Analysis of the conversion of indigo into indigo carmine dye using
SERRS
Iqbal T. Shadi, Babur Z. Chowdhry,* Martin J. Snowden and Robert Withnall*
Vibrational Spectroscopy Centre, Medway School of Sciences, University of Greenwich, Chatham Maritime
Campus, Pembroke, Chatham, Kent, UK ME4 4TB. E-mail: R.Withnall@gre.ac.uk; Fax: + 44 (0) 20 8331
9983; Tel: + 44 (0) 20 83318691
Received (in Cambridge, UK) 10th March 2004, Accepted 29th April 2004
First published as an Advance Article on the web 19th May 2004
In a novel application SERRS has been used, by employing a
silver sol, to monitor and analyse the conversion of indigo into
the indigo carmine dye.
photon correlation spectroscopy20 and were in accord with those
reported in the scientific literature. For SERRS analysis of indigo
the silver sol was aggregated using 35 mL of 1 mol dm23 HCl.
However, in order to analyse both synthesized and reference
samples of indigo carmine, aggregation of the silver sol was
achieved by addition of 150 mL of a 0.01% aqueous solution of
poly(l-lysine). Typically the foregoing were added to 2 mL of silver
colloid, which was prepared by diluting 1 mL of the stock silver sol
with 1 mL of deionized water. SERRS reference spectra of indigo
and indigo carmine, solubilized in methanol, were obtained at
concentrations of ~ 1026 mol dm23. The synthesis of indigo
carmine from indigo21 was conducted by adding 7 mg of indigo to
5 mL of concentrated sulfuric acid at room temperature. The
reaction mixture was left to stand for 1 h and the pH of the solution
adjusted to 2.3. 150 mL of this solution was then diluted five-fold
with methanol and a 150 mL aliquot added to the pre-prepared
aggregated silver sol and the SERRS spectrum collected. The
SERRS spectra were collected at 5–10 min intervals during the
conversion of indigo to indigo carmine. SERRS spectra were
obtained using a Labram Raman spectrometer (Instruments S. A.,
Ltd.) equipped with a 1800 grooves/mm holographic grating, a
holographic super-notch filter (Kaiser), an Olympus BX40 micro-
scope, and a Peltier-cooled CCD (MPP1 chip) detector. A helium–
neon laser provided exciting radiation of 632.8 nm. The laser beam
was attenuated by a 10% neutral density filter, resulting in a laser
power of 0.8 mW at the static sol and solution samples in all
experiments. All SERRS spectra were collected by using a 180°
back-scattering geometry. An Olympus microscope objective,
having a magnification of 310 and a numerical aperture of 0.25,
was used both to focus the incident laser light and to collect the
back-scattered Raman light.
Fig. 2 shows the SERRS spectra of indigo, indigo carmine
synthesized from indigo and indigo carmine. Spectral assignments
for selected vibrational bands, in the 1800–200 cm21 region, of the
SERRS spectra of a reference sample of indigo carmine and indigo
carmine synthesized from indigo are given in Table 1. It is
immediately obvious, both from the data in Table 1 and the SERRS
spectra shown in Fig. 2, that the synthetic and reference samples of
indigo carmine are identical. This conclusion was reinforced by
mass spectroscopy/NMR data (not shown) of the two com-
pounds.
The synthesis of indigo carmine from indigo is, under the
experimental conditions employed, complete within ~ 30 min (Fig.
3).
In Fig. 3 the ratio A1 : A2 is plotted versus time, where A1
represents the area (calculated using the Labram software) of the
vibrational bands between 522 and 626 cm21 for indigo carmine
and A2 represents the area between the same wavenumber limits for
indigo.
Surface enhanced resonance Raman spectroscopy (SERRS) has
found wide utility as a sensitive analytical tool in a multitude of
interdisciplinary scientific investigations, including e.g. clinical
and drug analysis, forensic, environmental and biological sciences
and archaeological/historical applications. In fact there has been a
growing interest in the uses of SERRS for highly sensitive chemical
analyses, in a wide range of matrices, down to the single molecule
level.1,2 The inability to interrogate intrinsically highly fluorescent
molecules, in solution, by dispersive (visible) Raman spectroscopy
can be overcome by using several alternative Raman analytical
techniques. These include e.g. FT-Raman (i.e. analysis at a
wavelength where the fluorescent molecules do not absorb),3
shifted subtractive Raman spectroscopy4 and the use of the Kerr
gate effect in time resolved Raman spectroscopy.5 In addition
SERRS [a combination of SERS (surface enhanced Raman
spectroscopy) and RRS] can also be employed, both to overcome
fluorescence and the analytically (relative) low-sensitivity gen-
erally associated with Raman scattering of molecules in solution.
Interestingly different kinds of media/substrates (e.g. silver and
gold colloids,6 nanoshell colloids7 and “gelcolls” which employ a
hydrophilic swelling polymer (such as polyacrylic acid) in
combination with a metal colloid8) have been and are being
developed for obtaining SERRS signals of analytes. Although the
theory of SERRS is still being developed, the observed SERRS
signal enhancement over RRS is currently hypothesised to be
caused by a combination of effects: the “classical electromagnetic
field (EM) enhancement”9 and the “chemical effect”.10 For
maximum sensitivity, SERRS requires controlled aggregation of
the colloidal sol used.11 In fact surface enhancement of the Raman
signals is dependent on the size of the colloidal particles as well as
the exciting wavelength employed. This is because the surface
plasmon absorption bands of metals such as silver and gold show
wavelength dependent shifts for metal particles with sizes > l/20
(l = wavelength of the incident light) and surface enhancement is
achieved by choosing the Raman excitation wavelength to coincide
with the plasmon band.12
Vat dyes such as indigo (or 2,2A-bis-indole; (1)), besides being of
significant historical interest in e.g. Coptic textiles13 and icons,14
hold a large part of the current dyestuff market ( ~ 3% of the total
production of dyes)15 for the coloration of cellulosic fibres. Its
major industrial application is the dyeing of clothes (blue jeans) and
other blue denim products. Indeed new, contemporary methods for
their synthesis16 or those of their analogues are of industrial,
economic and academic importance. The dye indigo carmine (2) is
used extensively as e.g. a food colorant (E.C. number E132) and as
a diagnostic tool in a number of medical applications.17 In an
attempt to develop novel applications of SERRS the technique has
been used to monitor and analyse the synthesis of the dye indigo
carmine from indigo (see Fig. 1) using a silver sol as the SERRS
substrate/medium.
A silver sol, for use as a SERRS substrate, was prepared via a
modified Lee–Meisel procedure.12,18 The physico-chemical char-
acteristics of the silver sol obtained were determined by UV–VIS
spectroscopy,19 transmission electron spectroscopy as well as
Fig. 1 Chemical structures of indigo (1) and indigo carmine (2). The
reaction conditions for conversion of (I) to (2) are indicated.
1436
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T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4