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Lecture 5

BIOL 1208 Lecture Notes - Lecture 5: Paper Chromatography, Elution, Tae Buffer


Department
Biological Sciences
Course Code
BIOL 1208
Professor
Abel
Lecture
5

Page:
of 4
PRACTICAL PORTION
Pipettes
Mechanical/Mircopipette: used for small volume transfers (less than 1mL).
Volume adjusted by turning adjustment rings. To fill click plunger first to stop.
To release click plunger all the way.
Volumetric: Designed to deliver a single volume precisely. The most accurate, but
there is only one measurement.
Mohr: Cheap to make and least accurate. Marking at tip is maximum volume
Serological: Many gradations, not as accurate as volumetric, but more accurate
than Mohr.
Spectrophotometer
1. Turn it on (left hand knob on the front of the instrument) and allow to warm up
for 15 minutes
2. Adjust the wavelength to the appropriate value. The knob on the top right of the
instrument controls the wavelength, which is indicated on the digital display.
3. With the same holder empty and lid closed, adjust the Zero Adjust Knob (left
front knob) until the instrument reads 0% on the transmittance scale. BE SURE IT
IS ON TRANSMITTANCE! If not, push mode button
4. Carefully insert the tube containing the blank solution into the sample holder
(after using Kimwipe) and close the cover. Line up the white markings with the
notch.
5. Adjust the 100% adjust knob (right front knob) until the display reads 100% on
the transmittance.
6. Remove the blank and immediately insert the same cuvette as described in step 4.
Do not change any instrument setting. Switch the display to read absorbance by
pushing the mode button.
7. The number shown on the display is the absorbance.
Beers Law: concentration and absorbance are directly related
As concentration increases, absorbance increases
Paper Chromatography and Electrophoresis
Chromatography:
Separates complex mixtures into their component parts
Consists of a Mobile Phase and a Stationary Phase
o Mobile Phase: molecules with a strong attraction for this phase move with
it (ether-nonpolar)
o Stationary Phase: molecules with a strong attraction for the stationary
phase are inhibited from moving in the mobile phase (paper-polar)
Based on solubility
The pigments move at different rates because they are not equally soluble in the
solvent and because they are attracted, to different degrees, to the cellulose in the
paper
o The most nonpolar pigment will travel with the solvent first
o The most polar pigment will stay in place (move last), bound to the paper
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Chlorophyll A: blue-green pigment
Chlorophyll B: yellow-green pigment (most polar/least soluble)
Xanthrophyll: yellow pigment (most nonpolar/most soluble)
Caretene: yellow-orange pigment
Gel Electrophoresis:
Agarose Gel is placed in the holder with the lanes on the NEGATIVE side so that
DNA will travel towards positive side (DNA is negative)
Gel is covered in a TAE buffer, which allows linear DNA to run faster
Separate DNA samples are placed in the lanes of the gel
There is a lambda, which is used as a ladder. It is used as a DNA size marker.
Agarose Gel: stationary phase
o Its a matrix, allows larger molecules to move slower than smaller
molecules
Electric field: mobile phase
Separates charged molecules using an electric field
Fermentation Tubes
Tubes are inverted into substrate and allowed to sit
WRITTEN PORTION
Enzymes Lab
Many reactions require a catalyst to occur
o Catalysts increase the rate of a chemical reactions without being consumed
or changed
o Catalysts reduce the activation energy
Enzymes are highly specific
Most enzymes are proteins and function depends a lot on shape
Substrate is attracted tto active site on the enzyme and interacts chemically with
the amino acids in the active site. Once it becomes the product it is no longer
attracted to the enzymes site so it leaves
Reaction: Catechol + Catechol Oxidase Benzoquinone (Product)
Measuring the Rate of Reaction:
o Increase of product or decrease of reactant
o Rate= concentration of product/time
Blank: sample that contains all components except for the absorbing molecule.
Blanks calibrate specs for the specific experiment.
Spec measures absorbance, use standard curve to measure concentration
Movement of Materials Across Cell Membrane
Osmosis: net movement of water from high to low H2O concentration
Diffusion: movement of other materials across membrane
Cell membrane composition:
o Phospholipid bilayer (polar phosphate head and fatty acid tail)
What can pass through the membrane?
o Small>larger
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o No charge > charged
o Nonpolar > polar
Photosynthesis
6 H20 + 6 CO2 C6H1206 + 6 O2
Respiration and Photosynthesis:
o Cycling carbon in the environment
o In photosynthesis, carbon is fixed to produce organic biomolecules
o In respiration, organisms utilize carbon based molecules to produce ATP
and CO2
o Photosynthesis involves the Calvin Cycle and Dark Reactions
Fluorescence
o When a chlorophyll solution is exposed to white light, the pigment
molecules absorb light and become chemically excited
o However, since there are now no molecules to accept the absorbed energy,
it is reemitted as light energy of a longer wavelength
o When extract is view from above (light on bottom) then there is still some
green on top but the rest is red.
Chromatography (see above)
Longer wavelength lower energy
Shorter wavelength higher energy
Net rate of photosynthesis: the amount of oxygen produced by photosynthesis
minus the amount consumed by aerobic respiration
Rate of photosynthesis will slow in the dark
Light Saturation:
o At higher light intensities, there is less of an increase in photosynthetic
rate per unit increase in light intensity, and eventually photosynthesis
reaches light saturation, when the plant cant increase its rate of
photosynthesis
Fermentation
Yeast: S. Cereviciae
Fermentation: processing of organic nutrients and produce energy in the absence
of oxygen. Responsible for production of alcohol! Beer!
Anaerobic (oxygen not present) Conditions:
o Glucose Pyruvate Lactic Acid and Alcohol + CO2
o Regenerate NAD+ from pyruvate
Variables Used
o Glucose (monosaccharide): CO2 produced
o Sucrose (disaccharide): CO2 produced
o Lactose (disaccharide): no CO2 produced
o Lactose + lactic acid (enzyme)
o Starch (polysaccharide): no CO2 produced
Lactose with lactaid would be expected to produce carbon quicker because lactaid
is an enzyme, which speeds up the rate of reaction. Yeast uses lactaid to help
breakdown lactose.
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