reproducibility. The extract is then partitioned against chloroform. The methanol-water phase containing
betaines is collected for further purification. Both fresh and dried plant materials are suitable for betaine
analysis [87]. Care should be taken if plant material is homogenized in MCW mixture because the heat
generated by homogenization could result in the breakdown of chloroform and acidification of the extract.
The performance of the ion-exchange chromatography step (described in the following) will be very low
if the extract has a pH below 5.0. It is important to homogenize tissue in extraction mixture over an ice
bath or otherwise adjust the pH between 5 to 7 with 1 M NaOH.
B. Purification
Most of the available quantification methods require removal of interfering compounds. The most widely
used purification protocols are based on the unique charge properties of betaines, a permanent positive
charge on the quaternary ammonium group with a carboxyl group of low pKa. Betaines are not retained
on either strong anion- or weak cation-exchange resin. This allows excellent separation from impurities
by passing through strong anion- and weak cation-exchange resins in series. Strong anionic resin removes
all anions, amino acids, and zwitterions except OHand betaines. All cations are retained on the weak
cation resin. The effluent of this column combination contains betaines, where amines are retained on the
884 SUBBARAO ET AL.
Figure 1 Analytical approaches for the determination of glycine betaine in plant material.