Peracetylated samples

As a general rule, peracetylation is most useful for compounds below Mr 2000, particularly those that have been reduced with sodium borohydride and still contain some salt. The best procedure for peracetylation is based on the method of Bourne and coworkers. Samples are dissolved in 2 1 (v/v) trifluoroacetic anhydride-acetic acid and the solutions kept for 10 min at room temperature. Reagents are removed under a stream of nitrogen, and a solution of the product in chloroform is washed with water to remove salts, and dried the peracetylated sample is dissolved in methanol for the f.a.b. analysis. 

Subsequent conversion of dextran derivatives with acetic acid or propionic acid anhydride is an effective method for revealing structural features on the molecular level. This is illustrated on a dextran propionate (Mw 5430 gmol x) which can be completely acetylated with acetic anhydride/pyridine in a separate step yielding a peracetylated sample (dextran propionate acetate, DPA). The assignment of the chemical shifts of DPA is carried out via 2D NMR (Fig. 17). 

The compositions of high-mannose oligosaccharides isolated from urine of animals suffering from genetic or chemically induced mannosidoses have been assigned from their f.a.b.-mass spectra. After chromatography, some samples were sufficiently pure to be examined underivatized. Others required conversion into their peracetylated derivatives. 

In a study completed during the early development of f.a.b.-m.s., both f.d. and f.a.b. were used to characterize 101 fractions containing neutral oligosaccharides isolated from human milk. Samples were examined as their peracetylated alditols. In subsequent work, the structures of two minor acidic oligosaccharides from human milk were investigated. The per-methylated derivatives were analyzed by f.a.b.-m.s., and their compositions and sequences were defined by the f.a.b. data. Methylation analysis and partial formolysis were the other principal methods used. 

A fourfold decrease in the IDIIM ratio was observed for the 5.3% peracetylated pyrenylmethyl polyethylenimine derivative in glycerol compared to methanol. The higher viscosity of the glycerol limits the mobility of the attached pyrene group necessary to form excimer, decreases the association rate, and hence lowers ID/IM. These samples at 77°C showed essentially no excimer emission. Clearly, diffusion of the pyrene moieties attached to the polymer side chains is necessary for excimer formation. 

Both samples, the peracetylated p-D-glucose (30mg) and the peracetylated oligosaccharide of unknown structure (6mg) were dissolved in CDCI, (99.8% D), doped with a trace of tetramethylsilane (TMS) as the internal standard. 

Fig. 3.23 The 2D spectrum of peracetylated glucose from a 2D TOCSY experiment. The same sample has been used and the expansion is the same as for the 2D phase-sensitive, DQ-filtered COSY spectrum (Fig. 3.21). Note the additional cross peaks obtained with the TOCSY experiment.

Figure 6.1 gives an overview of ID and 2D NMR data available for this peracetylated oligosaccharide. The sample was dissolved in CDCl, and all the experiments where measured on a 500 MHz Bruker DRX 500 spectrometer. 

The choice between peracetylation and permethlyation is dependent on the experiment being performed. When dealing with samples in complex, salty matrices, acid-catalyzed peracetylation proves to be the right choice107,145 while permethylation tends not to be as effective under such conditions.143... 

Figure 2. Influence of peracetylation on ionization response. Sample free and peracetylated phenyl P-/)-glucopyranoside ESI positive HPLC RP-18 200 pl/min MeOH-HzO-S mM NH4AC.

Figure 3. Influence of peracetylation on product ion spectra. Sample firee and peracetylated 3-Z)-glucopyranosyl-anthranilate ESI positive CID 1.8 mTorr Ar, -15eV HPLC RP-18 2 x 100 nun, 200 pl/min MeCN-H20-5 mM TFA.

On principle, pre- and post-column derivatization can be performed. Derivatization of complex samples prior to chromatographic separation is more problematic because matrix effects may alter reactions. Therefore, pre-column derivatization procedures are less suitable for quantitative analysis. Nevertheless quantitative procedures are described in the literature. Piretti et al. [71-72,247] analyzed peracetylated flavan-3-ol monomers and procyanidin dimers among other compounds in apple tissue after acetylation on a nitrile stationary phase under normal-phase conditions. Tarnai et al. [13] used the same approach in the analysis of procyanidins from cherry tissue. Incomplete acetylation was never observed [71], but so far validation data are not available. 

The adsorption experiments were performed by immersing the slides into the peracetylated or deacelylated saccharide solutions (10 mL). Following the immersion times indicated in the text, the slides were removed from the solution and rinsed with several milliliters of methylene chloride or in the case of the deprotected layers in a bath of methanol and methylene chloride. A third set of samples was made by adsorbing peracetylated saccharide from solution, followed by a deprotection of the adsorbed monolayers in place, on the substrates. The adsorbed monolayers were deprotected by immersing the sample in a 0.02% NaOMe/MeOH solution (10 mL), shaking for 24 h, and rinsing in a bath of methanol and methylene chloride. 

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