Carotenoids column

Alternate Protocol 2 Gradient Separation Using C 30 Carotenoid Column F2.3.9... 

Most of the early published work on the separation of carotenoids by HPLC employs C18 stationary phases. It has been shown that polymeric surface modified Cl8 columns are more selective toward isomers compared to monomeiically bound columns (Figure 4.3) (Sander et ah, 1994). In the 1980s and 1990s, a 30 carbon bound stationary phase (C30 column) was developed and coined the carotenoid column, because of... 

Figure 4.3 Comparison of separation of carotenoid standards on monomeric Cl8, polymeric C18, and C30 carotenoid column. Chromatographic conditions are 81 15 4 to 6 90 4 MeOH/MTBE/water over 90 min at ImL/min at 20 °C (Sander et al, 1994).

Gradient HPLC Analysis Using C30 Carotenoid Column... 

C30 carotenoid columns (329). However, a potential Umitation of electrospray is the relatively narrow range of linearity of response it shows for carotenoid quantitation (331). 

More specific methods involve chromatographic separation of the retinoids and carotenoids followed by an appropriate detection method. This subject has been reviewed (57). Typically, hplc techniques are used and are coupled with detection by uv. For the retinoids, fluorescent detection is possible and picogram quantities of retinol in plasma have been measured (58—62). These techniques are particularly powerful for the separation of isomers. Owing to the thermal lability of these compounds, gc methods have also been used but to a lesser extent. Recently, the utiUty of cool-on-column injection methods for these materials has been demonstrated (63). 

High performance liquid chromatography (HPLC) has been by far the most important method for separating chlorophylls. Open column chromatography and thin layer chromatography are still used for clean-up procedures to isolate and separate carotenoids and other lipids from chlorophylls and for preparative applications, but both are losing importance for analytical purposes due to their low resolution and have been replaced by more effective techniques like solid phase, supercritical fluid extraction and counter current chromatography. The whole analysis should be as brief as possible, since each additional step is a potential source of epimers and allomers. 

Because plants present chlorophylls and carotenoids simultaneously, it may be useful to separate both groups from each other in a laboratory or preparative scale in order to avoid contamination in further purification steps, mainly when they are prepared in large amounts. Clean-up procedures using an open column packed with absorbents such as alumina, magnesia, polyethylene powder, powdered sucrose, DEAE-Sepharose, starch, cellulose, or MgO HyfloSupercel are good approaches. MgO HyfloSupercel in a proportion of 1 1 or 1 2 is the usual adsorbent. Sucrose and cellulose are interesting as they do not alter the chlorophylls, but they are tedious to work with. 

The most common mobile phase is a gradient of petroleum ether or hexane with increasing concentrations of acetone or diethyl ether. Development of the column should be optimized for each sample to afford a quick and effective separation to avoid band broadening. The separation can be followed visually. The most non-polar a- and 3-carotenes are eluted first as a yellow band followed by the chlorophylls and other more polar carotenoids like cryptoxanthin, lutein, and zeaxanthin that frequently fuse together and appear as a single band. ... 

Although saponification was found to be unnecessary for the separation and quantification of carotenoids from leafy vegetables by high performance liquid chromatography (HPLC) or open column chromatography (OCC), saponification is usually employed to clean the extract when subsequent purification steps are required such as for nuclear magnetic resonance (NMR) spectroscopy and production of standards from natural sources. 

However, complete hydrolysis of carotenoid esters sometimes is not achieved in 1 to 3 hr. The saponification degree can be verified easily by the presence of carotenol ester peaks eluting later than the peaks of P-carotene on reversed phase columns. Retinol palmitate, added as an internal standard to orange juice, also serves to indicate whether saponification is complete, since it is converted to retinol which elutes at lower retention time. The mixture is subsequently washed with water until free of alkali in a separatory funnel. Other more polar solvents such as CH2CI2 or EtOAc, and diethyl ether alone or mixtured with petroleum ether can be used to increase the recovery of polar xanthophylls from the water phase. 

No expensive equipment is required for OCC however, the separation efhciency depends on the analyst s experience since a new column has to be packed for each analysis. In addition, depending on the packing type (powder or slurry), stationary phase, and purpose of the separation, the separation can take from 30 min to 4 hr. The AOAC official method for the determination of carotenoids still uses OCC." Separation of carotenoids from many foods was developed on a column packed with a mixture of MgO and HyfloSupercel (or celite or diatomaceous earth) at 1 1... 

An open column packed with neutral aluminium oxide (grade III) slurry is generally used for semi-preparative separation of large amounts of carotenoid extract, revealing three broad bands (1) carotenes and epoxy-carotenes constitute the first fraction to elute with petroleum ether, (2) monohydroxy and keto-carotenoids with 50 to 80% diethyl ether in petroleum ether are next, and (3) finally, the polyhydroxy carotenoids elute with 2 to 5% diethyl ether in ethanol or... 

Although some normal phase methods have been used, the majority of carotenoid separations reported in the literature were carried out by reversed phase HPLC. Among the Cjg columns employed for determination of complete carotenoid compositions in foods, the polymeric Vydac brand is preferably used for separation of cis isomers. Several examples of different C,g columns and mobile phases are cited in the literature, but not aU carotenoids are baseline separated in most systems. Table 6.2.1 shows some examples employing different brands of Cjg columns." Acetonitrile did not improve selectivity toward separation of carotene isomers in a Vydac 201TP column and resolution was strongly dependent on the Vydac column lot. ... 

Most laboratories now employ C30 columns for separation of carotenoids from complex matrices. There are several examples for separation of carotenoids from foods such as orange, watermelon, mango, camu-camu, carrot, spinach, tomato, " sweet com, and potato. The C30 column systems shown in Table... 

HPLC Systems Employing Reversed Phase C,8 Columns for Separation of Carotenoids... 

In recent years, the methods for carotenoid determination without saponification have increased. Independently of the mobile phase and food composition, there are similar patterns of chromatographic separation on reversed phase columns. A chromatograph can be divided roughly into four zones the first zone corresponds to free xanthophyUs, the second zone to monoesterified pigments, the third zone contains carotenes, and finally the fourth zone corresponds to diesterified carotenoids. - ... 

Independently of the reversed phase column, the addition of TEA to the mobile phase increases carotenoid recovery from the column." Increased recoveries of 18% lutein, 33% zeaxanthin, 33% P-cryptoxanthin, 53% lycopene, 30% a-carotene, and 42% P-carotene in a Vydac column were observed after the addition of 0.1% TEA to the mobile phase. Recovery on a C30 column was also enhanced by the addition of 0.1% TEA to the mobile phase, with the peak area of lutein increasing by 26%, that of zeaxanthin by 42%, that of P-cryptoxanthin by 55%, that of lycopene by 21%, and those of a-carotene and P-carotene by 47 and 64%, respectively. ... 

Temperature has an influence on the retention and consequently on the capacity factors of carotenoids in HPLC columns. Usually, as the column temperature increases, the retention decreases however, in a polymeric C30 column, after an initial decrease of the t values of cis isomers of carotenoids, the retention of cis isomers actually increases at temperatures above 35°C. This different behavior can be explained by the increased order and rigidity of the C30 stationary phase at lower temperatures that in turn induce preferential retention of long, narrow solutes as the trans isomer and partial exclusion of bent and bulky cis isomers. The greater chain mobihty and less rigid conformation of the C30 at higher temperatures may increase the contact area available for interaction with the cis isomers and also may lower... 

APCl in positive mode ionization and triple quadrupole detection was used for determination of free and bound carotenoids in paprika, obtaining the [M + H]+ and losses of fatty acids as neutral molecules from the [M + H]+ with MeOH, MTBE, and H2O as eluent from the C30 column. The positions of the fatty acids on unsymmetrical xanthophylls could not be established by the MS data. 

Epler, K.S. et al.. Evaluation of reversed-phase liquid chromatographic columns for recovery and selectivity of selected carotenoids, J. Chromatogr, 595, 89, 1992. 

Emenhiser, C. et al.. Separation of geometrical carotenoid isomers in biological extracts using a polymeric Cjq column in reversed-phase liquid chromatography, J. Agric. Food Chem., 44, 3887, 1996. 

Figure 4.7 shows the structures of important carotenoids (all-E) lutein, (all-E) zeaxanthin, (all-E) canthaxanthin, (all-E) p-carotene, and (all-E) lycopene. Employing a self-packed C30 capillary column, the carotenoids can be separated with a solvent gradient of acetone water=80 20 (v/v) to 99 1 (v/v) and a flow rate of 5 pL min, as shown in Figure 4.8 (Putzbach et al. 2005). The more polar carotenoids (all-E) lutein, (all-E) zeaxanthin, and (all-E) canthaxanthin elute first followed by the less polar (all-E) p-carotene and the nonpolar (all-E) lycopene. Figure 4.9 shows the stopped-flow II NMR spectra of these five carotenoids. The chromatographic run was stopped when the peak maximum of the compound of interest reached the NMR probe detection volume.

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. 

Several researchers have tried to isolate cellular CBPs from the silkworm. In Nakajima s study (1963), the whole midgut mucosa was homogenized and the proteins separated with a gel-filtration chromatography column. Carotenoids were found in certain fractions containing proteins, suggesting the existence of CBPs in the midgut. Jouni and Wells purified a 35 kDa protein containing lutein... 

A Waters 2690 Alliance HPLC equipped with a 996 photodiode array and a 896 IJV/Vis detector was used for carotenoid analysis. The column (Phenomenex, Torrance, CA) was a 250x4.6mm Ultracarb 3 pm C-18 stationary phase and elution was carried out isocratically at a flowrate of l.OmL/min with 85 15 (v v) acetonitrileimethanol (HPLC grade) containing 0.1% triethyl amine to prevent on-column carotenoid decomposition. 

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