Razi Vaccine & Serum Research Institute

Transcription

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2 2008 Razi Vaccine & Serum Research Institute

3 ph definition ph describes amount of H+ cations in solution. Concentration of these ions can change in very wide range - most often it has values lying somewhere between 1M and M, although sometimes even higher and lower concentrations can be observed ph is a unit of measure which describes the degree of acidity or alkalinity (basic) of a solution. The formal definition of ph is the negative logarithm of the hydrogen ion activity.( ph = -log[h+])

4 ph value & ph scale The ph value of a substance is directly related to the ratio of the hydrogen ion and hydroxyl ion concentrations. ph definition automatically introduced ph scale. As in most water solutions ph is in the 0-14 range it is commonly believed that ph scale is limited to this range, but in some cases ph values can be negative or higher than 14. 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

5 ph- meter history First ph meter was constructed in 1934 by Arnold beckman. Glass ph electrode that have potential dependent on activity of H+ ions was constructed much earlier, in 1906 by Haber and Klemensiewicz. But the technical difficulties were preventing large scale use of potentiometric measurements of the ph (note that before 1909 ph was not called this way). Main problem was caused by large internal resistance of glass electrodes, which make measurements very difficult.

6 To obtain reliable results one was forced to use very sensitive galvanoscope - expensive and difficult to maintain. To overcome the problem Arnold Beckman proposed to use simple high-gain amplifier made using two vacuum tubes. Amplified current was much easier to measure with cheap miliamperometers. Although 74 years passed since then, almost all ph meters follow the same general idea - external ph and reference electrodes (often in the same housing), high gain amplifier, and amperometer, all in one portable box.

7 ph- meter construction A ph measurement system consists of two parts: a high input meter and ph measuring electrode: Voltmeter : In the probe measures the difference between the voltages of the two electrodes.the meter then translates the voltage difference into ph and displays it on the screen. Temperature probe: sometimes built into the electrode. ph electrode: Most often used ph electrodes are glass electrodes. Typical glass (Corning 015) consisting of approximately 22% Na 2 O, 6% CaO and 72% SiO 2.The systematic representation of this cell contains two reference electrode: the external calomel electrode and the internal silver/silver chloride electrode.

8 The majority of ph electrodes available commercially are combination electrodes that have both glass H+ ion sensitive electrode. Active part of the electrode is the glass bubble. While tube has strong and thick walls, bubble is made to be as thin as possible. Surface of the glass is protonated by both internal and external solution till equilibrium is achieved. Both sides of the glass are charged by the adsorbed protons, this charge is responsible for potential difference.

9 This potential in turn is described by the Nernest equation. Nernst equation : E = E +RT/F ln a n As show in figure the potential of glass electrode has five components: Reference electrode (Ag/AgCl), Standard electrode (SCE), Boundary potential, Junction potential and Asymmetry potential. E cell =E Ag/AgCl E SCE + E j + E asy + E b

10 Junction potential :This potential exists across the salt bridge that separates the calomel electrode from the analyte solution. Boundary potential : This potential varies with the ph of analyte solution that depends on the H ion activities of solution o ether side of membrane. Asymmetry potential : This potential include such causes as differences in strain on two surfaces of membrane imparted during manufacture, mechanical abrasion on surface during use and chemical of the outer surface.

11 Choosing ph electrode If you are working with aqueous solutions with very low ionic strength or you work with solutions containing organic material, proteins, heavy metals, or you are working with solutions that can clog normal electrode junction (like oils, foods or paints), there are many types of ph glass electrodes. If you measure cations, anions or gas, In some specific applications you should be very careful when selecting one.

12 Aqueous application in general : Acids & alkalise Fast measurement Hot sample Emulsion and suspensions: Soil Oily samples Milk Surface measurement : Skin Agar dish Leather paper-drop size sample Organic :Alcohols & aldehyde, ketons Non aqueous media Hydrocarbons TRIS buffer Pure and ultra- pure water: Soft surface water Sterile purified water Distilled water Water for injection drinking water

13 Micro and semi-micro electrodes : Vials Serum Test tubes Enzyme solution Solid and semi-solid : Fruit Meat & fish Viscous or greasy sample : Proteinaceous media Biological media Honey & jam Fats & Cremes Sterilizable ph electrode: Fermenting broth Other electrodes Gas - sensing probes: CO 2 -NO 2 H 2 S SO 2 Crystalline Membrane : Sulphates, Halides Glass electrode for cat ion : Na +, K +, NH4 +, Rb +, CS + and Ag +

14 ph calibration When to calibrate? All ph electrodes require calibration from time to time. Depending on the ph meter type you must be calibrate daily or weekly. After connecting another electrode After a voltage interruption e.g. battery change When the sensor symbol flashes.

15 Why do I calibrate my ph meter? In order to eliminate the determinate errors caused by asymmetry potential all membrane electrode must be calibrated against one ore more standard solution. Calibration determines the current values of the slop (mv /ph) of electrode and stored in the instrument. Calibrate can be performed using one,two or three buffer solutions. single points calibration,two-point and three-point calibration.

16 - The alkaline error : Glass electrodes respond to concentration of both hydrogen ion and alkali metal ions in basic solution. (negative error) ph < ph r - The acid error: ph readings tend to be too high in this region. (positive error) ph > ph r In single point asymmetry be came zero. In two - point asymmetry and slop value are determined. In three- point two separate asymmetry and slop value are determined for two ranges between the three buffers. Most commonly used commercially available calibration buffers have ph of 4.01, 7.00 and

17 How do I calibrate my ph meter? 1) Rinse the electrode thoroughly with DI water to remove all traces of storage solution. Thoroughly rinse the electrode after each buffer test to prevent carry over contamination of the ph buffer solutions. 2) Use the instrument in calibration mode. 3) Insert the electrode and the automatic temperature compensator (ATC) in 7.00 ph buffer solution. Allow 30 seconds for the electrode/atc to reach thermal equilibrium with the buffer solution. Adjust the ph meter with the standardize/zero control for a ph indication equal to ) Repeat Step 1, and insert the electrode and the ATC in the buffer solution that automatically recognized by instrument. Allow 30 seconds before adjusting the ph meter with the slope/span control for a ph indication equal to 4.01 or ) In three - point calibration use three technical buffer solutions. Your ph meter is now calibrated

18 Calibration buffer Substance(s) hydrochloric acid Concentration M ph Potassium trihydrogen oxalate Potassium hydrogen phethalate Potassium hydrogen tartrate Na2HPO4 KH2PO4 Disodium tetraborate NaHCO3 Na2CO m m saturated in 25 C m m m m m

19 Notes: a) Always use fresh ph buffer solutions for the most accurate results. b) All ph buffer solutions above 7.00 ph are less stable and have a limited life. These high ph buffers will more readily absorb CO 2 from the atmosphere and will typically change to a lower ph value when left open. For this reason, a 4.01 buffer solution is recommended to perform a reliable two point calibration. Also, the buffers should bracket the desired ph range. c) When a ph electrode is calibrated with an auto calibration meter, consult the meter s operation manual for the required calibration procedure. d ) ph of buffer solutions change with temperature, so you can't assume - especially using warm and hot buffer solutions. ph values of calibration buffers should be tabularized by manufacturer. ph of potassium hydrogen phethalate solution rises to 4.16 at 80 C. Many ph meters doesn't take these changes into account automatically, even if they allow automatic temperature compensation during measurements.

20 ph measurements temperature compensation According to the Nernest equation cell potential depends on the temperature. Many commercially available ph meters, have built in temperature compensation. The temperature compensation may be either manual or automatic. With manual compensation, a separate temperature measurement is required, and the ph meter manual compensation control can be set with the approximate temperature value. With automatic temperature compensation (ATC), the signal from a separate temperature probe (sometimes built into the electrode) is fed into the ph meter, so that it can accurately determine ph value of the sample at that temperature.

21 ph electrode maintenance Handle electrode with care - it is fragile! Gently blot the electrode on a soft tissue to remove the excess rinse water. Do not rub the bulb since it can cause a static charge build-up. Keep electrode always wet. Use the solution recommended by manufacturer or solution of KCl (3M-4M). Never store them in DI water. Remember to always keep internal level of filling solution above the level of measured solution.

22 If you are using the electrode in solution containing substances able to clog the junction or stick to the glass bubble, clean the electrode as soon as possible after use. Fill electrode with correct filling solution (as recommended by manufacturer - usually KCl solution, 3M to saturated) to not let it dry internally. Don't put electrode in solutions that can dissolve glass such as hydrofluoric acid, acidified fluoride solution, concentrated alkalis, etc. Don't put electrode into dehydrating solution such as ethanol, sulfuric acid, etc.

23 ph electrode cleaning General Soak in 0.1M HCl for half an hour. Drain and refill the reference solution. Soak the electrode in filling solution for one hour. Inorganic: Soak in 0.1M tetrasodium EDTA solution for 15 minutes. Drain and refill the reference solution. Soak the electrode in filling solution for one hour. Protein: Soak in 1% pepsin / 0.1M HCl for 15 minutes. Drain and refill the reference solution. Soak the electrode in filling solution for one hour.

24 Grease and Oil: Rinse with detergent. Drain and refill the reference solution. Soak the electrode in filling solution for one hour. Electrode response may be enhanced by substituting a mixture of 1:1 ph 4 buffer and filling solution for the soaking solution. Pollution by sulfides: Use a solution of 8% thiocarbamide in 1 mol/l HCl. Keep the electrode in the above solution till junction's color turns pale. Pollution by silver chloride: Use concentrated ammonia solution. Keep the electrode in the above solution for about 12 hours. Rinse and put into ph 4 buffer for at least 1 hour.

25 Rejuvenating ph electrode First of all - clean the electrode as described in electrode cleaning. Soak the electrode for 4-8 hours in 1M HCl solution. Rinse it and move to ph 7 buffer for an hour. Give it a try. If the electrode is still not working: Fill the electrode with filling solution. Place the electrode in the 10% nitric acid solution on a hotplate. Heat to boiling, and keep it in the solution for 10 minutes. Place 50 ml of filling solution in a second clean beaker. Heat, although boiling is not necessary. While the electrode is still hot, transfer it to the beaker of heated filling solution. Set aside to cool.

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