Tools of the Trade 2 Weighing Femtomoles of DNA M13 bacteriophage circular DNA (MW 2.4 ✕ 106, Hydrogen bonding 0 0 7 249 nucleotide between phage Additions of base pairs) DNA and synthetic noncomplementary DNA nucleotide chain 200 200 T-G-C-T-T-A-A-G-G-G- 400 400 Additions of Mass of DNA (ng) complementary DNA 600 600 C-C-C-T-T-A-A-G-C-A 10-unit -G-G-G-A-A-T-T-C-G-Tsynthetic S S -G-G-G-A-A-T-T-C-G-TS S -G-G-G-A-A-T-T-C-G-T nucleotide Change in crystal vibrational frequency (Hz) 012 345 Au electrode bound to Au Time (h) Quartz crystal electrode through a Quartz sulfur atom crystal Left: Synthetic nucleotides attached to a gold electrode on a vibrating quartz crystal immersed in solution at 50°C. The synthetic chain can bind a complementary segment of DNA by formation of hydrogen bonds between G and C and between A and T. Right: Metal electrodes Arrows indicate addition of equal portions of DNA. Additions of complementary DNA lower the vibrational frequency of the crystal but give a smaller response as the electrode becomes saturated with DNA. Upper curve shows no response to noncomplementary DNA. [Y. Okahata, Y. Matsunobu, K. Ijiro, M. Mukae, A. Murakami, and K. Makino, t “Hybridization of Nucleic Acids Immobilized on a Quartz Crystal Microbalance,” J. Am. Chem. Soc. 1992, Electrical 114, 8299.] contacts A vibrating quartz crystal of the type that keeps time in your wristwatch and computer If pressure is applied to a quartz is an exquisitely sensitive tool to measure tiny masses. When a substance becomes crystal, a voltage develops between adsorbed (bound) on the surface of a quartz crystal microbalance, the vibrational fre- the faces. This phenomenon is called the piezoelectric effect. Conversely, a quency of the crystal changes in proportion to the adsorbed mass. sinusoidal voltage applied between The figure above shows how we can detect nanogram quantities of one specific the metal electrodes causes the deoxyribonucleic acid molecule (the genetic material, DNA) from a bacteriophage (a crystal to vibrate with frequency Hzиm)/t, where t is the 679 1) ؍ virus that attacks bacteria). The crystal is coated with a synthetic chain of 10 nucleo- F tides (designated C, G, A, and T) chosen to bind to a complementary sequence on the thickness. A layer of mass m (grams) 2 bacteriophage DNA. When the crystal is immersed in a solution containing many differ- spread over surface area A (m ) ؍ decreases the frequency by ⌬F ent DNA molecules, the complementary molecules bind to the crystal and lower its vi- ؋ 10؊10)F2m/A. A 10-MHz crystal 2.3) brational frequency. A frequency change of 560 Hz corresponds to 590 ng (0.25 pmol) coated with 1 ␮g of material over of DNA. The smallest quantity that could be detected by this system is approximately 1 cm2 changes its vibrational 25 ng—or about 11 fmol.1,2,3 frequency by 230 Hz. Analytical chemistry extends from simple “wet” chemical procedures to elaborate instrumental methods. This chapter describes basic laboratory apparatus and manipula- tions associated with chemical measurements. 23 24 2-1 Safe, Ethical Handling of Chemicals and Waste 2 Tools of the Trade Chemical experimentation, like driving a car or operating a household, creates hazards. The primary safety rule is to familiarize yourself with the hazards and then to do noth- ing that you (or your instructor or supervisor) consider to be dangerous. If you believe that an operation is hazardous, discuss it first and do not proceed until sensible precau- tions are in place. Preservation of a habitable planet demands that we minimize waste production and responsibly dispose of waste that is generated (Box 2-1).4 Recycling of chemicals is practiced in industry for economic as well as ethical reasons; it should be an important component of pollution control in your lab. Before working, familiarize yourself with safety features of your laboratory. You should wear goggles or safety glasses with side shields (Figure 2-1) at all times in the Figure 2-1 Goggles or safety lab to protect your eyes from liquids and glass, which fly around when least expected. glasses with side shields should be Contact lenses are not recommended in the lab because vapors can be trapped between worn at all times in every lab. the lens and your eye. You can protect your skin from spills and flames by wearing a flame-resistant lab coat. Use rubber gloves when pouring con- centrated acids. Never eat food in the lab. Limitations of gloves: In 1997, popular Dartmouth College Organic solvents, concentrated acids, and concentrated chemistry professor Karen Wetterhahn, age 48, died from a ammonia should be handled in a fume hood. Air flowing into drop of dimethylmercury absorbed through the latex rub- the hood keeps fumes out of the lab. The hood also dilutes ber gloves she was wearing. Many organic compounds fumes with air before expelling them from the roof. Never gen- readily penetrate rubber. Wetterhahn was an expert in the erate large quantities of toxic fumes that are allowed to escape biochemistry of metals and the first female professor of through the hood. Wear a respirator when handling fine pow- chemistry at Dartmouth. She was a mother of two children ders, which could produce a cloud of dust that might be inhaled. and played a major role in bringing more women into sci- Clean up spills immediately to prevent accidental contact ence and engineering. by the next person who comes along. Treat spills on your skin first by flooding with water. In anticipation of splashes on your body or in your eyes, know where to find and how to operate the emergency shower and eyewash. If the sink is closer than an eyewash, use the sink first for splashes in your eyes. Know how to operate the fire extinguisher and how to use an emergency blanket to extinguish burning clothing. A first aid kit should be available, and you should know how and where to seek emergency medical assistance. Label all vessels to indicate what they contain. An unlabeled bottle left and forgot- ten in a refrigerator or cabinet presents an expensive disposal problem, because the con- tents must be analyzed before it can be legally discarded. National Fire Protection Association labels, shown in Figure 2-2, identify hazards associated with chemical Box 2-1 Disposal of Chemical Waste If carelessly discarded, many chemicals that we use are A few examples illustrate different approaches to manag- 5 2Ϫ 3ϩ harmful to plants, animals, and people. For each experiment, ing lab waste. Dichromate (Cr2O7 ) is reduced to Cr with your instructor should establish procedures for waste dis- sodium hydrogen sulfite (NaHSO3), treated with hydroxide to posal. Options include (1) pouring solutions down the drain make insoluble Cr(OH)3, and evaporated to dryness for disposal and diluting with tap water, (2) saving the waste for disposal in a landfill. Waste acid is mixed with waste base until nearly in an approved landfill, (3) treating waste to decrease the neutral (as determined with pH paper) and then poured down Ϫ Ϫ hazard and then pouring it down the drain or saving it for a the drain. Waste iodate (IO3 ) is reduced to I with NaHSO3, landfill, and (4) recycling. Chemically incompatible wastes neutralized with base, and poured down the drain. Waste Pb2ϩ should never be mixed with each other, and each waste con- solution is treated with sodium metasilicate (Na2SiO3) solution tainer must be labeled to indicate the quantity and identity of to precipitate insoluble PbSiO3 that can be packaged for a land- its contents. Waste containers must indicate whether the con- fill. Waste silver or gold is treated to recover the metals.6 Toxic tents are flammable, toxic, corrosive, or reactive, or have gases used in a fume hood are bubbled through a chemical trap other dangerous properties. or burned to prevent escape from the hood. FIRE HAZARD (RED) 25 4. DANGER: Flammable gas or 2. CAUTION: Combustible liquid. extremely flammable liquid. Flash point of 100˚ F to 200˚ F. 2-2 The Lab Notebook 3. WARNING: Flammable 1. Combustible if (YELLOW) (BLUE) liquid. Flash point heated. INSTABILITY HEALTH below 100˚F. 0. Not combustible. HAZARD HAZARD 4. DANGER: Explosive 4. DANGER: May be material at room fatal on short exposure. temperature. Specialized protective RED 0 equipment required. 3. DANGER: May be explosive if shocked, 3. WARNING: heated, under Corrosive or toxic: confinement, 30 Avoid skin or mixed with contact water. or inhalation. BLUE YELLOW ACID 2. WARNING: 2. WARNING: Unstable, May be or may react harmful if mixed with if inhaled or water. Label on absorbed. % WHITE 37 wt HCl 1. CAUTION: May react if 1. CAUTION: May heated, or mixed cause with water. irritation. Avoid use W of water ALK – Alkali 0. Stable. 0. No unusual COR. – Corrosive Not hazard. Radiation OXY – Oxidizing Chemicals reactive when ACID – Acid mixed with water. Chemical Name Chemical Abstracts Service No. or MSDS No. Cas. No. Cas. No. Cas. No. Cas. No. Figure 2-2 Chemical hazards label used by the National Fire Protection Association. The number at the top of the diamond indicates a fire hazard rating from 0 (not combustible) to 4 (flammable gas or extremely flammable liquid). The number at the left gives a health hazard from 0 (safe) to 4 (fatal on short exposure), and the number at the right rates the stability from 0 (stable) to 4 (explosive at room temperature).