Intracavity Optogalvanic Spectroscopy, A New Ultra-sensitive Analytical Technique for ¹⁴C Analysis

Abstract

We show a new ultra-sensitive laser based analytical technique, intracavity optogalvanic spectroscopy, allowing extremely high sensitivity for detection of ¹⁴C labeled carbon dioxide. Capable of replacing large accelerator mass spectrometers, the technique quantifies attomoles of ¹⁴C in submicrogram samples. Based on the specificity of narrow laser resonances coupled with the sensitivity provided by standing waves in an optical cavity and detection via impedance variations, limits of detection near 10^{-15 14}C:¹²C ratios are obtained. Using a 15W ¹⁴CO₂ laser, a linear calibration with samples from 10^-15 to >2 × 10^-12 in ¹⁴C:¹²C ratios, as determined by accelerator mass spectrometry, is demonstrated. Possible applications include microdosing studies in drug development, individualized sub-therapeutic tests of drug metabolism, carbon dating and real time monitoring of atmospheric radiocarbon. The method can also be applied to detection of other trace entities.

Carbon 14 (radiocarbon) is an ideal organic tracer having an extremely low natural abundance in living systems, near 1 ppt, and a long half life, 5730 years, ideal for clinical and laboratory tracer experiments. Until recently almost all quantitation of ¹⁴C content has been by scintillation detection of the low energy beta particle emitted in its decay. At present there is great interest in the drug development community for pharmacokinetic information on new drug entities using non therapeutic microdoses of labeled drugs, which require much higher analysis sensitivity than is possible with scintillation counting¹. Accelerator Mass Spectroscopy (AMS), first developed to extend carbon dating to smaller and older samples, has become the standard method for ¹⁴C atom counting in such sensitive bioanalytical tracer studies^{2, 3}. Limitations to wide use of AMS for bioanalytical studies include size, cost and complexity of the analysis system as well as the fact that samples must contain of the order of 0.5 mg total carbon. The laser assisted ratio analyzer (LARA) technique⁴ is based on the existence of large isotope shifts in molecular spectra, the use of fixed frequency isotopic lasers and sensitive detection via the laser optogalvanic effect (OGE). Research and development during the past decade has demonstrated that LARA’s sensitivity and versatility make new classes of isotope ratio measurement systems possible^5,6.

The basic concept of OGE detection has long been used in atomic and molecular spectroscopy⁷. It is based on the electrical response of a gas discharge to an optical perturbation. If a laser of intensity I, and frequency ν is incident on a cylindrical (length L and radius R) weak electrical discharge, the electrical response, S, of the discharge can be expressed by an integral over the laser-discharge interaction volume:

$$S={\int }_0^{L}dz{\int }_0^{R}rdr{\int }_0^{2\pi }d\theta \left[n(r,\theta )I(r,\theta ,z,\nu )\sigma \left(\nu \right)K\right]$$ (1)

The density of interacting species is n and σ(ν) refers to the laser-species interaction cross section. S can be any discharge parameter related to conductivity and K is a corresponding optogalvanic proportionality constant that depends on the details of the electrical discharge. To a good approximation, on resonance, for cases where light absorption is small and K is independent of n, the average electrical response simplifies to a product:

$$\mathrm{S}=\mathrm{nLIA}\sigma \mathrm{K}$$ (2)

Here, n represents the average molecular density of interacting particles, L, the length of the interaction region, I the average laser intensity and A the average area of the laser beam. Physically, what happens is that the light changes the equilibrium distribution of species, including excited species in the discharge. This affects collision rates, including those between molecules and electrons, leading to a measurable conductivity change, S, of the ionized gas. Optogalvanic spectroscopy for isotopic analysis makes use of the fact that laser resonances in CO₂ are isotope dependent⁸.

In practice, a periodically switched cw laser in resonance with a specific molecular transition changes the population of excited molecular states, which, in turn, changes the electron density and/or temperature in the discharge giving rise to an impedance change varying with the laser intensity. There exist several significant advantages of OGE measurements compared to ion beam and other spectrometric stable isotope measurement technologies:

1. The measured parameter is electrical and periodic so that filtering and averaging techniques can be used to achieve extremely low noise and background.

2. No optical measurement is required, thus eliminating the need for collection and dispersion optics and light transducers.

3. The measured signal is proportional to the product of analyte density, path length and laser power. In contrast with laser absorption techniques or ion counting, the laser power provides a gain, or sensitivity enhancement per molecule, so that signals for dilute isotopes can be amplified relative to the majority species.

4. The measurement system can be varied, within limits, in order to enhance signal to background ratios by controlling gas mixtures (ranging from 100% to 1% or less CO₂, balance N₂, CO₂ free air or other inert buffer) pressure (typically 1 to 5 mbar) and electrical discharge power (typically 2 to 5 Watts).

Previous success using LARA for breath testing^{9,10,11,12,13,14,15} and environmental monitoring¹⁶ has led to the development of the technique for the detection of ¹⁴C reported here. When the laser operates at a particular transition in ¹⁴CO₂, any resonant change in the impedance of the discharge system is due to the presence of ¹⁴CO₂ in the discharge. The laser transition chosen for ¹⁴CO₂ must be well separated in wavelength from any transition in ¹²CO₂ or ¹³CO₂ (1% natural abundance) and from any transition in a CO₂ molecule with ¹⁸O (.02% natural abundance) or ¹⁷O (.004% natural abundance). The discreteness of energy levels and the large isotope shifts in vibrational-rotational transitions allow a line specific ¹⁴CO₂ laser to be built. The strongest lasing transitions observed in ¹⁴CO₂ are at 11.8 microns and 11.3 microns, significantly longer in wavelength than lasing transitions of the other “stable” isotope CO₂ lasers. Any ¹⁴CO₂ molecule in the lower or upper laser level is automatically in resonance (absorption or stimulated emission) with the narrow band laser, providing the sharp specificity required for isotope ratio analysis. The nearest ¹³CO₂ and ¹²CO₂ lines are separated by more than 500 linewidths, leading to non resonant cross sections reduced by approximately 10 orders of magnitude. The nonresonant interaction is further reduced for ¹³CO₂ and ¹²CO₂ by rotational state Boltzmann population ratios for the two isotopes. In any case, this small background effect is pressure dependent and measurable and does not seriously limit the ultimate ¹⁴CO₂ detection limit.

Extension of the LARA technique to ¹⁴CO₂ was previously established with highly enriched (nano-Curie) ¹⁴C samples¹⁷. In those studies, an isotope ratio (¹⁴C/¹²C) sensitivity of order 10^-9 was demonstrated with a very simple external cavity configuration. A ¹⁴CO₂ laser at 11.8 microns with a maximum output power of 2 Watts was used. The measurement yielded signal-to-noise ratios of several hundred with short averaging times and a sensitivity limit of about 1 picomole ¹⁴CO₂. Measurements with non-enriched carbon dioxide confirmed that no memory effect was present and that the system sensitivity was far too low for detection at Modern (1.180 × 10^{-12 14}C/C) levels of ¹⁴CO₂.

Enhancement of sensitivity in laser absorption experiments often involves increased absorption length in optical cavities-most notably cavity ringdown spectroscopy (CRDS)¹⁸ and laser intracavity absorption spectroscopy (ICAS)¹⁹. The new standing wave technique reported here is analogous to, but with key differences from, ICAS. The most significant difference from ICAS is that detection is via the OGE rather than optical. The analyte in this case is the same as the lasing species and an OGE exists for stimulated emission and/or absorption.

EXPERIMENTAL SECTION

The layout of the measurement system is shown in Figure 1. The sample cell is placed inside a laser cavity (LaserTech Group Inc. Model LTG250, 15 W output with sealed ¹⁴CO₂ fill) thus subjecting it to the full saturated laser power. Also inside the laser cavity is a computer controlled shutter used for modulating the ¹⁴CO₂ laser beam. The laser output beam can be used to monitor power and wavelength as well as for parallel external cell measurements if required. A small electronically modulated ¹²CO₂ laser (Synrad Model 48-1, 5 W output) beam passes through the ¹⁴CO₂ laser output coupler and the sample cell for single pass ¹²CO₂ measurement. This ¹²CO₂ signal is used for normalization. The quartz sample cell, 10 cm in length and 2.5 cm OD, with ZnSe Brewster angle windows is pumped with an oil free vacuum pump to low pressure. In these studies, a radio frequency (rf) glow discharge is utilized because of its stability and low inherent noise²⁰. With sample pressure held constant-typically between 1 and 5 mbar, a low power (2 to 5 Watt) rf discharge is ignited and maintained via external copper electrodes. The oscillator power supply circuit also monitors the average rf voltage amplitude across the discharge for the OGE signal. The ¹²CO₂ and ¹⁴CO₂ laser beams simultaneously passing through the sample cell are chopped at different frequencies, and the resulting voltage variation across the cell is measured. The Fourier Transform values of the voltage signal at the two different frequencies yield the ¹²CO₂ and ¹⁴CO₂ OGE signals.

Figure 1.

Experimental configuration: The OGE cell inside the cavity has Brewster windows to reduce losses. The C12 laser incident on the OGE cell provides a “C12 signal” that is used for normalization of the C14 signal. The shutter inside the laser cavity is for modulating the ¹⁴CO₂ laser. M1: High reflective mirror & grating, M2: 85% reflective output coupler, M3: Gold plate mirror, PS: Pressure Sensor, FC: Flow Controller, RGA: Residual Gas Analyzer, DAQ: Data Acquisition Board

The sample consists of CO₂ with a buffer (95 to 99%) of nitrogen or CO₂ free air, or 100% CO₂. Calibration samples consist of tanks of research grade CO₂ (Air Products) with ¹⁴CO₂ less than 10^-12 (1 Modern) level as determined by AMS measurements at Lawrence Livermore National Laboratory (LLNL). Aliquots of CO₂ with varying ¹⁴CO₂ concentrations were also supplied by LLNL. Measurements could be run in a sealed cell (batch mode) or continuous flow mode with flow rates of 0.1 to 2 sccm.

Time averaged OGE signals were obtained using a National Instruments Data Acquisition System. The virtual instrument designed controlled the shutter, and monitored pressure, ¹²CO₂ concentration and laser wavelength data. An OGE output from the ¹⁴CO₂ laser tube was used to stabilize laser wavelength and for laser power normalization.

RESULTS and DISCUSSION

Figure 2 shows the OGE response to an enriched sample (9 M) of 22 micromoles CO₂ containing 223 attomoles ¹⁴CO₂. The figure is a 5 second coherent averaged waveform with the ¹⁴CO₂ laser chopped at 63 Hz. Figure 3 shows a resonance curve obtained by tuning the laser through its gain profile. The optogalvanic effect line shape shows the Voigt profile expected for the discharge conditions. The non resonant background (and the peak intensity) varies with different laser lines, also as expected. Under all conditions, the observed signal is indicative of a greatly enhanced interaction length due to the intracavity standing wave. The maximum enhancement observed over the single pass system is of order 10 million which was initially surprising as in ICAS studies with a single mode laser gain enhancements only of order 100 are expected.

Figure 2.

The OGE signal in response to a laser modulated at 63 Hz. The sample is 5% CO₂ in N₂ at 10^{-11 14}C enrichment. The waveform is a 5-second coherent averaged waveform.

Figure 3.

Resonance curve for intracacity optogalvanic effect. The solid line is a best fit Voigt Profile, The width, 48 MHZ is expected for ¹⁴CO₂ in the 5 mbar discharge at 385C.

We believe that the observed mega-enhancement is due to the nature of the optogalvanic effect in the optical cavity. In equations 1 and 2 above I is the circulating internal laser power, around 50 W in our case, and L the interaction length is now greatly enhanced due to the standing wave. A simple estimate would have L enhanced only by the number of reflections of a single photon, where the major loss is due to the output coupler. However, for the cw laser system, loss in the OGE cell is balanced by the gain of the lasing medium keeping the internal circulating power constant. Now, the effective interaction length L_eff is given by L_eff=cΔt, where Δt is given by the time that the laser is on. For a modulation frequency of 63 Hz, Δt is 15.8 msec yielding an effective interaction length of almost 5000 km. This value is close to our observed maximum enhancement of ∼10⁷ for the intracavity configuration compared to single pass. In a multimode ICAS experiment, Δt is given by the spectral saturation time and can be as long as several seconds²¹. In the ICAS experiment what is observed is a change in laser output. The effect on laser output of the few thousand ¹⁴CO₂ molecules in the OGE analysis cell is negligible compared to the 10¹⁹ active ¹⁴CO₂ molecules in the laser cavity. However, all photon interactions in the intracavity cell contribute to the OGE.

The sensitivity is so great that the natural abundance of ¹⁴C in ambient air can easily be detected. An estimate of the present limit of detection is of order 10^{-15 14}C/¹²C ratio, similar to AMS. It is known that nitrogen is an ideal buffer gas for the OGE in CO₂ for much the same reason that nitrogen is used in the CO₂ laser. However, unlike the situation in a laser, where helium is also present to depopulate lower levels, helium is found to reduce the OGE²². Further, we have found that air is a better buffer gas than pure nitrogen, as it inhibits CO₂ dissociation. The optogalvanic proportionality factor for ¹⁴CO₂ in equations 1 and 2 is a complicated function of partial pressure for all gas constituents as well as discharge conditions,²³ even changing sign as the system goes from absorption to gain conditions. Also, equations 1 and 2 assume thermal equilibrium, a situation only easily achieved in tens of ms for low ¹⁴CO₂ concentration. These effects make calibration of the system over large changes in ¹⁴CO₂ concentration difficult. For radiocarbon dating of small samples and drug development studies near modern enrichment a linear calibration region over more than an order of magnitude has been found using pure CO₂ as the carrier gas, at approximately 1 mbar pressure with a Δt near 25msec. Figure 4 shows the calibration from 4.9 ×10^-3 Modern to 1.5 Modern samples with the OGE signal plotted against AMS results. A 12 Modern sample showed saturation effects under the same measurement conditions. The slope of the calibration curve, 0.4 is in agreement with calculations due to the non-resonant signal from ¹³C and ¹²C discussed above. A detailed model of the intracavity OGE must include laser saturation effects as well as equilibration time constant effects on the OGE parameter K.

Figure 4.

Calibration Curve: OGE signals obtained with the ICOGS system plotted against the AMS results. The vertical error bars are determined by voltage averaging statistics.

The system can run with continuous flow as shown in figure 5 for 1ml CO₂ injected into a gas stream of CO₂ free air as carrier. The buildup and loss of ¹²C and ¹⁴C is obvious. The saturation effect present when the concentration reaches around 1% is also obvious. Modeling of this effect using ICAS theory (References ¹⁸ and ²⁰) and OGE theory ²⁴ is in progress.

Figure 5.

Variation of OGE for ¹²C (top) and ¹⁴C (bottom) as 1 ml is injected into the CO₂ free buffer gas flowing at 10 SCCM. As shown in the inset, the signals scale at low concentrations, but the ¹⁴C signal saturates at a much lower concentration than the ¹²C.

CONCLUSION

It is believed that the sensitivity shown in these and certain AMS experiments will allow more rapid drug development by permitting early safe metabolic testing with microdoses of test compounds²⁵. Further, such microdosing studies can be used to customize doses for individual metabolic differences. Though both ¹⁴C LARA and AMS aim to quantify ¹⁴C in biological samples, there are several significant differences in the technologies. For both techniques, a carbon containing compound is first oxidized to CO₂. The analyte in the LARA technique remains in the form of CO₂, while AMS machines, at present, require an additional step to reduce the CO₂ sample to elemental carbon. There are significant efforts, however, to enable Bio-AMS machines to run effectively with a gas ion source utilizing CO ²⁶₂. Reasons for these efforts include improving efficiency via fewer processing steps, and higher throughput, as well as enabling smaller sample size. Ultimately it is desired to couple an input separation device such as a gas or liquid chromatograph with fractions oxidized to CO₂ and continuously flowed through the system. .

The ¹²C and/or ¹³C normalization is straightforward with our system for samples of any enrichment, using ¹²CO₂ and ¹³CO₂ lasers as is now done with existing breath and environmental monitoring instruments. Furthermore, the small sample required and the possibility of continuous flow or batch processing should permit development of combined instrumentation with gas chromatographs or liquid chromatographs coupled to an oxidizer as a front end^{27, 28}. The measurement sensitivity is so great that carbon dating using microgram samples can easily be envisioned.

Limits of precision and sensitivity are yet to be determined but should equal or exceed those of AMS. Use of an external calibration cell and double ratio measurements as is done to determine ¹³C variations at the part per 10 thousand⁶ level should also be possible with ¹⁴C. The engineering challenge will be in the handling of such small samples and eliminating the ubiquitous background ¹⁴CO₂. We further note that the technique may be extended to other trace atom or compound analysis with tunable or fixed frequency lasers.