Sergei V. Bykov

Steady-State and Transient Ultraviolet Resonance Raman Spectrometer for the 193–270 nm Spectral Region

We describe a state-of-the-art tunable ultraviolet (UV) Raman spectrometer for the 193–270 nm spectral region. This instrument allows for steady-state and transient UV Raman measurements. We utilize a 5 kHz Ti-sapphire continuously tunable laser (∼20 ns pulse width) between 193 nm and 240 nm for steady-state measurements. For transient Raman measurements we utilize one Coherent Infinity YAG laser to generate nanosecond infrared (IR) pump laser pulses to generate a temperature jump (T-jump) and a second Coherent Infinity YAG laser that is frequency tripled and Raman shifted into the deep UV (204 nm) for transient UV Raman excitation. Numerous other UV excitation frequencies can be utilized for selective excitation of chromophoric groups for transient Raman measurements. We constructed a subtractive dispersion double monochromator to minimize stray light. We utilize a new charge-coupled device (CCD) camera that responds efficiently to UV light, as opposed to the previous CCD and photodiode detectors, which required intensifiers for detecting UV light. For the T-jump measurements we use a second camera to simultaneously acquire the Raman spectra of the water stretching bands (2500–4000 cm−1) whose band-shape and frequency report the sample temperature.

Raman studies of solution polyglycine conformations.

Polyglycine (polygly) is an important model system for understanding the structural preferences of unfolded polypeptides in solution. We utilized UV resonance and visible Raman spectroscopy to investigate the conformational preferences of polygly peptides of different lengths in water containing LiCl and LiClO(4). Lithium salts increase the solubility of polygly. Our study indicates that in solution the conformational ensemble of polygly, as well as central peptide bonds of gly(5) and gly(6), are dominated by the 3(1) extended helix, also known as the polyglycine II conformation (PGII). This preference of the polygly backbone for the PGII conformation in solution is likely a result of favorable interactions between carbonyl dipoles in these extended helices. We found that high concentrations of Li(+) stabilize the PGII conformation in solution, most likely by polarizing the peptide bond carbonyls that makes PGII-stabilizing carbonyl-carbonyl electrostatic interactions more favorable. This ability of Li(+) to stabilize 3(1)-helix conformations in solution gives use to the denaturing ability of lithium salts.

UV Resonance Raman Investigation of Electronic Transitions in α-helical and Polyproline II-like Conformations

UV resonance Raman (UVRR) excitation profiles and Raman depolarization ratios were measured for a 21-residue predominantly alanine peptide, AAAAA(AAARA) 3A (AP), excited between 194 and 218 nm. Excitation within the pi-->pi* electronic transitions of the amide group results in UVRR spectra dominated by amide vibrations. The Raman cross sections and excitation profiles provide information about the nature of the electronic transitions of the alpha-helix and polyproline II (PPII)-like peptide conformations. AP is known to be predominantly alpha-helical at low temperatures and to take on a PPII helix-like conformation at high temperatures. The PPII-like and alpha-helix conformations show distinctly different Raman excitation profiles. The PPII-like conformation cross sections are approximately twice those of the alpha-helix. This is due to hypochromism that results from excitonic interactions between the NV 1 transition of one amide group with higher energy electronic transitions of other amide groups, which decreases the alpha-helical NV 1 (pi-->pi*) oscillator strengths. Excitation profiles of the alpha-helix and PPII-like conformations indicate that the highest signal-to-noise Raman spectra of alpha-helix and PPII-like conformations are obtained at excitation wavelengths of 194 and 198 nm, respectively. We also see evidence of at least two electronic transitions underlying the Raman excitation profiles of both the alpha-helical and the PPII-like conformations. In addition to the well-known approximately 190 nm pi-->pi* transitions, the Raman excitation profiles and Raman depolarization ratio measurements show features between 205-207 nm, which in the alpha-helix likely results from the parallel excitonic component. The PPII-like helix appears to also undergo excitonic splitting of its pi-->pi* transition which leads to a 207 nm feature.

Solution and Solid Trinitrotoluene (TNT) Photochemistry: Persistence of TNT-like Ultraviolet (UV) Resonance Raman Bands

We examined the 229 nm deep-ultraviolet resonance Raman (DUVRR) spectra of solution and solid-state trinitrotoluene (TNT) and its solution and solid-state photochemistry. Although TNT photodegrades with a solution quantum yield of φ ∼ 0.015, the initial photoproducts show DUVRR spectra extraordinarily similar to pure TNT, due to the similar photoproduct enhancement of the –NO2 stretching vibrations. This results in TNT-like DUVRR spectra even after complete TNT photolysis. These ultraviolet resonance Raman spectral bands enable DUVRR of trace as well as DUVRR standoff TNT detection. We determined the structure of various initial TNT photoproducts by using liquid chromatography-mass spectrometry and tandem mass spectrometry. Similar TNT DUVRR spectra and photoproducts are observed in the solution and solid states.

High-Throughput, High-Resolution Echelle Deep-UV Raman Spectrometer

We constructed an ultrahigh-throughput, high-resolution ultraviolet (UV) Raman spectrograph that utilizes a high-efficiency filter-stage monochromator and a high-dispersion Echelle spectrograph. The spectrograph utilizes a total of six mirrors and two gratings, with an overall efficiency at 229 nm of ∼18%. The limiting resolution of our spectrometer is 0.6 cm−1 full width half-maximum (FWHM), as measured for 229 nm Rayleigh scattering. Use of a 1 mm–wide entrance slit gives rise to an approximately 10 cm−1 FWHM resolution at 229 nm. The ultrahigh spectrograph throughput enables ultrahigh signal-to-noise ratio, deep UV Raman spectra that allow us to monitor <1% changes in peptide bond composition. The throughput is measured to be 35-fold greater than conventional deep UV Raman spectrometers.

Dependence of Glycine CH2 Stretching Frequencies on Conformation, Ionization State, and Hydrogen Bonding

We experimentally and theoretically examined the conformation, pH, and temperature dependence of the CH2 stretching frequencies of glycine (gly) in solution and in the crystalline state. To separate the effects of the amine and carboxyl groups on the CH2 stretching frequencies we examined the Raman spectra of 2,2,2-d3-ethylamine (CD3-CH2-NH2) and 3,3,3-d3-propionic acid (CD3-CH2-COOH) in D2O. The symmetric (nusCH2) and asymmetric (nuasCH2) stretching frequencies show a significant dependence on gly conformation. We quantified the relation between the frequency splitting (Delta = nuasCH2-nusCH2) and the xi angle which determines the gly conformational geometry. This relation allows us to determine the conformation of gly directly from the Raman spectral frequencies. We observe a large dependence of the nusCH2 and nuasCH2 frequencies on the ionization state of the amine group, which we demonstrate theoretically results from a negative hyperconjugation between the nitrogen lone pair and the C-H antibonding orbitals. The magnitude of this effect is maximized for C-H bonds trans to the nitrogen lone pair. In contrast, a small dependence of the CH2 stretching frequencies on the carboxyl group ionization state arises from delocalization of electron density from carboxyl oxygen to C-H bonding orbitals. According to our experimental observations and theoretical calculations the temperature dependence of the nusCH2 and nuasCH2 of gly is due to the change in the hydrogen-bonding strength of the amine/carboxyl groups to water.

Compact Solid-State 213 nm Laser Enables Standoff Deep Ultraviolet Raman Spectrometer: Measurements of Nitrate Photochemistry

We describe a new compact acousto-optically Q-switched diode-pumped solid-state (DPSS) intracavity frequency-tripled neodymium-doped yttrium vanadate laser capable of producing ~100 mW of 213 nm power quasi-continuous wave as 15 ns pulses at a 30 kHz repetition rate. We use this new laser in a prototype of a deep ultraviolet (UV) Raman standoff spectrometer. We use a novel high-throughput, high-resolution Echelle Raman spectrograph. We measure the deep UV resonance Raman (UVRR) spectra of solid and solution sodium nitrate (NaNO3) and ammonium nitrate (NH4NO3) at a standoff distance of ~2.2 m. For this 2.2 m standoff distance and a 1 min spectral accumulation time, where we only monitor the symmetric stretching band, we find a solid state NaNO3 detection limit of ~100 μg/cm2. We easily detect ~20 μM nitrate water solutions in 1 cm path length cells. As expected, the aqueous solutions UVRR spectra of NaNO3 and NH4NO3 are similar, showing selective resonance enhancement of the nitrate (NO3−) vibrations. The aqueous solution photochemistry is also similar, showing facile conversion of NO3− to nitrite (NO2−). In contrast, the observed UVRR spectra of NaNO3 and NH4NO3 powders significantly differ, because their solid-state photochemistries differ. Whereas solid NaNO3 photoconverts with a very low quantum yield to NaNO2, the NH4NO3 degrades with an apparent quantum yield of ~0.2 to gaseous species.

Solution and Solid Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) Ultraviolet (UV) 229 nm Photochemistry

We measured the 229 nm deep-ultraviolet resonance Raman (DUVRR) spectra of solution and solid-state hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). We also examined the photochemistry of RDX both in solution and solid states. RDX quickly photodegrades with a solution quantum yield of u ∼ 0.35 as measured by high-performance liquid chromatography (HPLC). New spectral features form over time during the photolysis of RDX, indicating photoproduct formation. The photoproduct(s) show stable DUVRR spectra at later irradiation times that allow standoff detection. In the solution-state photolysis, nitrate is a photoproduct that can be used as a signature for detection of RDX even after photolysis. We used high-performance liquid chromatography-high-resolution mass spectrometry (HPLC-HRMS) and gas chromatography mass spectrometry (GCMS) to determine some of the major solution-state photoproducts. X-ray photoelectron spectroscopy (XPS) was also used to determine photoproducts formed during solid-state RDX photolysis.

Ultraviolet Raman Wide-Field Hyperspectral Imaging Spectrometer for Standoff Trace Explosive Detection:

We constructed the first deep ultraviolet (UV) Raman standoff wide-field imaging spectrometer. Our novel deep UV imaging spectrometer utilizes a photonic crystal to select Raman spectral regions for detection. The photonic crystal is composed of highly charged, monodisperse 35.5 ± 2.9 nm silica nanoparticles that self-assemble in solution to produce a face centered cubic crystalline colloidal array that Bragg diffracts a narrow ∼1.0 nm full width at half-maximum (FWHM) UV spectral region. We utilize this photonic crystal to select and image two different spectral regions containing resonance Raman bands of pentaerythritol tetranitrate (PETN) and NH4NO3 (AN). These two deep UV Raman spectral regions diffracted were selected by angle tuning the photonic crystal. We utilized this imaging spectrometer to measure 229 nm excited UV Raman images containing ∼10–1000 µg/cm2 samples of solid PETN and AN on aluminum surfaces at 2.3 m standoff distances. We estimate detection limits of ∼1 µg/cm2 for PETN and AN films under these experimental conditions.

Raman Hyperspectral Imaging Spectrometer Utilizing Crystalline Colloidal Array Photonic Crystal Diffraction

We fabricated a novel hyperspectral Raman imaging spectrometer that, for the first time, uses a photonic-crystal wavelength-selecting device to select a narrow-wavelength spectral interval. The photonic crystal consists of an array of highly charged, monodisperse polystyrene particles that self-assemble into a face-centered cubic crystal. The photonic crystal Bragg-diffracts a narrow spectral interval that can be tuned by altering the incident angle of collimated Raman scattered light. Our prototype spectrometer diffracts a ∼200 cm−1 interval of the 488 nm excited visible Raman spectrum of Teflon. This enabled us to select a close-lying triplet of Teflon Raman bands. We imaged the Teflon surface by focusing this narrow region onto a charge-coupled device to create a Raman image of the sample surface that spectrally details the chemical composition.