Source: Laboratory of Dr. Paul Bower - Purdue University
The method of standard additions is a quantitative analysis method, which is often used when the sample of interest has multiple components that result in matrix effects, where the additional components may either reduce or enhance the analyte absorbance signal. That results in significant errors in the analysis results.
Standard additions are commonly used to eliminate matrix effects from a measurement, since it is assumed that the matrix affects all of the solutions equally. Additionally, it is used to correct for the chemical phase separations performed in the extraction process.
The method is performed by reading the experimental (in this case fluorescent) intensity of the unknown solution and then by measuring the intensity of the unknown with varying amounts of known standard added. The data are plotted as fluorescence intensity vs. the amount of the standard added (the unknown itself, with no standard added, is plotted ON the y-axis). The least squares line intersects the x-axis at the negative of the concentration of the unknown, as shown in Figure 1.
1Department of Neurology, McKnight Brain Institute, University of Florida, 2Department of Entomology and Nematology, University of Florida, 3Genetics Institute, Department of Molecular Genetics and Microbiology, University of Florida, 4McKnight Brain Institute, Department of Neuroscience, Genetics Institute, Center for Translational Research on Neurodegenerative Diseases, and Center for Movement Disorders and Neurorestoration, University of Florida
Source: Laboratory of Dr. B. Jill Venton - University of Virginia
Sample preparation is the way in which a sample is treated to prepare for analysis. Careful sample preparation is critical in analytical chemistry to accurately generate either a standard or unknown sample for a chemical measurement. Errors in analytical chemistry methods are categorized as random or systematic. Random errors are errors due to change and are often due to noise in instrument. Systematic errors are due to investigator or instrumental bias, which introduces an offset in the measured value. Errors in sample preparation are systematic errors, which will propagate through analysis, causing uncertainty or inaccuracies through improper calibration curves. Systematic errors can be eliminated through correct sample preparation and proper use of the instrument. Poor sample preparation can also sometimes cause harm to the instrument.…
1Breast Surgery Group, Division of Medical Sciences and Graduate Entry Medicine, School of Medicine, Queen's Medical Centre, University of Nottingham, 2Medical Microbiology and Immunology Department, Faculty of Medicine, Mansoura University, 3Nottingham Digestive Diseases Centre and NIHR Nottingham Digestive Diseases Biomedical Research Centre, School of Medicine, Queen's Medical Centre, University of Nottingham
Immunology and Infection
1Department of Pathology, Hershey Medical Center, Pennsylvania State University, 2Department of Chemistry, Pennsylvania State University, 3Departments of Pathology, and Biochemistry and Molecular Biology, Hershey Medical Center, Pennsylvania State University, 4Materials Research Institute, Pennsylvania State University
Source: Laboratory of Dr. Neal Abrams — SUNY College of Environmental Science and Forestry
Determining the composition of a solution is an important analytical and forensic technique. When solutions are made with water, they are referred to as being aqueous, or containing water. The primary component of a solution is referred to as the solvent, and the dissolved minor component is called the solute. The solute is dissolved in the solvent to make a solution. Water is the most common solvent in everyday life, as well as nearly all biological systems. In chemistry labs, the solvent may be another liquid, like acetone, ether, or an alcohol. The solute can be a liquid or a solid, but this experiment only addressesthe determination of solids.…
Source: Laboratory of Dr. B. Jill Venton - University of Virginia
Gas chromatography (GC) is used to separate and detect small molecular weight compounds in the gas phase. The sample is either a gas or a liquid that is vaporized in the injection port. Typically, the compounds analyzed are less than 1,000 Da, because it is difficult to vaporize larger compounds. GC is popular for environmental monitoring and industrial applications because it is very reliable and can be run nearly continuously. GC is typically used in applications where small, volatile molecules are detected and with non-aqueous solutions. Liquid chromatography is more popular for measurements in aqueous samples and can be used to study larger molecules, because the molecules do not need to vaporize. GC is favored for nonpolar molecules while LC is more common for separating polar analytes.
The mobile phase for gas chromatography is a carrier gas, typically helium because of its low molecular weight and being chemically inert. Pressure is applied and the mobile phase moves the analyte through the column. The separation is accomplished using a column coated with a stationary phase. Open tubular capillary columns are the most popular columns and have the stationary phase coated on the walls of the capillary. Stationary phases a…
Two-dimensional gel electrophoresis (2DGE) is a technique that can resolve thousands of biomolecules from a mixture. This technique involves two distinct separation methods that have been coupled together: isoelectric focusing (IEF) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). This physically separates compounds across two axes of a gel by their isoelectric points (an electrochemical property) and their molecular weights.
The procedure in this video covers the main concepts of 2DGE and a general procedure for characterizing the composition of a complex protein solution. Three examples of this technique are shown in the applications section, including biomarker detection for disease initiation and progress, monitoring treatment in patients, and the study of proteins following posttranslational modification (PTM).
Two-dimensional, or 2D, gel electrophoresis is a technique utilizing two distinct separation methods which can separate thousands of proteins from a single mixture. One of the techniques, SDS-PAGE or sodium dodecyl sulfate polyacrylamide gel electrophoresis, cannot fully separate complex mixtures alone. 2D gel electrophoresis couples the SDS-PAGE to a second method, isoelectric focusing or IEF, which separates based on isoelectric points, allowing for the resolution of potentially a…
1NMR Surgical Laboratory, Department of Surgery, Massachusetts General Hospital, Harvard Medical School, 2Shriners Burn Institute, 3Department of Radiology, Athinoula A. Martinos Center of Biomedical Imaging, Harvard Medical School, 4Molecular Surgery Laboratory, Department of Surgery, Massachusetts General Hospital, Harvard Medical School