**9. The use of chromatographic techniques for determining DA and DE**

DE is usually determined, together with galacturonic acid content, by titration. However, the presence of acetyl can contribute to an overestimation of DE, since the acetic acid released during the saponification will be titrated. Chromatographic methods can be used for determination of DE and DA. An HPLC method for simultaneous determination of DE and DA was proposed by Voragen et al (1986) [47] and modified by Levigne et al (2002) [31]. In the former, an Aminex HPX-87H column is used while the second method uses a C18 column. The pectin is saponified and precipitated followed by methanol and acetic acid separation from the supernatant by HPLC and quantification by refractometry using an internal standard. The method allows accurate determination in a single run and within a short time, the DE and DA using low amounts of pectins (~ 5mg). This method presents some advantages when compared to that using FT-IR for DE calculation. FT-IR method requires pectins standards with different DEs in order to construct a calibration curve and the presence of acetyl can contribute to an overestimation of the DE.

Huisman et al. (2004) [48] proposed a method for DE determination using head-space GC. The approach is similar to that used in the HPLC method. Head-space GC is used for the quantification of methanol released from pectin by saponification. A lower amount of pectin is needed (~2mg) and the chromatogram shows a symmetrically shaped methanol peak which is very easy to integrate. However, the method is only applicable for the determination of DE and another approach has to be used in order to estimate DA.
