Tetrabutylammonium phosphate

This experiment focuses on developing an HPLG separation capable of distinguishing acetylsalicylic acid, paracetamol, salicylamide, caffeine, and phenacetin. A Gjg column and UV detection are used to obtain chromatograms. Solvent parameters used to optimize the separation include the pH of the buffered aqueous mobile phase, the %v/v methanol added to the aqueous mobile phase, and the use of tetrabutylammonium phosphate as an ion-pairing reagent. 

Mobile Phase A 3 mM tetrabutylammonium phosphate, 15 mM ammonium phosphate, pH 7.50... 

Fig. 3.3e shows the separation of weak acids on a C-18 column with a mobile phase of methanol/water 50 50 + tetrabutylammonium phosphate. The pH of the mobile phase is about 7.5, as at this pH... 

Counter-ions which are frequently used include tetrabutylammonium phosphate for the separation of anions and hexane sulphonic acid for cations. The appropriate counter-ions are incorporated in the solvent, usually at a concentration of about 5 mmol 1" and the separation performed on the usual reverse phase media. This ability to separate ionic species as well as non-polar molecules considerably enhances the value of reverse-phase chromatography. 

Much effort has been devoted to the applicaton of electrophoretic techniques for the analysis of dyes and dye decomposition products in industrial waste-waters. CZE with DAD and MS detection was employed for the determination of reactive dyes in spent dye-baths and waste-waters. The chemical structure of the dyes included in the experiments are shown in Fig. 3.149. Liquid samples were purified and preconcentrated by SPE. ODS cartridges were conditioned by washing with two bed volumes of methanol, four bed volumes of water and two bed volumes of 5 mM tetrabutylammonium phosphate. Aliquots of 10 - 30 ml were loaded and the cartridges were washed with 2 ml of water. Analytes were eluted with 2 ml of methanol-water (70 30, v/v). Untreated fused-silica capillaries (110 cm and 57 cm X 50 /im i.d.) were coupled to MS and DAD. The running... 

Ion-pair chromatography has also been used for the separation of aspartame from other sweeteners. The ion-pair reagents commonly used are triethylammonium phosphate (32), tetra-ethylammonium hydroxyde (47), tetrapropylammonium hydroxide (40), pentanesulfonate (52), tetrabutylammonium phosphate (34), tetrabutylammonium hydrogen sulfate (66), and tetrabutyl-ammonium p-toluenesulfonate (24). 

Tetrabutylammonium phosphate was used for the extraction of dyes from grape beverages. Sample containing TBA was passed through a Sep-Pak Cl 8 cartridge. The synthetic colors were retained on the column while most natural colors were not. The cartridge is washed with water and the synthetic colors are eluted with methanol water, 1 1 v/v (159). 

An alternative approach for MOCA analysis by HPLC, also involving reverse phase chromatography on a yBondapak Cis column, utilizes the paired ion technique. Paired ion chromatographic (PIC) analysis is effective for the determination of compounds which may exist as ionic species in the polar mobile phase. A counter ion, such as an alkyl sulfonate for cations or tetrabutylammonium phosphate for anions, is added to the mobile phase at a concentration of approximately 0.005 M. This technique generally affords Improved efficiencies in comparison to ion exchange chromatography. For the analysis of MOCA by PIC, the following conditions apply ... 

Abbreviations DMF, dimethylformamide MeCN, acetonitrile THF, tetrahydrofuran DMSO, dimethylsulfoxide PC, polycarbonate TBAI, tetrabutyl-ammonium iodide TBAP, tetrabutylammonium phosphate TBABF4> tetrabutylammonium tetrafluoroborate TRAP, tetraethylammonium phosphate. Vsersus Ag Ag+ (0.01 M) in NH3 at -50°C. Versus Ag AgN03 (sat d) in SO2 at -40°C. 

For reversed-phase ion-pair chromatography a non-polar surface (e.g. Cg or C- g) is used as a stationary phase and an ionic alkyl compound is added to the aqueous mobile phase as a modifier. For the separation of acids, an organic base (e.g. tetrabutylammonium phosphate) is added to the eluent for the separation of bases, an organic acid (e.g. octane sulphonate) is used. Reversed-phase ion pairing is presently the most popular approach because of the simpler technical requirements and very high column performance. It is however essential to operate the system only after equilibrium of the mobile phase and the stationary phase has occurred in order to obtain reproducible analyses. 

Figure 1.4 Separation of substrates and products of an adenosine kinase reaction on ion-paired reversed-phase HPLC. The separation was carried out on a prepacked Ctg (/xBondapak) column with a mobile phase of 65 mAf potassium phosphate (pH 3.7) containing 1 mAf tetrabutylammonium phosphate and 5% methanol. The column was eluted isocratically, and the detection was at 254 nm. Four relative elution positions (elution times) arc shown.

The separation of product from substrate was accomplished using ion-paired, reversed-phase HPLC on a C18 (/xBondapak) column with a mobile phase of 65 mM potassium phosphate and 1 mM tetrabutylammonium phosphate adjusted to pH 3.6 with phosphoric acid and 1.5% acetonitrile. The column was eluted isocratically and monitored at 254 nm. The separations obtained are shown in Figure 9.97. 

Figure 9.97 Separation of adenine nucleotides and adenosine by ion-paired, reversed-phase HPLC. Standards of AMP, adenosine, ADP, ATP, and cAMP (approximately 2 nmol of each) in Tris-HCl (pH 7.4) were injected onto a Cis /xBondapak reversed-phase column (300 mm X 7.8 mm) and eluted with 65 mM KH2P04 (pH 3.6), 1 vaM tetrabutylammonium phosphate, and 2% acetonitrile. The flow rate was 2 mL/min, and detection was at 254 nm. (From Rossomando, 1987.)...

Figure 9.112 Separation of substrates and products of reaction catalyzed by adenylosuccinate synthetase. Column Prepacked C18 /xBondapak, 10 /im particle size. Mobile phase 65 mM potassium phosphate, 1 mM tetrabutylammonium phosphate, 10% methanol at pH 4.4. Absorbance was measured at 254 nm. (From Rossomando, 1987.)...

Separation of CMP and CMP-NeuAc was by reversed-phase chromatography on a Beckman C]8 Ultrasphere-IP column (4.6 mm x 250 mm, 5 /xm). The column was eluted at 2.0 mL/min using a mobile phase composed of 9% acetonitrile in 5 mM sodium phosphate buffer (pH 7.5) containing 5 mM tetrabutylammonium phosphate as an ion-pairing agent. The column eluate was monitored at 270 nm. 

Column. Amino-propyl bonded silica (Spherisorb S5NH2, 5 qm, 25 cm x 4 mm internal diameter). Eluent. Acetonitrile tetrabutylammonium phosphate, 0.005M, pH 7.5 (85 15). 

Sulphuric Acid-Ethanol Reagent add gradually 10 ml of sulphuric acid to 90 ml of ethanol Tetrabutylammonium Phosphate, 0.005M, pH 7 5 PIC Reagent A Marketed by Waters Associates Inc, Milford, MA01757, USA, Waters Associates (Instruments) Ltd, Northwich, Cheshire, UK... 

The following abbreviations have been used P, phosphate or phosphoric acid TMAP, tetramethylammonium phosphate TEAP, triethylam-monium phosphate TBAP, tetrabutylammonium phosphate Pyr, pyridine TEA, trifluoroacetic acid HFBA, heptafluorobutyric acid DDAP, dodecylammonium phosphate 2-ME, 2 methoxylethanol. 

Fig. 1.15. Dependence of retention factors, k, of dye iniermcdiaies on the concentration, ip i9< vol. x 10 -), of methanol in 0.005 M tetrabutylammonium phosphate, pH 7.5, on a Lichrosorb SI 100 ODS (10 pm) column (300 x 4.0 mm i.d.). Sample compounds naphthalene-1-sulphonic acid (/). 8-aminonaphthalene-l-sulphonic acid (2), 7-hydroxynaphthaIene-IJ-disuIphonic acid (3), 6-aminonaphlhalene-2-sulphonic acid (4), 4-tolucnesulphonic acid (5) and 4-nitroiolucne-2-sulphonic acid (6). Points experimental data lines best-fit plots of two-parameter Eq. (1. 18).

Reversed-phase chromatography can be used in this instance. For example, an alkylsulfonate is added to cationic analytes and tetrabutylammonium phosphate to anionic analytes. A sample containing both anionic and cationic components has one type masked by a counter ion and the other suppressed by a suitable pH level. 

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