CEU Malolos BSMT 2023 CC1L - M5 • 1 CLINICAL CHEMISTRY LABORATORY INSTRUMENTATION: SPECTROPHOTOMETRY OUTLINE • Introduction o Analytical method o Colorimetry • Spectrophotometry o Principles o Types of Spectrophotometry o Introduction o Spectrophotometry o Components of a Spectrophotometer • INTRODUCTION ANALYTICAL METHOD • Light energy, wavelength and radiant energy spectrum • Energy - is transmitted via electromagnetic waves that are characterized by their frequency and wavelength. • Wavelength- is the distance between 2 successive peaks and it is expressed in term of nanometer (nm) o 400-700 nm- visible spectrum o <400 ultraviolet region o >700 infrared region • Frequency - is the number of vibrations of wave motion per second. COLORIMETRY • Photoelectric Colorimetry o The primary analytical utility of spectrophotometry is the isolation of discreet portions of the spectrum for purpose of measurement • Spectrophotometric measurement- measurement of light intensity in narrower wavelength • Photometric measurement- measurement of light intensity SPECTROPHOTOMETRY PRINCIPLE • Spectrophotometry takes advantage of the property of colored solutions to absorb light of specific wavelength. • Measure light intensity as a function of wavelength • It diffracts the light beam into a spectrum of wavelengths, detecting the intensities with a charge-coupled device, and displaying the results as a graph on the detector and then on the display device TYPES OF SPECTROPHOTOMETRY • Single Beam • Double Beam o Double-beam in space o Double-beam in time INTRODUCTION • Light is a form of electromagnetic energy o Transmitted via electromagnetic waves o Waves is measured in nanometer between the peaks and valleys (wavelength). • Beer’s Law o The concentration of a substance is: o Directly proportional to the amount of light absorbed o Inversely proportional to the amount of transmitted light o Formula: A = 2 - log %T SPECTROPHOTOMETRY • Measures the light transmitted by a solution to determine the concentration of the substance in the solution COMPONENTS OF A SPECTROPHOTOMETER 1. Light Source 2. Entrance Slit 3. Monochromator 4. Exit Slit 5. Sample cell 6. Photodetector
CEU Malolos BSMT 2023 CC1L - M5 • 2 LIGHT SOURCE • Provide incident light for the system • Incandescent Tungsten or Tungsten iodide lamp o For visible and near infrared region spectrum (320 to 700nm) • Deuterium-discharge lamp and Mercury arc lamp o For UV spectrum (below 350nm) • Silicone carbide o For infrared spectrum ENTRANCE SLIT • Minimizes unwanted or stray light • Prevents the entrance of scattered light into monochromator system. • Exclude unwanted or “stray light MONOCHROMATOR • Isolates specific wavelength from the light source • Interference Filter - Based on constructive interference of waves • Prism - Separates white (visible) light into a continuous spectrum. • Diffraction gratings o Bends light and form wave fronts. Light that are in phase reinforce one another, if not it cancels out o Used for UV, visible or infrared measurements EXIT SLIT • Controls the width of the light beam SAMPLE CELL • Also known as cuvette or analytical cell • Holds the solution of which absorption is to be measured • Glass cuvette - for visible range • Quartz or fused silica - for UV range PHOTODETECTOR • Converts transmitted radiant energy into an equivalent amount of electrical energy. • Photocell (Barrier layer cell, selenide cell) o Generates electromotive force (no external voltage) o Output is not amplified • Phototube o Similar to photocell but requires external voltage • Photomultiplier tube o Detects and amplifies radiant energy (200x sensitive) • Phototransistors and Photoiodide o Uses a photosensitive positive-negative junction diode to produce a photocurrent. READ-OUT DEVICE • A moving needle on a dial or a digital display which indicates the amount of light passing through a sample.
CEU Malolos BSMT 2023 CC1L - M5 • 3 SPECTROPHOTOMETRY (DISCUSSION) SPECTROPHOTOMETRY • Spectroscopic method that studies the realtionship between light and a matter o Diffferent substance emit and absorbs different wavelengths of light • Most commonly used analytical method in clinical chemistry • Measures the absorbance of a substance by measuring the amount of light that is not absorbed (% Transmittance) o Absorbed light - light that do not pass through o Transmitted light/transmittance (I t )- light that passed through the substance o Incident light (I 0 )- light that strikes the sample o %T = It / I0 x 100 PARTS OF SPECTROPHOTOMETRY • Light source o Provides polychromatic light (light with all visible spectrum of wavelengths o Most improtant part ▪ Without light, absorbed light & transmitted light cannot be measured o Monochromatic light should be narrowed • Entance slit o Aka Collimator o Minimizes stray light (unwanted light, light not from light source) o Collimates the polychromatic light ▪ Arrange the wavelength in parallel before it passes through monochromator • Monochromator o Isolates specific wavelengths from polychromatic light o Allows light to pass through the exit slit • Exit slit o Aka wavelength selector o Will only allow the specific/selected wavelength to reach the sample o Adjustable for specific wavelengths • Incident light (I 0 ) - light that strickes the sample • Cuvette o Analytical cell o Contains the sample o Only utilizes one type (standardizes) of dimension of cuvette ▪ Absorbance is also directly proportional to the light path ▪ Short path = low absobrance ▪ Long path = high absorbance • Transmitted light (It) - light passed through the sample in the cuvet o Converted into phtoelectric energy by the action of photodetector • Photodetector o Converts light into energy • Read-out device o Visualizes converted energy BEER LAW • The absorbance of a substance is directly proportional to its concentration • A = εlc o ε - molar concentration absorbdibity (varies per subs.) o l - light path o c - concentration of sample • Alternative method is getting absorbance of different concentrations of specific solutions