Macroalgal Phycocolloids 155
FTIR spectroscopy. Infrared (IR) spectroscopy was, until recently the most frequently used vibrational
technique for the study of the chemical composition of phycocolloids. This technique presents two
main advantages: it requires minute amounts of sample (milligrams), and it is non-aggressive method
with reliable accuracy (Pereira et al. 2003). However, conventional IR spectroscopy requires laborious
procedures to obtain spectra with a good signal/noise ratio (Chopin and Whalen 1993). This limitation
was overcome with the development of interferometric IR techniques (associated with the Fourier
transform algorithm), known as FTIR spectroscopy (Fourier Transform IR). More recently, Pereira and
collaborators used a technique of analysis on the basis of FTIR-ATR (from Attenuated Total Reflectance)
spectroscopy, allowing for determination of the composition of the different phycocolloids from dried
ground seaweed, without having to prepare tablets of KBr (Pereira and Mesquita 2004; Pereira 2006;
Pereira et al. 2009a).
Raman spectroscopy. In contrast with FTIR, the application of traditional Raman spectroscopy was
limited until recently, due to the laser-induced fluorescence (strong background signal that is detected
when some samples, such as biochemical compounds, are excited with visible lasers) and risk of sample
destruction by light energy. The use of Nd:YAG lasers operating at 1064 nm has been generalized to
decrease the fluorescence level. Opto-electronic devices have progressed dramatically in the past decade,
because of major achievements in solid-state technology. As a result, compact, efficient and reliable diode
lasers are now available from visible to infrared light and have been shown to work correctly on Raman
instruments in combination with suitable filter sets (Pereira 2006; Pereira et al. 2009b).
Raman spectroscopy comprises the family of spectral measurements made on molecular media based
on inelastic scattering of monochromatic radiation. During this process, energy is exchanged between the
photon and the molecule such that the scattered photon is of higher or lower energy than the incident
photon. The difference in energy is made up by a change in the rotational and vibrational energy of the
molecule, and gives information on its energy levels. The modern FT-Raman spectrometers have been
used to produce good quality Raman spectra from seaweed samples (Matsuhiro 1996; Pereira et al. 2003;
Dyrby et al. 2004; Pereira 2006).
Four different FT-Raman spectra are presented in Fig. 3 (Chondrus crispus, female gametophytes),
corresponding to the different tests of depigmentation to reduce the fluorescence caused by the laser beam
in Raman spectroscopy. The spectrum “a” corresponds to the ground seaweed treated with a mixture of
acetone and methanol; this presents some fluorescence, particularly in the spectral area 600–875 cm
–1
and the peaks are ill-defined. The spectrum “b” corresponds to the fresh seaweed treated with calcium
hypochlorite 4% (30 s), then dried and milled. The spectrum “c” concerns the ground seaweed (obtained
from a herbarium sample) treated with calcium hypochlorite 4% (30 s). Finally, the spectrum “d” was
obtained from the native carrageenan (C. crispus water-extracted) analysis. The last three spectra (b, c, d)
so not exhibit fluorescence, with peaks well-defined and without background noise (after Pereira et al.
2009b).
NMR spectroscopy
Since natural carrageenans are mixtures of different sulfated polysaccharides, their composition differs
from batch to batch. Therefore, the quantitative analysis of carrageenan batches is of greatest importance
for both ingredient suppliers and food industries to ensure ingredient quality. From the pioneering work
of Usov and coworkers (Yarotsky et al. 1977; Usov 1984), NMR-spectroscopy is nowadays one of the
preferred techniques to determine and quantify the composition of carrageenan batches (van de Velde
et al. 2002). Starting with their early work, chemical shifts of carrageenan resonances are generally
converted to values relative to tetrametylsilane (TMS) via an internal dimethylsulphoxide (DMSO)
or methanol (MeOH) standard (Usov et al. 1980; Usov 1984; Knutsen et al. 1994). The use of DMSO
or MeOH as internal standard resulted in a generally accepted set of chemical shifts for different types
of carrageenans as summarized by van de Velde et al. (2002). However, to convert chemical shifts
from aqueous internal DMSO or MeOH to values relative to TMS is not obvious as TMS is only
sparingly soluble in highly polar solvents, such as water or D 2 O. Therefore, the IUPAC commission
for molecular structure and spectroscopy recently recommended the use of 2,2-dimethyl-2-silapentane-
FTIR spectroscopy. Infrared (IR) spectroscopy was, until recently the most frequently used vibrational
technique for the study of the chemical composition of phycocolloids. This technique presents two
main advantages: it requires minute amounts of sample (milligrams), and it is non-aggressive method
with reliable accuracy (Pereira et al. 2003). However, conventional IR spectroscopy requires laborious
procedures to obtain spectra with a good signal/noise ratio (Chopin and Whalen 1993). This limitation
was overcome with the development of interferometric IR techniques (associated with the Fourier
transform algorithm), known as FTIR spectroscopy (Fourier Transform IR). More recently, Pereira and
collaborators used a technique of analysis on the basis of FTIR-ATR (from Attenuated Total Reflectance)
spectroscopy, allowing for determination of the composition of the different phycocolloids from dried
ground seaweed, without having to prepare tablets of KBr (Pereira and Mesquita 2004; Pereira 2006;
Pereira et al. 2009a).
Raman spectroscopy. In contrast with FTIR, the application of traditional Raman spectroscopy was
limited until recently, due to the laser-induced fluorescence (strong background signal that is detected
when some samples, such as biochemical compounds, are excited with visible lasers) and risk of sample
destruction by light energy. The use of Nd:YAG lasers operating at 1064 nm has been generalized to
decrease the fluorescence level. Opto-electronic devices have progressed dramatically in the past decade,
because of major achievements in solid-state technology. As a result, compact, efficient and reliable diode
lasers are now available from visible to infrared light and have been shown to work correctly on Raman
instruments in combination with suitable filter sets (Pereira 2006; Pereira et al. 2009b).
Raman spectroscopy comprises the family of spectral measurements made on molecular media based
on inelastic scattering of monochromatic radiation. During this process, energy is exchanged between the
photon and the molecule such that the scattered photon is of higher or lower energy than the incident
photon. The difference in energy is made up by a change in the rotational and vibrational energy of the
molecule, and gives information on its energy levels. The modern FT-Raman spectrometers have been
used to produce good quality Raman spectra from seaweed samples (Matsuhiro 1996; Pereira et al. 2003;
Dyrby et al. 2004; Pereira 2006).
Four different FT-Raman spectra are presented in Fig. 3 (Chondrus crispus, female gametophytes),
corresponding to the different tests of depigmentation to reduce the fluorescence caused by the laser beam
in Raman spectroscopy. The spectrum “a” corresponds to the ground seaweed treated with a mixture of
acetone and methanol; this presents some fluorescence, particularly in the spectral area 600–875 cm
–1
and the peaks are ill-defined. The spectrum “b” corresponds to the fresh seaweed treated with calcium
hypochlorite 4% (30 s), then dried and milled. The spectrum “c” concerns the ground seaweed (obtained
from a herbarium sample) treated with calcium hypochlorite 4% (30 s). Finally, the spectrum “d” was
obtained from the native carrageenan (C. crispus water-extracted) analysis. The last three spectra (b, c, d)
so not exhibit fluorescence, with peaks well-defined and without background noise (after Pereira et al.
2009b).
NMR spectroscopy
Since natural carrageenans are mixtures of different sulfated polysaccharides, their composition differs
from batch to batch. Therefore, the quantitative analysis of carrageenan batches is of greatest importance
for both ingredient suppliers and food industries to ensure ingredient quality. From the pioneering work
of Usov and coworkers (Yarotsky et al. 1977; Usov 1984), NMR-spectroscopy is nowadays one of the
preferred techniques to determine and quantify the composition of carrageenan batches (van de Velde
et al. 2002). Starting with their early work, chemical shifts of carrageenan resonances are generally
converted to values relative to tetrametylsilane (TMS) via an internal dimethylsulphoxide (DMSO)
or methanol (MeOH) standard (Usov et al. 1980; Usov 1984; Knutsen et al. 1994). The use of DMSO
or MeOH as internal standard resulted in a generally accepted set of chemical shifts for different types
of carrageenans as summarized by van de Velde et al. (2002). However, to convert chemical shifts
from aqueous internal DMSO or MeOH to values relative to TMS is not obvious as TMS is only
sparingly soluble in highly polar solvents, such as water or D 2 O. Therefore, the IUPAC commission
for molecular structure and spectroscopy recently recommended the use of 2,2-dimethyl-2-silapentane-