Patai S., Rappoport Z. 1996 The chemistry of functional groups. The chemistry of amino, nitroso, nitro and related groups. Part 2 / 1041 1168

(161)

Primary amines form fluorescent Schiff base complexes in the presence of salicylaldehyde (163) and Be(II) ions, that can be measured in a FIA-FLD system. The reaction is

fast, allowing up to 30 determinations per hour. Analytical range: MeNH2 6 ð 10 6 to 6 ð 10 3 M, RSD 3%; EtNH2, n-PrNH2 and n-BuNH2 3 ð 10 5 to 8 ð 10 3 M. Secondary and tertiary amines do not react. This was applied to the determination of traces of MeNH2 (0.007 0.008%) in commercial Me2NH352.

A fast and sensitive method for determination of 4-aminoantipyrine (164) consists of coupling this compound with diazotized p-nitroaniline in a FIA system and measuring spectrophotometrically at 380 nm. About 50 determinations per hour could be carried out; LOD was 0.05 ppm (SNR 3), RSD 0.61% for 4 ppm and 0.27% for 50 ppm, with linearity up to 50 ppm353.

Surface-enhanced Raman scattering using a silver-coated alumina support selectively enhanced the spectrum of p-aminobenzoic acid. This allowed the determination of this compound at low ppm levels in vitamin B complex354,355.

LIF excitation spectra were recorded for alkyl aminobenzoates (165) under free jet conditions. The partially resolved band contours were different for the various compounds

Links

(a)

(b)

and could be assigned to conformers of the molecule. Differences in band contour were ascribed to changes in hybridization caused by the conformational structure356.

G. Enzymatic Biosensors

Amperometric biosensors incorporating certain enzymes on the electrode for the determination of D- and L-amino acids were investigated. The parameters included enzyme immobilization procedure, composition of the immobilizing matrix, amount of enzyme,

pH, flow rate and injection volume. The immobilized enzymatic system consisted of a D- or an L-amino acid oxidase producing hydrogen peroxide, and hydrogen peroxide reductase. The efficiency of the electrocatalytic reduction of hydrogen peroxide starts to increase from C600 mV towards negative potentials, and levels off at 100 mV, measured against a Ag/AgCl electrode. The 20 most common L-amino acids could be detected357. Specific amperometric amino acid sensors were based on a Clark oxygen electrode, with specific or nonspecific enzymes immobilized on the gas-permeable membrane. L-Glutamic acid was determined using L-glutamate oxidase, by measuring the oxygen consumption of reaction 18. For L-lysine, L-arginine and L-histidine the corresponding decarboxylases catalyzed reactions 19 21. The liberated carbon dioxide was consumed by autotrophic bacteria leading to oxygen consumption that was measured in the detector358.

Polyaniline-modified electrodes allow electrometric determination of hydrogen peroxide produced in aminooxidase systems, without interference of electroreactive amino acids, such as cysteine, histidine, methionine, tyrosine and tryptophan359.

An interdigitated array of microelectrodes in a small volume helped to significantly reduce the LOD of electrochemically reversible redox materials360,361. This was applied to the determination of p-aminophenol by small volume immunoassay, by sandwiching layers of a supported enzyme with the microelectrodes. A steady-state signal was obtained for the array, showing a linear relationship between the concentration and the limiting current over the range of 1 1000 mM. Less than 1 min detection time was required for 2 10 mL samples362.

A biosensor was designed where a dehydrogenase and an enlarged coenzyme are confined behind an ultrafiltration membrane. The amino acid is determined indirectly, by measuring the fluorescence of the reduced coenzyme ( ex 360 nm, fl 460 nm) produced in reaction 22, with the aid of an optical fiber. The coenzyme is regenerated with pyruvate in a subsequent step, as shown in reaction 23. This biosensor was proposed for determination of L-alanine and L-phenylalanine for monitoring of various metabolic diseases and for dietary management363.

Amino acids may be determined by measuring the amines obtained after the action of a carboxylase with a specific electrode for amines, which is based on a poly(vinyl chloride) membrane containing sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate (166) as ion exchanger and tricresyl phosphate as solvent mediator. LOD was 20 and 50 mM for tyroxine and phenylalanine, determined as tyramine (5) and phenethylamine (33), respectively364.

F3 C

B− Na+

F3 C 4

(166)

The hydrogen peroxide produced in a FIA system coupled with a bioreactor containing an amino acid peroxidase can be determined by the chemiluminescence it produces in the presence of phenyl 10-methylacridinium-9-carboxylate (167). Thus, for example, a throughput of 200 samples of glutamate per hour was achieved with LOD 0.5 mM (SNR 3)365. Hydrogen peroxide, generated on degradation of amino acids by L-amino acid oxidase immobilized in a reactor, can be determined by measuring the chemiluminescence in the presence of luminol (69) and hexacyanoferrate ions. The method was applied to the determination of free amino acids in cheese, with a throughput of 40 samples per hour366. This method was preferable to derivatization procedures with either trinitrobenzenesulfonic acid or by applying reaction 7367.

CO2 Ph

N+

Me

(167)

A sensitive method of determination of H2O2 is by the so-called peroxalate reaction luminescence (reaction 24) by which hydrogen peroxide reacts with an aryl oxalate

forming 1,2-dioxetanedione (168); this reacts with a fluorophore , leading to an excited state (169), that eventually returns to the ground state emitting a photon368. The effectiveness of the method depends on the readines by which 169 is formed and the nature of the fluorophore. For example, 2,4,6-trichlorophenyl oxalate (42) catalyzed by imidazole (170) is frequently used. The method was reviewed369. A modification claimed to be 10 times more sensitive uses 1,10-oxalyldiimidazole (171) as reagent and an immobilized form of 3-aminofluoranthene (172); LOD 10 nm of H2O2 in water, for 0.5 pmol (50 mL) injection370.

Me

(172)

H. Miscellaneous Methods

Carbon paste and graphite epoxy electrodes modified with RuO2 can be used for detection of amino acids and peptides in FIA systems. Optimal conditions are in strongly alkaline solutions at C0.45 V vs Ag/AgCl electrode, with a fast and linear response. Carbon paste electrodes can be modified also with Co3O4371. Colorimetric methods for the determination of amino groups attached to a solid support may give erroneous values

due to nonspecific adsorption of chromophores on the solid surface. An amperometric determination of primary amino groups was based on derivatization with glutaraldehyde followed by oxidation of the resulting dienamine (reaction 25). The amino group concentration is proportional to the oxygen consumption, that is monitored by a Clark oxygen electrode; RSD is 3.2% for AH-Sepharose 4B372.

R

Amino acids enhance the oxidation peak of Cu(0) obtained with a carbon paste electrode incorporating Cu(II) cyclohexylbutyrate. The increased current is proportional to the amino acid concentration at trace levels in the mM range373. The behavior of such electrodes was investigated for cysteine (115). On scanning potentials in the positive direction, the amino acid is accumulated on the electrode as the Cu(I) complex at C0.90 V vs a

standard calomel electrode (SCE), in acetate buffer at pH 4.5; linear range is 2 ð 10 9 to 1 ð 10 7 M, 1 min accumulation, RSD 3% (n D 5)374,375.

Amino acids can be determined in a two-step process (reaction 26). The SO2 produced

Ar D 2,4,6-(O2N)3C6H2

Radioimmunoassay (RIA) may sometimes be the method of choice for certain amines. Thus an 125I RIA method was developed for the specific detection of D-amphetamine (28) and D-methamphetamine (29) in urine, with LOD of approximately 25 mg/L. The method was compared with GC-MS and other commercially available amphetamine assays. Other drugs gave erroneous positive identification as 28 with the latter methods, whereas the results of RIA were negative377.

Amino acids accelerate and proteins retard the rate of Cu(II)-catalyzed oxidation of di- 2-pyridyl ketone hydrazone (173) yielding fluorescent compounds. This has been applied for the analysis of amino acids and proteins378.

1,4-Dihydropyridines and their N-alkyl derivatives undergo anodic oxidation in basic medium to the corresponding pyridines (reaction 27). The process may be complicated by the presence of other moieties; for example, a nitro group may reductively condense

N N

NNH2

(173)

with nearby cyano or ester functions to yield products such as 174 and 175379 382.

The fate of dissolved amines during disinfection of water by chlorination was determined by membrane injection MS. Aliphatic amines undergo N-chlorination to exhaustion of the N H atoms by one of the tentatively proposed paths shown in reaction 28. Aromatic amines undergo mainly ring substitution; however, the possible intervention of N Cl intermediates is not excluded. At pH 10.6 aniline chlorination is much slower than that of n-butylamine383.

Nonionic surfactants of general structure 176, used in off-shore drilling (e.g. Nonidet AT 85), are toxic and slowly biodegradable. They can be determined in an FIA system by

measuring the chemiluminescence produced on oxidation of the tertiary amino group by sodium hypochlorite at ca pH 11, in the presence of rhodamine B (177) as sensitizer. Only tertiary amines, including Me3N, Et3N and Pr3N, show chemiluminescence; however, the simpler amines can be distinguished by their faster kinetics; LOD ca 5 ppm of 176384.

CO2 −

Primary amino groups covalently attached to the surface of glass were determined by derivatization with 14C-labelled acetyl chloride and measurement of the radioactivity348. The interaction of hydrolyzed (3-aminopropyl)triethoxysilane with E-glass results in the formation of a thin coating about 6 nm thick. Investigation of this film by time of flight secondary ion MS (TOF-SIMS)385,386 and by X-ray photoelectron spectroscopy (XPS)387 led to the conclusion that the film consists of three main structures as depicted in 178, where the open bonds on silicon atoms represent oxygen bridges to other silicon atoms.

The structure of the two-dimensional molecular aggregates formed at the air water interface of aqueous solutions of amino acids carries precise enantioselective information that influences the direction of growth of glycine crystals at that interface. Thus, solutions of valine, leucine, phenylalanine, norleucine, isoleucine or 2-aminooctanoic

N

acid of S-configuration induce fast growth of the (010) face of glycine crystals at the air water interface, while that of the 010 phase is induced in the presence of those of R-configuration. Hexafluorovaline, neopentylglycine and t-butylglycyne fail to show this induction388. Amino acid enantiomers in association with the NaCl H2O eutectic were investigated by differential scanning calorimetry (DSC) and NMR. Thus, a solution of amino acid in 0.1 M NaCl was heated in the DSC apparatus at a rate of 1 °C/min, starting at 60 °C, and the 1H NMR spectrum was recorded at 20 °C. The combined DSC and NMR results showed that the L and D forms of the amino acids could be differentiated, based on the singlet and doublet bands389.

The bonding forms of nitrogen in several Australian coals were determined by XPS and predominantly assigned to pyrrolic and pyridinic forms. Amino forms appear to be absent390.

I. Derivatization

Amines are converted quantitatively to dithiocarbamates (reaction 29), that can be determined by nonaqueous titration with Ce(IV); accuracy 0.8%, RSD 0.7%391.

Section IV.D.4, more direct methods are afforded by NMR spectroscopy, especially for the products of synthesis. Ephedrine (179), pseudoephedrine (180a) and its Me ether (180b) yield stable epimeric N ! BH3 adducts on treatment with borane. The configuration of the nitrogen moiety was established by NMR, taking into account the conformational analysis of the molecule392.

Various chiral derivatization reagents containing phosphorus have been proposed for determination of enantiomer excess. Measurement of 31P NMR has the advantage of large peak separation. Reaction 30 takes place quantitatively with alcohols, thiols and amines in the NMR tube, at room temperature, in C6D6 or CDCl3 solution. Reagents 181 have C2 symmetry and yield diasteroisomers 182, with excellent 31P NMR peak separation for accurate integration and determination of enantiomer excess. Other spectra such as 1H NMR can also be taken, but they may be too complex for enantiomer analysis393.