Presentation on theme: "Terms Used in Biochemistry & Calculations. ■ Tips to Success in Biochemistry Lab 1.Write down a clear plan before you start working. 2.Keep very careful."— Presentation transcript:
■ Tips to Success in Biochemistry Lab 1.Write down a clear plan before you start working. 2.Keep very careful notes so you can recheck what you did if you get unexpected results. 3.Label all tubes and samples so they don't get mixed up. 4.Work slowly and carefully. Accurate, reproducible results in a biochemical test like this requires care on your part. If you are sloppy or careless your results will suffer.
■ Terms used in biochemistry lab Liter-------volume unit. 1L = 1000ml = 1000000 μl Gram--------mass unit (weight). 1g = 1000mg = 1000000 μg
Units of Concentrations 1- Mass unit / volume unit: Example: mg/ml, mg/l, g/l, g/dl, mg%, etc.
Units of Concentrations 2- Molarity (M): Another way of expressing concentration is called molarity. Molarity is the number of moles of solute dissolved in one liter of solution. The units, therefore are moles per liter, specifically it's moles of solute per liter of solution. Molarity = moles of solute liter of solution
Units of Concentrations 2- Molarity (M): Molarity = moles of solute liter of solution Rather than writing out moles per liter, these units are abbreviated as M or M. M = wt × 1000. MW × V(ml)
Units of Concentrations 2- Molarity (M): To convert from mg% to mmol/l: mmol/l = mg × 10. MW substance
Units of Concentrations 3- Normality (N): When you need to compare solutions on the basis of concentration of specific ions or the amount of charge that the ions have, a different measure of concentration can be very useful. It is called normality. No. of equivalent weight per liter of solution. Eq.wt = molecular weight (MW) Valence N = wt × 1000. Eq.wt × V(ml)
Problem: 1- How many grams of glucose are needed to make 100 ml of a 0.6 mol/l solution? (MW glucose = 180).
Problem: 2- How can you prepare 0.1 M NaOH solution?
Problem: 3- Describe the preparation of 5 L of 0.1 M Na2CO3 (MW = 105.99) from the primary standard solid.
Units of Concentrations 4- Percent Concentration (%): 1-Volume percent is usually used when the solution is made by mixing two liquids. The use of percentages is a common way of expressing the concentration of a solution. The percentages can be calculated using volumes as well as weights, or even both together. One way of expressing concentrations is by volume percent. Another is by weight percent. Still another is a hybrid called weight/volume percent. Volume percent (v/v) = volume of solute × 100. volume of solution
Example: Rubbing alcohol is generally 70% by volume isopropyl alcohol. That means that 100 ml of solution contains 70 ml of isopropyl alcohol. That also means that a liter (or 1000 ml) of this solution has 700 ml of isopropyl alcohol plus enough water to bring it up a total volume of 1 liter, or 1000 ml.
Units of Concentrations 4- Percent Concentration (%): 2- Weight percent: is expressing the concentration of a solution in weight percent (or mass percent). Weight percent (w/w) = weight of solute × 100. weight of solution
Question: What is the weight percent of glucose in a solution made by dissolving 4.6 g of glucose in 145.2 g of water? To get weight percent we need the weight of the solute and the total weight of the solution. Determine total weight of solution: 4.6g + 145.2 g = 149.8 g Calculate percent: Weight % glucose = 4.6g glucose × 100 = 3.1% glucose 149.8 g solution
Units of Concentrations 4- Percent Concentration (%): 3- Weight- volume percent: Another variation on percentage concentration is weight/volume percent or mass/volume percent. This variation measures the amount of solute in grams but measures the amount of solution in milliliters. Weight-Volume percent (w/v) = weight of solute × 100 volume of solution An example would be a 5 %( w/v) NaCl solution. It contains 5 g of NaCl for every 100 mL of solution.
Balance Rules and Instructions The Figure illustrates one type of top loading electronic balance. Refer to this figure when following the steps and precautions for using the balance listed below:
Balance Rules and Instructions 1. Never pour or transfer chemicals over the balance. Spilled chemicals can damage the balances, which are very expensive to repair or replace. Never weigh warm or hot objects; if you can feel any heat, the weighing will not be accurate. Always use a container such as a vial, beaker, flask, or watch glass to weigh a solid or liquid chemical on the balance to protect the balance pan.
Balance Rules and Instructions 2. Make sure your hands are clean and dry before you handle containers or objects that are to be weighed. The outside of these containers or objects must also be clean and dry. Clean up any spills on the balance pan or lab bench around the balance immediately.
Balance Rules and Instructions 3. First open or remove the draft lid or cover (if there is one) and check to make sure that the balance pan is clean. If the pan is dirty, have your TA show you how to clean it and gently place it back on the balance.
Balance Rules and Instructions 4. Close or put the draft lid back on the balance and zero the balance by pressing the "T" or "on/tare" button (re- zero bar on Mettler balances). Wait 5-10 seconds for the weight display to stabilize. (If the object to be weighed is so large that the draft lid can't be used, do this step without the draft lid in place.)
Balance Rules and Instructions 5. Open or remove the draft lid and place the object to be weighed on the balance pan. Then close or place the draft lid back on the balance. (As long as it does not touch the object to be weighed, leave the lid off if it does touch the object.) After 5-10 seconds the weight display will stabilize and you can record the mass to ±0.001 g.
Balance Rules and Instructions 6. Never unplug the balance.
Dilution Diluted solutions can be prepared from concentrated solutions. Mconc. × Vconc. = M dil. × V dil. Moles taken from concentrated solution = Moles placed in diluted solution
Problem: How can we prepare 100 ml of 0.04M K2Cr2O7 from 0.2M K2Cr2O7?
Serial Dilution This technique involves the removal of a small amount of an original solution to another container which is then brought up to the original volume using the required buffer or water.
Serial Dilution Example: if you have 1 mL of your original solution and you remove 10 µL and place it in a tube containing 990 µL of water or media you have made a 1:100 dilution.
Serial Dilution Here is an example of how to do a series of serial dilutions: 1 ml extract + 4 ml water = 1/5 dilution 1 ml 1/5 dilution + 4 ml water = 1/25 dilution 1 ml 1/25 dilution + 4 ml water = 1/125 dilution Dilution factor = Final volume Initial volume
Standard Curve A standard curve is a quantitative research tool, a method of plotting assay data that is used to determine the concentration of a substance.
Standard Curve The assay is first performed with various known concentrations of a substance similar to that being measured. For example a standard curve for protein concentration is often created using known concentrations of bovine serum albumin.
Standard Curve The assay procedure may measure - absorbance, - optical density, - luminescence, - fluorescence, - radioactivity, or something else.
Standard Curve This data is used to make the standard curve, plotting concentration on the X axis, and assay measurement on the Y axis. The same assay is then performed with samples of unknown concentration.
Standard Curve To analyze the data, one locates the measurement on the Y-axis that corresponds to the assay measurement of the unknown substance and follows a line to intersect the standard curve. The corresponding value on the X-axis is the concentration of substance in the unknown sample.
Standard Curve Here is an example of how construct a standard curve. Concentration mg/ml Absorbance 545nm 0.50.1 1.00.2 1.50.3 2.50.4 5.00.5 10.00.6 25.00.7 PROTEIN STANDARD CURVE... a plot of absorbance @ 545nm vs. protein concentration
Definition of pH pH: is a measure of acidity and the alkalinity of a solution in terms of hydrogen ion H+ or (hydronium ion concentration) pH= -log[H 3 O+] Thus, it is evident that the pH is inversely proportional to the acidity. Lower the pH, higher the acidity or hydrogen ion concentration while higher the pH, the acidity is lower.
Definition of pH Just as pH is convenient way to represent the concentration of H 3 O+, pOH is convenient way to express the concentration of OH-. pOH= -log[OH-]
Definition of pH A solution is acidic if its pH is less than 7 A solution is basic if its pH is grater than 7 (base is any substance that accept H+) A solution is neutral if its pH is equal to 7 pH value of some common materials Material pH Gastric juice 1-3 Vinegar 2.4- 3.4 Urine 5.5- 7.5 Milk 6.3- 6.6 Saliva 6.5- 7.5 Pure water 7 Blood 7.35- 7.45 Sea water 8- 9 Ammonia 11.7
Definition of pH There are 2 ways to measure the pH: 1.By using a pH paper which is a plane paper soaked with mixture of pH indicator. Some acid-base indicators.
Definition of pH There are 2 ways to measure the pH: 2. By using pH meter this method is more accurate and more precise.
Definition of Buffer Is a solution that resists change in pH when limited amounts of an acid or a base are added to it. buffers consist of weak acids + corresponding salt Or weak base + corresponding salt.
Definition of Buffer * Applications Their resistance to changes in pH makes buffer solutions very useful for chemical manufacturing and essential for many biochemical processes. Buffer solutions are necessary to keep the right pH for enzymes in many organisms to work. Many enzymes work only under very precise conditions. Industrially, buffer solutions are used in fermentation processes and in setting the correct conditions for dyes used in coloring fabrics. The acid-base balance or pH of the body fluids is maintained by a closely regulated mechanism. This involves the body buffers, the respiratory system and the kidney.
Definition of Buffer * Selection of the buffer: The selection of a particular buffer for a given application is based on two consideration: 1.the desired pH 2.chemical compatibility of the buffer components with the sample
Definition of Buffer Example of the Preparation of phosphate buffer Preparation of 0.1M sodium phosphate buffer at 25ºC. Phosphate buffer is composed of NaH2PO4 (acid) and Na2HPO4 (base). Preparation of 0.1M sodium phosphate buffer at 25 º C Volume of 1M NaH2PO4 (mL) Volume of 1M Na2HPO4 (mL) Desired pH 92.1 7.95.8 88.012.06.0 18.104.22.168 74.525.56.4 64.835.26.6 53.746.36.8 42.357.77.0 31.668.47.2 22.6 77.4 7.4 15.584.57.6 10.489.67.8 6.893.28.0 Samar A. Damiati