The uncertaintyMANAGER facilities the user to calculate the measurement uncertainty for HPLC/LC-MS, GC/GC-MS and IC/IC-MS including various steps from sample preparation. Because we have developed for all these analytical techniques an extensive library with individually adopted sample preparation steps.
HPLC/LC-MS, GC/GC-MS and IC/IC-MS are like all other measurements a relative method. i.e. the signal of the sample is compared with the signal of a reference, whereas both measurements, sample and reference, are performed with the same HPLC/LC-MS, GC/GC-MS or IC/IC-MS in the own laboratory. This arrangement has direct effects on the strategy of measuring and on the calculation of the measurement uncertainty.
HPLC/LC-MS s a form of column chromatography used frequently in biochemistry and analytical chemistry. HPLC/LC-MS is used to separate components of a mixture by using a variety of chemical interactions between the substance being analyzed and the chromatography column. It is one of the most important analytical techniques, among other it is used for production control in pharmaceutical industry.
The sample to be analyzed is introduced in small volume to the stream of mobile phase and is retarded by specific chemical or physical interactions with the stationary phase as it traverses the length of the column. The amount of retardation depends on the nature of the analyte, stationary phase and mobile phase composition. The time at which a specific analyte elutes is called the retention time and is considered a reasonably unique identifying characteristic of a given analyte. The use of pressure increases the linear velocity giving the components less time to diffuse within the column, leading to improved resolution in the resulting chromatogram. Common solvents used include any miscible combinations of water or various organic liquids.
A further refinement to HPLC/LC-MS has been to vary the mobile phase composition during the analysis, this is known as gradient elution. A normal gradient for reversed phase chromatography might start at 5 % methanol and progress linearly to 50 % methanol over 25 minutes, depending on how hydrophobic the analyte is. The gradient separates the analyte mixtures as a function of the affinity of the analyte for the current mobile phase composition relative to the stationary phase. This partitioning process is similar to that which occurs during a liquid-liquid extraction but is continuous, not step-wise. The choice of solvents, additives and gradient depend on the nature of the stationary phase and the analyte.
GC/GC-MS is a type of chromatography in which the mobile phase is a carrier gas, usually an inert gas such as helium or an unreactive gas such as nitrogen, and the stationary phase is a microscopic layer of liquid or polymer on an inert solid support, inside glass or metal tubing, called a column. Gas Chromatographs are different from other forms of chromatography (HPLC/LC-MS, IC/IC-MS, etc.) because the solutions travel through the column in a gas state. The interactions of these gaseous analytes with the walls of the column, which are coated by different stationary phases depending on the various needs, causes different compounds to elute at different times called retention time. The comparsion of these retention times is the analytical power to GC. This makes it very similar to HPLC/LC-MS.
In a GC/GC-MS analysis, a known volume of gaseous or liquid analyte is injected into the head of the column, usually using a microsyringe (or, solid phase microextraction fibers, or a gas source switching system). As the carrier gas sweeps the analyte molecules through the column, this motion is inhibited by the adsorption of the analyte molecules either onto the column walls or onto packing materials in the column. The rate at which the molecules progress along the column depends on the strength of adsorption, which in turn depends on the type of molecule and on the stationary phase materials. Since each type of molecule has a different rate of progression, the various components of the analyte mixture are separated as they progress along the column and reach the end of the column at different retention times. A detector is used to monitor the outlet stream from the column; thus, the time at which each component reaches the outlet and the amount of that component can be determined. Generally, substances are identified qualitatively by the order in which they elute from the column and by the retention time of the analyte in the column.
Ion-exchange chromatography (IC/IC-MS) is a process that allows the separation of ions and polar molecules based on the charge properties of the molecules. It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids. Therefore it is often used in protein purification, water analysis, and quality control.
Ion exchange chromatography (IC/IC-MS) retains analyte molecules based on coulombic interactions. The stationary phase surface displays ionic functional groups (R-X) that interact with analyte ions of opposite charge. This type of chromatography is further subdivided into cation exchange chromatography and anion exchange chromatography. The ionic compound consists of the cationic species or of the anionic species, which are retained by the stationary phase.
A sample is introduced, either manually or with an autosampler, into a sample loop of known volume. A buffered aqueous solution known as the mobile phase carries the sample from the loop onto a column that contains some form of stationary phase material. This is typically a resin or gel matrix consisting of agarose or cellulose beads with covalently bonded charged functional groups. The analyte, either anions or cations, are retained on the stationary phase but can be eluted by increasing the concentration of a similarly charged species that will displace the analyte ions from the stationary phase. For example, in cation exchange chromatography, the positively charged analyte could be displaced by the addition of positively charged sodium ions. The analyte is then detected using typically conductivity or UV/Visible light absorbance.
A special module for the simulation of peak overlap permits the user to estimate its size and then its effect onto the measurement uncertainty. The user performs this simulation using the information from the chromatograms.
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