cause trimethyl QACs yield TMA in addition to GB upon pyrolysis and the absolute yields of individual
trimethyl QACs differ, the method measures trimethyl QACs in only a semiquantitative manner. The
identity of betaines present in plant extract must be verified through other techniques. In addition, the ex-
pense of pyrolysis probes, which require frequent replacement (an average of 300 analyses), makes this
technique less attractive and thus not readily adapted by the researchers working on GB.
HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY. HPLC is by far the most widely used
method because of its low cost, sensitivity, specificity, accuracy, and versatility. Separation of QACs and
their esters can be accomplished on strong cation [85,86,99,100], weak cation [88], amino-bonded silica
gel [87,101], and reverse-phase columns [37,82]. Most betaines do not have significant ultraviolet (UV)
absorbance above about 210 nm and thus require either low wavelengths (190–200 nm) or a differential
refractometer for direct quantification. The differential refractometer requires a larger sample (about 20
times higher betaine content) than UV detectors. The estimated minimum detection limit for GB by low-
wavelength UV varies from 0.5 to 4.4 nmol, and that is dependent on the wavelength and instrument used
for analysis [85,100]. Optimum peak sensitivity is achieved at 195 nm, and increasing the wavelength
above 195 nm decreases the peak area; e.g., peaks are three times larger at 195 nm than at 200 nm [85].
The sensitivity is better than that reported for pyrolysis-GC [46] and TLE with scanning densitometry
[97].
Derivatization increases selectivity and sensitivity by conferring particular physical or chemical
properties to the compounds. Methyl esters of -alaninebetaine, GB, trigonelline, and -aminobutyric
acid betaine are quantified by mobile phase ion chromatography using suppressed conductivity detec-
tion having a detection limit less than 0.42 nmol for GB [102]. Glycine betaine can also be esterified
with,p-dibromoacetophenone,p-nitrobenzyl bromide, or -bromo-p-tolunitrile. These aromatic
derivatives are then quantified either by directly measuring the absorbance at high wavelength (e.g. 262
nm for the p-bromophenacyl ester) in aqueous phase [103] or by HPLC separation on a strong cation-
exchange column followed by UV detection. Complete esterification (99%) of several betaines and
betaine analogues with ,p-dibromoacetophenone is observed. Generally the p-bromophenacyl esters
are preferred because of their higher molar absorptivity (e1.968 104 mol^1 cm^1 for GB phenacyl
ester). The HPLC-UV analysis of betaine phenacyl esters can detect 1 nmol of betaines. A combina-
tion of capillary electrophoresis and UV detection of betaine phenacyl esters was introduced by Zhang
et al. [84]. Estimation of betaines as either the methyl or p-phenacyl esters has several advantages over
low-wavelength UV detection. It is more reliable because of less interference at high wavelength and
more sensitive; thus it is able to detect smaller concentrations in plant tissues.
- Spectrometric Method
(^1) H NMR. This method utilizes the proton nuclear magnetic resonance signal of the three N-methyl
groups in GB [81]. The signal from the N-methyl resonance of GB (arising from nine protons) occurs in
a region of the^1 H nuclear magnetic resonance (NMR) spectrum that is generally free from interfering sig-
nals and allows the detection of 85 nmol (16 or 32 data acquisitions, a total data acquisition time of 2
min) of the compound. Greater sensitivity can be achieved by using a superconducting NMR spectrome-
ter of high field strength [104]. This method can be used for simultaneous determination of other N-
methyl compounds with different sensitivities depending on the number of protons and coupling with
neighboring protons (mutiplet or singlet). Another feature of NMR spectroscopy is that the radio-fre-
quency radiation used in the method can penetrate “opaque” objects such as seed coat, cell walls, and cel-
lular membranes, and thus this method can be used for determination of GB levels of intact organisms and
organelles [43].
NATURAL ABUNDANCE^13 C NMR. As with^1 H NMR, the resonance signal of a selected car-
bon isotope (^13 C) in the compound of interest can be used for quantification. The three carbons in
the trimethylated quaternary ammonium group of GB give rise to a very strong and distinct signal at
56.4 ppm. Signals from methylene groups on proline (26.54, 31.76, 48.83 ppm) and the trimethylated
ammonium group on betaine (56 ppm) are distinguishable [105]. Crude extract and plant tissue in vivo
can be analyzed because of the specificity of the chemical shift of a chosen carbon in the compound of
interest. Also, this method has potential for nondestructive analysis of solutes extracted from the plant. It
allows identification of solute molecules (concentrations of 1 mol/g fw) relevant to osmotic adjustment
in vivo.
886 SUBBARAO ET AL.