Phosphate Buffer with Ammonium Ions

At pH 7.0, ammonium phosphate buffer was found to give similar retention but somewhat improved peak shape as compared with potassium phosphate buffers, presumably because of the superior deactivating effect of the ammonium ion on ionized silanols [72], In contrast, this effect was hardly observed at low pH, probably... 

For most reactions, one can use 1-2 mM acetyl phosphate and 1-2 international units of acetate kinase. The enzyme is usually supplied as a crystalhne suspension in 3 M ammonium sulfate, and 10 mM ammonium ion is inhibitory. Therefore, a useful practice is to snip off 0.5 cm from a disposable Eppendorf micropipette tip to facih-tate removal of 10-20 microliters of the crystalline suspension then spin down the enzyme in a 1.5 ml disposable conical plastic centrifuge tube, and remove the ammonium sulfate solution with a wick of twisted Kim-wipe. The enzyme precipitate can now be taken up directly into your working buffer. Note Acetate kinase is inactivated by cold exposure, but incubation with 10 M ATP or GTP reactivates the enzyme if warmed to room temperature for 5-10 min. 

Gradients of aqueous and organic mobile phases are typically used for LC-MS/MS analysis of drug compounds and metabolites. The most common aqueous solvents are water with 0.1 % formic acid or 0.1 % acetic acid (v/v) or volatile buffers like 5 mM ammonium-acetate or ammonium-formate. Often adjusted to a certain pH value with the corresponding acid or base (the pH of the eluents will have to be optimized with respect to the polarity of the analytes, since ionic species will have very low or no retention on the reversed pahse LC-columns). Other volatile buffers can be used as well. Phosphate buffers should be avoided, since they will cause suppression of the ionization and thus lead to very bad analytical performance (Venn 2000). Reagents like triethyl-amine should also be avoided as mobile phase or as part of mobile phases. They induce ion suppression as well. In terms of the organic solvents, methanol and acetonitrile are very widely used and they are very well suitable for LC-MS. Other solvents can be used as well, as long as they are compatible with the materials used in the LC-MS system. 

Fig. 34. Chromatographic separation of dopamine and derived salsolinoids. Salsoiinol-1-carboxylic acid (1), dopamine (2), saisolinol (3), and 7-methylsalsolinol-l-carboxylic acid (4) were separated by ion-exchange chromatography on a Nucleosil 10 SA column (4.6 x 250 mm, 10-/Ltm particle size) with 8% methanol in a citrate-ammonium phosphate buffer at I.O ml/min as the mobile phase (inlet pressure 1100 psi). Detection was at 280 nm.

Type I CSPs have also been used with aqueous mobile phases. Pirkle et al. (32) have reported on the resolution of N-(3,5-dinitrobenzoyl) derivatives of M-amino adds and 2-aminophosphonic adds on an (l )-N-(2-naphthyl)-alanine-derived CSP using a mobile phase composed of methanol-aqueous phosphate buffer. The utility of achiral alkyltrimethylammoruum ion-pairing reagents was also investigated. Other examples include the following (1) The recently commercialized ot-Burke 1 CSP resolves the enantiomers of a number of underivatized p-blockers using an ethanol-dichlorornethane-ammonium acetate mobile phase (33) (2) an (R)-l-naphthylethylurea CSP was used to resolve N-(3,5-dinitrobenzoyI)-substituted amino adds and 3,5-dinitrobenzoyl amide derivatives of ibuprofen, naproxen, and fenoprofen with acetonitrile-sodium acetate mobile phases (34). 

Octadecyl (octyl) bonded phase with low percentage of free silanol groups ION-SUPPRESSION MODE methanol (acetonitrile) -water containing ca. 0.01 - 0.1 M phosphate buffer, ammonium carbonate or sodium acetate (pH 4-7). 

Thomson reported the reversed-phase ion-pair separation of psilocybin and psilocin. Because both alkaloids exist as zwitter-ions, cationic and anionic pairing ions can be used. Alkyl sulfonates (Cg-Cg) and tetraalkyl ammonium (C3-Cg) ions were found unsatisfactory for psilocybin. Good results were obtained with a long chain quaternary ammonium ion, cetrimonium. Optimal conditions for quantitative analysis on an octadecyl stationary phase were 0.15% pairing ion in methanol - 0.4% aqueous phosphate buffer (pH 7.Z). Some other quaternary indole alkaloids have also been separated by means of ion-pair HPLC. Parkin6 analyzed the bisquater-nary alkaloid alcuronium in biological fluids. After an ion-pair extraction, the alkaloid was analyzed on an octadecyl column with the mobile phase methanol - water (4 1) containing O.Z5% acetic acid and 0.005 M dodecylsulfate. 

Substantial efforts have been devoted to the development of molecular sensors for dopamine. Raymo et al.70 reported a two-step procedure to coat silica particles with fluorescent 2,7-diazapyrenium dications sensing toward dopamine. The analysis of the fluorescence decay with multiple-equilibria binding model revealed that the electron deficient dications and the electron-rich analytes form 1 1 and 1 2 complexes at the particle/water interface. The interfacial dissociation constants of the 1 1 complexes were 5.6mM and 3.6mM for dopamine and catechol, respectively. Dopamine was dominated by the interaction of its electron-rich dioxyarene fragment with the electron-deficient fluorophore in neutral aqueous environments. Ahn et al.71 reported tripodal oxazoline-based artificial receptors, capable of providing a preorganized hydrophobic environment by rational design, which mimics a hydrophobic pocket predicted for a human D2 receptor. A moderate binding affinity, a dissociation constant of 8.2 mM was obtained by NMR titrations of tripodal oxazoline-based artificial receptor with dopamine in a phosphate buffer solution (pH 7.0). Structurally related ammonium ions, norepinephrine, 2-phenylethylamine,... 

Ion-exchange chromatography is used most frequently, because of its simplicity and low cost. Extracts usually containing [ HJinositol-labeled inositol phosphates are placed on a Dowex formate column from which GPI, IP, IP2 (inositol bisphosphate), and IP3 are eluted sequentially with buffers of ammonium formate of increasing molarity. 

The major source of acid is CO2, which reacts with water to produce carbonic acid. To maintain the pH of body fluids in a range compatible with life, the body has buffers such as bicarbonate, phosphate, and hemoglobin (see Fig 4.1). Ultimately, respiratory mechanisms remove carbonic acid through the expiration of CO2, and the kidneys excrete acid as ammonium ion (NHfi) and other ions. 

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