a dielectrophoretic mode where nonuniform fields are produced by employing arrays of insulating structures. This study presents the concentration and fractionation of a mixture of bacteria and yeast cells employing direct current-iDEP in a microchannel containing an array of cylindrical insulating structures. Negative dielectrophoretic trapping of both types of microorganisms was demonstrated, where yeast cells exhibited a stronger response, opening the possibility for dielectrophoretic differentiation. Simultaneous concentration and fractionation of a mixture of both types of cells was carried out analogous to a chromatographic separation, where a dielectropherogram was obtained in less than 2 min by applying an electric field gradient and achieving concentration factors in the order of 50 and 37 times the inlet concentration for Escherichia coli and Saccharomyces cerevisiae cells, respectively. Encouraging results were also obtained employing a sample of water taken from a pond. The findings demonstrated the great potential of iDEP as a rapid and effective technique for intact microorganism concentration and separation.

Peak assignment in multi-capillary column–ion mobility spectrometry using comparative studies with gas chromatography–mass spectrometry for VOC analysis

Over the past years, ion mobility spectrometry (IMS) as a well established method within the fields of military and security has gained more and more interest for biological and medical applications. This highly sensitive and rapid separation technique was crucially enhanced by a multi-capillary column (MCC), pre-separation for complex samples. In order to unambiguously identify compounds in a complex sample, like breath, by IMS, a reference database is mandatory. To obtain a first set of reference data, 16 selected volatile organic substances were examined by MCC-IMS and comparatively analyzed by the standard technique for breath research, thermal desorption-gas chromatography-mass spectrometry. Experimentally determined MCC and GC retention times of these 16 compounds were aligned and their relation was expressed in a mathematical function. Using this function, a prognosis of the GC retention time can be given very precisely according to a recorded MCC retention time and vice versa. Thus, unknown MCC-IMS peaks from biological samples can be assigned-after alignment via the estimated GC retention time-to analytes identified by GC/MS from equivalent accomplished data. One example of applying the peak assignment strategy to a real breath sample is shown in detail.

Single Cell Analysis of Complex Thymus Stromal Cell Populations: Rapid Thymic Epithelia Preparation Characterizes Radiation Injury

Thymic epithelial cells (TECs) and dendritic cells are essential for the maintenance of thymopoiesis. Because these stromal elements define the progenitor niche, provide critical survival signals and growth factors, and direct positive and negative selection, detailed study of these populations is necessary to understand important elements for thymic renewal after cytotoxic injury. Study of TEC is currently hindered by lengthy enzymatic separation techniques with decreased viability. We present a new rapid separation technique that yields consistent viable TEC numbers in a quarter of the prior preparation time. Using this new procedure, we identify changes in stroma populations following total body irradiation (TBI). By flow cytometry, we show that TBI significantly depletes UEA+ medullary TEC, while sparing Ly51+ CD45- cells. Further characterization of the Ly51+ subset reveals enrichment of fibroblasts (CD45- Ly51+ MHCII-), while cortical TECs (CD45- Ly51+ MHCII+) were markedly reduced. Dendritic cells (CD11lc+ CD45+) were also decreased following TBI. These data suggest that cytotoxic preparative regimens may impair thymic renewal by reducing critical populations of cortical and medullary TEC, and that such thymic damage can be assessed by this new rapid separation technique, thereby providing a means of assessing optimal conditioning pretransplantfor enhancing thymic-dependent immune reconstitution posttranspiant.

A simple and rapid technique for radiochemical separation of iodine radionuclides from irradiated tellurium using an activated charcoal column

A simple and inexpensive method for the separation of medically useful no-carrier-added (nca) iodine radionuclides from bulk amounts of irradiated tellurium dioxide (TeO 2) target was developed. The β − emitting 131I radionuclide, produced by the decay of 131Te through the natTe(n, γ) 131Te nuclear reaction, was used for standardization of the radiochemical separation procedure. The radiochemical separation was performed by precipitation followed by column (activated charcoal) chromatography. Quantitative post-irradiation recovery of the TeO 2 target material (98–99%), in a form suitable for reuse in future irradiations, was achieved. The overall radiochemical yield for the complete separation of 131I was 75–85% ( n=8). The separated nca 131I was of high, ∼99%, radionuclidic and radiochemical purities and did not contain detectable amounts of the target material. This method can be adopted for the radiochemical separation of other different iodine radionuclides produced from tellurium matrices through cyclotron as well as reactor irradiation.

Dielectrophoresis of Functionalized Lipid Unilamellar Vesicles (Liposomes) with Contrasting Surface Constructs

Dielectrophoresis (DEP) has been exceedingly exploited recently as an effective technique for rapid manipulation, separation, and assembly of biological particles including cells, proteins, and DNAs. However, optimizing of the DEP process with biocolloidal suspensions remains challenging, mainly due to inadequate understanding of AC polarization of complex biological structures. In this paper, we examine the DEP behavior of model functionalized lipid unilamellar vesicles (liposomes) with distinct exterior surface properties and compare the measured crossover frequency, omegac, with the theoretical prediction based on the Maxwell-Wagner interfacial polarization mechanism for a shell structure. With a uniform shell coating of calcium phosphate, we observe a drastic decrease in omegac compared to the measured value for plain liposome particles. In sharp contrast, with the patchy surface created by the adsorption of oppositely charged nanoparticles on liposome outer surfaces, we observe that omegac is independent of nanoparticle surface coverage, despite the considerable change in liposome surface conductivity by increasing adsorbed nanoparticles. Our results indicate the particle construct, rather than surface conductivity, plays a critical role in the DEP behavior of a shell particle, which is remarkably different from a solid dielectric particle.

Ultrasound Assisted Engineering of Lactose Crystals

To engineer lactose crystals of desired size, shape, surface and particle size distribution (PSD) as a carrier for dry powder inhalers (DPI) by ultrasound assisted in-situ seeding. Lactose crystals were obtained from solution by ultrasound assisted in-situ seeding, followed by growth in viscous glycerin solution. The crystals were characterized for physical properties and 63-90 mum size fractions of different batches were mixed with salbutamol sulphate (SS) and compared for in-vitro deposition. Cooling crystallization with stirring for 10-20 h resulted in crystals with wide PSD and varied shape. Application of ultrasound resulted in rapid and complete crystallization in 5 min with rod-shaped fine crystals (15-30 microm) and narrow PSD. In-situ seeded batches yielded micro-fine rod-shaped seed crystals. Seeding followed by growth in glycerin showed desirable size, high elongation ratio, smooth surface and narrow PSD, while growth under stirring showed high elongation ratio with rough surface. Crystals grown in glycerin showed highest dispersibility and fine particle fraction (FPF) of SS. Ultrasound assisted in-situ seeding, followed by ordered growth in glycerin offers rapid technique for separation of nuclei induction from crystal growth yielding desirable characteristics for better dispersion and in-vitro deposition when employed as DPI carrier.

A simple and rapid method for the determination of taxol produced by fungal endophytes from medicinal plants using high performance thin layer chromatography.

Taxol is an important anticancer drug used widely in the clinical field. In this study, some endophytic fungi were isolated from selected medicinal plants, and were screened for their potential in the production of taxol, using a rapid separation technique of high performance thin layer chromatography (HPTLC). Of the 20 screened fungi, only 13 fungal species produced taxol in the artificial culture medium. The results of HPTLC showed that the 13 fungal species had identical ultraviolet (UV) characteristics, positive reactivity with a spray reagent, yielding a blue spot, which turned to dark gray after 24 hours, and had Rf values identical to that of the authentic taxol. The amount of taxol was also quantified by comparing the peak area and the peak height of the fungal samples with those of authentic taxol.

High‐Performance Liquid and Ion Chromatography: Separation and Quantification Analytical Techniques for Rare Earth Elements

High‐performance liquid chromatography (HPLC) and ion chromatography (HPIC) have been widely used as cheap, relatively rapid, precise and accurate separation and quantification analytical techniques for the rare earth elements (REE). In this “Back to Basics” review, a general description of the basic components of instrumentation and the most common detectors is presented, with special attention to separation conditions and chromatographic quality parameters for the REEs. Also included is a brief survey of the literature on the use of HPLC and HPIC in the determination of REEs in synthetic solutions as well as other matrices such as nuclear fuels and geochemical reference materials. For a proper evaluation of HPLC and HPIC applications, and use of these techniques by other researchers it is recommended that parameters such as the amount and uncertainty estimates of an element injected, eluents and post‐column reagents and information on linear or complex gradients and detector system set‐up, should always be reported or relevant literature references provided. Finally, a brief account is also presented on state‐of‐the‐art liquid chromatography known as ultra high‐ or extremely high‐performance liquid chromatography, which has high potential for a diversity of applications in all scientific and engineering fields including the Earth Sciences.

Applications of field‐flow fractionation in proteomics: Presence and future

Field-flow fractionation (FFF) represents a group of elution separation methods where external force fields act perpendicularly on analytes in a carrier liquid flows with nonuniform velocity profiles. It is an elution separation method that enables to separate analytes in relatively short times and collect fractions for further characterization or for investigation of their properties. Other advantages of FFF are small consumption of samples and gentle experimental conditions. These make FFF uniquely qualified for separation and purification of biological samples. The most promising are applications of different variants of flow FFF utilizing a cross flow through membrane channel walls to separate proteins. The separation is based on differences in protein diffusion coefficients, which allows calculating the size of macromolecules. Other FFF techniques (e.g., electrical, isoelectric, and sedimentation FFF) were also used for separation of biomolecules. FFF appears to be not only promising rapid technique for protein separation but it offers some other advantages in sample preparation, especially, focusing (hyperlayer) FFF techniques that enable preparation of homogeneous fractions of cells. Selected applications of FFF to protein analysis are described and future trends in application of FFF to proteomics are discussed.

Evaluating the utility of ion mobility separation in combination with high‐pressure liquid chromatography/mass spectrometry to facilitate detection of trace impurities in formulated drug products

Many formulated products contain complex polymeric excipients such as polyethylene glycols (PEGs). Such excipients can be readily ionized by electrospray and may be present at very high concentrations, thus making it very difficult to identify trace level impurities such as degradants in samples, even if hyphenated techniques such as liquid chromatography/mass spectrometry (LC/MS) are used. Ion mobility (IM) spectrometry is a very rapid gas-phase separation technique and offers additional separation capability within the LC timeframe. This work investigates the use of an IM separator in combination with high-pressure liquid chromatography (HPLC) and MS, to improve the separation of drug-related materials from excipients, thus aiding the identification of trace-level impurities in an anti-HIV medication, Combivir.

Characterization of polyether mixtures using thin‐layer chromatography and matrix‐assisted laser desorption/ionization mass spectrometry

Thin-layer chromatography (TLC) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) were combined to achieve characterization of polyether mixtures. Three polyethers, polyethylene glycol (PEG), polypropylene glycol (PPG) and polytetramethylene glycol (PTMG), or mixtures of these compounds, were studied. One shortcoming of mixture analysis of synthetic polymers using MALDI-MS is that individual polymers in the mixture may display different detection sensitivities. For example, the MALDI mass spectrum of an equimolar mixture of PEG, PPG and PTMG displayed a high intensity of PPG ions, while no PTMG ions were detectable; however, PTMG ions were detected after the mixture had been separated by TLC. This combined TLC and MALDI-MS analysis of a PPG polymer bearing reactive epoxy groups showed that the polymer contained byproducts with different end-groups. These byproducts were identified as chloro-substituted polymers formed during polymer synthesis. Our study shows TLC to be a rapid and low-cost separation technique, and that it can be combined with MALDI-MS to achieve effective analysis of synthetic polymers.