L5 - LABS1000 Spectrophotometry PDF

Objectives Introduction to Absorbance Spectrophotometry Standards Wavelength Transmittance & Absorbance Beer’s Law Spectrophotometer & Cuvettes Absorbance Curve  Clinical Methodologies In the clinical laboratory – we use quantitative techniques of measurement. The methodolog...

Objectives Introduction to Absorbance Spectrophotometry Standards Wavelength Transmittance & Absorbance Beer’s Law Spectrophotometer & Cuvettes Absorbance Curve  Clinical Methodologies In the clinical laboratory – we use quantitative techniques of measurement. The methodologies used in the technological automated analyzers and manual assays are based on traditional technologies: photometry, spectrophotometry, ion-selective electrodes, electrophoresis, nephelometry, immunoassays, mass spectrometry, fluorescence, and molecular diagnostic techniques. Analytical techniques and instrumentation provide the foundation for all measurements made in a modern clinical laboratory.  Absorbance Spectrophotometry The process of measuring the concentration of an unknown solution using the properties of light energy (electromagnetic spectrum). A concentration of an unknown solution is determined by measuring its absorbance of light (at a particular wavelength) and comparing it to a solution of known concentration of the same chemical makeup (standard) at the same wavelength. Concentration of juice The intensity of color, is directly proportional to the concentration of substance, or solute Calibration Standards Chemical standards are materials containing a known concentration of an analyte. They provide a reference to determine unknown concentrations or to calibrate analytical instruments.  Absorbance Spectrophotometry Known Standard unknown unknown [10%] solution A solution B < 10% > 10% Absorbance Spectrophotometry Every chemical compound absorbs, transmits or reflects light of a certain wavelength or range of wavelengths. Light energy levels that are visible to the human eye are referred to as the visible light spectrum (400 – 750 nm). 400 nm 750 nm Wavelength ( λ ) To understand how Absorbance Spectrophotometry works Understand the fundamentals about color. To understand color, we must know the nature of light. Light is a type of radiant energy that travels in waves.  White Light Every color is seen has their own particular wavelength A combination or mixture of wavelengths White light or polychromatic light White light passes through a Prism, filter, or diffraction grating, it can be broken into a visible spectrum 750nm Infrared Wavelengths, not visible to human eye Transmitted or Absorbed When light strikes an object, some wavelengths will be transmitted by the object and some will be absorbed The wavelengths of light that are transmitted or reflected, determine the colour we see Colored Solutions & Light When white light is shone at a colored solution, certain energies of light are transmitted (pass through the solution), while other energies of light are absorbed (taken in by the solution). Io It light detector Io = intensity of incident light, It = light transmitted Transmittance is the fraction of light that passes through the sample. Transmittance equation: T = It / Io Absorbance is the fraction of light absorbed by the sample. Absorbance equation: -log (T)  -log (It / Io) Transmittance T = It / Io Io = intensity of incident light, It = light transmitted E.g. if you have an intensity of incident light of 0.49 W/m2 and the intensity of the light entering the detector is 0.30 W/m2 what is the transmittance? T = 0.30 W/m2 / 0.49 W/m2 T = 0.6122 Transmittance & Absorbance Transmittance (T) = 0.6122 Absorbance (A) = -log(T) or -log (It / Io) A = -log(0.6122) A = 0.213 Absorbance of the solution is 0.213 Absorbance ↑ Transmittance ↓ Absorbance ↓ Transmittance ↑  Beer-Lambert Law (Beer’s Law) Is the basis for the use of spectrophotometry in quantitative measurement. States that there is a linear relationship between absorbance and the concentration of the solution:  the concentration of a substance in a solution is directly proportional to the amount of light absorbed or inversely proportional to the logarithm of the transmitted light. Known Standard [10%] unknown solution A 10%  Beer’s Law: A = ε b C Represented by the equation: A = ε b C A = absorbance ε = molar absorptivity coefficient (*) b = path length (distance light travels through a solution *) C = concentration of the solute A=εbC If a compound can be converted into a soluble color its concentration can be determined through absorbance spectrophotometry. Since pathlength and molar absorptivity are constant for a given wavelength, absorbance is directly proportional to concentration A~C Absorbance Spectrophotometry This technique is used in the clinical laboratory to determine quantity of various solutes in solution. Ex. Protein, iron, electrolytes, hemoglobin, bilirubin, RBCs, drug levels, antibodies, liver enzymes, cardiac markers, hormone levels, complement levels,. Etc Found in many analytical instruments. Ranges from visible light to UV. Limitations The solution needs to be colored Spectrophotometer A device used to measure the light transmitted by a solution to determine the concentration of the light-absorbing substance in the solution. QUANTITATIVE measurement which is dependent on 2 main factors: 1. the color itself 2. the intensity of the color Spectrophotometer: Basic Components Light Source 1. Lamp – light source different lamps depending on the spectral range required for analysis 1. Tungston-Halogen for visible light 2. Xenon & Mercury Lamp – Covers UV & visible light 3. Deuterium lamp – UV Light 2. Collimator– to collect the light A lens to concentrate the light May or may not have this, It may just be a slit Choosing a wavelength 3. Monochromator – Produced by a prism or diffraction grating: to split white light into separate colors of the visible spectrum (or wavelengths) When we change the wavelength on the instrument, it is the part that moves 4. Wavelength selector (Slit) – To select a specific color- wavelength Remains stationary Sample holder 5. Sample Cell (Cuvette or Test Tube) – is used to hold the solution Cuvettes are a type of vessel necessary for the precise spectral analysis of liquid samples. Generally used in spectrophotometry, designed to hold liquid substances during chemical analysis. Can have two or four optical windows Scratches or marks on these windows = discard, not usable Can be made of plastic, glass, or quartz Plastic and glass for visible light Quartz is for UV, Typically 1cm Displaying results 6. Detector (photocell)– convert light into an electrical signal 7. Amplifier – to amplify the electrical signal Photomultiplier & photodiodes amplifies low intensity light 8. Meter – shows the data from the detector Results can be printed out and displayed on the screen How the Spectrophotometer works Orientation of the cuvette  Light source Detector  Light source Detector Green Dye Determination Absorption Curve: - determines wavelength of max absorbance of a known standard - Greatest sensitivity for method - Minimize interferences 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 400 410 420 430 440 450 460 470 480 490 500 510 520 530 540 550 560 570 580 590 600 610 620 630 640 650  Absorption Curve: Green Dye Solution (green dye + dH O) 2 Materials: spectrophotometer, green dye standard (10%), blank (dH2O) Procedure: Set the spectrophotometer to 400nm (violet). Place the blank (dH2O) in the sample holder, press zero absorbance  display reads 0.000 Place an aliquot of green dye standard (10%) in the sample holder  Absorbance reading (ex. 0.581) record reading. Set the spectrophotometer to 410nm. Place the blank (dH2O) in the sample holder, press zero absorbance  display reads 0.000 Place an aliquot of green dye standard (10%) in the sample holder  Absorbance reading (0.637) record reading. Set the spectrophotometer to 420nm…….  650nm. Monochromator set to 400 nm (violet) Blank of distilled water Monochromator set to 400 nm (violet) 10% green dye standard Absorbance Readings: Green Wavelength (nm) Dye Absorbance Wavelength (nm) Absorbance 400 0.581 530 0.034 410 0.637 540 0.049 420 0.625 550 0.071 430 0.596 560 0.111 440 0.562 570 0.167 450 0.515 580 0.227 460 0.418 590 0.289 470 0.312 600 0.347 480 0.202 610 0.485 490 0.098 620 0.695 500 0.043 630 0.802 Max Abs 510 0.026 640 0.650 520 0.025 650 0.371 Max Transmittance  Absorption Curve – Green Dye Max absorbance 630nm 0.9 0.8 0.7 0.6 0.5 Abs 0.4 0.3 Max transmittance 520 nm 0.2 0.1 0 400 410 420 430 440 450 460 470 480 490 500 510 520 530 540 550 560 570 580 590 600 610 620 630 640 650 Wavelength (nm) Max Transmittance Max Transmittance corresponds to the point where the absorbance is the lowest, indicating the sample is allowing the most light to pass through at that specific wavelength Can determine the color of the solution. Colors of the Visible Spectrum & Corresponding Wavelengths Max Absorbance = 630nm What color of the visible spectrum is 630nm? Red Max Transmittance = 520nm What color of the visible spectrum is 520nm? Green Yes, you need to memorize this chart Relationship Between Colors Max Absorbance = 630nm Red Represents the complementary color wavelength of the colored solution being tested. Max Transmittance = 520nm Green Represents the color wavelength of the colored solution being tested. You are able to determine the color of the solution if I find out the max absorbance Absorption Curve Question If we were to plot this as an Wavelength Absorbance Wavelength (nm) Absorbance (nm) absorption curve… 400 0.102 530 0.670 What is the Max Transmittance In this 410 0.029 540 0.750 absorption curve? 420 0.019 550 0.850 430 0.030 560 0.900 What is the Max Absorbance? 440 0.104 570 1.090 450 0.150 580 0.950 What is the color of the solution being tested? 460 0.240 590 0.840 470 0.310 600 0.760 480 0.350 610 0.680 What is the color of the wavelength that provides the most sensitivity for 490 0.391 620 0.550 the method? 500 0.430 630 0.490 510 0.501 640 0.380 520 0.590 650 0.300 Next week: Bring this lecture to help understand how to make an absorption curve. In class assignment worth 5% Make an absorption curve & unknown determination using over & down method