Bengkel Keselamatan Makmal (Laboratory Safety Workshop)

Goh Hoe Han, PhD

16 MAY 2013

Talk outline � pH meter � Weighing scale/ balance

� Fume cupboard, laminar flow & biosafety cabinet

Measuring the power of [H+]

Background Working principles of pH electrode

Considerations

pH= - Log [H+]

Background pH meter

• pH = pondus hydrogenii – means the weight of hydrogen – introduced in 1909 by the Danish

biochemist S. P. L. Sørensen

• A pH meter measures essentially the electro-chemical potential between a known liquid inside the glass electrode (membrane) and an unknown liquid outside.

� When a pH sensing electrode comes in contact with a sample, a potential develops across the sensing membrane surface and that membrane potential varies with pH.

Working principles of pH electrode

pH meter

Working principles of pH electrode

pH meter pH meter pH meter

Working principles of pH electrode

pH meter

� Because the thin glass bulb allows mainly the agile and small hydrogen ions to interact with the glass, the glass electrode measures the electro-chemical potential of hydrogen ions or the potential of hydrogen � active hydrogen ion concentration

pH meter pH meter

Working principles of pH meter

• measurements = comparative readings with that of pre-defined pH standards (buffers)

pH meter

� The pH meter detects the pH sensing bulb signal, reference signal and temperature signal and uses these values to calculate the pH using the Nernst equation

temperature compensation function will automatically correct for the error caused by temperature variations.

measured potential

potential ofthe reference electrode

Nernst factor

Temperature

pH meter pH meter

pH meter

pH meter

Other considerations

7.01 - 4.01

7.01

- 10

.01

pH meter pH meter

pH meter do and don’t ! signal can and will change over time for all electrodes � Needs periodic calibration � Older electrode (������������more frequent calibration � End of life:

o displays different pH values with the same solution o will not calibrate o exhibits an offset of > -30mV (~0.5 pH) o when drift/ take unusually long to settle (>2 min) normally 30 s – 1 min To know the offset, simply place the electrode in a pH 7 buffer and read the raw reading from the probe in millivolts by switching the reading from pH to mV � need to change if greater than [-30mV] i.e. [-36mV]

� keep the electrode hydrated! • salts in the solution are at the same concentration as the salts inside the

electrode � use 3M KCl not DIH2O, relatively ionless water "sucks" ions out of the probe membrane

• life of an electrode will vary depending upon the type of solutions, temperatures, immersion times and cleaning cycles � Typically 1-2 years

pH meter

Measuring the power of [H+] Hands-on practical

METTLER TOLEDO Delta 320

RM 1,285

pH meter

METTLER TOLEDO Delta 320 pH meter Part No. : MB-LE438Item Name : 3 in 1 Epoxy Combination Electrode

With epoxy body and gel electrolyteFixed cable with BNC/CINCH connection – for portable pH meters with temperature sensor pH Range: 0...14 Temperature Range: 0...80 deg.C Temperature Probe: NTC 30K Type of Membrance: U Glass Type of Junction: Open Junction Connections: BNC /RCA (CINCH) Shaft Material: Polysulfone(Transparent)Ele.Storage Solution: 3 mol/L KCI

Shaft Length/Diameter: 120mm/12mm

Applications: General pH measurement with automatic temperature compensation Tris compatible

> RM 700

~ RM 250

pH meter

1. Turn on pH meter

2. Wait ca. 30 minutes for the electronics to warm up.

3. Take electrode out of storage solution.

4. Rinse the electrode with de-ionised water. Do not wipe the electrode membrane, dab the electrode with Kimwipe® instead.

5. Submerse the electrode into the pH 7 buffer.

6. Press the calibrate button.

7. Wait until the pH icon stops flashing and press the calibrate button again.

8. Rinse the electrode with de-ionised water and dab with Kimwipe®.

9. Submerse the electrode into the pH 4 buffer.

10. Wait until the pH icon stops flashing and press the measure button.

11. Rinse the electrode with distilled water and dab dry with Kimwipe®.

12. Submerse the electrode into your sample.

13. Press the read button.

14. Wait until the pH icon stops flashing and record the pH of your sample.

Basics step-by-step pH meter

e.

Advanced practical

pH meter

Referring to the operational manual

Troubleshooting Three common problems might be encountered when calibrating a pH measuring system. These are:

1) difficulty in achieving a zero point calibration 2) no success in obtaining a slope calibration 3) a very long response time (longer than 3 minutes)

There could be various reasons for the above mentioned problems. The most frequent ones :

a) contaminated or out-dated buffers – never store buffer solutions in unmarked bottles/ label with date b) worn electrode - the electrolyte of the electrode is contaminated and/ blocked diaphragm - cracks in the membrane of the measuring electrode c) an electrode not been hydrated long enough (after dry storage or after cleaning with an acid

solution) d) electrostatic charge on the electrode - caused by rubbing the electrode shaft with cloth vs.

gently dabbing it with tissue paper e) a temperature difference between electrode and buffer solution of > 10 °C f) problem with the connection between electrode and measuring instrument : either open-circuit

in the cable or short-circuit in cable and/or connection plug (usually due to moisture ingress)

pH meter

Always keep the electrode clean and hydrated in 3 molar potassium

chloride (3M KCl)

Tap water is acceptable for short period but not distilled/ de-ionised water

Try to remove any deposits from the membrane /

diaphragm by rinsing the electrode with mild detergent (to be soaked in 3M KCl >12 hours before use)

pH meter

Analytical balance/ Weighing scale

METTLER TOLEDO PB-S/FACT

� fully automatic calibration with built-in load

RM 3,300

A&D GR-200

RM 4,797

210g capacity & 0.1mg resolution

Analytical balance/ Weighing scale

Do and don’t

Don't pick up tare containers with bare hands since your fingerprints add mass. Use Kimwipes or tongs to prevent this. Don't lean on the bench while weighing.

� Do record the mass of your container, if you will need

it later. � Do check the level indicator bubble before weighing.

The two rear balance feet serve as leveling screws. � Always clean up the weighing pan and work area

Analytical balance/ Weighing scale

Place creased, small weighing paper/ boat on the balance pan.

Close the sliding glass doors, wait for the reading to stabilise.

Carefully add the substance to be weighed up to the desired mass. Do not attempt to reach a particular mass exactly.

Before recording the mass, close the glass doors and wait until the reading stabilise.

Use a brush to clean spills in the weighing chamber. Discard any waste.

Clean-up

Weighing work flow

Turn the balance on Tur

Press the control bar / tare to cancel out the weight of the container.

nt

Analytical balance/ Weighing scale

More than just weighing

Laminar flow cabinet, Biological safety cabinet,

Fume cupboard/hood

Vertical laminar flow Airstream AVC-4A1 ESCO

Fume cupboard GPF-1200 Laboff

BSC Topsafe 1.2 Bioair

(1) Laminar flow cabinet (2) Biological safety cabinet (3) Fume cupboard

A. enclosed bench designed to maintain a working area devoid of contaminants (1, 2)

B. Local ventilation device that is designed to limit exposure to noxious hazards (2, 3)

C. protection for samples and processes (1, 2)

D. protect the user/ operator (2, 3) E. protect the environment (2, 3)

F. explosion protection, spill containment (3)

Quiz

Laminar flow cabinet

• process of laminar air flow - described as airflow where an entire body of air flows with steady, uniform velocity.

• work by the use of in-flow laminar air drawn through one or more ULPA (ultra-low penetration air)/HEPA (High-Efficiency Particulate Air) filters, designed to create a particle-free working environment and provide product protection.

• enclosed on the sides and constant positive air pressure is maintained to prevent the intrusion of contaminated room air.

• ! do not provide operator protection

Principle of laminar flow cabinet: Sterilize air though filter and blow it across work surface as a particle-free laminar air stream

Types of laminar flow cabinet

• Horizontal - direction of air flow which comes from above but then

changes direction and is processed across the work in a horizontal direction.

• Vertical - function equally well as horizontal with the laminar air

directed vertically downwards onto the working area. - Can provide greater operator protection

� constant flow of filtered air provides material and

product protection

Vertical laminar flow cabinet Airstream AVC-4A1 ESCO

RM 8,800

Functioning of fume hood • A type of ventilation system with the primary function

to exhaust chemical fumes, vapors, gasses, dust, mist and aerosol

• Also serve as physical barriers between reactions and the laboratory, offering a measure of protection against inhalation exposure, chemical spills, run-away reactions and fires.

� Air is removed from the workspace and dispersed into the atmosphere.

Hood body The visible part of the fume hood that serves to contain hazardous gases and vapors.

Baffles Moveable partitions used to create slotted openings along the back of the hood body. Baffles keep the airflow uniform across the hood opening, thus eliminating dead spots and optimizing capture efficiency.

Sash The sliding “door” to the hood. By using the sash to adjust the front opening, airflow across the hood can be adjusted to the point where capture of contaminants is maximized.

Face The imaginary plane between the bottom of the sash and the work surface

Airfoil Located along the bottom and side edges the airfoil streamlines airflow into the hood, preventing the creation of turbulent eddies that carry vapors out of the hood.

Work surface Generally a laboratory bench top, where the work is conducted.

Exhaust plenum The exhaust plenum helps distribute airflow evenly across the hood face.

Principle of fume hood: Discharge chemical vapor outside the building or absorb it with carbon filter

When must use fume hood?

When working with chemicals producing hazardous or noxious fumes, vapours, irritants

– Phenol, chloroform e.g. TRIzol – Strong acids

– Other volatiles

– Flammables

Specialty fume hood types Perchloric acid hood with Scrubber (an extract treatment system)• These units feature a waterwash system in the ductwork. Because perchloric acid fumes settle, and form

explosive crystals, it is vital that the ductwork is cleaned internally with a series of sprays. • Scrubber consist essentially of layers of pads to separate and trap fume particles (hydrofluoric and

perchloric acid), to allow them to be scrubbed. The chemicals are washed into a sump/ basin, which is often filled with a neutralizing liquid. The fumes are then dispersed, or disposed of, in the conventional manner.

• designed for handling hot perchloric acid, hot nitric acid, or for routine use of perchloric acid • Do not handle sulfuric acid, acetic acid, organic solvents, or any combustible materials in

perchloric acid hoods. • Perchloric acid reacts violently with organic materials. • Dried perchloric acid (perchlorate salt deposit) is highly explosive - detonate on contact. Radioisotope hood• This fume hood is made with a coved stainless steel liner and coved integral stainless steel countertop that

is reinforced to handle the weight of lead bricks or blocks. Acid digestion hood• These units are typically constructed of polypropylene in order to resist the corrosive effects of acids at

high concentrations. If hydrofluoric acid is being used in the hood, the hood's glass sash should be constructed of polycarbonate which resists etching. Hood ductwork should be lined with polypropylene or coated with PTFE (Teflon).

Maintenance of fume hood can involve daily, periodic, and annual inspections:

Daily fume hood inspection

– The fume hood area is visually inspected for storage of material and other visible blockages.

• If hood function indicating devices are not a part of the fume hood, a 1-inch (25 mm) by 6-inch (150 mm) piece of soft tissue paper should be placed at the hood opening and observed for appropriate directional flow into the hood.

Periodic fume hood function inspection

• Capture or face velocity is typically measured with a velometer or anemometer. Hoods for most common chemicals must have an average face velocity of 100 feet (30 m) per minute at sash opening of 18 inches (460 mm) or higher. Face velocity readings should not vary by more than 20%. A minimum of six readings shall be used determine average face velocity.

• Other local exhaust devices shall be smoke tested to determine if the contaminants they are designed to remove are being adequately captured by the hood.

Annual maintenance • Exhaust fan maintenance, e.g., lubrication, belt tension, fan blade deterioration and rpm,

shall be in accordance with the manufacturer’s recommendation or as adjusted for appropriate hood function.

Fume cupboard GPF-1200 Laboff

> RM 32,800

• Most fume hoods are fitted with a mains-powered control panel. Typically, they perform one or more of the following functions: – Warn of low air flow. – Warn of too large an opening at the front of the unit. Known as a "high

sash" alarm, this is caused by the sliding glass at the front of the unit being raised higher than is considered safe, due to the resulting air velocity drop.

– Provide a method of switching the exhaust fan on or off.

– Provide a method of turning the internal light on or off.

• Specific extra functions can be added, for example, a switch to turn a waterwash system on or off.

Good laboratory practices of fume hood

Good laboratory practices of fume hood

! Conventional 6 feet constant volume hood fume hoods can consume three times more energy than an average American home. Variable air volume (VAV) systems reduce lab ventilation costs, which reduce the volume of the air exhausted as the fume hood sash is closed. Often enhanced by an automatic sash closing device, which will close the fume hood sash when the user leaves the fume hood face � hoods are operating at the minimum exhaust volume whenever no one is actually working in front of them.

In case of a power outage, the hood sash should be lowered within an inch to maintain a chimney effect to keep some air flowing into the hood.

Functioning of BSC

Principle of BSC: Create inflow to protect the operator by exhausting air from cabinet through HEPA filter

Purpose of BSC: Operator protection from microorganisms. Most BSC also offer product protection from room contaminants

Types of Biological Safety Cabinet (BSC)

Bio Safety Level (BSL)

Types of Biological Safety Cabinet (BSC) Class I BSC

Types of Biological Safety Cabinet (BSC) Class II A1 BSC

Types of Biological Safety Cabinet (BSC) Class II A2 BSC

Class II A1 vs. Class II A2 BSC

Types of Biological Safety Cabinet (BSC) Class II B1 BSC

Types of Biological Safety Cabinet (BSC) Class II B2 BSC

Types of Biological Safety Cabinet (BSC) Class III BSC

Biological safety cabinet Topsafe 1.2 Bioair

RM 24,500 RRRRRRRRMMMMMMMMMMMMMMMMMMMMMM 222222222222222444444444Class II type A2 microbiological safety cabinets

Working safely in your biological safety cabinet

Working safely in your biological safety cabinet

Gloves

To protect yourself or to prevent sample contaminations

When is wearing gloves necessary? Which type?