Adulteration with Melamine

• 2008 adulterated milk powder and infant formula • Resulted in more than 300,000 people becoming ill • 3 infant deaths• WHO maximum residues

• 2.5 mg/L milk• 1 mg/L infant formula

• Economic adulteration• 90-4000 mg/L

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Milk adulterants

Melamine

Urea

Dicyandiamide

Ammonium sulfate

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Why Raman spectroscopy?

• Non-destructive• Potentially no sample preparation• Raman scattering selection rule

• 𝑑𝑑𝛼𝛼𝑑𝑑𝑑𝑑 𝑑𝑑−0

≠ 0

• Minimal interference from water• Strong Raman scattering from the molecules

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Raman spectroscopy

Image from R. Smith, K. L. Wright, L. Ashton, Raman spectroscopy: an evolving technique for live cell studies. Analyst, 141:35903600 (2016)

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Instrument configuration

• 785 nm excitation – why?

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Instrument configuration

• 785 nm excitation – why?

J. Qin, M. S. Kim, K. Chao, S. Dhakal, B.-K., Cho, S. Lohumi, C. Mo, Y. Peng, M. Huang, Advances in Raman spectroscopy and imaging techniques for quality and safety inspection of horticultural products, Postharvest Biology and Technology 149:101-117 (2019)

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Sample preparation

• 15 µl drops of samples were placed individually into 4 mm chrome based sampling wells

• The milk was allowed to dry over 1 hr to an average thickness of 112 µm

• Why chrome based sampling wells?• Why dry the milk first?• With this sample preparation is a 50x objective appropriate?

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Instrument configuration

• 50x objective – is it appropriate?• Sampling spot size

Spot diameter =1.22𝜆𝜆𝑁𝑁𝑁𝑁

Spot diameter =1.22(785 nm)

0.5

Spot diameter ≈ 2 μm• Depth of focus

∆𝑓𝑓 =𝜋𝜋 𝑑𝑑 2

2𝜆𝜆

∆𝑓𝑓 ≈ 8 μm

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Spectra from: M.K. Nieuwoudt, S. E. Holroyd, C. M. McGoverin, M. C. Simpson, D.E. Williams , Raman spectroscopy as an effective screening method for detecting adulteration of milk with small nitrogen-rich molecules and sucrose. Journal of Dairy Science 99:2520-2536 (2016)

Raman spectra9

Univariate calibrations – peaks used

Spectra from: M.K. Nieuwoudt, S. E. Holroyd, C. M. McGoverin, M. C. Simpson, D.E. Williams , Raman spectroscopy as an effective screening method for detecting adulteration of milk with small nitrogen-rich molecules and sucrose. Journal of Dairy Science 99:2520-2536 (2016)

Spectra of milk solutions with single adulterants added; concentrations added between 0 and 1000 mg/L.

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Univariate calibrations - results

M.K. Nieuwoudt, S. E. Holroyd, C. M. McGoverin, M. C. Simpson, D.E. Williams , Raman spectroscopy as an effective screening method for detecting adulteration of milk with small nitrogen-rich molecules and sucrose. Journal of Dairy Science 99:2520-2536 (2016)

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Partial least squares – spectra

M.K. Nieuwoudt, S. E. Holroyd, C. M. McGoverin, M. C. Simpson, D.E. Williams , Raman spectroscopy as an effective screening method for detecting adulteration of milk with small nitrogen-rich molecules and sucrose. Journal of Dairy Science 99:2520-2536 (2016)

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Partial least squares – regression coefficients

M.K. Nieuwoudt, S. E. Holroyd, C. M. McGoverin, M. C. Simpson, D.E. Williams , Raman spectroscopy as an effective screening method for detecting adulteration of milk with small nitrogen-rich molecules and sucrose. Journal of Dairy Science 99:2520-2536 (2016)

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Partial least squares - results

M.K. Nieuwoudt, S. E. Holroyd, C. M. McGoverin, M. C. Simpson, D.E. Williams , Raman spectroscopy as an effective screening method for detecting adulteration of milk with small nitrogen-rich molecules and sucrose. Journal of Dairy Science 99:2520-2536 (2016)

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Calibration comparison

M.K. Nieuwoudt, S. E. Holroyd, C. M. McGoverin, M. C. Simpson, D.E. Williams , Raman spectroscopy as an effective screening method for detecting adulteration of milk with small nitrogen-rich molecules and sucrose. Journal of Dairy Science 99:2520-2536 (2016)

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Partial least squares discriminant analysis

M.K. Nieuwoudt, S. E. Holroyd, C. M. McGoverin, M. C. Simpson, D.E. Williams , Raman spectroscopy as an effective screening method for detecting adulteration of milk with small nitrogen-rich molecules and sucrose. Journal of Dairy Science 99:2520-2536 (2016)

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How can we make this Raman method more practical?

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Fibre optic probe based Raman spectroscopy

Image from: http://bwtek.com/spectrometer-part-8-fiber-optic-probes/

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Sample well design

• Sample wells were made to maximise collection of scattered light

• Depth of focus of laser, δa

𝛿𝛿𝑎𝑎 =3𝜋𝜋 𝜔𝜔𝑖𝑖

2

𝜆𝜆

• Beam waist diameter, 2ωi2𝜔𝜔𝑖𝑖 = 𝑓𝑓𝜃𝜃𝑑𝑑

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Sample well design

• Beam waist• 2𝜔𝜔𝑖𝑖 = 𝑓𝑓𝜃𝜃𝑑𝑑• 2𝜔𝜔𝑖𝑖 = 5700 × 0.004 = 22.8 μm

• Depth of focus• 𝛿𝛿𝑎𝑎 = 3𝜋𝜋 𝜔𝜔𝑖𝑖

2

𝜆𝜆• 𝛿𝛿𝑎𝑎 ≈ 900 μm

450 µm

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Sample well manufacture

Images from: https://commons.wikimedia.org/wiki/File:Ball_Bearings.jpg; https://commons.wikimedia.org/wiki/File:Aluminium-4.jpg; https://en.wikipedia.org/wiki/Vise#/media/File:ViceBench-insetSoftJaws.jpg

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Sample wells

M.K. Nieuwoudt, S. E. Holroyd, C. M. McGoverin, M. C. Simpson, D.E. Williams , Rapid, sensitive, and reproducible screening of liquid milk for adulterants using a portable Raman spectrometer and a simple, optimized sample well. Journal of Dairy Science 99:7821-7831 (2016)

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Raman spectra from different sized wells

M.K. Nieuwoudt, S. E. Holroyd, C. M. McGoverin, M. C. Simpson, D.E. Williams , Rapid, sensitive, and reproducible screening of liquid milk for adulterants using a portable Raman spectrometer and a simple, optimized sample well. Journal of Dairy Science 99:7821-7831 (2016)

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Partial least squares results

M.K. Nieuwoudt, S. E. Holroyd, C. M. McGoverin, M. C. Simpson, D.E. Williams , Rapid, sensitive, and reproducible screening of liquid milk for adulterants using a portable Raman spectrometer and a simple, optimized sample well. Journal of Dairy Science 99:7821-7831 (2016)

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Partial least squares discriminant analysis

M.K. Nieuwoudt, S. E. Holroyd, C. M. McGoverin, M. C. Simpson, D.E. Williams , Rapid, sensitive, and reproducible screening of liquid milk for adulterants using a portable Raman spectrometer and a simple, optimized sample well. Journal of Dairy Science 99:7821-7831 (2016)

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Immersion fibre optic probe

R. A. Schwarz, D. Arifler, S. K. Chang, I. Pavlova, I. A. Hussain, V. Mack, B. Knight, R. Richards-Kortum, A. M. Gillwater, Ball lens coupled fiber-optic probe for depth-resolved spectroscopy of epithelial tissue. Optics Letter, 30(10):1159-1161 (2005)

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Partial least squares discriminant analysis results

M.K. Nieuwoudt, S. E. Holroyd, C. M. McGoverin, M. C. Simpson, D.E. Williams , Screening for adulterants in liquid milk using a portable Raman miniature spectrometer with immersion probe. Applied Spectroscopy 71(2):308-312 (2017)

Sensitivity =TN

TN + TP× 100 = 89%

Specificity = TPTP+TN

× 100 = 92%

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