Here are the steps to prepare the buffers from the given stock solutions:
1. To prepare 100mL of 0.1M Tris-Cl, 0.01M EDTA, and 1M NaCl:
- Take 1mL of 1M Tris-Cl stock, 0.1mL of 0.5M EDTA stock, and 1mL of 1M NaCl stock.
- Bring the final volume to 100mL with distilled water.
2. To prepare 500mL of TENS buffer from stock solutions:
- Take 5mL of 1M Tris-Cl, 0.5mL of 0.5M EDTA, 2.5mL of 2N NaOH, and 10mL of 10
Here are the steps to prepare the buffers from the given stock solutions:
1. To prepare 100mL of 0.1M Tris-Cl, 0.01M EDTA, and 1M NaCl:
- Take 1mL of 1M Tris-Cl stock, 0.1mL of 0.5M EDTA stock, and 1mL of 1M NaCl stock.
- Bring the final volume to 100mL with distilled water.
2. To prepare 500mL of TENS buffer from stock solutions:
- Take 5mL of 1M Tris-Cl, 0.5mL of 0.5M EDTA, 2.5mL of 2N NaOH, and 10mL of 10
Here are the steps to prepare the buffers from the given stock solutions:
1. To prepare 100mL of 0.1M Tris-Cl, 0.01M EDTA, and 1M NaCl:
- Take 1mL of 1M Tris-Cl stock, 0.1mL of 0.5M EDTA stock, and 1mL of 1M NaCl stock.
- Bring the final volume to 100mL with distilled water.
2. To prepare 500mL of TENS buffer from stock solutions:
- Take 5mL of 1M Tris-Cl, 0.5mL of 0.5M EDTA, 2.5mL of 2N NaOH, and 10mL of 10
Here are the steps to prepare the buffers from the given stock solutions:
1. To prepare 100mL of 0.1M Tris-Cl, 0.01M EDTA, and 1M NaCl:
- Take 1mL of 1M Tris-Cl stock, 0.1mL of 0.5M EDTA stock, and 1mL of 1M NaCl stock.
- Bring the final volume to 100mL with distilled water.
2. To prepare 500mL of TENS buffer from stock solutions:
- Take 5mL of 1M Tris-Cl, 0.5mL of 0.5M EDTA, 2.5mL of 2N NaOH, and 10mL of 10
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Buffers and
Buffer Preparation What is a Buffer?
• A buffer is a solution that can resist a
marked change in pH on the addition of small quantities of acid or bases • They are important in maintaining the pH of living systems. Buffering Capacity • A buffer or buffering capacity is the efficiency of a buffer in maintaining a constant pH when a small quantity of an acid or base is added. • It is a measure of the magnitude of resistance to pH change on the addition of an acid or a base. • Buffer capacity is also referred to as buffer value, buffer efficiency, buffer index, or buffer coefficient. • A buffer in its buffering zone can resist a pH change up to ±1 of its buffering zone • Buffering capacity = Amount of acid or base added change in pH Applications of buffer • In the Laboratory • To enable in vitro analysis to occur as they would in biological system by maintaining the normal pH of different physiological conditions • In the Pharmaceutical industry • Buffers are added in drug preparation to minimize drug degradation • Drugs applied through injections are maintained at an ideal pH of 7.4, same as that of blood • In the Cosmetic industry • Use to ensure stability of creams and ointments • They are used in shampoos to counteract the basicity of the detergents present in the shampoo. This is because alkaline pH affects hair fibre surface leading to cuticle damage. • In the Textile Industry • Used to provide the appropriate conditions for dyeing fabrics • The colour strength of dyes is related to a particular pH which is maintained by using different buffer system.
• In the Food Industry
• Set up appropriate conditions for the fermentation process • Used to maintain the acidity of foods in order to preserve its flavour and appearance Buffer Preparation Units of concentration used in Buffer Preparation • Concept of strength (x) and stock • Molarity (M) solutions • Normality (N) • Preparing buffers from stock • Parts solutions • Part per hundred (Percentage) • Parts per thousand (milligram/mL) • Part per million (microgram/mL) • Parts per billion (microgram/µL) Molarity (Molar concentration) • The molarity of a solution is the gram molecular weight of a solute per liter of solution. • Solute + solvent = Solution • A one molar solution is a solution that contains the gram molecular weight of the solute in a litre (1dm3) of the solution • Unit is g/dm3 (M, mM, µM) • Gram molecular weight is also referred to as gram molecular mass or gram formular weight • The molecular mass is always written on the container of every solute and solvent with its unit as g/mol. • The molecular weight can also be calculated if the chemical composition of the compound is known • Examples • Hydrochloric acid – HCl has one Hydrogen ion and and a chloride ion. Their mass number is 1 and 35.5 respectively. To calculate the molecular mass of HCl = (1 x 1) + (1 x 35.5) = 36.5g/mol • Sodium Hydroxide (NaOH) [Na = 23, O = 16, and Hydrogen = 1]. The molecular mass of NaOH = (1 x 23)+ (1 x 16) + (1 x 1) = 40g/mol • It is advisable to always check the molecular weight on the container of the reagent and only attempt to calculate if the information is not available or supplied. • NaCl = (1 x 23) + (1 x 35.5) = 58.5 Preparing buffers with molar concentration 1. Prepare a buffer containing 1M Tris • Molecular weight 2. Prepare a buffer containing • Tris = 121.14g/mol 100mM Tris • Na2 EDTA = 372.24g/mol 3. Prepare 100mls of 100mM Tris • NaCl = 58.44g/mol 4. Prepare 100mL of a buffer containing 50mM Tris and 10mM NOTE EDTA The result should always be written in prose, 5. Prepare 250mL of TE buffer describing the preparation process. (10mM Tris, 1mM EDTA) [pH 8.0) When preparing a buffer, use a volumetric flask to make up the volume Normality (Normal concentration) • The normality of a solution is the gram equivalent weight of a solute per liter of solution. • A one normal solution is a solution that contains the gram equivalent weight of the solute in a litre of the solution • Gram equivalent weight = gram molecular weight number of replaceable hydrogen or hydroxide ion • Unit is eqg/dm3 (N, eq/L, or meq/L) • Like Molarity, the reference volume for normality is also 1 litre • It is important to note the relationship between molar concentration and normal concentration • Normal concentration = Molar concentration number of replaceable hydrogen or hydroxide ion • For HCl, normality = molarity • For H2S04, normality = molarity divided by 2. • Molecular weight of H2S04 = (2 x 1) + (1 x 32) + (4 x 16) = 98g/mol • Equivalent weight of H2S04 = 98 ÷ 2 (since H2S04 has 2 replaceable hydrogen ion) = 49g/mol • Hence 1N H2S04 = 0.5M H2S04 Part per hundred (Percentage) • PPH is also referred to as percentage • Calculations involving concentration in parts does not require using the gram molecular weight or the gram equivalent weight • The reference volume for percentage is 100 millilitres • Hence to prepare a 2% solution. You weight out 2g of the solute and dissolve and make up to 100mL in a volumetric flask Part Per Thousand (PPT) • PPT is also known as milligram per millilitre (mg/mL) • The reference volume for mg/mL is 1 millilitre (1000 microlitres) • For a 20mg/mL solution. You weigh out 20mg and dissolve and make up to 1000µL PPM and PPB • Part per million (microgram/mL) • Parts per billion (microgram/µL) • Note • Preparing buffers with PPM and PPB can be quite dauting without an analytical weighing balance that can weigh micrograms (6 decimal place) • It may be necessary to prepare the buffer as a higher stock concentration (strength) and dilute to the working concentration when needed. Concept of Stock Solutions (X solutions) • Concentrated solutions, that may then be diluted as needed for laboratory applications, are often stored. These are referred to as stock solutions • Concentrated stocks of buffer solutions are prepared to save time and space. • These concentrated stocks can last for a long period of time (if stored properly) and can be easily diluted for use. • Typical stock solutions include 1mTris [pH 8.0], 0.5M EDTA [pH 8.0], and 5M NaCl • When a buffer contains more than one component, then the unit of concentration is the X factor. The X-factor indicates that the solution is concentrated (x = concentration times the number indicated). • In preparing such solutions, you need to measure N times the amount of each solute to come up with a highly concentrated stock solution. • X factors solutions are commonly labeled as 10X, 5X, 100X etc. and must be diluted usually with water to a 1X concentration for use. • Examples include 10x TBE, 50x TAE, 100x TE buffer. Preparing buffers from Stock Solution Tris SDS • C1V1 = C2V2 C1 = 1M = 1000mM, V1 = ?, C1 = 10% = 1000mM, V1 = ?, • Where C1 is the stock concentration. C2 = 10mM, V2 = 1L = 1000mL C2 = 1%, V2 = 1L = 1000mL • V1 is the volume required from the C1V1 = C2V2 C1V1 = C2V2 stock V1 = (C2V2) / C1 V1 = (C2V2) / C1 • C2 is the concentration of the stock V1 = (10 x 1000) / 1000 V1 = (1 x 1000) / 10 solution. V1 = 10mL V1 = 100 mL • V2 is total volume needed for the experiment. • Answer • Example - Make 1 Litre of a solution • Mix 10 mL of 1M Tris and 100 mL which contains 10mM Tris and 1% of 10% SDS and bring to 1 L with SDS, using a 1 M Tris and a 10% SDS ddH20 stock. Examples 1. Prepare 100mL of a buffer containing 0.1 M Tris-Cl (pH 8.0), 0.01 M EDTA (pH 8.0), and 1 M NaCl 2. Prepare 500 mls of TENS buffer • (TENS = 50mM Tris-Cl (pH 8.0), 10mM EDTA (pH 8.0), 1 N NaOH, and 2% SDS) 3. Prepare the above buffer from a 1M Tris-Cl [pH 8.0], 0.5 M EDTA [pH 8.0], 2N NaOH, and 10% SDS) 4. Prepare 10X TE buffer (TE buffer = 10mM Tris [pH 8.0] and 1mM EDTA [pH 8.0])