Spectrophotometry/Chromatography

Question 1

Spectrophotometry

Answer 1

The measure of the interaction of light with matter

Question 2

Practical outline

Answer 2

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

Question 3

Chromatography

Answer 3

A method for separating the individual components f a mixture on the basis of differences in physical or chemical charachteristics

Question 4

gel-filtration chromatography

Answer 4

separation on the basis of molecular mass

Question 5

ion-exchange chromatography

Answer 5

separation on the basis of charge

Question 6

the elctromagnetic spectrum

Answer 6

consists of diverse types of radiation associated with the transmission of energy in waveform

Question 7

wavelengths

Answer 7

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).

Question 8

absorption spectrum

Answer 8

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.

Question 9

tyrosine and tryptophan absorbance max

Answer 9

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).

Question 10

ability of a compound to absorb light, and chemical structure

Answer 10

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

Question 11

spectral shifts

Answer 11

chemical modification of a compound can produce spectral shifts that are of practical value

Question 12

spectral shift in nicotinamide coenzyme

Answer 12

in the spectrophotometry practical we identify the spectral shifts that occur when a NAD+ is reduced.

Question 13

the beer lambert law

Answer 13

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.

Question 14

NAD+

Answer 14

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

Question 15

reduction of NAD+

Answer 15

results in a distinct change in the absorption spectrum.

Question 16

how can we follow the conversion of NADH back to NAD+

Answer 16

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+.

Question 17

alcohol dehydrogenase

Answer 17

catalyzes the oxidaton of ethanol to acetaldehyde, with simultaneous reduction of NAD+ to NADH

Question 18

how is the rate of reaction calculated

Answer 18

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

Question 19

progress of an enzymatic reaction

Answer 19

The rate is linear at the start of the reaction but after some
time the reaction may begin to slow down.

Question 20

why may the reaction begin to slow down after the start

Answer 20

The rate is linear at the start of the reaction but after some
time the reaction may begin to slow down.

Question 21

initial rate

Answer 21

the initial linear portion of the reaction

Question 22

what happens when a strong acid, eg hydrochloric acid, is dissolved completely in water

Answer 22

it dissociates completely into ions

Question 23

how are protons present in the solution

Answer 23

in their hydrated form, as hydronium ions.

H30+

Question 24

essential components of a chromatography system

Answer 24

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

Question 25

experimental setup for column chromatography

Answer 25

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.

Question 26

the sample - chromatography

Answer 26

the mixture to be separated - is introduced to the top of the column and then carried through the column by the mobile phase.

Question 27

gel filtration chromatography

Answer 27

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.

Question 28

the stationary phase in this practical

Answer 28

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.

Question 29

why was haemoglobin chosen for this practical

Answer 29

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.

Question 30

why does haem change color

Answer 30

as a result of changes in the oxidation state

Question 31

haem structure

Answer 31

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.

Question 32

porphyrin ring system

Answer 32

A porphyrin is a large ring molecule consisting of 4 pyrroles, which are smaller rings made from 4 carbons and 1 nitrogen

Question 33

the function of the nitrogen atoms in haem

Answer 33

the coordinated nitrogen atoms help prevent the conversion of iron to the ferric (Fe3+) state

Question 34

PH

Answer 34

pKa + log10[A-][AH]

Question 35

DEAE cellulose

Answer 35

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

Question 36

what will happen to ionizable groups depending on the pKa of a solution

Answer 36

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.

Question 37

Isoelectric point

Answer 37

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.

Question 38

what happens when a mixture of proteins is passed through a column of DEAE cellulose

Answer 38

positively charged proteins will pass straight through, whereas negatively charged proteins stick to the column

Question 39

how can we elute the bound protein from the column of DEAE cellulose

Answer 39

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.

Question 40

conjugated double bonds

Answer 40

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.

Question 41

Transmittance

Answer 41

(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

Question 42

What does 100% transmittance mean

Answer 42

That no light was absorbed, since all the light passed through the sample

Question 43

Absorbance

Answer 43

The amount of light that does not pass through

A = log101/T = log1010/I

Question 44

Beer Lamber Law

Answer 44

relates the absorbance of a solution to the concentration of the solute and the thickness of the solution as follows.