Derivatization

little dispersive properties can be used and thus the dispersive interactive properties of the stationary phase can be exploited. Conversely, if dispersive solvents are used, the dispersive character of the stationary phase is swamped, and the polar and or ionic interactive character of the stationary phase can be utilized. These polar interactions will take place with the many hydroxyl groups that are intrinsically present or with other polar or ionic centers that may have been introduced by derivatization. The sites of derivatization are shown in figure 41 together with an example of a naphthyl carbamate derivative attached to the 2 or 3 positions. Courtesy of ASTEC Inc. Figure 41. The Bonding and Derivative Positions on the Cyclodextrin Structure

nbsp; The water sample was pumped through the extraction tube at a rate of 100 ml/min. The solutes removed were displaced from the extraction tube with 10 ml of methanol followed by 10 ml of (MTBE) and dried over anhydrous sodium sulfate. It is seen that all the chlorinated pesticides were extracted and concentrations down to 1 ppb could be easily identified. Derivatization GC samples are usually derivatized to render highly polar materials sufficiently volatile so that they can be eluted at reasonable temperatures without thermal decomposition or molecular re-arrangement. Examples of such materials that need to be derivatized are the organic acids, amides, poly hydroxy compounds, amino acids etc. In order to render such materials more volatile, they are either esterified, silanated or acetylated using one of a number of different methods

about 1 mg. Helium was used as the carrier gas at a linear velocity of 20 cm/sec. The value of the open tubular column is clearly demonstrated. Free Fatty Acids from Milk An example of the use of the packed column in natural product analysis is the separation and determination of the free fatty acids in whole milk. An example of such an analysis is shown in figure 42. This analysis requires a rather lengthy procedure for sample preparation but, at the same time, avoids a derivatization procedure that can easily give incorrect, low values for the fatty acid content. Due to their relatively high volatility, the lower fatty acids can be lost as vapor during the procedure. Losses can also occur as a result of their incomplete derivatization. The sample preparation developed by Supelco involved mixing 10 ml of the milk with 10 ml ethanol, 3 ml of 28% ammonium hydroxide, 25 ml of petroleum ether and 25 ml of diethyl ether. The mixture is then well shaken and

acetonitrile, the tetrabutyl ammonium salt would be completely removed and the interactions of the solutes with the stationary phase would become almost exclusively dispersive. This is an example where the phase system is complex and is required to be so, because limited column efficiency demanded clever phase selection to obtain the necessary differential retention. Analysis of b -blockers Another liquid chromatography application that involves some novel derivatization is the separation of some ‘b blockers’, the enantiomers of propranolol, metroprolol and atenalol as fluorescent enhanced derivatives on cellulose tris(3,5�dimethylphenylcarbamate) coated on silica gel. This technique was developed by Yang et al. [14] developed and involved the synthesis of their fluorescent derivatives, which were formed by reacting them with an electrophilic fluorogenic reagent, 4-(N-chloroformethyl-N- methylamino-7-N- N

Another motor actuates the piston by a different system of gearing to refill the syringe rapidly when required. The solvent is sucked into the cylinder through a hole in the center of the piston and between the piston and the outlet there is a coil that acts as a dampener. This type of pump is still occasionally used for the mobile phase supply to microbore columns that require small volumes of mobile phase to develop the separation. It is also sometimes used for reagent delivery in post column derivatization as it can be made to deliver a very constant reagent supply at very low flow rates. The Single Piston Reciprocating Pump The single piston reciprocating pump was the first of its type to be used with high efficiency LC columns (columns packed with small particles) and is still very popular today. It is simple in design and relatively inexpensive. A diagram of the single piston pump is shown in figure 7. Figure 7. A Single Piston Reciprocating Pump

Figure 20. The Structure of a – Cyclodextrin The phenyl-methyl-polysiloxane confers onto the column an intermediate level of polarity so the separations are basically enthalpic due to the dispersive and polar interactions that take place largely with the polymer but also entropic resulting from the chiral selectivity of the cyclodextrins. Derivatization of the base cyclodextrin structure can introduce groups to which only one enantiomer can interact, while the other(s) are partially or wholly entropically hindered from interaction. This increases the differential interaction between the enantiomers and the stationary phase, thus, increasing the separation ratio and hence the resolution. Courtesy of Supelco, Inc. Solute Retention Time (min