Lipids mobile phase

An excellent example of PLC applications in the indirect coupling version is provided by the works of Miwa et al. [12]. These researchers separated eight phospholipid standards and platelet phospholipids from the other lipids on a silica gel plate. The mobile phase was composed of methylacetate-propanol-chloro-form-methanol-0.2% (w/v) potassium chloride (25 30 20 10 10, v/v). After detection with iodine vapor (Figure 9.2), each phospholipid class was scraped off and extracted with 5 ml of methanol. The solvent was removed under a stream of nitrogen, and the fatty acids of each phospholipid class were analyzed (as their hydrazides) by HPLC. The aim of this study was to establish a standardized... 

Jansson et al. [189] were able to use the SX3 Biobeads and a mobile phase of dichloromethane in hexane (1 1) to make a further separation of the chloro-paraffins from lipids and other organochlorine pollutants. Using diethylhexyl phthalate (DEHP) as a marker, they collected the appropriate fractions to isolate the chloroparaffins and other pollutants. 

The idea of using a nonpolar stationary phase and a polar mobile phase was first put forward in the literature by Boscott ( ), who advocated the use of cellulose acetate as the stationary phase. Shortly thereafter Bol-dingh (4) separated Cg-Cu fatty acids by using moderately vulcanized rubber saturated with benzene as the stationary phase and water-methanol as the mobile phase. This was rapidly followed by other work (5-7) and the method was widely practiced by lipid chemists until the advent of HPLC. The main problem with the use of rubber for the stationary phase is that the degree of swelling is extremely critical in determining the... 

Polyether antibiotics are hydrophobic compounds that are characterized chemically by their low polarities and their instability under acidic conditions. These antibiotics can be quantitatively extracted from the primary organic extract into carbon tetrachloride (393-395). When partitioning from a sodium chloride solution into an organic solvent, high yields have been achieved using dichloromethane (396, 397), carbon tetrachloride (391, 399), and chloroform (14, 398) as extraction solvents. In a different approach, water extracts containing lasalocid residues have been purified by partitioning into the mobile phase, which was a complex mixture of tetrahydrofuran, methanol, n-hexane, and ammonia (387, 389, 390, 392). To remove lipids, sample extracts have often been partitioned with n-hexane. 

According to the modified procedure (602), milk is thoroughly mixed in its storage container immediately before transfer of the 1 ml aliquot in the extraction tube. This is necessary because approximately 50% of phenylbutazone in milk is associated with the cream. The sample is extracted with 2.4 ml diethyl ether and 2.4 ml petroleum ether in presence of 1 ml ethanol and 100 1 25% ammonia solution. The organic layer that contains the milk lipids is discarded. Five ml hexane-tetrahydro furan (4 1) is added to the aqueous layer, which is tiien acidified with hydrochloric acid and the layers are mixed. Under the acidic conditions, phenylbutazone partitions quantitatively into tlie organic layer, which is collected, evaporated, and dissolved in the mobile phase to be analyzed by liquid chromatography. Separation is performed on a reversed-phase column using an isocratic 0.02 M phosphate buffer/methanol mobile phase, and determination is by ultraviolet detection at 264 nm (Fig. 29.18.2). The limit of detection and limit of quantification were 3.0 and 5.4 ppb, respectively (Table 29.17). 

Aromatic amino acids, lipids, and many other materials can be separated on reversed-phase columns in which nonpolar groups, usually long-chain alkyl groups, are covalently attached to silica gel, alumina, or other inert materials.66 80 The mobile phase is a more polar solvent, often aqueous, and gradually made less polar by addition of an organic solvent. 

Figure D1.6.2 TLC-FID separation of lipids recovered from the gastric contents of a hooded seal pup. The mobile phase was 91 6 3 1 (v/v/v/v) hexane/ethyl acetate/diethyl ether/formic acid. Time refers to scanning time of the Chromarod. Abbreviations DG, 1,2-diglyceride FFA, free fatty acid MG, monoglyceride IS, internal standard TG, triglyceride. Reproduced from Ackman and Heras (1997) with permission from AOCS Press.

Samples, even at moderate concentrations, injected into the HPLC column may precipitate in the mobile phase or at the column frit. In addition, the presence of other compounds (e.g., lipids) in the injection sample may drive the carotenoids out of solution or precipitate themselves in the mobile phase, trapping carotenoids. It is best to dissolve the sample in the mobile phase or a slightly weaker solvent to avoid these problems. Centrifugation or filtration of the samples prior to injection will prevent the introduction of particles that may block the frit, fouling the column and resulting in elevated column pressure. In addition to precipitation, other sources of on-column losses of carotenoids include nonspecific adsorption and oxidation. These can be minimized by incorporating modifiers into the mobile phase (Epler et al., 1993). Triethylamine or diisopropyl ethylamine at 0.1% (v/v) and ammonium acetate at 5 to 50 mM has been successful for this purpose. Since ammonium acetate is poorly soluble in acetonitrile, it should be dissolved in the alcoholic component of the mobile phase prior to mixing with other components. The ammonium acetate concentration in mobile phases composed primarily of acetonitrile must be mixed at lower concentration to avoid precipitation. In some cases, stainless steel frits have been reported to cause oxidative losses of carotenoids (Epler et al., 1992). When available, columns should be obtained with biocompatible frits such as titanium, Hastolloy C, or PEEK. 

Modern LSD detectors yield good results even under gradient elution. No disturbance is observed when solvent composition changes. Organic solvents (acetone, propanol, chloroform) can be used in the mobile phase. In reversed-phase mode, water content up to 25% and small amounts of buffers are not a problem. Typical applications are lipids, phospholipids, sugars, and vitamins. 

Low-wavelength UV detection (200-210 nm) is more sensitive and permits the use of gradients but precludes the use of certain common lipid solvents, such as chloroform and acetone, which are opaque in the UV region of interest. With low-wavelength UV detection, the response will also be somewhat dependent on fatty acid composition. For these reasons the mobile phases used in lipid analysis by HPLC may seem rather strange to workers familiar with the Thin Layer Chromatography (TLC) or open column separations. 

Neff et al. (99) applied Ag-HPLC with an FID to the quantitative detection of triacylglyc-erols (TAGs) of Crepis alpina oil (CrAO). The Ag-HPLC column was a Chromsphere Lipids (4.6-mm ID X 250 mm 5 micron). All TAGs were eluted in 120 min by an isocratic mobile phase of 0.5% acetonitrile in hexane at a flow rate of 1.0 ml/min. The FID block temperature was set at 110°C, the cleaning flame hydrogen at 300 ml/min, and the oxygen flow at 175 ml/min. 

Fig. 30 Silver ion high-performance liquid chromatography (Ag-HPLC-FID) with flame ionization detector (FID) analysis of the triacylglycerols of chromatographed Crepis alpina seed oil. Ag-HPLC-FID conditions 0.5-mg sample 5-micron Chromspher Lipids column (Chrompack International, Middelburg, The Netherlands) (4.6 X 250 mm) mobile phase 0.5% acetonitrile in hexane (v/v) flow rate 1.0 ml/min FID. Chromatogram peak triacylglycerol fatty acid abbreviations S, saturated (palmitic and stearic) O, oleic L, linoleic and Cr, crepenynoic fatty acids.

Actually, solid-phase extraction is used not only as a rough preliminary fractionation procedure. Prieto et al. described the complete fractionation of the total lipids from wheat into eight neutral lipid, two glycolipid, and four phospholipid classes in addition to PC and LPC, TV-acyl PE and A-acyl LPE were detected (37). However, two separate stationary phases (silica and aminopropyl) as well as seven different mobile phases were needed. Moreover, 14% crosscontamination of PC and LPC was observed, and the recovery of the phospholipids was limited to about 85%. Hence, SPE is a rapid and efficient technique for preliminary fractionation, but loses its advantages if more complex separations are tried. 

Stationary phase Mobile phase Detection (Phospho)lipids Ref. 

In this case, a binary gradient is used, which is meant mainly to yield a better resolution of glycolipid classes. When the lipid extracts do not contain glycolipids, this phase of the separation can be omitted, and hence an isocratic mobile phase can be used. Also, for extracts poor in molecular species, the silica gel column may be omitted, for the diol column on its own provides resolution of the major phospholipid classes. 

Silica columns can tolerate relatively heavy loads of triglyceride and other nonpolar material. Such material is not strongly adsorbed and can easily be washed from the column with 25% diethyl ether in hexane after a series of analyses (83). Procedures for determining vitamins A and E have been devised in which the total lipid fraction of the food sample is extracted with a non-aqueous solvent, and any polar material that might be present is removed. An aliquot of the nonpolar lipid extract containing these vitamins is then injected into the liquid chromatograph without further purification. Direct injection of the lipid extract is possible because the lipoidal material is dissolved in a nonpolar solvent that is compatible with the predominantly nonpolar mobile phase. Procedures based on this technique are rapid and simple, because there is no need to saponify the sample. 

For the determination of supplemental vitamin E in infant formulas, Woollard and Blott (222) employed a radially compressed Radial-PAK cartridge. This enabled lipid material to be rapidly cleared by stepping up the mobile-phase flow rate from 2 ml/min to 10 ml/min after elution of the a-tocopheryl acetate. Fluorescence detection, using a filter-type fluorometer, allowed the indigenous a-tocopherol to be conveniently estimated, while UV absorbance detection was used to quantify the a-tocopheryl acetate. Supplemental retinyl acetate could be assayed simultaneously with either added or indigenous vitamin E using the appropriate detection mode. With the aid of a dual-monochromator spectrofluorometer, a-tocopheryl acetate and a-tocopherol could be determined simultaneously with wavelengths of 280 nm (excitation) and 335 nm (emission), but the increased selectivity eliminated detection of the vitamin A esters (233). 

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