**2.1 Pulsed laser irradiation**

150 Olive Oil – Constituents, Quality, Health Properties and Bioconversions

The use of an electronic nose for quality evaluation as a means of olfactory sensing is becoming widespread due to its advantages of low cost, reliability and high portability. Electronic noses based on different sensor technologies and using different recognition

When samples of olive oil are analyzed with an electronic nose, the standard procedure is to put a fixed quantity in a vial and sense the headspace. The main drawback of this method is that the concentration of some compounds in the headspace may be quite different from their concentration in the liquid phase. For example, the concentration of methanol and ethanol is usually much higher in the vapor phase than in the liquid. However, these substances have been found to be irrelevant in the definition of the olive oil characteristics (S. de Koning, 2008). On the other hand, substances such as hexanal and trans-2-hexanal which are responsible for the organoleptic properties, are more abundant than methanol and ethanol (Cosio et al, 2006, C. Di Natale et al, 2001) in the oil, but are scarcely present in the headspace. It is well known that the odour activity of hexanal and trans-2-hexanal is higher than that of those alcohols because of their low odour thresholds (Morales et al., 2005, J. Reiners, W. Grosch 1998, A. Runcio et al., 2008). Despite the abovementioned drawback, several efforts have been made to use the electronic nose for olive oil quality control (Guadarrama et al., 2000). The combination of electronic nose fingerprinting with multivariate analysis enabled the study of the profile of olive oil in relation to its geographical origin (Ballabio et al., 2006; Cosio et al., 2006). García Gonzalez and Aparicio, 2002, detected the vinegary defect in Spanish VOO with the use of metal oxide sensors. They used an Alpha MOS e-nose equipped with 18 MOS sensors distributed in three chambers, and heated the samples to 34 °C during 10 minutes before testing the headspace. Servili et al., 2009, reported the first study of the use of an Electronic Olfactory System (EOS 835) online to control the formation and evolution of the volatile compounds that characterize the

most important sensory notes of VOO during the malaxation process.

sample was incubated at 37°C for 7 minutes before injection.

methods (C. A. Rinaldi and J. C. Ferrero, 2001).

**1.4 Laser irradiation effect** 

In 2010, M. J. Lerma-García et al. compared the response of an electronic nose (EOS 507) to classify oils containing the five typical virgin olive oil sensory defects with that of a sensory panel. They demonstrated the usefulness of this tool when combined with panels to perform a fast screening of a large set of samples with the aim of discriminating defective oils. Each

In the same year, Massacane et al. proposed a method to improve the electronic nose performance for discriminating among different olive oils without changing the properties of the original oil sample. This task is carried out by IR laser vaporization (IRLV) which seems to be a promising technique that modifies only slightly the headspace by volatilizing certain organic compounds or by cracking them. Thus IRLV improves the selectivity of the overall response of the electronic nose. Due to the extremely low sample vaporization that it produces this method can be considered non-destructive as most ablation laser Analytical

Normal vaporization occurs when the vapour pressure in the ambient gas is lower than the saturation pressure of the liquid at the liquid temperature (Xu, X., and D. A. Willis, 2002). As the liquid's temperature increases, so do the saturation pressure and the rate of vaporization.

schemes have been employed for this task.

In this experiment the Infrared Laser Vaporization, IRLV, properties to improve the e-nose selectivity are investigated. The role of the laser wavelength is additionally analyzed. This is due to the fact that the quality and the quantity of the chemical compounds incorporated the headspace depend on the laser parameters, particularly, the fluence and the pulse length, as it was mentioned in Section 1.4.

Two extra virgin olive oils produced in the same geographical region of Argentina (San Juan) classied as A and B, were tested. Three samples of 15 ml of each oil were subjected to three different analytical methods in order to compare the effects of the laser vaporization.

All analytical methods were carried out under the same temperature and humidity conditions, of 25 °C and 43%, respectively. The samples were introduced in 100 ml T-shaped Pyrex test tubes with screw-caps in air inlet and outlet channels and a CaF2 window in order to allow the laser beam admission, referred to as vials.

The following analytical methods were undertaken:

**Method I:** Vial with oil sample A is kept closed during 2 minutes. Immediately afterwards the vial headspace is subjected to 35 seconds sampling with a Cyranose® 320. This procedure is repeated 5 times. The same operation is performed with oil sample B.

**Method II:** Vial with oil sample A is kept closed for 1min. The sample is subsequently irradiated with Nd:YAG laser pulses of 1064 nm at a repetition rate of 10 Hz during 1

Innovative Technique Combining Laser Irradiation Effect and

Electronic Nose for Determination of Olive Oil Organoleptic Characteristics 153

as may be calculated from the volume of the sample and its average heat capacity. Therefore, the temperature of the sample remains constant throughout the experiment.

(a) (b)

(c)

Nd:YAG laser, and (c) with CO2 laser. (Massacane et al., 2010) Permission

IRLV and that it is rather insensitive to the recognition pattern employed.

evident by this result.

(Massacane et al., 2010 )

Fig. 2. PCA for olive oils (■:A; ▲: B) performed for experiments (a) without laser, (b) with

Not only the S/N ratio is increased by IRLV but also the discrimination of samples corresponding to different oils is dramatically increased. This fact can be verified by the results shown in Figures 2(a), 2(b) and 2(c). Thus the modification of the headspace is made

IR spectra of the liquid oil samples were registered both before and after methods II and III were performed. There were no significant differences between them indicating that IRLV does not produce changes in the liquid oil. On the other hand, IRLV does modify the vapour-liquid equilibrium conditions improving the selectivity of the electronic nose overall response. The only effect of IRLV is to increase the vapor concentration of the olive oil

It has been shown that the electronic nose selectivity is dramatically increased by the use of

minute. The laser is turned off and the vial headspace is immediately subjected to 35 seconds sampling with a Cyranose® 320. This procedure is repeated 5 times. The same operation is then performed with oil sample B.

The Nd:YAG laser (Continuum, Surelite I) has a pulse length of 5 ns and an output energy of 80 mJ. The laser beam is focused in order to obtain a spot size of 0.037 cm2 at the surface of the sample so that a fluence of 2.14 J/cm2 is therefore achieved.

**Method III:** The vial with oil sample A is kept closed for 1 minute. The sample is subsequently irradiated with a homemade TEA CO2 laser (D. Petillo, J. Codnia, M. L. Azcárate, 1996) operating at 10.59 m with a repetition rate of 1 Hz during 1 minute. The laser is turned off and the vial headspace is immediately subjected to 35 seconds sampling with a Cyranose® 320. This procedure is repeated 5 times. The same operation is made with oil sample B.

The TEA CO2 laser has a pulse length of 100 ns and output energy of 1.45 ± 0.04 J/pulse. The beam is focused in order to obtain a spot size of 0.68 cm2 at the surface of the sample; a uence of 2.14 J/cm2 is therefore achieved. The software provided by the Cyrano® 320TM electronic nose allowed the processing of the raw data given by the 32 sensors responses.

Fig. 1. Sensor response time dependence. (▪) Without laser, (▲) Nd:YAG laser, and (•) CO<sup>2</sup> laser. (Massacane et al., 2010) Permission?

Figure 1 shows the signals measured with one sensor corresponding to the samples of oil A subjected to the three analysis methods. We observe that the signal-to-noise ratio (S/R), is considerably increased by the laser vaporization. The highest S/N ratio is obtained when vaporization is performed with the CO2 laser.

As it is well known, the uence and the pulse length determine the laser radiation absorption mechanisms and these parameters modify the laser power. In this work the same power was used in both IRLV methods although different total energies were delivered to the sample in each analysis method: 47.5 and 87 J. in Methods II and III, respectively. This energy range produces a negligible temperature increment. Even assuming an ideal oil absorption of 100% of laser energy the sample temperature increment would be about 3°C,

minute. The laser is turned off and the vial headspace is immediately subjected to 35 seconds sampling with a Cyranose® 320. This procedure is repeated 5 times. The same

The Nd:YAG laser (Continuum, Surelite I) has a pulse length of 5 ns and an output energy of 80 mJ. The laser beam is focused in order to obtain a spot size of 0.037 cm2 at the surface

**Method III:** The vial with oil sample A is kept closed for 1 minute. The sample is subsequently irradiated with a homemade TEA CO2 laser (D. Petillo, J. Codnia, M. L. Azcárate, 1996) operating at 10.59 m with a repetition rate of 1 Hz during 1 minute. The laser is turned off and the vial headspace is immediately subjected to 35 seconds sampling with a Cyranose® 320. This procedure is repeated 5 times. The same operation is made with

The TEA CO2 laser has a pulse length of 100 ns and output energy of 1.45 ± 0.04 J/pulse. The beam is focused in order to obtain a spot size of 0.68 cm2 at the surface of the sample; a uence of 2.14 J/cm2 is therefore achieved. The software provided by the Cyrano® 320TM electronic nose allowed the processing of the raw data given by the 32 sensors responses.

Fig. 1. Sensor response time dependence. (▪) Without laser, (▲) Nd:YAG laser, and (•) CO<sup>2</sup>

Figure 1 shows the signals measured with one sensor corresponding to the samples of oil A subjected to the three analysis methods. We observe that the signal-to-noise ratio (S/R), is considerably increased by the laser vaporization. The highest S/N ratio is obtained when

As it is well known, the uence and the pulse length determine the laser radiation absorption mechanisms and these parameters modify the laser power. In this work the same power was used in both IRLV methods although different total energies were delivered to the sample in each analysis method: 47.5 and 87 J. in Methods II and III, respectively. This energy range produces a negligible temperature increment. Even assuming an ideal oil absorption of 100% of laser energy the sample temperature increment would be about 3°C,

operation is then performed with oil sample B.

laser. (Massacane et al., 2010) Permission?

vaporization is performed with the CO2 laser.

oil sample B.

of the sample so that a fluence of 2.14 J/cm2 is therefore achieved.

as may be calculated from the volume of the sample and its average heat capacity. Therefore, the temperature of the sample remains constant throughout the experiment.

Fig. 2. PCA for olive oils (■:A; ▲: B) performed for experiments (a) without laser, (b) with Nd:YAG laser, and (c) with CO2 laser. (Massacane et al., 2010) Permission

Not only the S/N ratio is increased by IRLV but also the discrimination of samples corresponding to different oils is dramatically increased. This fact can be verified by the results shown in Figures 2(a), 2(b) and 2(c). Thus the modification of the headspace is made evident by this result.

IR spectra of the liquid oil samples were registered both before and after methods II and III were performed. There were no significant differences between them indicating that IRLV does not produce changes in the liquid oil. On the other hand, IRLV does modify the vapour-liquid equilibrium conditions improving the selectivity of the electronic nose overall response. The only effect of IRLV is to increase the vapor concentration of the olive oil (Massacane et al., 2010 )

It has been shown that the electronic nose selectivity is dramatically increased by the use of IRLV and that it is rather insensitive to the recognition pattern employed.

Innovative Technique Combining Laser Irradiation Effect and

the laser beam admission referred to as vials.

seconds to allow the stabilization of the samples:

sampling.

sampling.

Fig. 4. Experimental set up

spectrum of each EVOO used before being irradiated.

Method I: The vial headspace is subjected to a 15 seconds sampling.

Figure 4 shows a brief scheme of the experimental set up.

Laser

Electronic Nose for Determination of Olive Oil Organoleptic Characteristics 155

All the analytical methods were carried out under the same temperature and humidity conditions: 22 ºC and 36 %, respectively. The samples were introduced in 100 ml T-shaped test tubes with screw caps, air inlet and outlet channels and BK7 upper windows to allow

The three analytical methods differ in whether the samples are irradiated or not and in the wavelength of the laser used for the irradiation. The vials were kept closed for about 90

Method II: After the headspace stabilization takes place, the closed vial is irradiated during one minute with a continuous wave diode laser emitting radiation of 98 mW at 450 nm. Immediately after the laser is turned off, the vial headspace is subjected to a 15 seconds

Method III: After the headspace stabilization takes place, the closed vial is irradiated during one minute with a continuous wave diode laser emitting radiation of 98 mW at 650 nm. Immediately after the laser is turned off, the vial headspace is subjected to a 15 seconds

Sample Patagonia

We have measured the V-UV spectra of the liquid oil samples before and after being irradiated during different time periods. We have noticed significant differences in the spectra of the liquid samples that had been irradiated during 5 minutes. Figure 5 shows the

Nose

Air inlet

#### **2.2 Continuous wave laser irradiation**

A homemade portable electronic nose, Patagonia nose, was used in this study. The instrument comprises three parts: the automatic sampling system, the sensors' chamber with the sensor array, and the software for the e-nose control, data recording and processing. The first two are integrated in the same device and the software can be installed on any notebook (Figure 3)

(a)

(b)

Fig. 3. a) Homemade e-nose (Patagonia) b) Sensor chamber

The chamber contains 2 MOS commercial sensors (Silsens MSGS 4000, sensor array). Each sensor has four thin SnO2 films, one of them is doped with Pd. Each thin film is maintained at the temperature range between 300 and 500 °C during all the measurements.

Two EVOO produced in neighboring geographical regions of Argentina (San Juan) were classified as A and B. About 15 samples of both A and B EVOO were tested. Each sample was divided into three 15 ml samples so that three sets of the 15 samples were obtained to be subjected to three different analytical methods.

A homemade portable electronic nose, Patagonia nose, was used in this study. The instrument comprises three parts: the automatic sampling system, the sensors' chamber with the sensor array, and the software for the e-nose control, data recording and processing. The first two are integrated in the same device and the software can be installed

(a)

(b)

The chamber contains 2 MOS commercial sensors (Silsens MSGS 4000, sensor array). Each sensor has four thin SnO2 films, one of them is doped with Pd. Each thin film is maintained

Two EVOO produced in neighboring geographical regions of Argentina (San Juan) were classified as A and B. About 15 samples of both A and B EVOO were tested. Each sample was divided into three 15 ml samples so that three sets of the 15 samples were obtained to

at the temperature range between 300 and 500 °C during all the measurements.

Fig. 3. a) Homemade e-nose (Patagonia) b) Sensor chamber

be subjected to three different analytical methods.

**2.2 Continuous wave laser irradiation** 

on any notebook (Figure 3)

All the analytical methods were carried out under the same temperature and humidity conditions: 22 ºC and 36 %, respectively. The samples were introduced in 100 ml T-shaped test tubes with screw caps, air inlet and outlet channels and BK7 upper windows to allow the laser beam admission referred to as vials.

The three analytical methods differ in whether the samples are irradiated or not and in the wavelength of the laser used for the irradiation. The vials were kept closed for about 90 seconds to allow the stabilization of the samples:

Method I: The vial headspace is subjected to a 15 seconds sampling.

Method II: After the headspace stabilization takes place, the closed vial is irradiated during one minute with a continuous wave diode laser emitting radiation of 98 mW at 450 nm. Immediately after the laser is turned off, the vial headspace is subjected to a 15 seconds sampling.

Method III: After the headspace stabilization takes place, the closed vial is irradiated during one minute with a continuous wave diode laser emitting radiation of 98 mW at 650 nm. Immediately after the laser is turned off, the vial headspace is subjected to a 15 seconds sampling.

> Air inlet Sample Patagonia Nose Laser

Figure 4 shows a brief scheme of the experimental set up.

#### Fig. 4. Experimental set up

We have measured the V-UV spectra of the liquid oil samples before and after being irradiated during different time periods. We have noticed significant differences in the spectra of the liquid samples that had been irradiated during 5 minutes. Figure 5 shows the spectrum of each EVOO used before being irradiated.

Innovative Technique Combining Laser Irradiation Effect and

Electronic Nose for Determination of Olive Oil Organoleptic Characteristics 157

EVOO A

EVOO B

The discrimination ability of the three methods mentioned above was analyzed. For the data processing the absorption and desorption rates were taken into account in addition to the ratio of the peak value of each sensor response to the base-line value. Since different sensors have distinct desorption times, for each chemical compound, we have considered the

The usual PCA of the data obtained with Methods II and III was performed. Figures 8 (a) and 8 (b) show the score plot obtained with both methods. It is evident that better

Fig. 7. V-UV spectra of EVOO (A) and (B)- Method I (\_\_\_\_\_) - Method II (- - - -) –

integral of each time signal to the base line integral ratio.

discrimination is achieved with Method III.

Method III (- - - -)

Fig. 5. V-UV Spectra of liquid EVOO A( - - - - ) and liquid EVOO B (. . . . ) before irradiation

Figure 6 shows the Principal Components Analysis (PCA) of the Patagonia Nose results obtained with Method I.

Fig. 6. PCA of EVOO A(GV) and B(IN) with Method I

Figure 7 shows the V-UV spectra registered for each liquid EVOO after the application of the three analytical methods. The experiment was repeated 5 times for each sample. It can be observed that the spectra of the irradiated samples of EVOO A exhibit significant changes with respect to the non-irradiated sample. The largest effect is produced by the irradiation with blue light (450 nm). This more energetic radiation affects the sample composition probably due to a photochemical effect. On the other hand, irradiation with the red light, gives rise to a thermal effect which causes the introduction of more molecules into the gas phase.

Fig. 5. V-UV Spectra of liquid EVOO A( - - - - ) and liquid EVOO B (. . . . ) before irradiation

Figure 6 shows the Principal Components Analysis (PCA) of the Patagonia Nose results

Figure 7 shows the V-UV spectra registered for each liquid EVOO after the application of the three analytical methods. The experiment was repeated 5 times for each sample. It can be observed that the spectra of the irradiated samples of EVOO A exhibit significant changes with respect to the non-irradiated sample. The largest effect is produced by the irradiation with blue light (450 nm). This more energetic radiation affects the sample composition probably due to a photochemical effect. On the other hand, irradiation with the red light, gives rise to a thermal

effect which causes the introduction of more molecules into the gas phase.

obtained with Method I.

Fig. 6. PCA of EVOO A(GV) and B(IN) with Method I

Fig. 7. V-UV spectra of EVOO (A) and (B)- Method I (\_\_\_\_\_) - Method II (- - - -) – Method III (- - - -)

The discrimination ability of the three methods mentioned above was analyzed. For the data processing the absorption and desorption rates were taken into account in addition to the ratio of the peak value of each sensor response to the base-line value. Since different sensors have distinct desorption times, for each chemical compound, we have considered the integral of each time signal to the base line integral ratio.

The usual PCA of the data obtained with Methods II and III was performed. Figures 8 (a) and 8 (b) show the score plot obtained with both methods. It is evident that better discrimination is achieved with Method III.

Innovative Technique Combining Laser Irradiation Effect and

**3. Conclusions** 

some light on this question.

**4. Acknowledgements** 

*Chem*. 46, 648–653.

financial support

**5. References** 

Electronic Nose for Determination of Olive Oil Organoleptic Characteristics 159

*<sup>T</sup>* <sup>1</sup> *m (x) = (x k kk k <sup>μ</sup> ) <sup>Σ</sup> (x <sup>μ</sup> )*

where *mk(x)* is the statical or Mahalanobis distance, *µk* is the mean value of the

An easy to implement method to improve electronic nose discrimination ability of a priori similar odours has been presented. This technique has been then applied to the case of virgin olive oils. The way laser vaporization of the samples improves this task has been additionally explored. The behaviour of the gas phase headspace following pulsed and cw

The use of pulsed IR lasers increases the sensitivity of the e-nose performance. Furthermore, the use of a CO2 laser allows a better discrimination than the use of a Nd:YAG laser. When using the CO2 laser, the signal-to-noise ratio (S/N) is increased by an order of magnitude with respect to the S/N without laser vaporization effect. The IR laser wavelength influences the discrimination capabilities of the method, probably due to the different IR absorption properties of the sample compounds. Further experiments in progress may shed

The use of continuous wave visible diode lasers (methods II and III) produces significant changes in the V-UV spectrum of one of the EVOO, (EVOO A). Irradiation with the diode laser at 450 nm produces larger changes than those produced by the 650 nm diode laser irradiation. The 450 nm laser induces chemical reactions in the liquid oil surface and as a result precludes the discrimination. On the other hand, Laser Vaporization at 650 nm modifies the vapour-

It is important to emphasize that although the discrimination obtained with IRLV is larger than that resulting from LV at 650 nm it is insensitive to the recognition pattern used. On the other hand, diode lasers are considerably cheaper than high power TEA CO2 lasers and they

The authors acknowledge, Agencia Nacional de Promoción Cientíca y Tecnológica (ANPCyT) and the Comisión Nacional de Energía Atómica (CNEA) and CONICET, for

Angerosa, F., Camera, L., d'Alessandro, N. & Mellerio, G., (1998). Characterization of seven

Angerosa, F., Mostallino, R., Basti, C. & Vito, R., (2000). Virgin olive oil odour notes: their

new hydrocarbon compounds present in the aroma of virgin olive oil. *J. Agric. Food* 

relationship with volatile compounds from the lipoxygenase pathway and

liquid equilibrium conditions improving the selectivity of the electronic nose.

produce very good results considering the benefit-cost ratio.

secoiridoid compounds. *Food Chem.* 68, 283–287.

corresponding class and *µk* is the covariance matrix within each class sample.

laser irradiation with different wavelengths has been analyzed.

A classification following the discrimination step was designed for Method III using about 70% of the available samples of both A and B oils.

Fig. 8. PCA (a) Method II (b) Method III (discriminating regions are shown).

For validation purposes, 30% of the measured samples were disregarded. Within the resultant restricted space, an unsupervised PCA was performed. It was then possible to establish multivariate confidence regions for the two identified classes: olive oil A and olive oil B. The assignment of a new multivariate measurement *x* to a given category *k* occurs when the quadratic discriminant *dk (x)* is maximized:

$$d\_k(\mathbf{x}) = -\frac{1}{2} \ln \left| \Sigma\_k \right| - \frac{1}{2} m\_k(\mathbf{x})$$

$$m\_k(\mathbf{x}) = (\mathbf{x} - \boldsymbol{\mu}\_k)^T \boldsymbol{\Sigma}\_k^{-1} (\mathbf{x} - \boldsymbol{\mu}\_k)^T$$

where *mk(x)* is the statical or Mahalanobis distance, *µk* is the mean value of the corresponding class and *µk* is the covariance matrix within each class sample.
