The texts, tables and images contained in this lecture notes are not my own, they can be found on: References supplied

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1 The texts, tables and images contained in this lecture notes are not my own, they can be found on: References supplied Atlases or The web sites Source: p.html How to prepare a buffer A compound can buffer the ph of a solution only when its concentration is sufficient and when the ph of the solution is close (within about one ph unit) to its pk a. To make a buffer you must first pick a compound whose pk a is close to the ph you want for the solution, and then decide what the buffer concentration should be. Typically, buffer concentrations are between 1 mm and 200 mm, depending on the desired ionic strength and the buffering capacity required. If the ph is expected to decrease during the experiment, choose a buffer with a pk a slightly below the working ph. Conversely, if the ph is expected to increase during the experiment, select a buffer with a pk a slightly above the working ph. Having decided on the total buffer concentration, you must adjust the ratio of the protonated and unprotonated forms of the buffer in your solution so as to give the desired ph. Typically, buffers are composed of weak acids and their salts, or weak bases and their salts. If the protonated form is uncharged, it is an acid (like acetic acid), and its unprotonated form is a salt (e.g., sodium acetate). Conversely, if the unprotonated form is uncharged it is a base (like Tris base), and its protonated form is a salt (e.g., TrisHCl). Four practical ways to make a buffer are described below: (1) The Slow and Stupid Method To avoid adding extra salt to a solution, prepare a buffer composed of an acid and its salt by dissolving the acid form of the buffer in about ~60% of the water required for the final solution volume. Adjust the ph using a strong base, such as NaOH. When preparing a buffer composed of a base and its salt, start with the base form and adjust the ph with strong acid, such as HCl. After the ph is correct, dilute to just under the final solution volume. Check the ph and correct if necessary, then add water to the final volume. Advantages: Easy to understand. Disadvantages: Slow. May require lots of base (or acid). If the base (or acid) is concentrated, it is easy to overshoot the ph. If the base (or acid) is dilute, it is easy to overshoot the volume. Ionic strength will be unknown. Adding a strong acid or base can result in temperature changes, which will make ph readings inaccurate (due to its dependence to temperature) unless the solution is brought back to its initial temperature. (2) The Mentally Taxing Method 1

2 Using the buffer pk a, calculate the amounts (in moles) of acid/salt or base/salt present in the buffer at the desired ph. If both forms (i.e., the acid and the salt) are available, convert the amount required from moles to grams, using the molecular weight of that component, and the weigh out the correct amounts of both forms. If only one form is available, you can prepare the buffer by adding all of the buffer as one form, and then adding acid or base to convert some of the added buffer to the other form. Decide what the total concentration of buffer will be in the solution, and convert the concentration to amount (in moles) using the volume of solution, and then to grams, using the molecular weight of the buffer form available. Then calculate the amounts (in moles) of each form that will be present in the final solution, using the buffer pk a and the desired ph. Then calculate how much strong acid or base must be added to convert enough of the buffer form added to the other form, to give the correct amounts of each form at the ph of the final solution. Dissolve the buffer and strong acid or base in slightly less water than is required for the final solution volume. Check the ph and correct if necessary. Add water to the final volume. Advantages: Fast. Easy to prepare. Additional ph adjustment is rarely necessary, and when necessary, the adjustment is small. Ionic strength easily calculated. Disadvantages: Requires the buffer pk a and solving two equations. (3) The Two Solution Method Make separate solutions of the acid form and base form of the buffer, both solutions having the same buffer concentration (and ionic strength, if required) as the concentration of total buffer in the final solution. To obtain the desired ph, add one solution to the other while monitoring the ph with a ph meter. Advantages: Easy to do. Disadvantages: Requires both forms of buffer. The required solution volumes are proportional to the ratio of buffer components in the final solution at the desired final ph, so making equal amounts of each form may waste a lot of one solution. (4) The Completely Mindless Method Find a table of the correct amounts of acid/salt or base/salt required for different ph's, and dissolve the components in slightly less water than is required for the final solution volume. Check that the ph and correct if necessary. Add water to the final volume. Advantages: Easy to do (with appropriate table). Convenient for frequently prepared buffers. Disadvantages: May be impossible to find table. Table may be incorrect. Requires both forms of buffer. Component amounts from table will need to be adjusted to give the buffer concentration and volume in your solution. Ionic strength is unknown. The choice of buffer is based on: 1. The buffering capacity in the desired ph range with the ability to maintain constant ph during fixation. 2. The side effects which vary with the tissue type: a. Suitable osmolarity so that cells and organelles neither swell nor shrink during fixation. b. Suitable ionic concentration so that materials are neither extracted nor precipitated during fixation. 2

3 c. The toxicity of the buffer. Criteria of a good buffer: 1. pka: usually between 6 and 8 desired for biological specimens. 2. Maximum solubility in water and minimum solubility in all other solvents. 3. Reduced ion effects. 4. Dissociation of buffer least influenced by buffer concentration, temperature and ionic composition. 5. Resistance to oxidation (stable). 6. Inexpensive and easy to prepare. 7. No reaction with fixation. Common Buffers I. Phosphate Buffer (Sorenson's buffer) ph Advantages: 1. Most physiological of common buffers. Mimics certain components of extracellular fluids. 2. Non-toxic to cells. 3. ph changes little with temperature. 4. Stable for several weeks at 4 C. Disadvantages: 1. Precipitates more likely to occur during fixation. Tends to form precipitates in presence of calcium ions. Precipitates uranyl acetate and tends to react with lead salts. 2. Becomes slowly contaminated with micro-organisms Preparation of Buffer of Phosphate Buffer Stock solutions: 0.2 M dibasic sodium phosphate 1 liter Na 2 HPO 4 *2H 2 0 (MW = ) g or Na 2 HPO 4 *7H 2 0 (MW = ) g or Na 2 HPO 4 *12H 2 0 (MW = ) g 0.2 M monobasic sodium phosphate 1 litter NaH 2 PO 4 *H 2 0 (MW = ) 27.6 g 3

4 or NaH 2 PO 4 *2H 2 0 (MW = ) g Working buffer: 0.1M 100 ml Mix X ml of 0.2 M dibasic sodium phosphate with Y ml monobasic sodium phosphate. Dilute to 100 ml with ddh 2 0 or dilute 1:1 with fixative. ph (25 C) X ml Y ml Osmolarity is adjusted by varying the molarity of phosphates or by the addition of sucrose, glucose or sodium chloride. At ph 7.2: 0.10 M = 226 mos (milliosmoles) 0.05 M = 118 mos M = 180 mos 0.15 M = 350 mos II. Cacodylate Buffer (arsenate buffer) ph Advantages: 4

5 1. Easy to prepare. 2. Stable during storage for long periods of time. 3. Does not support growth of microorganisms. 4. Precipitates usually do not occur. Precipitates do not occur at low concentrations of calcium. Disadvantages: 1. Toxic. Contains arsenic 2. Unpleasant smell Preparation of Buffer: Stock solutions: 0.2 M sodium cacodylate 1 liter Na(CH 3 ) 2 As0 2 *3H 2 0 (MW = ) 42.8 g 0.2 M HC1 Conc. HC1 (36-38%) ddh ml 603 ml Working buffer: 0.1 M 100 ml Adjust 50 ml of 0.2 M sodium cacodylate to desired ph with 0.2 M HC1. Dilute to 100 ml with ddh 2 0 or dilute 1:1 with fixative. ph 0.2 M HC1 (ml) Buffer may also be made with cacodylic acid Stock solutions: 5

6 0.2 M cacodylic acid 1 liter (CH 3 ) 2 AsO 2 H (MW = 138.0) 27.6 g 0.2 M NaOH 100 ml NaOH (MW = 40) 0.8 g + ddh 2 0 to make 100 ml Working buffer: 0.1 M 100 ml Adjust 50 ml of 0.2 M cacodylic acid to desired ph with 0.2 M NaOH. Dilute to 100 ml with ddh 2 or dilute 1:1 with fixative. III. Veronal-acetate Buffer (Michaelis buffer) Advantages: Useful for block staining with uranyl acetate since precipitates do not form. Disadvantages: 1. Reacts with aldehydes. 2. Poor buffer at physiological ph. 3. Supports growth of micro-organisms. 4. Contains barbiturate. Preparation of Buffer: Stock solution: 0.28 M 100 ml Sodium veronal (barbitone sodium) C 8 H N 2 Na (MW = ) 2.89 g Sodium acetate (anhydrous) CH 3 C00Na (MW = 82.03) 1.15 g or Sodium acetate (hydrated) CH 3 C00Na*3H 2 0 (MW = ) 1.90 g + ddh 2 H 2 0 to make 100 ml Solution is stable and may be stored for some months at 4 C. Working buffer: Veronal acetate stock solution ddh ml 15 ml 6

7 Add 0.1 HC1 gradually to desired ph. Solution cannot be stored. Supports growth of bacteria and molds even at 4 C. Crystallizes in absence of osmium tetroxide. IV. Collidine Buffer ph Advantages: 1. Maximum buffering capacity about Stable indefinitely at room temperature. 3. Useful for fixation of large tissue blocks. Aids penetration of fixative due to extractive effects (see disadvantage 1). Disadvantages: 1. Not suitable as buffer during primary fixation with osmium tetroxide due to considerable extraction of tissue components. 2. Use leads to lysis of cytoplasmic matrix and extensive membrane destruction when used with paraformaldehyde fixatives. 3. Use gives poorer results with glutaraldehyde than those obtained with phosphate or cacodylate buffer. Preparation of Buffer: Stock solution: 0.4M 100 ml Pure s-collidine 5.34 g 2,4,6(CH 3 ) 3 (C 2 H 5 N) (MW = ) + ddh 2 0 to make 100 ml Working buffer: 0.2M 100 ml Adjust 50 ml of s-collidine stock solution to desired ph with 1N HC1. Dilute to 100 ml with ddh 2 0. ph 1N HC1 (ml)

8 V. Tris buffer Advantages: 1. Good buffering capacity at higher ph required for some tissues and some cytochemical procedures. 2. "More or less" physiologically inert. Disadvantages: 1. ph changes with temperature. Must be measured at desired temperature. 2. ph must be measured with certain type of electrode. Preparation of Buffer: A. Tris Buffer ph Stock solution 0.2 M 1 liter Tris(hydroxymethyl)aminomethane 24.2 g H 2 NC(CH 2 0H) 3 (MW = ) Working buffer: 0.1M 100 ml Adjust ph of 50 ml of stock solution with 0.1 M NaOH. Dilute to 100 ml with ddh 2 0. B. Tris-maleate Buffer ph Stock solution: 0.2 M 1 liter Tris(hydroxymethyl)aminomethane 24.2 g Maleic acid 23.2 g HO 2 CCH:CHCO 2 H (MW = ) or Trizima-maleate (MW = 237.2) 47.4 g 8

9 Working buffer: 0.2 M 100 ml Adjust 50 ml of stock solution to desired ph with 0.1M NaOH. Dilute to 100 ml with ddh 2 0. VI. Special Buffers Used for Cytochemical Reactions. A. Acetate Buffer (sodium acetate-acetic acid buffer) ph Sodium acetate 0.2 M = 27.2 g/l CH 3 CO 2 Na*3H 2 0 (MW ) Acetic acid 0.2 M CH 3 COOH (MW = 60) Add sodium acetate to acetic acid to give desired ph. Dilute with ddh 2 0 to desired molarity. B. Borate Buffer ph Borax (sodium tetraborate) 0.2 M = 76.2 g/ml Na 2 B *120H 2 0 (MW = ) Boric acid 0.2 M = g/l H 3 BO 3 (MW = 61.83) Add boric acid to borax solution until desired ph is reached. Dilute to desired molarity with ddh 2 0. C. Citrate Buffer (sodium citrate-citric acid buffer) ph Sodium citrate 0.2 M = 58.8 g/l Na 3 C 6 H *H 2 0 (MW = ) Citric acid 0.2 M = g/l C 6 H *H 2 0 (MW = ) Mix citric acid and sodium citrate to give desired ph. Dilute with ddh 2 0 to desired molarity. D. Dimethylglutarate Buffer ph Dimethylglutaric acid 0.1M = g/l C 7 H (MW = 160.2) Add 0.2 N NaOH to give desired ph. Dilute with ddh 2 0 to desired molarity. E. Succinate Buffer ph Succinic acid 0.2 M = g/l C 4 H (MW = ) Add 0.2 M NaOH to desired ph. Dilute with ddh 2 0 to desired molarity. 9

10 F. Maleate Buffer (sodium hydrogen maleate buffer) ph Stock solution: 0.2 M 1 liter Maleic acid (MW = ) 23.2 g Adjust ph with 0.1 M Na0H. Dilute with ddh 2 0 to desired molarity. G. Imidazole Buffer ph Imidazole 0.2 M = 13.62/L C 3 H 4 N 2 (MW = 68.08) Adjust 0.2 N HC1 to imidazole solution until desired ph is reached. Dilute to desired molarity with ddh 2 0. H. AMPd Buffer ph amino-methyl-1,3-propanediol 0.2 M = g/l C 4 H 11 NO 2 (MW = ) Add 0.2M HC1 until desired ph is reached. Dilute with ddh 2 0 to desired molarity. 10