Week 1 To Week 3 Laboratory
Week 1 To Week 3 Laboratory
Rmt2023
PROFESSOR: BILLY JOE AFRICA
OUR LADY OF FATIMA UNIVERSITY I PAMPANGA CAMPUS I COLLEGE OF MEDICAL LABORATORY SCIENCE I CLIN CHEMISTR 1
STORAGE AND HANDLING OF CHEMICALS
CORROSIVE CHEMICALS
REACTIVE CHEMICALS
CARCINOGENIC CHEMICALS
ELECTRICAL SAFETY
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
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:
BEAKER
GRADUATED CYLINDER
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.
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