Lab One Weights Volume Solution and Dilutions Biochemistry quantitative science Fructose 6 phosphate ATP Fructose 1 6 biphosphate ADP Assays are 1ml or less of the final volume CuSO4 has a pale blue color due to the hydrated complex the amount of blue can be measured by a spectrometer Absorbance is related to concentration C1V1 C2V2 Water is precisely 1 gram per cubic centimeter 1 g ml 0 degrees C Or 9982 g ml 20 degrees C 1ul of water will weigh 1 milligram A 5 error in pipetting is normal Concentration amount of one substance uniformly dispersed within a known amount of a second substance expressed at molarity Weight and weight solutions mass of solute and solvent together 100 grams Weight and volume solutions final volume is 100 ml Volume and Volume solutions 100 ml final volume Dilutions C1V1 the concentration and volume of the initial undiluted stock solution C2V2 is the final desired concentration and volume Example pg 8 Volume Ratios and Serial Dilutions Examples page 9 Lab Acivity 2 UV Visible Absoprtion Spectroscopy Gamma 5 X Rays 3 Ultraviolent 10 1 1 10 102 Visible light between 102 and 103 Infared 104 Microwaves 106 Radio 109 and up Visible between ultraviolet and infrared Violet 380 435 Blue 435 500 Cyan 500 520 Green 520 565 Yellow 565 590 Orange 590 625 Red 625 740 Unit of light photon When a molecule absorbs a unit of light an outer orbital valence electron is temporarily premoted to a higher energy level energized or excited state A substance wills absorbs wavelengths proportional to the amount of energy it takes to boost the electron to the outer energy level A graphical plot of the amount of energy absorbed over a range of different wavelengths is called and Absorbance spectrum Most electronic tansitions for biomolecules lie in the visible and ultraviolet region 200 700 nm of the electromagnetic spectrum Spectrometers Polychromatic Light Source incandescent or tungsten lamps emit wavelengths of visible light 400 800 nm while hydrogen or deuterium lamps emit wavelengths of light in the UV range 200 360 nm Modern spectrometers often use xenon light which is close to sunlight The emit wavelengths of light from 190 1100 nm EMITS LIGHT Monochrometer a device that both splits polychromatic light into its component wavelengths and a slit or wavelength selector that allows only selected wavelengths of light to reach the sample The dispersive device it typically a prism SPLITS LIGHT Detector A photometric device that measures and or amplifies the amount of transmitted light that emerges from the sample MEASURES THE WAVELENTGH Recorder Computer compares the intensity of the incident vs transmitted beams performs simple mathematic calculations and records stores or plots the data from the output detector Beer Lambert Law a mathematical expression that relates absorbance concentration and path length When an incident beam of light I0 if the right wavelength passes through and excites a substance i e is absorbed the energy or the intensity of the transmitted light that emerges from the substance I is measurably reduced The amount of a given wavelength absorbed by a given substance Absorbance A is defined as A log10 I I0 I transmitted I0 absorbed Or A log10 I0 I Absorbance is unitless but may be expressed as OD The amount of light absorbed by a given substance depends on 1 Concentration 2 Distance or path length L of sample through which the light must pass If the concentration of a light absorbing compound doubles so will the amount of light absorbed over the same path length Concentration and absorption are proportional moles liter A ecl e extinction coefficient c concentration l path length the extinction coefficient is a measure of the efficiency with which a compound absorbs light of a specific wavelength Is a constant when all other parameters are held constant Expressed as inverse concentration of the inverse path length M 1 x cm 1 C refers the the concentration of the light absorbing compound M molar Path length is most often 1 cm Determining the concentration of an unknown substance C A E E 340 is 6220 Transmittance T 100 I I0 A log 100 T Blanks can absorb 50 of light Polystryene and glass curvettes quickly become opaque in the UV range less then 320nm Qaurtz is the best curvette but the most exspensive Acrylic curvettes are intermediate in their spectral properties Plastic curvettes cannot be used for organic solvents Our spectrophotometer is a xenon light source costing 5000 1500 Chemical reactions are carried out by biological catalyst enzymes Reactivity measured by the increasing concentration of a product given a product of a given enzymic reaction as it is formed or the decreasing concentration of a reactant in that same reaction as it is consumed Enzyme Kinetics measurement of enzyme activity Rate of reaction measured in umoles mins ml Lactate Dehydrogenase is an enzyme that catalyzes colorimetric reactions Present in most body tissues where it catalyzes the reversible oxidation of lactic acid to pyruvic acid while using nicotinamide adenine dinuelcotide NAD as the hydrogen electron acceptor Reaction equilibrium strongly favors the reverse reaction namely the reduction of pyruvic acid to lactic acid NADH absorbs UV radiation 340nm while NAD A E x C x l Lab Activity Three Acids Bases Buffers and Titrations Acids bases buffers and titrations all affect the hydrogen ion concentration in aqueous solutions Acid dissociates in water to release or donate Hydrogen Ions Bases release OH ions or accept H Strong acids and bases disassociate completely pH log10 H pOH log10 OH pH scale is inversely logarithmic for every increase in pH by a unit there is a 10 fold decrease in the H Any change in acidity or alkalinity is always accompanied by a corresponding but opposite change in alkalinity and acidity Measurement and control of pH Buffers are weak acids or bases that are used to control pH Buffers Weak acid or base buffer equation HA H AB H BH Physiological pH pH optimal for blood is 7 4 HA is the weak acid and A is its conjugate base and B is the weak base and BH is its conjugate acid KA H A HA Ka is the disassociation constantan The smaller the Ka the weaker the acid Ka is constant pKa log10 Ka Henderson hasselbach relates pH pKa and the acid base components of a buffer systems pH pKa log10 A HA A is the conjugate base HA is the undissociated acid Used to predict the pH of a weak acid pKa is the pH of a weak acid or base 50 dissociated i e A HA log10 of 1 0 pH pKa