Recently, the field of molecular spectroscopy has expanded into new and exciting biological, medical, and sensing applications.
This expansion resulted from both improvements in existing instrumentation and the development of new techniques in the fields
of Raman, fluorescence, and infrared spectroscopy. Of particular interest, is how these techniques have moved beyond previous
limitations, such as the diffraction limit of light or direct imaging through difficult media like plastic or bone. Although
much of this work was carried out in an academic setting, the results provide important directions for future industrial endeavors.
A broad field encompassing several techniques, molecular spectroscopy has revealed fundamental chemical and biological information
in a variety of applications. Although there are many different types of molecular spectroscopy, these processes involve how
analytes interact with light. The following sections highlight recent advancements in three major areas of molecular spectroscopy:
infrared (IR), Raman, and fluorescence. Improvements in instrumentation and the development of new techniques have greatly
enhanced each field in imaging, sensing, and understanding fundamental chemical principles. Each one has certain advantages
and problems that confine them to certain disciplines, and in some cases, these limitations have been overcome for future
IR spectroscopy uses infrared light sources to irradiate molecules, which absorb at specific frequencies. These frequencies
depend on the molecular size, structure, and composition, with the measured frequencies corresponding to certain motions.
Furthermore, the vibrational and rotational motions of the molecule also depend on the functional groups, and these can be
used to differentiate similar compounds with small differences. The broad vibrational bands of water are one of the primary
limitations in IR spectroscopy, but this limitation has been addressed by advancements in spectral processing. Other alternatives
include working with solid samples and collecting reflectance measurements using techniques such as attenuated total reflectance
IR spectroscopy. Although the details of IR spectroscopy have been reviewed and highlighted elsewhere, the following section
describes some recent advances in sensing, protein folding, and new applications through coupling to another instrument.
The most common IR technique, Fourier transform infrared (FT-IR) spectroscopy, is a widely used approach in fields ranging
from forensics to art conservation. Recently, it has become a popular analytical tool in detecting adulterated food. This
field encompasses industry as well as government in testing the authenticity and safety of different foods, plants, and herbs.
Some of the advantages of FT-IR for food detection are ease of use, quick read out, small waste, and retention of the original
sample (1). The primary concerns in food safety include watered-down products, cheap unnatural fillers, and, most importantly,
toxic contaminants. Melamine is a common industrial plastic that can react with cyanuric acid or other additives and become
toxic and lead to kidney damage. The Food and Drug Administration (FDA), European Union (EU), and World Health Organization
(WHO) have set standards on the amount of melamine that can be consumed, which became an international health concern when
it was discovered in infant formula. Although mass spectrometry (MS) can identify melamine, it requires longer processing
times with a relatively large sample volume. In the case of trying to test several shipments of a product entering a country,
such as formula, this would be difficult to implement. Researchers used FT-IR and multivariate analysis to detect melamine
in tainted formula based on stretches from the amino groups (1).
To explore complex biological processes such as protein folding, two-dimensional (2D) IR spectroscopy uses ultrafast spectroscopy
to probe mechanical motions on picosecond timescales (2). Two-dimensional IR is still an emerging technique, but it has successfully
determined the structure of small-unknown peptides and has similar capabilities to 2D nuclear magnetic resonance (NMR) spectroscopy.
With the addition of isotopic labeling, Buchanan and coworkers (3) directly explored the misfolding and kinetics of an amyloid
peptide, amylin, associated with type II diabetes. By monitoring a carbonyl backbone isotope, they discovered important information
related to the secondary structure of this peptide in forming fibrils, which has further implications for other diseases such
as Alzheimer's disease and Parkinson's disease. Although NMR provides structural information with good resolution, it cannot
determine intermediates on this timescale.
Even though IR spectroscopy has biological limitations, new progress in ionic sources and techniques are making IR more competitive
and universal (4). One such example is IR ion spectroscopy coupled with MS, which has the capability of discerning small molecules.
An area of high interest for identifying such compounds is metabolomics, which involves the study of residual small analytes
to gain new insight about intracellular processes. The addition of IR to MS aided in identifying a group of peptides that
all have a mass at ~669 Da, but contain unique functional groups such as a phosphate or amide stretch in the molecule. Such
subtle details hidden within the mass spectrum were actually quantified using IR spectroscopy.
Although the examples outlined here describe the broad applications of IR, several challenges remain. In the case of FT-IR,
the overtones associated with water — especially in the near-infrared region — completely overwhelm the spectra, and this
can prevent moisture-rich foods from being detected. Moreover, in detecting melamine, while MS was more time consuming, the
detection limit was ~250 ppb, but FT-IR only reached ~75 ppm. While efficiency and sample preparation have some importance,
the primary aim remains sensitivity. As the 2D IR technique develops and researchers continue to explore problems like protein
misfolding, it still requires careful labeling and only a few bands or stretches can be monitored at a time. Furthermore,
2D NMR still provides better structural resolution and ease in assignment of spectral peaks even at slower timescales. The
coupling of IR with MS bridges the advantages of both techniques, but the difficulty lies in the ionization of biomolecular
analytes. In addition, the complex nature of such a high concentration of metabolites requires an initial screening with a
high-resolution combination MS technique before IR. Some of the challenges discussed here can be addressed or avoided using
other techniques in Raman or fluorescence spectroscopy.