Thin-Layer Chromatography
TLC is a widely-used chromatography technique used to separate chemical compounds. It involves a stationary phase consisting of a thin layer of adsorbent material, usually silica gel, aluminium oxide, or cellulose immobilised onto a flat, inert carrier sheet.
A liquid phase consisting of the solution to be separated dissolved in an appropriate solvent is drawn through the plate via capillary action, separating the experimental solution.
It can be used to determine the pigments a plant contains, to detect pesticides or insecticides in food, in forensics to analyze the dye composition of fibers, or to identify compounds present in a given substance, among other uses. It is a quick, generic method for organic reaction monitoring.
TLC Analysis
As the chemicals being separated may be colorless, several methods exist to visualize the spots.
Often a small amount of a fluorescent compound, usually Manganese-activated Zinc Silicate, is added to the adsorbent that allows the visualization of spots under a blacklight (UV254).
The adsorbent layer will thus fluoresce light green by itself, but spots of analyte quench this fluorescence. Iodine vapors are a general unspecific color reagent Specific color reagents exist into which the TLC plate is dipped or which are sprayed onto the plate.
Once visible, the Rf value of each spot can be determined by dividing the distance traveled by the product by the total distance traveled by the solvent (the solvent front). These values depend on the solvent used, and the type of TLC plate, and are not physical constants.
Gas-Liquid Chromatography
Gas-liquid chromatography (GLC), or simply gas chromatography (GC), 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.
The instrument used to perform gas chromatographic separations is called a gas chromatograph (also: aerograph, gas separator).
Spectroscopy
Spectroscopy is the study of the interaction between radiation (electromagnetic radiation, or light, as well as particle radiation) and matter. Spectroscopy is often used in physical and analytical chemistry for the identification of substances through the spectrum emitted from or absorbed by them.
Atomic Absorption Spectroscopy
Atomic absorption spectroscopy is a technique for determining the concentration of a particular metal element in a sample. Atomic absorption spectroscopy can be used to analyse the concentration of over 62 different metals in a solution.
Three steps are involved in turning a liquid sample into an atomic gas:
Desolvation – the liquid solvent is evaporated, and the dry sample remains
Vaporisation – the solid sample vaporises to a gas
Volatilisation – the compounds making up the sample are broken into free atoms
A beam of light passes through this flame at its longest axis (the lateral axis) and hits a detector.
The light that is focused into the flame is produced by a hollow cathode lamp. Inside the lamp is a cylindrical metal cathode containing the metal for excitation, and an anode.
When a high voltage is applied across the anode and cathode, the metal atoms in the cathode are excited into producing light with a certain emission spectra. This amount of energy is specific to a particular electron transition in a particular element.
As the quantity of energy put into the flame is known, and the quantity remaining at the other side (at the detector) can be measured, it is possible to calculate how many of these transitions took place, and thus get a signal that is proportional to the concentration of the element being measured.
DNA & protein eletrophoresis
Gel electrophoresis is the separation of deoxyribonucleic acid, ribonucleic acid, and protein through an electric charge. It is usually performed for analytical purposes, but may be used as a preparative technique to partially purify molecules prior to use of other methods such as mass spectrometry, PCR, cloning, DNA sequencing, or immuno-blotting for further characterization.
"Electrophoresis", refers to the electromotive force (EMF) that is used to push or pull the molecules through the gel matrix; by placing the molecules in wells in the gel and applying an electric current, the molecules will move through the matrix at different rates, towards the anode if negatively charged or towards the cathode if positively charged.
After the electrophoresis runs, when the smallest molecules have almost reached the anode, the molecules in the gel can be stained to make them visible. Ethidium bromide, silver, or coomassie blue dye can be used. Other methods can also be used to visualize the separation of the mixture's components on the gel. If the analyte molecules fluoresce under ultraviolet light, a photograph can be taken of the gel under ultraviolet light. If the molecules to be separated contain radioactive atoms, an autoradiogram can be recorded of the gel.
