T have a wide variety of lipid classes and 2D-TLC is

T have a wide variety of lipid classes and 2D-TLC is commonly used in plant lipid analysis. The presence of glycolipids and phospholipids with similar polarities make their separation through one-dimension chromatography difficult. The combination of solvent systems for 2D-TLC is chosen based on lipid class to be isolated. Although the quality of separation is highly improved by 2D-TLC, this technique presents disadvantages compared to one-dimensional: Since only a single sample can be applied on the plate, the simultaneous application of samples and standards is not possible, thus 2D-TLC is more time-consuming [101]. An alternative to 2D-TLC is to use a multiple order GW610742 development in a single dimension, as applied by Olsen and Henderson [102] in lipid separation of all major algae lipid classes. Olsen and Henderson used as a first solvent system methyl acetate-isopropanol-chloroform-methanol-0.25 KCl (25:25:25:10:4, v/v/v/v); after drying, the plates were developed with the second solvent system hexane-diethylether-acetic acid (70:30:2, v/v/v) [102]. In these types of experiments, the high performance TLC plates are commonly used, since they have high CI-1011 site resolution and sensitivity, presenting a good performance and reproducibility [103]. The analysis of lipids can be performed by observation of the intensity of the spots after spraying with a solution of primuline in acetone and visualization under a UV lamp, or by placing the plate in iodine vapor. After visualization of the spots, the identification is based on the comparison with migration of pure lipid standards applied to the same TLC plate. The relative quantification of lipid classes can be achieved by densitometry, based on specific colorimetric methods that can reflect the intensity of the spots or by separation of each spot and then using a specific colorimetric method. Quantification of phospholipids spots is based on phosphorous amounts, determined by Bartlett and Lewis [104]. Glycolipids can be quantified by sugar estimation using, for example, 5-methylresorcinol method or 5-hydroxy-1-tetralone, which forms a fluorescent product [105]. Analysis of the molecular species can also be achieved by scraping the spots of each lipid class, extracting the lipids in each spot with organic solvents and then analyzing the extract by MS-based approaches [106], which has the advantages of identifying all molecular species within each lipid class. The main advantage of the TLC is the possibility of obtaining a rapid screening of the sample being analyzed without the sophisticated equipment to separate lipids with different polarities. Apart from being time-consuming, other major disadvantages of this approach are its low resolution and sensitivity. TLC has been widely used to ascertain the polar lipidome of marine macrophytes, generally followed by off-line structural characterization; the majority of this characterization is performed by gas chromatography (GC) [21,26,29,45,60,61,63,65] or MS [39,52,57,107,108]. 4.2.2. Gas Chromatography (GC) Methods encompassing GC are usually employed to analyze fatty acid methyl esters (FAMEs) and are typically coupled to MS (GC-MS) or flame-ionization detection (GC-FID). GC methods are sensitive to compound polarity and need derivatization steps to improve volatility. Since FAs are mainly esterified to TGs, PLs and GLs, their derivatization is performed by transmethylation in the presence of alkaline or acid catalyst, with further analysis of FAMEs [109]. BF.T have a wide variety of lipid classes and 2D-TLC is commonly used in plant lipid analysis. The presence of glycolipids and phospholipids with similar polarities make their separation through one-dimension chromatography difficult. The combination of solvent systems for 2D-TLC is chosen based on lipid class to be isolated. Although the quality of separation is highly improved by 2D-TLC, this technique presents disadvantages compared to one-dimensional: Since only a single sample can be applied on the plate, the simultaneous application of samples and standards is not possible, thus 2D-TLC is more time-consuming [101]. An alternative to 2D-TLC is to use a multiple development in a single dimension, as applied by Olsen and Henderson [102] in lipid separation of all major algae lipid classes. Olsen and Henderson used as a first solvent system methyl acetate-isopropanol-chloroform-methanol-0.25 KCl (25:25:25:10:4, v/v/v/v); after drying, the plates were developed with the second solvent system hexane-diethylether-acetic acid (70:30:2, v/v/v) [102]. In these types of experiments, the high performance TLC plates are commonly used, since they have high resolution and sensitivity, presenting a good performance and reproducibility [103]. The analysis of lipids can be performed by observation of the intensity of the spots after spraying with a solution of primuline in acetone and visualization under a UV lamp, or by placing the plate in iodine vapor. After visualization of the spots, the identification is based on the comparison with migration of pure lipid standards applied to the same TLC plate. The relative quantification of lipid classes can be achieved by densitometry, based on specific colorimetric methods that can reflect the intensity of the spots or by separation of each spot and then using a specific colorimetric method. Quantification of phospholipids spots is based on phosphorous amounts, determined by Bartlett and Lewis [104]. Glycolipids can be quantified by sugar estimation using, for example, 5-methylresorcinol method or 5-hydroxy-1-tetralone, which forms a fluorescent product [105]. Analysis of the molecular species can also be achieved by scraping the spots of each lipid class, extracting the lipids in each spot with organic solvents and then analyzing the extract by MS-based approaches [106], which has the advantages of identifying all molecular species within each lipid class. The main advantage of the TLC is the possibility of obtaining a rapid screening of the sample being analyzed without the sophisticated equipment to separate lipids with different polarities. Apart from being time-consuming, other major disadvantages of this approach are its low resolution and sensitivity. TLC has been widely used to ascertain the polar lipidome of marine macrophytes, generally followed by off-line structural characterization; the majority of this characterization is performed by gas chromatography (GC) [21,26,29,45,60,61,63,65] or MS [39,52,57,107,108]. 4.2.2. Gas Chromatography (GC) Methods encompassing GC are usually employed to analyze fatty acid methyl esters (FAMEs) and are typically coupled to MS (GC-MS) or flame-ionization detection (GC-FID). GC methods are sensitive to compound polarity and need derivatization steps to improve volatility. Since FAs are mainly esterified to TGs, PLs and GLs, their derivatization is performed by transmethylation in the presence of alkaline or acid catalyst, with further analysis of FAMEs [109]. BF.