HPLC
HPLC (High Performance Liquid Chromatography) is an analytical technique which separates a complex mixture of components into its specific individual components. It is a powerful tool in analysis, as it combines high speed with extreme sensitivity compared to traditional methods of chromatography because of the use of a pump which creates a high pressure and forces the mobile phase to move with the analyte in high speed. It is been used as a principle technology in various automated analyzers used for diagnostic purpose.
Principle:
HPLC technology works on the principle of conventional chromatography where in there is a stationary phase and a mobile phase. The sample containing the mixture of components is introduced in a column packed
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Mobile phase:
The choice of mobile phase depends on the chemical nature of the compound of interest and could be purely organic, inorganic or a mixture of both in gradient. Most commonly used mobile phases are organic solvents like acetonitrile or methanol. Some HPLC analysis require the use of water free solvents as mobile phase and in such cases acids like formic acid, phosphoric acid, trifluoroacetic or salts which will assist in separation of components in the sample are used.
Stationary phase:
Stationary phase is of extreme importance in an HPLC analysis, as the chemical nature of the same and its compatibility with the analyte of interest is extremely significant for efficient separation. The most commonly used stationary phase is silica packed column which acts as a adsorbent. Each component in the sample interacts with these silica particles and gets eluted out in different time intervals. These silica columns may be of C14 or C18 type depending on the component of interest and also the columns themselves come in various dimensions each with a specific purpose of analysis.
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The working begins with an auto-sampler which picks up a definite amount of sample as programmed from the defined tube and passes in to the pump wherein it is mixed with the mobile phase in definite proportion. This is followed by entry of the mixture into the column under high pressure which aids in separation. The individual analytes in the mixture will interact with the stationary phase and finally be eluted out at a definite retention time. The retention time of each eluting component is recorded and based on this data the output is displayed in the graphical format. Peaks are seen on the graph and each peak corresponds to a particular component in the mixture, while the area under the curve of the peak denotes the concentration of the analyte. The higher the number of peaks, more is the number of analytes present in the
In order to separate the mixture of fluorene, o-toluic acid, and 1, 4-dibromobenzene, the previously learned techniques of extraction and crystallization are needed to perform the experiment. First, 10.0 mL of diethyl ether would be added to the mixture in a centrifuge tube (1) and shaken until the mixture completely dissolved (2). Diethyl ether is the best solvent for dissolving the mixture, because though it is a polar molecule, its ethyl groups make it a nonpolar solvent. The compounds, fluorene and 1, 4-dibromobenzene, are also nonpolar; therefore, it would be easier for it to be dissolved in this organic solvent.
electrophoresis. The way the PCR method works is by first mixing a solution containing the
The dietary goal for total fat intake is 20 to 35% of total calories. How did your fat intake compare to the stated goal? (6 pts.)
Furthermore, an additional method to use other hydrochloric acids that have different concentration levels such as 1 M and 2.5 M ones, can improve the outcome of the results. This increases the variation of the independent variable, which accordingly increases the precision of results.
...lications in the future. This is due to the fact that this method has become rough, not complicated and it can be performed in a conventional way without being mandatory the investigation into depth for every application (Tetala and van Beek, 2010). New forms are going to be operated in order to recognize bacteria and also aptamers are going to be used more often. Moreover, the investigation of new types of monoliths will also include the study of present or alterative types of polymers, in order to come out with a wider range of pore sizes, surface areas and new morphologies that can be used in this type of affinity chromatography (Pfaunmiller et al., 2013). Finally, monolithic stationary phases are expected to have a great impact on future applications, for instance if organic monolithic supports will be combined with hybrids of silica (Pfaunmiller et al., 2013).
Paper chromatography is the ability to separate specific parts of a mixture, in order to identify its content. There are many forms of chromatography, but paper chromatography tends to work with substances such as dyes, inks, or any colored chemical. In the fields of biology, paper chromatography benefits police that need to test blood. It can also be used by chemists to test substances in their labs. Lastly, it can be used to identify compounds that may be in a plant substance.
developing chambers and place the lid on. c. Mark each chromatography strip 1cm from each end with a pencil. d. Using the capillary tube, apply three drops of the chlorophyll
In exercise 1, the photosynthetic pigments in spinach leaf extract will be separated using chromatography paper. Chromatography is a technique used by scientist to separate substances from a mixture in order to determine what that mixture is made up of. A small sample of the mixture will be added to the chromatography paper and put in a solvent tank so the bottom of the paper makes contact with the solvent. The chromatography paper has capillaries so once the
A gas chromatography (GC) is used for a quantitative analysis to separate mixtures with low melting point. Essentially a gas chromatography analysis use a thermal conductivity (TC) detector that will produce an electronic signal whose voltage is proportional to the amount of each component. Each component will reach the detector at different time. The components separate according to the polarity and volatility. Some components will move faster than others. The GC will print out a graph which plots the changes in voltage as a function of time.
Given that the shapes of the peaks are not ideal, they are different from the percentage height for each component. As such, the area measurement is used, to determine the concentration. For the first peak, the percent area of total was 49.65% and for the second peak, the percent area of the total was 50.35%, which reflects the quantity of the analyte that exists in the chromatogram. The retention time in both cases is constant and the pattern of peaks is constant which reflects that the conditions of testing are constant. Based on the percentages it can be seen that the major product was 2-isopropyl-1,4-dimethylbenzene at 50.35% and the minor product was 1,4-Dimethyl-2-propylbenzene at 49.65%. Therefore, on Figures 2 and 3 the peak at 2.93 is the major peak and it represents 2-isopropyl-1,4-dimethylbenzene and the peak at 2.58 is the minor peak and represents
Components of a solution with polarity similar to that of the solvent used in chromatography will move further across the adsorbant used, while components with different polarity will move less because they are not as dissolved by the developing liquid. With thin layer chromatography, a TLC plate is prepared coated with an adsorbant. Samples are dotted at the bottom of the plate on a line called the origin, and then are set in a consciously chosen devloping liquid in a jar so that the adsorbent picks up the liquid and moves up the plate. As the liquid moves and the plate develops, different components are dissolved or not dissolved in each sample dot, and the ones that are more dissolved move up the plate. The location where the solvent stops when the plate is removed from the developing jar is called the solvent front.
The solvent used was acetone, as indicated by the multiplet at around 2.05 ppm. A strong signal resembling a multiplet at around 0 ppm suggests that tetramethylsilane was used as the standard for 0 ppm. Traces of the starting materials, such as maleic acid, are present, as suggested by the singlet at around 7.3 ppm with an integration of 0.03. Additionally, there are traces of 3-sulfolene, indicated by the signal at 6.1 ppm with an integration of 0.09, and another signal at 3.72 ppm with an integration of 0.09.
Cut up a handful of leaves (reasonably small handful) with the scissors and place in the mortar.
... of the column (Wilson & Walker, 2010). Factors that influence the separation and rate of elution include polarity of the solvent. With increasing polarity of the mobile phase, the substrate travels at a faster rate. Another factor that influences the separation include the substrate interaction with stationary phase in which the stronger the interaction, the slower the movement of the substance (Totah, 2011). The major advantage of column chromatography is the ability to separate mixtures at a large volume however it would require a long period of time. Besides Thin layer chromatography and column chromatography, other separation techniques such as gas chromatography, paper chromatography, high pressure liquid chromatography, ion exchange chromatography, gel permeation chromatography, hydrophobic interaction chromatography and affinity chromatography are also used.
It is where it uses biomolecules from organisms such as enzymes, antibodies, nucleic acid and a cell as a whole. It is design to interact with the specific analyte of interest to produce an effect measurable by the transducer.