Clinical Chemistry Lab - 03 Spectrophotometry

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 • 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 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 • SPECTROPHOTOMETRY Measures the light transmitted by a solution to determine the concentration of the substance in the solution 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 COMPONENTS OF A SPECTROPHOTOMETER TYPES OF SPECTROPHOTOMETRY • • Single Beam Double Beam o Double-beam in space o Double-beam in time CEU Malolos BSMT 2023 1. 2. 3. 4. 5. 6. Light Source Entrance Slit Monochromator Exit Slit Sample cell Photodetector CC1L - M5 • 1 LIGHT SOURCE PHOTODETECTOR • • • • • 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 • • • • 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. 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 CEU Malolos BSMT 2023 READ-OUT DEVICE • A moving needle on a dial or a digital display which indicates the amount of light passing through a sample. CC1L - M5 • 2 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 o o o 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 Absorbed light - light that do not pass through Transmitted light/transmittance (I t)- light that passed through the substance Incident light (I0)- light that strikes the sample %T = It / I0 x 100 PARTS OF SPECTROPHOTOMETRY BEER LAW • • • • • • • 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 (I0) - light that strickes the sample CEU Malolos BSMT 2023 • 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 CC1L - M5 • 3