Isocratic carotenoid separation

Basic Protocol 1 Isocratic Carotenoid Separation Using Wide-Pore,... 

ISOCRATIC CAROTENOID SEPARATION USING WIDE-PORE,... 

ISOCRATIC CAROTENOID SEPARATION CAPABLE OF SIMULTANEOUS SEPARATION OF RETINOL AND TOCOPHEROL USING SPHERISORB ODS2... 

Figure F2.3.1 Isocratic HPLC separation of carotenoid standards using Basic Protocol 1. Conditions 5-pm x 250-mm x 4.6-mm Vydac 201TP column, 90 10 methanol/acetonitrile mobile phase, 1.0 ml/min flow rate, visible detection at 450 nm, column temperature 25°C.

Nyambaka, H. and Ryley, J., An isocratic reversed-phase HPLC separation of the stereoisomers of the provitamin A carotenoids (a- and (3-carotene) in dark green vegetables, Food Chem., 55, 63, 1996. 

NMR spectroscopy is essential for the structure determination of carotenoid isomers because the TI-NMR signals of the olefinic range are characteristic for the arrangement of the isomers. The stereoisomers of astaxanthin, as shown in Figure 4.16, can be separated on a shape-selective C30 capillary column with methanol under isocratic conditions. 

The effect of temperature on the RP-HPLC behaviour of /(-carotene isomers has been extensively investigated and the results were employed for the separation of carotenoids of tomato juice extract. Carotenoids were extracted from food samples of 2g by adding magnesium carbonate to the sample and then extracted with methanol-THF (1 1, v/v) in a homogenizer for 5min. The extraction step was repeated twice. The collected supernatants were evaporated to dryness (30°C) and redissolved in methanol-THF (1 1, v/v). Separations were performed on a polymeric ODS column (250 X 4.6 mm i.d. particle size 5/.an). The isocratic mobile phase consisted of methanol-ACN-isopropanol (54 44 2, m/m). The flow-rate was 0.8 or 2.0 ml/min. The effect of temperature on the retention times of lycopene and four /(-carotene isomers is shown in Table 2.11. The data indicated that the temperature exerts a considerable influence on the retention time and separation of /(-carotene isomers. Low temperature enhances the efficacy of separation. 

Extracts were further purified on neutral alumina cartridges conditioned by passing through 5 ml of hexane. Extracts were loaded in hexane and washed by 5 ml of hexane. The at- and /1-carotenes were removed by 3.5 ml of acetone-hexane (10 90, v/v), other carotenoids were eluted with acetone-hexane 30 70 and 70 30 v/v. Prepurification of pigments was performed in subdued light under a stream of nitrogen. Analyses were carried out in a C30 column (250 X 4.6 mm i.d., particle size 5/tm) using isocratic mobile phase composed of methyl-ferf-butyl ether (MTBE)-methanol (3 97 and 38 62, v/v) at a flow rate of 1 ml/min. The column was not thermostated separations were achieved at room temperature (about 23°C). Carotenoids were detected at 453 and 460 nm (lutein). The... 

Calcium hydroxide is an excellent adsorbent for resolving carotenoid cis-trans isomers compared with alumina, it is less retentive and less sensitive to temperature and moisture content of the mobile phase. However, calcium hydroxide columns are not commercially available, and separations using them can be difficult to reproduce, owing to extreme sensitivities to mobile-phase composition and temperature. Nine cis-/3-carotenes were characterized from a chromatogram of 18 peaks after isocratic elution of a mixture obtained by thermal isomerization and photoisomerization of /3-carotene (162). Chandler and Schwartz (10), using a calcium hydroxide column and a mobile phase of hexane containing 0.3% acetone, separated six carotene isomers from canned carrots. These were, in order of elution, two cw-a-carotenes, all-tram-a-carotcnc, 13-m-/3-carotcnc, all- ram-/3-carotene, and 9-cw-/3-carotene. A mobile phase of hexane modified with 2% p-methylanisole separated all-rram-/3-carotcnc and its 9-, 13-, and 15-cis isomers in an iodine isomerized mixture (163). 

Nelis and De Leenheer (180) used isocratic NARP-HPLC with Zorbax ODS (a monomeric ODS stationary phase with a 20% carbon loading) and a mobile phase of acetonitrile/dichlorometh-ane/methanol (70 20 10) to separate nine carotenoids spanning a wide polarity range. This classic separation was achieved by virtue of the fact that the Zorbax ODS material supplied at that time was nonendcapped. The carotenes were retained by hydrophobic interaction with the ODS... 

HPLC is commonly used to separate and quantify carotenoids using C18 and, more efficiently, on C30 stationary phases, which led to superior separations and improved peak shape.32 4046 An isocratic reversed-phase HPLC method for routine analysis of carotenoids was developed using the mobile phase composed of either methanol acetonitrile methylene chloride water (50 30 15 5 v/v/v/v)82 or methanol acetonitrile tetrahydrofuran (75 20 5 v/v/v).45 This method was achieved within 30 minutes, whereas gradient methods for the separation of carotenoids can be more than 60 minutes. Normal-phase HPLC has also been used for carotenoid analyses using P-cyclobond46 and silica stationary phases.94 The reversed-phase methods employing C18 and C30 stationary phases achieved better separation of individual isomers. The di-isomers of lycopene, lutein, and P-carotene are often identified by comparing their spectral characteristic Q ratios and/or the relative retention times of the individual isomers obtained from iodine/heat-isomerized lycopene solutions.16 34 46 70 74 101 However, these methods alone cannot be used for the identification of numerous carotenoids isomers that co-elute (e.g., 13-ds lycopene and 15-cis lycopene). In the case of compounds whose standards are not available, additional techniques such as MS and NMR are required for complete structural elucidation and validation. 

Reversed-phase packing materials (Cig and Cg) are utilized especially for the separation of the different retinyl esters and for combined vitamin A and carotenoid mixtures such as those in food extracts and clinical samples. Most semiaqueous mobile phases, consisting of mixtures of methanol or acetonitrile and water, result in chromatographic runs of more than 1 h to elute retinyl stearate. Gradient elution shortens this elution time but results in a loss of separation of two predominant esters, retinyl palmitate and retinyl stearate. More recently, isocratic non-aqueous reversed-phase (NARP) conditions with an acetonitrile-dichloromethane mixture resulted in a successful separation of the different esters within 15 min. The big advantage of NARP conditions over semiaqueous systems is the increased solubility of lipids. To compensate for this increased affinity of the compounds of low polarity for the eluent, highly retentive stationary phases are recommended. 

Gradient elution should only be employed when the analysis cannot be done isocratically. Isocratic separation is rapid, can be performed with simple equipment (a single high-pressure pump and premixed solvent), and results in a stable baseline and more reproducible retention times. It is usually sufficient for the determination of provitamin A carotenoids or the principal carotenoids of natural samples. 

Because cis isomers have different biological potencies than their trans counterpart, it is necessary to separate and quantify cis isomers when they are present in appreciable amounts. This level of detail, however, makes the analysis even more complicated. The polymeric C30 column was developed specifically for this purpose [101, 102]. This colunm, with an isocratic solvent system consisting of methanol methyl-tert-bufyl ether (89 11), was used for the quantificatirm of cis-trans isomers of provitamin A carotenoids in fresh and processed fruits and vegetables [103]. 

This sectiOTi describes several liquid chromatographic methods for separating and analyzing carotenoids. The first procedure incorporates a reversed-phase separation using a wide-pore, polymerically synthesized C18 column with visible detection at 450 nm (see Sect. 4.2.1). The first alternate procedure is also isocratic Cl8 reversed phase but permits simultaneous analysis of retinol, tocopherols, and... 

Craft NE, Wise SA, Soares JH Jr (1992) Optimization of an isocratic high-performance liquid chromatographic separation of carotenoids. J Chromatogr 589 171... 

Milk. Simultaneous analysis of retinol and carotenoids present in human milk was carried out on a YMC RP 5-p.m ODS column (0.46 X 25 cm) by isocratic elution with tetrahydrofuran/methanol (90 10) (216). Thirty-four carotenoids, including 13 geometric isomers and eight metabolites, along with vitamins A and E in milk of lactating mothers, were separated by a combination of normal and RP-HPLC (217). 

Many spices also contain carotenoids. The composition and changes in concentration of different carotenoids during the ripening process in different species of pepper were studied by use of a binary gradient of water/acetone and a C18 RP column (245-247). An isocratic RP HPLC separation of capsanthin and capsorubin in Capsicum annum paprika and oleoresin on a Merck LiChros-pher 100 C18 5-p.m (0.4 X 25 cm) column with CH3CN/2-propanol/ethyl acetate (80 10 10) has been described (248). The carotenoids in saponified extracts of irradiated and ethylene oxide-treated red pepper were determined by HPLC on... 

Some of the methodologies used are outlined. For the separation of carotenes, interesting methods have been described. O Neil et al. evaluate the resolving capacity and quantihcation of Z/S-P-carotene isomers in four chromatographic columns and with various solvent systems, finding the Ca(OH)2 column the best. Biacs et al. employed the isocratic mixture acetonitrile/2-propanol/water (39 57 4) to separate esterified carotenoids of paprika. Khachik et al. quantify the major carotenoids and their esters in apricot, peach, melon, and grape. Heinonen et al. use this last method to analyze the carotenoid content of 69 types of vegetables and fruits, both fresh and processed. ... 

Bushway, R.J., Determination of a- and p-carotene in some raw fmits and vegetables by high-performance liquid chromatography, J. Agric. Food Chem., 34, 409, 1986. LeseUier, E. et al.. Optimization of the isocratic non-aqueous reverse phase (NARP) HPLC separation of trans/cis a- and p-carotenes, J. High Res. Chromatogr., 12,447,1989. Saleh, M.H. and Tan, B., Separation and identification of cis/trans carotenoids isomers, J. Agric. Food Chem., 39, 1438, 1991. 

Numerous works have been cited that use normal-phase silica columns and isocratic mixtures of solvents such as acetone/ligroin (20 80, v/v) for the quantification of chlorophylls and derivatives in phytoplankton [223], or iso-octane/98% ethanol (9 1, v/v) in spinach [224]. Watanabe et al. [225] separated chlorophylls and pheophytins using the isocratic mixture 2-propanol/n-hexane (3 97, v/v). This method yields chlorophylls with levels of purity above 99%. Abaychi and Riley [226], using the mixture petroleum ether/acetone/dimethylsulfoxide/diethylamine (75 23.25 1.5 0.25, v/v) as mobile phase, detected and quantified 16 pigments of chlorophylls, derivatives, and carotenoids. 

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