INDEX reversed-phase

Picomole (10 mol) Polyaromatic hydrocarbon Refractive index Reversed phase Sodium dodecyl sulfate Signal-to-noise ratio Solute retention time Void volume of system Triethylamine Trifluoroacetic acid Tetrahydrofuran Ultraviolet... 

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 ... 

A useful guide when using the polarity index is that a change in its value of 2 units corresponds to an approximate tenfold change in a solute s capacity factor. Thus, if k is 22 for the reverse-phase separation of a solute when using a mobile phase of water (P = 10.2), then switching to a 60 40 water-methanol mobile phase (P = 8.2) will decrease k to approximately 2.2. Note that the capacity factor decreases because we are switching from a more polar to a less polar mobile phase in a reverse-phase separation. 

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. 

If the mixture to be separated contains fairly polar materials, the silica may need to be deactivated by a more polar solvent such as ethyl acetate, propanol or even methanol. As already discussed, polar solutes are avidly adsorbed by silica gel and thus the optimum concentration is likely to be low, e.g. l-4%v/v and consequently, a little difficult to control in a reproducible manner. Ethyl acetate is the most useful moderator as it is significantly less polar than propanol or methanol and thus, more controllable, but unfortunately adsorbs in the UV range and can only be used in the mobile phase at concentrations up to about 5%v/v. Above this concentration the mobile phase may be opaque to the detector and thus, the solutes will not be discernible against the background adsorption of the mobile phase. If a detector such as the refractive index detector is employed then there is no restriction on the concentration of the moderator. Propanol and methanol are transparent in the UV so their presence does not effect the performance of a UV detector. However, their polarity is much greater than that of ethyl acetate and thus, the adjustment of the optimum moderator concentration is more difficult and not easy to reproduce accurately. For more polar mixtures it is better to explore the possibility of a reverse phase (which will be discussed shortly) than attempt to utilize silica gel out of the range of solutes for which it is appropriate. 

Solvent strength determines the value, but not the selectivity. The mobile phase can be established by using the polarity index P proposed by Snyder. The highest values of P represent the strongest solute adsorbed in conventional TLC but represent the weakest for the separation in reversed phases. Sometimes aqueous polar mixtures cannot totally wet the chemically bonded layer. For this reason, checking... 

White et al.23 devised a reverse phase high performance Tiquid chromatographic procedure for erythromycin. Refractive index detection was used since the compound absorbs weakly in the UV. 

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. 

Multiangle light-scattering detectors are increasingly used to obtain on-line information on protein molecular weight. However, they must be used in combination with refractive index detectors, and so this technique is not compatible with reversed-phase gradient elution. 

Kuronen P, Volin P, Laitalainen T. 1998. Reversed-phase high-performance liquid chromatographic screening method for serum steroids using retention index and diode-array detection. J Chromatogr B 718(2) 211-224. 

Endo, Y., Tagiri-Endo, M., Seo, H. S., and Fujimoto, K. (2001). Identification and quantification of molecular species of diacylglyceryl ether by reversed-phase high-performance liquid chromatography with refractive index detection and mass spectrometry. J. Chro-matogr. A 911, 39-45. 

Many scales, either empirical or measured, have been proposed for the hydrophobicity of amino acid residues in proteins (Nakai and Li-Chan, 1988). The most extensive study on tlje hydrophobicity index of amino acids was published by Wilce et al. (1995). The authors derived four new scales of coefficients from the reversed-phase high-performance liquid chromatographic retention data of 1738 peptides and compared them with 12 previously published scales. 

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)... 

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-... 

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. 

General procedure for deprotection of mono- and polymethyl-aryl ethers with boron tribromide.41 To a 10-ml flask fitted with a septum and magnetic stirrer bar are added reactant (3.6 mmol) and 5 ml of dichloromethane. An inert atmosphere is established and maintained. This mixture is cooled in a dry ice/propan-2-ol bath and boron tribromide [0.13 ml, 1.32 mmol (for monomethyl ethers), or 0.38 ml, 4 mmol (for dimethyl ethers)] is added through the septum by use of a syringe. The cold bath is removed and the mixture stirred for 30 minutes, poured into ice water, stirred for 30 minutes, saturated with salt and extracted with dichloromethane. The extract is dried and concentrated. The purity of the product is established by h.p.l.c. analysis on a Waters Associates 6000A model using both refractive index and u.v. absorbance detectors with a Waters 3.9mm i.d. x 30cm p-Bondapack Ci8 reverse phase column. 

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