Spectrophotometry/Chromatography Flashcards
Spectrophotometry
The measure of the interaction of light with matter
Practical outline
Use a spectrophotometer to monitor the presence/absence of the cofactor NADH and to follow the course of an enzyme catalyzed reaction that involvs the production of NADH
Chromatography
A method for separating the individual components f a mixture on the basis of differences in physical or chemical charachteristics
gel-filtration chromatography
separation on the basis of molecular mass
ion-exchange chromatography
separation on the basis of charge
the elctromagnetic spectrum
consists of diverse types of radiation associated with the transmission of energy in waveform
wavelengths
The different types of radiation that make up the electromagnetic spectrum (Figure 1) are characterized by their wavelengths (), measured in metres and sub-multiples of a metre [millimetre (mm or 10-3 m), micrometre (µm or 10-6 m), nanometre (nm or 10-9 m) and picometre (pm or 10-12 m)]. Wavelength is related to the energy of the radiation: the longer the wavelength the less energy is contained in the radiation. The wavelengths most useful to biochemists are within the 200 – 800 nm range (UV to visible wavelengths).
absorption spectrum
the fraction of radiation absorbed by the material over a range of frequencies
shown as a plot of incident light absorbed by the compound as a function of wavelength.
tyrosine and tryptophan absorbance max
Both tyrosine and tryptophan have an absorbance maximum centred on a wavelength of 280 nm (Figure 2). The wavelength of maximum absorption is defined as the max (lambda max).
ability of a compound to absorb light, and chemical structure
because the ability of a compound to absorb light is a function of its chemical structure, absorption spectra often show unique absorption maxima and minima that can be used to identify a compound
spectral shifts
chemical modification of a compound can produce spectral shifts that are of practical value
spectral shift in nicotinamide coenzyme
in the spectrophotometry practical we identify the spectral shifts that occur when a NAD+ is reduced.
the beer lambert law
states that there is a linear relationship between the concentration and the absorbance of the solution, which enables the concentration of a solution to be calculated by measuring its absorbance.
NAD+
Nicotinamide adenine dinucleotide (NAD+) is a coenzyme present in all living
cells, where it is involved in redox reactions, transferring electrons from one
molecule to another. The coenzyme is, therefore, found in two forms: the oxidized
form NAD+ accepts electrons from other molecules and is reduced to NADH, which
can then be used as a reducing agent to donate electrons
reduction of NAD+
results in a distinct change in the absorption spectrum.
how can we follow the conversion of NADH back to NAD+
The reduction of NAD+ results in a distinct change in the absorption spectrum. The
oxidized form (NAD+) has an absorption maximum at wavelength 260 nm.
Reduction results in the emergence of a second absorption maximum at
340 nm (Figure 2). The disappearance of the absorption peak at 340 nm can be
used to follow the conversion of NADH back to NAD+.
alcohol dehydrogenase
catalyzes the oxidaton of ethanol to acetaldehyde, with simultaneous reduction of NAD+ to NADH
how is the rate of reaction calculated
in the enzme assay the rate of reaction was monitored by following the change in absorbance of one of the reactants eg NADH. The change in absorbance per minute, is a measure of the enzyme activity
progress of an enzymatic reaction
The rate is linear at the start of the reaction but after some
time the reaction may begin to slow down.
why may the reaction begin to slow down after the start
The rate is linear at the start of the reaction but after some
time the reaction may begin to slow down.
initial rate
the initial linear portion of the reaction
what happens when a strong acid, eg hydrochloric acid, is dissolved completely in water
it dissociates completely into ions
how are protons present in the solution
in their hydrated form, as hydronium ions.
H30+
essential components of a chromatography system
A stationary phase, i.e a gel a solid or an immobilised liquid.
A chromotographic bed, a column or thin layer of the
stationary phase material
a mobile phase, a liquid or a gas that acts as a solvent carrying the components of the mixture through the stationary phase
experimental setup for column chromatography
The stationary phase is packed into a column, and the liquid mobile phase flows down through the stationary phase.
Collected as a series of fractions as it leaves the column outlet.
the sample - chromatography
the mixture to be separated - is introduced to the top of the column and then carried through the column by the mobile phase.
gel filtration chromatography
the order of elution is inversely
related to size: large molecules are eluted before the small ones. Molecules of
intermediate sizes may penetrate only part of the gel water, and so may be
separated both from larger and from smaller molecules.
the stationary phase in this practical
Sephadex is a 1-6 linked glucose polymer
You will use Sephadex G-25, which is
very highly cross-linked and excludes molecules with Mr greater than 5000.
Most proteins are larger than Mr = 5000 and therefore most proteins are
excluded from Sephadex G-25. However inorganic salts such as NaCl (Mr =
58.5) will enter the gel particles.
why was haemoglobin chosen for this practical
Haemoglobin has been chosen as a specimen protein for this practical
because it is coloured and changes in its colour can be brought about by reaction
with reagents during chromatography.
why does haem change color
as a result of changes in the oxidation state
haem structure
consists of a porphyrin ring system, bound to a single ferrous ion (Fe2+)
The ferrous ion has 6 coordination bonds, four to nitrogen atoms in the plane of the porphyrin ring, and two perpendicular to the plane of the ring.
porphyrin ring system
A porphyrin is a large ring molecule consisting of 4 pyrroles, which are smaller rings made from 4 carbons and 1 nitrogen
the function of the nitrogen atoms in haem
the coordinated nitrogen atoms help prevent the conversion of iron to the ferric (Fe3+) state
PH
pKa + log10[A-][AH]
DEAE cellulose
the cellulose has been modified so it carries an ionizable tertiary amine group
the pKa of DEAE cellulose is ~ 10, therefore it will be protonated at pH 7
hence, anions carrying a negative charge will bind to DEAE cellulose at pH 7
what will happen to ionizable groups depending on the pKa of a solution
1 pH unit below the pKa an ionizable group will be protonated , and more than 1 pH unit above the pKa an ionizable group will be deprotonated.
Isoelectric point
The overall charge a protein carries at a particular pH will depend on the pKa values of its constituent charged amino acids.
The isoelectric point of a protein (pI) is the pH at which the protein carries no net charge.
If the pH is above the pI, the protein will be negatively charged.
If the pH is below the pI, the protein will be positively charged.
what happens when a mixture of proteins is passed through a column of DEAE cellulose
positively charged proteins will pass straight through, whereas negatively charged proteins stick to the column
how can we elute the bound protein from the column of DEAE cellulose
Lowering the pH to neutralise the negative charges on the protein molecules
raising the concentration of anions in the mobile phase will also elute protein from the column, since then there will be more negative ions in the medium to compete with the protein for the positive charges of DEAE cellulose.
conjugated double bonds
At visible wavelengths, radiation is relatively weak, and the electrons whose energy levels are closest together are the ones that are excited.
These electrons are usually found in systems of conjugated double bonds, which are alternating double and single bonds.
Transmittance
(T)
When light is passed through a solution a proportion may
be absorbed, the remainder is transmitted.
The intensity of the light incident on the sample is I0, and that coming through is the transmittance
The transmittance is given by the equation -
T = I/I0
percentage transmission = 100 x T
What does 100% transmittance mean
That no light was absorbed, since all the light passed through the sample
Absorbance
The amount of light that does not pass through
A = log101/T = log1010/I
Beer Lamber Law
relates the absorbance of a solution to the concentration of the solute and the thickness of the solution as follows.
