Fundamentals and Theory of HPTLC-Based Separation

Abstract High-performance thin-layer chromatography (HPTLC) is a form of thin-layer chromatography (TLC) that provides superior separation power using optimized coating material, novel procedures for mobile-phase feeding, layer conditioning, and improved sample application. It promotes for higher separation efficiencies, shorter analysis time, lower amounts of mobile phase, and efficient data acquisition and processing. The major parameters that influence separation of the constituents within a mixture are the partition coefficients, retention factor (R f ), and capacity factor of the individual constituents on the plate, selectivity of the mobile and stationary phase to the solutes, and the plate height that decide the separation efficiency as well as resolution of the individual constituents within a mixture. The partition coefficient is defined as the molar concentration of the analyte in the stationary phase to that in the mobile phase. R f , a fundamental qualitative value, is expressed as the ratio of migration distances of an individual components of a mix relative to the mobile phase. Capacity factor (k) is a fundamental characteristic of a substance that determines its qualitative chromatographic behavior. It can be expressed as the ratio of the retention time of the substance in the stationary to that in the mobile phase and is influenced by the chemical nature of the two phases. The separation number (SN) that influences separation power of HPLC is defined as the highest possible number of components that are completely separated in a mixture under a gradient-free isocratic TLC. The efficacy of separation of two components of a mixture in a chromatogram is termed as resolution and is influenced by the selectivity of the components between the stationary and the mobile phase, mobile phase flow rate influenced by particle size and solvent strength that influence capacity factors

Approaches in biotechnological applications of natural polymers

Natural polymers, such as gums and mucilage, are biocompatible, cheap, easily available and non-toxic materials of native origin. These polymers are increasingly preferred over synthetic materials for industrial applications due to their intrinsic properties, as well as they are considered alternative sources of raw materials since they present characteristics of sustainability, biodegradability and biosafety. As definition, gums and mucilages are polysaccharides or complex carbohydrates consisting of one or more monosaccharides or their derivatives linked in bewildering variety of linkages and structures. Natural gums are considered polysaccharides naturally occurring in varieties of plant seeds and exudates, tree or shrub exudates, seaweed extracts, fungi, bacteria, and animal sources. Water-soluble gums, also known as hydrocolloids, are considered exudates and are pathological products; therefore, they do not form a part of cell wall. On the other hand, mucilages are part of cell and physiological products. It is important to highlight that gums represent the largest amounts of polymer materials derived from plants. Gums have enormously large and broad applications in both food and non-food industries, being commonly used as thickening, binding, emulsifying, suspending, stabilizing agents and matrices for drug release in pharmaceutical and cosmetic industries. In the food industry, their gelling properties and the ability to mold edible films and coatings are extensively studied. The use of gums depends on the intrinsic properties that they provide, often at costs below those of synthetic polymers. For upgrading the value of gums, they are being processed into various forms, including the most recent nanomaterials, for various biotechnological applications. Thus, the main natural polymers including galactomannans, cellulose, chitin, agar, carrageenan, alginate, cashew gum, pectin and starch, in addition to the current researches about them are reviewed in this article.. }To the Conselho Nacional de Desenvolvimento Cientfíico e Tecnológico (CNPq) for fellowships (LCBBC and MGCC) and the Coordenação de Aperfeiçoamento de Pessoal de Nvíel Superior (CAPES) (PBSA). This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit, the Project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462) and COMPETE 2020 (POCI-01-0145-FEDER-006684) (JAT)

Temporal and spatial changes in ion homeostasis, antioxidant defense and accumulation of flavonoids and glycolipid in a halophyte <i>Sesuvium portulacastrum</i> (L.) L.

<div><p>Salinity is an important environmental constraint limiting plant productivity. Understanding adaptive responses of halophytes to high saline environments may offer clues to manage and improve salt stress in crop plants. We have studied physiological, biochemical and metabolic changes in a perennial, fast growing halophyte, <i>Sesuvium portulacastrum</i> under 0 mM (control), 150 mM (low salt, LS) and 500 mM (high salt, HS) NaCl treatments. The changes in growth, relative water content, cation, osmolyte accumulation, H₂O₂ and antioxidant enzyme activity (SOD, CAT and APX) were observed under different treatment conditions. A positive correlation was revealed for sodium ion accumulation with malondialdehyde (r² = 0.77), proline (r² = 0.88) and chlorophyll content (r² = 0.82) under salt treatment while a negative correlation was observed with relative tissue water content (r² = -0.73). The roots and leaves showed contrasting accumulation of potassium and sodium ions under LS treatment. Temporal and spatial study of sodium and potassium ion content indicated differential accumulation pattern in roots and leaves, and, high potassium levels in root. Higher H₂O₂ content was recorded in roots than leaves and the antioxidant enzyme activities also showed significant induction under salt treatment conditions. Gene expression profiling of sodium transporters, <i>Sodium proton exchanger</i> (<i>NHX3</i>), <i>Vacuolar ATPase</i> (<i>vATPase</i>) and <i>Salt overly sensitive1</i> (<i>SOS1</i>) showed up regulation under salt stress after 6–24 hr of NaCl treatment. Metabolite changes in the salt stressed leaves showed increased accumulation of flavonoids (3,5-dihydroxy-6,4’-dimethoxy-flavone-7-O-[α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside], and3,5-dihydroxy-6,3’,4’-trimethoxy-flavone-7-O-[α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside] in both LS and HS treatments, while a glycolipid, 1-O-linolenyl-2-O-(palmitoyl)-3-O-galactopyranosyl glycerol, accumulated more in LS over HS treatments and control. The results suggest that differential spatial and temporal cation levels in roots and leaves, and accumulation of flavanoid and glycolipid could be responsible for salt adaptation of <i>S</i>. <i>portulacastrum</i>.</p></div

Effect of different NaCl treatments on MDA content of roots and leaves of <i>S</i>. <i>portulacastrum</i>.

<p>Different letters over bar with same color indicates significant difference in treatment according to Duncan's multiple range test DMRT <i>p</i>≤ 0.05).</p

Effect of different NaCl treatments on proline content in roots and leaves of <i>S</i>. <i>portulacastrum</i>.

<p>All the values are means ±S.E. (n = 3). Different letters over bar with same color indicates significant difference in treatment according to Duncan's multiple range test (DMRT p≤ 0.05).</p

Effect of different NaCl treatments on root and shoot length of <i>S</i>. <i>portulacastrum</i> plants.

<p>Different letters over bar of same color indicates significant difference in treatment according to Duncan's multiple range test DMRT <i>p</i>≤ 0.05).</p

Temporal and spatial changes in ion homeostasis, antioxidant defense and accumulation of flavonoids and glycolipid in a halophyte <i>Sesuvium portulacastrum</i> (L.) L. - Fig 8

<p>Effect of different NaCl) treatments on sodium, potassium and calcium ion content A) in roots, B) in leaves of <i>S</i>. <i>portulacastrum</i>. Different letters over bar with same color indicates significant difference in treatment according to Duncan's multiple range test DMRT <i>p</i>≤ 0.05).</p

Effect of different NaCl treatments on % relative water content RWC) of leaves of <i>S</i>. <i>portulacastrum</i>.

<p>Different letters over bar indicates significant difference between treatments according to Duncan’s multiple range test <i>p</i>≤ 0.05).</p