Spectrophotometry and Chromatography Flashcards
What is gel filtration chromatography ?
A method of separating substances according to their molecular sizes
What are the two other names for gel filtration chromatography ?
- Gel permeation chromatography
2. Size exclusion chromatography
What happens to substances that interact weakly with the stationary phase ?
They will pass through with minimal delay
What happens to substances that interact strongly with the stationary phase ?
They will be slowed the the greatest extent
What is the liquid mobile phase collected as as it leaves the column ?
Fractions
What flows through the column ?
The liquid mobile phase
Where is the stationary phase packed into ?
A column
What is the mobile phase ?
A liquid or gas that acts as a solvent carrying the components of the mixture through the stationary phase
What is a chromatographic bed ?
A column or a thin layer of the stationary phase material
What is the stationary phase ?
A solid, gel or an immobilised liquid
What is chromatography ?
A method for separating the individual components of a mixture on the basis of differences in physical or chemical characteristics
Does the Beer-Lambert law have units ?
No
What is the Beer-Lambert law ?
Relates the absorbance of a solution to the concentration of the solute and thickness of the solution
What is the absorbance of a compound in a solution directly proportional to ?
The concentration of the compound
What are the two light sources of spectrophotometers and what do they emit ?
- A tungsten filament lamp - Visible light
2. Deuterium lamp - UV light
What are the essential components of a single beam spectrophotometer ?
- Light source
- Monochromator
- Cuvette containing sample
- Detector
Give an example of a spectral shift in the human body ?
Oxygenation of haemoglobin causes a colour change from the purplish hue characteristic to the brilliant scarlet colour of arterial blood
What can chemical modification of a compound produce ?
Spectral shifts
Why is nucleic acids absorbance maximum at 260nm ?
Due to the presence of aromatic bases
Why is proteins maximum absorbance at 280nm ?
Due to the presence of tyrosine and tryptophan
What is the characteristic absorbance of DNA ?
260nm
What is the characteristic absorbance of proteins ?
280nm
What can be used to identify a compound ?
The unique absorption maxima and minima
What is the ability of a compound to absorb light a function of ?
Its chemical structure
Why do haemoglobin solutions appear red ?
The blue-green wavelengths are absorbed by haemoglobin and the red wavelengths are transmitted to the eye
What wavelengths are human eye photoreceptors sensitive to ?
400 - 700nm
What are two photoreceptors in the human eye ?
- Rhodopsin
2. Visual pigments
Why do substances that absorb visible radiation appear coloured ?
The selective absorption of certain wavelengths from the complete spectrum present in ordinary white light
Where do conjugated double bonds appear in the human body ?
Many of the compounds that appear coloured to the eye, for example chlorophyll and haemoglobin
At visible wavelengths, what way are electrons whose energy levels are closest together ?
The ones that are excited
At visible wavelengths, what strength is the energy of the radiation ?
Relatively weak
What are conjugated double bonds ?
Alternating and single double bonds
What is the wavelength of maximum absorption defined as ?
Lambda max
What is the absorbance maximum of tyrosine and tryptophan centered on ?
A wavelength of 280nm
What is the absorption spectrum of a compound shown by ?
As a plot of incident lights absorbed by the compound as a function of wavelength
What is the absorption spectrum of a compound ?
The fraction of radiation absorbed by the material over a range of frequencies
What are two examples of biologically important aromatic compounds ?
Tyrosine and tryptophan
What are transitions between the pi orbitals in aromatic compounds induced by ?
UV radiation
What do visible and UV radiation induce changes in ?
The energy of the molecules outer electrons
What effects how radiation interacts with matter ?
- The energy of the radiation
2. The properties of the molecules involved
What wavelengths are most useful to biochemists and why ?
200 - 800nm range
These are the UV to visible wavelengths
When is their less energy contained in radiation ?
The longer the wavelength
What happens to energy, the longer the wavelength ?
The less energy is contained in the radiation
What is the wavelength related to ?
The energy of the radiation
What is the symbol for wavelengths ?
Lambda
What can wavelengths be measured in ?
- Metres
- Millimetre
- Micrometre
- Nanometre
- Picometre
What are the different types of radiation that make up the electromagnetic spectrum characterized by ?
Their wavelengths
What is the electromagnetic spectrum associated with ?
The transmission of energy in waveform
What does the electromagnetic spectrum consist of ?
Diverse types of radiation
At visible wavelengths, what strength is the energy of the radiation ?
Relatively weak
In gel filtration chromatography, what is the stationary phase ?
A synthetic polymer with a molecular structure that contains a range of molecular sized holes
In gel filtration chromatography, what does the term excluded mean ?
When the size of the molecule is greater than the largest of the molecular holes in the stationary phase. They move down a column at the greatest speed and elute from the column first.
In gel filtration chromatography, what does the term included mean ?
Small molecules can penetrated the buffer inside the gel particles and therefore constantly move into and out of the the gel particles and partition between the volumes of buffer that are outside and inside the gel network
Why do smaller molecules spend half their time in the moving interstitial liquid and what does this result in ?
The volumes of the buffers that are outside and inside the gel network are approximately equal.
They move at half the speed of the larger molecules
What is gel filtration chromatography often used for ?
Investigating relative molecular mass of a molecule
In the practical, what is the stationary phase used ?
Sephadex
What is sephadex ?
1-6 linked glucose polymer (dextran)
What determines the range of the size of molecules that can penetrate the gel (in the practical) ?
The degree of cross linking of the dextran and the resultant pore size in the gel beads
We used sephadex G-25, what is it used for ?
Very highly cross linked and excludes molecules with a relative molecular mass greater than 5000
What molecule has a relative molecular mass greater than 5000 and what happens to them in gel filtration (practical)?
- Proteins
2. Proteins are excluded from sephadex G-25
What does sephaadex G-25 also do ?
Removes inorganic salts from protein preparations
What is desalting ?
Removing inorganic salts from protein preparation
Other than separation, what can gel filtration also be used for ?
Treating protein with a reagent
How is a protein treated with a reagent in gel filtration ?
The protein moves through a zone of the reagents and it reacts efficiently, since at each layer it meets fresh reagent after it has partly reacted. It is then separated from the reagent by continuation of the elution.
Why was haemoglobin chosen in this practical ?
It is coloured and changes in its colour can be brought about by the reaction with reagents in chromatography
Why do colour changes occur in haemoglobin during gel filtration chromatography and what do they illustrate ?
- The changes in oxidation state of the haem moiety
2. Haemoglobin function and dysfunction
What is a porphyrin ring system ?
Conjugate acids of ligands that binds metals to form complexes
What does haem consist of ?
A porphyrin ring system to which is bound a single ferrous ion (fe^2+)
How many bonds does the ferrous ion have ?
Six coordination bonds
Where are the bonds located in the ferrous ion ?
4 to nitrogen atoms in the plane of the porphyrin ring and two perpendicular to the plane of the ring
What helps prevent conversion of the ferrous iron to the ferric state ?
The coordinated nitrogen atoms which have an electron donating character
When can we see a single broad absorption band in the visible region, which is a purple/red colour ?
When a histidine residue of a polypeptide chain fills one of the plane coordination positions and the sixth position is empty
When can we see two very sharp absorption bands and a scarlet colour is visible on haemoglobin ?
The last valency is filled by an oxygen molecule (oxyhaemoglobin)
When is methaemoglobin formed ?
Ferricyanide can be used to oxidise the the ferrous iron of haemoglobin to ferric ion
What does ion-exchange chromatography depend on ?
Electrostatic attraction between species of opposite charge
What do ion exchangers usually consist of ?
Modified derivatives of some support material such as cellulose
How has DEAE-cellulose been modified ?
So it carries an ionizable tertiary amine group
What is the pKa of DEAE cellulose and where will it be protonated ?
- Roughly 10
2. pH of 7
When is an ionizable group protonated ?
1 pH unit below the pKa
When is an ionizable group deprotonated ?
1 pH unit above the pKa
What binds to DEAE cellulose at pH of 7
Anions
How is CM-cellulose been chemically modified ?
So it carries a carboxymethyl group
What is the pKa of CM-cellulose ?
Roughly 4
What happens to CM-cellulose at a pH of 7 ?
The carboxyl group is fully ionized and therefore negatively charged
What is CM-cellulose called and what does it bind ?
- Cation exchanger
2. Positively charged speices
What is an anion exchanger ?
Binds negatively charged species
What are some ionizable molecules ?
- Several side chain groups of the amino acid residues on proteins, for example lysine and glutamic acid
- The N-terminal amino and the C-terminal carboxyl groups
What does the overall charge a protein carries at a particular pH depend on ?
The pKa values of its constituent charged amino acids
What is the isoelectric point of a protein?
Is the point at which the protein carries no net charge
When is a protein negatively charged ?
If pH is greater than pI
When is a protein positively charged ?
If pH is less than pI
What is the pI of acidic proteins ?
pI less than 7
What is the pI of basic proteins ?
pI greater than 7
What happens when a mixture of proteins is passed through a column of DEAE cellulose ?
Positively charged proteins pass straight through whereas negatively charged proteins stick to the column
How could you elute the bound proteins from the column ?
- Lowering the pH will neutralize the negative charges on the protein molecules and cause them to fall off
- Raising the concentration of anions in the mobile phase will elute protein from the column since there will be more negative ions in the medium to compete with the protein for the positive charges of DEAE cellulose
What are three applications of gel filtration chromatography ?
- Determination of relative molecular size of proteins
- Separation of molecules on the basis of size
- Removal of inorganic ions from preparations of protein
What are three applications of ion exchange chromatography ?
- Separation of molecules on the basis of charge
- Water softening, removal of Ca ^2+
- Analytical chemistry- high performance liquid chromatography
What are protons present as in solutions ?
There hydrated forms, as hydronium ions
Do weak acids dissociate completely in water ?
no
What are present in a solution at equilibrium ?
Both acid and conjugate base
What is defined by the dissociation constant Ka ?
The strength of the acid HA
What do square brackets indicate ?
Concentration in moles per litre
The stronger the acid…….
The larger the value of the dissociation constant
How can the pKa of any weak acid be determined ?
Titration with a strong base
What is a buffer region ?
The flat region of a curve which extends 1 pH unit either side of the pH of the acid, there is resistance to a change in pH
Explain Le Chatelier’s Principle.
If a dynamic equilibrium is disturbed by changing the conditions, the position of equilibrium shift to counteract the change to re-establish an equilibrium
What is the pH of the equimolar solution equal to ?
The pKa of acid
What is a buffer solution ?
A mixture of a weak acid and its conjugate base or a weak base and its conjugate acid
Where does the innate resistance of biological fluids to changes in pH come from ?
The presence of cellular constituents that acts as buffers eg. proteins
What is the shape of a titration curve of any weak acid described as ?
Henderson-Hasselbalch equation
What is the pKa of amino acid carboxyl groups ?
1.8-2.4
What is the pKa of amino acid amino group ?
8-11
What is the pKa of the aspartate group ?
3.7
What is the pKa of the lysine group ?
10.5
What is nicotinamide adenine dinucleotide (NAD+) ?
A coenzyme present in all living cells
What is nicotinamide adenine dinucleotide involved in ?
Involved in redox reactions, transferring electrons from one molecule to another
What are the two forms nicotinamide adenine dinucleotide found in ?
- The oxidised NAD+ accepts electrons from other molecules
2. Reduced to NADH which can be used as a reducing agent
What is the absorption maximum of NAD+ ?
260nm
What is the second absorption maximum of NAD+ ?
340nm
What is the measure of enzyme activity ?
The change in absorbance per minute
What is the initial rate ?
The initial linear portion of the reaction
What is the function of glucose oxidase in ion exchange chromatography ?
Catalyzes the oxidation of glucose to glucono-1,5-lactone, which hydrolyzes to gluconic acid
What enzyme is required to catalyze the oxidation of glucose to glucono-1,5-lactone which then hydrolyzes to gluconic acid ?
Cofactor flavin adenine dinucleotide (FAD)
What colour is FAD ?
Yellow
What colour is FADH2 ?
Colourless
What does FAD work as ?
Initial electron acceptor
