Specific Sample Preparation Techniques Research Articles

Reflecting on the Influence of the Current State of Sample Preparation on GC, Part 1

Nearly all gas chromatographic analyses are preceded by sample preparation, which is often the major determiner of success. Recently, LCGC surveyed its readers about their work in sample preparation, covering the overall state of the field, and the results were presented in the “Sample Prep Perspectives” column. In this installment, we discuss the impact of part 1 of this survey, which covers broad questions about who does sample preparation, the fields they work in, and their most important aspects and goals for sample preparation on the gas chromatographic analysis that follows. We see that sample preparation has been (and remains) a critical step in any gas chromatographic method. In a second installment, we will discuss the impact of specific sample preparation techniques and their management on gas chromatography, following the release of part 2 of the sample preparation survey.

Development and validation of a dried plasma spot LC-MS/MS method for therapeutic monitoring of vancomycin and comparison with enzyme-multiplied immunoassay.

Vancomycin is used as an antimicrobial agent for the treatment of severe gram-positive infections. The importance of therapeutic monitoring of antimicrobials has led to the development of more specific sample preparation techniques capable of identifying with accuracy the concentration of this substance in the organism. An aliquot of 10μl of plasma was transferred to Whatman 903 paper and dried at room temperature. The extraction method was performed by cutting and transferring the paper to a microtube and adding sodium phosphate buffer and internal standard. The mixture was shaken and centrifuged, and a 5-μl aliquot was injected into the analytical system. The optimization of the main parameters that can influence the extraction efficiency was performed using multivariate approaches to obtain the best conditions. The method developed was validated, providing coefficients of determination higher than 0.994 and a lower limit of quantification of 1mg/L. Within- and between-run precision ranged from 11.4 to 17.30% and from 6.65 to 13.51%, respectively. This method was successfully applied to 75 samples of patients undergoing vancomycin therapy. The method was rapid, simple, and environmentally friendly with satisfactory analytical performance and was advantageous over the laborious and time-consuming methodologies used in therapeutic drug monitoring routine analyses.

An in-situ method for protecting internal cracks/pores from ion beam damage and reducing curtaining for TEM sample preparation using FIB

Focused ion beam (FIB) milling has evolved to be one of the most important Transmission Electron Microscope (TEM) site specific sample preparation techniques. However, this technique still poses challenges, such as the structural damage and potential curtaining issues often observed for thin TEM lamella. These artefacts can negatively affect the TEM analysis results. In particular, structures such as internal cracks and pores in FIB prepared TEM samples can often be damaged during sample preparation. This is commonly regarded as an unavoidable problem, even though microstructurally intact thin lamellae TEM samples are widely needed for the investigation of crack tips or pore morphologies in many different materials. This presents a strong driver for the development of innovative methods to overcome damage and curtaining issues during FIB sample preparation. Here we report on a new methodology developed to protect internal cracks and pores from ion beam damage. Our proposed method also mitigates curtaining issues, which often make TEM analysis more difficult. This method uses the FIB to sputter and redeposit material onto the edges of any cracks or pores in order to fill these features in-situ prior to lamella thinning. Case studies showcasing this method are presented, demonstrating the approach on a modular pure iron sample and on a porous laser treated Al/B4C composite sample. Our proposed ‘filling’ method has demonstrated a two key benefits; it preserves the integrity of the edges of any cracks and pores and it reducing curtaining. The results also demonstrate that this technique can be an alternative to conventional Gas Injection System (GIS) deposition for protecting the external top surface.

Radiocarbon Dating of Dolomitic Mortars from the Convent Saint John, Müstair (Switzerland): First Results

ABSTRACTThe monastery of St. John in Müstair, a UNESCO world heritage site, preserves archaeological remains and stone structures dated from the 8th century to the present. It has been extensively studied archaeologically so that numerous samples of historical materials, including mortar, are available for study. In addition to that, some of the structures have been precisely dated with dendrochronology. The monastery is located in a region characterized by dolomite rocks and the mortars are therefore of dolomitic nature, being perfectly suited to test the possibility of being dated with 14C. Furthermore, the presence of embedded carbon fragments has provided additional independent data to support or deny the results of mortar dating. A comparison of the results obtained from radiocarbon (14C) dating of bulk mortars, sieved fractions enriched in binder, lime lumps and carbon fragments, for two samples is presented, in relation to the petrographic characterization and the mineralogical phase content. This preliminary study shows that the dating of 14C can potentially be applied to the mortar of Müstair, as results in accordance with the established chronologies have been obtained for one sample. However, if the dolomitic sand contamination is very high, further studies are needed to develop a specific sample preparation technique.

Electron backscatter diffraction of Nb3Sn coated niobium: Revealing structure as a function of depth

Over the last two decades, advances in Electron Backscatter Diffraction (EBSD) have moved the technique from a research tool to an essential characterization technique in many fields of material research. EBSD is the best suited technique for determining structure as a function of depth. This characterization is critically important but has been previously absent from Nb3Sn efforts. While EBSD is the technique of choice, obtaining quality data can be difficult. Sample preparation in particular is nontrivial. Here, we summarize the general principles of EBSD, discuss specific sample preparation techniques for Nb3Sn coated SRF cavity materials, and give examples of how EBSD is being used to understand fundamental growth mechanisms for Nb3Sn coatings.

Adapted methods for scanning electron microscopy (SEM) in assessment of human sperm morphology.

Infertility is a widespread problem, and in some cases, the routine basic semen analysis is not sufficient to detect the cause of male infertility. The use of the scanning electron microscope (SEM) could provide a detailed insight into spermatozoa morphology, but it requires specific sample preparation techniques. The purpose of this study was to select, adjust, and optimize a method for the preparation of spermatozoa samples prior to SEM analysis, and to establish the protocol required for its use in clinical practice. We examined sperm samples of 50 men. The samples were fixed with modified iso-osmolar aldehyde solution followed by osmium post-fixation. In the first method, dehydration of the cells and subsequent critical point drying (CPD) were performed on a coverslip. In the second method, the samples were dehydrated in centrifuge tubes; hexamethyldisilazane (HMDS) was used as a drying agent instead of CPD, and the samples were air-dried. The third procedure was based on a membrane filter. The samples were dehydrated and dried with HMDS in a Gooch crucible, continuously, without centrifugation or redispersion of the sample. Our results showed that the fixation with modified iso-osmolar aldehyde solution followed by osmium post-fixation, and combined with dehydration and CPD on a coverslip, is the most convenient procedure for SEM sample preparation. In the case of small-size samples or low sperm concentration, dehydration and drying with HMDS on the membrane filter enabled the best reliability, repeatability, and comparability of the results. The presented procedures are suitable for routine use, and they can be applied to confirm as well as to correct a diagnosis.

Heating scans of stretched polystyrene films with stable baselines: Preparation method of samples for thermal analysis

Differential scanning calorimetry (DSC) examinations were carried out for stretched polystyrene (PS) films. A specific sample preparation technique was developed to overcome the entropic shrinkage, which makes DSC baselines unstable and divergent with each other. As a result, the data were acquired for stretched PS film with stable baselines overlayable below and above glass transition temperature shoulder. This method enables us to observe shoulder shift and evaluate the area bounded by two heat flow curves with and without both thermal ageing and stretching.

A single lysis solution for the analysis of tissue samples by different proteomic technologies

Cancer, being a major healthcare concern worldwide, is one of the main targets for the application of emerging proteomic technologies and these tools promise to revolutionize the way cancer will be diagnosed and treated in the near future. Today, as a result of the unprecedented advances that have taken place in molecular biology, cell biology and genomics there is a pressing need to accelerate the translation of basic discoveries into clinical applications. This need, compounded by mounting evidence that cellular model systems are unable to fully recapitulate all biological aspects of human dissease, is driving scientists to increasingly use clinically relevant samples for biomarker and target discovery. Tissues are heterogeneous and as a result optimization of sample preparation is critical for generating accurate, representative, and highly reproducible quantitative data. Although a large number of protocols for preparation of tissue lysates has been published, so far no single recipe is able to provide a “one-size fits all” solubilization procedure that can be used to analyse the same lysate using different proteomics technologies. Here we present evidence showing that cell lysis buffer 1 (CLB1), a lysis solution commercialized by Zeptosens [a division of Bayer (Schweiz) AG], provides excellent sample solubilization and very high 2D PAGE protein resolution both when using carrier ampholytes and immobilized pH gradient strips. Moreover, this buffer can also be used for array-based proteomics (reverse-phase lysate arrays or direct antibody arrays), allowing the direct comparison of qualitative and quantitative data yielded by these technologies when applied to the same samples. The usefulness of the CLB1 solution for gel-based proteomics was further established by 2D PAGE analysis of a number of technically demanding specimens such as breast carcinoma core needle biopsies and problematic tissues such as brain cortex, cerebellum, skeletal muscle, kidney cortex and tongue. This solution when combined with a specific sample preparation technique – cryostat sectioning of frozen specimens – simplifies tissue sample preparation and solves most of the difficulties associated with the integration of data generated by different proteomic technologies.

Hyphenation of capillary high-performance liquid chromatography to microcoil magnetic resonance spectroscopy—determination of various carotenoids in a small-sized spinach sample

The development of miniaturized hyphenated systems such as capillary high-performance liquid chromatography–and nuclear magnetic resonance spectroscopy (HPLC–NMR) remains challenging in the field of structure elucidation. In combination with a highly specific sample preparation technique, matrix solid-phase dispersion (MSPD), and a highly selective C 30 reverse phase HPLC–NMR enables the identification of small amounts of natural compounds. Here, the investigation of five carotenoids in a standard solution and two carotenoids from a spinach sample demonstrate the potential of this new development. The separation of the carotenoids is performed with self-packed fused-silica capillaries with a binary solvent gradient consisting of acetone and water. The miniaturized system allows the use of fully deuterated solvents for on-line HPLC–NMR coupling. The 1H NMR spectra of the various carotenoids obtained in stopped-flow mode gave a high signal-to-noise ratio with a sample amount in the low nanogram range. All necessary parameters for structure elucidation such as multiplet structure, coupling constants and integration values can be detected unambiguously.

A phase II study of navelbine ® (NVB) and cisplatin (CDDP) in advanced non-small cell lung cancer stage IIIb–IV (NSCLC): interim results

Cancer, being a major healthcare concern worldwide, is one of the main targets for the application of emerging proteomic technologies and these tools promise to revolutionize the way cancer will be diagnosed and treated in the near future. Today, as a result of the unprecedented advances that have taken place in molecular biology, cell biology and genomics there is a pressing need to accelerate the translation of basic discoveries into clinical applications. This need, compounded by mounting evidence that cellular model systems are unable to fully recapitulate all biological aspects of human dissease, is driving scientists to increasingly use clinically relevant samples for biomarker and target discovery. Tissues are heterogeneous and as a result optimization of sample preparation is critical for generating accurate, representative, and highly reproducible quantitative data. Although a large number of protocols for preparation of tissue lysates has been published, so far no single recipe is able to provide a “one-size fits all” solubilization procedure that can be used to analyse the same lysate using different proteomics technologies. Here we present evidence showing that cell lysis buffer 1 (CLB1), a lysis solution commercialized by Zeptosens [a division of Bayer (Schweiz) AG], provides excellent sample solubilization and very high 2D PAGE protein resolution both when using carrier ampholytes and immobilized pH gradient strips. Moreover, this buffer can also be used for array-based proteomics (reverse-phase lysate arrays or direct antibody arrays), allowing the direct comparison of qualitative and quantitative data yielded by these technologies when applied to the same samples. The usefulness of the CLB1 solution for gel-based proteomics was further established by 2D PAGE analysis of a number of technically demanding specimens such as breast carcinoma core needle biopsies and problematic tissues such as brain cortex, cerebellum, skeletal muscle, kidney cortex and tongue. This solution when combined with a specific sample preparation technique – cryostat sectioning of frozen specimens – simplifies tissue sample preparation and solves most of the difficulties associated with the integration of data generated by different proteomic technologies.

Some potential developments for trace element and surface analysis using a grazing incident X-ray beam

Total-reflection X-ray fluorescence (TXRF) spectrometry has proved to be a powerful tool in trace element and microanalysis. The performance in these fields might be increased further using higher brightness X-ray sources such as rotating anodes. Moreover, surface specific sample preparation techniques, such as electrochemical enrichment procedures, open up new applications. For pure surface analysis, the strengths and weaknesses of TXRF are discussed. In order to overcome shortcomings m lightweight (Z < 13) element and chemical state determination, it is proposed to replace the semiconductor by an appropriate wavelength dispersive or ß-detector. Initial attempts have been made to improve lateral resolution and depth profiling capacity by a particular combination of TXRF analysis and sputtering. A further important step would be the development of a standardized, computer guided data analysis procedure to evaluate roughness, elemental composition, layer thickness and density from fluorescence signals as a function of the incident X-ray angle.

Fourier transform ir spectroscopic study of the preparation of a carbon-coupled porphyrin catalyst structure

It has been demonstrated by means of Fourier Transform infrared spectroscopy, a specific sample preparation technique, and a background correction procedure, that the chemical coupling of tetra ( p-carboxyl)porphyrin to an oxidized carbon surface is accomplished via an ethylenediamide bridge.