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+
ΔA=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
Amino acids are weak acids and bases at the same time
Amino acids are the building blocks of proteins
Corn is deficient in lysine and tryptophan
Legumes are deficient in methionine
Meats are balanced in amino acids
Amino acids have an amino and a carboxyl group
Has an alpha carbon
Amino acids are amphoteric (can act as an acid or base) depending on the pH
Zwitterions because they both carry a – and a + charge (dependent on pH)
The structure of the R group varies from one AA to another
A physiological pH (7.4) AA’s are neutral because carboxyl group loses a hydrogen
giving a -1 charge and the amino group accepts hydrogen giving it a +1 for a net
charge of 0.
If a certain AA has an extra amino or carboxyl group it can be slightly basic or acidic
The pH that results in an overall net charge of 0 is the isoelectric charge of that AA
pI is the number of dissaccociable groups
pKa can be determined through titrations
Ninhydrin and the detection of AA’s
Only reacts with free amino acids not AA’s in proteins
Proteins must 1st be hydrolyzed to get AA content
Ninhydrin is a chromogenic reagent that produces an intensely colored blue purple
product when it reacts with the AMINE groups of AA’s
Decarboxylates the alpha amino acids in a reaction that releases CO2, water, and
aldehydes derived from the carbon skeleton of the AA
Can detect fingerprints
Organic solvents solution make it blue when dried
When reacts with imino groups (proline) it turns yellow
The thiol containing AA cycstenine cannot be detected with it unless mixed with HCl
Under controlled conditions the color change is quantitative and can be used to
measure the amount of amino acids in a solution
6N of HCl for 24hrs @ 110 C= hydrolization
Titrations: find the concentration of an unknown reactant by adding a second
known reagent until it is consumed in the reaction
Acid base reactions the amount of known reagent is expressed as equivalents
An equivalent is the amount required for one mole of hydrogen ions in the reaction
Base equivalent is the amount of base required to neutralize 1 mole of Hydrogen
ions to form water
Acetic acid is an example of an acid base titration (vinager and oil)
Results in a pH of 8.5 with 10.8mEq
Titrations provide information on:
# of titratable groups: the “steps”
pKa of each titratable group: flat points in the titration curve
original titrand concentration:
Buffer capacity: horizontal line implies that a solution is resisting pH changes in this
range. The amount of acid or base that can be neutralized by a buffer system is
buffer capacity.
B max= .567c c=buffer concentration
Useful buffering range:
Isoelectric point: the point at which the pH rises the fastest between two pK’s
Buffers:
Solutions of weak acids or bases that resist changes in pH when strong acids or
bases are added to the solution
Each titratable group has its own pKa
Factors to take into account
The pKa of the buffer: normally a buffer with a pKa at or near the desired pH is
chosen because this will be the pH which buffering is strongest usually + or – 1 pH
unit from the pKa value for that buffer.
Non-specific buffer interactions or toxicity
Interfere and can contribute to background absorbance
Change their pH/pKa at different temperatures or concentrations
Decompose under certain conditions
Cross biological membranes
Become involved in the process
(at a given pH one buffer may be more or less problematic, toxic, or inhibitory than
another buffer.)
N-substituted glycine and tuarine buffers were developed to help allieviate this
problem by Norman Good
Lab Activity Four
Carbs: the extractions, quantifications, and analysis of simple
sugars
Iodine reaction for the detection of starch:
Starch- made up of amylose and amylopectin
(alpha 1-4 linkages)
Amylose- 15-30% branched
Amylopectin- 70-85% branched with alpha 1-6
I2 and KI the tri-iodide anion spontaneously forms and inserts into the center of the
helix. This interaction shifts the orbitals of electrons of the iodine atoms so they are
easily reached by light. The amylose tri-iodine turns blue. Positive test for starch.
A quantitative test for glucose:
Glucose is the only sugar found in starch, cellulose, and glycogen
Enzymed coupled reaction quantifies glucose: generates a product that consumes
NADH was consumed in the reduction of pyruvic acid to lactic acid
Glycerides: two fatty acids that are esterified on a glycerol backbone (palmatic,
stearic, oleic)
Sterol: cholesterol short chain hydrocarbon tail and a hydroxyl group
Sphingolipids: ceramide long hydrocarbon chain polar head group
Fluid-mosaic model: how closely lipids are packed depends on temperature and cis
unsaturation
Up in saturation causes solidity
Cooler temperatures less kinetic energy more solidity
Palmitic Acid: 16:0
Stearic 18:0
Oleic Acid 18:1
Linoleic Acid 18:2
Linolenic Acid 18:3
Storage Lipids: contain twice as much energy as carbohydrates
Differ from membrane lipids in that they have a 3rd esterfied fatty acid on the
glycerol backbone instead of a polar head group
Referred to as triglycerides: found in fatty meats
Animals: enriched saturated fatty acids (16 and 18:0) solid at room temp
Plants: polyunsaturated (18:2 and 18:3) liquid at room temp
Lipid Analyses: sample must first be extracted from non lipid components
Involves mixing sample with aqueous solvents like chloroform, methanol, acetic
acid, hexane/isopropyl, diethyl ether
Most common techniques
Thin Layer Chromatography:
Gas Liquid Chromatography
Chromatography: originally developed to separate plant pigments in petroleum
Has 3 components
The solute: mixture of components to be separated
Mobile phase (solvent): moves past stationary phase differentially carrying some
sample with it
Stationary phase: retards the forward movement of some sample components
Techniques distinquied b/w on how the stationary phase retards the development
of the movement phase
Adsorption c: stationary phase is nonpourous or gel, only the surface reacts
Retarding force is a temporary reversilble binding
Partition: can be porous or oily liquid that coats an inert solid
Retarding force is temporary or reversible removal of of the sample components
from the mobile phase
(the sample components differentially partition out into and out of the stationary
phase)
pg 55