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Amazing Biology 290 Exam Notes.doc

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Department
Biology
Course
Biology 2290F/G
Professor
All Professors
Semester
Fall

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Biology 290 Exam NotesDean Unit17 Spectrophotomety Transmittance T is the fraction of the incident light that passes through is transmitted by the sample T II where I is the amount shined on the sample and I is the amount of light that passes through the sample OOAbsorbance A is the amount of light absorbed by the sample Lamberts Law Thin layers of materials will absorb a constant amount of light regardless of how much light is shined on it Light quality decreases exponentially as it passes through the sample Therefore absorbance A is a logarithmic function of transmittance A log 1Tlog IIOIf all the incident light is transmitted though the sample A log 11 0 The amount of radiant energy transmitted by a substance at different wavelengths and therefore the amount of light absorbed by it can be measured by placing a solution of the substance in a spectrophotometer We usually use absorbance values instead of transmittance values because there is a linear relationship between absorbance and solution concentrationBeers Law absorbance of a solution is proportional to the number of absorbing particles or concentration of the absorbing material in solution A E x l x cA absorbance no unitsE absorption coefficient Lmolcm E is a measure of the probability that a given type of photon will be absorbed by a given type of molecule E varies with wavelength and the solvent used c concentration of the solution molLl path length through the spectrophotometer tube usually 1 cmIn the Spectronic 20 radiant energy of wavelengths 340 900 nm is supplied by a tungsten lamp The light is focused onto a diffraction grating which like a glass prism splits the light into its component wavelengths Different wavelengths are selected by changing the position of the diffraction grating so that light of a very narrow range of wavelength passes through a slit before it reaches the sample It is the wavelength cam that is responsible for changing the direction of the diffraction grating Any light transmitted by the sample reaches a phototube which transduces the light energy to an electric current The current produced is measured by a galvanometer amp meter which is calibrated in terms of how much light of a given wavelength the sample transmits and how much is absorbed by the sample The spec 20 does not measure absorbance even though it gives an absorbance reading it measures the amount of light transmitted by a sample The transmittance is then compared with the transmittance of a reference blank to determine how much light has been absorbed by the sample The blank should contain all of the substances in the sample except for the material whose absorbance you wish to measure The blank is inserted into the sample holder and the meter is adjusted to 100 T This compensates for any absorbance by the solvent in which the sample is dissolved and for the reflection of light from the surface of the tube which holds the sample Therefore the difference between the reference and the sample is the absorbance The major uses of the spectrophotometer are1to produce absorption spectra that can be used to characterize or identify compounds in solution2to make quantitative determinations of the concentration of materials in solution by reference to your own standard curves3to study the rate at which biochemical reactions proceed Absorption SpectrumsSpectrophotometry can be used as a means to identify or characterize compounds in solution by producing a characteristic absorption spectrum An absorption spectrum is a graph of the absorption values for a substance measured at a series of different wavelengths Generate a graph of absorbance vs concentration The graph will exemplify Beers law which states the absorbance of light is proportional to the number of absorbing particles in solution Spectrophotometry can therefore be used to measure an unknown solution concentration given a standard curve of known concentrations of that substance vs absorbance The absorbance coefficient E can be calculated from the slope of the standard curve E Ac0504mn 00036 ta ecna02brosbAStandard Curve of Absorbance Versus Various Concentrations of DCPIP01000511522533545Concentration of DCPIP M x 10 The spectrophotometer can also allow us to determine the rate of a biochemical reaction Calculate the DA values at a given point 1 min This DA value represents a change in oxidized DCPIP concentration Given the absorption coefficient E the change in DCPIP concentrationminute Dcmin can be calculatedDcmin DAminE molesL of DCPIP reducedminMultiply Dcmin x total volume of reaction mixture mL 1000mL used to get the moles of DCPIP reducedminute which is the average rate As the initial concentration of the reducing agent Sodium Hydrosulphite increases the average rate increases linearly 45 84E0The relationship between moles of DCPIP reduced per minute and the initial concentration 1 35x etunof Sodium Hydrosulphite 3im reni25p mdleoc2umder P15ICD f1o selo05M0012345Initial concentration of Sodium Hydrosulphite x 10E5 molL8 Resolution in Microscopyd Resolution is the smallest distance by which two objects must be separated in order to allow them to be imaged as two separate objects instead of one A When a particle is examined with a light microscope its periphery appears blurry at high magnification because of diffraction 0202 B When two adjacent particles are separated by about 02 m their images will overlap but their individual character can still be seen ie they can be resolvedC When two adjacent particles are separated by less than 02 m the images of the two particles fuse The same image would appear if the particle was actually a single oblong structure These particles therefore cannot be resolved Abbes equation gives the theoretical limit of resolution in an optical systemdthe limit of resolution ie the limiting distance between two points such that they are still resolvable as separate pointsd0612n sin 0612a constant derived from experimental studies of diffraction at the plane of the specimenwavelength of imageforming radiation light in this casen sin numerical aperture of the lens It is a measure of the light gathering capacity of the lens The aperture angleis the angle subtended by the optical axis and the outermost rays that enter the objective lens The numerical aperture value is obtained by multiplying the sin ofby the refractive index n of the medium filling the space between the cover slip and the lens ie numerical aperturen sinAir has a refractive index of 1 n1 so it can be ignored when dry objective lenses are used Immersion oil can be used to fill the space between the cover slip and the objective of an oil immersion lens The oil has a refractive index of 1515 and this can produce a considerable gain in numerical aperture From Abbes equation it is clear that to attain maximum resolution with a light microscope ie a minimum value for d the value forshould be minimized and the value for n sinshould be maximized To do this1 n must be increased as far as possible beyond the value of that for air n1 by using an immersion lens and immersion oil2must be as close to 90 as possible although this limiting angle cannot be obtained because the specimen must be placed at some finite distance from the lens 3must be near 400nm violet light which is the shortest wavelength to which the average eye is sensitive In practice yellowgreen light 520580 nm is used since the eye is more sensitive to this part of the spectrumExample If n1515 and sin 087 and 400 nm the theoretical resolution attainable isd0612400 186 nm or 0186 m1515087The actual practical limit of resolution for biological specimens in a light microscope is about 0510 m 9 Estimating the number of cellsi HaemocytometerDirect counts of the number of cells in a sample of culture medium can be made using a particular kind of microscope slide the haemacytometer The haemacytometer is a thick glass slide with lines etched precisely on the surface to form two counting grids At the centre of each grid is a 1mm x 1mm square This square is divided into 25 smaller squares 5 rows x 5 columns that are surrounded by triple lines Each smaller square is again divided into 16 even smaller squaresTo optimize the sampling of cells with the haemacytometer you need to find a dilution of your cell suspension that will produce about 20 cells per square to be counted 1015 cells per square is OK 4060 cells per square is OK This is done empirically Place the cover on the haemacytometer and then draw up a small amount of cell suspension into a pipette Place the pipette tip carefully at the edge of the cover and gently expel the suspension so that both counting chambers are filled by capillary action Do not overfill or underfill the chambers Allow the cells to settle for 1 minute
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