Carbohydrates separation

Sugar analysis by hplc has advanced greatly as a result of the development of columns specifically designed for carbohydrate separation. These columns fall into several categories. (/) Aminopropyl-bonded siHca used in reverse-phase mode with acetonitrile—water as the eluent. (2) Ion-moderated cation-exchange resins using water as the eluent. Efficiency of these columns is enhanced at elevated temperature, ca 80—90°C. Calcium is the usual counterion for carbohydrate analysis, but lead, silver, hydrogen, sodium, and potassium are used to confer specific selectivities for mono-, di-, and... 

Alkaline conditions are used so frequently in carbohydrate separations on CarboPac columns that it should be pointed out that acidic conditions are suitable for separation of acidic sugars. Figure 24 shows the separation of sialic acid containing oligosaccharides.256 Alkaline conditions were used for neutral milk oligosaccharides and mucin oligosaccharide alditols were characterized similarly.257 The carbohydrates released from yeast mannopro-tein with N-acetyl-P-D-glucosaminidase were also fractionated on CarboPac ... 

This chapter reviews the adsorptive separations of various classes of non-aromatic hydrocarbons. It covers three different normal paraffin molecular weight separations from feedstocks that range from naphtha to kerosene, the separation of mono-methyl paraffins from kerosene and the separation of mono-olefins both from a mixed C4 stream and from a kerosene stream. In addition, we also review the separation of olefins from a C10-16 stream and review simple carbohydrate separations and various acid separations. 

It is assumed that the reader is familiar with such common chromatographic concepts as efficiency, selectivity, capacity factors, and theoretical plates, and how these parameters affect and effect chromatographic resolution. Excellent descriptions of these general chromatographic principles have been published. Other reviews on various aspects of carbohydrate separations will be cited in the appropriate Sections. 

In the literature, isocratic solvent systems are found for monosaccharides, and gradient systems are described for oligosaccharides. Table 3.4 gives some gradient systems for carbohydrate separation Table 3.5 illustrates programs for pulsed amperometric detectors. 

The reaction of carbohydrates with ethylenediamine sulfate produces a stable fluorescence with excitation at 394 nm and emission at 470 nm. The reaction is specific for aldehydes and aliphatic polyhydroxyl compounds. It is used for the spray detection of carbohydrates separated by paper chromatography [109]. 

Analytical Properties Polar phase useful for sugar and carbohydrate separation not recommended for samples that contain aldehydes and ketones Reference 2, 3... 

Solms and Deuel95 initially prepared a wholly synthetic, borylated polymer by using m-phenylenediamine, p-aminophenylboron dichloride, and formaldehyde, and they investigated carbohydrate separations on it, but addition polymers have usually been favored. Thus, ribonucleosides and deoxyribonucleosides have been efficiently separated on a column of a mixed copolymer of the methacroyl derivatives96 51 and 52, and the method has been extended to... 

Because the two regioisomeric products 8a and 8b have almost the same molecular dimensions, it is difficult to discriminate between the two isomers with the geometric constraints imposed by the zeolite pores. Considering that calcium ions are apt to form mainly five-membered chelate complexes with polyhydroxy compounds (Fig. 4b) 32,33) and that calcium zeolites have also been employed as sorbents in carbohydrate separations (ii), it is possible to speculate that in the CaY-supported NaN3 system the epoxy alcohol first forms a coordinated structure around a calcium ion, as shown in Fig. 4a, followed by ring opening with an azide anion at the C-3 position of the epoxy alcohol, giving a stable, five-membered chelate complex with the calcium ion. 

Liquid separation Liquid drying Trace impurity removal Xylene, cresol, cymene isomer separation Fructose-glucose separation Fatty chemicals separation Breaking azeotropes Carbohydrate separation... 

Sugars are typically separated on propylamino bonded phases using iso-cratic elution with about 70-80% acetonitrile, depending on the stationary phase. Oligosacharides require a larger concentration of water for elution. A polymeric packing such as Asahipak NHj, which does not have the hydrolysis problems of silica-based propylamino phases, is an excellent substitute. Commonly, carbohydrate separations are carried out isocratically, since a refrac-tometer is used for detection. But one can also use a UV detector at <210 run. 

Precolumns can also be used to condition the mobile phase. In this case they are usually operated in a position upstream from the sample injection point. For example, a silica column loaded with a large amount of amine modifier can be used to equilibrate the mobile phase with the amine modifier. This technique has been employed in carbohydrate separations on silica columns that are dynamically coated with an amine modifier. Silica columns have been used to saturate the mobile phase with silica in an attempt to prolong the column life of silica-based columns in alkaline mobile phases. That this technique actually works has, however, not yet been demonstrated unambiguously. 

The retentivity of normal silica-based reversed-phase packings is barely enough to achieve retention of sugars. Also, not all C g bond phases are useful for carbohydrate analysis. A typical mobile phase is 100% water. In this mobile phase, well-endcapped packings often undergo hydrophobic collapse, namely, a sudden loss in retention. Nonendcapped Cjg bonded phases are therefore more suitable for carbohydrate separation by reversed-phase chromatography. 

Silica coated with a crosslinked polyimine serves the same purpose. Yet packings based on an organic polymer are hydrolytically more stable. An example is Asahipak NHj, which is a polyamine-derivatized polyvinylalcohol-based packing. Since there is no concern about any dissolution of the polymer, this packing is currently probably the best choice ot carbohydrate separations via HILIC. 

Numerous research papers and reviews on carbohydrate separations by CE have been written for the past several years. Researches have successfully addressed problems, such as tremendous diversity and complexity of this class of compounds, polar and neutral nature of most carbohydrates, their low ultraviolet (UV) extinction coefficients, and lack of functional groups. In the previous edition of this book, Olechno and Nolan [16] published a comprehensive overview of the CE separation techniques, attempted and developed for intact and derivatized carbohydrates, charged and neutral, as well as detection approaches by UV, indirect fluorescence, electrochemical (e.g., amperometric) detection, refractive index, and laser-induced fluorescence (LIE). A variety of buffer systems were... 

Sheath-flow and nanospray ESI interfaces have successfully coupled CE to almost every mode of MS including the use of quadrupole, time-of-flight, ion-trap, and magnetic sector instruments. CE-MS has been employed in the analysis of a wide range of analytes but has been especially successful in biological assays. The mass selectivity of MS detection is especially useful in complex DNA, protein, and carbohydrate separations. 

In chromatography it is always necessary to adjust conditions so that retention times of the analytes fall within a convenient time period. This is usually accom-pHshed by adjusting the ionic concentration of the mobile phase, but water is commonly used as the mobile phase for carbohydrate separations, so other means... 

Foster [198] has used2,3,4,6-tetramethyl-D-glucose as non-migrating substance and glucose as reference substance for carbohydrate separations... 

The major advantage of anion-exchange chromatography for carbohydrate separations is the ability to experimentally affect both the retention times and the elution order. Figure 3.209 shows the dependence of retention on the hydroxide ion concentration, exemplified by some selected carbohydrates. This figure reveals that optimal resolution between raffmose, stachyose, and maltose is obtained with 0.15 mol/L NaOH on this column. 

Figure 3.228 Influence of Ba(ll) on carbohydrate separation by anion-exchange. Separator column CarboPac PA1 eluent (a) 0.1 mol/L NaOH, (b) 0.1 mol/L NaOH +1 mmol/L Ba (0H)2, flow rate 1 mL/min detection pulsed amperometry on a gold working electrode ...

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