10 / 07BRUINS INSTRUMENTS1 Training Near Infrared Transmission Whole Grain Analyzer

10 / 07 BRUINS INSTRUMENTS 2 Targets: History and Basics of NIR Calibration development Toubleshooting & Maintainance

10 / 07 BRUINS INSTRUMENTS 3 History of NIR First detected by William Herschel beginning of 19th century First instrument built by W.W. Coblentz about 1900 First practical use in the 1930s First agriculture usage by Karl Norris 1968 First commercial scanning instruments 1978 First BRUINS NIR spectrophotometer 1979 First BRUINS NIT spectrophotometer 1982

10 / 07 BRUINS INSTRUMENTS 4 Basics of NIR NIR = Near InfraRed Spectroscopy Wavelength of Light UV> VIS> NIR>(M)IR nm / (NIT / NIR) Energy of Light

10 / 07 BRUINS INSTRUMENTS 5 Basics of NIR Typical properties analysed by NIR are organic compounds like Oil Protein Starch And Water Useful concentration are in % range, ppm or ppb level are normally impossible.

10 / 07 BRUINS INSTRUMENTS 6 Basics of NIR NIR active groups O-HWater, Alcohol C-HCarbohydrates (Starch, Sugar, Cellulose) Fat / Oil N-HProtein

10 / 07 BRUINS INSTRUMENTS 7 Basics of NIR Molekular Vibrations O H H

10 / 07 BRUINS INSTRUMENTS 8 Basics of NIR Molekular Vibrations

10 / 07 BRUINS INSTRUMENTS 9 Basics of NIR Molekular Vibrations

10 / 07 BRUINS INSTRUMENTS 10 Calibration Reference Analysis Sample selection Sampling Sample Preparation / Homogenisation Sample Presentation

10 / 07 BRUINS INSTRUMENTS 11 Calibration: Reference Analysis NIR calibration will be dependend from reference analysis, so Select best possible method Investigate „real“ method error Make sure that the same sample material will be analysed Use at least double analysis Comment all specialities of actual sample

10 / 07 BRUINS INSTRUMENTS 12 Calibration: Lab Error Influence

10 / 07 BRUINS INSTRUMENTS 13 Calibration: Lab Error Influence

10 / 07 BRUINS INSTRUMENTS 14 Calibration: Sample Selection The complete interesting concentration range must be covered by calibration samples. The calibration set must also include samples with changes from other constituents, eg. low protein with high moisture, low protein with low moisture etc. Further samples must be included which are representing influences from climate, soil, cultivars etc. Due to the effect of temperature to NIR also cold and warm samples should be added.

10 / 07 BRUINS INSTRUMENTS 15 Calibration: Sampling The sample must be representative for the total amount. Take subsamples from different locations. Thoroughly mix these to get a homogene sample.

10 / 07 BRUINS INSTRUMENTS 16 Calibration: Sample Preparation No extra preparation needed for most grain and oil seeds. Samples with thick shell must be ground (e.g. sunflower seeds). The same sample preparation must be used for calibration and routine analysis. By grinding a sample the moisture content will decrease. Select the best grinder to reduce this moisture loss. If a grinder with a sieve will be used, control the size of the mesh.

10 / 07 BRUINS INSTRUMENTS 17 Calibration: Sample Presentation OmegAnalyzer and AgriCheck uses the NIR transmission, so for such meal the MultiCheck will be the better choice As an alternative, but less efficient, a micro sample cup for meal or powders can be used

10 / 07 BRUINS INSTRUMENTS 18 Calibration: Sample Presentation In all cases, if fine powder or ground sample: Thoroughly mix the sample before filling In cases of components with different density make sure, that no material separate If a micro sample cup is used: Make sure that the sample is filled without wholes Knock a bit, that the sample will slide together, but not to often because different components may separate

10 / 07 BRUINS INSTRUMENTS 19 Calibration: Standardisation Before using the instrument in routine, it must be compared against the reference method with a set of samples. These samples should cover the range of concentrations and normal variations in moisture, area and genotypes. In order to get a good adjustment, the sample must be well mixed that the same material will be analysed with both methods. The data for the reference analysis need to be well controlled, at least a double analysis must be performed.

10 / 07 BRUINS INSTRUMENTS 20 Calibration: Standardisation The easiest comparison of accuracy and necessary adjustment can be made with Excel. Just two columns need to filled, one with reference analysis the second with predicted NIR data. Using the Excel functions for mean, standard error xy and correlation will calculate all necessary information. Additional the Excel graph can be used to show how good the predicted values fit to the reference analysis.

10 / 07 BRUINS INSTRUMENTS 21 Calibration: Bias ActualPredicted

10 / 07 BRUINS INSTRUMENTS 22 Calibration: Bias ActualPredicted ActualPredicted Mean SEP0.121 R²0.999 Bias Slope1.001

10 / 07 BRUINS INSTRUMENTS 23 Calibration: Bias

10 / 07 BRUINS INSTRUMENTS 24 Calibration: Bias, 0.43 added ActualPredicted

10 / 07 BRUINS INSTRUMENTS 25 Calibration: Bias

10 / 07 BRUINS INSTRUMENTS 26 Calibration: Slope While the bias will move all data parallel, the slope will change the extreme values more than the values around the middle. So adjusting the slope will much more influence the predicted result. For the slope adjustment the sample set must really cover the full calibration range. Normally this adjustment should not be used. If it seems to be necessary, look for the reason. It might be better to select or built another calibration.

10 / 07 BRUINS INSTRUMENTS 27 Calibration: Slope ActualPredicted

10 / 07 BRUINS INSTRUMENTS 28 Calibration: Slope

10 / 07 BRUINS INSTRUMENTS 29 Calibration: corrected Slope ActualPredicted

10 / 07 BRUINS INSTRUMENTS 30 Calibration: corrected Slope

10 / 07 BRUINS INSTRUMENTS 31 Calibration: Slope ActualPredicted

10 / 07 BRUINS INSTRUMENTS 32 Calibration: Slope

10 / 07 BRUINS INSTRUMENTS 33 Calibration: Slope

10 / 07 BRUINS INSTRUMENTS 34 Calibration: corrected Slope? ActualPredicted

10 / 07 BRUINS INSTRUMENTS 35 Calibration: corrected Slope?