Eluents phenolate

The chemical stability of Toyopearl HW-40 resin to organic eluents allows this material to be used for a variety of applications, including the purification of synthetic functionalized surfactants, polyphenolics, and phenolic glycosides (50,51). 

An ECD measures the current generated by electroactive analytes in the HPLC eluent between electrodes in the flow cell. It offers sensitive detection (pg levels) of catecholamines, neurotransmitters, sugars, glycoproteins, and compounds containing phenolic, hydroxyl, amino, diazo, or nitro functional groups. The detector can be the amperometric, pulsed-amperometric, or coulometric type, with the electrodes made from vitreous or glassy carbon, silver, gold, or platinum, operated in the oxidative or reductive mode. Manufacturers include BSA, ESA, and Shimadzu. 

Retention volumes of monosubstituted benzenes, benzoic acid, phenols, and anilines have been measured in RPLC [76]. Buffered acetonitrile/water and tetrahydrofuran/water eluents were used with an octadecylsilica adsorbent. From the net retention volumes, a substituent interaction effect was calculated and described with the linear free energy relationship developed by Taft. The data was interpreted in terms of hydrogen bonding between the solutes and the eluent. 

Figure 2.2 Separation of aromatic compounds using isocratic elution. Conditions column, 5 pm Cis-bonded silica gel, 15 cm x 4.6 mm i.d. eluent, 0.001 M phosphoric acid in 55% aqueous acetonitrile flow rate, 1ml min-1 temperature, ambient, detection, UV 254 nm. Peaks 1, phenol, 2, 4-methylphenol 3, 2,4-dimethylphenol 4, 2,3,5-trimethylphenol 5, benzene, 6, toluene, 1, ethylbenzene, 8, propylbenzene and 9, butylbenzene.

Figure 4.7 Anion exchange separation of carboxylic acids in red wine. Column, Shodex C811, 100 cm x 7.6 mm i.d. eluent, 3 mM perchloric acid flow rate, 0.9 ml min-1 temperature, 60 °C detection, reaction detection using chloro-phenol red at 430 nm. Peaks 1, citric acid 2, tartaric acid 3, malic acid 4, succinic acid 5, lactic acid 6, formic acid and 1, acetic acid.

Due to the popularity of THF as an eluent for GPC, this sort of "differential solvation" must be kept in mind, particularly when polar solutes are analyzed. This effect can also work against resolution in SMGPC, as demonstrated in Figure 1. Here BHA and BHT are fully resolved in CHCI3 but coelute in THF. Both BHA and BHT have phenolic sites, but the site on BHT is sterically hindered and apparently does not form a hydrogen bond with THF. The hydrogen bonded BHA/THF complex which does form... 

Fio. 23. Dependence of ralentioa bctor on eolvent oompoiitkm. using methanol-water mixture as the eluent. The data, plotted on a togaritlunic scaie, were obtained with butanol, jwntanol, and phenol as the ehute on Si-100 which had been treated, with trichlorobu-tylsilane (—) or with trichlorpoctadecylsilane (——). Reprinted with permission from Karch el al. (143). 

Phenolic acids are often found in plant tissue, and have been implicated in many cases of allelopathy (4). Figure 6 shows a separation of three free phenolic acids and Figure 7 shows mass spectra obtained from these compounds. These spectra give both molecular weight and structural information. Phenolic acids can easily be thermally decarboxylated. The height of the molecular ion peak varies owing to ion source temperature. The variation depends also to some extent on the composition of the LC eluent, and this will be further examined. 

It is used in IC systems when the amperometric process confers selectivity to the determination of the analytes. The operative modes employed in the amperometric techniques for detection in flow systems include those at (1) constant potential, where the current is measured in continuous mode, (2) at pulsed potential with sampling of the current at dehned periods of time (pulsed amperometry, PAD), or (3) at pulsed potential with integration of the current at defined periods of time (integrated pulsed amperometry, IPAD). Amperometric techniques are successfully employed for the determination of carbohydrates, catecholamines, phenols, cyanide, iodide, amines, etc., even if, for optimal detection, it is often required to change the mobile-phase conditions. This is the case of the detection of biogenic amines separated by cation-exchange in acidic eluent and detected by IPAD at the Au electrode after the post-column addition of a pH modiher (NaOH) [262]. 

One disadvantage of using salt solution as eluent is that the lignin sulfonates tend to adsorb onto the gel matrix, resulting in a resolution inferior to that obtained by elution with water. On the other hand, elution behavior with water is adversely affected by the polyelectrolyte properties of the lignin sulfonates. Adsorption, which is caused by the phenolic hydroxyl... 

With 0.5M sodium hydroxide as eluent, Sephadex G-50 effects fractionation in the molar mass range 1000-15000 dalton and can be used for a period of 3-4 weeks with a single calibration carried out with proteins and polypeptides of known molar mass, as revealed by Figure 10. Relative retention volumes 0.0 and 1.0 are defined with Blue Dextran and phenol, respectively. 

Figure 25-12 Isocratic HPLC separation of a mixture of aromatic compounds at 1.0 mL/min on a 0.46 x 25 cm Hypersil ODS column (C,8 on 5-jxm silica) at ambient temperature ( 22 C) (1) benzyl alcohol (2) phenol (3) 3, 4 -dimethoxyacetophenone (4) benzoin (5) ethyl benzoate (6) toluene (7) 2,6-dimethoxytoluene (8) o-methoxybiphenyl. Eluent consisted ot aqueous buffer (designated A) and acetonitrile (designated B). The notation 90% B in the first chromatogram means 10 vol% A and 90 vol% B. The buffer contained 25 mM KH2P04 plus 0.1 g/L sodium azide adjusted to pH 3.5 with HCI.

A study of phenol red binding under different pH conditions may be completed by changing the pH of the reaction mixtures and Sephadex gel column. For each pH to be studied, the column must first be equilibrated with the proper buffer. Several buffers are available including acetate buffer (pH 4.0, 4.5, and 5.0) and phosphate buffer (pH 6.0, 7.0, and 8.0). Equilibrate the column with approximately 20-25 mL of the new buffer. To be assured of the proper pH, check the column eluent with a pH indicator strip or collect a fraction for measurement with a pH meter. Prepare the reaction mixtures by mixing the protein with the appropriate buffer and phenol red. Be sure to note that solutions of phenol red are prepared in different buffers. Load the reaction mixture on the equilibrated Sephadex G-25 column and develop and analyze the column fractions as described above. 

High-performance LC is also a suitable method for separating BHA isomers. Commercially available BHA is a mixture of two positional isomers, an approximately 85 15 ratio of 3-BHA and 2-BHA. The former is approximately 2.4 times more effective as a food antioxidant than is 2-BHA, and half as effective as an inhibitor of benzo(a)pyrene-induced for stomach neoplasia in mice. For the separation, Ansari (116) used isocratic elution with 7% of 2-propanol in hexane on a Pirkle Type I-A column packed with 5- 01 y-aminopropyl packing, modified with lV-3,5-dinitrobenzoyl derivative of D-phenylglycine. Column eluent was monitored at 288 nm, with a detection limit between 1 and 2 ng. Under these conditions, isomers were separated without derivatization, where the phenolic group of 3-BHA was sterically hindered by an o-rm-butyl group and therefore could not interact with stationary phase that resulted in its rapid elution. 

The variety of detection modes available for HPLC analysis that provide additional information about the eluent as it exits the column greatly facilitates unknown characterization. The majority of analytical methods for phenolic compounds includes HPLC with spectrophotometric-based detection techniques (UV absorption, fluorescence, photo diode array—PDA) as well as HPLC with electrochemical detection. 

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