The use of a new, modified Dittmer–Lester spray reagent for phospholipid determination by the TLC image analysis technique

Abstract

A new phospholipid-specific spray reagent is described. A new phospholipid-specific spray reagent, which is a modification of the Dittmer-Lester reagent, is described in authors' studies. The difference between these two reagents is in the addition of tin (II) chloride to the proposed spray reagent. The use of the described spray reagent together with an image analysis technique allows not only for qualitative, but also for quantitative, determination of major phospholipid classes. Separation of phosphatidylserine (PS), phosphatidylethanolamine (PE) and phosphatidylcholine (PC) was conducted on an HPTLC plate with a mixture of chloroform, methanol and 25% ammonia solution in a volume ratio of 65:25:4 as mobile phase. The calibration curves were linear in the ranges of 5.0-25.0, 1.5-15.0 and 1.0-20.0 µg/spot for PC, PS and PE, respectively. The use of the new spray reagent resulted also in lower limits of detection than the standard molybdenum method for the investigated phospholipids. The proposed method was used to determine the amount of PS in the dietary supplement 'Session', and of PS, PE and PC in biological samples, with good results.

Structure Characterization of Phospholipids and Lipid a of Pseudomonas Putida KT2442

“…The dried lipid A and phospholipids were dissolved in a solvent of chloroform and methanol (4 : 1, v/v), spotted onto silica 60 TLC plates. The lipid A plates were developed in a solvent of chloroform, methanol, water and ammonia (40 : 25 : 4 31 while the phospholipid plates were developed in a solvent of chloroform, methanol, water and ammonia (65 : 25 : 32 Next, the plats were dried and sprayed with 10% sulfuric acid in ethanol; bands of lipid A or phospholipids on the plates could be visualized by charring at 150°C.…”

Capturing Membrane Protein Ribosome Nascent Chain Complexes in a Native-like Environment for Co-translational Studies

“…Plates were imaged using blue fluorescence once they had cooled. Alternatively, the dried plate was stained with iodine vapor or sprayed with a molybdenum blue stain (commercial Dittmer-Lester stain 45 ) to specifically detect phospholipid. ImageJ 42 was used for densitometry analysis, where the raw spot intensity was converted to mole fraction, and the percentage of total lipid composition was plotted with SD for at least three biological repeats (new growth and preparation of RNCs, lipid extraction, and running of TLC plates).…”

Unsaturated fatty acids as high-affinity ligands of the C-terminal Per-ARNT-Sim domain from the Hypoxia-inducible factor 3α

“…The plates were then left exposed to air to allow for evaporation of the mobile phase residual. For differential detection of neutral lipids and phosphorous-containing lipids, iodine vapor and a modified Dittmer–Lester reagent 13 were employed for dual staining. The plates were scanned immediately after drying.…”

“…Aiming at experimental confirmation of the identity of the proposed monoacylglycerol, the hydrophobic fraction was carefully isolated from pure protein samples in solution and analyzed by thin-layer chromatography (TLC). By applying two mobile phases distinct in polarity, as well as dual staining to allow for differential detection of neutral and phosphorous-containing lipids 13 , we surprisingly observed the dominant presence of two neutral lipids and two phospholipids as ligands of PASb-3α ( Fig. 2a ).…”