Lipid analysis by thin-layer chromatography--a review of the current state

High-performance thin-layer chromatography (HPTLC) is a widely used, fast and relatively inexpensive method of separating complex mixtures. It is particularly useful for smaller, apolar compounds and offers some advantages over HPLC. This review gives an overview about the special features as well as the problems that have to be considered upon the HPTLC analysis of lipids. The term "lipids" is used here in a broad sense and comprises fatty acids and their derivatives as well as substances related biosynthetically or functionally to these compounds. After a short introduction regarding the stationary phases and the methods how lipids can be visualized on an HPTLC plate, the individual lipid classes will be discussed and the most suitable solvent systems for their separation indicated. The focus will be on lipids that are most abundant in biological systems, i.e. cholesterol and its derivates, glycerides, sphingo- and glycolipids as well as phospholipids. Finally, a nowadays very important topic, the combination between HPTLC and mass spectrometric (MS) detection methods will be discussed. It will be shown that this is a very powerful method to investigate the identities of the HPTLC spots in more detail than by the use of common staining methods. Future aspects of HPTLC in the lipid field will be also discussed.     

Capabilities and Disadvantages of Combined Matrix-Assisted Laser-Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) and High-Performance Thin-Layer Chromatography (HPTLC): Analysis of Egg Yolk Lipids

JPC – Journal of Planar Chromatography – Modern TLC - Lipids are important natural products and essential in nutrition, cosmetic formulations, pharmaceuticals, etc. Lipids and,...     

Automated NMR assignment of protein side chain resonances using automated projection spectroscopy (APSY)

This paper describes an automated method for sequence-specific NMR assignment of the aliphatic resonances of protein side chains in small- and medium-sized globular proteins in aqueous solution. The method requires the recording of a five-dimensional (5D) automated projection spectroscopy (APSY-) NMR experiment and the subsequent analysis of the APSY peak list with the algorithm ALASCA (Algorithm for local and linear assignment of side chains from APSY data). The 5D APSY-HC(CC-TOCSY)CONH experiment yields 5D chemical shift correlations of aliphatic side chain C-H moieties with the backbone atoms H(N), N, and C'. A simultaneous variation of the TOCSY mixing times and the projection angles in this APSY-type TOCSY experiment gives access to all aliphatic C-H moieties in the 20 proteinogenic amino acids. The correlation peak list resulting from the 5D APSY-HC(CC-TOCSY)CONH experiment together with the backbone assignment of the protein under study is the sole input for the algorithm ALASCA that assigns carbon and proton resonances of protein side chains. The algorithm is described, and it is shown that the aliphatic parts of 17 of the 20 common amino acid side chains are assigned unambiguously, whereas the remaining three amino acids are assigned with a certainty of above 95%. The overall feasibility of the approach is demonstrated with the globular 116-residue protein TM1290, for which reference assignments are known. For this protein, 97% of the expected side chain carbon atoms and 87% of the expected side chain protons were detected with the 5D APSY-HC(CC-TOCSY)CONH experiment in 24 h of spectrometer time, and all these resonances were correctly assigned by ALASCA. Based on the experience with TM1290, we expect that the approach presented in this work is routinely applicable to globular proteins with sizes up to at least 120 amino acids.     

Characterization of short-time dealumined HZSM-5 zeolites

Temperature-programmed desorption (TPD) of water was applied to characterize short-time dealuminated HZSM-5 zeolites. Using a regularization method, distribution functions of the effective desorption energy of water were calculated. The results clearly show that during dealumination a new adsorption site is formed which can be attributed to non-framework or transient aluminium species. The highest concentration of these sites was observed for a dealumination time of 25-30 min. NO adsorption studies support this result. Furthermore, it could be concluded that the heterogeneity and the average acid strength of the remaining Si-OH-Al groups of the dealuminated samples do not change compared to the Si-OH-Al groups of the parent HZSM-5 zeolite. This revised version was published online in July 2006 with corrections to the Cover Date.     

Phosphatidylcholines and -ethanolamines can be easily mistaken in phospholipid mixtures: a negative ion MALDI-TOF MS study with 9-aminoacridine as matrix and egg yolk as selected example

Phospholipids (PL) are increasingly analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). As in the case of polar molecules, however, the careful selection of the matrix is crucial for optimum results. 9-Aminoacridine (9-AA) was recently suggested as the matrix of choice to analyze PL mixtures because of (a) the improved sensitivity and (b) the reduction of suppression effects compared to other matrices. However, the distinction of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in the negative ion mode is obscured as PC is also detectable as –CH₃⁺ ion if 9-AA is used as matrix. This may result in the erroneous assignment of PC as a PE species. Using an organic extract from hen egg yolk as example it will be shown that the contribution of PC must be taken into consideration if the negative ion mass spectra are used to evaluate the fatty acyl compositions of PE mixtures. 9-AA can as well be used in hyphenated thin-layer chromatography (TLC)-MALDI-TOF MS where PC and PE are chromatographically well separated for unequivocal assignments.      

A simple method to identify ether lipids in spermatozoa samples by MALDI-TOF mass spectrometry

Plasmalogens (alkenylacyl glycerophospholipids) are important lipid constituents of many tissues and cells (e.g., selected spermatozoa). Since the molecular weights of plasmalogens overlap with that of diacyl- or alkyl acyl lipids, sophisticated mass spectrometry (MS; including MS/MS) analysis is normally used for the unequivocal identification of plasmalogens. We will show here that a simple matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (without MS/MS capability) in combination with acidic hydrolysis and subsequent derivatization with 2,4-dinitrophenylhydrazine (DNPH) and/or digestion with phospholipase A₂ (PLA₂) is sufficient to determine the contributions of ether lipids in spermatozoa extracts. As neither diacyl nor alkylacyl lipids are sensitive to acids and do not react with DNPH, the comparison of the mass spectra before and after treatment with acids and/or DNPH addition readily provides unequivocal information about the plasmalogen content. Additionally, the released aldehydes are readily converted into the 2,4-dinitrophenylhydrazones and can be easily identified in the corresponding negative ion mass spectra. Finally, PLA₂ digestion is very useful in confirming the presence of plasmalogens. The suggested method was validated by analyzing roe deer, bovine, boar, and domestic cat spermatozoa extracts and comparing the results with isolated phospholipids.