INTRODUCTION:

Reagents are substances used for the detection, identification, analysis, etc. of chemical, biological, or pathologic processes or conditions. Indicators are substances that change in physical appearance, e.g., colour, at or approaching the endpoint of a chemical titration, e.g., on the passage between acidity and alkalinity. Reagents are substances used for the detection or determination of another substance by chemical or microscopical means, especially analysis. Types of reagents are precipitants, solvents, oxidizers, reducers, fluxes, and colorimetric reagents. Reagent is a “substance or compound that is added to a system in order to bring about a chemical reaction, or added to see if a reaction occurs.” In organic chemistry, reagents are compounds or mixtures, usually composed of inorganic or small organic molecules that are used to effect a transformation on an organic substrate. Examples of organic reagents include the Collins reagent, Fenton’s reagent, and Grignard reagent. There are also analytical reagents which are used to confirm the presence of another substance.

Received on 28.11.2014 Modified on 11.12.2014

Research J. Pharm. and Tech. 8(2): Feb. 2015; Page 110-117

DOI: 10.5958/0974-360X.2015.00020.7

Advantages²:

Analytical reagents are very cheaper to use

Time consuming is less

More reliable

Compatible with most of drugs

No harmful reactions occurs

Easy of preparation

List of Reagents:

1. Bratton-Marshall Reagent²:

It is chemically N-1-naphthyl ethylene diamine dihydrochloride. It is widely used for the determination of drugs and pharmaceuticals containing free primary aromatic amino group. At present, it is employed for the determination of sulpha drugs and local anaesthetics.

Synonyms:

Monomethanolate

Bratton’s reagent

Marshall’s reagent

Bratton-Marshall reagent

Chemical Name:

N-(1-Naphthyl) ethylenediamine dihydrochloride

Molecular Formula: C₁₂H₁₆Cl₂N₂

Formula weight: 259.17

Structure:

Mechanism of action:

The primary aromatic amino group is first diazotized with sodium nitrite and hydrochloric acid. The excess nitrous acid (HNO₂) is neutralized by treating with ammonium sulphamate reagent. Finally, the diazonium ion is allowed to couple with BM reagent to produce a highly colored azodye complex measured at 550 nm.

Applications:

BM reagent is used to estimate the sulpha drugs like sulphanilamide, sulphamethoxazole.

· Estimation of antipsychoticseg:ziprosidone

· Estimation of antiamoebics eg: Metronidazole

2. 2, 6-Dichloroquinone-4-chloroimide^2,1

Synonyms

· 2,6-dichloro-p-benzoquinone-4-chloroimine

· N,2,6-trichloroquinoneimine

· N,2,6-trichlorobenzoquinoneimine

· Gibbs reagent

IUPAC Name:

2,6-Dichloro-4-(chloroimino)cyclohexa-2,5-dienone

Molecular formula: C₆H₂Cl₃NO

Appearance: Yellow crystals

Structure:

Use:

In determination of phenols, for detection of antioxidants, primary and secondary aliphatic amines, secondary and tertiary aromatic amines, aromatic hydrocarbons, pharmaceuticals, phenoxyaceticacid herbicides, etc.

Principle and Mechanism of action:

· When phenolic compounds reacts with Gibbs reagent, coupling reaction may occur

· The first step of reaction is formation of the corresponding quinine imines

· Quinone imines are condensation products of quinine chlorimines with phenols in aqueous alkaline media

· Imides portion of Gibbs reagent reacts with phenolic compounds gives corresponding products coupling with nucleophilic sites by the elimination of chlorine.

3. Dimedone^2,4

Dimedone is a cyclic diketone used in organic chemistry to determine whether a compound contains an aldehyde group

Synonym:

Cyclomethone

5,5-dimethyl-1,3-cyclohexanedione

Dimethyldihydroresorcinol

Methone

Systematic Name: 5,5-Dimethyl-1,3-cyclohexanedione

Molecular Formula: C₈H₁₂O₂

Structure:

Synthesis:

Dimedone is prepared from Mesityloxide and Diethylmalonate

Physical properties:

Dimedone usually comes in the form of white crystals. It is stable under ambient conditions and soluble in water, as well as ethanol and methanol. It has a melting point range of 147-150°C (420-423 K)

Applications:

Cyclohexanedionescan be applicable for the industry of

· Transition-metal complex catalyst chemistry

· Luminescence chemistry and spectrophotometric analysis

· Organic synthesis

· Crystallography and Crystal Chemistry

· Organic low electrical resistance chemistry

· Colorimetry

4, 4-(Dimethylamino) Cinnamaldehyde⁵

DMACA is an histological dye used to detect indole production in cells. It is used for the rapid identification of bacteria containing tryptophanase enzyme systems. It is also particularly useful for localization of proanthocyanidins compound in plants resulting in a blue staining. It has been used for grapevine fruit legumes foliage histology.

Synonyms: p-Dimethylaminocinnamaldehyde

DMACA Reagent

4-Dimethylcinnamaldehyde

Chemical Name: 4-(Dimethylamino) cinnamaldehyde

Molecular Formula: C₁₁H₁₃NO

Formula weight: 175.23

Structure:

Principle:

Certain amines condense with various aldehydes in strongly acidic media to give products that are oxidisable to give color. Among many, the following aldehydes are widely used. They are p-dimethylaminobenzaldehyde, vanillin, formaldehyde etc.

Mechanism of action:

The mechanism of aldehydes which condenses with the aromatic amines involves the condensation of the aldehydes to release the oxygen molecule and then it combines with the amine group to form the Schiff’s base in the presence of acidic medium such as HCl or H₂SO₄.

5. Deniges’ Reagent²

The Deniges’ reagent was developed in 1898 by G.Deniges. Is a reagent used for qualitative analysis.

Structure:

Use:

Deniges’ reagent is used to detectolefin or tertiary alcohols which can be easily dehydrated to form is oolefin in the presence of acid. Treatment of solutions containing either is olefin or tertiary alcohols with this reagent will result in the formation of a solid yellow or red precipitate.

6. Emmerie-Engel Reagent⁴

Chemical Formula: 2,2’-bipyridine-iron(III)chloride reagent

Molecular Formula: C₃₀H₂₄Cl₂FeN₆

Molecular Weight: 595.2

Structure:

Preparation of the Reagent:

(In the presence of Orthophosphoric acid)

Solution I: Dissolve 0.1g iron(III)chloride hexhydrate in 50ml ethanol

Solution II: Dissolve 0.25g 2,2’-bipyridine(αα dipyridyl) in 50ml ethanol

Mix equal quantities of solutions I and II immediately before use.

Storage:

Solution I should be stored in the dark.

Principle:

Drugs containing groups like phenols, vitamins, Hydroxymic esters, secondary amines and phenolic hydroxyl group in their molecule react with emmerie-engel reagent.

The following are the reactions involved in the estimation of drugs by using emmerie-engel reagent

Drug +Fe(III) → oxidized form of drug

Applications:

Estimation of Lornoxicam using Emmerie-Engel Reagent

Spectrophotometric determination of ornidazole in pure and pharmaceutical formulations

Spectrophotometric determination of Acyclovir.

7. Folin-Ciocalteu Reagent^2,4

The folin-ciocalteu reagent / folin phenol reagent / folindenis reagent also called the gallic acid equivalence method (GAE), is a mixture of phosphomolybdate and phosphotungstate used for the colorimetric invitro assay of phenolic and polyphenolic antioxidants. It is named after otto folin, vintila ciocalteu, and willey gloverdenis.

Appearance:

Clear bright yellow solution

Storage:

FC should be stored tightly capped at room temperature. The reagent can be diluted with deionized water.

Method of preparation:

Dissolve 10g of sodium tungstate and 2.5g of sodium molybdate in 70ml of water add 5ml 85% phosphoric acidand 10ml conc. Hydrochloric acid reflux for 10 hrs. Add 15g lithium sulphate 5ml waterand 1drop bromine. Reflux for 15min. cool to room temperature and bring to 100ml with water.

Molecular formula:

3H₂O. P₂O_5.13WO₃. 5M_OO_3.10H₂O

3H₂O. P₂O_5.14WO_3.4M_OO_3.10H₂O

Principle: “Reduction”

When FC reagent reacts with drug in presence of reducing agents like sncl₂, ascorbic acid hydrazine, probably drug effects reduction of 1 or more oxygen atoms from tungstate or molybdate in the F-C reagent, there by producing one or more possible reduced species which have characteristic intense blue colour.

Applications:

Folin-ciocalteu reagent was widely used in the analysis of the following drugs

Analgin, Amidopyrine, Acetazolamide, Acetanilide, Aspirine, Ampicillin, Astemizole, Allopurinol, Ascorbic acid, Azathiopurine, BrucineBarbituric acid, Diacerecin, Diazepam

8. Froehde Reagent²

The Froehde Reagent is used as a simple spot-test to presumptively identify alkaloids, especially opioids, as well as other compounds. It is composed of a mixture of molybdic acid or sodium molybdate and hot, concentrated sulfuric acid, which is then dripped onto the substance being tested. A presumptive test for opioids.Froehde’s reagent consists of 0.5 gram of sodium molybdate (Na₂MoO₄) dissolved in 100 ml of concentrated sulphuric acid. LSD gives a blue-green colour, heroin gives purple to olive green, and mescaline gives a greenish colour.

9. Hydroxylamine²

Hydroxylamine is an inorganic compound with the formula NH₂OH. The pure material is a white, unstable crystalline, hygroscopic compound. However, hydroxylamine is almost always provided and used as an anaqueous solution. It is used to prepare oximes, an important functional group. It is also an intermediate in biological nitrification. The oxidation of NH3 is meditated by the enzyme hydroxylamine oxidoreductase (HAO).

Structure:

Structure Of Hydroxylamine

IUPAC name: Hydroxylamine

Other names: Aminol, Azanol, Hydroxylamine, Hydroxyazane, Hydroxylazane

Uses: Hydroxylamine and its salts are commonly used as reducing agents in myriad organic and inorganic reactions. They can also act as antioxidants for fatty acids. Some non-chemical uses include removal of hair from animal hides and photography developing solutions.

10. Lucas Reagent²:

Luca’s Reagent is a solution of anhydrous zinc chloride in concentrated hydrochloric acid. This solution is used to classify alcohols of low molecular weight. The reaction is a substitution in which the chloride replaces a hydroxyl group. A positive test is indicated by a change from colourless to turbid, signaling formation of achloroalkane. The test was reported in 1930 and became a standard method in qualitative organic chemistry. The test has since become somewhat obsolete with the availability of various spectroscopic and chromatographic methods of analysis. It was named after Howard Lucas (1885-1963), an American chemist.

Lucas test:

Lucas test in alcohols is a test to differentiate between primary, secondary and tertiary alcohols. It is based on the difference in reactivity of the three classes of alcohols with hydrogen halides.

ROH + HCl →RCl + H₂O

The differing reactivity reflects the differing ease of formation of the corresponding carbocation’s. Tertiary carbocation’s are far more stable than secondary carbocation’s, and primary carbocation’s are the least stable.

An equimolar mixture of ZnCl₂ and HCl is the reagent. The alcohol is protonated by this mixture, and H₂O group attached to carbon isreplaced by the nucleophile Cl^-, which is present in excess. Tertiary alcohol react immediately with Lucas reagent as evidenced by turbidity owing to the low solubility of the organic chloride in the aqueous mixture. Secondary alcohols react within five or so minutes (depending on their solublty).primary alcohols donot react appreciably with Lucas reagent at room temperature. Hence, the time taken for turbidity to appear is a measure of the reactivity of the class of alcohol, and this time difference is used to differentiate between the three classes of alcohols.

No visible reaction at room temperature and cloudy only on heating: primary such as normal amyl alcohol (1-Pentanol)

Solution turns cloudy in 3-5 minutes: secondary, such as sec-amyl alcohol (2-pentanol)

Solution turns cloudy immediately, and/or phases separate: tertiary, such as tert-amyl alcohol (2-Methyl-2-butanol).

11. 3-Methyl-2-Benzothiazolin Hydrazone^2,3

Synonyms: Mbth

Mbth monohydrate

Sawicki's reagent

Besthorn'shydrazone

Mbth hydrochloride hydrate

3-Methyl-2-benzothialinone

Structure:

Uses:

Phenols, Aldehydes, Aromatic amines.

Principle:

“Oxidation followed by coupling”. In general MBTH undergoes oxidative coupling reaction catalyzed by iron. Under reaction conditions MBTH loses 2 electrons and one proton forming an electrophilic intermediate, which is the active coupling species. This intermediate undergoes electrophilic substitution with the phenols, amines, aldehydes to form coloured product.

Applications:

Estimation of Ezetimibe

Estimation of Lamotrigine

Estimation ofMoprolol

Estimation of Pregabalin

12. Metol^4,5

Introduction:

Metol is the chemical compound with the name monomethyl-p-aminophenol hemi sulfate. It is a developing agent used in blackand white photographic developers, antioxidant, antibacterial, agent and corrosion inhibitor. It is also used as an intermediate for fur and hair dyes and for a drug called diloxanide. In its pure form, it is a solid rather light-sensitive chemical which is the half sulfate (hemi sulfate) salt of N-methyl-p-aminophenol.

Synonym:

Elon, Genol,Armol, Metol(Tm), Methol, Ecol(R), Planetol, Pictol(Tm), Photo-Rex(R), Rhodol, Enel, Viterol, Scalol, Satrapol.

Other names:

N-methyl-p-aminophenol sulfate, p-(methylamino) phenol sulfate.

Chemical Name:

4-(Methylamino) phenol sulfate

Molecular Formula: C₁₄H₂₀N₂O₆S

Molecular Weight: 344.38

Structure:

Properties:

Melting point: 260⁰C

Density: 1.577

Color: White to faintly Brown

Solubility: Freely soluble in water

Storage temp.: -20⁰C

Mechanism of Metol:

Metol forms complexes in its oxidized form i.e., Quinone form and it is oxidized by oxidizing agents like NBS, tannic acid etc. The drugs are estimated by reacting with the oxidizing agents, the unreacted oxidizing agents oxidize metol which forms complex with sulphanilic acid or other complexing agent.

Drug + oxidizing agentàOxidized form of drug +

unreacted oxidizing agent

Unreacted oxidizing agent +MetolàOxidized form of Metol

Oxidized form of Metol + sulphanilic acidàColored

complex

Applications:

· Estimation of zidovudine

· Estimation of Ceftiofur

· Estimation of capecitabine

13. Molybdenum Blue²

Molybdenum blue is a term applied to

· Reduced heteropolymolybdate complexes, polyoxometalates containing Mo(V), Mo(VI) and a hetero atom such as phosphorus or silicon

· Reduced isopolymolybdate complexes, polyoxometalates containing Mo(V), Mo(VI) formed when solutions of Mo(VI) are reduced

· A blue pigment containing molybdenum (VI) oxide The “heteropoly-molybdenum blues”, are used extensively in analytical chemistry and as catalysts. The formation of “isopropyl-molybdenum blues” which are intense blue has been used as a sensitive test for reducing reagents. They have recently been shown to contain very large anionicspecies based on the so-called “big wheel” containing 154 Mo atoms, with a formula [Mo₁₅₄O₄₆₂H₁₄(H₂O)₇O]

Principle:

Heteropoly-molybdenum blues. The first heteropolymolybdate and first hetero polymetallate, yellow ammonium phosphomolybdate, (NH₄)₃PMo₁₂O₄₀ was discovered by Berzelius in 1826. The phosphorus atom in the anion is termed the hetero-atom; other hetero-atoms are silicon and arsenic. The heteropoly-molybdenum blues have structures based on the Keg gin structure. The blue color arises because the near-colorless anion, such as the phosphomolybdate anion, PMo₁₂O₄₀^3-,can accept more electrons (i.e. be reduced) to form an intensely colored mixed-valence complex. This can occur in one electron or two electron steps. The reduction process is reversible and the structure of the anion is essentially unchanged. The structure of the anion, PMo₄Mo₈O₄₀^7-_,has been determined in the solid state and is a β-isomer (i.e. with one of the four groups of edge-shared octahedra on the α -Keg gin ion rotated through 60 degrees) Similar structures have been found with silicon, germanium or arsenic hetero-atoms. The intense blue color of the reduced anion is the basis for the use of heteropoly-molybdenum blues in quantitative and qualitative analytical techniques.

14. NQS reagent⁴

Chemically is 1,2-naphthoquinone-4-sulfonate sodium

It is discovered by Folin so it is also called Folin’s reagent

Synonym: beta-naphthoquinone-4-sulfonate sodium salt

IUPAC name: sodium 3,4-dioxo-3,4,dihydronaphthalene-1-sulfonate

Molecular formula: C₁₀H₅NaO₅S

Molecular weight: 260.20 g/mol

Melting point: 289⁰C

Structure:

Principle:

· NQS reacts with primary and secondary amines, by a nucleophilic substitution reaction, in relatively mild conditions.

· The derivative can be detected by UV-VIS and by fluorimetry (Ex. 260 nm, Em. 435 nm)

Applications: NQS reagent is used for the

· Determination of drugs which contain primary or secondary amines

· Determination of aromatic amines in industrial waste water

· Determination of amphetamine and metamphetamine in urine by reverse-phase HPLC

15. Ninhydrin⁴

Synonym: 2,2-Dihydroxyindane-1, 3-Dione

Molecular formula:C₉H₆O₄

Molecular weight: 178.14

Structure:

Melting point: 240-245⁰C

Description: White to brownish white crystals or crystalline powder

Solubility: Soluble in water and alcohol, slightly soluble in ether and CHCl₃

Storage: Protected from light

Mechanism:

Ninhydrin is shown to tautomerize 1, 2, 3-indantrione which forms a Schiff’s base with the amino acid, the ketimine formed undergoes decarboxylation yielding the aldehyde and an intermediate amine (2-amino-1, 3-indandione, VI), which was isolated by Rheumann. Condensation of this intermediate amine with another molecule of ninhydrin follows to form the expected chromophore, Ruhemann’s purple(VII).

Applications:

Estimation of Lisinopril

Estimation of Cefditorenpivoxil

Estimation of Topiramate

16. Para – Dimethyl Amino Benzaldehyde²

Para- Dimethylaminobenzaldehyde is a bifunctional aromatic skeleton possessing the aldehyde\ (CHO) Para to an activating substituent dimethyl amino group [-N(CH₃)₂].

Synonyms:4-(dimethylamino) benzaldehyde

p-(dimethylamino)-benzaldehyde

Ehrlich’s Reagent

p-Formyl-N,N dimethyl aniline

PDAB

N,N-Dimethyl-4-aminobenzaldehyde

Wasickys reagent.

Structure:

Principle:

“Formation of condensation products referred as Schiff’s base”.

Uses:

Para-Dimethylaminobenzaldehyde is an organic compound containing amine and aldehyde moieties which is used in Ehrlich’s reagent for determination of hydrazine and Kovac’s reagent for microbiology’s indole test

Applications:

· Urobilinogen determination

· Hydrazine determination

17. Phosphomolybdic Acid²

Phosphomolybdic acid, also known as dodecamolybdo phosphoric acid or PMA is a component of Masson’ strichrome stain. It is a yellow-green compound, freely soluble in water and polar organic solvents such as ethanol. It is used as a reagent in thin layer chromatography for staining, phenolics, hydrocarbon waxes, alkaloids and steroids conjugated, unsaturated compounds reduce PMA to molybdenum blue. The color intensifies with increasing number of double bonds in the molecule being stained phosphomolybdic acid is primarily used in spectrophotometric determination of phosphate ions. It is also component in the 3-color histology stain called Masson’s trichrome employed in the differentiation step. It is one of the solutions fixed tissues are immersed in prior to subsequent staining.

Synonym: PMA

Molecular formula: HMoO₂P

Molar mass: 159.92 g/mol

Appearance: Liquid

Structure:

Use:

Phosphomolybdic acid is also used as reagent for simple and rapid colorimetric determination of phenothiazine derivatives.

18. Simon’s Reagent²

Simon’s reagent is used as a simple spot-test to presumptively identify alkaloids as well as other compound. It reacts with secondary amines like MDMA and methamphetamine to give a blue solution

Chemistry:

The reagent is composed of a mixture of sodium nitroprusside, sodium carbonate and acetaldehyde, which is dripped onto the substance being tested. The amine and acetaldehyde produce the enamine, which subsequently reacts with sodium nitroprusside to the imine. Finally, the immonium salt is hydrolysed to the bright cobalt-blue Simon-Awe complex.

Use:

The primary use of this reagent is for detecting secondary amines, such as MDMA and methamphetamine and is typically used after the mecke or marquis reagents to differentiate between the two mentioned and amphetamine or MDA.

19. 2, 3, 5 TriphenylTetrazolium salt^4,5

Synonym: Urocheck

TTC(TTZ)

Uroscreen

Vita stain

Tetrazole red

Chemical name: 2, 3, 5-Triphenyltetrazolium chloride

Molecular formula: C₁₉H₁₅ClN₄

Molecular Weight:334.8

Structure:

Solubility: water, ethanol and acetone soluble, Ether insoluble

Color: white

Principle:

Triphenyltetrazolium chloride TTC is a redox indicator commonly used in biochemical experiments especially to indicate cellular respiration in the TTC assay, TTC is used to differentiate between metabolically active and inactive tissues. The white compound is enzymatically reduced to red TPF (1, 3, 5-triphenylformazane) in living tissues due to the activity of various dehydrogenases, while it remains as white TTC in areas of necrosis since these enzymes have been either denatured or degraded. For this reason, TTC has been employed in autopsy pathology to assist identification of post mortem myocardial infractions. Healthy viable heart muscle will stain deep red from the cardiac lactate dehydrogenase, while areas of potential infractions will be more pale.

Applications:

Colorimetric determination of catecholamine’s by 2, 3, 5triphenyltetrazolium chloride

Estimation of epinephrine, leva isoproterenol and methyldopa

20. TPTZ Reagent⁵

Chemical name:2, 4, 6-Trypyridyl-s-triazine

Tri-2-pyridyl-s-triazine

Molecular formula: C₁₈H₁₂N₆

Molecular weight:312.33

Structure: