Naturally abundant polysaccharides, in particular starch and cellulose, are increasingly being considered as renewable and potentially biodegradable starting materials in many traditional industries [1
]. In many applications, the properties of natural polysaccharides are not adequate for their intended uses and physical or chemical modification is employed in order to obtain a material with improved properties. It has been found that even small changes in the structure of the polysaccharide can lead to extensive alterations of chemical and physical properties [1
]. Chemical modification usually aims to hydrolyse glucoside links in the chain, or to attach different types of molecules on to the chains, taking advantage of the three hydroxyls present in each anhydroglucose unit. This has led modified starches to become important commercial products, with a range of uses most notably in the food industry [2
Biocatalysis is an attractive option for chemical modification of starch, and has been extensively used for partial hydrolysis to generate oligomers. However, the enormous range and diversity of available enzymes offers the possibility to obtain completely novel products with new or improved functionalities that were impossible to produce through traditional means. The use of enzymes to acylate the starch molecule, and generate compounds with commercial importance, is a relatively unexplored field [3
]. Even where the modifications have been made previously, enzymatic routes may be preferable because the chemical processes include extreme pH conditions, solvents that push the limits of acceptability for health and other reasons, and substrates such as anhydrides and acid chlorides [2
]. New possible uses of modified starches in fields such as the pharmaceutical or biomedical industries and even traditional uses are challenged by new waves of strict health and safety laws and regulations. This results in the need to update or replace traditional methods of modified starch formation with "cleaner" methods. Biocatalysis is a possible solution considered in situations where milder reaction conditions are required, with fewer by-products and higher selectivity.
Starch fatty acid esters have traditionally been dominated by starch acetates which presented more desirable properties for the intended uses. In recent years however starch acylates of medium or longer chain acids have emerged as candidates for novel uses, mainly in the biodegradable and renewable materials industries. One of the main advantages of these materials is the ability of longer chain esters to work as internal plasticizers of starch's glucan matrix [8
]. The extent of acylation is characterised by the degree of substitution (DS), defined as the average number of acyl groups per anhydroglucose unit.
When starch has been esterified, whether by conventional chemical or enzymatic routes, product analysis is important. The analysis of esterified starch samples has been approached in a few different ways. The oldest and most common method however has been a titrimetric one, first proposed by Genung and Mallatt [9
]. The principle of the method is that if modified starch is saponified with a known amount of hot aqueous NaOH, the ester bonds will be hydrolysed and sodium acylates will form. When this solution is back-titrated with a standard strong acid (e.g. HCl), the amount of NaOH used for saponification can be calculated and consequently the acyl group substitution can be quantified (Figure ). This method is still widely employed, as shown by recent references [6
]. Recently this method has also been used to quantify starch esters of fatty acids [6
]. The case of cellulose has been approached with the same methodology. Saponification followed by titration has been used to a great extent, even in recent years [14
Figure 1 Reactions and side-reactions of starch ester analyses. a) Titration Analysis: 1 Hydrolysis of starch ester (circle denotes starch), 2 Saponification of acid, 3 Titration of residual hydroxide ions, 4 Titration of saponified acid, b) Transesterification/GC (more ...)
Other more recent methods include the quantification based on the absorption band assigned to the vibration of the carbonyl in the ester group, with FTIR [15
] and a method based on NMR spectroscopy [16
]. However due to the need for instrumentation and the uncertainty of analysis of polymers with these methods, the titration analysis has been the method of choice in the past years. In recent years and with the introduction of specialised high throughput titration tools, there has been, to our knowledge, no attempt at a more insightful look at the parameters of this method. We attempted to perform this analytical approach, with an automatic titrator, which allows for precise readings and accurate titration of samples of small volume. These experiments revealed flaws that led us to seek another approach for an easy benchtop analytical method. Alkaline methanolysis is used in many cases to produce methyl esters via a transesterification reaction. The products are usually suitable for GC analysis, especially in the case of esters of medium chain length fatty acids. Methanolysis of cellulose acetates has been reported, followed by GC analysis of the methyl acetate [17
]. Here we propose an analysis scheme for starch esters of fatty acids, based on treatment of the starch ester with sodium methoxide, followed by GC analysis of the resulting fatty acid methyl ester.