BMS481 Buffers and Solutions PDF

CHAPTER 2.0 BUFFERS AND SOLUTIONS Syllabus Content 2.1 Water for biological reactions and cell culture 2.2 pH and buffer systems 2.3 Commonly used buffers in molecular biology 2.4 Calculations in Molecular Biology BMS481 NURUL...

CHAPTER 2.0 BUFFERS AND SOLUTIONS Syllabus Content 2.1 Water for biological reactions and cell culture 2.2 pH and buffer systems 2.3 Commonly used buffers in molecular biology 2.4 Calculations in Molecular Biology BMS481 NURUL AILI ZAKARIA (Ph.D)  COURSE LEARNING OUTCOME At the end of the course, students should be able to: Describe the fundamentals concepts of analytical techniques e.g. accuracy, concentrations, molarity. Illustrate the principles and applications of basic centrifugation, chromatographic and spectroscopic analytical techniques in the isolation and characterization of biological molecules. Perform scientific experiments on biomolecules separation, characterization and analysis.  “Buffers often are overlooked and taken for granted by laboratory scientists until the day comes when a bizarre artifact is observed and its origin is traced to a bad buffer. Although mistakes in the composition of buffers have led occasionally discoveries such as the correct number of human chromosomes (Arduengo, 2010), using the proper buffer, correctly prepared, can be key to success in the laboratory.” Arduengo, P.M. (2010) Sloppy technicians and the progress of science. Promega Connectionshttp://promega.wordpress.com/2010/03/15/sloppy-technicians 2.0 Water for biological reactions and cell culture Why water is important? Most biochemical processes essential for living organism takes place in presence of water Water is the best solvent known – universal solvent Water organize nonpolar molecules Water causes hydrophobic molecules to aggregate or assume specific shapes.  Lets discuss; Can you list and describe the biological important of water due to its polarity? 1. Universal solvent. Describe the example? 2. Water is liquid at room temperature. Describe. 3. Cohesion and Adhesion. Describe. 4. Water form freeze at below 4 degree celcius. Describe. 5. Water posses thermal stability. Describe. 6. Water takes part as reactant in metabolic reactions. Describe. Classification for water purity  H+ and H3O+ is often used interchangeably to represent the Dissociation of water hydrated proton. Water is amphoteric in nature, hence it dissociates. The dissociation is an equilibrium process (water equilibrium) H2O ⇋ OH- + H+ (hydroxide ion) (hydrogen ion) As the H+ ions are formed, they bond with H2O molecules in the solution to form H3O+ 2H2O (ℓ) ⇋ OH- (aq) + H3O+ (aq) (hydronium ion) Remember, pH of a solution is dependent on the concentration of hydronium ions (H3O+) or simply put as H+  Dissociation of water Kw = [H+] [OH-]= 1 x 10 -14 M2 The water equilibrium constant (Kw) is defined as: Kw = [H+] [OH-] where [H+] and [OH-] are concentrations of H+ and OH- ions in the solution. * Experimentally, it has been found that the concentrations: [H+] = [OH-] = 10-7 Therefore Kw = [10-7][ 10-7] = 10-14 (1) The equation (1) shows that the multiplication of the concentrations [H+] and [OH-] is a constant value and equals to 10-14 at 250C. **The sum of exponents in equation (1) must always be equal to 10-14. How to calculate dissociation of water? Eg 1: What is the concentration of OH- in a solution of 0.5 M NaOH? Solution: We begin with the equation for the ion product of water Kw = [H+] [OH-] = 1 x 10 -14 M2 With [OH-] = 0.5 M; solving for [H+] gives [H+] = Kw/ [OH-] = 1 x 10 -14 M2/ 0.5 M = ??? How to calculate dissociation of water? Eg 2: What is the concentration of OH- in a solution with an H+ concentration of 1.3 x 10-4 M? Solution: We begin with the equation for the ion product of water Kw = [H+] [OH-] = 1 x 10 -14 M2 With [H+] = 1.3 x 10-4 M; solving for [OH-] gives [OH-] = Kw/ [H+] = 1 x 10 -14 M2/ 1.3 x 10-4 M = ???  The acidity or alkalinity of solutions 2.2 pH and buffer system can be described by means of hydrogen ion concentrations – 10- 2.2.1 The hydrogen ion exponent (pH) 12, 10-10, 10-4 and etc. But this way is unsuitable and pH values have been introduced in 1924 Definition: pH is defined as the negative decadic logarithm of the hydrogen ion concentration. The equation is: pH = - log [H+] where - [H+] or [H3O+] is hydrogen ion concentration; **The purpose of the negative sign is to give a positive pH value. Kw = [H+] [OH-]= 1 x 10 -14 M2 pKw = pH + pOH = 14 2.2 pH and buffer system 2.2.1 The hydrogen ion exponent (pH) Example If an acid has an H+ concentration of 0.0001, find the pH. Solution: First convert the number to exponential notation, find the logarithm, then solve the pH equation. pH = - log [H+] [H+] = 0.0001 = 10-4; log of 10-4: log (10-4)= - 4; pH = - log [ H+] = - log (10-4) = - (-4) = +4 pH = 4 2.2 pH and buffer system 2.2.1 The hydrogen ion exponent (pH) Example What is the pH of a 3.41 x 10-4 M NaOH solution? Solution: Use this equation. Kw = [H+] [OH-]= 1 x 10 -14 M2 pKw = pH + pOH = 14  2.2 pH and buffer system 2.2.1 The hydrogen ion exponent (pH) pH scale The hydrogen ion concentration [H+], pH numbers and hydroxide ion concentration are represented in the table: pH values: Acidic pH7 Kw = [H+] [OH-] = 1 x 10 -14 M2 2.2 pH and buffer system Solution which resist a change in pH when small BUFFER? amounts of a strong acid or strong base are added A mixture of weak acid and its Conjugate Pair conjugate base (or a mixture of a weak base and its Acid + Base Base + Acid conjugate acid) Conjugate Pair Conjugate Base: remains after H+ is lost (donors) Eg: acid: HCI; conj. Base : CI- Conjugate acid: remains after H+ is gained (receptors)  2.2 pH and buffer system Acidic buffer What buffers do? A buffer functions to resist changes in pH (H+ concentration) However, biologists often hy think of buffers as doing much T..W are more: BU fers t?? f n Providing essential cofactors bu orta p Providing critical salts im Providing essential nutrients for cells and tissues Alkaline buffer  Why can’t we used HCI What make a buffer system to make buffer? A buffer consist of a weak acid (HA) and its conjugate base (A-) or weak base and its conjugate acid Weak acids and bases do not dissociate completely in water, but instead exist in solution as an equilibrium of dissociated and undissociated species. For acetic acid, we would express this equilibrium like this: (HA) (A-)  “A buffer solution has to remove any H+ or OH- that can cause pH change” How buffers work?  Pick buffers by choosing the nearest pKa to the pH we want! Buffer Capacity ”is the amount of acid or base that can be added to a given volume of a buffer solution before the pH changes significantly, usually by one unit.” Ø It depends on the amount of weak acid and its conjugate base that are in the buffer mixture 1L of buffer containing 1.0 M acetic acid and 1.0 M sodium acetate has a greater buffer capacity than a 1L buffer with 0.1 M acetic acid and 0.1 M sodium acetate, even though both solutions have the same pH. **The first solution has more buffer capacity because it contains more acetic acid and acetate ion Buffered & unbuffered solution How to calculate pH of a buffer? Determine by two factors: 1. The equilibrium constant (pKa) of the weak acid 2. The ratio of weak base (A-) to weak acid (HA) in the solution 1. The equilibrium constant (pKa) of the weak acid Different weak acids have different Ka. The Ka tells us what proportion of HA will be dissociated into H+ and A- in solution. The more H+ ions created, the more acidic and lower the pH of the resulting solution. (pKa = -log Ka) 2. The ratio of weak base (A-) to weak acid (HA) in the solution If a buffer has more base than acid, more OH- ions are likely to be present and the pH will rise (vice versa). When the concentrations of A- and HA are equal, the H+ is equal to Ka Formalized in HHE  useful for estimating ***Henderson-Hasselbalch equation the pH of a buffer pKa can be determined using the Henderson-Hasselbalch equation. – With the use of this equation, it is possible to calculate the concentration of acid and conjugate base at all points of titration curves – The equation may be used for: to determine the amount of acid and conjugate base needed to make a buffer solution of a certain pH.  ED! AC K t C R ’s ge Let Solve the following problems… 1. What is the pH of a solution consisting of 0.75M HC2H3O2 (acetic acid) and 0.5M NaC2H302 (sodium acetate) ? Ka of HC2H3O2 is 1.8 x 10^-5 2. What is the pH of a solution consisting of 0.15M NH4CI (Ammonium chloride) and 1.5M NH3? The Kb of NH3 is 1.8 x 10^-5. What makes a “Good’’ buffer In 1996, Norman Good and colleagues developed criteria for buffers for biological systems. A pKa between 6 and 8. Solubility in water. Exclusion by biological membranes. Minimal salt effects. Minimal effects on dissociation from changes in temperature and concentration. Minimal interactions between buffer components and critical reaction components. Chemical stability. Light absorption outside of wavelengths used for assay readout. Components should be easy to obtain and prepare. Good, N.E. et al. (1966) Hydrogen ion buffers for biological research. Biochemistry. 5, 467–77.  1. Optimal buffering at a neutral pH — Most biochemical reactions have an optimal pH in the range of 6–8, so buffers for these reactions need to have pKa(s) that support buffering at these pH values. — Some buffers and their pKa values (in water at 25°C): – Acetate 4.76 – PIPES 6.76 – MOPS 7.20 Buffer is effective in a range about +/- 1 pH – HEPES 7.48 unit of the pKa value – Tris 8.06 – Borate 9.23  2. Solubility in water Most biochemical reactions occur in aqueous conditions, so your buffering components should be soluble in water. If for some reason, you will be using a solvent other than water, make sure you understand how that solvent affects the dissociation of your buffer components. 4. Minimal salt interactions If the system to be studied requires salts, appropriate ions can be added. However, using an ionic buffer can adversely affect the reaction if reaction studied is affected by salts. In other words, the buffer components should not interact or affect ions involved in the biochemical reactions being explored.  5. Minimal effects on the dissociation from changes in concentration Changes in dissociation resulting from changes in concentration are usually small, and most buffers can be made as stock solutions that are diluted to working solutions. However, some buffers do show a significant change in pH upon dilution. For instance, the pH of Tris decreases approximately 0.1 pH unit per tenfold dilution, and the pH could change dramatically if you dilute a working solution and are at the limits of the optimal buffering range of the Tris. 5. Minimal effects on the dissociation from changes in temperature Temperature changes can be a problem too. Eg: Tris exhibits a large shift in dissociation with a change in temperature. (Tris is not one of Good’s buffers) - If you prepare a Tris buffer at pH 7.0 at 4.0°C and perform a reaction in that same buffer at 37°C, the pH will drop to 5.95. - If you have a Tris buffer prepared at 20°C with a pKa of 8.3, it would be an effective buffer for many biochemical reactions (pH 7.3–9.3), but the same Tris buffer used at 4°C becomes a poor buffer at pH 7.3 because its pKa shifts to 8.8. 5. Minimal effects on the dissociation from changes in temperature So the take-home message: Make the buffer at the temperature you plan to use it, and if your experiment involves a temperature shift, select a buffer with a range that can accommodate any shift in dissociation as a result. 6. Minimal interactions between buffer components and critical reaction components If a complex forms between the buffer and a required cofactor, say a metal cation like zinc or magnesium, your reaction might be compromised. For example calcium precipitates as calcium phosphate in phosphate buffers. Not only would any Ca2+-requiring reactions be compromised, but the buffering capacity of the phosphate buffer also is affected. Having excessive amounts of a chelating agent in an enzymatically driven reaction could cause problems (e.g., a high concentration of EDTA in a PCR amplification). Citrate is a calcium chelator, so avoid citrate buffers in situations where calcium concentrations are critical. Watch buffer components that have reactive R groups. For instance Tris has a reactive amine group. Remember buffers are not inert! 7. Chemical stability The buffer should be stable and not break down under working conditions. It should not oxidize or be affected by the system in which it is being used. Try to avoid buffers that contain participants in reactions (e.g., metabolites). Some buffers, such as MOPS, must be protected from light, but when they are stored properly they are still extremely useful buffers in biochemical reactions and laboratory protocols like RNA electrophoresis.  8. Light absorption outside of wavelength used for assay readout Buffer should not absorb UV light at wavelengths that may be used for readouts in photometric experiments. Should not absorb any light at wave-lengths longer than 230 nm, since many spectrophotometric investigations are performed in this range (determination of the concentrations of DNA, RNA and proteins) Preparing buffers Prepare buffers at the appropriate temperature and concentration. If you dilute a buffer or use it at a different temperature than the one at which it was prepared, measure the pH after dilution and equilibration to the new temperature. Adjust the pH of the buffer system correctly. Not all buffers are prepared the same way. Be sure you understand how your buffer system works and that you do not introduce any new ions into the system during pH measurement. Make sure you know how to use and care for the pH meter. Preparing buffers Some buffering components may have to be heated or put in alkaline or acidic conditions before they will dissolve. Some buffers cannot be autoclaved because they will degrade upon heating (so they will need to be filter sterilized). When working with acids and bases be sure to wear the appropriate protective clothing and eyewear. Do not try to neutralize strong acids with strong bases. If you are using a solvent other than water, be sure you know how that solvent affects the pKa of the buffer system.  if they look cloudy or discolored, do not use them. Using buffers Such solutions may have microbial contamination or may have become chemically unstable Check all stored buffers before use. Swirl the bottle to check for any contaminants that may have settled to the bottom during storage. Remeasure pH if you are diluting a buffer before using it in your reaction. Keep detailed notes on buffer preparation so that you can replicate your experiments (or troubleshoot confusing data). Indicate grade of materials used, supplier, and lot no. if known. Indicate what acid or base was used to pH the buffer and its concentration. If additional components were added to the buffer indicate at what point pH was measured. 3.3 Common buffer in biology The decision for or against a buffer is also dependent on the method for which it is used Buffer Application Tris Electrophoresis H2PO4-/HPO42- Protein purification H2CO3/HCO3- Blood buffer Can you name a few? pH and buffers in living systems Common examples of how pH plays a very important role in our daily lives are given below: Water in swimming pool is maintained by checking its pH. Acidic or basic chemicals can be added if the water becomes too acidic or too basic. Whenever we get a heartburn, more acid build up in the stomach and causes pain. We needs to take antacid tablets (a base) to neutralize excess acid in the stomach. The pH of blood is slightly basic. A fluctuation in the pH of the blood can cause in serious harm to vital organs in the body. Certain diseases are diagnosed only by checking the pH of blood and urine. Certain crops thrive better at certain pH range. Using pH meter A pH meter basic principle is to measure the concentration of hydrogen ion. A pH meter is a voltmeter which measures the voltage of an electrode sensitive to the hydrogen ion concentration relative to another electrode which exhibits a constant voltage. This is translated into pH by the instrument and reading is displayed. higher voltages signalling acidic pH levels and lower voltages signalling basic Calibrating pH meter Why? To be certain of accurate and reliable measurements, you need to perform pH meter calibration. Over time, aging and coating of pH electrodes can cause changes in the measurement This is generally done by measuring different buffer solutions with standardized, well-defined values, and then adjusting the pH meter based on any deviations from the buffer’s known pH value. Calibration at three or more pH values (i.e. pH 3, 7, 10) increases the measurement range of the device without recalibration being required.  3.4 Calculation in molecular biology Solution Concentration Dilution Serial Dilutions Metric Unit 0.00000005 g too many 000!; better express as a number between 1 but less than 1000 So ; 50 ng rather than 0.05 mg or 50000 pg.  Solution: Concentration and Calculation — Solution: a homogenous mixture of two or more substances in the same phase. ¡ Solute: substance dissolved in another substance (the solvent) ¡ Solvent: substance that dissolves a solute, resulting in a solution ÷ Water is the “universal solvent” ÷ Other common lab solvents: Ethanol, Methanol, and Acetone Solubility -the ability of one compound to dissolve in another; maximum amount of solute capable of being dissolved by a solvent.  Solution: Concentration and Calculation — Concentration: the quantity of solute present per unit volume of the solvent  Solution: Concentration and Calculation Concentrations for solutions may be expressed in multiple ways (units): 1. Molarity (unit = M; Moles/Liter) - Concentration based on the no of moles of solute per 1 liter of solution Ex: 1 M solution of the alanine (mw=89.1) contains 1 mole or 89.1 g of alanine in a 1L volume. - In molecular biology, typical concentration ranges; mM (1x10-3 M), μM (1x10-6 M), nM (1x10-9 M) Ex: 1 mM solution of alanine has 0.089 g or 89 mg (89.1 x 0.001) alanine in 1 L solution volume.  Solution: Concentration and Calculation 2. Percent by weight (unit = % w/w) - Concentration based on the no of grams of solute per 100 g solution Ex: A 5 % w/w alanine solution contains 5 g of alanine in a 100 g of solution 3. Percent by volume (unit = % w/v) - Concentration based on the no of grams of solute per 100 ml solution Ex: A 5 % w/v alanine solution contains 5 g of alanine in a 100 ml of solution 4. Weight per volume (unit = w/v) - Concentration based on the no of grams, mg, μg of solute per unit volume Ex: mg/ml, g/l, mg/100 ml A 5 mg/ml alanine solution contains 5 mg alanine in a 1 ml of solution  Why would I ever need Dilution to make a dilution? — Dilution: the process of decreasing the concentration of a stock (original) solution by adding more solvent to the solution The equation for dilution is M1V1=M2V2 stock solution= diluted solution M1= molarity of the stock solution M2= molarity of the diluted solution  Type of Dilution 1. Simple Dilution: dilution of a single solution to make a diluted solution 2. Serial Dilution: series of dilutions resulting in progressively diluted versions of the Primary (“original” or “stock”) solution.  Dilution Simple Dilution: dilution of a single solution to make a diluted solution + + 4 cans of water Dilution Factor: A dilution of 1 can to 5 can (Volume of stock solution) Dilution factor (Volume of stock solution) + (Volume of solvent) = 1:5 or 1/5  Serial Dilution — Serial Dilution: series of dilutions resulting in progressively diluted versions of the stock (“original”) solution. — Typically made in increments of 1000, 100, or 10 (logarithmic scale) Dilution factor When you dilute, the concentration change! Ex: if stock solution concentration is 0.5 M then 1:10 diluted solution has concentration of ? M. HOW TO FIND THE DILUTED CONCENTRATION? MOLARITY (M) MOLARITY BY SOLUTION *Mol of solute = mass of solute / MW of solute Calculation dilution: Example Calculate the molarity of a 5L solution containing 126g of HNO3 (63 g/mol). Calculate the number of moles: 126g HNO3 Mass of solute / MW (126 g / 63 g/mol) M= moles of solute liters of solution M= 2mol HNO3 5L M= 0.4 mol/L Molarity : Learning Check 1) Calculate the mass of NaOH (MW: 40 g/mol) needed to prepare 1.0L of a 1.5M solution? 2) 960g of NaOH is used in preparing a 1.5M solution. What volume of solution can be made?  Molarity : Learning check 3) Describe how you would prepare 30 ml of 70 % ethanol solution from 95 % ethanol stock solution? 4) You have 1 L of a 5 M solution. How would you make this into a 2.5 M solution? Molarity : Learning check 5) If 8 ml of distilled water is added to 2 ml of 95 % ethanol solution, what is the concentration of the diluted solution. 6) Calculate the volume should you dilute 133 mL of an 7.90 M CuCI2 solution so that 51.5 mL of the diluted solution contains 4.49 g CuCI2? (MW CuCI2: 134.45 g/mol) THANK YOU QUIZIZZ TIME!!!