Reversed phase HPLC chain length

Reverse phase HPLC describes methods that utilize a polar mobile phase in combination with a nonpolar stationary phase. As stated above, the nonpolar stationary phase structure is a bonded phase—a structure that is chemically bonded to the silica particles. Here, typical column names often have the carbon number designation indicating the length of a carbon chain to which the nonpolar nature is attributed. Typical designations are C8, C18 (or ODS, meaning octadecyl silane), etc. Common mobile phase liquids are water, methanol, acetonitrile (CH3CN), and acetic acid buffered solutions. 

The above-given Martin equation form the basis for the Kovats retention index system in gas chromatography as well as for several HPLC retention prediction schemes. It must be noted here that the relationships between retention parameters and carbon numbers are usually linear at some limited range of the aliphatic chain length up to 6-8 carbon atoms in reversed-phase HPLC [491. 

High-performance liquid chromatography can be used as a separation method of volatile compounds, followed by GC or GC-MS for further separation and analysis. The most common assay is performed by reversed-phase HPLC, usually on Cig, when the separation of the compounds is according to their hydrophobicity, i.e., according to chain length and polar groups. 

AEO can be sensitively determined in the form of these corresponding UV-active phenylisocyanate derivatives by UV detection. In this case, the residue of the extraction of a water sample or a solid matrix is dissolved in dichloromethane or dichloroethane. This solution is mixed with phenylisocyanate as well as 1-octanol and/or 1-eicosanol as internal standards and heated to 55 to 60°C for 45 to 120 min. Then the AEO derivatives are separated either by reversed-phase HPLC with regard to different alkyl chain lengths or by normal phase HPLC with regard to different ethoxylate oligomers.The addition of the internal standard is imperative for quantitative determination because derivatization is not completed even after 2 h. ... 

A series of alternative reversed phase systems have been used for the separation of individual triglyceride species on the basis of their chain length and degree of unsaturation. For example, the first conventional reversed phase HPLC separation utiUsed a mobile phase of 60% aqueous methanol (Pei et al., 1975). It should be noted that the substitution of a methanol-acetone mobile phase for an acetonitrile-acetone phase causes small changes in retention time, with the introduction of one double bond in a fatty acid being... 

HPLC analysis of oligosaccharides from animal tissues or body fluids has been used to determine their molecular weight distribution. The majority of complex oligosaccharide separations has been carried out using chemically bonded amine columns which separate molecules on the basis of chain length, although reversed phase HPLC has been used to separate human milk oligosaccharides (Dua and Bush, 1983). 

Examples of reversed-phase HPLC bonded phases are given in Table 1. These include alkyl bonded silica, where the R group is an alkyl chain, the length of which determines the degree of hydrophobicity (C18, C8, C4, etc.) the phenyl stationary phase. 

Determination of the alkyl chain length distribution is readily performed by reversed-phase HPLC, as described in Chapter 7 (134). Taurates behave as strongly anionic surfactants and can be separated according to the ion exchange methods given in Chapter 6 (135). Taurates can be separated from other surfactants by TLC, as covered in Chapter 9 (136). 

Fatty acids are easily determined by gas chromatography, either high temperature GC of the free acids or lower temperature analysis of ester derivatives. All vendors of GC col-unrns will provide application information. The alkyl chain length may also be determined by reversed-phase HPLC, as described in Chapter 7. 

TLC allows determination of the main components and impurities simultaneously (Chapter 9). Reversed-phase HPLC analysis is most useful for the rapid determination of chain length distribution of the fatty acid portion of the alkanolamide. The materials are injected without deiivatization, and detected by UV absorbance at 210 nm (Chapter 7). Although some impurities appear in the chromatogram, the analysis is not as comprehensive as that obtained with GC. 

Alkyl polyglucosides are separated according to alkyl chain length by reversed-phase HPLC, reversed-phase TLC, and high-temperature GC, as described in the respective chapters later in this volume. Alternatively, alcohols can be liberated by acid hydrolysis of the APG and determined by HPLC (140). 

FIG. 1 Reversed-phase HPLC of LAS, with elution according to increasing alkyl chain length. (Reprinted with permission from Ref. 33. Copyright 1991 by Elsevier Science.)... 

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