Absolute optical chiral analysis using cavity-enhanced polarimetry

Chiral analysis is central for scientific advancement in the fields of chemistry, biology, and medicine. It is also indispensable in the development and quality control of chiral compounds in the chemical and pharmaceutical industries. Here, we present the concept of absolute optical chiral analysis, as enabled by cavity-enhanced polarimetry, which allows for accurate unambiguous enantiomeric characterization and enantiomeric excess determination of chiral compounds within complex mixtures at trace levels, without the need for calibration, even in the gas phase. Our approach and technology enable the absolute postchromatographic chiral analysis of complex gaseous mixtures, the rapid quality control of complex mixtures containing chiral volatile compounds, and the online in situ observation of chiral volatile emissions from a plant under stress.


S2. CCP experiment
We employ a frequency metrology scheme 3-6 for the measurement of the central frequencies of each cavity mode (RCW, LCW, RCCW, LCCW), and, hence, of f ) . In particular, in Fig. S1A we present a schematic of our experimental setup, showing the CCP device that consists of a ring (four-mirror) optical cavity with the mirrors arranged in a bowtie configuration 1,2 . We split the laser beam into two parts which are then coupled, through separate optical paths, to the CW and CCW propagation modes of the cavity. We use two separate acousto-optic modulators (AOM) (Gooch & Housego 3270) to independently control and servo-lock the frequency of each of the CW and CCW laser beams into a corresponding cavity mode pair, i.e., RCW/RCCW or LCW/LCCW. The frequency stabilization/servo-locking is achieved using the Pound-Drever-Hall technique. Both AOMs are driven by a double-channel RF synthesizer (Moglabs Agile XRF421). Measurement of the servocontrolled frequency outputs (possible via the measurement of the supplied RF frequencies to the AOMs), provides us the central frequency of each mode. By resonantly coupling light into the RCW/RCCW mode pair we can record directly a frequency difference equal to 2f ) only in the presence of a chiral signal (Fig.S1B). Similarly, resonantly coupling light into the LCW/LCCW mode pair yields a frequency difference equal to −2f ) . The measurements of the AOM RF frequencies are performed using frequency counters (Stanford Research Systems; SR620) that allow us to control the measurement integration time (typically ~1s).
Finally, measurements of the average refractive index are possible through the direct measurement of the central frequencies of the cavity modes. To do so accurately, we additionally servo-lock the emission frequency of the laser to a cavity resonance. In this case, the laser's wavelength is fixed while its frequency tracks the changes in refractive index, since: δf f 6 = − δ 6 7,8 . From the gas optical refractivity measurements, we can then extract the gas density of each compound 9 .

S3.Specific optical rotation of chiral compounds
In Table 1 we provide the values for the specific optical rotation (SOR) [ ] /0123 01 ! of all the different chiral compounds we present and are relevant to our work.

S4.GC/CCP analysis of enantiopure (±)-limonene
In Fig.S2 we present GC/CCP-based analysis of enantiopure (+)-and (-)-limonene. For our analysis we use splitless injection (Agilent HP 6890A GC, Agilent Technologies, USA) of mixture solutions in ethanol (1μL of solution injected). The chiral separations were achieved using a 30m β-DEX™ 120 column (Sigma-Aldrich GmbH, Germany) with 0.25 mm internal diameter and a 0.25 μm film thickness. The column flow was chosen to be 8ml/min and the temperature programme to be 40 °C for 2 minutes then 40 °C to 210 °C at 12 °C min -1 . We use these results, under the 'GC' label, in our comparison with direct total sample measurements of enantiopure limonene in Fig.2C of the main text.

S5. Offline chiral analysis of perfumes using GC-MS
We select the commercially available low-cost fragrance clones according to advertised information provided by their respective production companies, in connection to publicly available information concerning fragrance clones. 3 In the following figures we present the GC/MS analysis of all the perfumes used in this work, both the authentic and the low-cost fragrance clones. We use an identical separation method for all perfumes, designed specifically for the optimal separation of chiral monoterpenes and monoterpenoids. The chiral separations were achieved using a 30m β-DEX™ 120 column (Sigma-Aldrich GmbH, Germany) with 0.25mm internal diameter and a 0.25 μm film thickness. The column flow was chosen to be 1ml/min and the temperature programme consists of an initial 5min step where the oven temperature is held at 40 °C, after which it was increased at a rate of 4 °C/min from 40 °C to 220 °C and then held again for 5 minutes. In Figs.S3-S5, the presented labelled peaks correspond to: 1 (-)-α-pinene; 2 (+)-α-pinene; 3 (-)-β-pinene; 4 (+)-limonene; 5 (-)-limonene; 6 (-)-linalool followed by (+)-linalool (retention times of 23.54 min and 23.79 min, respectively, for the chromatogram of 'CM' shown in Fig. S3); and 7 linalyl acetate (achiral).

a. Temperature and humidity measurements
We use a 25L Tedlar® sampling bag (ANALYT-MTC GmbH, Germany) secured around the tree stem to create an open-bottom sampling chamber that encloses the plant's upper branches. This open-bottom design allows air to move between the interior and exterior of the enclosed space to avoid possible suffocation of the plant during sampling (we do not implement any active mixing of both air volumes). A heating tape supported around the sampling bag allows us to actively control the temperature within the sampling chamber. We use a temperature and humidity logger device (TSP01, Thorlabs GmbH) to record the relative humidity in the laboratory and the temperature inside and outside the sampling chamber. We present our recordings for the whole duration of our measurements in Fig.S6, showing an overall stability of the laboratory humidity and the temperature inside the chamber during the measurements.

b. Estimation of overall concentration changes and effective specific optical rotation signal from GC/MS analysis
To sample the plant emissions, we use a ¼ inch teflon sampling line placed inside the sampling chamber ( Fig.4 main text). Samples are collected onto sorbent cartridges at 200 ml min -1 using a GilAir Plus Personal Air Sampling Pump (Sensidyne®, USA). We collect four samples before the mechanical wounding, i.e., branch cut, with a sampling time of ~10min. Following wounding, we collect two or three consecutive samples approximately every hour for 11 hrs, and two additional samples after 24hrs (sampling time ~10min). For the resin data, we collect two samples directly from the exposed wounding site (sampling time ~5min). In Fig.S7 we reproduce the chromatogram of a sample collected before wounding (as shown also in Fig.4 of the main text).
We analyze the samples using the offline GC/MS analysis procedure described in the main text. We use a non-linear, least-square minimization method to fit the observed time-dependent behavior of the concentration of each of the dominant monoterpene VOCs observed (pre-and postwounding), as this extracted from the GC/MS analysis, using the following model function:  (Table I) for each of the dominant chiral monoterpenes observed in the plant emissions, we can then calculate an effective expected specific optical rotation signal from the GC/MS analysis as follows: In Fig.4C and Fig.4D of the main text we present the results for :;:<$ ( ) = ∑ 4 ( ) 4 and 67/89 ( ), respectively, that are in agreement with our CCP-based online chiroptical measurements. We reproduce these results in Fig.S8 for clarity.

c. Concentration changes as observed from the CCP-based refractometric measurements
In Fig.S9 we present the CCP-based refractometric raw-data for each online sampling measurement using the thermal desorption unit (TDU, Fig.4B main text). One of these (for the online sample recorded at Δt=0.3hr) is presented as an inset in Fig.4B.
From these data we extract the measurements of the overall concentration of the emitted compounds from the coniferous plant, pre-and post-wounding, as presented in Fig.4C. CCP measurements combined with our sampling approach, which based on the use of the thermal desorption unit, allows us to observe directly in the refractometric data differences in dynamics between the dominant (monoterpene) chiral compounds. In particular, in accord with the GC/MS analysis (Fig.S8), the α-pinene/camphene enantiomers decay much faster (blue points Fig.S9) than (-)-limonene (green points Fig.S9) following mechanical wounding.

S7. Photo of the damaged plant branch
In Fig.S10 we present a photo of the damaged branch of the Pinus heldreichii we used for the measurements (shown also in Fig.4 of the main text). The resin covering the wounding site can be clearly seen in the photo. Samples of the emissions of the resin (Fig.4C,D of the main text) are taken directly from the exposed wounding site. Fig. S1. Schematic and operation principles for chirality sensing using cavity-enhanced chiral polarimetry and frequency metrology. A. Schematic diagram of the experimental setup. The laser beam is being split into two orthogonal linearly polarized parts, which couple into the fourmirror cavity in opposite directions (CW and CCW). Quarter-wave plates (λ/4) allow us to convert the linear polarizations of the CW and CCW beams into circular polarized beams and vice versa. AOM: Acousto-optic modulator. The Faraday crystal, generating an intracavity Faraday polarization anisotropy ( ), is located on one arm of the cavity while the chiral sample, generating an intracavity chiroptical polarization anisotropy ( ) is located on the other. We analyze the chiroptical signal from gaseous samples using either gas chromatography or direct dynamic air headspace sampling. B. Cavity frequency polarization spectrum of the optical cavity in the absence and presence of an intracavity chiroptical ( ) polarization anisotropy. Measuring the frequency difference between different mode pairs we extract the chiroptical signal ( ) through a direct measurement of = ( • ). C. Time record of frequency difference between the RCW/RCCW modes ( = − ), and D its associated (overlapping) Allan deviation.