**2. Material and methods**

#### **2.1 Samples**

Samples archived in the Soil Labs at Cranfield University were originally collected from the top 0-20 cm of the soil layer from five fields in Silsoe experimental farm at Bedfordshire, United Kingdom. Figure 1 illustrates the location of these fields, namely, Avenue Field (#A), Orchard (#B), Ivy Ground (#C), Showground (#D) and Copse Field (#E). According to the soil descriptions presented on www.landis.org.uk, managed by the National Soil Resources Institute (MSRI), Cranfield University, these fields belong to two major soil World Reference Base (WRB) classifications, namely, Cambisol and Luvisol. Of them, Ivy Ground (Sample codes: C15, C21-C39), Orhcard (B01-B25 ) and Copse Field (E01- E23) are Cambisols and Showground (D01-D35 ) is Luvisol, while Avenue Filed (A01-A12, A14-A20) comprises of both soils. The parent material underlying these fields is mainly siliceous stones.

A total of 122 bulk soil samples used in this study are with various proportions of sand, slit and clay (Table 1) and hence belong to three soil textures, e.g. sandy loam, clay loam and clay, according to the United States Department of Agriculture (USDA) triangular diagram

Fig. 1. Location of the five fields targeted in the study (www.landis.org.uk)


a according to USDA triangular diagram relating particle size distribution to soil texture. b mixture of Cambisol and Luvisol types.

Table 1. Description of the five targeted farm fields

of soil texture classification. Soil samples were air-dried and crushed at first. Plant residues and stones were then removed. After that, the samples were sieved to pass a 2 mm mesh and air-dried again at 40℃ for 48h. A small amount of soil was used for chemical analysis, whereas the majorities were left for spectrophotometer measurement.

#### **2.2 Reference methods**

186 Infrared Spectroscopy – Life and Biomedical Sciences

d. Applying Vis-NIR spectroscopy to predict soil properties needs no special sample preparation. However, MIR spectra are traditionally obtained by a FT-IR spectrometer with samples pressed in KBr pellets, which requires labour and specific skills. Fortunately, newly-developed ATR (attenuated total reflection) and DRIFT (diffuse reflectance infrared Fourier Transform) accessories are becoming the predominant FT-IR sample analysis tool. This is because sample handling is greatly simplified and sample preparation is eliminated. Hence, Vis-NIR-MIR spectrometry without sample

e. This study investigated the potential of calibrating Vis-NIR, ATR-FTIR and DRIFT spectra to soil N and C concentrations with an aim of comparing the performance of two spectrometers, namely, a Vis-NIR spectrometer vs. a FT-IR spectrometer with ATR and DRIFT accessories. The models developed for N and C were then compared to those developed with the combination of Vis-NIR and ATR-FTIR spectra (Vis-NIR-ATR) and the combination of Vis-NIR and DRIFT spectra (Vis-NIR-DRIFT) for investigating whether the combination of Vis-NIR spectrometer and FT-IR spectrometer could improve the prediction accuracy of soil N and C. For each spectrometer, spectral data were subjected to various spectral transformation approaches before model

Samples archived in the Soil Labs at Cranfield University were originally collected from the top 0-20 cm of the soil layer from five fields in Silsoe experimental farm at Bedfordshire, United Kingdom. Figure 1 illustrates the location of these fields, namely, Avenue Field (#A), Orchard (#B), Ivy Ground (#C), Showground (#D) and Copse Field (#E). According to the soil descriptions presented on www.landis.org.uk, managed by the National Soil Resources Institute (MSRI), Cranfield University, these fields belong to two major soil World Reference Base (WRB) classifications, namely, Cambisol and Luvisol. Of them, Ivy Ground (Sample codes: C15, C21-C39), Orhcard (B01-B25 ) and Copse Field (E01- E23) are Cambisols and Showground (D01-D35 ) is Luvisol, while Avenue Filed (A01-A12, A14-A20) comprises of

A total of 122 bulk soil samples used in this study are with various proportions of sand, slit and clay (Table 1) and hence belong to three soil textures, e.g. sandy loam, clay loam and clay, according to the United States Department of Agriculture (USDA) triangular diagram

both soils. The parent material underlying these fields is mainly siliceous stones.

preparation will bring about new wave of soil research.

calibration, aiming at model optimization.

**2. Material and methods** 

**2.1 Samples** 

2002; Yang*, et al.*, 2011a).

2010), attention is being given to possible alternatives such as Vis-NIR and/or MIR spectroscopy. Numerous analyses of soil N and C have been conducted during the past decades using this technique, for examples, to predict the soil C and N mineralization rates (Fystro, 2002; Mutuo*, et al.*, 2006), to derive spectral characteristics for classifying conventional and conservation agricultural practices (Haché*, et al.*, 2007), to assess soil changes due to site disturbance during forest harvesting (Ludwig*, et al.*, 2002), to evaluate the recovery of microbial functions during soil restoration (Schimann*, et al.*, 2007), to determine carbon inventories (Reeves III*, et al.*, 2002), to determine (*in situ*) organic matter composition of coatings at crack surfaces and linings of earthworm burrow walls (Reeves III*, et al.*, 2002) and others (Chang*, et al.*, 2001; Chang & Laird,

> Particle size distribution was determined by a combination of wet sieving and hydrometer tests using the USDA soil texture classification system. Reference values of N and C were analyzed through a sequence of processes. First, a 50 mg sample was used for the measurement of TN and TC by a TrusSpecCNS spectrometer (LECO Corporation, St. Joseph, MI, USA) using the Dumas combustion method. Next, another 50 mg from each soil sample was mixed with 5% HCl and then oven-dried at 90℃ for 4 h in order to remove IC. Then, OC in IC-free samples were measured by the same Dumas combustion method. Finally, IC was calculated by the difference between TC and OC.

Vis/Near- and Mid- Infrared Spectroscopy for Predicting Soil N and C at a Farm Scale 189

When measuring solids by ATR, it is essential to ensure good optical contact between the sample and the crystal. The accessories have devices that clamp the sample to the crystal surface and apply pressure. This works well with elastomers and other deformable materials, but many solids give very weak spectra because the contact is confined to small areas. The effects of poor contact are the greatest at shorter wavelengths where the depth of penetration is the lowest. The issue of solid sample/crystal contact has been overcome to a great extent by the introduction of ATR accessories with very small crystals, typically about 2 mm across. The most frequently-used small crystal ATR material is diamond because it has the best durability and chemical inertness. These small area ATR crystal top-plates generally provide only a single reflection but this is sufficient, given the low noise levels of PerkinElmer's modern FT-IR spectrometers. Much higher pressure with limited force can now be generated onto these small areas. As a result, spectra can be obtained from a wide

After the crystal area has been cleaned and the background collected, the soil material is placed onto the small crystal area. Then the pressure arm should be positioned over the crystal/sample area. Force is applied to the sample, pushing it onto the diamond surface. It is good practice to apply pressure until the strongest spectral bands have an intensity which extends beyond 70%T, namely, from a baseline at 100%T down to 70%T. Then, the data are collected in the normal manner. Unlike transmission measurements, ATR sampling does not produce totally absorbing spectral bands because the effective pathlength is controlled by the crystal properties thereby minimizing sample re-preparation time. After the spectrum has been collected, the crystal area must be cleaned before placing the next sample on the crystal. A 100%T line with no spectral features should be seen if the crystal is clean, if spectral features are seen, the crystal should be cleaned again

In the case of a solid sample, it is pressed into direct contact with the crystal. Because the evanescent wave into the solid sample is improved with a more intimate contact, solid samples are usually firmly clamped against the ATR crystal, so that trapped air is not the medium through which the evanescent wave travels, as that would distort the results.

Diffuse reflectance occurs when light impinges on the surface of a material and is partially reflected and transmitted. Light that passes into the material may be absorbed or reflected out again. Hence, the radiation that reflects from an absorbing material is composed of surface-reflected and bulk re-emitted components, which summed are the diffuse

Fig. 2. A multiple reflection ATR system (www.perkinelmer.com)

variety of solid materials including minerals.

using a solvent soaked tissue.

**2.4.2 Principles of DRIFT** 

#### **2.3 Vis-NIR spectra acquisition**

The soil samples were equilibrated to room temperature (20-25℃) and carefully mixed before spectral measurement. A sub-sample of ~5g was loaded into a static ring cup and measured using a LabSpec 2500 spectrophotometer (Analytical Spectral Devices Inc. Boulder, CO, USA) equipped with a fibre-optic probe. The light source was a quartzhalogen bulb of 3000K°. The light source and reflectance fibre were gathered with a certain angle of 35°. One Si photodiode array in the range of 350-1000 nm and two Peltier cooled InGaAs detectors in the ranges of 1000-1800 nm and 1800-2500 nm were used. All spectra were recorded in diffuse reflection mode over the wavelength range of 400-2500 nm at 1 nm data spacing interval, which resulted in 2101 wavelengths per spectrum. The reflectance spectra were transformed into absorbance spectra using Log(1/R), as absorbance is directly proportional to the concentration of an absorber according to Beer-Lambert Law. The actual spectra resolution was 3 nm at 700 nm and 10 nm at 1400 and 2100 nm. Before sample spectral acquisition, twenty five reference scans were taken on a ceramic standard supplied with the spectrophotometer. Ten photometric scans were conducted for each sample, followed by another ten scans of the refilled sample cup. The twenty scans were then averaged in one spectrum for each sample.

#### **2.4 MIR spectra acquisition**

MIR spectra were collected by an ALPHA Fourier transform infrared (FT-IR) spectrometer (Bruker Optics, Billerica, MA, USA) with wavelength range of 7500–375 cm-1, equipped with two exchangeable QuickSnapTM sampling modules. This instrument acquired spectra with two sampling accessories, namely, ATR and DRIFT. ATR is an easy-to-use FT-IR sampling method that is ideal for both solids and liquids and does not require any sample preparation. The Eco ATR is a single reflection ATR sampling module equipped with a versatile high throughput ZnSe ATR crystal for the analysis of powders, solids, pastes and liquids. The DRIFT module is an economical analysis option for a broad variety of solid samples: powders, inorganic material, gem stones, papers, textiles and others. The DRIFT module is designed for easy sampling and high light-throughput.

#### **2.4.1 Principles of ATR-FTIR**

An attenuated total reflectance accessory operates by measuring the changes that occur in a totally internally reflected infrared beam when the beam comes into contact with a sample (Fig.2). An infrared beam is directed onto an optically dense crystal with a high refractive index at a certain angle. This internal reflectance creates an evanescent wave that extends beyond the surface of the crystal into the sample held in contact with the crystal. It can be easier to think of this evanescent wave as a bubble of infrared that sits on the surface of the crystal. This evanescent wave protrudes only a few microns (0.5μ − 5μ) beyond the crystal surface and into the sample. Consequently, there must be good contact between the sample and the crystal surface. In regions of the infrared spectrum where the sample absorbs energy, the evanescent wave will be attenuated or altered. The attenuated energy from each evanescent wave is passed back to the IR beam, which then exists at the opposite end of the crystal and is passed to the detector in the IR spectrometer. The system then generates an infrared spectrum (www.perkinelmer.com).

The soil samples were equilibrated to room temperature (20-25℃) and carefully mixed before spectral measurement. A sub-sample of ~5g was loaded into a static ring cup and measured using a LabSpec 2500 spectrophotometer (Analytical Spectral Devices Inc. Boulder, CO, USA) equipped with a fibre-optic probe. The light source was a quartzhalogen bulb of 3000K°. The light source and reflectance fibre were gathered with a certain angle of 35°. One Si photodiode array in the range of 350-1000 nm and two Peltier cooled InGaAs detectors in the ranges of 1000-1800 nm and 1800-2500 nm were used. All spectra were recorded in diffuse reflection mode over the wavelength range of 400-2500 nm at 1 nm data spacing interval, which resulted in 2101 wavelengths per spectrum. The reflectance spectra were transformed into absorbance spectra using Log(1/R), as absorbance is directly proportional to the concentration of an absorber according to Beer-Lambert Law. The actual spectra resolution was 3 nm at 700 nm and 10 nm at 1400 and 2100 nm. Before sample spectral acquisition, twenty five reference scans were taken on a ceramic standard supplied with the spectrophotometer. Ten photometric scans were conducted for each sample, followed by another ten scans of the refilled sample cup. The twenty scans were then

MIR spectra were collected by an ALPHA Fourier transform infrared (FT-IR) spectrometer (Bruker Optics, Billerica, MA, USA) with wavelength range of 7500–375 cm-1, equipped with two exchangeable QuickSnapTM sampling modules. This instrument acquired spectra with two sampling accessories, namely, ATR and DRIFT. ATR is an easy-to-use FT-IR sampling method that is ideal for both solids and liquids and does not require any sample preparation. The Eco ATR is a single reflection ATR sampling module equipped with a versatile high throughput ZnSe ATR crystal for the analysis of powders, solids, pastes and liquids. The DRIFT module is an economical analysis option for a broad variety of solid samples: powders, inorganic material, gem stones, papers, textiles and others. The DRIFT

An attenuated total reflectance accessory operates by measuring the changes that occur in a totally internally reflected infrared beam when the beam comes into contact with a sample (Fig.2). An infrared beam is directed onto an optically dense crystal with a high refractive index at a certain angle. This internal reflectance creates an evanescent wave that extends beyond the surface of the crystal into the sample held in contact with the crystal. It can be easier to think of this evanescent wave as a bubble of infrared that sits on the surface of the crystal. This evanescent wave protrudes only a few microns (0.5μ − 5μ) beyond the crystal surface and into the sample. Consequently, there must be good contact between the sample and the crystal surface. In regions of the infrared spectrum where the sample absorbs energy, the evanescent wave will be attenuated or altered. The attenuated energy from each evanescent wave is passed back to the IR beam, which then exists at the opposite end of the crystal and is passed to the detector in the IR spectrometer. The system then generates an

**2.3 Vis-NIR spectra acquisition** 

averaged in one spectrum for each sample.

module is designed for easy sampling and high light-throughput.

**2.4 MIR spectra acquisition** 

**2.4.1 Principles of ATR-FTIR** 

infrared spectrum (www.perkinelmer.com).

Fig. 2. A multiple reflection ATR system (www.perkinelmer.com)

When measuring solids by ATR, it is essential to ensure good optical contact between the sample and the crystal. The accessories have devices that clamp the sample to the crystal surface and apply pressure. This works well with elastomers and other deformable materials, but many solids give very weak spectra because the contact is confined to small areas. The effects of poor contact are the greatest at shorter wavelengths where the depth of penetration is the lowest. The issue of solid sample/crystal contact has been overcome to a great extent by the introduction of ATR accessories with very small crystals, typically about 2 mm across. The most frequently-used small crystal ATR material is diamond because it has the best durability and chemical inertness. These small area ATR crystal top-plates generally provide only a single reflection but this is sufficient, given the low noise levels of PerkinElmer's modern FT-IR spectrometers. Much higher pressure with limited force can now be generated onto these small areas. As a result, spectra can be obtained from a wide variety of solid materials including minerals.

After the crystal area has been cleaned and the background collected, the soil material is placed onto the small crystal area. Then the pressure arm should be positioned over the crystal/sample area. Force is applied to the sample, pushing it onto the diamond surface. It is good practice to apply pressure until the strongest spectral bands have an intensity which extends beyond 70%T, namely, from a baseline at 100%T down to 70%T. Then, the data are collected in the normal manner. Unlike transmission measurements, ATR sampling does not produce totally absorbing spectral bands because the effective pathlength is controlled by the crystal properties thereby minimizing sample re-preparation time. After the spectrum has been collected, the crystal area must be cleaned before placing the next sample on the crystal. A 100%T line with no spectral features should be seen if the crystal is clean, if spectral features are seen, the crystal should be cleaned again using a solvent soaked tissue.

In the case of a solid sample, it is pressed into direct contact with the crystal. Because the evanescent wave into the solid sample is improved with a more intimate contact, solid samples are usually firmly clamped against the ATR crystal, so that trapped air is not the medium through which the evanescent wave travels, as that would distort the results.

#### **2.4.2 Principles of DRIFT**

Diffuse reflectance occurs when light impinges on the surface of a material and is partially reflected and transmitted. Light that passes into the material may be absorbed or reflected out again. Hence, the radiation that reflects from an absorbing material is composed of surface-reflected and bulk re-emitted components, which summed are the diffuse

Vis/Near- and Mid- Infrared Spectroscopy for Predicting Soil N and C at a Farm Scale 191

spectral features. Furthermore, PLSR takes as well variations of the absorbance as variations of the calibration data into account. PLSR is a rapid analysis, can handle co-linear data, and

Before the absorbance spectra were calibrated to predict soil properties, PCA was conducted to detect sample outliers in raw data set of Vis-NIR spectra, ATR spectra and DRIFT spectra. The identified sample outlier/s was/were excluded from further investigation. The remaining spectra were then subjected to various spectral pre-processing algorithms to reduce or eliminate noise, offset and bias in raw spectra. The investigated spectral preprocessing techniques included Savitzky-Golay smoothing, standard normal variate (SNV), multiplicative scatter correction (MSC), baseline offset correction (BOC), centre & scale, 1stand 2nd- detrendings, and 1st- and 2nd- derivatives. Several spectral normalizations were also included. They were conducted according to maximum, range, mean and quantile values. Details of these algorithms are available in www.camo.com. PLSR algorithm was used to decompose both raw and transformed spectra matrix into 10 LVs. All PLSR models were validated with full cross-validation approach in which each spectrum was in turn excluded from the calibration sample set and was predicted by the PLSR model calibrated for the remaining spectra. By decomposing the spectra into 10 LVs, it was assumed that the PLSR model would be over-fitted because signal noise of the spectral measurements could also be correlated with the property vector. The optimal number of LVs was determined by minimizing the predicted residual error sum of squares (PRESS). For better understanding the importance of different wavelength ranges in the prediction of soil N and C, PLSR models were also developed for the combinational Vis-NIR-ATR and Vis-NIR-DRIFT spectra. Spectral transformation and model calibration were conducted using the

The validation accuracy of PLSR models is given by the root mean squared error (RMSE):

where X� is the predicted value, Y� the measured (reference) value and N the number of soil samples. To compare model performance, we recorded the residual predictive deviation (RPD), which is the ratio of standard deviation of reference values to RMSE of the calibration set during cross-validation. The criteria adopted for RPD classification (Mouazen*, et al.*, 2006) was that an RPD value below 1.5 indicates very poor model/predictions and that such as value could not be useful; an RPD value between 1.5 and 2.0 indicates a possibility to distinguish between high and low values, while a value between 2.0 and 2.5 makes approximate quantitative predictions possible. For RPD values between 2.5 and 3.0 and above 3.0, the prediction is classified as good and excellent, respectively. Meanwhile, we compared the coefficient of determination (*R*2) in crossvalidation of calibration models. Generally, a good model would have high values of *R*2 and

<sup>N</sup>� �X� − Y�)� �

RMSE = �<sup>1</sup>

can provide useful qualitative information.

UnscramblerX10.1® (CAMO, Oslo, Norway).

**2.6 Model assessment criteria** 

RPD for cross-validation.

**2.5.3 Procedure of spectral processing and model calibration** 

reflectance of the sample (www.uksaf.org). DRIFT analysis of powders is conducted by focusing infrared light onto the powder (sometimes diluted in a non absorbing matrix, e.g. KBr) and the scattered light is collected and relayed to the IR detector.

In practice, DRIFT is most conveniently and rapidly used for soil analysis in diffuse reflection mode, where the incoming radiation is focused onto the soil sample surface, often in the form of a dry powder or <2 mm micro-aggregates, and the reflected radiation is passed back into the spectrophotometer (Fig.3, www.clw.csiro.au). In this study, infrared spectra were recorded in diffuse reflection mode with an Alpha spectrometer with DRIFT accessory. Bulk soil samples were scanned 20 times in the range from 4000 to 400 cm-1. DRIFT spectra were corrected against atmospheric CO2 and water vapour. Finally, the infrared reflectance spectra were transformed into absorbance spectra using Log(1/R).

Fig. 3. A description of the method of acquiring a DRIFT spectrum (www.clw.csiro.au)
