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Week 1 To Week 3 Laboratory

This document outlines various laboratory safety regulations and procedures. It identifies potential hazards in the laboratory such as electric shock, toxic vapors, and biological hazards. It emphasizes the importance of safety equipment, biological safety practices like proper protective equipment and spill cleanup. Regulations discussed include OSHA bloodborne pathogens standard, hazard communication program, and electrical safety precautions. The primary goal is to raise awareness of safety risks and ensure compliance with required safety protocols.

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Jerome Lising
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© © All Rights Reserved
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0% found this document useful (0 votes)
61 views9 pages

Week 1 To Week 3 Laboratory

This document outlines various laboratory safety regulations and procedures. It identifies potential hazards in the laboratory such as electric shock, toxic vapors, and biological hazards. It emphasizes the importance of safety equipment, biological safety practices like proper protective equipment and spill cleanup. Regulations discussed include OSHA bloodborne pathogens standard, hazard communication program, and electrical safety precautions. The primary goal is to raise awareness of safety risks and ensure compliance with required safety protocols.

Uploaded by

Jerome Lising
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
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LABORATORY SAFETY AND REGULATIONS

Rmt2023
PROFESSOR: BILLY JOE AFRICA

➢ Vaccination against HBV (medical technologists,


LABORATORY SAFETY AND REGULATIONS phlebotomists, pathologists)
➢ Appropriate signs to identify hazard
POTENTIAL HAZARDS
➢ electric shock, toxic vapors, compressed gases, flammable SAFETY EQUIPMENTS
liquids, radioactive material, corrosive substances, ➢ Developed specifically for use in the laboratory
➢ mechanical trauma ➢ Safety showers, eyewash stations, fire extinguishers
➢ poisons ->periodically tested and inspected
➢ Inherent risks of handling biologic materials ➢ Mechanical pipetting device must be used to manipulate
liquids

PRIMARY CAUSE OF ACCIDENTS


➢ Unsafe acts (not always recognized by personnel) BIOLOGICAL SAFETY
➢ Unsafe environmental condition ➢ All samples and other body fluids should be transported,
handled, and processed using strict precautions
➢ First rule of self-protection -> alertness at all times ➢ Gloves, gowns and face protection must be used if splash or
➢ Stay informed splattering is likely to occur
➢ Use common sense ➢ Specimens should be “capped” during centrifugation
➢ LISTEN to any instructions ➢ Any blood, body fluid, or other potentially infectious material
spill must be cleaned up

PSYCHOLOGY OF SAFETY IN CLEANING POTENTIALLY INFECTIOUS MATERIAL SPILL,


➢ Laboratory safety necessitates the effective control of all YOU MUST…
hazards that exists in the clinical laboratory at any time.
➢ Wear appropriate protective equipment
➢ Safety begins with the recognition of hazards and is achieved ➢ Use mechanical devices to pick-up broken glass or other
through: sharp objects
✓ Application of common sense ➢ Absorb spills with paper towels, gauze pad, or tissue
✓ Safety-focused attitude ➢ Clean the spill site using a common aqueous detergent
✓ Good personal behavior ➢ Disinfect the spill site using approved disinfectant or 10%
✓ Good housekeeping in all laboratory work and bleach using appropriate contact time
storage areas ➢ Dispose all materials in appropriate biohazard containers
✓ Continual practice of good laboratory technique
BIOLOGICAL SAFETY
➢ Inexperience may cause some accidents; others are results of
ignoring known risks, haste carelessness, fatigue or mental ➢ OSHA Blood-Borne Pathogens standard requires written
preoccupation “Exposure Control Plan”
➢ Categories of Exposure:
➢ Preventive Measures: ✓ Category I – daily exposure to blood and body fluids
✓ Category II – regular exposure to blood and body fluids.
✓ Annual safety reviews ✓ Category III – no exposure to blood and body fluids
✓ Safety drills
✓ General consciousness ✓ Employers must offer HBV to all personnel (Category I
✓ Appropriate orientation to safety rules and II)
✓ Safe work environment
➢ Biological Safety Cabinets should be installed to facilitate
SAFETY AWARENESS FOR CLINICAL LABORATORY manipulations of infectious materials
PERSONNEL ➢ Safety against exposure to toxic chemicals
➢ Precautions: ✓ Hazard Communication Standard
✓ Appropriate barriers (gloves, gowns or laboratory ▪ Hazard communication program
coats) ▪ Chemical Hygiene Plan
➢ Universal Practices: ▪ Inventory of hazardous substances
✓ Wearing of gloves -> reusing is not allowed ✓ Toxic chemicals (Communication):
✓ Handwashing • Labelling of containers
✓ Laboratory coats -> on site • Information and training
✓ Prohibited: eating, drinking, smoking, applying • Program of Hazard communication
cosmetics, touching contact lense ✓ MSDS -> properties and effects of each chemical
➢ Inactivation: ✓ NFPA hazard labelling system
✓ Heat sterilization (250oC for 15 minutes) ✓ Fume hoods – reagent preparation
✓ Ethylene Oxide (450-500 mg/L at 55-60oC ) • Required to expel
✓ 2% Glutaraldehyde noxious and
✓ 10% hydrogen peroxide hazardous fumes from
✓ 5.25 hypochlorite (bleach) chemical reagents
✓ 10% (v/v with tap water) of common household
bleach) -> HBV (10 minutes), HIV (2 minutes)

OUR LADY OF FATIMA UNIVERSITY I PAMPANGA CAMPUS I COLLEGE OF MEDICAL LABORATORY SCIENCE I CLIN CHEMISTR 1
STORAGE AND HANDLING OF CHEMICALS

FLAMMABLE / COMBUSTIBLE CHEMICALS

➢ Classified according to flash point the temperature at which


sufficient vapor is given off to form an ignitable mixture with
air

CORROSIVE CHEMICALS

➢ injurious to the skin or eyes by direct contact or to the tissue


of the respiratory and gastrointestinal tracts if inhaled or
ingested

REACTIVE CHEMICALS

➢ spontaneously explode or ignite or that evolve heat or


flammable or explosive gases

CARCINOGENIC CHEMICALS

ELECTRICAL SAFETY

➢ Lock-out or tag malfunctioning electrical


SAFETY AWARENESS FOR CLINICAL LABORATORY or mechanical equipment until serviced
PERSONNEL ➢ Know how to knock a shocked person
loose using a non-conductive material
➢ Product name and identification ELECTRICAL PRECAUTIONARY PROCEDURES
➢ Hazardous ingredients
➢ Permissible exposure limit (PEL) ➢ Use only explosion-proof equipment in hazardous
➢ Physical and chemical data atmospheres.
➢ Health hazard data and carcinogenic potential ➢ Be particularly careful when operating high-voltage
➢ Primary routes of entry equipment, such as electrophoresis apparatus.
➢ Fire and explosion hazards ➢ Use only properly grounded equipment (three-prong plug).
➢ Reactivity data ➢ Check for frayed electrical cords.
➢ Spill and disposal procedures ➢ Promptly report any malfunctions or equipment
➢ PPE recommendations ➢ Do not work on “live” electrical equipment
➢ Handling ➢ Never operate electrical equipment with wet hands.
➢ Emergency and first aid procedures ➢ Know the exact location of the electrical control panel for the
➢ Storage and transportation precautions electricity to your work area.
➢ Chemical manufacturer’s name, address, and telephone ➢ Use only approved extension cords and do not overload
number circuits. (Some local regulations prohibit the use of any
➢ Special information section extension cord.)
➢ Have ground checks and other periodic preventive
maintenance performed on equipment

OUR LADY OF FATIMA UNIVERSITY I PAMPANGA CAMPUS I COLLEGE OF MEDICAL LABORATORY SCIENCE I CLIN CHEMISTR 2
RADIATION SAFETY CLASS C FIRES
➢ Electrical equipment
➢ Radiation safety policy should include environmental and ➢ Motors
personnel protection. ➢ Switches
➢ All areas where radioactive materials are used or stored must ➢ TYPES OF FIRE EXTIGUISHER;
be posted with caution signs, and traffic in these areas should ✓ Carbon Dioxide
be restricted to essential personnel only. ✓ Halon
➢ Records must be maintained for the length of employment ✓ Dry Chemical
plus 30 years
➢ Radiation monitoring utilizes film badge or survey meter CLASS D FIRES
->maximum permissible dose is 5000 mrem/year whole body ➢ Rammable metals
➢ The wipe test (leak test) ➢ Magnesium
involves wiping laboratory ➢ TYPE OF AGENT
surfaces with moistened ✓ Metal X
absorbent material and the ➢ Cover burining material with extinguishing agent (scoop,
radiation contained in each wipe sprinkle)
is counted
CRYOGENIC MATERIAL HAZARDS
COMPRESSED GASES HAZARDS ➢ Liquid nitrogen -> most widely used cryogenic fluids (liquefied
gases) in the laboratory
➢ Danger of fire ➢ It may cause fire or explosion, asphyxiation, pressure buildup,
➢ Explosion embrittlement of materials, and tissue damage similar to that
➢ Asphyxiation of thermal burn
➢ Mechanical injuries
MECHANICAL HAZARDS
GENERAL REQUIREMENTS IN HANDLING COMPRESSED
GASES ➢ Centrifuges ->must be balanced to distribute the load equally.
✓ Never open the lid until the rotor has come to a
➢ Know the gas that you will use. complete stop
➢ Store tanks in a vertical position. ✓ Safety locks on equipment should never be
➢ Keep cylinders secured at all times. rendered inoperable
➢ Never store flammable liquids and compressed gases in the ➢ Glass beads – help eliminate bumping/boilover when liquids
same area. are heated
➢ Use the proper regulator for the type of gas in use. ➢ Infectious sharps - disposed in OSHA-approved containers
➢ Do not attempt to control or shut off gas flow with the pressure
relief regulator.
➢ Keep removable protection caps in place until the cylinder is DISPOSAL OF HAZARDOUS MATERIALS
in use
➢ Make certain that acetylene tanks are properly piped (the gas 4 BASIC WASTE DISPOSAL TECHNIQUE
is incompatible with copper tubing). Do not force a “frozen” or
stuck cylinder valve. Use a hand truck to transport large ➢ Flushing down the drain
tanks. Always check tanks on receipt and then periodically for ➢ Incineration
any problems such as leaks. Make certain that the cylinder is ➢ Landfill burial
properly labeled to identify the contents. Empty tanks should ➢ Recycling
be marked “empty”
CHEMICAL WASTE

FIRE SAFETY ➢ Flush water-soluble substances down the drain with large
➢ PULL PIN quantities of water
➢ AIM NOZZLE ➢ Strong acids and bases should be neutralized before disposal
➢ SQUEEZE TRIGGER ➢ Foul smelling chemicals should never be disposed down the
➢ SWEEP NOZZLE drain
CLASS A FIRES ➢ Flammable solvents -> collected in approved containers
➢ Flammable material -> specially designed incinerators
➢ Ordinary ➢ Solid chemicals -> landfill
➢ Combustibles; Wood, paper, cloth
➢ TYPES OF FIRE EXTIGUISHER; RADIOACTIVE WASTE
✓ Pressurized Water
✓ Dry Chemical ➢ depends on the type of waste (soluble or nonsoluble), its level
of radioactivity, and the radiotoxicity and half-life of the
CLASS B FIRES isotopes involved
➢ Flammable
➢ Liquid BIOHAZARDOUS WASTE
➢ Grease
➢ Gasoline ➢ Medical waste -> special waste from health care facilities
➢ Paints ✓ solid waste that, if improperly treated or handled,
➢ Oils “may transmit infectious diseases.”
➢ TYPES OF FIRE EXTIGUISHER; ✓ Blood and blood products, microbiologic waste,
✓ Dry Chemical pathologic waste and sharps
✓ Carbon Dioxide ➢ Steam sterilization, incineration, thermal
inactivation, burial, chemical disinfection,
encapsulation in a solid matrix

OUR LADY OF FATIMA UNIVERSITY I PAMPANGA CAMPUS I COLLEGE OF MEDICAL LABORATORY SCIENCE I CLIN CHEMISTR 3
LABORATORY EQUIPMENT
Rmt2023
PROFESSOR: BILLY JOE AFRICA

BASIC LABORATORY EQUIPMENT AND SUPPLIES BASED ON DRAINAGE


BLOW OUT
PIPETS
➢ Pipettes are glass or plastic tubes, usually open at both ends, ➢ Has a continuous etched ring or two small continuous rings
which are used to transfer specific amounts of liquid from one located near the top of the pipet
container to another. ➢ Last drop should be expelled into receiving vessel
➢ They are usually used for volumes between 1 and 100 ➢ Ex: serologic, ostwald folin
milliliters ➢ The frosted band should not be confused
with thicker colored rings or colored dots,
CLASSIFICATION OF PIPETS which are a manufacturer’s code for the
➢ Depends on the amount of liquid needed to wet the interior maximum volume of the pipette
surface of the ware and the amount of any residual liquid left
in the pipet tip:
✓ Base on Design
• To contain (TC)
• Tp deliver (TD)
✓ Base on Drainage
• Blow out
• Self-draining
SELF DRAINING
✓ Base on use
➢ Allows the content to drain by gravity
• Measuring / Graduated ➢ The tip of the pipet should not be in contact with
• Volumetric / Transfer ✓ The accumulating fluid in the receiving vessels
✓ During drainage except mohr pipet
BASED ON DESIGN ➢ Ex: volumetric, mohr
TO CONTAIN PIPET (TC) ➢ Remember, only blowout a serological pipette if it has a
frosted band or two thin rings
➢ Contains a particular volume but does not dispense the exact
volume BASED ON USE
➢ Rinse out pipet (diluting fluid) MEASURING OR GRADUATED
➢ A small amount of fluid will cling to the inside wall of the pipet
➢ Example : Sahli – Hemoglobin, Lang-levy ➢ Deliver the amount of liquid contained between two calibration
➢ Not often used in clin chem marks
➢ Used in hematology section as it is used for viscous fluid ➢ Not calibrated with sufficient tolerance to use in measuring
standard or control solutions.
➢ Measuring pipettes are divided into:
✓ Serologic or serological -
the graduation marks
continue to the tip
✓ Mohr - the graduations on
these always end before
the tip
✓ Bacteriologic
✓ Ball, kolmer, or kahn
✓ Micropipet
Sahli hemoglobin-used in hematology section to measure
hemoglobin TRANSFER PIPET
• Volumetric and Ostwald folin is usually calibrated with only
TO DELIVER PIPET (TD)
one specific volume
➢ Dispense the amount of volume indicated
➢ VOLUMETRIC PIPET
➢ Designed to be drain by gravity ✓ Used to deliver a single specific volume of liquid,
➢ Must be held vertically and the tip placed against the side of usually between 1 and 100 ml.
the container and must not touch the liquid in it
✓ Shaped like rolling pins with a large belly, one blunt
➢ A small amount of fluid will remain in the tip of the pipette
end, the neck, and one tapering end, the tip
➢ Meet requirements of transfer pipets
✓ TD, self-draining
➢ Ex: mohr, serologic, volumetric transfer pipets
✓ Higher degree of accuracy than in measuring
pipettes

OUR LADY OF FATIMA UNIVERSITY I PAMPANGA CAMPUS I COLLEGE OF MEDICAL LABORATORY SCIENCE I CLIN CHEMISTR 4
➢ OSTWALD- FOLIN pipet PIPETTING VOLUMES
✓ For viscous fluids (hematology) DISPENSER AND DILUTOR (AUTOMATIC PIPET)
✓ Blow out ➢ The dilutor often combines sampling and dispensing functions
✓ Has bulb near the tip
✓ Not class A

➢ PASTEUR PIPETS
✓ Do not have calibration marks
✓ Use to transfer solutions without consideration of a
specific volume

PIPET BULBS
➢ A pipette bulb is used to draw liquid up into the pipette.
➢ There are many types of pipette bulbs:
✓ Rubber bulb
✓ ✓ Pipet filler
✓ Pipet aid
➢ AUTOMATIC MACROPIPETS OR MICROPIPETS ✓ Pipet pumper
✓ Air displacement
• Relies on piston for suction creation to
draw the sample into a disposable tip that
must be changed for use.
• The piston does not come in contact with
the liquid
✓ Positive-displacement
• by moving the piston in the pipet tip.
• Like syringe.
• Doesn’t require different tips
• Rinsing and blotting between samples
maybe required PROCEDURE ON USING THE PIPET
✓ Dispenser and dilutor/dispenser
• Obtain the liquid from common reservoir ➢ Hold the pipette about 8 cm below the mouthpiece with one
and dispense it repeatedly. hand. Then with your other hand squeeze the bulb and touch
• bottle-top the opening to the mouth of the pipette.
• Motorized ➢ Insert no more than one-half cm of the pipette into the bulb.
• Handheld ➢ Place the tip into the colored liquid and slowly release the
• Attached to a dilutor pressure on the bulb
➢ The liquid will be drawn up into the pipette and will form a
curved surface against the glass
➢ This surface is called the meniscus. Pull the bottom of the
meniscus up about 1 cm past the desired level
➢ Then quickly, but carefully, remove the bulb as you slip your
free index finger over the tip of the mouthpiece hole
✓ Never use your thumb--your index finger will allow
better control and will also enable you to hold other
items with your free fingers when necessary
➢ Then with your finger still on the end of the pipette, gently lift
the pipette out of the solution
➢ Then raise your finger just enough to allow the bottom of the
meniscus to line up with the desired graduation mark. You
should observe the meniscus at eye-level while doing this.
✓ When the meniscus is at the desired level, touch the
tip of the pipette to the inside of the container
➢ LEGEND: (A & C) – AIR DISPLACEMENT AUTOMATIC
holding the colored water, to remove any drops of
PIPET
liquid on the end of the pipette. Now, there is
➢ (B & D) - POSITIVE DISPLACEMENT AUTOMATIC PIPET
precisely (0.645 + 0.001) ml of colored water in your
pipette
➢ Keeping your finger on the end of the pipette, wipe the sides
of the pipet with tissue paper & gently move it to the waste
container.
➢ Touch the tip to the inside of the tilted container, lift your
finger off the end and allow the liquid to drain out of the
pipette.
➢ Hold the pipette in this position for a few seconds after it stops
draining. Wipe the pipet again before disposal or cleaning the
pipet

OUR LADY OF FATIMA UNIVERSITY I PAMPANGA CAMPUS I COLLEGE OF MEDICAL LABORATORY SCIENCE I CLIN CHEMISTR 5
MENISCUS ➢ Electronic thermometer/ thermistor probe
➢ Curvature in the top surface of the liquid. Pipette should be ✓ advantage: size and millisecond response time
held that the calibration mark is at the eye level. ✓ disadvantage: expensive
✓ Lower meniscus – clear solutions ➢ Digital thermometer
✓ Upper meniscus- colored or viscous solutions
Note:

BASIC LABORATORY EQUIPMENT AND SUPPLIES micropipette


LABORATORY VESSELS
➢ Calibration – glassware or other apparatus used in minimum-maximum volume (ul)
quantitative measurement is checked to determine its exact p2-0.2 to 2 um
volume p20- 2-20um
➢ National bureau of standards calibrated by weight using p200-20-200
distilled water and analytic balance p1000-100-1000

VOLUMETRIC FLASKS 052 in p1000 is 520 ul

➢ Calibrated to hold one exact p20 and p200-yellow tip


volume of liquid (TC) p100-blue tip
➢ Used for known volume filter tip-prevent contamination
rather than determining the
unknown volume mohr pipetting
-only calibrated to the last graduation line
-excess reagent must be discarded and not deposited back to the
ERLENMEYER FLASKS reagent=contamination

➢ Designed to hold different


volumes rather than one exact
amount
➢ Less accurate than volumetric
flask
➢ Used for known volume rather
than determining the unknown volume

BEAKER

➢ Hold different volumes


rather than one exact
amount
➢ Griffin beakers
➢ Berzeleus beakers

GRADUATED CYLINDER

➢ Used to measure liquid when high degree of


accuracy is not essential
➢ Calibrated to deliver
➢ Can be used to measure specified volume of
liquid
➢ Higher accuracy than Erlenmeyer flask

THERMOMETER
➢ Liquid-in-glass Use color liquid or mercury encased in plastic
or glass material with bulb at one end and a graduated stem
Usually measures temperature between 20 C ̊ to400 C
̊
➢ Liquid-in-glass (3 types)
✓ A.partial immersion- used for
measuring temp in units such
as heating blocks and water
bath. Should be immersed to
the proper height as indicated
by the continuous line etched
in the thermometer
✓ Total immersion- used for
refrigeration application.
✓ Surface thermometers- for
flat surface such as oven or
Incubator

OUR LADY OF FATIMA UNIVERSITY I PAMPANGA CAMPUS I COLLEGE OF MEDICAL LABORATORY SCIENCE I CLIN CHEMISTR 6
SpectrophotometRY (week 3: lab)
Rmt2023
PROFESSOR: BILLY JOE AFRICA

SPECTROPHOTOMOTER
➢ Very important in clinical chemistry
ELECTROMAGNETIC ENERGY
➢ Radiant energy (light) from short wavelength gamma rays to
long wavelength radio waves
o Theory of light- the radiant energy is travelling in a
wavelength matter
o Peak/ wave crest-highest point of wavelength
o Through-lowest part of wavelength
o Amplitude-distance between the peak and through
o Wavelength- Distance between two successive
peaks ➢ Violet: 400 - 420 nm
▪ Lead to characteristics of light ➢ Indigo: 420 - 440 nm
▪ The shorter the wavelength, emits more ➢ Blue: 440 - 490 nm
radiant energy ➢ Green: 490 - 570 nm
▪ The longer the wavelength, the poorer the ➢ Yellow: 570 - 585 nm
➢ Orange: 585 - 620 nm
light emitted
➢ Red: 620 - 780 nm
▪ Visible region-type of light that can be
seen in the naked eye
COLORIMETRY
▪ Invisible region- cannot be detected the
➢ use of light to determine the concentration or presence or
eye
absence of analyte.
➢ They are photons of energy traveling in a wavelike manner.
➢ There are two primary considerations in every colorimetric
The shorter the wavelength, the lighter the electromagnetic
analysis:
energy.
✓ quality of the color and intensity of the color=dependent
TYPES OF ELECTROMAGNETIC ENERGIES
on the light source
Invisible Region-below 340 nm (UV REGION)
➢ Cosmic rays
KINDS OF COLORIMETRY:
➢ Gamma rays
➢ Visual Colorimetry – uses the eyes in determining end point
➢ X-rays
➢ Photoelectric Colorimetry
➢ Ultra-violet (UV)
PHOTOELECTRIC COLORIMETRY: 2 CATEGORIES
➢ Visible- 340-700 nm
✓ Spectrophotometry (Spectrophotometric measurements)
Invisible Region- greater than 700 nm (INFRARED REGION)
✓ Filter photometry (Photometric measurements)
➢ Infrared (IR)
➢ Radio, TV, microwave, etc.
SPECTROPHOTOMETRY
➢ measurement of light intensity in a much narrower
ENERGY
wavelength. It uses a device (prisms and/or gratings) to
➢ transmitted via electromagnetic waves characterized by
disperse the source of light into a continuous spectrum.
frequency and wavelength
o Frequency-number of vibration (wave) in one
FILTER PHOTOMETRY
second (shorter the wavelength=more waves
➢ measurement of light intensity of multiple wavelengths. It
produces)
uses filter to isolate part of the spectrum.
➢ Planck’s formula – shows the relationship between
wavelength and energy
BEER’S LAW OR BEER-LAMBERT’S LAW
➢ E = hv
➢ States that the concentration of a substance is directly
✓ h ->constant (6.62 x 10-27 erg sec)
proportional to the amount of the light absorbed or inversely
✓ v -> frequency
proportional to the logarithm of transmitted light.
WAVELENGTH
TRANSMITTANCE
➢ distance between peaks as light is envisioned to travel in
➢ ratio of the radiant energy transmitted, divided by the radiant
wavelike manner.
energy incident on the sample.
➢ Kinds of wavelength:
✓ visible spectra – 340 nm – 700 nm
ABSORBANCE
✓ invisible spectra – below 340 nm (ultraviolet region
➢ the amount of light absorbed
(UV) above 700 (infrared region [IR])
➢ Proportional to the inverse log of transmittance
➢ Frequency – number of vibrations of wave motion per second
➢ Mathematically derived from %T
✓ A = 2 – log%T

OUR LADY OF FATIMA UNIVERSITY I PAMPANGA CAMPUS I COLLEGE OF MEDICAL LABORATORY SCIENCE I CLIN CHEMISTR 7
✓ A = -log%T = 1/log%T TYPES OF MONOCHROMATOR:
➢ Prisms – wedge-shaped pieces of glass, quartz, NaCl, or
✓ A = abc some other material that allows transmission of light.
✓ Where: o Disperses white light into a continuous spectrum of
✓ A = absorbance colors based on variation of refractive index for
✓ a = molar absorptivity (absorptivity of the compound different wavelength.
under standard conditions) ➢ Gratings -has small grooves cut at such as angle that each
✓ b = length of light through the solution (light path) groove behave like a very small prism.
✓ c = concentration of absorbing molecules/solution o Separates white light into various color component.
o Based on the principle that wavelengths are bent as
COMPONENTS OF SPECTROPHOTOMETER they pass a sharp corner.
➢ Colored filters – made of a glass that absorbs some portion
LIGHT SOURCE of the electromagnetic spectrum and transmit others.
✓ Provides radiant energy in the form of visible or non- o Light energy is absorb by dye compounds on the
visible light that may pass through the monochromator. glass and is dissipated as heat.
The light of proper wavelength will be made incident on o It is used in filter photometer and not on
the analytical cell. spectrophotometer
✓ Tungsten Light bulb – commonly used light source in ➢ Interference filter – utilizes the wave character of light to
the visible and near infrared region (up to 1,200 nm) enhance the intensity of the desired wavelength by
✓ LASER (Light Amplification by Stimulated Emission of constructive interference and eliminates others by destructive
Radiation) interference and reflections.

Factors for choosing a light source EXIT SLIT


✓ Range ➢ controls the width of light beam (bandpass; accurate->
✓ Spectral <1/5 the natural bandpass of the spectrophotometer)
distribution- free ANALYTICAL CELL OR CUVETTE
from stray light ➢ used to hold the solution in the instrument whose
✓ Source-depends concentration is to be measured. It is made of glass, quartz or
on the type of plastic.
light bulb
✓ Stability-shelf-life TYPES OF CUVETTE
(life source) ➢ Borosilicate glass cuvette – for solutions that do not etch
glass.(acids can etch the glass)
➢ Quartz or plastic – does not absorb UV radiation at
TYPES OF LIGHT SOURCE: wavelength below 320
➢ Tungsten Iodine lamp – produces energy wavelength from ➢ Alumina silica glass – good for 340 nm and above (visible)
340 to 700 nm (visible region).
➢ Quartz Halide lamp – contains small amounts of halogen
such as iodine to prevent the decomposition of the vaporized
tungsten from the very hot filament.
➢ Deuterium Discharge lamp – provides energy source with
high output in the UV range (down to 165 nm).
➢ Infrared Energy source – used above 800 nm.

EXAMPLES:
➢ Merst glower – an electrically heated rod of rare earth DETECTORS (PHOTODETECTORS)
element oxides. ➢ electron tube amplifying a current that can convert transmitted
➢ Globar – uses silicon carbide energy into an equivalent amount of electrical or photoelectric
➢ Mercury Vapor lamp – exits narrow bands of energy at well- energy.
defined places in the spectrum (UV and visible)
➢ Hollow Cathode lamp – consists of a gas-tight chamber
containing anode, a cylindrical cathode, and insert gas such
as helium of argon.

ENTRANCE SLIT
➢ minimizes unwanted or stray light and prevents the
entrance of scattered light into the monochromator system
➢ Stray light causes systematic error; causes loss of linearity
TYPES OF DETECTORS
MONOCHROMATOR ➢ Barrier layer cells (Photocell or Photovoltaic cell)
➢ Isolate specific wavelength of light. o Composed of a film of light sensitive material like
➢ Monochromatic light – light radiation of a single wavelength. selenium on iron plate with transparent layer of
silver.

OUR LADY OF FATIMA UNIVERSITY I PAMPANGA CAMPUS I COLLEGE OF MEDICAL LABORATORY SCIENCE I CLIN CHEMISTR 8
o Requires no external voltage sources. (it has its own
source of electricity)
➢ Photoemissive tube or Phototube
o Has photosensitive material that gives off electron
when light energy strikes it.
o Consists of 2 electrodes (cathode and anode)
sealed in an evacuated glass to prevent scattering
of photoelectrons by collision with gas molecules.
o Require an outside voltage for operation.
➢ Photomultiplier tube
o Uses a series of electrodes to internally amplify the
photo signal before leaving the tube.
o Most common type ->measures visible and UV
regions
o Excellent sensitivity and rapid response
o Must never be exposed to room light (

METER
➢ simplest method of displaying output of the detection system.
Also called read-out device
➢ Galvanometer/ammeter

QUALITY CONTROL
➢ Used to determine whether the spectrophotometer is
calibrated
✓ Didymium and holmium oxide filter – used to check
wavelength accuracy
✓ Neutral density filters and dichromate solution – verify
absorbance accuracy on linearity

DOUBLE-BEAM SPECTROPHOTOMETER
➢ Splits monochromatic light into two components
➢ 2 Types:
✓ Double-beam in space -> uses 2 photodetectors, for the
sample beam and reference beam
✓ Double-beam in time ->uses 1 photodetector and
alternately passes the monochromatic light through the
sample cuvette and then reference cuvette using a
chopper

USE OF SPECTROPHOTOMETER
➢ Turn on the spectrophotometer, allow to warm up for 30 mins
➢ Choose absorbance and transmittance by pressing button
mode
➢ Absorbance; set the wavelength needed
➢ Place the cuvettes to the compartment (4 compartment)
➢ 1st Reagent Blank (RB)-used to zero the absorbance of
spectrophotometer=erase the previous reading of
spectrophotometer
➢ Before placing of cuvette, wipe it down first
➢ Cuvette has an arrow, masure to face the arrow in front of the
detector
➢ 2nd Standard (S) and 3rd Unknown Sample (U)
➢ Close the lid
➢ Press the blanking
➢ Get the standard absorbance by pulling the tab once
➢ Pull the tab again to get the absorbance of unknown sample
➢ The push the lever back and get the cuvettes
➢ Turn off the spectrophotometer

OUR LADY OF FATIMA UNIVERSITY I PAMPANGA CAMPUS I COLLEGE OF MEDICAL LABORATORY SCIENCE I CLIN CHEMISTR 9

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