Phenolic acid separation

Table 3.2 Different reversed-phase HPLC mobile phase gradients used in phenolic acid separation. 

The columns used for the separation of phenolic acids are mainly reversed phase (RP), other sihca-based chemically bound phases, and non-silica polymers or mixed inorganic-organic phases. Special silica sorbents with reduced metallic residue contents and minimum residual silanol groups on the surface could positively influence peak symmetry without the strict demands for the successful separation of acidic analytes. Almost exclusively, RP C18 phases ranging from 100 to 250 mm in length and usually with an internal diameter of 3.9 to 4.6 mm are recommended. Particle sizes are in the range of 3-10 pm. Short 50- 100-mm columns with 3-pm particles have also been tested for fast phenolic acid separations. Narrow bore columns (internal diameter 2 mm) are recommended especially for HPLC-MS applications.Some problems could arise with the applications of narrow or microbore columns in the direct injections of plant extracts there is the possibility of plugging the column after repeated injections. In these cases, an additional clean-up step has to be applied instead of just the simple extraction... 

Fig. 4.2 Chromatogram of standard phenolic acids separated as their methyl ester-trimethylsilyl ether derivatives on a 6 ft column (0.004 m i.d.) packed with 10 per cent F-60 on silanized Gas Chrom P (80-100 mesh) using temperature programming from 100°C to 240°C at 2°C min" Peak identifications are 1, 2-hydroxyphenylacetate 2, 3-hydroxyphenylacetate 3, 4-hydroxyphenylacetate 4, indoleacetate 5, homovanil-late 6, homogentisate 7, vanillylmandelate 8, 5-hydroxyindoleacetate 9, nonadecanoate (standard). (Redrawn with modifications from Horning etal, 1966)...

Concentrate the mother liquors from this recrystallisation and combine with the oily filtrate dissolve in 250 ml. of 10 per cent, sodium hydroxide solution, and extract with two 50 ml. portions of ether to remove non-phenolic products. Acidify the alkaline solution with hydrochloric acid, separate the oily layer, dry it over anhydrous magnesium sulphate, and distil under diminished pressure, preferably from a Claisen flask with fractionating side arm (Figs. II, 24, 2-5). Collect the o-propiophenol (65 g.) at 110-115°/6 mm. and a further quantity (20 g.) of crude p-propiophenol at 140-150°/ 1 mm. 

P-Hydroxy-a-naphthaldehyde, Equip a 1 litre three-necked flask with a separatory funnel, a mercury-sealed mechanical stirrer, and a long (double surface) reflux condenser. Place 50 g. of p-naphthol and 150 ml. of rectified spirit in the flask, start the stirrer, and rapidly add a solution of 100 g. of sodium hydroxide in 210 ml. of water. Heat the resulting solution to 70-80° on a water bath, and place 62 g. (42 ml.) of pure chloroform in the separatory funnel. Introduce the chloroform dropwise until reaction commences (indicated by the formation of a deep blue colour), remove the water bath, and continue the addition of the chloroform at such a rate that the mixture refluxes gently (about 1 5 hours). The sodium salt of the phenolic aldehyde separates near the end of the addition. Continue the stirring for a further 1 hour. Distil off the excess of chloroform and alcohol on a water bath use the apparatus shown in Fig. II, 41, 1, but retain the stirrer in the central aperture. Treat the residue, with stirring, dropwise with concentrated hydrochloric acid until... 

In the case of low temperature tar, the aqueous Hquor that accompanies the cmde tar contains between 1 and 1.5% by weight of soluble tar acids, eg, phenol, cresols, and dihydroxybenzenes. Both for the sake of economics and effluent purification, it is necessary to recover these, usually by the Lurgi Phenosolvan process based on the selective extraction of the tar acids with butyl or isobutyl acetate. The recovered phenols are separated by fractional distillation into monohydroxybenzenes, mainly phenol and cresols, and dihydroxybenzenes, mainly (9-dihydroxybenzene (catechol), methyl (9-dihydtoxybenzene, (methyl catechol), and y -dihydroxybenzene (resorcinol). The monohydric phenol fraction is added to the cmde tar acids extracted from the tar for further refining, whereas the dihydric phenol fraction is incorporated in wood-preservation creosote or sold to adhesive manufacturers. Naphthalene Oils. Naphthalene is the principal component of coke-oven tats and the only component that can be concentrated to a reasonably high content on primary distillation. Naphthalene oils from coke-oven tars distilled in a modem pipe stiU generally contain 60—65% of naphthalene. They are further upgraded by a number of methods. 

In the second half of the 1960s, at the same time but independently, three basically different plastic separators were developed. One was the polyethylene separator [16] already referred to in starter batteries, used only rarely in stationary batteries, but successful in traction batteries. The others were the microporous phenolic resin separator (DARAK) [18] and a microporous PVC separator [19], both of which became accepted as the standard separation for stationary batteries. They distinguish themselves by high porosity (about 70 percent) and thus very low electrical resistance and very low acid displacement, both important criteria for stationary batteries. 

Many of the phenols which are used in household and other commercial disinfectant products are produeed from the tar obtained by distillation of coal or more recently petroleum. They are known as the tar acids. These phenols are separated by fractional distillation according to their boiling point range into phenol, cresols, xylenols and high boiling point tar acids. As the boiling point increases the properties of the products alter as shown ... 

Typically lipids, chlorophyll, and phenolic acids can be separated by liquid-liquid partition. Lipids and chlorophyll can be removed from acetone-water extracts by chloroform while phenolic acids have higher affinities for ethyl acetate at a pH close to nentral and water. °°... 

Males et al. [103] used aqueous mobile phase with formic acid for the separation of flavonoids and phenolic acids in the extract of Sambuci flos. In a cited paper, authors listed ten mobile phases with addition of acids used by other investigators for chromatography of polyphenolic material. For micropreparative separation and isolation of antraquinone derivatives (aloine and aloeemodine) from the hardened sap of aloe (Liliaceae family), Wawrzynowicz et al. used 0.5-mm silica precoated plates and isopropanol-methanol-acetic acid as the mobile phase [104]. The addition of small amounts of acid to the mobile phase suppressed the dissociation of acidic groups (phenolic, carboxylic) and thus prevented band diffusions. 

HPLC) for phenolic acids analysis. When procedure (ii) was applied, the ion-exchange resin was separated from the methanol phase and eluted with three 40 ml aliquots of 80% methanol. The resin bead eluates were evaporated to dryness and subjected to spectrophotometry (Shimadzu UV 160 spectrophotometer) for total phenolics and high-performance liquid chromatography (HPLC) for phenolic acids analysis. 

Procedure Phenolic acids were detected between 210 and 360 nm using a Hewlett Packard diode array detector (HP 1100 HPLC system). The separation was achieved with a Nucleosil 100-5 C18 column 5 pm 4.0x250 mm (Agilent Technologies, USA) at a flow rate of 1.0 ml/min and injection volume of 5 mL. For the elution, a discontinuous acetonitrile-water gradient was used 15% acetonitrile (5 min), 30% acetonitrile (20 min), 40% acetonitrile (25 min), 60% acetonitrile (30 min), 60% acetonitrile (35 min) and... 

Stalikas C. 2007. Extraction, separation, and detection methods for phenolic acids and flavonoids. J Sep Sci 30(18) 3268-3295. 

RP-HPLC has been employed for the determination of flavonoids and other phenolic compounds in cranberry juice. The neutral and acidic analytes were preconcentrated octadecyl silica SPE cartridges conditioned with distilled water (neutral analytes) or with 0.01 M HC1 (acidic compounds). Hydrolysis of samples was carried out in aqueous methanol solution acidified with 6 M HC1 at 35°C for 16h. Chromatographic separation was performed in an ODS column (150 X 4.6mm i.d. particle size 5/.an). Solvents A and B were water-acetic acid (97 3, v/v) and methanol, respectively. The gradient started with 0 per cent B (flow rate, 0.9 ml/min), reached 10 per cent B in lQmin (flowrate, 1.0 ml/min) and increased to 70 per cent B in 40min (flowrate, 1.0 ml/min). Analytes were detected at 280 and 360 nm. Some typical chromatograms are presented in Fig. 2.71. The concentrations of flavonoids and phenolic acids are compiled in Table 2.69. It was stated that the SPE-HPLC procedure makes possible the simultaneous determination of phenolic compounds and flavonoids, therefore, it can be employed for the measurement of these classes of analytes in other fruit juices [188],... 

Many nonvolatile and thermally labile allelochemicals can be well separated by liquid chromatography (LC). Identification of the separated components on-line by mass spectrometry (MS) is of great value. Fused-silica LC columns of 0.22 mm ID packed with small-particle material are used in the described LC/MS system. The shape of the column end allows direct connection to a electron impact ion source of a magnetic sector mass spectrometer. Separations by LC are reported and LC/MS mass spectra are shown for monoterpenes, diterpene acids, phenolic acids and cardiac glycosides. The LC/MS system provides identification capability and high-efficiency chromatography with a universal detector. 

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. 

Figure 6. Separation of free phenolic acids. 1, Caffeic acid 2, p-coumaric acid 3, sinapic acid. Column 18 cm x 0.22 mm I.D. 3-um Spherisorb ODS. Mobile phase methanol-water-acetic acid (20 75 5). Detection TIC (ions of m/z <60 suppressed). Ion source temperature 210 C.

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