The fraction 1000mL/1L is a conversion factor -- a number where the numerator and denominator are equal to the same amount but in different units.

Transcription

1 Technicians must learn how to use precision instruments with accuracy to make measurements and prepare solutions. Volume is a measurement of the amount of space something occupies. In a lab, liquids are typically measured in liters (L), milliliters (ml) or microliters (μl). 1 L = 1000 ml = 1,000,000 μl Lab equipment used to measure volume graduated cylinders, pipets, micropipets. Use the smallest instrument possible to measure the volume needed --- to give the least error in measurement. To measure volumes larger than 10 ml, use graduated cylinder. To measure volumes from 10 ml to.5 ml, use a pipet. To measure volumes less than 1 ml use a micropipette. Converting UNITS B S Bigger unit to smaller unit to the right and multiply S B Smaller unit to bigger unit to the left and divide Example: Convert 1.25 L x 1000 ml/1 L = 1250 ml The fraction 1000mL/1L is a conversion factor -- a number where the numerator and denominator are equal to the same amount but in different units. Review: 1. Convert the following: a μl = L b ml = L c ml = μl d. 1.7 L = ml e μl = ml f ml = μl

2 Making SOLUTIONS Solution are prepared daily in most labs virtually all reactions involves proteins or nucleic acids in an aqueous solution. Solutions are mixtures in which substances are dissolved in another substance. The substance being dissolved is the solute and the solvent is dissolving the solute. Water is the solvent in most solutions either deionized or distilled water is used. Mineral impurities are removed that if present might interfere with reactions. Some organic molecules are not water soluble to dissolve these solutes use ethanol, acetone, etc.. as a solvent. Solid solutes are measured by balances used to measure mass or weight of a substance. The standard unit of mass is gram (g). Mass is the amount of matter an object contains. Weight is the force exerted on something by gravity. The amount of solute added to a solvent depends on the solution to be made this proportion is called the concentration. Concentration is measured several ways in biotech labs mass/volume, volume/volume, %mass/volume, molarity and normality. Molarity is a measure of concentration that represents the number of moles of a solute in a liter solution. Normality is a measurement of concentration used for acids and bases that is expressed in gram equivalent weights of solute per liter of solution represents the amount of ionization of an acid or base. Concentration mass/volume Common Unit(s) of measurement g/l mg/ml μg/ml μg/μl % % molarity M mm μm What happens to the ratio of solute molecules to solvent molecules as a solution becomes more concentrated? Making Mass/Volume Solutions The concentration of a solution can be expressed as the amount of mass per unit of volume. For example, a concentration of 1mg/mL means that for every 1mL of solution contains 1 mg of solute. Calculating how much solute is used in a solution: g/ml x ml = g of solute to be weighed out, concentration volume dissolved in the solvent desired desired Example: To make 100 ml of.05 g/ml glucose solution..05 g/ml x 100 ml = g of glucose Answer: 5g Measure out 5 g of glucose and mix it with enough solvent to reach a final volume of 100 ml. PRACTICE making mass/volume solutions Total Concentration Calculation of Mass Needed (g) NaCl Solution Volume mg/ml 5.0 ml of 300 mg/ml 5.0 ml 300 mg/ml 5.0 mlx300mg = 1500 mg = 1.5 g 4.5 ml of 150 mg/ml 4.5 ml 150 mg/ml 4.0 ml of 75 mg/ml 4.0 ml 75 mg/ml 3.5 ml of 37.5 mg/ml 3.5 ml 37.5 mg/ml 3.0 ml of mg/ml 3.0 ml mg/ml

3 Making Solutions of Differing % Mass/Volume Concentrations Rule a 1% solution means 1g solute in 100 ml solvent Step 1: Convert the % to a decimal by dividing % by 100. This changes the units to g/ml. Step 2: Use mass/volume equation. Example: To make 37 ml of 5% NaCl solution. 5g/100 ml =.05 g/ml.05 g/ml x 37 ml = x Answer: x = 1.9 g of NaCl Weigh out 1.9 g of NaCl and dissolve in approximately 20 ml of water. Then bring the volume up to 37 ml. PRACTICE making %mass/volume solutions Solutions Amount of Solute (g) Volume ml Calculations 5.0 % CuSO 4 5 ml 7.0 % gelatin 15 ml 2.5 % NaCl 150 ml 3% protein 25 ml Making Solutions of Differing Molar Concentrations The concentration of many solutions is reported as moles/liter (mol/l or M; the M is spoken molar ). This concentration measurement is called molarity. To understand how to make a molar solution, you need to know what a mole is. o The unit 1 mole is the mass (g), equal to its molecular weight (MW), also called the formula weight (FW). o A mole s worth of molecules is not only defined by mass, but also by evidence that a mole contains 6x10 23 of atoms or molecules. Measure out the molecular weight (MW) in grams of the chemical and you will have a mole of that compound. The MW can be determined by using a Periodic Table and adding the atomic weights of the atoms in the molecule. An easy way, though, is to just read the label of a chemical reagent bottle, which lists the MW or FW. (Molecular weight is the sum of all the atomic weights of the atoms in a given molecule.) Example: The molecular weight of NaCl is 58.5 atomic mass units (amu), since the Na atom weighs 23 amu, and a Cl atom weighs 35.5 amu. Review: 1. What is the molecular weight of each compound? a. NaOH b. MgCl 2 c. MgO

4 It is valuable to know the molecular weight for two reasons. 1. Molecular weight can be used to identify the molecule. Using a mass spectrometer, you can determine the molecular weight of a compound so that it can be recognized in a mixture. 2. Molecular weight can be used to determine how to make up molar solutions. Molarity concentrations are reported as the number of moles per liter (mol/l or M). The unit M is used as a base unit, just like meters, liters, or grams, so you can have millimolar (mmol/l, or mm) and micromolar (μmol/l, or μm) concentrations, both of which are used frequently in biotech labs. How to prepare 1 M solutions? Example: If you wanted a 1 M NaCl solution, you would measure out 1 mole of NaCl (58.5 g) and dissolve it in water to a total volume of 1 L. This gives you 1 mole of NaCl per liter of solution, or 1 M NaCl. How to prepare solutions of differing molarity concentration? A liter of solution is a large volume for most purposes in biotechnology labs. Instead, ml or μl quantities are usually used. To determine how to mix up a smaller volume of solution, follow the equation below. Molarity Concentration Equation Volume desired x Molarity desired x MW of solute = the # of grams to be (L) (mol/l) (g/mol) dissolved in solvent, up to the total volume of solution desired Example: Make 20 ml of a.5 M CaCl 2 (calcium chloride) solution. 1. Convert 20 ml =.02 L 2. Find MW of CaCl 2 ( =111 g/mol) 3. Use formula..02l x.5 M x 111g/mol = 1.1g of CaCl 2 with solvent to a total of 20 ml PRACTICE making molar solutions Molarity (M = mol/l) Volume (L) MW (g/mol) Mass Calculations 1.0 M CuSO4 5 L.5 M NaCl 500 ml.25 M MgO 200 ml 125 mm NaOH 750 L

5 Proportional Method Example: If g of CaCl 2 in 1L is a 1M solution, then 11.1g of CaCl 2 in 100 ml is also a 1M solution g CaCl 2 = g CaCl 2 = 11.1g CaCl 2 1L 1000 ml 100 ml Dilutions of Concentrated Solutions When solutions are too concentrated to use the way they are initially prepared dilutions (addition of more solvent) of a concentrated solution is needed. Very often in biotech labs, you will dilute a concentrated stock solution to use as a working solution. This is done to save time, as well as space in the lab. Also, it is often more accurate to prepare a concentrated stock solution because it involves weighing larger masses of chemicals (therefore less error in measurements). Dilution Equation C 1 V 1 = C 2 V 2 C 1 = the concentration of the concentrated stock solution V 1 = the volume to use of the stock solution in the diluted sample C 2 = the desired concentration of the diluted sample V 2 = the desired volume of the diluted sample Example: How would you make 1L of 1mg/mL protein solution from 100 mg/ml concentrated stock solution? 1. Convert 1 L to 1000 ml. 2. Use formula 100mg/mL x V 1 = 1mg/mL x 1000mL V 1 = 10 ml To dilute the concentrated solution to make 1L of 1mg/mL solution, take 10 ml of concentrated stock and add 990 ml of solvent. Protein and nucleic acids are stored in buffered solutions. Buffers resist changes in ph, and help preserve the structure and function of molecules dissolved in the buffer. Depending on the kind of buffer needed, different salts at various concentrations are used. A common buffer salt is TRIS a complex organic molecule used to maintain the ph of a solution. TRIS is used in TE buffer (a buffer for storing DNA) and in TAE buffer (used for running DNA samples on agarose gels). When TAE buffer is prepared at a concentration that is ready to use, it is called 1XTAE. 1X is the working concentration. (TAE has 3 ingredients -- TRIS, acetic acid, EDTA.) Example: How is 600 ml of 1XTAE buffer made from 40XTAE buffer stock solution? 40XTAE x V 1 = 1XTAE x 600mL V 1 = 15 ml So to dilute the 40XTAE to working concentration of 1XTAE, mix 15 ml of 40XTAE with 585mL of deionized water. This makes 600mL of 1XTAE.

6 Practice dilutions Calculations Volume stock to use Volume H2O to add Prepare 40mL of a 2mg/mL protein solution from 10mg/mL protein solution. Prepare 200mL of 2X enzyme buffer from 10X enzyme buffer. Prepare 3L of 1XTAE buffer from 50XTAE buffer stock solution. Serial Dilutions When a number of dilutions must be made, and each is proportionally the same dilution as the one before, it is called a serial dilution. Doing a serial dilution makes sense for many experiments when many samples of varying concentrations are needed. A serial dilution is also useful for preparing very dilute solutions that are hard to make from scratch, because the solute masses can be too small to measure on a balance.

7 Significant Figures Rule 1: The number of significant figures is the number of digits to the right of the first non-zero number &.01 all have one significant figure. Zeros to the right of a whole number are not significant. 1, 40 & 6000 all have one significant figure, while 420 & 7,200 have two significant figures. If zeros are between two numbers, they are significant. 402 & 1040 have three significant figures. If the number has a decimal point, the zeros to the right of the decimal point are significant has six significant figures. Rule 2: When adding or subtracting numbers, the resulting numbers cannot have any more significant figures to the right of the decimal point than any value used in the calculation. Example: 21.3g 0.232g = g is rounded to 21.1g. The value 21.3 is the least precise measurement and has one significant figure to the right of the decimal. Rule 3: When multiplying or dividing numbers, the answer cannot have any more significant figures than the value in the calculations with the fewest significant figures. Example: 30.5μL x 2.223mM = μl is rounded to 13.6μL 5.00mM The values 30.5 and 5.00 have the fewest significant figures (3) than other values in the calculation. Scientific Notation Method to display numbers that convey their level of precision. Use a number that is multiplied by 10 raised to a positive or negative number. Example: 1,200,000 = 1.2 x 106 or.169 = 1.69 x 10-1 Prepping Glassware 1. Add small amount of laboratory soap and use a brush to make sure all surfaces are washed 2. Rinse with tap water until no suds are evident, usually at least 3 times 3. Rinse twice with distilled water 4. Air-dry or use oven to dry Labeling Solutions o Name of solution o Concentration o Date made o Name of person who made it o Potential hazards if necessary BASIC SKILLS In Biotechnology Standards: HS-EB-5: Demonstrate how concepts of physical science connect to biochemical applications and techniques. 5.1 Calculate and prepare buffers, stock solutions, and reagents. 5.2 Apply the concepts of homeostasis, normality, and molar relationships to biochemical reactions. 5.6 Demonstrate proficiency in the use of basic laboratory equipment, electronic and analytical balances, autoclave, micropipetting, pouring agarose/agar, etc.