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p H METER

pH meters are essential tools for measuring the acidity or basicity of a solution. This article explains how pH is measured, the components of a pH meter, and the process of calibration. Additionally, it discusses the sedimentation principle and working of a laboratory centrifuge.

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p H METER

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  1. pH METER

  2. pH • pH is a unit of measure which describes the degree of acidity or basicity of a solution. • It is measured on a scale of 0 to 14. • The formal definition of pH is the negative logarithm of the hydrogen ion activity. • pH = -log[H+]

  3. pH value • The pH value of a substance is directly related to the ratio of the hydrogen ion and hydroxyl ion concentrations. • If the H+ concentration is higher than OH- the material is acidic. • If the OH- concentration is higher than H+ the material is basic. • 7 is neutral, < is acidic, >7 is basic

  4. pH METER • pH meter has two basic components: the digital meter (with a numeric display), and either one or two probes that we insert into the test solution. • The electrode that does the most important job, is the glass electrode, having a silver based electrical wire suspended in a solution of hydrogen chloride, contained inside a thin bulb made from a special glass containing metal salts (typically compounds of sodium and calcium). • The other electrode is called the reference electrode and has a silver chloride wire suspended in a solution of potassium chloride.

  5. A pH meter is essentially a voltmeter which measures the voltage of an electrode sensitive to the hydrogen ion concentration, relative to another electrode which exhibits a constant voltage. • The key feature of the pH-sensitive electrode is a thin glass membrane whose outside surface contacts the solution to be tested. The inside surface of the glass membrane is exposed to a constant concentration of hydrogen ions

  6. PH meter Electrode holding arm Combination electrode Display Keypad Slide out card

  7. Glass Electrode • Inside the glass electrode assembly, a silver wire, coated with silver chloride and immersed in the HCl solution, is called an Ag/AgCl electrode. This electrode carries current through the half-cell reaction. The potential between the electrode and the solution depends on the chloride ion concentration, but, since this is constant (0.1 M), the electrode potential is also constant.

  8. REFERENCE ELECTRODE • A reference electrode is needed to complete the electrical circuit. A common choice is to use another Ag/AgCl electrode as the reference. The Ag/AgCl electrode is immersed in an 0.1 M KCl solution which makes contact with the test solution through a porous fiber which allows a small flow of ions back and forth to conduct the current.

  9. Glass electrode Reference electrode Combination electrode

  10. WORKING • To measure pH the pH sensor is immersed in test solution. The H+ ions are able to penetrate the boundary area of glass membrane which is called gel layer and results in charge separation. • The same process occurs inside sensor with neutral solution buffered at pH 7 that has constant concentration of H+ ions • If the H+ ion concentration hence the pH value on inside differs from concentration on outside then measurable potential difference forms.

  11. If H+ ion concentration on inside is lower than on outside , the measured solution is acidic pH>7 . • If H+ ion concentration is identical on both sides , NO potential difference forms, measured solution is neutral with pH value 7. • Measured solution is basic if H+ ion concentration inside glass bulb is higher than in test solution.

  12. Accuracy • For pH meters to be accurate, they have to be properly calibrated, so they usually need testing and adjusting before we start to use them. • To calibrate a pH meter we dip it into buffers (test solutions of known pH) and adjust the meter accordingly. • Another important consideration is that pH measurements made this way depend on temperature. Some meters have built-in thermometers and automatically correct their own pH measurements as the temperature changes; those are best if fluctuations in temperature are likely to occur while making a number of different measurements.

  13. Centrifuge

  14. INTRODUCTION • A laboratory centrifuge • is laboratory equipment. • driven by a motor. • spins liquid samples at high speed. • There are various types of centrifuges, depending on the size and the sample capacity. • They vary widely in speed and capacity • Work by the sedimentation principle, where the centrifugal force is used to separate substances of greater and lesser density. • Comprise a rotor containing two, four, six, or many more numbered wells within which the samples containing centrifuge tips may be placed.

  15. Centrifuge 5804 R Centrifuge lid Monitoring Glass Rotor Operator panel with display The centrifuge which we have in our lab is eppendorf 5804R refrigerated . In this both fixed angle and swinging bucket rotors can be used.

  16. WORKING • Increasing the effective gravitational force will more rapidly and completely cause the precipitate to gather on the bottom of the tube as a "pellet". • The remaining solution is called the "supernate" or "supernatant". • The supernatant liquid is then either • decanted from the tube without disturbing the pellet, or • withdrawn with a pipette. • The rate of centrifugation is specified by the acceleration applied to the sample, typically measured in revolutions per minute (RPM) or relative centrifugal force (RCF).

  17. WORKING • The particles settling velocity in centrifugation is a function of their • size and shape, • centrifugal acceleration, • the volume fraction of solids present, • the density difference between the particle and the liquid, • and the viscosity. • More-dense components of the mixture migrate away from the axis of the centrifuge, while less -dense components of the mixture migrate towards the axis.

  18. ROTORS Generally spoken, there are two main types of rotors: • Fixed-angle rotor • The rotor (mainly made of aluminium) is very compact. • There are boreholes with a specific angle (like 45°) within the rotor. • These boreholes are used for the sample tubes. Tube holder.

  19. Swing-out rotor (= horizontal rotor) • The rotor looks like a cross with bucket. • Within these buckets, different tubes can be centrifuged. • For a safe centrifugation, a specific adapter for every tube shape is mandatory.

  20. TYPES OF CENTRIFUGE • Preparative centrifuge: This technique is concerned with the actual separation, isolation and purification of, for example, whole cells, subcellular organelles, plasma membrane, polysomes, ribosomes, chromatin, nucleic lipoprotein and viruses, for subsequent biochemical investigation. • Analytical centrifuge:This techniques are devoted mainly to the study of pure or virtually pure macromolecules or particles. It is primarily concerned with the study of the sedimentation characteristics of biological macromolecules and molecular structure, rather than with the actual collection of particular fractions

  21. CENTRIFUGE TUBES • Centrifuge tubes or centrifuge tips are tapered tubes of various sizes made of glass or plastic. • They may vary in capacity from tens of mm, to much smaller capacities used in micro-centrifuges used extensively in molecular biology laboratories. • The most commonly encountered tubes are of about the size and shape of a normal test tube (~ 10 cm long). • Micro-centrifuges typically accommodate micro-centrifuge tubes with capacities from 250 μl to 2.0 ml These are exclusively made of plastic.

  22. Glass centrifuge tubes can be used with most solvents, but tend to be more expensive. They can be cleaned like other laboratory glassware, and can be sterilized by autoclaving. • Plastic centrifuge tubes, especially micro-centrifuge tubes tend to be less expensive. They are more difficult to clean thoroughly, and are usually inexpensive enough to be considered disposable .

  23. SAFETY ASPECTS… • The load in a laboratory centrifuge must be carefully balanced. • The rotor should be closed by a rotor lid. • Before starting a centrifuge, an accurate check of the rotor lockage as well as the lid lockage is mandatory. • Centrifuge rotors should never be touched while moving, because a spinning rotor can cause serious injury.

  24. Biological Safety cabinets

  25. Biological Safety Cabinets • Designed to contain biological hazards • Inward airflow for personnel protection • HEPA filtered exhaust air for environmental protection • Supply air HEPA filter for product protection (except Class I) • Separated into Classes and Types – Class I – Class II • Type A1, A2 • Type B1, B2 – Class III

  26. BSC • A biological safety cabinet consists of : • a filter pad, • a fan and • a HEPA filter • a UV light. • The fan sucks air through the filter pad where dust is trapped. Then the pre filtered air has to pass the HEPA filter where contaminating fungi, bacteria, dust, spores are removed .

  27. Laminar flow cabinets work by the use of in flow laminar air drawn through one or more HEPA filters, designed to create a particle free working environment. • Biological safety cabinet is applied in microorganism operation and testing to provide safety protection for laboratory personnel, objects and environment.

  28. CLASS II TYPE A2 • Ventilated cabinet • Provides personnel, product, and environmental protection • Open front with inward airflow for personnel protection • Downward HEPA filtered laminar airflow for product protection • HEPA filtered exhaust air for environmental protection .

  29. 30% Exhaust, 70% Re-circulate • Inflow velocity 100 fpm minimum • BSL 1 –3 Usage • Personnel and Product protection. • Typical uses today: Bacterial, Viral, Fungal, Parasitic

  30. Operator panel Movement of HEPA filtered air Working bench

  31. Class II Type A2 can’t be used for chemical vapours. Chemical vapour build-up in cabinet & lab is dangerous. Thimble duct: have holes for room air

  32. Air flow pattern Pump Filtered Air

  33. Conditioned Air Out plus Rejected Heat Fan Air / Rejected Heat Fan Control / Rejected Heat Light / Rejected Heat Outlet / Process Use Power In Conditioned Air In

  34. application • Biological safety cabinet is applied in microorganism operation and testing to provide safety protection for laboratory personnel, objects and environment. • The negative pressure air curtain at the opening of cabinet prevents contaminated aerosol from extravasations , so to protect operation personnel.

  35. Milli-Q

  36. MILLI-Q • Quality water is the key to all laboratory applications. • Consistent pure water quality is crucial for successful reproducibility in laboratory applications. • Depending on the activity , regulatory bodies have defined several levels of water purity as • Pure Type I • Pure Type II • Ultrapure water

  37. Pure water is ideal for wide range of uses: • Microbiological media preparation • Buffer preparation • Reagent preparation • Milli-Q system produces both pressurized pure and ultrapure water directly from tap water. • The Milli-Q Integral system provides perfect convenience through separate Points-of-Delivery (PODs).

  38. STEPS IN WATER PURIFICATION • Pretreatment: This is the first purification step and it removes • Particles (1µm filter) • Free chlorine and colloids from tap water • An anti-scaling compound which prevents the reverse osmosis membrane from scaling in hard water. • Reverse Osmosis: • This is the second step removes 95-99% of inorganic ions and 99% of all dissolved organic substances as well as microorganisms and particles.

  39. Ion removal: This is third step which removes the remaining ions. Ion exchange resins contained in module are continuously regenerated by means of an electric current. This self regeneration technique eliminates the need of costly resin replacement. • Ultraviolet lamp: Last treatment step , water is sanitized through 254 nm and 185 nmUV lamp in a stainless steel cartridge.

  40. Milli-q Integral 3 Main display Progard pack Quantum cartridge

  41. Q-pod unit Q-POD Plunger Point of delivery Display POD Pak Keypad

  42. Components Of Milli-Q • Progard Pack : The Progard Pack protects the RO Cartridge in order to increase its lifetime. The Progard Pack prevents mineral scaling, organic fouling and chlorine oxidation of the RO Cartridge(s). • UV 254 nm lamp: The UV 254 nm Lamp emits light at 254 nm. The UV 254 nm Lamp is used to kill bacteria.

  43. UV 185 nmLamp: The dual wavelength UV 185 nm Lamp emits light at 185 nm and at 254 nm. The UV 185 nm Lamp kills bacteria and reduces the level of organic molecules in the water. • POD Pak The POD Pak is the final water purification device. It is attached to the Point of Delivery outlet. The POD Pak provides additional quality and insurance that trace contaminants related to specific applications are removed just before ultrapure water is delivered.

  44. Quantum cartridge:

  45. Water quality The water derived from Q-POD unit has following characteristics:

  46. THANK YOU

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