Analytical Chemistry Lab - Key Concepts and Techniques

ANALYTICAL CHEMISTRY LABORATORY INTRODUCTION TO ANALYTICAL CHEMISTRY BACKGROUND • Definition: o Analytical Chemistry: The Science of Chemical Measurements. • Types of Questions Asked in Analytical Chemistry o What is in the sample? (qualitative analysis) o How much is in the sample? (quantitative analysis) o What is it ? o How much is there? o How pure is it? o What are the impurities? • Techniques used in Analytical Chemistry: o Wet Chemical Methods : titrations, color-forming reactions, precipitations, etc. o Instrumental Methods : spectrometry, chromatography, etc ANALYTICAL CHEMISTRY • Is a branch of science which deals with the study of the laws and principles which have to do with the processes of determining the composition and identification of a material • Quantitative Analysis o Measures and determines the amount of the different chemicals in the substance being analyzed. • Qualitative Analysis o Identifies or determines the various types of elements and compounds that are in a substance. QUALITATIVE CHEMICAL ANALYSIS • It starts with the determination of large constituents like determining if protein is present in a certain food item. • This could extend to the determination of smaller constituents like what amino acids are present. • In qualitative analysis of ions, o analysis is first done as groups of ions o separated into subgroups o finally, each member ion is confirmed present by specific test ▪ Precipitation reactions ▪ Flame test SCALE OF OPERATIONS • Macro Analysis o utilizes volumes of solutions ranging from 5 to 100 ml • Semi-micro Analysis o uses volumes ranging from 1 drop to approx. 1 ml AREAS OF CHEMICAL ANALYSIS AND QUESTIONS THEY ANSWER • Quantitation : How much of substance X is in the sample? • Detection : Does the sample contain substance X? • Identification : What is the identity of the substance in the sample? • Separation : How can the species of interest be separated from the sample matrix for better quantitation and identification? LESSON 1 - BASIC LABORATORY PROCESSES MEASUREMENT LIQUID • Measuring volumes of liquid o Graduated cylinder o Pipet o Biuret • Liquid assumes a curve with a lowest point at the center called the meniscus. o The volume is read relative to the lower meniscus for colorless and light colored liquids. o For dark colored liquids, the volume is read relative to the upper meniscus. • In reading the meniscus, the eyes line of sight must be perpendicular to the scale as shown in the figure above. o Avoid Parallax Error SOLID • Analytical Balance o Is a class of a balance that has a readability of 0.1 mg or better. o It is required for the balance to have a draft shield. ▪ The purpose of the draft shield is to prevent drafts from influencing the weighing results. ▪ Most analytical balances are so accurate, even just minor breezes caused by someone walking by would disrupt the reading. • Top loading Balance o These balances typically have a readability of 1 mg or less. ▪ This readability range puts them into a class of precision balances. o It is a balance that does not have a draft shield. ▪ The reference to "top-loading" simply demonstrates that there is no draft shield. ▪ It is a balance with an open weighing pan loaded directly from the top. TRANSFERRING LIQUID • Glass wares o Graduated cylinder and Beaker ▪ have an indentation in their mouth o Pair of Funnel and Erlenmeyer or Volumetric Flask

SEPARATION TECHNIQUE • Separation techniques o They are necessary in carrying out qualitative analysis. ▪ From large molecule to micromolecule • Laboratory Materials o Decantation/Filtration Set –up o Evaporation set –up o Distillation set –up o Crystalization o Separatory Funnel o Centrifuge o Spectroscopy o Chromatorgraphy LESSON 2 - APPARATUS AND UNIT OPERATIONS OF QUALITATIVE ANALYSIS CENTRIFUGE • Centrifuges are used in various laboratories to separate fluids, gases, or liquids based on density. • Separation is achieved by spinning a vessel containing material at high speed; the centrifugal force pushes heavier materials to the outside of the vessel. o Centrifugation is a technique used for the separation of particles from a solution according to their size, shape, density, viscosity of the medium & rotor speed. o The particles are suspended in a liquid medium and placed in a centrifuge tube. The tube is then placed in a rotor and spun at a define speed. PRINCIPLES OF CENTRIFUGATION • Centrifugation takes advantage of the density of substance o Within a solution, gravitational force will cause particles of higher density than the solvent to sink, and those less dense than the solvent to float to the top. • As the rotor spins around a central axis, it generates a centrifugal force acting to move particles away from the axis of rotation. If the centrifugal force exceeds the buoyant forces of liquid media and the frictional force created by the particle, the particles will sediment. WHAT SAFETY PRECAUTIONS SHOULD BE TAKEN WHEN WORKING WITH A CENTRIFUGE? • Ensure a sturdy, level worksurface o Always ensure the centrifuge is on an appropriate surface prior to operation. • Balance the centrifuge o Running an unbalanced centrifuge may cause significant damage, and injure the operator and other laboratory personnel. The total mass of each tube should be as close as possible- this becomes increasingly important at very high rotor speeds. Balancing masses to the nearest 0.1 gram is advisable, and it is important to balance tubes by mass, not volume. For example, do not balance a sample consisting of liquid with a higher or lower density than water with an equal volume of water. • Do not open the lid while the rotor is moving o Many centrifuges have a “safety shutoff”. However, this will only stop power to the rotor, which will still spin due to its own inertia for some time until it is slowed to a stop by friction. • If the centrifuge is wobbling or shaking, pull the plug o A little vibration is normal, but excessive amounts can mean danger. First, double check that the tubes are correctly balanced. If this does not resolve the issue, do not operate the centrifuge until it has been serviced by the manufacturer or dealer. HOW DO YOU BALANCE A CENTRIFUGE? • Why you need to balance a centrifuge o Prior to starting the centrifuge, it is necessary to load it correctly. Balancing the centrifuge prevents potential damage to the instrument, and is crucial for safe operation. • How to balance a centrifuge 1. Ensure all sample tubes are evenly filled. If additional tubes are required for balancing, fill them with water or a liquid of similar density to the sample, and ensure the mass is balanced to the nearest 0.1 grams. 2. For each tube inserted in the rotor, add a tube of equal weight directly opposite it. This will ensure the center of gravity remains in the center of the rotor. 3. Rotate the rotor 90° and add two additional tubes directly opposite one another. 4. Repeat. VOLUMETRIC FLASK • Volumetric flasks are used to make solutions with very accurately known concentrations. • The volumetric flask, available in sizes ranging from 1 mL to 2 L, is designed to contain a specific volume of liquid • The flask has a calibration line engraved on the narrow part of its neck. It is filled with liquid so the bottom of the meniscus is on this engraved line. o The calibration line is specific to a given flask; a set of flasks built to contain the same volume will have lines at different positions. • Volumetric flasks are not used for storage of solutions. Once the solution is prepared, it is transferred to a clean, labeled bottle or beaker. o The flask is then washed and rinsed well. The last few rinsings should be with deionized water • Using the Volumetric Flask

BURETS • A buret is a long, narrow tube with a stopcock at its base. • It is used for accurately dispensing variable volumes of liquids or solutions. • It is graduated in 0.1 mL increments, with the 0.00 mL mark at the top and the 50.00 mL mark near the bottom. • The marks do not go all the way to the stopcock. HOW TO USE BURETS • Rinse the burette with the standard solution to be used, and align burette tube vertically. • Fill the burette slightly above the zero mark. To prime the stopcock, drain the burette no further than the nominal capacity. • Refill the burette with titrant free of air bubbles to approx. 5 mm above the zero mark. • Drain liquid to set the zero point accurately. o Important: Meniscus must be read at eye level (parallax-free level). o Automatic burettes: Fill to approximately 5 mm above the zero mark This is adjusted automatically after air release. • Wipe off any drops adhering to the discharge tip. • Open the stopcock and slowly add titrant to the sample (containing the indicator). The discharge tip must not touch the wall of the vessel. Keep swirling the sample vessel lightly while adding titrant, or place it on a magnetic stirrer. • Read the discharged volume at eye level. • Any drops remaining on the tip of the stopcock should be wiped against the vessel wall and rinsed down. It is part of the titrated volume. PIPETS • A pipet is designed to deliver a known volume of a liquid. ◦ • Their volumes range from less than 1 mL to about 100 mL. • Types of pipet o Air Displacement Micropipettes o Positive Displacement Pipette o Volumetric Pipette o Pasteur Pipette o Mohr and Serological Pipettes AIR DISPLACEMENT MICROPIPETTES • These pipettes operate by piston driven air displacement. A vacuum is generated by the vertical travel of a metal or ceramic piston within an airtight sleeve. • These pipettes are capable of being very precise & accurate. • Since they rely on air displacement, they are subject to inaccuracies caused by the changing environment, particularly temperature and user technique. • For these reasons this equipment must be carefully maintained and calibrated, and users must be trained to exercise correct and consistent technique. POSITIVE DISPLACEMENT PIPETTE • These are similar to air displacement pipettes, but are less commonly used and are used to avoid contamination and for volatile or viscous substances at small volumes, such as DNA. • The major difference is that the disposable tip is a microsyringe (plastic), composed of a plunger which directly displaces the liquid. VOLUMETRIC PIPETTE • Volumetric pipettes or bulb pipette allow the user to measure a volume of solution extremely precisely (precision of four significant figures). • Typical volumes are 10, 25, and 50 mL. • Volumetric pipettes are commonly used to make laboratory solutions from a base stock as well as prepare solutions for titration. PASTEUR PIPETTE • Pasteur pipettes are plastic or glass pipettes used to transfer small amounts of liquids, but are not graduated or calibrated for any particular volume. • The bulb is separate from the pipette body. • Pasteur pipettes are also called teat pipettes, droppers, eye droppers and chemical droppers. MOHR AND SEROLOGICAL PIPETTE • The graduations on Mohr Pipette always end before the tip. • Mohr Pipettes are not designed to be completely drained. • The graduation marks on a Serological Pipette always continue till the tip.

USING THE PIPETTE HEATING DEVICES • Utilized for heating and drying • Ovens - Electrically heated ovens are commonly used in the laboratory to remove water or other solvents from chemical samples and to dry laboratory glassware. • Hot Plates - normally used for heating solutions to 100o C or above when inherently safer steam baths cannot be used. • Heating Mantles - commonly used for heating round-bottomed flasks, reaction kettles and related reaction vessels. These mantles enclose a heating element in a series of layers of fiberglass cloth. As long as the fiberglass coating is not worn or broken, and as long as no water or other chemicals are spilled into the mantle, heating mantles pose no shock hazard • Water Bath - used to heat samples in the lab. Some applications include maintaining cell lines or heating flammable chemicals that might combust if exposed to open flame. A water bath generally consists of a heating unit, a stainless steel chamber that holds the water and samples, and a control interface. • Steam Bath - It is a relatively safe way to heat flammable organic liquids. They are designed to heat beakers, Erlenmeyer flasks, and round-bottomed flasks, and have a series of concentric rings that can be removed to adjust to the size of the flask. • Sand Bath - A sand bath is a common piece of laboratory equipment made from a container filled with heated sand. It is used to provide even heating for another container, most often during a chemical reaction. A sand bath is most commonly used in conjunction with a hot plate or heating mantle. LESSON 3 AND 4 - OPERATIONS AND TECHNIQUE INVOLVED IN QUALITATIVE ANALYSIS METHODS OF SEPARATING SOLIDS FROM LIQUIDS • Precipitation • Filtration • Centrifugation • Evaporation • Heating a Solution • Making a Solution Acid, Alkaline or Ammoniacal PRECIPITATION • The common method used in group separations. • Materials: o Test tubes o Medicine dropper o Glass rod • Check for complete precipitation • Large excess of the reagent should be avoided. • Digestion o Process which involves heating the mixture in a water bath for few minutes to allow coagulation to occur. o Commonly employed to colloidal suspension FILTRATION • Is the act of pouring a mixture onto a membrane that allows the passage of liquid and results in the collection of the solid. CENTRIFUGATION • Centrifuge o A device by which a centrifugal force produced by an electric motor speeds up the rate of settling of a precipitate. • Removal of Supernatant Liquid o Hold the tube at an angle. o Draw the centrifugate from tube using capillary pipet. o Transfer the centrifugate to another tube. • Do not disturb the precipitate. • Washing of Precipitate o Add the required amount of distilled water. o Mix o Centrifuge o Collect the centrifugate and transfer it to the tube containing the (previously) separated centrifugate. o Do the procedure twice, but discard the next liquid. • Dissolving Precipitates o Centrifuge o Decant the mixture o Save the solution EVAPORATION • Used to reduce the volume of the solution • Heating in a Water Bath Heating a Solution

MAKING A SOLUTION ACID, ALKALINE OR AMMONIACAL • Making a Solution Acid or Alkaline o Add from a graduated cylinder an amount of acid or alkaline solution less than what is needed to neutralize the solution. o Add some more using a dropper. o Test the solution using litmus paper. • Making a Solution Ammoniacal o Add NH3 or NH4OH o Shake o Observe for the odor of the vapor. • Using pH Paper o Place a piece of pH paper on a clean watch glass. o Use a stirring rod to remove a drop of solution from your test tube. o Touch the stirring rod to the paper and compare the color to the pH key on the package. o Note that the sample should be brought to the paper, not the other way around. This keeps your sample from being contaminated by the dye in the pH paper.