Fluorescence spectroscopy is a type of electromagnetic spectroscopy that analyzes fluorescence from a sample. It involves using a beam of light, usually ultraviolet light, that excites the electrons in molecules of certain compounds and causes them to emit light; typically, but not necessarily, visible light. A complementary technique is absorption spectroscopy. In the special case of single molecule fluorescence spectroscopy, intensity fluctuations from the emitted light are measured from either single fluorophores, or pairs of fluorophores.
Ninhydrin (2,2-dihydroxyindane-1,3-dione) is an organic compound with the formula C6H4(CO)2C(OH)2. It is used to detect ammonia and amines. Upon reaction with these amines, ninhydrin gets converted into deep blue or purple derivatives, which are called Ruhemann's purple. Ninhydrin is most commonly used to detect fingerprints in forensic cases, as the terminal amines of lysine residues in peptides and proteins sloughed off in fingerprints react with ninhydrin.
Laser-induced fluorescence (LIF) or laser-stimulated fluorescence (LSF) is a spectroscopic method in which an atom or molecule is excited to a higher energy level by the absorption of laser light followed by spontaneous emission of light. It was first reported by Zare and coworkers in 1968.
Bis-tris propane, or 1,3-bis(tris methylamino)propane, also known as BTP, is a chemical substance that is used in buffer solutions. It is a white to off-white crystalline powder that is soluble in water. It has a wide buffering range, from 6 to 9.5 due to its two pKa values which are close in value. This buffer is primarily used in biochemistry and molecular biology.
In analytical chemistry, a chiral derivatizing agent (CDA), also known as a chiral resolving reagent, is a derivatization reagent that is a chiral auxiliary used to convert a mixture of enantiomers into diastereomers in order to analyze the quantities of each enantiomer present and determine the optical purity of a sample. Analysis can be conducted by spectroscopy or by chromatography. Some analytical techniques such as HPLC and NMR, in their most commons forms, cannot distinguish enantiomers within a sample, but can distinguish diastereomers. Therefore, converting a mixture of enantiomers to a corresponding mixture of diastereomers can allow analysis. The use of chiral derivatizing agents has declined with the popularization of chiral HPLC. Besides analysis, chiral derivatization is also used for chiral resolution, the actual physical separation of the enantiomers.
Reversed-phase liquid chromatography (RP-LC) is a mode of liquid chromatography in which non-polar stationary phase and polar mobile phases are used for the separation of organic compounds. The vast majority of separations and analyses using high-performance liquid chromatography (HPLC) in recent years are done using the reversed phase mode. In the reversed phase mode, the sample components are retained in the system the more hydrophobic they are.
Tetrasodium tris(bathophenanthroline disulfonate)ruthenium(II) (Na4Ru(bps)3) is a sodium salt of coordination compound. In this form, it is the salt of a sulfonic acid. This compound is an extension of the phenanthroline series of coordination compounds. Ruthenium(II) tris(bathophenanthroline disulfonate), referring to the anionic fragment, is used as a protein dye in biochemistry for differentiating and detecting different proteins in laboratory settings.
Phthalaldehyde (sometimes also o-phthalaldehyde or ortho-phthalaldehyde, OPA) is the chemical compound with the formula C6H4(CHO)2. It is one of three isomers of benzene dicarbaldehyde, related to phthalic acid. This pale yellow solid is a building block in the synthesis of heterocyclic compounds and a reagent in the analysis of amino acids. OPA dissolves in water solution at pH < 11.5. Its solutions degrade upon UV illumination and exposure to air.
Fluorogenic describes a property of chemical compounds which are initially not fluorescent, but become fluorescent through a chemical reaction, typically through an intermolecular covalent reaction which binds the now fluorescent compound to a target molecule. IUPAC uses a broader definition of fluorogenic, wherein a enhancement of fluorescence via a chemical reaction is not required, however in contrast to the IUPAC definition common use of fluorogenic does not refer to non-reaction effects like the enhancement of fluorescence from a fluorophore being in different solvents. Fluorogenic labeling reagents are often used in analytical chemistry procedures, particularly in HPLC or CE to derivative target compounds, thereby allowing enhanced sensitivity through fluorescence based detection.
Folin's reagent or sodium 1,2-naphthoquinone-4-sulfonate is a chemical reagent used as a derivatizing agent to measure levels of amines and amino acids. The reagent reacts with them in alkaline solution to produce a fluorescent material that can be easily detected.
Dansyl chloride or 5-(dimethylamino)naphthalene-1-sulfonyl chloride is a reagent that reacts with primary amino groups in both aliphatic and aromatic amines to produce stable blue- or blue-green–fluorescent sulfonamide adducts. It can also be made to react with secondary amines. Dansyl chloride is widely used to modify amino acids; specifically, protein sequencing and amino acid analysis. Dansyl chloride may also be denoted DNSC. Likewise, a similar derivative, dansyl amide is known as DNSA.
Dansyl amide is a fluorescent dye that forms in a reaction between dansyl chloride and ammonia. It is the simplest representative of the class of dansyl derivatized amines, which are widely used in biochemistry and chemistry as fluorescent labels. The dansyl amide moiety is also called a dansyl group, and it can be introduced into amino acids or other amines in a reaction with dansyl chloride. The dansyl group is highly fluorescent, and it has a very high Stokes shift. The excitation maximum is essentially independent on the medium, whereas the emission maximum strongly depends on the solvent and varies from 520 to 550 nm.
Fluorescamine is a spiro compound that is not fluorescent itself, but reacts with primary amines to form highly fluorescent products, i.e. it is fluorogenic. It hence has been used as a reagent for the detection of amines and peptides. 1-100 µg of protein and down to 10 pg of protein can be detected. Once bound to protein the excitation wavelength is 381 nm and the emission wavelength is 470 nm (blue). This method is found to suffer from high blanks resulting from a high rate of hydrolysis due to requiring a large excess concentration. Alternative methods are based on ortho-phthalaldehyde (OPA), Ellman's reagent (DTNB), or epicocconone.
A chromatography detector is a device that detects and quantifies separated compounds as they elute from the chromatographic column. These detectors are integral to various chromatographic techniques, such as gas chromatography, liquid chromatography, and high-performance liquid chromatography, and supercritical fluid chromatography among others. The main function of a chromatography detector is to translate the physical or chemical properties of the analyte molecules into measurable signal, typically electrical signal, that can be displayed as a function of time in a graphical presentation, called a chromatograms. Chromatograms can provide valuable information about the composition and concentration of the components in the sample.
Chiral analysis refers to the quantification of component enantiomers of racemic drug substances or pharmaceutical compounds. Other synonyms commonly used include enantiomer analysis, enantiomeric analysis, and enantioselective analysis. Chiral analysis includes all analytical procedures focused on the characterization of the properties of chiral drugs. Chiral analysis is usually performed with chiral separation methods where the enantiomers are separated on an analytical scale and simultaneously assayed for each enantiomer.
3-(2-Furoyl)-quinoline-2-carboxaldehyde (FQ) is a fluorogenic amine labeling dye that is not fluorescent itself, but reacts with primary amines to form fluorescent products. It was first reported in 1990. Cyanide, typically provided via KCN or NaCN salts, is a required co-substrate in the fluorogenic reaction. It has been used for the detection of amines and peptides, largely in CE-SDS, where it is recognized to reach a silver stain-like high sensitivity via laser-induced fluorescence. Once bound to protein the excitation wavelength is 480 nm (blue) and the emission wavelength is ~600 nm (orange).
6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) is a fluorogenic, amine labeling dye that is not fluorescent itself, but covalently reacts with secondary amines to form a fluorescently labeled product. It has a fluorescence excitation wavelength of 250 nm (UV-C), and emission wavelength of 395 nm.
3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) is a fluorogenic amine labeling dye that is not fluorescent itself, but covalently reacts with primary amines to form fluorescent products. It was first reported in 1991. Today, it is largely used in the context of quantifying peptides or proteins. Either cyanide or thiols are required as a co-substrate in the fluorogenic reaction, although thiols also react with & mask the CBQCA aldehyde thereby preventing the fluorogenic reaction against the targeted primary amines. Once bound to protein the excitation wavelength is 465 nm (blue) and the emission wavelength is ~550 nm (green).
The colloidal gold protein assay is a highly sensitive biochemical assay for determining the total concentration of protein in a solution. It was first described in 1987 by two groups who used commercially available "Aurodye" colloidal gold solutions. Notably, the formulation of Aurodye changed between 1987 and 1990 such that it became incompatible with protein assays, however vendors such as Bio-Rad & Diversified Biotech starting offering colloidal gold formulations that were suitable for protein assays. These products have since been discontinued and there are no vendors that currently explicitly sell colloidal gold for the assay, however detailed synthetic procedures were published to produce the ~17-40 nm gold nanoparticles that are suitable for the assay, along with modifications to increase the shelf stability of the colloidal gold & adapt the assay to microplate format & increase its sensitivity. Gold nanoparticles in the ~17-40 nm size range that are presumably compatible with the assay are currently commercially available.
Pyrylium-1 (Py-1) is a fluorogenic salt of a pyrylium derivative and tetrafluoroborate. It is an amine-labeling dye that is not fluorescent itself, but reacts with primary amines to form fluorescent products. It is within the "chameleon labels" class, so named due to their clear color-changing properties upon conjugation. Py-1 was first reported in 2004. It has been used for the detection of amines and peptides, largely in CE-SDS, where it is recognized to reach a high sensitivity via laser-induced fluorescence. Once bound to protein the excitation wavelength is 503 nm (green) and the emission wavelength is 603 nm (orange). Similar to FQ, these fluorescence wavelengths makes Py-1 suitable for excitation with a 488 nm argon-ion laser.
