HPLC lecithin

The total phosphoms content of the sample is determined by method AOCS Ja 5-55. Analysis of phosphoUpid in lecithin concentrates (AOCS Ja 7-86) is performed by fractionation with two-dimensional thin-layer chromatography (tic) followed by acid digestion and reaction with molybdate to measure total phosphorous for each fraction at 310 nm. It is a semiquantitative method for PC, PE, PI, PA, LPC, and LPE. Method AOCS Ja 7b-91 is for the direct deterrnination of single phosphoHpids PE, PA, PI, PC in lecithin by high performance Hquid chromatography (hplc). The method is appHcable to oil-containing lecithins, deoiled lecithins, lecithin fractions, but not appHcable to lyso-PC and lyso-PE. 

Fig. 6.3 Separation of lecithin phospholipids by HPLC with evaporative light scattering...

Up to now, however, HPLC has remained the method of choice. This is due mainly to the fact that this technique is much easier to automate as compared to TLC. Besides, a wide variety of stationary and mobile phases are available, so the technique is highly flexible. Besides, the investment cost is much lower as compared to P-NMR, whereas quantitation is more straightforward than in MEKC. Hence, the more recent official methods for the analysis of phospholipids, as proposed by the American Oil Chemists Society (AOCS), by the International Union for Pure and Applied Chemistry (IUPAC), and by the International Lecithin and Phospholipid Society (ILPS), all use HPLC. In this chapter, a review is presented of HPLC procedures that have been described during the past 10 years the older literature was discussed in a previous edition of this handbook (21). 

Fig. 6 ELSD chromatogram of a commercial soy lecithin following HPLC on a 5-/tm Ultrasphere Si stationary phase with a gradient of chloroform, methanol, and 28.7% NH4OH. 1 = nonpolar lipids, 2 = PE, 3 = PI, 4 = PC, 5 = PA. (Reproduced from Ref. 73 with the permission of the Journal of High-Resolution Chromatography.)...

R Szucs, K Verleysen, GS Duchateau, P Sandra, BGM Vandeginste. Analysis of phospholipids in lecithins. Comparison between MEKC and HPLC. J Chromatogr A 738 25-29,1996. 

C Hanras, JL Perrin. Gram-scale preparative HPLC of phospholipids from soybean lecithins. J Am Oil ChemSoc 68 804-808, 1991. 

SL Melton. Analysis of soybean lecithins and beef phospholipids by HPLC with an evaporative light scattering detector. J Am Oil Chem Soc 69 784-788, 1992. 

B Hersloef, U Olsson, P Tingvall. Characterization of lecithins and phospholipids by HPLC with light scattering detection. In I Hanin, G Pepeu, eds. Phospholipids. New York Plenum Press, 1990, pp 295-298. 

BH Klein, JW Dudenhausen. Simultaneous determination of phospholipid classes and the major molecular species of lecithin in human amniotic fluid by HPLC. J Liq Chrom 17 981-998, 1994. 

A wide range of data has been published showing the variability in the composition of phospholipids and fatty acids in soybean lecithin (Tables 6 and 7) (32). Older data were often determined by qualitative TLC, whereas today 31P-NMR, quantitative Li-Sc HPLC, and HPTLC methods have been developed. 

Zhang et al. (143) reported the effects of an expander process on the phospholipids in soybean oil by comparing the differences in phospholipid compositions of the oils and the lecithins produced from expander and conventional processes by HPLC. The phosphorus content indicated that the expander-processed oil contained more phosphorus (985 ppm) than the conventionally processed oil (840 ppm). However, the phospholipids in the expander-processed oil were more hydratable than those in the conventionally processed oil. After degumming, the phosphoms content in the expander-processed oil and conventionally processed oil were reduced by 93.2% and 78.6%, respectively. The expander-processed lecithin contained 74.3% AI matter, and the conventionally processed lecithin contained 65.8%. There was also more phosphatidylcholine in the expander-processed lecithin (39.8%,... 

International Lecithin and Phospholipid Society (ILPS), Anal)4ical Method AM 101, HPLC Determination of Lecithins (1998). 

P NMR spectroscopy is the method of choice for phospholipids or any other phosphorus-containing compound. Most phospholipids contain only one phosphorus atom, so the P NMR spectrum of lecithin reads like an HPLC chromatogram. There are some advantages in eomparison with HPLC specific detection of the phosphorus nucleus, high dispersion and hi dynamics. The role of P NMR spectroscopy will be discussed later in detail. 

Many HPLC methods for phospholipids have been developed, but chromatographic resolution and dynamics of detection are not always satisfactory. For each source of phospholipids, special standards are needed due to the different distribution of fatty acids. These standards are expensive and in some cases are not available. Another problem is represented by the analysis of phospholipids in complex matrices. In many cases, separation is impossible or very difficult, not least due to the surface activity, which is desired in the application of phospholipids, but which complicates the analysis of these compounds. Therefore, a method is needed which is selective in the detection of phospholipids in order to avoid a separation from the matrix. The P NMR spectroscopy of phospholipids meets these requirements. The I.L.P.S. (Internationa Lecithin and Phospholipid Society) has chosen the P NMR method as the reference method [62],[63],[64]. It has been tested world-wide by round robin tests in comparison to various HPLC and TLC methods. With triphenylphosphate as internal standard, a pulse angle of 15°, 10-s relaxation delay, and 32-256 accumulations, the method has a precision of <0,5%. 

P. A. Scindler, C. A. Settineri, X. Collet, C. J. Fielding, and A. L. Burlingame, Site-specific detection and stmctnral characterization of the glycosylation of human plasma proteins lecithin cholesterol acyltransferase and apolipoprotein D using HPLC/ electrospray mass spectrometry and sequential glycosidase digestion. Protein Sci. 4, 791-803 (1995). 

Diehl, B. W. K. and Stein, J. (1994) Calibration standards for lecithin analysis using HPLC with light scattering detector. 84th AOCS Annual Meeting and Exposition, Atlanta, GA, May 1994 copy available from author. 

Figure 20a. HPLC profile of lecithin components prior to supercritical fluid processing.

Figure 20b. HPLC profile of preparative SFC-extract (fraction 5) from lecithin.

Fig. 20. Elution patterns of choline-containing phospholipids for each lipoprotein fraction isolated by the ultracentrifugation. Column G4000SW+G3000SW. Sample A, normal female B, acute hepatitis C, lecithin cholesterol acyltrans-ferase deficiency. Other HPLC conditions as in Fig. 14.

Also the HPLC-analysis of phospho- and glycolipids is of growing importance. Figure 3.11 for example demonstrates the separation of soya raw lecithin . 

Trade names Amlno-Cerv, B-50, Cardene, Lecithin, Mega-B, Megadose, Sclerex Use Lipotropic HPLC LiNHj 70D 30E 9.0 GC ... 

Acetone-insoluble (AI). The amount of AI matter (%AI) is the approximate indication for the amount of phospholipids, glycolipids and carbohydrates, because the oil and fatty acids dissolve in acetone. In crude lecithin AI is synonymous with activity, i.e. functional or nutritional properties. The AI is a commercial and legal specification. However, with NMR and HPLC the more exact amount and composition of true phospholipids can be analysed. 

Widely distributed in biol. tissues. Component of lecithin. Chemical intermed., org. base. Removes CO2 and H2S from gases. Reagent for fluorimetric anal, of carbohydrates by hplc. Viscous, hygroscopic liq. Misc. H2O spar. sol. C H, Et20. df 1.012. Bp 171°. pA 9.47. < 1.4539. 

HPLC is the most common technique applied to the determination of the chemical composition of lecithin. Normal phase HPLC is convenient for the determination of the major constituents (i.e., phosphatidylcholine, phosphatidylethanolamine, etc), as described in Chapter 7. P NMR is also suitable for this analysis, as discussed in Chapter 14. The biochemical literature contains many enzymatic methods, mainly for specific determination of phosphatidylcholine and its hydrolysis product, choline (32). For instance, phosphatidylcholine can be hydrolyzed by phospholipase C to a diacylglycerol and the phosphate ester of choline, which itself can be hydrolyzed by alkaline phosphatase to form choline and phosphate ion. Alternatively, action of phospholipase D on phosphatidylcholine yields phosphatidic acid and choline. These methods are not applied to analysis of the commercial lecithin used as a surfactant. 

Preparative HPLC of 100 mg soybean lecithin varying ratios of /i-hexane/2-Pr0H/H20, monitoring UV absorbance at 214 nm. 

TABLE IS HPLC Analysis of Phosphatides from Egg Yolk and Soy Lecithin... 

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