Pair Reagent

TFA is the most common ion-parr reagent. The low pKs value (0.8) protonizes the carboxyl groups and, therefore, retention is higher than with formic acid or acetic acid, with pKs values of 3.75 or 4.76. The typical concentration is 0.1%. An increase in the ion-parr concentration results in the peptides and proteins tending to have a higher retention. Because this increase is different for the respective peptides or proteins, concentration is an important parameter for the method development. 

Theoretically, other acids, such as phosphoric acid or hydrochloric acid, can also be used, because these can also positively influence selectivity. The reason for their rare use is, in the case of hydrochloric acid, its high potential for corrosion, and phosphates are not volatile, which is an exclusion criterion in the case of MS or preparative apphcations. 

In the neutral pH range, ammonium formiate, ammonium acetate, and triethy-lamine phosphate and in the alkaline range ammonia, trimethylamine, and tri-ethylamine are used. 

Columns that are to be used with ion-pair reagents should be reserved for this application only. Otherwise, there is a very high risk of a change in selectivity and associated with this, bad reproducibility. 

Amino acids, as components of peptides and proteins, are zwitterions, that is, they have both positive (-NHR2 ) and negative charges (-COO ) depending on the solutions pH value. If the amino acid has the same number of positive and negative charges, namely at the pi value, it is neutral. Below the pi value, the peptides are cations and above it they are anions. The pi value of acidic amino acids is approximately 3, that of the polar, non-polar, and aromatic amino acids is 5-6.5 and that of the basic ones is greater than 7 (see Table 5.1). 

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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. 

An alternative way of eliminating water in the RPLC eluent is to introduce an SPE trapping column after the LC column (88, 99). After a post-column addition of water (to prevent breakthrough of the less retained compounds), the fraction that elutes from the RPLC column is trapped on to a short-column which is usually packed with polymeric sorbent. This system can use mobile phases containing salts, buffers or ion-pair reagents which can not be introduced directly into the GC unit. This system has been successfully applied, for example, to the analysis of polycyclic aromatic hydrocarbons (PAHs) in water samples (99). 

To determine secondary alkanesulfonates in sewage wastewaters, solid phase extraction (SPE) and a single-step procedure which combines elution and injection port derivatization for analysis with GC-MS were developed [36]. Again a tetrabutylammonium ion pair reagent was employed both to elute the secondary alkanesulfonates as their ion pairs from CI8-bonded silica disks and to derivatize sulfonate ion pairs under GC injection port conditions. Secondary alkanesulfonates were effectively recovered from samples of raw sewage (>92%) and from primary (>98%) and secondary (>85%) effluents. No... 

A method offering the possibility for the separation, identification, and determination of alkyl- and alkylphenol ether carboxylates, even in mixtures with other nonionic and amphoteric substances, is carried out by HPLC using a reverse phase RP18 column and a mixture of methanol, water, and acetonitrile with the addition of an ion-pairing reagent as mobile phase working under isocratic conditions [242]. 

Finally, ion chromatography can be used to determine the a-sulfo fatty acid esters. The chromatographic column is a nonpolar poly sty rene/divinylbenzene column and the ion pair reagent is 0.005 M ammonia. In order to reduce the elution time, acetonitrile is added as a modifier with increasing concentration. This gradient technique makes it possible to separate simultaneously ester sulfonates and disalts by chain length. Determination is achieved by standards with defined chain length [107]. 

Factors may be classified as quantitative when they take particular values, e.g. concentration or temperature, or qualitative when their presence or absence is of interest. As mentioned previously, for an LC-MS experiment the factors could include the composition of the mobile phase employed, its pH and flow rate [3], the nature and concentration of any mobile-phase additive, e.g. buffer or ion-pair reagent, the make-up of the solution in which the sample is injected [4], the ionization technique, spray voltage for electrospray, nebulizer temperature for APCI, nebulizing gas pressure, mass spectrometer source temperature, cone voltage in the mass spectrometer source, and the nature and pressure of gas in the collision cell if MS-MS is employed. For quantification, the assessment of results is likely to be on the basis of the selectivity and sensitivity of the analysis, i.e. the chromatographic separation and the maximum production of molecular species or product ions if MS-MS is employed. 

A method for the development of a generic LC-electrospray-MS method for the analysis of acidic compounds using experimental design has been reported [5], From an HPLC perspective, this type of analysis often requires the use of an ion-pairing reagent to obtain separation however, many of these, such as tetraalkylammonium ions, are involatile and have undesirable effects on the performance of the mass spectrometer and more volatile alternatives have to be found - in this case, triethylamine was used. 

The factors chosen for study were the concentration of the ion-pairing reagent, the solution pH ( quantitative factors) and the acid chosen for pH adjustment (formic, acetic, propionic and trifluoroacetic acids) ( quahtative factor). The effect of these factors was assessed by using responses that evaluated both the HPLC (the number of theoretical plates and the retention time) and MS performance (the total peak area and peak height) for each of the four analytes studied, i.e. 1-naphthyl phosphate (1), 1-naphthalenesulfonic acid (2), 2-naphthalenesulfonic acid (3) and (l-naphthoxy)acetic acid (4). 

Why was a volatile ion-pairing reagent used in this work ... 

An involatile ion-pairing reagent would be deposited in the electrospray interface and lead to a reduction in performance. Some interfaces have been specifically designed to minimize this by removing the line-of-sight between the spray and the entrance to the mass spectrometer, and are thus more tolerant to involatile buffers. The performance of the interface will be improved by the use of volatile alternatives. 

Ion-pairing reagent A compound that forms a complex with an ionic compound to allow its analysis using HPLC. 

High-performance liquid chromatography (HPLC) with a micellar mobile phase or with a selective pre-column or reaction detection system has also been used to determine alkylenebis(dithiocarbamaes). ° Zineb and mancozeb residues in feed were determined by ion-pair HPLC with ultraviolet (UV) detection at 272 nm. These compounds were converted to water-soluble sodium salts with ethylenediaminetetra-acetic acid (EDTA) and sodium hydroxide. The extracts were ion-pair methylated with tetrabuthylammonium hydrogensulfate (ion-pair reagent) in a chloroform-hexane solvent mixture at pH 6.5-8.S. The use of an electrochemical detector has also been reported. ... 

Several extractions are required when the distribution constant is small. The addition -of salts, pH adjustment, ion-pairing reagents, etc., can be used to isprove the distribution of organic solutes into the extracting solvent. 

Typical ion-pairing reagents are, for cations, alkyl sulphonic acids, eg pentane, hexane, heptane or octane sulphonic acid, and for anions, tetrabutylammonium or dibutylamine ammonium salts. In ion-pair chromatography the retention of solutes can be controlled in a number of ways ... 

Conditions as Fig. 3.3f(i) except that hexane sulphonic acid is used as the ion-pair reagent. 

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