weak cation resin exchanger. With a strong cation exchanger in combination with weak cation exchanger,
betaines or beatines plus amines are retained and are subsequently eluted with HCl or NH 4 OH, depend-
ing on the analytes of interest. While GB may be eluted with HCl or NH 4 OH, choline, betaine aldehyde,
-alaninebetaine, -butylrobetaine, and -dimethylsulfoniopropionate are not recovered quantitatively
under alkaline conditions [8]. In some quantification techniques, one-step purification using strong
cation-exchange resin is sufficient. Detailed information on purification using three-resin, two-resin, and
one-resin systems can be found elsewhere [23,88,89].
Recovery of quaternary ammonium compounds (QACs) can be determined by adding known
amounts of betaine (either radiolabeled or not) as an internal standard to the sample. Hitz and Hanson [46]
reported an average recovery of 78% of radioactive label following extraction and ion-exchange chro-
matography. Studies conducted in our laboratory (unpublished) indicate 70–74% recovery after passing
the aqueous phase of the extract directly through a mixture of two resins. The recovery is improved by
washing the resin several times with water. Guy et al. [87] did not find noticeable differences in chro-
matography between the two- and three-resin purification systems in 27 species examined (mostly halo-
phytes). It is recommended that an internal standard is added during extraction and purification steps.
Dimethylsulfonioacetate (the sulfur analogue of glycine betaine) is a good candidate.
C. Separation and Detection
- Spectrophotometric Method
Early analyses of QACs were based on nonspecific precipitation with periodide [90,91], reineckate salts
[92], or modified Dragendorff reagent [93]. These methods are not specific, and quantification of indi-
vidual compounds in mixtures requires prior separation. The periodide method was further advanced to
enable determination of both betaine and choline [19]. In this modified method, choline and betaine are
selectively precipitated at different pHs. The betaine or choline-periodide complex is extracted with 1,2-
dichloroethane and absorbance is measured at 365 nm. Betaine can be subsequently derived from QACs
(pH 2.0) after subtracting choline (pH 8.0). When these methods are employed, care should be taken to
minimize the background absorbance and remove other naturally occurring nitrogenous substances.
These deficiencies have promoted the development of quantification methods based on chromatographic
separation in conjunction with more sophisticated detection techniques.
- Chromatographic Method
TLC AND TLE. The advancement of betaine analysis is largely dependent on the analytical tools
available at the time. Thin-layer chromatography (TLC) or paper chromatography followed by visualiza-
tion of separated compounds with Dragendorff’s reagent was used widely before high-performance liq-
uid chromatography (HPLC). Paper chromatography or TLC is insufficient for complete separation of all
QACs [94,95]. Separation is further improved by adopting high-voltage electrophoresis [51,96]. In the
early 1980s, thin-layer electrophoresis (TLE) in combination with scanning densitometry was widely
used for the determination of a number of ammonium compounds [97]. Muller and Eckert [89] improved
sensitivity by using methyl orange as the visualization reagent. Although reasonably rapid, adaptable, and
free from interference, this approach is not particularly sensitive. Also, a standard curve is required for
every plate.
GAS CHROMATOGRAPHY (GC). Glycine betaine is a zwitterion and nonvolatile and thus cannot
be directly analyzed by gas chromatography. Esterification of its carboxylic group [98], pyrolysis of
glycine betaine [46], is necessary for the volatilization of GB. The esterification product of GB with N-
O-bis-trimethylsilyl-trifluoracetamide is not well characterized. Ranfft and Gerstl [98] reported a detec-
tion limit of 0.42 nmol and a reproducibility of 1.79% (relative standard deviation, RSD) for a concen-
tration of 41 ppm. The low-temperature pyrolysis–gas chromatography technique introduced by Hitz and
Hanson [46] is much more sensitive (minimum detection limit, MDL, is about 2 nmol). This technique is
based on pyrolytic dealkylation and deamination of the ammonium group in QACs. Low-temperature py-
rolysis of the OHform of GB favors deamination, giving trimethylamine (TMA) as the major product
that can be separated by gas chromatography (GC) and determined by flame ionization detection (FID).
The absolute TMA yields varied with pyrolysis probes, temperature, and concentration ranges. Hitz and
Hanson [46] reported 42–51% of the theoretical yield over a concentration range of 20–150 nmol GB. Be-
GLYCINE BETAINE IN STRESS RESISTANCE 885