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Biology 2290F/G
Irene Krajnyk

Biology 2290 Dean Unit Spectronic 20 Optical System and Spectrophotometry - Radiant energy is focused onto a diffraction grating which splits the light into its component wavelengths - Different wavelengths can be selected by changing the position of the grating, allowing only a narrow range of wavelength to pass through the slit and reach the sample - Any light not absorbed (transmitted) by the sample reaches a phototube that converts it to electrical current - This current is measured by a galvanometer (amp meter) - Output information is both transmittance and absorbance, but the machine only measures transmittance - It compares measured transmittance to that of a reference blank to determine absorbance - Absorbance is the amount of light absorbed by a sample - Transmittance is the fraction of light that passes through the sample - Lambert’s Law: for a thin layer of ample, the same proportion of light is absorbed regardless of the radiant power of the incident light o Imagine a sample divided into many thin layers, the first layer absorbs half the light (and transmits half), the second layer will also absorb half the light it receives, etc… o Layers of infinite thinness would exponentially decrease light quantity o Absorbance is a logarithmic function of transmittance o (o = radiant power of incident light) - Beer’s Law: the absorbance of a solution is proportional to the number of absorbing particles (or concentration of the absorbing material) in solution o A = E x l x c  A = absorbance (no units)  E = absorption coefficient (litres/mole/cm)  probability that a given type of photon will be absorbed by a given type of molecule, varies with wavelength and solvent used  C = concentration of solution (mol/L)  L = path length through the spectrophotometer tube (usually 1cm) - The blank used in experiments should contain all of the substances in the sample except for the material whose absorbance you wish to measure - Spectrophotometers are important in experiments where observation is not always possible, allows you to follow the course of a biological process - To make a standard curve of absorbance and concentration you make a number of tubes with different concentrations of your material of interest and record the absorptions o Graph this, the slope of the line will be your E value for that material - A at a chosen time gives the change in concentration of the solution, which can be used with a standard curve to find the average rate of reaction o Slope of the standard curve = E o You can then use the volume used in the experiment to get moles/minute Microscopy - Resolution (d) is the smallest distance by which two objects must be separated in order to allow them to be imaged as two separate objects. - Abbe’s equation gives the theoretical limit of resolution in an optical system o (0.612 = constant, n sin  = numerical aperture of lens, = wavelength of light) o N = refractive index of material medium,  = aperture angle o To attain maximal resolution want to increase n (use immersion lens and immersion oil) and make  as close to 90 as possible, decrease to about 400nm (shortest to which eye is sensitive) Counting Cells - There are two ways to count cells: haemocytometer and spectrometry - Using the haemocytometer you place the cells on the plate and count the outer four squares and the middle square o Cells on the bottom or right borders count, top and left borders do not o Do this for both grids - Find the average number of cells per square 3 - Each square counted has a volume of 0.004mm - Multiply the average cell count by 250 (= 1/0.004) and then by 1000 (1000mm in 1cm = 1mL) to get cells/mL - To use the spectrometer you first need to use the haemocytometer to count cells and then make a standard curve of A vs. # of cells/mL 400 - The slope of this graph can then be used to determine subsequent readings o #cells/mL = A / slope 400 Photon Fluence Rate - The number of photons in the 380-700nm range incident per unit time on a unit surface - Direct relationship between number of molecules that are photochemically altered and number of photons within 380-700nm range that are absorbed, regardless of photon energy, useful to know how many photons hit surface and not how much energy they deliver - Measured with a quantum sensor, units: mols of photons per second per square metre) 17 - 1 mol of photons contains 6.02 x 10 photons pH Calculations - [H ][OH] = 1 x 10 -14 + + -pH - pH = -log[H ] and [H ] = 10 - pOH = -log[OH] and [OH] = 10 -pOH - pH + pOH = 14 - Buffer Solutions - + - HA  A + H weak acid and it’s conjugate base in solution creates this equilibrium - If hydroxide were added it would combine with some of the hydrogen ions to make water, the equilibrium would be pushed to the right and more hydrogen ions would be made, maintaining the pH - If hydrogen ions were added it would combine with A and form HA, using the hydrogen ions and keeping the pH steady - When [HA] = [A] the pH = pK oa the buffer, this is when buffering capacity is at its greatest o The buffer will be effective as long as its pH is within 1 pH unit of the pK a - To make: dissolve Tris and KCl in sorbitol and MgCl and then (using the pH electrode) add NaOH dropwise until the bugger reaches a pH of 7.5 - Many factors affect the decision on which buffer is best to use o pH of use should be within 1 pH unit of the pKa for that buffer o all buffer components should be compatible (no reactions happening within the buffer) o buffer should not affect the system being studied o if weak acid is positive dilution will lower pH and if it is negative dilution will increase the pH o pH of most buffers decreases with increasing temperature, make buffer at temperature to be used at o if additional salts included in buffer add them before adjusting the pH and volume Krajnyk Unit Definitions - Null hypothesis: statement saying that tested variables will have no effect on subject - Alternate hypothesis: tested variables will have an effect on subject - Descriptive statistics: tell you something about the data (mean, variance, standard deviation, etc…) - Protocol: sequentially detailed account of an experiment, always numbered - Repetition: an identical treatment regimen o Important to check for consistency and discount ‘flukes’ - Control: an organism or system with no treatment applied, how it reacts through the protocol alone o Important in determining relevant observed changes, those attributed to the treatment - Significance level: provides scientists with an objective criterion for accepting/rejecting null hypothesis - Randomization: showing no preference to which organisms or treatments go where o Important in ensuring that the sample size is truly representative of the population The Scientific Method - A logical, orderly, arranged approach used to study nature, also a process used to gain new knowledge - Observation: first step is to make observations about things in na
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