White Paper: Important Aspects of Sample Preparation of Biological Materials
From cell disruption to homogenization and pulverization of a great variety of biological samples
Biological samples exist in all shapes and sizes: hard bones, tough and fibrous plants, tough and viscous sputum, soft muscles, tumor or liver tissue. Not to mention the millions of cells such as yeast, bacteria or algae, which have to be disrupted for applications such as DNA or RNA isolation or protein extraction. For research in genomics, transcriptomics or metabolomics, all kinds of biological samples are prepared. Sample preparation is the first step of every analytical process. Retsch offers a range of mills and grinders for easy and reproducible pulverization of solid sample materials some of which are also suitable for cell disruption and homogenization of biological sample materials.
White Paper: Cryogenic Preparation of Sample Materials
A solid sample material should always be sufficiently prepared by size
reduction and homogenization before it is subjected to chemical or physical analysis. Care should be taken that the analysis sample fully represents the original material and that the sample preparation process is carried out reproducibly. Only then are meaningful results guaranteed. Most sample materials can be reduced to the required analytical fineness at room temperature by choosing a mill with a suitable size reduction principle (impact, pressure, friction, shearing, cutting).
White Paper: How to minimize standard deviations by correct sample preparation
Modern analytical methods increase precision and push detection limits to make even the smallest traces of sample components detectable. Despite this development sample preparation, which is carried out prior to the actual analysis, is frequently neglected. Errors caused by lacking accuracy in sample preparation have a much bigger impact than errors made during analysis. Just like an iceberg which is mostly hidden under water, only a small part of the sum of errors is perceived whereas the major part of potential errors is not taken into account (fig. 1). One of the reasons may be the fact that sampling and sample preparation have always been done in a traditional way which has become a routine over the years and is no longer considered as having a critical influence on the subsequent analyses.
White Paper: Ultrafine Grinding with Laboratory Ball Mills
How are nano particles produced? The “Bottom-Up” method synthesizes particles from atoms or molecules. The “Top-Down” method involves reducing the size of larger particles to nanoscale, for example with laboratory mills. Due to their significantly enlarged surface in relation to the volume, small particles are drawn to each other by their electrostatic charges. Nano particles are produced by colloidal grinding which involves dispersion of the particles in liquid to neutralize the surface charges. Both water and alcohol can be used as dispersion medium, depending on the sample material. Factors such as energy input and size reduction principle make ball mills the best choice for the production of nanoparticles.
Il rilevamento di droghe e farmaci illegali è importante in settori quali la scienza forense, il traffico o competizioni sportive. Sostanze chimiche si possono riscontrare nel sangue, nella saliva, nelle urine e nel capello, quest’ultimo possiede il vantaggio di memorizzare le sostanze per un periodo più o meno lungo, a seconda della sua lunghezza. Oltre al rilevamento delle droghe, i capelli sono utilizzati anche per le analisi del DNA.
Per la riduzione di dimensioni di molti materiali è consigliabile utilizzare un mulino criogenico invece di un mulino che opera a temperature ambiente. Il campione viene infragilito con l'azoto liquido che migliora il suo comportamento di rottura quando viene sottoposto a impatto, pressione e attrito. Inoltre, in questo modo vengono conservati i componenti volatili del campione. Il mulino Cryomill non solo è il mulino criogenico più sicuro e moderno sul mercato ma fornisce anche eccellenti risultati di macinazione.
Por lo general se asocia a la noción "fragmentación", el machacamiento, de sustancias sólidas mediante fuerza mecánica. Pero también la división de líquidos en gotas o de gases en bollas representa un proceso de fragmentación.
Los más precisos resultados de trituración en el más breve tiempo se consiguen con los nuevos molinos planetarios de bolas, de Retsch. Con el PM100 y el PM200, Retsch presenta una nueva generación de molinos planetarios de bolas con uno o dos unidades de trituración, que establecen nuevos estándares de rendimiento en este segmento de la producción.
El nuevo molino ultracentrífugo ZM 200 de RETSCH es un molino de rotor no sólo extremadamente rápido y cuidadoso con el material molido, sino también de uso universal gracias a su amplia gama de accesorios.
Die meisten Probenmaterialien lassen sich durch die Wahl des geeigneten Zerkleinerungsgerätes problemlos mahlen. Die Beanspruchungsmechanismen wie Prall, Druck, Scherung, Schneiden, Reibung reichen bei Raumtemperatur aus, um das Material auf die benötigte Partikelgröße zu zerkleinern. Was aber kann man tun, wenn die mechanische Beanspruchung allein nicht ausreicht, um das Probenmaterial in möglichst kleine Partikel zu überführen? Eine Lösung dieser Problematik bietet der Einsatz von Flüssigstickstoff, der das Bruchverhalten solcher Materialien begünstigt.
Das Michael Rout Labor, welches an der New Yorker Rockefeller Universität beheimatet ist, nahm erstmals 2006 Kontakt zu RETSCH in den USA auf, um die Möglichkeit der kryogenen Vermahlung von Hefezellen mit einer Planeten-Kugelmühle zu diskutieren. Das Ziel ihrer Untersuchungen war die Erforschung der „Bauweise“ von Komplexen, welche an den Zellwänden von Hefezellen zu finden sind. Für den Einsatz einer Planeten-Kugelmühle sprach in erster Linie, dass sehr feine Partikelgrößen erreicht werden können, was eine wichtige Voraussetzung für fundierte Analysen an den Hefezellen ist. Eine Partikelgröße im Submikron-Bereich begünstigt eine hohe Ausbeute für die nachfolgende Proteinreinigung.
Die Anwendung stabilisotopenanalytischer Methoden als Werkzeug zur Aufklärung von unklaren Tatbeständen hat sich in der Forensik etabliert. Am Institut für Rechtsmedizin der Universität München gehen Anfragen zu Erstellung von Isotopengutachten aus dem In- und Ausland ein. Der Großteil der Isotopengutachten beantwortet die Frage nach der geogra-phischen Herkunft und den Aufenthaltsorten unbekannter Toter. Die Ergebnisse der Analyse der Stabilisotopenhäufigkeitsverhältnisse von Bio- und Geoelementen in Körpergeweben lie-fern Ansatzpunkte für polizeiliche Ermittlungen. Die Grundidee der geographischen Her-kunftszuordnung von Personen mittels Isotopenanalyse liegt in der Beobachtung, dass über Ernährung und Umwelt unterschiedliche Isotopenverhältnisse chemischer Elemente in den Körper eingebaut werden, die eine regionale Komponente aufweisen (Tabelle). Menschliche Gewebe zeigen dann ebenso bestimmte Isotopensignaturen, über die sich Rückschlüsse auf die Aufenthaltsorte und die Ernährungsweise des Konsumenten ziehen lassen.
Sometimes the preparation and homogenization of biological samples can be as tough as the material itself. The widely used 2 ml single-use Eppendorf tubes are often not large enough to accommodate the whole sample volume; hence, the sample needs to be divided and reunited after the homogenization process which means an additional time-consuming working step in the lab routine. While it is true that usually larger sized grinding jars, e. g. of stainless steel, are available which accommodate the complete sample volume, these have the drawback of requiring cleaning after use.
Use of laboratory grinders for size reduction of human bones and bioceramics
Bone implants and substances for bone regeneration are used in surgery to replace degenerated bone material by implants or to “re-build” it with specific substances. The material used in implants varies from autogeneic (supplied by the patient) through allogeneic (supplied by a donor) bones to replacement materials such as hydroxylapatite (HA) and tricalcium phosphate (TCP). Bovine bones and corals are used in conjunction with synthetically produced foamed materials to form a basis for the regeneration of bone substance. Various RETSCH mills are suitable for the preliminary and fine grinding of human bones as well as bioceramic materials.
A faultless and comparable analysis is closely linked to an accurate sample handling. Only a sample representative of the initial material can provide meaningful analysis results. Rotating dividers and rotary tube dividers are an important means to ensure the representativeness of a sample and thus the reproducibility of the analysis. Correct sample handling consequently minimizes the probability of a production stop due to incorrect analysis results. Thus correct sample handling is the key to effective quality control.
Some sample materials have properties which make size reduction at ambient temperature impossible. If, for example, very elastic materials need to be ground or volatile components have to be preserved for further analysis, it is essential to carry out cryogenic grinding. The use of liquid nitrogen helps to embrittle the sample, thus improving its breaking properties, and preventing volatile substances from escaping due to the frictional heat produced by the grinding process.
The detection of illegal drugs and pharmaceuticals plays a role in various fields, for example in forensic science, road traffic accidents, in competitive sports or at the workplace. Chemical substances can be detected in blood, saliva, urine and in hair. Hair has the great advantage of storing the substances for a long period, which means that detection is still possible several months after consumption of the drug. In addition to the detection of drugs, hair samples are also used for DNA analysis as well as for the analysis of heavy metals and minerals.
Cell disruption of bacteria, yeast, filamentous fungi or microalgae is a standard procedure in basic biological research, applied biotechnology or medical research to get access to nucleic acids (DNA, RNA) or cell proteins. For the isolation of DNA or RNA usually less than 1 ml of cell material is needed. For the extraction of proteins, however, larger amounts of cell suspension are required. A very efficient method of cell disruption is the co called “bead beating” where cells in suspension are mechanically disrupted by glass beads in single-use reaction vials.
Cashmere wool is the best known precious wool. It is won from the cashmere goat which originates from the high mountain region of the same name. Due to its properties such as softness and warmth, cashmere wool gains more and more popularity in the manufacture of clothing. Genuine cashmere is won solely from the goat’s downy hair and must possess a certain hair structure with an exactly defined length and thickness.
The following situation is typical for many production plants: After a routine quality check, the production process is stopped or an already produced batch is suspended, because the analysis results were not within the relevant critical values. But does the tested product really deviate from the specifications? The quality control managers are convinced of this because modern analysis instruments provide results with very low tolerances. The sample in question was tested several times and the result was confirmed. The question is why the product does not match the specifications although the production parameters have not been changed in any way. The possibility that the tested product is indeed deficient cannot be excluded. However, it is often not the product itself which causes irregular analysis results but a lack of understanding of the steps which come before the analysis.
Cryogenic disruption of yeast cells - Rockefeller University, USA