INDEX reversed-phase HPLC

A reverse-phase HPLC separation is carried out using a mobile-phase mixture of 60% v/v water and 40% v/v methanol. What is the mobile phase s polarity index ... 

PTFE polytetrafluoroethylene PUFA polyunsaturated fatty acid PV peroxide value PVDF polyvinylidene difluoride PVP polyvinylpyrrolidone PVPP polyvinylpolypyrolidone RAS retronasal aroma stimulator RDA recommended dietary allowance RF radio frequency RFI relative fluorescence intensity RI retention index RNU relative nitrogen utilization ROESY rotational nuclear Overhauser enhancement spectroscopy RP-HPLC reversed-phase HPLC RPER relative protein efficiency ratio RS resistant starch RT retention time RVP relative vapor pressure S sieman (unit of conductance)... 

Reversed-phase HPLC has been used to analyze the oxidation products of triacylglycerols in edible oils. The detection is often based on monitoring the conjugated dienes with an ultraviolet detector (234-235 nm). However, the UV detector provides no information about oxidation products without a conjugated diene structure, e.g., products of oleic acid. Information about these compounds is important when oils with a high oleic acid content are studied. The most common universal detector types—refractive index and flame ionization detectors—are not sensitive enough to detect small amounts of oxidation products. 

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. 

The polarity index or the solubility parameter may be used as a measure of solvent strength, which would be a measure of polarity in those cases. For reversed phase HPLC, solvent strength parameters have been proposed for the four most common solvents used, i.e. water (Si = 0), methanol (Si = 2.6), acetonitrile (Si = 3.2) and THF (Si = 4.5). Using these values water makes no contribution to the eluting power of the mobile phase and the solvent strength is measured by the volume fraction of organic modifier. 

D. Bushec. I.S. Krull. R.N. Savage and S.B. Smith. Jr., Metal calion/anion speciation via paired-ion, reversed phase HPLC with refractive index and/or inductively coupled plasma emission spee-troseopic detection methods, / Eiq. Chromatogr.. S. 463. 1982. 

Slip point (ISO 6321, 2005) and solid fat index (AOCS method Cd 10-57, 2005) can provide information as to the suitability of an oil for use in manufacturing margarines and shortenings. Triacylglycerol (TAG) composition is an additional compositional analysis that can provide information on the potential functionality of an oil as well as its potential oxidative stability. Reversed-phase HPLC with various detection methods such as flame ionization, refractive index, evaporative light scattering, or atmospheric chemical ionization (coupled with mass spectrometry) can be used to determine TAG composition (Neff et d., 1994 Neff et al., 2001). 

Autoxidized trilinolein was separated into mono-, bis- and tris-hydroperoxides by preparative reversed phase HPLC using a 5 m C-18 column with UV at 235 nm and refractive index detectors (Chapter 2, F Figure 6.9). The monohydroperoxides of trilinolein were further resolved into the positional isomers components by normal phase HPLC on a 5 m silica column with UV detection (Figure 6.10). Complete and partial separation of various cis,trans- and trans,trans-2- and l(3)-mono-13- and 9-hydroperoxides were identified by lipolysis and capillary GC. The ratio of 1(3)- to 2-monohydroperoxides estimated by normal phase HPLC averaging 2.0 indicated that oxidation of trilinolein was not selective toward either the 1(3)- or 2-position. 

Figure 6.9. Preparative reversed-phase HPLC of autoxidized trilinolein with a 5 fjm C-18 column, 30 70 methylene chloride acetonitrile (v/v) as eluting solvent broken line, UV detection at 235 nm solid line, refractive index detection. From Neff etal. (1990). Courtesy of the American Oil Chemists Society.

It is noteworthy that cis- and /rans-isomers are well resolved, and reversed-phase HPLC has been suggested as a means of estimating such fatty acids (as the methyl esters) in hydrogenated fish oils [894]. In this instance, an isocratic mobile phase, consisting of methanol-water (89 11, v/v), was used with refractive index detection. Some positional isomers can also be resolved, and as an example various conjugated trienoic acids have been separated by the technique [900]. 

C refractive index (20°C) 1.4010 density (20°C) 0.73g/mL viscosity (25°C) 0.36 cP polarity index (P) 1.8 solubility in water (20°C) 5.5% water solubility in triethylamine (20°C) 4.6%. TWethy-lamine is commonly used in reversed-phase HPLC as a basic mobile phase modifier. TEA interacts with residual silanol groups on the support surface and limits the interaction of a basic analyte with these sites thereby decreasing peak taUing. 

The most common application of critical conditions in reversed-phase HPLC is the determination of PEG in ethoxylates. This is easily carried out with most reversed-phase media, using 95 5 methanol/water and refractive index or evaporative light scattering detection (90,91). PEG elutes prior to the surfactants. PPG can be determined in propoxy-lates in the same manner (92). 

FIG. 6 Reversed-phase HPLC analysis of an alkylpolyglycoside commercial mixture. Peak assignment as indicated. Differential refractive index detection. (Reprinted with the author s permission from Ref. 281.)... 

Amine oxides and various amphoteric surfactants can be determined by reversed-phase HPLC using a Cis column with a mobile phase of 90 10 or 80 20 methanol/water. Differential refractive index detection is suitable (179)... 

Hplc techniques are used to routinely separate and quantify less volatile compounds. The hplc columns used to affect this separation are selected based on the constituents of interest. They are typically reverse phase or anion exchange in nature. The constituents routinely assayed in this type of analysis are those high in molecular weight or low in volatility. Specific compounds of interest include wood sugars, vanillin, and tannin complexes. The most common types of hplc detectors employed in the analysis of distilled spirits are the refractive index detector and the ultraviolet detector. Additionally, the recent introduction of the photodiode array detector is making a significant impact in the analysis of distilled spirits. 

Separation and quantitation of carbohydrate mixtures may be achieved using HPLC, a method that does not necessitate the formation of a volatile derivative as in GLC. Both partition and ion-exchange techniques have been used with either ultraviolet or refractive index detectors. Partition chromatography is usually performed in the reverse phase mode using a chemically bonded stationary phase and acetonitrile (80 20) in 0.1 mol U1 acetic acid as the mobile phase. Anion- and cation-exchange resins have both been used. Carbohydrates... 

Various liquid chromatographic techniques have been frequently employed for the purification of commercial dyes for theoretical studies or for the exact determination of their toxicity and environmental pollution capacity. Thus, several sulphonated azo dyes were purified by using reversed-phase preparative HPLC. The chemical strctures, colour index names and numbers, and molecular masses of the sulphonated azo dyes included in the experiments are listed in Fig. 3.114. In order to determine the non-sulphonated azo dyes impurities, commercial dye samples were extracted with hexane, chloroform and ethyl acetate. Colourization of the organic phase indicated impurities. TLC carried out on silica and ODS stationary phases was also applied to control impurities. Mobile phases were composed of methanol, chloroform, acetone, ACN, 2-propanol, water and 0.1 M sodium sulphate depending on the type of stationary phase. Two ODS columns were employed for the analytical separation of dyes. The parameters of the columns were 150 X 3.9 mm i.d. particle size 4 /jm and 250 X 4.6 mm i.d. particle size 5 //m. Mobile phases consisted of methanol and 0.05 M aqueous ammonium acetate in various volume ratios. The flow rate was 0.9 ml/min and dyes were detected at 254 nm. Preparative separations were carried out in an ODS column (250 X 21.2 mm i.d.) using a flow rate of 13.5 ml/min. The composition of the mobile phases employed for the analytical and preparative separation of dyes is compiled in Table 3.33. 

Earlier work in the HPLC analysis of TGs used a differential refractometer as the detector a number of papers have detailed isocratic systems combined with refractive index (RI) detectors, often with acetonitrile/acetone mobile phases. Although aqueous mobile phases were generally used with alkyl-bonded phase columns, due to the lipophilicity of TGs, water could not be used in the mobile phase for this particular application therefore the mobile phases generally employed consisted of mixtures of acetone and acetonitrile and occasionally tetrahydrofuran, methylene chloride, or hexane (the conspicuous absence of water in the mobile phase prompted the term nonaqueous reverse phase, or NARP, to describe these systems). 

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-... 

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