Carbohydrates mobile phase additives

Mobile-phase additives can also influence the relative abundance of the various adduct ions. Karlsson [105] performed post-column addition of alkali cations to enhance ESI-MS of carbohydrates and other compormds without nitrogen atoms. For most analytes, the adduct formation increased with the size of the cation. Optimum concentration of the cation in the solution was ca. 5x10 mol/1. Alkali-metal affituties and alkali-metal influence on fragmentation in MS-MS have been studied by others as well [106-107]. 

The selectivity of separation of some closely related carbohydrates can be modified by mobile phase additives. Typical examples are boric acid, phenylboronic acid and 2-aminoethyl dipheny borinate (NST) (64,65,67,68). 

It is used in IC systems when the amperometric process confers selectivity to the determination of the analytes. The operative modes employed in the amperometric techniques for detection in flow systems include those at (1) constant potential, where the current is measured in continuous mode, (2) at pulsed potential with sampling of the current at dehned periods of time (pulsed amperometry, PAD), or (3) at pulsed potential with integration of the current at defined periods of time (integrated pulsed amperometry, IPAD). Amperometric techniques are successfully employed for the determination of carbohydrates, catecholamines, phenols, cyanide, iodide, amines, etc., even if, for optimal detection, it is often required to change the mobile-phase conditions. This is the case of the detection of biogenic amines separated by cation-exchange in acidic eluent and detected by IPAD at the Au electrode after the post-column addition of a pH modiher (NaOH) [262]. 

Most liquid chromatographic experiments performed with PAD employ alkaline mobile phases or use postcolumn addition of base to get the electrode at the appropriate pH for the formation of the oxide. The exceptions to this are the detection of carbohydrates and alcohols in acidic media and the detection of sulfur compounds. The oxidation of carbohydrates and alcohols is not oxide catalyzed, and since they exhibit a stronger adsorption to piatinum than gold, they can be determined under acidic conditions. Sulfur compounds are adsorbed at oxide-free surfaces, and the kinetics for detection are favorable even at pH values below 7. 

The use of NCW as the mobile phase in liquid chromatography was recently reviewed. In this area, in addition to its green credentials, NCW is compatible with a range of detection methods flame ionization detection, mass spectrometry (MS) and UV (to short wavelengths). The reason for the recent growth in this area is the development of more thermally stable stationary phases. It has been used to analyse a growing number of analytes (alkylbenzenes, phenols, ketones, carboxylic acids, amino acids, carbohydrates and some pharmaceuticals). For example, carbohydrates (monosaccharides, disaccharides and sugar... 

Probably the application of system peaks of most practical importance is the detection and quantitation of analytes which cannot be detected directly. Then, an additive that is easy to detect and whose concentration can readily be monitored is added to the mobile phase. In most cases, this method is applied for the detection of compounds that have no UV chromophores in the range of conventional UV absorption photometry (e.g., triglycerides and other lipids, carbohydrates), and a UV detector is used with a conjugated aromatic compoimd as additive. The method has been used also with fluorescence [24] and electrochemical detection [25]. 

Most chiral HPLC analyses are performed on CSPs. General classification of CSPs and rules for which columns may be most appropriate for a given separation, based on solute structure, have been described in detail elsewhere. Nominally, CSPs fall into four primary categories (there are additional lesser used approaches) donor-acceptor (Pirkle) type, polymer-based carbohydrates, inclusion complexation type, and protein based. Examples of each CSP type, along with the proposed chiral recognition mechanism, analyte requirement(s), and mode of operation, are given in Table 3. Normal-phase operation indicates that solute elution is promoted by the addition of polar solvent, whereas in reversed-phase operation elution is promoted by a decrease in mobile-phase polarity. 

Sluny packing is the oldest and most reliable way for the novice to pack a column. It is the only packing mode available for materials that swell in the mobile phase, such as the carbohydrate packings. The desired amount of adsorbent is placed in a beaker and a solvent added see Fig. 12). The mixture is stirred and additional solvent added if needed, to obtain a pourable slurry. The slurry must not be so thick that air bubbles are trapped in the column or so thin that the column cannot be packed in one pour. For adsorbents that swell, sufficient time has to be allowed for the adsorbent to be fully solvated. Typically, one might add enough solvent to wet the adsorbent and produce a thick slurry and then wait overnight before adding additional solvent to produce the pourable slurry. 

Amberlite resin prior to injection into a dilute sodium hydroxide mobile phase. This provides the alkaline environment suitable for pulsed amperometry, although if reversed-phase chromatography provides better resolution of the carbohydrates under study, postcolumn addition of hydroxide can be carried out. 

The sensitivity increase for some selective carbohydrates that is caused by the addition of Ba(II) is shown in Figure 3.229. While retention of the first three components remains practically the same when adding barium acetate, a slight retention decrease is observed for fructose and sucrose. At first view, this seems surprising one might suppose that the complete removal of carbonate would lead to a retention increase. However, since in this case Ba(II) has been added as acetate salt, and acetate is a stronger eluent than hydroxide, even traces of acetate in the mobile phase will result in a retention decrease. The sensitivity increase is attributed by Cataldi et al [219] to the inhibition of the gold oxide formation by alkaline-earth metals in the order Ca(ll) > Sr(II) > Ba(II) that, in turn, results in an increase of the electrocatalytic activity of the electrode. 

A significantly better separation between the two disaccharides is obtained with a more complex NaOH gradient. Instead of zinc acetate, small amounts of sodium acetate are added to the mobile phase in order to elute more strongly retained saccharides within an acceptable time frame. Figure 3.236 shows a respective chromatogram with carbohydrates that are relevant for the analysis of urine to assess kidney functionality. In addition to lactose and lactulose, these carbohydrates include weso-erythritol, mannitol, sucrose, and turanose. Under these chromatographic conditions, resolution between lactose and lactulose is so large that even an excess of lactose does not prohibit clear quantitation of lactulose. 

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