Reversed-phase liquid chromatography compositional analysis

An additional technique that has been found useful in analysis of the composition of polymers and blends is liquid chromatography. Pasch and Rode used the critical point of adsorption of the least polar component of a blend to determine the liquid chromatographic conditions for separating blends of polymethacrylates into components (50). High pressure liquid chromatography (HPLC) in combination with mass spectroscopy was used to analyze the components of an epoxy resin (51). HPLC has also been used with a precipitation-redissolution technique to separate polymer molecular weights for several polymers as a shorter technique compared to SEC (52). Reverse-phase liquid chromatography with UV detection was useful in qualitative determination of brominated flame retardants in polymeric waste materials (53). 

Normal-phase (NP) and reversed-phase (RP) liquid chromatography are simple divisions of the LC techniques based on the relative polarities of the mobile and stationary phases (Figure 4.10). Both NPLC and RPLC analysis make use of either the isocratic or gradient elution modes of separation (i.e. constant or variable composition of the mobile phase, respectively). Selection from these four available separation techniques depends on many variables but basically on the number and chemical structure of the compounds to be separated and on the scope of the analysis. 

The chemical properties of HVA, 5HIAA and 3-MD make them amenable to reverse-phase high-performance liquid chromatography (HPLC) with electrochemical detection. Furthermore, the composition of CSF means that little, if any, sample preparation is required prior to analysis. However, the susceptibility of these metabolites to oxidation means that careful sample collection and storage is required in order to minimise analyte degradation. 

A nonaqueous reversed-phase high-performance liquid chromatography (NARP-HPLC) with refractive index (RI) detection was described and used for palm olein and its fractions obtained at 12.5°C for 12-24 h by Swe et al. (101). The objective of their research was to find the optimum separation for analysis of palm olein triglycerides by NARP-HPLC, and to find a correction factor to be used in calculating CN and fatty acid composition (FAC). The NARP-HPLC method used to determine the triglyceride composition was modified from the method of Dong DiCesare (88). Palm olein was melted completely at 70°C in an oven for 30 min prior to crystal-... 

Analysis of DCLs by a combination of high-performance liquid chromatography (HPLC) and MS (LC-MS) has proven very powerful, provided the library members are stable enough to allow chromatographic separation. LC-MS analysis can yield both the quantity and the identity of individual library members. Care should be taken that the exchange between library members is slow on the timescale of the experiment. This is usually the case for hydrazone and disulfide libraries, without requiring special precautions. It has recently been reported that even the composition of DCLs of hydrolytically labile imines can be analyzed directly by HPLC, provided an acidic mobile phase is used. Beau et al. successfully used a common reversed-phase HPLC solvent system based on acetonitrile/water mixtures containing 0.1% trifluoroacetic acid [10]. Imine hydrolysis is slow under these conditions because at low pH the zwitterionic hemi-aminal intermediate that is believed to be important in the hydrolysis process is more difficult to attain (Scheme 2.1). 

Reversed-phase high-performance liquid chromatography (RP-HPLC) is more popular for analysis of carotenoids than is normal-phase HPLC because (1) retention is very little affected by small variations in the mobile-phase composition, and (2) the risk of artifact formation on passage through the column is minimal as solute-support interactions on non-polar-bonded phases only involve weak forces. A variety of stationary phases of various polarities are available, such as C18, C8, C4, C2, Cl, phenyl, and cyano derivatives the C18 phase is the most popular. 

In reverse phase high-pressure liquid chromatography (RP HPLC), the mobile phase is usually an aqueous-organic mixture, permitting the phenomenological theory to be applied. LePree and Cancino carried out this analysis. The composition-dependent variable is the capacity factor k, defined by eq. [8.2.51],... 

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