《英语教学》（英文版）The Preparation of Buffers and Other Solutions-A Chemist's Perspective

Molecular Biology Problem Solver: A Laboratory Guide. Edited by Alan S Gerstein opyright◎2001 ISBNS:0-471-37972-7( Paper);0-471 (Electronic) The Preparation of Buffers and other solutions: A Chemist's Perspective Edward A. Pfannkoch 32 Why buffer Can You substitute one buffer for another? How Does a Buffer Control the pH of a Solution? When Is a Buffer Not a Buffer? 33 What Are the Criteria to Consider When Selecting a Buffer What Can Generate an Incorrect or Unreliable Buffer? What ls the Storage Lifetime of a Buffer? Editors note: Many, perhaps most, molecular biology procedures don't require perfection in the handling of reagents and solution preparation. When procedures fail and logical thinking produces a dead end, it might be worthwhile to carefully review your experimental reagents and their preparation. The author of this discussion is an extremely meticulous analytical chemist, not a molecular biologist. He describes the most frequent mistakes and misconceptions observed during two decades of experimentation that requires excruciating accuracy and reproducibility in reagent preparation 3 1

31 3 The Preparation of Buffers and Other Solutions: A Chemist’s Perspective Edward A. Pfannkoch Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 Why Buffer?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 Can You Substitute One Buffer for Another?. . . . . . . . . . . . . 32 How Does a Buffer Control the pH of a Solution? . . . . . . . 32 When Is a Buffer Not a Buffer? . . . . . . . . . . . . . . . . . . . . . . . . 33 What Are the Criteria to Consider When Selecting a Buffer? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 What Can Generate an Incorrect or Unreliable Buffer? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 What Is the Storage Lifetime of a Buffer? . . . . . . . . . . . . . . . 37 Editor’s note: Many, perhaps most, molecular biology procedures don’t require perfection in the handling of reagents and solution preparation. When procedures fail and logical thinking produces a dead end, it might be worthwhile to carefully review your experimental reagents and their preparation. The author of this discussion is an extremely meticulous analytical chemist, not a molecular biologist. He describes the most frequent mistakes and misconceptions observed during two decades of experimentation that requires excruciating accuracy and reproducibility in reagent preparation. Molecular Biology Problem Solver: A Laboratory Guide. Edited by Alan S. Gerstein Copyright © 2001 by Wiley-Liss, Inc. ISBNs: 0-471-37972-7 (Paper); 0-471-22390-5 (Electronic)

R Which Grade of Reagent Does Your Experiment Require? Should You Question the Purity of Your Reagents?........ 39 What Are Your Options for Storing Reagents 40 Are All Refrigerators Created Equal? Safe and Unsafe Storage in Refrigerators What Grades of Water Are Commonly Available in he lab? 42 When is 8MQ Water not 8 MQ Water What ls the Initial ph of the Water? 44 What Organics Can Be Present in the Water? What Other Problems Occur in Water Systems? 46 BUFFERS Why Buffer? e The primary purpose of a buffer is to control the pH of the solu- tion. Buffers can also play secondary roles in a system, such as controlling ionic strength or solvating species, perhaps even affect- ng protein or nucleic acid structure or activity. Buffers are used to stabilize nucleic acids, nucleic acid-protein complexes, proteins, and biochemical reactions(whose products might be used in subsequent biochemical reactions). Complex buffer systems are used in electrophoretic systems to control pH or establish ph grad Can You substitute one Buffer for Another? It is rarely a good idea to change the buffer type-that is, amine-type buffer(e.g, Tris) for an acid-type buffer(e.g, phos- phate). Generally, this invites complications due to secondary effects of the buffer on the biomolecules in the system. If the purpose of the buffer is simply pH control, there is more latitude to substitute one buffer for another than if the buffer plays other Important roles in the assay. low Does a Buffer Control the pH of a Solution? Buffers are solutions that contain mixtures of weak acids and bases that make them relatively resistant to pH change Concep tually buffers provide a ready source of both acid and base to either provide additional H* if a reaction(process)consumes H* or combine with excess H if a reaction generates acid

Reagents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Which Grade of Reagent Does Your Experiment Require?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Should You Question the Purity of Your Reagents?. . . . . . . . 39 What Are Your Options for Storing Reagents? . . . . . . . . . . . 40 Are All Refrigerators Created Equal? . . . . . . . . . . . . . . . . . . . 41 Safe and Unsafe Storage in Refrigerators . . . . . . . . . . . . . . . . 41 What Grades of Water Are Commonly Available in the Lab? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 When Is 18MW Water Not 18MW Water? . . . . . . . . . . . . . . 44 What Is the Initial pH of the Water?. . . . . . . . . . . . . . . . . . . . 44 What Organics Can Be Present in the Water? . . . . . . . . . . . 45 What Other Problems Occur in Water Systems?. . . . . . . . . 46 Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 BUFFERS Why Buffer? The primary purpose of a buffer is to control the pH of the solu￾tion. Buffers can also play secondary roles in a system, such as controlling ionic strength or solvating species, perhaps even affect￾ing protein or nucleic acid structure or activity. Buffers are used to stabilize nucleic acids, nucleic acid–protein complexes, proteins, and biochemical reactions (whose products might be used in subsequent biochemical reactions). Complex buffer systems are used in electrophoretic systems to control pH or establish pH gradients. Can You Substitute One Buffer for Another? It is rarely a good idea to change the buffer type—that is, an amine-type buffer (e.g., Tris) for an acid-type buffer (e.g., phos￾phate). Generally, this invites complications due to secondary effects of the buffer on the biomolecules in the system. If the purpose of the buffer is simply pH control, there is more latitude to substitute one buffer for another than if the buffer plays other important roles in the assay. How Does a Buffer Control the pH of a Solution? Buffers are solutions that contain mixtures of weak acids and bases that make them relatively resistant to pH change. Concep￾tually buffers provide a ready source of both acid and base to either provide additional H+ if a reaction (process) consumes H+ , or combine with excess H+ if a reaction generates acid. 32 Pfannkoch

The most common types of buffers are mixtures of weak acid and salts of their conjugate bases, for example, acetic acid/sodium acetate. In this system the dissociation of acetic acid can be written CH3COOH→CH3COO+H where the acid dissociation constant is defined as Ka=HI [CH_ COO[ COOH Rearranging and taking the negative logarithm gives the more familiar form of the Henderson-Hasselbalch equation DH=pk+log ICHsCOo- ICH3 COOH Inspection of this equation provides several insights as to the functioning of a buffer When the concentrations of acid and conjugate base are equa log(1)=0 and the pH of the resulting solution will be equal to the pKa of the acid. The ratio of the concentrations of acid and con jugate base can differ by a factor of 10 in either direction, and the resulting pH will only change by l unit. This is how a buffer main- tains ph stability in the solution To a first approximation, the ph of a buffer solution is inde- pendent of the absolute concentration of the buffer; the pH depends only on the ratio of the acid and conjugate base present However, concentration of the buffer is important to buffer capac ity, and is considered later in this chapter When Is a Buffer not a Buffer? Simply having a weak acid and the salt of its conjugate base present in a solution doesnt ensure that the buffer will act as a buffer Buffers are most effective within t 1 pH unit of their pK Outside of that range the concentration of either the acid or its salt is so low as to provide little or no capacity for pH control Common mistakes are to select buffers without regard to the pKa of the buffer. Examples of this would be to try to use K,HPO/KH2PO4(pKa=6.7)to buffer a solution at pH 4, or to use acetic acid(pKa=4.7)to buffer near neutral pH. What Are the Criteria to Consider When Selecting a Buffer? Target pH Of primary concern is the target pH of the solution. This narrows the possible choices to those buffers with pka values within 1 pH unit of the target pH The Preparation of Buffers and Other Solutions

The most common types of buffers are mixtures of weak acids and salts of their conjugate bases, for example, acetic acid/sodium acetate. In this system the dissociation of acetic acid can be written as CH3COOH Æ CH3COO- + H+ where the acid dissociation constant is defined as Ka = [H+ ] [CH3COO- ]/[H3COOH]. Rearranging and taking the negative logarithm gives the more familiar form of the Henderson-Hasselbalch equation: Inspection of this equation provides several insights as to the functioning of a buffer. When the concentrations of acid and conjugate base are equal, log(1) = 0 and the pH of the resulting solution will be equal to the pKa of the acid. The ratio of the concentrations of acid and con￾jugate base can differ by a factor of 10 in either direction, and the resulting pH will only change by 1 unit. This is how a buffer main￾tains pH stability in the solution. To a first approximation, the pH of a buffer solution is inde￾pendent of the absolute concentration of the buffer; the pH depends only on the ratio of the acid and conjugate base present. However, concentration of the buffer is important to buffer capac￾ity, and is considered later in this chapter. When Is a Buffer Not a Buffer? Simply having a weak acid and the salt of its conjugate base present in a solution doesn’t ensure that the buffer will act as a buffer. Buffers are most effective within ± 1 pH unit of their pKa. Outside of that range the concentration of either the acid or its salt is so low as to provide little or no capacity for pH control. Common mistakes are to select buffers without regard to the pKa of the buffer. Examples of this would be to try to use K2HPO4/KH2PO4 (pKa = 6.7) to buffer a solution at pH 4, or to use acetic acid (pKa = 4.7) to buffer near neutral pH. What Are the Criteria to Consider When Selecting a Buffer? Target pH Of primary concern is the target pH of the solution. This narrows the possible choices to those buffers with pKa values within 1 pH unit of the target pH. pH pK CH COO CH COOH = + [ ] [ ] - log 3 3 The Preparation of Buffers and Other Solutions 33

Concentration or Buffer Capacity Choosing the appropriate buffer concentration can be a little tricky depending on whether pH control is the only role of the buffer, or if ionic strength or other considerations also are impor tant. When determining the appropriate concentration for pH control, the following rule of thumb can be used to estimate a reasonable starting concentration 1. If the process or reaction in the system being buffered does not actively produce or consume protons(H), then choose a moderate buffer concentration of 50 to 100 mM 2. If the process or reaction actively produces or consumes protons(H,), then estimate the number of millimoles of H* that are involved in the process (if possible)and divide by the solu tion volume. Choose a buffer concentration at least 20x higher than the result of the estimation above The rationale behind these two steps is that a properly chosen buffer will have a 50: 50 ratio of acid to base at the target pH, therefore you will have 10x the available capacity to consume or supply protons as needed. A 10% loss of acid(and corresponding increase in base species), and vice versa, results in a 20% change n the ratio (CH3 COo][CH3COOH from the Henderson- Hasselbalch example above]) resulting in less than a 0.1 pH unit change, which is probably tolerable in the system. While most bio- molecules can withstand the level of hydrolysis that might accom- pany such a change(especially near neutral pH), it is possible that he secondary and tertiary structures of bioactive molecules might be affected Chemical Compatibility It is important to anticipate(or be able to diagnose) problems due to interaction of your buffer components with other solution components. Certain inorganic ions can form insoluble complexes with buffer components; for example, the presence of calcium wi cause phosphate to precipitate as the insoluble calcium phosphate, and amines are known to strongly bind copper. The presence of significant levels of organic solvents can limit solubility of some inorganic buffers. Potassium phosphate, for example, is more readily soluble in some organic solutions than the correspond ing sodium phosphate salt One classic example of a buffer precipitation problem occurred when a researcher was trying to prepare a sodium phosphate buffer for use with a tryptic digest, only to have the Ca(a nec-

Concentration or Buffer Capacity Choosing the appropriate buffer concentration can be a little tricky depending on whether pH control is the only role of the buffer, or if ionic strength or other considerations also are impor￾tant. When determining the appropriate concentration for pH control, the following rule of thumb can be used to estimate a reasonable starting concentration. 1. If the process or reaction in the system being buffered does not actively produce or consume protons (H+ ), then choose a moderate buffer concentration of 50 to 100 mM. 2. If the process or reaction actively produces or consumes protons (H+ ), then estimate the number of millimoles of H+ that are involved in the process (if possible) and divide by the solu￾tion volume. Choose a buffer concentration at least 20¥ higher than the result of the estimation above. The rationale behind these two steps is that a properly chosen buffer will have a 50 :50 ratio of acid to base at the target pH, therefore you will have 10¥ the available capacity to consume or supply protons as needed. A 10% loss of acid (and corresponding increase in base species), and vice versa, results in a 20% change in the ratio ([CH3COO- ]/[CH3COOH from the Henderson￾Hasselbalch example above]) resulting in less than a 0.1 pH unit change, which is probably tolerable in the system. While most bio￾molecules can withstand the level of hydrolysis that might accom￾pany such a change (especially near neutral pH), it is possible that the secondary and tertiary structures of bioactive molecules might be affected. Chemical Compatibility It is important to anticipate (or be able to diagnose) problems due to interaction of your buffer components with other solution components. Certain inorganic ions can form insoluble complexes with buffer components; for example, the presence of calcium will cause phosphate to precipitate as the insoluble calcium phosphate, and amines are known to strongly bind copper. The presence of significant levels of organic solvents can limit solubility of some inorganic buffers. Potassium phosphate, for example, is more readily soluble in some organic solutions than the correspond￾ing sodium phosphate salt. One classic example of a buffer precipitation problem occurred when a researcher was trying to prepare a sodium phosphate buffer for use with a tryptic digest, only to have the Ca2+ (a nec- 34 Pfannkoch

essary enzyme cofactor) precipitate as Ca3(PO4)2. Incompatibili ties can also arise when a buffer component interacts with a surface. One example is the binding of amine-type buffers (i.e Tris) to a silica-based chromatography packing Biochemical Compatibility Is the buffer applied at an early stage of a research project com- patible with a downstream step? A protein isolated in a buffer containing 10mM Mg* appears innocuous, but this cation con centration could significantly affect the interaction between a reg- ulatory protein and its target DNA as monitored by band-shift assay(Hennighausen and Lubon, 1987; Band Shift Kit Instruction Manual, Amersham Pharmacia Biotech, 1994). Incompatible salts can be removed by dialysis or chromatography, but each manipu- lation adds time, cost, and usually reduces yield. Better to avoid a problem than to eliminate it downstream What Can Generate an Incorrect or Unreliable buffer Buffer Salts All buffer salts are not created equal. Care must be exercised when selecting a salt to prepare a buffer. If the protocol calls for an anhydrous salt, and the hydrated salt is used instead the buffer concentration will be too low by the fraction of water present the salt. This will reduce your buffer capacity, ionic strength, and can lead to unreliable results. Most buffer salts are anhydrous, but many are hygroscopic they will pick up water from the atmosphere from repeated opening of the container. Poorly stored anhydrous salts also will produce lower than expected buffer concentrations and reduced buffering capacity. It is always wise to record the lot number of the salts used to prepare a buffer, so the offending bottle can be tracked down if an error is suspecte If a major pH adjustment is needed to obtain the correct pH of your buffer, check that the correct buffer salts were used the ratios of the two salts werent switched, and finally verify the calculations of the proper buffer salt ratios by applying the Henderson-Hasselbalch equation. If both the acid and base com ponents of the buffer are solids, you can use the Henderson Hasselbalch equation to determine the proper mass ratios to blend and give your target pH and concentration. When this ratio is actually prepared, your ph will usually need some minor adjust ment, which should be very minor compared to the overall con- centration of the buffer The Preparation of Buffers and Other Solutions

essary enzyme cofactor) precipitate as Ca3(PO4)2. Incompatibili￾ties can also arise when a buffer component interacts with a surface. One example is the binding of amine-type buffers (i.e., Tris) to a silica-based chromatography packing. Biochemical Compatibility Is the buffer applied at an early stage of a research project com￾patible with a downstream step? A protein isolated in a buffer containing 10 mM Mg2+ appears innocuous, but this cation con￾centration could significantly affect the interaction between a reg￾ulatory protein and its target DNA as monitored by band-shift assay (Hennighausen and Lubon, 1987; BandShift Kit Instruction Manual, Amersham Pharmacia Biotech, 1994). Incompatible salts can be removed by dialysis or chromatography, but each manipu￾lation adds time, cost, and usually reduces yield. Better to avoid a problem than to eliminate it downstream. What Can Generate an Incorrect or Unreliable Buffer? Buffer Salts All buffer salts are not created equal. Care must be exercised when selecting a salt to prepare a buffer. If the protocol calls for an anhydrous salt, and the hydrated salt is used instead, the buffer concentration will be too low by the fraction of water present in the salt. This will reduce your buffer capacity, ionic strength, and can lead to unreliable results. Most buffer salts are anhydrous, but many are hygroscopic— they will pick up water from the atmosphere from repeated opening of the container. Poorly stored anhydrous salts also will produce lower than expected buffer concentrations and reduced buffering capacity. It is always wise to record the lot number of the salts used to prepare a buffer, so the offending bottle can be tracked down if an error is suspected. If a major pH adjustment is needed to obtain the correct pH of your buffer, check that the correct buffer salts were used, the ratios of the two salts weren’t switched, and finally verify the calculations of the proper buffer salt ratios by applying the Henderson-Hasselbalch equation. If both the acid and base com￾ponents of the buffer are solids, you can use the Henderson￾Hasselbalch equation to determine the proper mass ratios to blend and give your target pH and concentration. When this ratio is actually prepared, your pH will usually need some minor adjust￾ment, which should be very minor compared to the overall con￾centration of the buffer. The Preparation of Buffers and Other Solutions 35