Modern Optical Spectroscopy http://www.scientific-web.com/en/Books/ModernOpticalSpectroscopyWithExercises.html Introduction & the BeerLambert Law Shu-Ping Lin, Ph.D. Institute of Biomedical Engineering E-mail: splin@dragon.nchu.edu.tw Website: http://web.nchu.edu.tw/pweb/users/splin/ Energy Absorption The mechanism of absorption energy is different in the Ultraviolet, Infrared, and Nuclear magnetic resonance regions. However, the fundamental process is the absorption of certain amount of energy. The energy required for the transition from a state of lower energy to a state of higher energy is directly related to the frequency of electromagnetic radiation that causes the transition. Wave Number (cycles/cm) X-Ray UV 200nm Visible 400nm IR 800nm Wavelength (nm) Spectral Distribution of Radiant Energy Microwave Region of the Electromagnetic Spectrum Electromagnetic Radiation V = Wave Number (cm-1) l = Wave Length C = Velocity of Radiation (constant) = 3 x 1010 cm/sec. u = Frequency of Radiation (cycles/sec) u C V = = l The energy of photon: h (Planck's constant) = 6.62 x 10- (Ergsec) 27 E = h u= h C l u = C l C = ul Spectral Properties, Application and Interactions of Electromagnetic Radiation Wave Number V Energy Kcal/mol 9.4 x 107 9.4 x 103 9.4 x 101 eV 4.9 x 106 4.9 x 102 4.9 x 100 cm-1 3.3 x 1010 3.3 x 106 3.3 x 104 Wavelength λ cm 3 x 10-11 3 x 10-7 3 x 10-5 Frequency υ Type Radiation Type spectroscopy Gamma ray Gamma ray emission Hz 1021 1017 X-ray 1015 Ultra violet 9.4 x 10-1 4.9 x 10-2 3.3 x 102 3 x 10-3 1013 9.4 x 10-3 4.9 x 10-4 3.3 x 100 3 x 10-1 1011 4.9 x 10-8 3.3 x 10-4 3 x 103 107 Infrared Nuclear X-ray absorption, emission Electronic (inner shell) UV absorption Electronic (outer shell) Visible 9.4 x 10-7 Type Quantum Transition IR absorption Microwave Microwave absorption Radio Nuclear magnetic resonance Molecular vibration Molecular rotation Magnetically induced spin states Dispersion of Polymagnetic Light with a Prism Prism - Spray out the spectrum and choose the certain wavelength (l) that you want by slit. Infrared monochromatic Ray Polychromatic Ray PRISM Red Orange Yellow Green Blue Violet Ultraviolet Polychromatic Ray Monochromatic Ray SLIT Electronic Spectroscopy Ultraviolet and visible Where in the spectrum are these transitions? Why should we learn this stuff? After all, nobody solves structures with UV any longer! Many organic molecules have chromophores that absorb UV UV absorbance is about 1000 x easier to detect per mole than NMR Still used in following reactions where the chromophore changes (useful) because timescale is so fast, and sensitivity is very high. Kinetics, esp. in biochemistry, enzymology. Most quantitative Analytical chemistry in organic chemistry is conducted using HPLC with UV detectors One wavelength may not be the best for all compound in a mixture. Affects quantitative interpretation of HPLC peak heights Ultra Violet Spectrometry The absorption of ultraviolet radiation by molecules is dependent upon the electronic structure of the molecule. So the ultraviolet spectrum is called electronic spectrum. The absorption of light energy by organic compounds in the visible and ultraviolet region involves the promotion of electrons in , , and norbitals from the ground state to higher energy states. This is also called energy transition. These higher energy states are molecular orbitals called antibonding. n n * Antibonding n * * * * Antibonding * Energy Electronic Molecular Energy Levels Nonbonding Bonding Bonding The higher energy transitions ( *) occur a shorter wavelength and the low energy transitions (*, n *) occur at longer wavelength. Spectrophotometer An instrument which can measure the absorbance of a sample at any wavelength. Light Lens Sample Slit Detector Monochromator Quantitative Analysis Slits Fluorometer Instrument to measures the intensity of fluorescent light emitted by a sample exposed to UV light under specific conditions. Emit fluorescent light as energy decreases Antibonding ' ' Antibonding n->' n-> ' Nonbonding n -> ' Bonding ->' Energy Bonding Ground state Electron's molecular energy levels UV Light Source Monochromator 90° C Sample Detector Monochromator Spectrophotometry Key Concepts • Lambert’s Law of Absorption • Beer’s Law • Beer-Lambert Law • Absorption Cross-Sections • Photometric quantities • Spectrophotometer • The Cary 50 Spectrophotometer OU NanoLab/NSF NUE/Bumm & Johnson Lambert‟s Law of Absorption Lambert described how intensity changes with distance in an absorbing medium. The intensity I0 if a beam of light decreases exponentially as it passes though a uniform absorbing medium with the linear decay constant α. Restatement: In a uniform absorbing medium, the intensity of a beam of light decreases by the same proportion for equal path lengths traveled. The linear decay constant α is a characteristic of the medium. It has units of reciprocal length. α is the path length over which the intensity is attenuated to 1/e. l α I0 I = I 0e- l I I ( x) = I 0e- x I(x) The distance traveled through the medium is called the path length. d I = - I d x I = I 0e x Johann Heinrich Lambert 1728-1777 - x dI = - I dx I - x =e I0 OU NanoLab/NSF NUE/Bumm & Johnson Photo: http://www-history.mcs.st-andrews.ac.uk/history/PictDisplay/Lambert.html Lambert’s Law of Absorption (base 10) Typically base 10 is used in photometry. I = I 0e - x = I 010 -kx k = ln 10 I - x -kx = e = 10 I0 k is the path length over which the intensity is attenuated to 1/10. I = 10 - k x I0 OU NanoLab/NSF NUE/Bumm & Johnson Lambert’s Law Example If one slab of absorbing material of thickness l reduces the intensity of a beam of light to half. l α I0 I 1 = 10 - k l = I0 2 I Then two slabs of the same absorbing material will then reduce the intensity of a beam of light to one quarter. l l α I0 α 2 I 1 1 = 10 - k 2l = = I0 4 2 I And three slabs will reduce the intensity of a beam of light to one eight. l I0 l α OU NanoLab/NSF NUE/Bumm & Johnson l α α 3 I I 1 1 - k 3l = 10 = = I0 2 8 Beer’s Law Beer found that Lambert’s linear decay constant k for a solution of an absorbing substance is linearly related to its concentration c by a constant, the absorptivity ε, a characteristic of the absorbing substance. Restatement: The linear decay constant k is linear in concentration c with a constant of proportionality ε. (August Beer, 1825-1863) k = c Typical units are: k cm−1; c M (moles/liter); ε M−1cm−1 A colored absorber has an absorptivity that is dependent on wavelength of the light ε(λ). The absorptivity is the fundamental property of a substance. This is the property that contains the observable spectroscopic information that can be linked to quantum mechanics (also see absorption cross section.) Photometric Quantities In photometry we measure the intensity of light and characterize its change by and object or substance. This change is typically expresses as percent transmittance or absorbance. Transmittance (T) Frequently when your primary interest is the light beam Absorbance (A) I T= I0 usually given in percent (AKA optical density, O.D.) Used almost exclusively when your interest concerns the properties of the material I A = - log = - log T I0 OU NanoLab/NSF NUE/Bumm & Johnson by convention, base 10 logs are used Beer – Lambert Law Light I0 I Glass cell filled with concentration of solution (C) As the cell thickness increases, the transmitted intensity of light of I decreases. T- Transmittance T= I I0 I0 - Original light intensity I- Transmitted light intensity I I0 % Transmittance = 100 x Absorbance (A) = Log I 1 T = Log I0 I = 2 - Log%T Log I0 is proportional to C (concentration of solution) and is also proportional to L (length of light path through the solution). Beer-Lambert Law #Beers Law states that absorbance is proportional to concentration over a certain concentration range Lambert‟s and Beer‟s Laws are combined to describe the attenuation of light by a solution. It is easy to see how the two standard photometric quantities can be written in terms of this law. I = I 010 Transmittance I T= I0 T = 10 - c x - c l http://en.wikipedia.org/wiki/Beer%E2%80%93Lambert_law Absorbance I A = - log = - log T I0 A = cl A = absorbance = molar extinction coefficient (M-1 cm-1 or mol-1 L cm-1) c = concentration (M or mol L-1) l = path length (cm) (width of cuvette) Beer-Lambert Law Beer‟s law is valid at low concentrations, but breaks down at higher concentrations For linearity, A < 1 1 Beer-Lambert Law If your unknown has a higher concentration than your highest standard, you have to ASSUME that linearity still holds (NOT GOOD for quantitative analysis) Unknowns should ideally fall within the standard range Quantitative Analysis A<1 If A > 1: Dilute the sample Use a narrower cuvette (cuvettes are usually 1 mm, 1 cm or 10 cm) Plot the data (A v C) to produce a calibration „curve‟ Obtain equation of straight line (y=mx) from line of „best fit‟ Use equation to calculate the concentration of the unknown(s) 25 Quantitative Analysis Absorbance ( no units) Calibration curve showing absorbance as a function of metal concentration 1.2 y = 0.9982x R2 = 0.9996 1 0.8 0.6 0.4 0.2 0 0 0.2 0.4 0.6 0.8 1 1.2 Concentration (mg L-1) 26 Steps in Developing a Spectrometric Analytical Method 2. Obtain a monochromatic wavelength for the maximum absorption wavelength. 3. Calculate the concentration of your sample using Beer Lambert Equation: A = ECL Absorbance 1. Run the sample for spectrum 2.0 0.0 200 250 300 350 400 Wavelength (nm) 450 Spectrometer Reading A C Slope of Standard Curve = x 1.0 x 0.5 x 1 4 2 3 Concentration (mg/ml) 5 There is some A vs. C where graph is linear. NEVER extrapolate beyond point known where becomes non-linear. Spectrometric Analysis Using Standard Curve 1.2 0.8 0.4 3 1 2 Concentration (g/l) glucose 4 Avoid very high or low absorbencies when drawing a standard curve. The best results are obtained with 0.1 < A < 1. Plot the Absorbance vs. Concentration to get a straight line Homeworks 1. Calculate the Molar Extinction Coefficient ε at 351 nm for aquocobalamin in 0.1 M phosphate buffer. pH = 7.0 from the following data which were obtained in 1 cm cell. Solution C x 105 M A 2.23 100 27 B 1.90 100 32 2. Io I The molar extinction coefficient (ε) of compound riboflavin is 3 x 103 Liter/Cm x Mole. If the absorbance reading (A) at 350 nm is 0.9 using a cell of 1 Cm, what is the concentration of compound riboflavin in sample? 3. The concentration of compound Y was 2 x 10-4 moles/liter and the absorption of the solution at 300 nm using 1 cm quartz cell was 0.4. What is the molar extinction coefficient of compound Y? 4. Calculate the molar extinction coefficient ε at 351 nm for aquocobalamin in 0.1 M phosphate buffer. pH =7.0 from the following data which were obtained in 1 cm cell. Solution C x 105 M A 2.0 I0 I 100 30 Cross-Sections and Absorptivity the connection to single particles and molecules The absorption of light by particles (and single molecules) is characterized by an absorption cross section C. In this model the particle is replaced by a perfectly absorbing sphere with a cross sectional area C. This cross section is a property of the particle and is not related to its geometric cross sectional area. The concentration of particles per unit volume is N. = NC k = NC ln 10 typical units are: C cm2; N cm−3 cross section can be directly related to the -3 liter The molar absorptivity. NA is Avagadro‟s number. = N A C ln 10 10 3 units are: C cm2; N cm−3; N mole−1; ε A cm −1 −1 M cm OU NanoLab/NSF NUE/Bumm & Johnson Efficiency The absorption efficiency Q of a particle is the ratio of its absorption cross section C to its geometric cross section Cgeo. Absorption efficiency is dimensionless. C Q= C geo OU NanoLab/NSF NUE/Bumm & Johnson Extension to Scattering and Extinction Attenuation of light by absorption and scattering both obey Lambert‟s Law. Thus we can extend our treatment of absorption to scattering and extinction. (Recall that extinction is the effect of absorption + scattering.) Cext = Cabs C sca Qext = Qabs Qsca The scattering efficiency can be much larger than unity. Extinction paradox: Qext = 2 (Qabs = 1; Qsca = 1) for an perfectly absorbing particle very large compared to the wavelength of light. ext = abs sca A = ext cx = abs sca )cx Note: •All of these quantities are in general wavelength dependent. •Our discussion has not included the mechanism (cause) of absorption and scattering. •There are many different mechanisms that cause of absorption and scattering. OU NanoLab/NSF NUE/Bumm & Johnson Instrumentation Spectrometer: measures I vs λ. Simply measures the spectrum of the light (e.g. emission spectroscopy). Spectrophotometer: measures I/I0 vs λ. Measures how the sample changes the spectrum of the light (e.g. transmission, reflection, scattering, fluorescence). All spectrophotometers contain a spectrometer. -meter: the detector is electronic -graph: light intensity recorded on film photometer: measures I/I0 without λ selection. OU NanoLab/NSF NUE/Bumm & Johnson The Spectrophotometer Measures absorbance as a function of wavelength Components: light source, monochromator, sample cell, detector, optical system. sample cell slit diffraction grating monochromator light source OU NanoLab/NSF NUE/Bumm & Johnson detector Cary 50 UV-Vis Spectrophotometer Computer controlled acquisition of absorption spectra monochromator balance the forces: detector sample light source OU NanoLab/NSF NUE/Bumm & Johnson Can you find the diffraction grating and the slit? www.varianinc.com Making a Measurement with the Cary 50 First, measure the baseline using a blank sample. This is raw I0. The blank sample is the cuvette with deionized water (everything but your nanoparticles). This corrects for any absorption due to the cuvette, water, and variations of the light intensity of the light source, monochromator, etc. Second, measure the zero by inserting the beam block. This corrects the instrument for the detector background. Third, measure your sample. This is the raw I. The Cary 50 automatically calculates the corrected intensities (I and I0) by subtracting the zero from each of the raw intensities. Subsequent measurements do not require re-measuring the blank and zero, simply repeat step 3. I raw I - zero T= = I 0 raw I 0 - zero A = - log T OU NanoLab/NSF NUE/Bumm & Johnson Applications of Spectrophotometry Spectroscopy Chemical Analysis: trace analysis, pH, forensic, in situ monitoring, remote monitoring, geology, astronomy, .... Particle size Thin film characterization Color matching Optics OU NanoLab/NSF NUE/Bumm & Johnson Sample Cells (Cuvettes) UV Spectrophotometer Quartz (crystalline silica): works in the UV region below 350 nm, can be employed across the whole UV-Visible wavelength range, 190-1,000 nm Visible Spectrophotometer Optical glass: employed in the region above 300nm Moulded plastic cells: used in the visible region above 350 nm, low cost Common size of cells is the 1cm rectangular cell, which has an optical path length of 1cm and path width of 1 cm. Typically, these cells hold 2-3 ml of sample. When sample volume is an important issue Consider micro cells Light Sources UV Spectrophotometer 1. Hydrogen Gas Lamp 2. Mercury Lamp Visible Spectrophotometer 1. Tungsten Lamp Uses for UV, continued Knowing UV can help you know when to be skeptical of quant results. Need to calibrate response factors Assessing purity of a major peak in HPLC is improved by “diode array” data, taking UV spectra at time points across a peak. Any differences could suggest a unresolved component. “Peak Homogeneity” is key for purity analysis. Sensitivity makes HPLC sensitive e.g. validation of cleaning procedure for a production vessel But you would need to know what compounds could and could not be detected by UV detector! (Structure!!!) One of the best ways for identifying the presence of acidic or basic groups, due to big shifts in l for a chromophore containing a phenol, carboxylic acid, etc. “hypsochromic” shift “bathochromic” shift l The UV Absorption process • * and * transitions: high-energy, accessible in vacuum UV (lmax <150 nm). Not usually observed in molecular UV-Vis. •n * and * transitions: non-bonding electrons (lone pairs), wavelength (lmax) in the 150-250 nm region. •n * and * transitions: most common transitions observed in organic molecular UV-Vis, observed in compounds with lone pairs and multiple bonds with lmax = 200-600 nm. •Any of these require that incoming photons match in energy the gap corrresponding to a transition from ground to excited state. •Energies correspond to a 1-photon of 300 nm light are ca. 95 kcal/mol What are the nature of these absorptions? π* π* π* π* π* π* n π π n π π -*; lmax=218 =11,000 π* π* π* n π π Example for a simple enone n-*; lmax=320 =100 Example: * transitions responsible for ethylene UV absorption at ~170 nm calculated with ZINDO semi-empirical excited-states methods (Gaussian 03W): h 170nm photon HOMO u bonding molecular orbital LUMO g antibonding molecular orbital How Do UV spectrometers work? Rotates, to achieve scan Matched quartz cuvettes Sample in solution at ca. 10-5 M. System protects PM tube from stray light D2 lamp-UV Tungsten lamp-Vis Double Beam makes it a difference technique Two photomultiplier inputs, differential voltage drives amplifier. Diode Array Detectors Diode array alternative puts grating, array of photosens. Semiconductors after the light goes through the sample. Advantage, speed, sensitivity, The Multiplex advantage Model from Agilent literature. Imagine replacing “cell” with a microflow cell for HPLC! Disadvantage, resolution is 1 nm, vs 0.1 nm for normal UV Experimental details What compounds show UV spectra? Generally think of any unsaturated compounds as good candidates. Conjugated double bonds are strong absorbers Just heteroatoms are not enough but C=O are reliable Most compounds have “end absorbance” at lower frequency. Unfortunately solvent cutoffs preclude observation. You will find molar absorptivities in L•cm/mol, tabulated. Transition metal complexes, inorganics Solvent must be UV grade (great sensitivity to impurities with double bonds) The NIST databases have UV spectra for many compounds An Electronic Spectrum Make solution of concentration low enough that A≤ 1 (Ensures Linear Beer‟s law behavior) 1.0 lmaxwith certain extinction UV Visible Even though a dual beam goes through a solvent blank, choose solvents that are UV transparent. Absorbance Can extract the value if conc. (M) and b (cm) are known 0.0 200 UV bands are much broader than the photonic transition event. This is because vibration levels are superimposed on UV. 400 Wavelength, l, generally in nanometers (nm) 800 Solvents for UV (showing high energy cutoffs) Water 205 THF CH2Cl2 CH3CN 210 220 235 C6H12 210 CHCl3 245 Ether 210 CCl4 265 EtOH 210 benzene 280 Acetone 300 Hexane 210 MeOH Dioxane 220 210 Various buffers for HPLC, check before using. Chemical Structure & UV Absorption Chromophoric Group ---- The groupings of the molecules which contain the electronic system which is giving rise to absorption in the ultraviolet region. Chromophoric Structure Group Structure nm Carbonyl >C=O 280 Azo -N = N- 262 Nitro -N=O 270 Thioketone -C =S 330 Nitrite -NO2 230 Conjugated Diene -C=C-C=C- 233 Conjugated Triene -C=C-C=C-C=C- 268 Conjugated Tetraene -C=C-C=C-C=C-C=C- 315 Benzene 261 Organic compounds (many of them) have UV spectra One thing is clear UVs can be very non-specific It is hard to interpret except at a cursory level, and hard to say that the spectrum is consistent with the structure Each band can be a superposition of many transitions Generally we don‟t assign the particular transitions. From Skoog and West et al. Ch 14 An Example--Pulegone Frequently plotted as log of molar extinction So at 240 nm, pulegone has a molar extinction of 7.24 x 103 Antilog of 3.86 O Can we calculate UVs? Molar Abs orptivity (l/mol-cm) Electronic Spectra 50243 40194 30146 20097 10049 nac indolA 0 220 Wavelength 230 240 250 260 270 280 290 300 Electronic Spectra Molar Abs orptivity (l/mol-cm) 51972 41578 Semi-empirical (MOPAC) at AM1, then ZINDO for config. interaction level 14 31183 20789 Bandwidth set to 3200 cm-1 10394 Nac etylindo 0 220 Wavelength 230 240 250 260 270 280 290 300 The orbitals involved Electronic Spectra Molar Absorptivity (l/mol-cm) 55487 44390 Showing atoms whose MO‟s contribute most to the bands 33292 22195 11097 Nacetylindol 0 200 Wavelength (nm) 210 220 230 240 250 260 270 280 290 300 The Quantitative Picture • Transmittance: T = P/P0 • Absorbance: A = -log10 T = log10 P0/P P0 (power in) P (power out) B(path through sample) • The Beer-Lambert Law (a.k.a. Beer‟s Law): A = bc Where the absorbance A has no units, since A = log10 P0 / P is the molar absorbtivity with units of L mol-1 cm-1 b is the path length of the sample in cm c is the concentration of the compound in solution, expressed in mol L-1 (or M, molarity) Beer-Lambert Law Linear absorbance with increased concentration-directly proportional Makes UV useful for quantitative analysis and in HPLC detectors Above a certain concentration the linearity curves down, loses direct proportionality--Due to molecular associations at higher concentrations. Must demonstrate linearity in validating response in an analytical procedure. Polyenes, and Unsaturated Carbonyl groups; an Empirical triumph R.B. Woodward, L.F. Fieser and others Predict lmax for π* in extended conjugation systems to within ca. 2-3 nm. Attached group increment, nm Extend conjugation +30 Homoannular, base 253 nm Addn exocyclic DB +5 Acyclic, base 217 nm O-Acyl 0 S-alkyl +30 O-alkyl +6 NR2 +60 Cl, Br +5 heteroannular, base 214 nm Alkyl +5 Similar for Enones b b O O O 227 202 215 Base Values, add these increments… X=H 207 x b 239 g d, Extnd C=C Add exocyclic C=C +30 +5 Homoannular diene +39 alkyl +10 +12 With solvent correction of….. OH +35 +30 Water +8 OAcyl +6 +6 +6 +6 EtOH 0 O-alkyl +35 +30 +17 +31 CHCl3 -1 Dioxane -5 Et2O -7 +15/+25 +12/+30 X=R 215 X=OH 193 X=OR 193 Hydrcrbn -11 NR2 S-alkyl Cl/Br +18 +18 +50 Some Worked Examples O Base value 2 x alkyl subst. exo DB total Obs. 217 10 5 232 237 Base value 3 x alkyl subst. exo DB total Obs. 214 30 5 234 235 Base value 2 ß alkyl subst. total Obs. 215 24 239 237 Distinguish Isomers! Base value 4 x alkyl subst. exo DB total Obs. 214 20 5 239 238 Base value 4 x alkyl subst. total Obs. 253 20 273 273 HO2C HO2C Generally, extending conjugation leads to red shift “particle in a box” QM theory; bigger box Substituents attached to a chromophore that cause a red shift are called “auxochromes” Strain has an effect… lmax 253 239 256 248 Interpretation of UV-Visible Spectra Transition metal complexes; d, f electrons. Lanthanide complexes – sharp lines caused by “screening” of the f electrons by other orbitals One advantage of this is the use of holmium oxide filters (sharp lines) for wavelength calibration of UV spectrometers. See Shriver et al. Inorganic Chemistry, 2nd Ed. Ch. 14 Benzenoid aromatics UV of Benzene in heptane From Crewes, Rodriguez, Jaspars, Organic Structure Analysis Group K band () B band() R band Alkyl 208(7800) 260(220) -- -OH 211(6200) 270(1450) -O- 236(9400) 287(2600) -OCH3 217(6400) 269(1500) NH2 230(8600) 280(1400) -F 204(6200) 254(900) -Cl 210(7500) 257(170) -Br 210(7500) 257(170) -I 207(7000) 258/285(610/180) -NH3+ 203(7500) 254(160) -C=CH2 248(15000) 282(740) -CCH 248(17000) 278(6500 -C6H6 250(14000) -C(=O)H 242(14000) 280(1400) 328(55) -C(=O)R 238(13000) 276(800) 320(40) -CO2H 226(9800) 272(850) -CO2- 224(8700) 268(800) -CN 224(13000) 271(1000) -NO2 252(10000) 280(1000) 330(140) Quantitative analysis Great for nonaqueous titrations Example here gives detn of endpoint for bromcresol green Binding studies Form I to form II Isosbestic points Single clear point, can exclude intermediate state, exclude light scattering and Beer‟s law applies Binding of a lanthanide complex to an oligonucleotide More Complex Electronic Processes • Fluorescence: absorption of radiation to an excited state, followed by emission of radiation to a lower state of the same multiplicity • Phosphorescence: absorption of radiation to an excited state, followed by emission of radiation to a lower state of different multiplicity • Singlet state: spins are paired, no net angular momentum (and no net magnetic field) • Triplet state: spins are unpaired, net angular momentum (and net magnetic field) UV Spectrometer Application Protein Amino Acids (aromatic) Pantothenic Acid Glucose Determination Enzyme Activity (Hexokinase) Flurometric Application Thiamin (365 nm, 435 nm) Riboflavin Vitamin A Vitamin C Visible Spectrometer Application Niacin Pyridoxine Vitamin B12 Metal Determination (Fe) Fat-quality Determination (TBA) Enzyme Activity (glucose oxidase)