Measurement of diffuse reflectance from combinatorial samples

Analytica Chimica Acta 677 (2010) 79–89

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Measurement of diffuse reflectance from combinatorial samples F.A. Saeed a , J.R.G. Evans b,∗ a b

Department of Materials, Queen Mary, University of London, Mile End Road, London, E1 4NS, UK Department of Chemistry, University College London, 20 Gordon Street, London, WC1H 0AJ, UK

a r t i c l e

i n f o

Article history: Received 20 May 2010 Received in revised form 8 July 2010 Accepted 13 July 2010 Available online 30 July 2010 Keywords: High throughput Combinatorial Colour measurement Fluorescence Diffuse reflectance

a b s t r a c t Using a luminescence spectrometer as a platform, a system of fibre-optic probes was created that allows full colour characterisation, fluorescence and phosphorescence spectra to be recorded in diffuse reflectance and in transmission from thick or thin film arrays of combinatorial samples of diameter down to 2 mm and from liquids. An integrating sphere is not required and the method is more versatile than the instrument’s fibre-optic plate reader which has conjoined fibre bundles set at a fixed angle. Incident and detected light is routed via separate optical fibre bundles which remain stationary above or below a two-axis table. The validation and calibration are described. A library of 25 members was scanned for both diffuse reflectance (colour) and fluorescence in less than an hour. The method thus combines techniques that conventionally rely on different instruments and makes them amenable for high throughput libraries. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Our initial purpose in constructing this device was to explore doped TiO2 which is one of the best candidates for photocatalysis on safety and economic grounds [1], the most active field being the photo-degeneration of organic compounds [2–16] including organics, viruses, bacteria, fungi, algae, and cancer cells, which can be decomposed to less harmful species [2,11,13–15,17]. TiO2 is also able to form complex inorganic colourants (CIC) [18] that present structural and chemical stability toward dissolution after firing [19,20]; the rutile lattice offering considerable scope for colour variation by metal ion doping [21]. The procedure described here can both characterise catalysts and measure catalytic behaviour. It can detect the effects of, for example transition metal dopants on ceramic colour and track colour changes in reactants during photodegradation or photocatalysis in a high throughput process. Reflectance and colour measurement are also of importance in the petroleum industry [22]. Another reason for the work was slightly more explorative. It is sometimes found in the emerging combinatorial studies of materials that apparently disconnected properties are related. However such relationships are not found until high throughput libraries are scanned for several apparently unrelated properties. For example, the electronic bandgap is partly responsible for colour and in turn the bandgap influences catalytic behaviour. The establishment of such correlations might speed up the exploration of large sam-

∗ Corresponding author. Tel.: +44 20 7679 4689; fax: +44 20 7679 4603. E-mail address: [email protected] (J.R.G. Evans). 0003-2670/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2010.07.007

ple spaces if inferences could be made from a simple, rapid and non-contact process such as high throughput colour measurement. We therefore describe the construction and validation of a diffuse reflectance optical probe (DROP) to record the diffuse reflectance and transmission spectra of very small samples without losing signal. It needs to scan solid as well as liquid samples for fluorescence, reflectance and transmission in the characterisation of films. The design criteria for DROP were therefore that it should be able to (i) scan small samples without compromising signal level, (ii) scan solid and liquid samples, (iii) scan for fluorescence, reflectance, transmission and absorption (thin film characterisation) and (iv) be integrated within a larger platform (the London University Search Instrument, LUSI) [23] via long optical cables in such a way that reciprocating cable movement is limited to avoid fibre surface abrasion and consequent optical deterioration. Common types of spectrophotometer either scan the illumination wavelength and use a polychromatic detector to receive the emitted or reflected light (e.g. UV–visible instrument) or they use a polychromatic illuminant such as a tungsten–halogen source and discriminate at the detector as in a diode array spectrometer [24]. The platform used here has two monochromators so that excitation and emission wavelengths are independently variable. This means that it can distinguish between intrinsic reflectance and fluorescence. For example, if a series of complete wavelength scans using the detector is made, one for each setting of the incident monochromator at intervals of say 10 nm, a complete matrix of intensities is produced for excitation vs emission wavelengths; a bispectral measurement. The reflectance is obtained from the diagonal and the reflectance plus fluores-

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Fig. 1. Schematic diagram of the goniometer in three positions. (A) Collection of specular reflection, (B) position where diffuse reflection is almost extinguished and (C) incident beam position where diffuse reflection is again attenuated at glancing incident angles.

cence is obtained by summing the excitation columns; generally, fluorescence occurs at wavelengths longer than the excitation wavelength. Four optical geometries are accepted for reflectance and colour measurement and all are intended to eliminate specular reflection [25]. In the integrating sphere, the sample is placed at a port of a spherical, internally white sphere and illuminated either through an orthogonal port or a slightly off-normal port so that multiple reflections occur. Internal baffles restrict specular reflection and the detector is fitted to whichever port is not used for illumination. A shortcoming is that if the sample fluoresces, the fluorescent emission also contributes to illumination. Another shortcoming of sphere-based measurements is the problem of gloss which adds a lightness component that is wholly dependent on illumination and viewing angle and is not fully extinguished. The geometries that avoid this confusion do so by making use of only one reflection. Either the illumination is normal and detection is at 45◦ which is designated 0◦ /45◦ geometry or the reverse is used 45◦ /0◦ , the first angle always being that of illumination. The present work therefore makes use of 0◦ /45◦ and 45◦ /0◦ bispectral measurement.

2. Experimental 2.1. Instrument platform The platform is a luminescence spectrophotometer (LS55, Perkin Elmer) able to measure fluorescence, phosphorescence, chemiluminescence and bioluminescence in which excitation and emission monochromators can be varied independently. For emission spectra, the excitation wavelength is fixed and the emission monochromator is scanned. For an excitation scan, the emission wavelength is fixed and the excitation wavelength is scanned. A synchronous scan is obtained by varying both excitation and emission monochromators at constant wavelength or constant energy difference. Sensitivity calibration was based on the signal-to-noise ratio at 10 nm slit width with both monochromators set to wavelengths to detect the Raman band of water. Emission spectra were recorded at seven excitation wavelengths and this test was run intermittently. Initial work on combinatorial solid discs of diameter 4 mm made use of the plate reader optical fibre accessory supplied with the platform instrument, which consisted of conjoined incident and detection fibre optics. This picked up background signal from the

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substrate. Furthermore its geometry was unsuitable for bispectral reflectance of solid samples because it was not set at 0◦ /45◦ or for transmission work because optical cables could not be separated. The excitation and emission beams are set orthogonally at the instrument port and to conduct initial trials of spectral reflectance factor measurement in the synchronous scan mode without the intervention of optical fibre transmission, a graduated rotatable jig was built from the vernier goniometer stage of a petrographic optical microscope and positioned in place of the cell holder. A commercial red pigment glaze (‘Post Office Red’ ex Potterycraft Ltd., Stoke on Trent, Staffs UK) applied to an alumina slide and fired at 700 ◦ C was placed on the turntable in front of the sample holder window (Fig. 1). A series of spectra were recorded in steps of 5◦ of ˛ in order to find the bispectral reflected radiance factor and shape of the diffuse reflectance spectrum. In a similar manner, spectra were recorded for the white reflectance standard (LabSphere, Shaker Street, North Sutton, NH 03260 USA) for colorimetric calculations.

2.2. The diffuse reflectance optical probe (DROP) Fibre bundles of 1 m length were assembled so that the probe could be integrated with the X–Y measurement table of the combinatorial robot LUSI described elsewhere [23]. The operating range for the fibres (Oxford Electronics Ltd., Hampshire, UK) was 180–1200 nm, extending beyond the instrument range. The diameter was 2 mm and the fibre count 295, producing a numerical aperture (NA) of 0.22. The core was pure silica fibres of 95 ␮m diameter and the cladding was doped silica fibres of diameter 100 ␮m. The coating was polyimide and the bundle was housed in a stainless steel jacket of 5 mm diameter. The optimum height and offset for illumination and viewing fibres were found using the NA and trigonometric identities. The main criterion is that the long axis of the emergence/acceptance ellipse of the viewing fibre bundle must be kept within the sample circumference. This sets the maximum height to the upper edge of the detector fibre bundle. The diffuse colour and neutral reflectance standards (LabSphere, North Sutton, NH 03260 USA) were used to calibrate the LS55 for colorimetric values. The bispectral reflectance spectra were recorded for the red, green, blue, and yellow standards using DROP in 45◦ /0◦ illumination/viewing geometry and from 380 nm to 700 nm. Spectra were also recorded for neutral reflectance standards white, light grey, dark grey and black with 99%, 50%, 20%, and 2% reflectance respectively. To validate the procedure for liquid samples, Rhodamine B solution of 5 ␮M was transferred to a 24-well plate positioned on the 128 mm × 96 mm plate reader accessory capable of being driven in continuous or stepwise mode.

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For experiments on high throughput samples of TiO2 , a TiO2 dispersion was printed as drops on silicone release paper to produce disc-shaped samples using the LUSI ink-jet printer. These were dried and preheated to 400 ◦ C to remove dispersants prior to doping with soluble salts of, inter alia, gadolinium and iron using a method described in full elsewhere [26]. After drying at ambient temperature for 86 ks (24 h), they were transferred by pneumatic tweezers to a coarse zirconia bed in an alumina crucible and heated to 600 ◦ C and 800 ◦ C.

3. Results and discussion 3.1. Design considerations The standards specified by CIE for the viewing and illumination conditions for colour measurement limit the geometrical considerations for measuring the spectral reflectance factor. The only two allowable geometries are spherical and bidirectional [25]. The variable nature of shape and sizes of combinatorial libraries under investigation further limit the design. The bidirectional geometry was chosen because it is specular-excluding, it allows the operator to record fluorescence, colour, and colour of a fluorescent sample in the same geometry. In addition, with the bidirectional geometry of the probe, very small solid as well as liquid samples can be scanned both for spectral reflectance and bispectral reflectance factor. If a solid sample is scanned using the synchronous scan mode with wavelength difference  = 0, the result is uncorrected bispectral reflected radiance (reflected component of total radiance) and the instrument records values only along the diagonal of the bispectral radiance matrix, the off-diagonal values, contributed by fluorescence, being excluded. The diagonal values correspond to the spectral distribution obtained by a UV–vis spectrometer when scanning a non-fluorescent sample. For bidirectional geometry, re-emission is not a problem, since there is no “re-radiation” as within a sphere but bidirectional geometries have difficulty with samples with different scattering characteristics. So, a 0◦ /45◦ instrument will work better for determining the colour of a matt paper sample or uniform plastic part than for a textured cloth sample or samples with effect-colourants included [27]. Another advantage of bidirectional geometry is that it excludes specular reflection by its configuration and so gloss is not a complicating factor [24,27]. The 45◦ /0◦ and 0◦ /45◦ geometries are optically the same but the former was preferred when working with combinatorial libraries of TiO2 discs because they are slightly dome-shaped and the setting of sample height without damaging the sample on the fibre-optic ferrule (Fig. 2a) was much easier. We designate this the “synchronous bispectral reflectance radiance” method (SBRR) and for measuring the colour of non-

Fig. 2. Diffuse reflectance optical probe configurations for (a) diffuse reflectance and (b) transmission. The configuration in (a) allows scanning in bidirectional geometries (both 45◦ /0◦ and 0◦ /45◦ ).

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Fig. 3. Bispectral reflectance radiance spectra of commercial red ceramic pigment recorded in synchronous mode. The specular part in the wavelength region 360–560 nm reduces as the angle ˛ (defined in Fig. 1) increases from 45◦ to 70◦ .

luminescent samples using the bispectral spectrometer, it is much more efficient than the method described in the literature [28]. The scan and data handling times are considerably reduced. For non-luminescent samples fluorescence is non-existent or negligible. Thus the whole matrix is created to determine the diagonal values and the number of scans required, Ns is: Ns =

End wavelength − Start wavelength +1 Wavelength interval

Thus to build a bispectral matrix from 300 nm to 700 nm with a wavelength interval of 10 nm, 41 separate scans would be required. With SBRR method, similar data are collected in one scan and only one spectrum is manipulated for obtaining diffuse reflectance and colour computation.

Ceramic combinatorial libraries of interest could well have high reflectivity (for example, in the search for new non-toxic ceramic red glazes) and luminescence (in searching for photoactive and optical materials) but these examples are not likely to have effectcolourants included. The bidirectional geometry was chosen for the probe, based on the advantages of being specular-excluding, allowing the operator to record fluorescence, fluorescence and colour, and colour only in the same position. In addition, with the bidirectional probe geometry, very small solid or liquid samples can be scanned for reflectance. Fig. 2(a) shows the arrangement for illumination and collection of light in the 45◦ /0◦ geometry. For transmission mode, the illuminating and receiving fibres were placed facing each other with the sample in the middle (filters were placed in the incident beam) exactly as employed in transmission or absorbance spectroscopy as shown in Fig. 2(b). In this way, coloured

Fig. 4. The diffuse reflectance spectra recorded by the goniometer for angle ˛ (defined in Fig. 1) of 60◦ and 30◦ , effectively exchanging incident and collected rays.

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Fig. 5. Excitation and emission spectra of 5 ␮M Rhodamine B solution. Excitation spectra recorded by (1) illuminating sample with plate reader fibres and viewing by DROP fibres and (2) illuminating sample with DROP fibres and viewing by plate reader fibres. Emission spectra recorded by (3) illuminating sample by plate reader fibres and viewing by DROP fibres and (4) illuminating sample by DROP fibres and viewing by plate reader fibres.

glazes could be characterised when fired on a silica glass substrate as could thin films for colour and absorbance when coated on a transparent substrate. The first stage of validation was done without optical fibre transmission. Using the vernier goniometer stage in position A (Fig. 1), corresponding to 45◦ /45◦ , the instrument collects specular reflection but as angle ˛ is increased, the goniometer allows the fraction of specular to decrease and diffuse reflectance is

collected until, as ˛ approaches 90◦ , the detected beam is attenuated. The bispectral reflected radiance spectra of red pigment obtained from the goniometer recorded at increasing angles ˛ are given in Fig. 3. At ˛ = 45◦ , there is significant specular reflectance over the whole range of wavelengths. The specular component reduces progressively as the angle of incidence changes and only the diffuse reflected radiance is observed in the red area with a peak at 640 nm. A similar trend was observed

Fig. 6. 3D projection of fluorescence intensity of Rhodamine B against concentration recorded by (a) plate reader fibres and (b) DROP. (c) Fluorescence intensity of Rhodamine B at 575 nm plotted as a function of concentration for two detection methods.

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Fig. 7. Normalized bispectral reflectance radiance spectra recorded for the minimum spot size measurements by DROP in 45◦ /0◦ geometry.

Fig. 8. Bispectral reflectance radiance of red pigment on fused silica glass substrate using DROP in 45◦ /0◦ geometry and transmission geometry.

when the angle of incidence of light changes in the other direction. Table 1 contains colour coordinates for the spectra in Fig. 3. The colour coordinates were calculated from the diffuse reflectance spectra that were obtained from the bispectral reflectance radiance using the equation below. A grey Spectralon with 20% reflectance

was used as the reference. D(, ω) =

ˇws (, ω) Lref () ˇref (, ω)

where D(, ω) is the reflectance of the sample, ˇws (, ω) is bispectral reflectance radiance distribution of the sample, Lref () is the

Table 1 Colour measurement values for commercial red ceramic pigment corresponding to bispectral spectra presented in Fig. 3. Angle ˛

45◦ 50◦ 55◦ 60◦ 65◦ 70◦

Colour coordinates X

Y

Z

x

y

L

a

b

C

H

71.88 50.63 27.36 22.92 21.62 18.86

64.19 42.20 17.68 13.32 13.36 19.03

55.93 34.71 8.89 3.60 4.60 15.83

0.3743 0.3969 0.5072 0.5751 0.5460 0.3511

0.3343 0.3308 0.3278 0.3343 0.3374 0.3542

84.06 71.01 49.11 43.25 43.30 50.73

22.03 28.34 47.91 54.36 48.14 2.65

-3.14 0.15 17.09 31.71 25.79 -0.30

22.26 28.34 50.87 62.93 54.62 2.67

351.86 0.31 19.63 30.25 28.17 353.52

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Fig. 9. Reflectance spectra of colour standards recorded in 45◦ /0◦ geometry using DROP.

Fig. 10. Fluorescent and reflected parts together with actual spectral distribution recorded from fluorescent green sample.

reflectance of the reference used and ˇref (, ω) is the bispectral reflectance radiance distribution of the grey reference. The CIE L* a* b* and CIE Yxy values were calculated for each sample from the reflectance spectrum using the D55 as CIE illuminant and CIE 1931 ¯ (2◦ ) colour matching functions x¯ (), y(), z¯ (). The colour of commercial pigment used was bright red and it can be seen in both tables that when ˛ = 45◦ (specular reflectance geometry) the x coordinate is closer to the coordinate of the illuminant (D55) used in the colour calculations, namely x = 0.33. The high specular component means the photomultiplier tube is seeing the sample as white rather than red. When ˛ is increased by rotating the sample towards the emission window, the x coordinates move towards the red area in the 1931 CIE chromaticity diagram for which x ≈ 0.6 [25]. Note that y changes little because both red and white light lie on the same ‘y’ coordinate on the chromaticity diagram. The brightest red colour is recorded in Fig. 3 at ˛ = 60◦ . The same trend was seen as the jig was rotated towards the excita-

tion window and the deepest red is then presented at ˛ = 30◦ . The colour coordinates recorded at the positions where ˛ = 60◦ and ˛ = 30◦ are similar; they correspond to interchanging the incident and collected beams. The slight difference is caused by small geometrical misalignments and the fact that colour measurement is dependent on the viewing and illumination conditions: a colour measuring instrument should be calibrated for the exact geometrical conditions used in practice. As ˛ is increased from 60◦ , the colour coordinates again move away from the red area in the 1931 chromaticity diagram. The diffuse reflectance spectra computed from the spectra recorded at ˛ = 60◦ and at ˛ = 30◦ by dividing by the spectrum for the 20% reflectance standard measured under identical conditions are shown in Fig. 4. Ratioing to the grey standard shows that almost no specular reflection is present and a typical red spectrum is revealed. This evidence validates our approach to a new SBRR method as developed in this paper for recording diffuse reflectance and colour. The next stage of validation examines the intrusion of fibre-optic bundles into the ray paths.

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Fig. 11. Comparison between the reflected component of spectral power distribution recorded by emission scans and by SBRR from the fluorescent green sample.

3.2. Validation of DROP by interchanging fibre bundles In order to validate the 45◦ /0◦ DROP fibre-optic assembly, the excitation and emission bundles were used separately in conjunction with the plate reader fibre-optic assembly supplied with the instrument but with emission and excitation fibre bundles interchanged. The fused silica fibres selected for DROP have, according to manufacturer’s data, very low fluorescence of their own and the same numerical aperture as the plate reader probe fibres (NA = 0.2). Emission and excitation spectra were then recorded under four configurations: 1. Plate reader fibre-optic probe was used. 2. Plate reader fibres were used for incident beam and DROP fibres for collection. 3. DROP fibres were used for illumination and plate reader fibres for collection. 4. DROP fibres were used for illumination and viewing. The spectra were collected in fluorescence mode with slit widths of 5 nm and scan speed 500 nm min−1 . Rhodamine B, having characteristic peaks for emission and excitation was used at 5 ␮M for recording spectra in these four configurations. The emission spectrum was taken from 555 nm to 700 nm with excitation at 552 nm and the excitation spectrum 500 to 575 nm with emission at 585 nm. The spectra recorded in all configurations had the same shape, the emission spectra were particularly close. In the coupled fibre-optic configurations 2 and 3 (Fig. 5) and in the DROP-only configuration 4, the emission and excitation peak positions for Rhodamine B fluorescence were observed at the same wavelengths and no other peaks were observed beside the solution fluorescence peak when compared with the spectra recorded with plate reader fibres. The plate reader fibres are made of silica with transmission characteristics down to approximately 260 nm. The fibre bundles are polished flat and permanently joined with an angle between axes of approximately 25◦ . The fibres have NA of about 0.2 which produces an emergence/acceptance angle of nominally 23◦ . These results show that shapes of both excitation and emission spectra, recorded by the new fibres and plate-reader fibres are almost identical. The peak positions are also consistent. It can be concluded that the DROP fibre assembly, when tested in all practical configurations, did not alter the fluorescence accuracy, no additional emission or shift in actual peak position was observed,

hence the optical fibres behave the same as the original fibre assembly. The active diameter of DROP fibres is much less than the plate reader fibres and the illuminating/viewing area is directly proportional to the active diameter of the fibre bundle. As a result, the intensity of the scans performed by DROP are less than those performed by the larger plate reader bundles. In spite of the difference in illumination and viewing geometries, both probes have identical fluorescence spectra. Fluorescence is Lambertian in nature and a luminescent sample radiates equally in all directions. In addition, these results show that the DROP setup is capable of scanning fluorescence from liquid samples. 3.3. Comparison of calibration curves The fluorescence of Rhodamine B at concentrations from 1 ␮M to 8 ␮M with an interval of 1 ␮M was recorded using both the plate-reader fibres and DROP and spectra are shown in Fig. 6a and b respectively. Comparing the main peak, it is noticeable that the spectra recorded by DROP have a smoother slope than those recorded by plate reader fibres. The corresponding calibration curves for both fibre-optic systems obtained by plotting the intensity at 575 nm against concentration of Rhodamine B are shown in Fig. 6(c). The calibration curve for fluorescence recorded by the DROP fibre system shows that a linear relationship is maintained throughout the concentration interval in good agreement with results obtained by the plate reader probe. The fluorescence intensity recorded by DROP is less than that of the plate reader probe because of smaller active diameter and less viewing area; the difference in slopes being partly because of the use of filters in the plate reader system to bring the intensity into range. The calibration curve produced by DROP approaches the origin on extrapolation unlike the plate-reader curve which, having a larger diameter, may have picked up scattered light. 3.4. Effect of sample size on colour To determine the smallest recordable sample size in a combinatorial library from which a valid spectrum can be obtained using the DROP in the 45◦ /0◦ geometry, the glaze was fired on silica glass and covered with a metal foil perforated with holes of different sizes so that the disc surround was reflective. All spectra were normalized to 100 intensity units at the highest peaks. Fig. 7 shows the normalized bispectral reflectance radiance recorded for

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Table 2 Colour coordinates for diffuse colour standards measured using SBRR by DROP in 45◦ /0◦ geometry.

Blue

Green

Yellow

Red

X

Y

Z

x

y

L

Ref 99% R 50% R 20% R

30.58 24.55 27.41

25.86 31.00 25.11 27.69

78.46 62.45 73.87

0.2180 0.2184 0.2190 0.2125

0.2280 0.2214 0.2240 0.2147

57.91 62.51 57.19 59.61

−0.11 3.47 2.28 2.85

−48.6 −54.21 −49.49 −62.39

Ref 99% R 50% R 20% R

25.40 20.56 22.44

34.07 34.95 28.51 30.81

22.55 17.86 21.19

0.3022 0.3064 0.3072 0.3014

0.4178 0.4216 0.4259 0.4139

65.01 65.71 60.35 62.35

−31.12 −30.86 −29.57 −30.17

14.07 15.78 15.88 7.96

Ref 99% R 50% R 20% R

83.09 67.30 72.88

78.52 83.84 68.41 73.35

10.50 8.39 9.81

0.4632 0.4683 0.4671 0.4671

0.4694 0.4725 0.4747 0.4701

91.02 93.38 86.21 88.61

4.79 5.56 4.11 4.53

85.23 91.61 86.25 82.02

Ref 99% R 50% R 20% R

35.70 28.59 30.82

18.11 20.66 16.66 18.02

6.35 5.05 5.96

0.5524 0.5693 0.5683 0.5623

0.3300 0.3294 0.3313 0.3288

49.63 52.57 47.84 49.52

57.99 64.39 59.11 59.45

30.71 36.24 34.09 29.64

the red pigment from sample discs of diameters 5, 4, 3, 2, 1.5, and 1 mm. The spectra for the 1 mm and 1.5 mm samples show high specular reflectance and noise in the 340–500 nm region. The remaining spectra, which are for 2, 3, 4, and 5 mm sample size are almost coincident. These results, based on comparison of the shapes of spectra, suggest that a fairly accurate measurement can be performed for a sample with diameter down to 2 mm using the DROP in 45◦ /0◦ geometry. Using these spectra, diffuse reflectance from all spots sizes was calculated using the grey diffuser with 20% reflectance as a reference and recording the reference spectra from the same spot sizes as the samples

a

b

using the same perforated mask. These spectra confirmed that the reflectance of samples taken from samples of diameter 2 mm and above were consistent with reflectance taken from larger spots. 3.5. Recording spectra in transmission mode Fig. 8 shows the bispectral reflectance radiance and bispectral radiance (as recorded spectra) of the red pigment applied to a fused silica glass substrate and fired at 700 ◦ C using DROP in 45◦ /0◦ and in transmission geometries respectively. The spectra are similar but that taken in 45◦ /0◦ geometry shows higher intensity in the red region compared to the spectrum measured in transmission. In the rest of the wavelength range the spectra are similar. The lower intensity of the spectrum in transmission mode is due to absorption in the film. Variation in intensity could be accommodated by calibration for different film thicknesses. The peak positioning and overall spectrum shapes are similar confirming that the modified spectrophotometer may also be calibrated to function in transmission mode. This is an important facility for work on thin films. 3.6. Calibration of DROP for reflectance

Fig. 12. A library of 25 members being scanned for diffuse reflectance and luminescence.

DROP was calibrated for reflectance/colour measurements in 45◦ /0◦ geometry using the neutral and colour diffuse reflectance reference standards and the SBRR method. The bispectral

Fig. 13. Diffuse reflectance spectra of un-doped TiO2 , 2 at.% Fe–TiO2 and 2 at.% Gd–TiO2 .

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reflectance radiance spectra for the coloured reflectance references were recorded. These curves, along with the bispectral reflectance radiance for the neutral references were used to find the deviation of the instrument from the reference values for colorimetric calculation over the entire visible range. The reflectance spectra were obtained by dividing bispectral reflectance radiance of samples with those of neutral references having 99%, 50% and 20% reflectance and then by multiplying by the given reflectance of the reference used. The computed reflectance spectra in terms of percent reflectance for the colour references are shown in Fig. 9. The colorimetric values using XYZ, xy, and Lab coordinates, calculated from the bispectral reflectance radiance for the coloured references are listed in Table 2. The corresponding E values comparing instrument and reference values were computed and are listed in Table 3 (see supplementary details). The colour difference between any two colours in CIELAB space is the distance between the colour locations. This distance can be expressed using CIE parameters L*a*b*(E) [29] as follows: ∗ Eab = (L∗2 + a∗2 + b∗2 )

1/2

where L∗ is the lightness difference, a∗ is the red/green difference, and b∗ is the yellow/blue difference. The least E values are observed for the colour coordinates calculated using the neutral reference with 20% reflectance and this reference was selected for colour computation in the following work. For the four standards, E was 0.9 (blue), 3.1 (green), 4.2 (yellow) and 1.7 (red). It is known that the difference between readings taken on spherical geometry and bidirectional geometries is reduced by using a reference with lower reflectance [27]. In practice, a E of 1 or less between two colours that are not contiguous is hardly distinguishable by the average human observer; a E between 3 and 6 is normally regarded as a satisfactory match in commercial reproduction on printing presses [30]. The reference colour coordinates for diffuse reflectance standards are calculated from reflectance spectra recorded in a hemispherical geometry (integrating sphere) rather than the 45◦ /0◦ geometry used here. In theory, colour computations from all these geometries should be the same because the reflectance is a ratio of the sample’s spectral distribution to that of the reference. Therefore the deviation obtained using dissimilar geometries should be automatically corrected. However in this case, there are a few factors that can account for slightly higher values of E particularly in the middle range of the visible spectrum. One of the main factors is the considerable difference in area of illumination and viewing for the measurement. The DR references have 50.8 mm (nominal 2 in.) diameter and in an integrating sphere all of this area is illuminated and viewed for the measurements. In contrast, the DROP instrument is designed for scanning very small samples and the diameter for viewing and illumination can be as small as 2 mm. Measurements made in bidirectional geometries are known to be very sensitive to surface irregularities [27]. Thus while variations arising from surface irregularities can be averaged out due to the larger viewing and illumination area for spectral measurements in the integrating sphere, this may not be the case in the DROP configuration. The slightly raised values of E could be due to the fact that in the integrating sphere, a sample with up to 625 times larger area is scanned compared to the DROP configuration. Of course, readings taken on two different instruments can also be a reason for somewhat higher E: the integrating sphere radiates directly into the spectrometer without optical fibre transmission. Nevertheless, the E values in Table 3 show that the colours perceived by the modified LS55 are an acceptable match with the colours of the references and the results show that it can be used for colour measurement.

3.7. Colour measurement of a fluorescent sample For the fluorescent green sample, a strong emission in the 480–560 nm region of the visible spectrum complements the intrinsic colour. The human eye cannot differentiate between the colour and the fluorescence and so the fluorescent green sample looks unusually radiant and vivid to the eye. The bispectral total radiance measured for bispectral colorimetry is expressed as a matrix in which the columns correspond to the excitation while the rows correspond to the emission wavelengths [28]. The values along the diagonal correspond to the reflected radiance component while the values off-diagonal correspond to fluorescent contributions. Conventionally [28] the values in each row are summed over the excitation waveband to give the stimulus function including the reflected radiance and luminescent contributions. The fluorescent and non-fluorescent parts add up to give the total bispectral radiance of the sample but the constituent parts of the spectrum can be separated arithmetically and Fig. 10 shows the bispectral radiance (as recorded spectrum) along with the separated fluorescent and non-fluorescent components. Standard colour measuring instruments are only capable of measuring the apparent colour of samples; the fluorescent component cannot be distinguished by a UV–vis colour measuring instrument and as a result, colour of a luminescent sample measured on such instruments is not accurate. The magnitude of error is presented in Fig. 10 by mathematically splitting the spectral distribution of the green fluorescent sample obtained conventionally using the matrix method for measuring colour on a two monochromator instrument [24,28]. The total radiance observed by a UV–vis colour measuring instrument, and by human eye, will include the fluorescent component and reflected component. This inaccuracy can be eliminated using a two monochromator instrument. The same sample was also evaluated for colour using the novel SBRR method. In Fig. 11, the reflected component recorded using the SBRR method is compared to the reflected component obtained from the matrix. The scanned and calculated reflected parts have similar shape however the calculated reflected part has fewer data points resulting in sharper peaks in comparison with the SBRR scanned spectrum. This comparison shows that SBRR method provides equivalent results to the matrix method with one scan instead of 41 and is much faster method to record colour of a nonluminescent sample using a two monochromator instrument.

3.8. Combinatorial libraries of doped TiO2 The working instrument was used to examine an array of Gdand Fe-doped TiO2 catalysts in two ways; capturing their diffuse reflectance spectra and deducing bandgap therefrom and tracking the fluorescence changes of Rhodamine B under UV illumination as it was photocatalytically degraded by each doped TiO2 sample. A two dimensional 5 × 5 array was adopted for co-doping as shown in Fig. 12. For every experiment, four replica libraries were synthesized. The libraries were investigated for photocatalysis, colour, photoluminescence and by XRD. The diffuse reflectance spectra for the libraries were obtained from the bispectral reflectance radiance recorded on the LS55 with DROP as shown in Fig. 13. The reflectance for each member and the CIE L* a* b* and CIE Yxy values were calculated for each sample from the reflectance spectrum using the D55 as CIE illuminant ¯ and CIE 1931 (2◦ ) colour matching functions x¯ (), y(), z¯ (). The full details of results of TiO2 combinatorial libraries co-doped with iron and gadolinium are given elsewhere [26] but Fig. 13 shows diffuse reflectance spectra of un-doped and 2 at.% Gd and 2 at.% iron doped TiO2 . Bandgap (Eg ) values can be calculated from these

F.A. Saeed, J.R.G. Evans / Analytica Chimica Acta 677 (2010) 79–89

spectra using the following equation [31]: Eg =

hc 

where h is Plank’s constant, c is velocity of light, and  is bandgap edge wavelength. The calculated bandgap values of un-doped, Fe–TiO2 and Gd–TiO2 are 3.15, 3.24 and 2.15 eV respectively. These values show an increase in bandgap by Gd doping and decrease by Fe doping into TiO2 lattice. These values including the bandgap of un-doped TiO2 are consistent with reported values in the literature [32,33]. 4. Conclusions These results demonstrate the validity of a new approach to measurement of optical properties of combinatorial libraries on sample diameters as low as 2 mm by modifying a conventional luminescence platform. As well as recording fluorescent or non-fluorescent samples, the independent fibre-optic arrangement can be used for 0◦ /45◦ and 45◦ /0◦ (i.e. CIE permitted) bispectral measurement geometries and measurement in transmission. Indeed it could equally be configured into a goniometer to record diffuse reflectance as function of angle. It is also able to measure spectra from well-plates. For colour measurements of non-luminescent samples the SBRR method not only qualifies but it is more efficient than the conventional method of colour measurement on two monochromator instruments. The DROP configuration is capable of measuring the colour of non-luminescent samples as efficiently as standard (e.g. UV–vis attached to integrating sphere) colour measuring instruments; it also has the advantage of measuring the actual colour of a fluorescent sample by eliminating the error introduced in standard colour measurements. The SBRR method offers advances in terms of efficiency and ease of use for diffuse reflectance and colour measurements of nonluminescent samples over conventional bispectrometer methods and is truly a high throughput method capable of being applied to conventional well-plate configurations and saving approximately 2 h of scanning time for one sample: in scanning a library of 25 samples, this novel method may save up to 2 days of operator time. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.aca.2010.07.007.

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