Off-Line Derivatization

1 Off-Line Derivatization MS identification of double-bond location in fatty acyl chains of lipids has also been performed with off-line derivatization followed by product-ion analyses of the derivatized lipids (see [12] for a recent review). For example, Moe and colleagues [13] pretreated GPL species and NEFA species with osmium tetroxide to generate hydroxyl-containing GPL species at the initial site(s) of unsaturation. The dihydroxylated lipids were analyzed with product-ion 

FRAGMENTATION PATTERNS OF FATTY ACIDS AND MODIFIED FATTY ACIDS 

MS analyses to locate the position of the initial double bond [14], The fragmentation of these derivatized fatty acids largely depends on the derivatizing reagents [12], 

---

The derivatization is commonly done for medium and low volatility compounds. The volatile compounds are more frequently analyzed by on-line techniques without derivatization, although on-line derivatization is occasionally utilized (such procedures were discussed in Section 2.7). When the off-line derivatization is done for the pyrolysate generated in a furnace, no particular precautions are recommended. For Curie-point or filament type pyrolysers, the pyrolysate can be collected in a deactivated piece of capillary column connected to the pyrolyser and cooled either in an ice bath or at lower temperatures needed for capturing more volatile compounds. 

Off line derivatization has also been utilized for the derivatization of pyrolysate to be analyzed by high-pressure liquid chromatography (HPLC) techniques [6,10, 28]. 

Fructose was also pyrolysed in similar conditions with glucose, with the purpose to identify similarities and differences between the two substances. The chromatographic trace for fructose pyrolysate is shown in Figure 7.1.4. The results were obtained as in the case of glucose by off-line derivatization and GC/MS analysis. 

In order to identify less volatile compounds, the pyrolysis of cellulose acetate sample (at 590° C) can be followed by an off-line derivatization with bis-(trimethylsilyl)-trifluoroacetamide (BSTFA). Typically the derivatized pyrolysate is analyzed by GC/MS on a non-polar column. The results of this type of analysis (separation on a 60 m DB-5 column 0.32 mm i.d. and 0.25 p film thickness) is shown in Figure 7.3.2 and the peak identification is given in Table 7.3.3. 

The same polymer was also analyzed for less volatile components by performing the pyrolysis off line at 600 C in a filament system followed by off-line derivatization with BSTFA. The silylated pyrolysate was analyzed by GC/MS on a DB-5 column (60 m long, 0.32 mm i.d., 0.25 urn film thickness). For this analysis the GC separation was done using a temperature gradient between 50° C and 300° C. The chromatogram is shown in Figure 11.3.2. 

An appropriate derivatization reaction should be. sought these are often described in the literature for classical off-line derivatization... 

The small inner diameter of the separation capillary used in CE implies a short optical pathway, and the consequent poor concentration sensitivity when conventional UV detectiOTi is used. To overcome this drawback several techniques have been developed some of them consist in application of general approaches that are not specifically addressed to CE analysis of alkaloids. One is the use of LIF detector for analysis of alkaloids with native fluorescence [68, 69] or after their off-line derivatization [64, 88, 112]. Sample pretreatment, a second major approach, is popularly employed in combination with sample extraction and can be conveniently applied in analysis of alkaloids because they can be easily retained in cationic-exchange sorbents in solid-phase extraction (SPE) mode [113, 114]. It may be interesting to focus on more specific aspects to detect very low levels of analytes using limited amoimts of samples to this regard chemiluminescence reactions and the use of online preconcentration methods will be considered. 

---