enzymes and chromatography Flashcards
mn what makes up an organisms metabolism?
all biochemical reactions in the body
what two types of reaction does metabolism include?
anabolic and catabolic
what is an anabolic reaction with examples
building up/synthesis
A+B->C
e.g. protein synthesis and photosynthesis
what is a catabolic reaction with examples
breaking down/ degradative
C->A+B
e.g. respiration and digestion
how many enzymes may an individual cell contain?
when are they made?
1000s
when required
general enzyme equation
enzyme+substrate <–> enzyme-substrate complex <–> enzyme-product complex <–>enzyme+product
example of how enzymes affect structure of organisms
polymers require enzymes for speedy formation e.g. starch, cellulose, DNA, collagen, muscle fibres
why are enzymes used in organisms rather than chemical catalysts
bio reactions wouldn’t occur quickly enough w/o enzymes
chem catalysts require specific conditions of high temps and/or pressure
bio enzymes work under less extreme conditions, so do not affect vital processes
features of enzymes
act as biological catalysts
are globular proteins
many end in -ase
possess an active site and can form ESCs
can catalyse forwards/backwards reactions
remain unchanged by reaction they catalyse
v small no. of enzyme molecules are needed
can be inhibited
may be denatured
some require presence of cofactors to function
how do enzymes act as biological catalysts?
proteins used in metabolism which speed up the rate of reaction by lowering the activation energy
how are enzymes globular proteins?
have precise/specific 3D shape, with polar/hydrophilic R groups pointing outwards and hydrophobic R groups pointing inwards
therefore they are soluble tertiary/quaternary proteins
examples of enzymes that end in -ase
lipase
amylase
protease
maltase
catalase
ATPase
what is an enzyme’s active site?
a cleft/depression in an enzyme
how does an ESC form
temporary bonds form between a substrate and an enzyme’s active site
what is the direction of reaction that an enzyme catalyses determined by?
substrate availability and other factors
explain why enzymes remain unchanged by the reaction they catalyse?
can be reused
constantly broken down and reformed only when needed
why are there only a very small number of enzyme molecules needed?
they can be reused
the rate at which substrate binds and is converted to products is very rapid
types of inhibitors
competitive inhibitors
non-competitive inhibitors
what is denaturing?
a permanent change in the tertiary and secondary structure of an enzyme
what can enzymes be denatured by?
high temperature of extreme pH
(NOT LOW TEMP; THIS ONLY MAKES ENZYMES INACTIVE)
what is a cofactor
any substance which is essential for efficient functioning of a enzyme
types of cofactor
prosthetic group
inorganic ions
coenzymes
prosthetic group cofactor:
fetaures
examples
non-protein part of an enzyme which is tightly bound on a permanent basis
usually metal ions e.g. harm (Fe 2+) found in catalase and zinc (Zn 2+) found in carbonic anhydrase
inorganic ions cofactor:
fetaures
examples
not permanently bound, may bind temporarily to an enzymes or substrate
e.g. Cl- for amylase
coenzyme (organic) cofactor:
fetaures
examples
bind to active site for short periods/at the same time as the substrate
temporarily bound
carry chemical groups between enzymes e.g. electrons
often vitamins e.g. NAD and FAD derived from vitamin B are involved in respiration
vitamin K involved in blood clotting
how do coenzymes, cofactors and prosthetic groups increase enzyme activity
bind to enzyme/AS
cofactors & coenzymes bind temporarily and change the shape of the AS
may affect charges on AS
may bind to substrate
increase likelihood of ESC formation
residues directly involved in enzyme action
contact residues
catalytic residues
what do contact residues do?
bind to the substrate and therefore it is these residues that determine enzyme specificity
what do catalytic residues do?
act on the bonds within the substrate that are broken by enzyme action
function of hydrophobic residues?
interact by pointing inwards to maintain the 3D tertiary structure of an enzyme
function of hydrophilic residues?
point outwards and maintain the solubility of enzyme so it can move and collide with the substrate
what reactions do intracellular enzymes catalyse?
examples
reactions inside the cell
catalase
respiratory enzymes
photosynthetic enzymes
enzymes acting inside nucleus
what is a metabolic pathway?
a series of consecutive reactions, every step catalysed by a specific enzyme that produces a specific product
why do endotherms maintain a stable internal temperature?
so that optimum temperature is maintained for enzyme activity
where is catalase found
liver, potato, yeast
in vesicles in eukaryotic cells called peroxisomes
what is hydrogen peroxide and what does catalase break it down into?
a toxic product of many metabolic pathways
catalase breaks it down into water and oxygen
how many reactions can catalase catalyse?
6 million per second
reaction for breakdown of hydrogen peroxide
H2O2<–>2H2O+O2
catalase primary structure
sequence of amino acids joined by peptide bonds
catalase secondary structure
folding into alpha helices and beta pleated sheets
held by hydrogen bonds
catalase tertiary structure
3D folding of secondary structure into a specific shape
held together by hydrogen bonds, hydrophobic/hydrophilic interactions, ionic bonds and disulphide bonds
catalase quaternary structure
more than one polypeptide chain interacting
held together by hydrogen bonds, hydrophobic/hydrophilic interactions, ionic bonds and disulphide bonds
catalase conjugated protein?
has 4 ham groups= prosthetic groups (cofactors) which allow it to interact what hydrogen peroxide
what can act as a non-competitive inhibitor of catalase
any heavy metal ion e.g. copper
the poison cyanide
what are respiratory enzymes responsible for?
the breakdown of glucose and the formation of ATP
examples of respiratory enzymes?
phosphorylases
decarboxylases
dehydrogenases
ATP synthase
what do phosphorylase do and where do they act?
act in cytoplasm
glucose is phosphorylated to keep it in the cell and make it more reactive
what is phosphorylation?
adding a phosphate group (PO4 3-)
what do decarboxylases and dehydrogenases do and where do they act?
act in matrix of mitochondria
decarboxylases remove CO2
dehydrogenases remove H2
what does ATP synthase do and where does it act
acts on inner mitochondrial membrane
synthesises ATP by converting ADP, Pi and energy to ATP
example of photosynthetic enzymes
ribulose biphosphate carboxylase
examples of enzymes acting inside the nucleus and their function
DNA polymerase (synthesis of DNA)
RNA polymerase (synthesis of RNA)
what do extracellular enzymes do?
catalyse reactions outside the cell
example of extracellular enzyme functioning
some organisms e.g. fungi release the enzymes outside their cells and sometimes their whole body e.g. flies, fungal hypha
saprotrophs secrete digestive enzymes from thread-like hyphae and reabsorb digested material
other organisms have internal digestive systems but many of their enzymes act extracellularly
decomposer saprotrophic nutrition example
decomposers break down cellulose in dead plants using enzyme cellulase to release beta glucose which they absorb and use for respiration
digestive enzymes in heterotrophs?
carbohydrases
proteases
lipases
carbohydrase
example
substrate molecule
bond to break
site of production and action
products
amylase
carbohydrate starch
glycosidic
salivary glands & mouth OR pancreas & small intestine
maltose
protease
example
substrate molecule
bond to break
site of production and action
products
pepsin
protein
peptide
stomach
peptides
lipase
example
substrate molecule
bond to break
site of production and action
products
lipase
lipids
ester
pancreas & small intestine
fatty acids & glycerol
what happens to soluble products of digestion?
they can be absorbed via epithelial cells of villi in small intestine
advantage of having an internal digestive system compared with secreting enzymes outside the organism?
enzymes not lost to the environment so they can be reused and recycled
internal environment can be regulated to give optimum temperature and pH for enzymes
amylase structure
1 polypeptide chain made of 496 amino acids
Cl- (inorganic ion cofactor)
calcium ion (prosthetic group cofactor)
step-by-step lock and key hypothesis
1: substrate arrived and collides with active site
2: substrate fits into active site, complementary binding
3: products leave active site
4: enzyme reused
step-by-step induced fit model
1: initial binding; temporary bonds form (e.g. H bonds, ionic bonds, hydrophobic/hydrophilic interactions)
2: conformation change in enzyme structure (shape change of active site might put strain on the chemical bonds in the substrate- lowers Ea) (catalytic R groups now interact with substrate and reaction occurs; lowers Ea)
how does the induced fit differ from the lock and key hypothesis?
IF: active site changes shape due to bonds acting on it when the substrate enters. once reaction is complete, AS returns to its original shape and next reaction can occur
LK: AS is complementary to substrate. substrate fits into AS like key in lock, no change in shape of AS
hexokinase glucose molecule catalyse
glucose induces change in shape in the enzyme
enzyme can enclose substrate to lower activation energy
what is necessary for a reaction to take place?
some or all of the chemical bonds need to be broken and new bonds formed
to get the bonds to a state that allows them to break the molecule must be deformed intern unstable state called the what?
transition state
what energy is required to reach the transition state?
the activation energy: the extra energy needed for the substrate to be converted into products
what can speed up the rate of reaction?
issue with this in living organisms?
how is this overcome?
increasing the temperature
there comes a point where further heating causes molecules to denature
they lower the activation energy required
reactions catalysed by enzymes take place at much lower temperatures that they would without them
how do enzymes lower activation energy?
1: catalytic R groups might donate or accept electrons or form bonds with substrate (helps substrate reach the transition state)
2: enzyme may create a particular environment by enclosing the substrate e.g. water may be excluded due to hydrophobic environment (or acidic environment)
3: on binding, bonds in the substrate may be strained (stretched/weakened), which will help molecules reach transition state (unstable)
4: enzymes orientate molecules so reacting bonds are near to each other
why do reactions begin swiftly as soon as enzymes and substrate Arte mixed and large amounts of oxygen are quickly released?
when enzyme and substrate are first mixed, there are large numbers of substrate molecules available.
at any moment, virtually every enzyme has a substrate molecule in its active site
why does the rate at which oxygen is released gradually plateau aa reaction continues?
rate is prevented from increasing further because fewer and fewer substrate molecules remain to bind with enzyme (limiting factor)
enzymes may be ‘waiting’ for a substrate molecule to hit their AS
increased product molecules get in the way of collision
eventually reaction stops and no more O2 is produced; this is because all substrate molecules have reacted
why is rate of an enzyme controlled reaction always fastest at the beginning
initial rate of reaction at t=0s
this is used to compare with each value of the independent variable
calculation for initial rate of reaction
change in y / change in x
OCR guidance when drawing tangent
draw tangents as long as possible
enzymes properties
soluble and reactive
enzymes structure due to solubility
hydrophilic R groups point out
hydrophobic R groups point in
spherical shape (folded)
globular proteins
all have tertiary structure, folding into precise 3D shape (held by H & ionic bonds and hydrophobic/philic interactions)
enzymes function which means they must be soluble?
enzymes need to be soluble to move around and collide with the substrate in order to catalyse reactions
have specific shape of active site where substrate binds to
enzymes structure due to reactivity
complementary active site
contact residues
catalytic residues
all have tertiary structure which is precise and specific to substrate
cofactors: inorganic ions, coenzymes, prosthetic groups
induced fit model
enzymes function which means that must be reactive
catalysts, so must be able to bind specifically to the substrate
put strain on bonds in substrate
2 ways to measure rate of enzyme reactions
measuring appearance of a product
measuring disappearance of a substrate
2 examples of measuring disappearance of a substrate
caesin -> amino acids using trypsin
starch -> maltose using amylase
where is caesin found and what colour is it
milk
white
are amino acids soluble or insoluble?
what colour solution do they form?
soluble
translucent
how to measure disappearance of a substrate to calculate rate of reaction: breakdown of starch to maltose
take samples at known intervals and to each sample add iodine in potassium iodide solution
starts blue-black. eventually when all the starch is broken down it would remain brown/orange
how to calculate initial rate from starch/maltose disappearance of substrate reaction
use a colorimeter to measure the intensity of blue colour and use this as a measure of the concentration of starch remaining, increasing accuracy of determining this
get colorimeter readings for known concentrations and plot a calibration curve
compare your data to thus
error that may have resulted form subjectiveness of end point of PAG
improvement and justification for this
random errors may lead to inaccuracy/anomalies
colorimeter could be used instead with samples taken at regular intervals
allows quantitative determination of I=end point an should increase the repeatability and precision of the data
limitations for catalase practical
only 1 measurement taken of volume at each time for each enzyme conc
whole exp. only conducted once
samples may have been take from different sources e.g. different potatoes
pH not kept constant
temp not kept constant
equipment not calibrated properly
low resolution of equipment
no negatives control experiment conducted
SA not controlled
syringe not large enough to collect all gas
difficult to cut all potatoes to same size
error of limitation: only 1 measurement taken of vol. at each time at each enzyme conc
improvement and justification
unable to identify anomalies and cannot assess precision or repeatability, no mean can be calculated or standard deviation so random errors have large impact on data
take 5 replicates and calc. mean
allows repeatability, precision e.t.c. and increases accuracy of mean, increasing confidence in the trends drawn from the results
error of limitation: whole experiment only conducted once
improvement and justification
unable to assess reproducibility
repeat whole exp. on a different day or with different people but using same equipment
increased confidence
error of limitation: samples may have been from different organisms
improvement and justification
different age/variety of environmental state so may introduce random errors
use same tissue source e.g. take smaller cylinders all from same potato or us potato clones
reduces effect of random differences in enzyme conc. of potatoes
error of limitation: pH not constant
improvement and justification
method not valid: pH change caused by a change in H+ conc. reduces the rate of an enzyme controlled reaction
use pH buffer solution to keep pH constant and use pH probe to check
ensures variables are controlled and that results obtained are valid
error of limitation: temp not constant
improvement and justification
method not valid. an increase in temp = increase in KE and more ESCs form. high temp denatures enzymes so no ESCs can form
use thermostatically controlled WB before and during reaction
ensures variables are controlled and results obtained are valid
error of limitation: equipment not calibrated properly
improvement and justification
systematic error due to displacement of air int he tube as syringe is pressed down, giving overestimation of rate
subtract the additional volume from each result. pilot study
ensures errors do not affect rate measurements
error of limitation: low resolution of syringe
improvement and justification
measurements have high uncertainty
choose equipment with high resolution and low uncertainty e.g. volumetric syringe
gives greater accuracy and precision of measurements
error of limitation: no negative control experiment conducted
improvement and justification
investigation not valid bc not clear that change in IV has caused change in DV. no baseline or reference value for comparison
conduct a control experiment e..g. boil to denature the enzymes or do without substrate
allows comparison to ensure external factors do not contribute to reaction.
error of limitation: SA not controlled
improvement and justification
investigation not valid bc SA;vol not controlled, which affects rate bc more molecules exposed
crush up sample to extract the enzyme-containing part and perform serial/proportional dilution to change enzyme conc. keep SA the same
increased validity
error of limitation: syringe not big enough to collect all of gas given off
improvement and justification
results not accurate bc some of gas not collected so volume collected was underestimated
use bigger/volumetric syringe
increased accuracy
why does increasing enzyme conc. increase rate?
increased no. of active sites available
increased frequency of successful collisions
increased no. of ESCs formed
increased product formed per second
increased initial rate
why does graph of enzyme conc. vs initial rate plateau if substrate conc. is fixed?
substrate= limiting factor
if enzyme conc increases further, rate doesn’t increase as there are active sites not occupied by substrate
V max. reached
why is enzyme conc. usually low in cells
enzymes can be reused
genes for enzymes can be switched on if required
why does increasing substrate conc increase initial rate?
increased frequency of successful collisions between active sites and substrates
increased no. of ESCs formed
increased product formed per second
increased initial rate
at high substrate concentrations with a fixed enzyme rate, rate plateaus because…
enzymes reach V max and every AS is occupied
turnover rate for each enzyme= different
what is an enzyme’s turnover rate?
the number of substrates broken down by a single enzyme per second
how to measure rate of breakdown of H2O2
measure volume of O2 produced per minute for 5 mins at each IV change
plot on a graph
dare tangents at t=0s
calculate initial rate for each IV change
effect of temp below/near freezing on enzymes
enzyme is inactivated
effect of low temp on enzymes
molecules move more slowly so have less KE so have a decreased frequency of successful collisions between AS and substrate so less ESCs are formed, decreasing rate
effect of increasing temp on enzymes
increased KE so increased frequency if successful collisions between AS and substrate so increased ESCs form, increasing rate
up to optimum, where there is the highest initial rate (V max)
what is Q10
temperature coefficient
a way of quantifying the effect of temp on the rate of reaction
the factor by which the rate increases for a 10C rise in temp
Q10 formula
rate at t +10C / rate at t
what is the value for Q10 in an enzyme reaction?
why?
taken as two
rate of reaction doubles with a 10C rise in temp (below the optimum)
above optimum temperature, what happens to rate of reaction as temp increases?
rate drops sharply
increasing temp causes molecules to vibrate more
initially, enzyme just starts to change shape, decreasing frequency of successful collisions (enzymes not yet denatured)
more vibrations can break H and ionic bonds
loss of 2ary and 3ary structure
(peptide bonds/1ary structure not broken)
AS changes shape and is no longer complementary to substrate
enzyme function is not restored
what is denaturing?
complete, irreversible change in the shape of an enzyme’s active site so it is no longer complementary to the substrate
rate = 0
effect of pH on enzymes
enzymes work within narrow ranges of pH
small changes either side of optimum pH decrease rate bc shape of AS is disrupted BUT NOT DENATURED
at extreme changes from optimum pH, ENZYME DENATURES so rate=0
how do changes from optimum pH affect enzyme structure? ACIDS
acids= proton donors
protons are attracted to - charged ions/molecules/R groups
excess H+ interfere with H and ionic bonds
can cause 2ary and 3ary structure to unravel
AS changes shape
substrate molecule no longer fits
CAN also alter charges on AS so more protons cluster around - charge R groups and stop substrate bonding
how do changes form optimum pH affect enzyme structure? ALKALI
base= proton/H+ acceptor
pos. charged ions/molecules/R groups accept a H+, lose charge and cannot form H/ionic bonds anymore
2ary and 3ary structure unravel
AS changes shape
substrate molecule no longer fits
what is a buffer?
a chemical substance that resists large changes in pH
types of enzyme inhibitors
competitive inhibitor
non-competitive inhibitor
end product inhibition
what do enzyme inhibitors do?
reduce are of enzyme-controlled fractions bc they have an effect on the enzyme molecule
competitive inhibitors:
structure
how they work
reversible/irreversible?
effect of increasing substrate conc.
have close structural resemblance to substrate of an enzyme
compete with substrate for active site
mostly reversible, and if binding irreversibly they are called inactivators
increasing substrate conc relative to inhibitor conc increases initial rate
example of competitive inhibitor
what enzyme does it inhibit? what is this enzyme’s function
function and use
statins
HMG-coA reductase (makes cholesterol in liver)
treats people with CHD as it decreases fatty deposit in arteries bc it decreases cholesterol production
non competitive inhibitors
structure?
where does it bind to?
how does it work?
no structural similarity to substrate (not competing with it for AS)
binds to allosteric site of enzyme (not AS)
distorts active site: conformational change in AS (change to 3ary structure) so is no longer complementary to substrate
equal affinity for enzyme and ESC
increasing substrate conc with a NC inhibitor…
doesn’t affect rate of reaction bc enzymes can be inhibited regardless of how saturated their active sites are (NC inhibitor can bind to enzyme regardless of if substrate has already bound. once bounds, enzyme cannot catalyse its substrate)
will a reaction reach its normal V max with a NC inhibitor?
what is this V max determined by?
no
subset of enzyme molecules will always be inactivated by inhibitor bc it can bind to enzymes regardless of whether they are bound to a substrate or not
lowers conc. of usable enzyme so lowers V max
conc. of inhibitor
when NC inhibitor saturation is reached, the rate of reaction is….
almost 0
bc all bound to enzymes
example of NC inhibitor
enzyme it inhibits
inhibitor function
cyanide (poison)
respiratory enzyme cytochrome oxidase
prevents formation of ATP
particularly dangerous bc it binds irreversibly
COMPETITIVE vs NON-COMPETITIVE inhibitors: reversible or irreversible
most competitive inhibitors do not bind permanently to AS (reversible); they bind for short period the leave. removal of inhibitor from reaction mixture leaves enzyme molecule unaffected
many non-competitive inhibitors bind permanently to enzyme molecules. inhibtion cannot be reversed and any molecules bound by inhibitor are inactivated
examples of positive inhibition
statins
naloxone
why is initial rate of reaction lower at low substrate concs when competitive inhibitor is added
some enzymes are bound to inhibitors at low substrate conc.
can the normal V max be reached with a competitive inhibitor?
yes it is possible at v high substrate concs
all AS bound to substrate at start
why does initial rate increase as substrate conc increases with non competitive inhibitor
some AS that have not changed shape as not bound to inhibitor but could not bind to substrate at low substrate conc as there were not enough
these AS are filled as substrate conc increases so rate increases
what is negative feedback?
change away from set point (e.g. conc of product) that leads to a reversal of this change
example of negative feedback?
end-product inhibition (end product of multienzyme complex inhibits an enzyme in a step of its synthesis to prevent further synthesis of itself)
why is product inhibition useful?
no over production, which could be a waste of resources
reversible, so low product amount means low inhibition
what is a multi enzyme complex?
usefulness
where do they take place
stable assembly of more than 1 enzyme
increase efficiency of metabolic reactions as they keep enzymes and substrate in same location (less diffusion needed)
some enzymes found close together in membrane so work together in multi-step reaction
why are some enzymes synthesised as inactive precursors in metabolic pathways?
prevent damage to cells producing them
how do inactive precursors work?
part of precursor molecule inhibits enzyme action. once this part is removed, the enzyme becomes active
sometimes changes in pH or temp or addition of cofactor can change 3ary stricture and activate precursor enzyme
example of inactive enzyme precursor
why is it synthesised in its inactive form?
pepsinogen (inactive) –> pepsin (active) in low pH environments (stomach HCl)
proteases are synthesised as inactive precursors to prevent damage to proteins in the cell
how does activation of inactive precursors by addition of cofactor work?
example process
apoenzyme (inactive precursor) + FAD/NAD (cofactor/coenzyme activator) -> holoenzyme (active)
cofactor fits into part of active site, without it, enzyme is unactivated and substrate cannot fit
example of cascade system of activation
BLOOD CLOTTING
platelets-> aggregated proteins release vit.K (cofactor for thromboplastin)
|->prothrombin -> thrombin
|->fibrinogen -> fibrin (formed mesh which traps RBC to form clot)
3 examples of metabolic poisons that act as enzyme inhibitors
cyanide
snake venom
malonate
CYANIDE/CN- ions
enzyme affected?
mechanism
cytochrome oxidase (final enzyme in aerobic respiration)
non-reversible inhibitor
aerobic resp. stops
SNAKE VENOM
enzyme affected
mechanism
acetyl cholinesterase (enzyme which breaks down neurotransmitters)
stops NT being broken down->constant muscle contraction->fatigue->paralysis->suffocation
MALONATE
enzyme affected
mechanism
inhibits succinate dehydrogenase
competitive inhibition of respiratory enzyme
6 examples of medicinal drugs that act as enzyme inhibitors
apsirin
cardiac glycosides
ACE inhibitors
protease inhibitors
nucleoside reverse transcriptase inhibitors
proton pump inhibitors
ASPIRIN
enzyme affected
mechanism/use
why should children not take aspirin?
enzymes that form prostaglandins which are produced when tissues are damaged
PG make nerve cells more sensitive to pain and increase swelling
less PG means less pain
can reduce risk of blood clots forming in blood vessels and strokes
can damage children stomach lining
CARDIAC GLYCOSIDES
examples
enzyme affected
mechanism/use
digitalis, digitoxin, digitalin, digoxin
ATPase (less ATP production with digitalis)
less ATP means Na+/K+ pumps in cell membrane of heart muscle cells are inhibited
more Ca2+ can enter cells and this increases contraction, strengthening heartbeat
ACE INHIBITORS
enzyme affected
mechanism/use
angiotensin converting enzyme which normally increases BP
lowers BP in patients with hypertension(high BP) who cannot take beta-blockers
treat heart failure
minimise risk of 2nd heart attack or stroke
PROTEASE INHIBITORS
examples
enzyme affected
mechanism/use
ritonavir and amprenavir
protease
prevent replication of virus particles inside host cells as protein coat cannot be made
competitive inhibition
NUCLEOSIDE REVERSE TRANSCRIPTASE INHIBITORS
examples
enzyme affected
mechanism/use
zidovudine and abacavir (treat HIV+ patients)
nucleoside reverse transcriptase
inhibit enzyme involved in making DNA using viral RNA as a template
PROTON PUMP INHIBITORS
enzyme affected
mechanism/ use
block H+/K+ ATPase enzyme that secretes H+ into the stomach
reduce production of excess stomach acid to prevent ulcers
what is chromatography an example of?
a separation technique
what can chromatography be used to separate
biological molecules e.g. amino acids, carbohydrates, nucleic acids in a mixture
the 2 types of chromatography
thin layer chromatography
paper chromatography
what is the stationary phase of chromatography?
what is it in TLC and paper chromatography?
property of stationary phase?
where the molecules cannot move
TLC plate: sheet of plastic coated with silica gel or aluminium hydroxide (adsorbent)
paper: made of cellulose
VERY POLAR
what is the mobile phase of chromatography?
examples and types
properties
what does it do?
where the molecules can move
solvent:
water (for polar molecules)
hexane, organic solvent, alcohols (e.g. ethanol, butan-1-ol) (for non-polar molecules)
flows over stationary phase carrying biological molecules with it
calculation for retention factor?
chromatography: what do amino acids separate out based on?
their solubility in solvent
adsorption to plate
size
polarity
stationary phase property and what does this mean?
VERY POLAR
hydrogen bonds form between the molecules and the paper (adsorption)
what is adsorption?
adhesion of atoms from a gas/liquid/solid onto a surface
what is the running solvent in chromatography usually a mixture of?
therefore what property does it have
butan-1-ol
ethanoic acid
water
RELATIVELY NON-POLAR
why do amino acids vary in polarity?
due to their different R groups
highly polar amino acids do what during a chromatography experiment?
why?
stick/adsorb to surface more and move slowly up plate
least soluble in non-polar solvent & more attracted to polar plate
less polar amino acids do what in a chromatography experiment?
why?
travel furthest
most soluble in non-polar solvent and less attracted to polar plate
how to carry out chromatography with amino acid sample due to their colourless property?
cannot see samples as they rise up paper
chemical is used to develop chromatogram
ninhydrin is sprayed on the paper after it has been allowed to dry
purple/brown spot appears, showing the location of the samples
use of reference materials in chromatography?
testing a sample alongside a set of reference samples can be used to identify the chemicals present
spots in the sample can be compared with spots from the known reference samples
or the Rf values can be compared with a reference table
why is the same Rf value as a reference sample not always achieved?
TLC plate/ paper might be different
different concentration of solvent
different size of plate
different temperature
may have used a different solvent
what do a high and low Rf value mean?
low Rf value means sample has moved a short distance
high Rf value means sample has moved a longer distance
example of a highly polar and less polar amino acid
aspartic acid/ glutamate
leucine
are paper and TLC qualitative or quantitative analyses?
why?
QUALITITATIVE
they don’t show how much of a chemical is present- instead they just show what is or isn’t present
advantages of TLC compared to paper chromatography
TLC gives better results for a wider range of chemicals
TLC is quicker, more sensitive and produces a clearer separation so it is easier to analyse
what is a TLC plate?
a sheet of glass coated with a thin layer of solid adsorbent, usually silica (polar)
equipment that could be used to accurately measure the volume of 1cm3 an enzyme solution
1cm3 syringe
volumetric pipette
graduated teat pipette
how does variegin (competitive inhibitor of thrombin) act as an inhibitor?
thrombin converts fibrinogen to fibrin
variegin has a similar shape to fibrinogen
so can occupy the active site of thrombin enzyme
variegin prevents fibrinogen binding to the active site/ enzyme-substrate complex formation
slower rate of conversion of fibrinogen to fibrin
action of variegin not permanent/ temporary
things to talk about whenadating procedures of experiments
APPARATUS AND METHOD e.g. test tubes, apparatus for volume measurements, distilled water, water bath, stopclock/timer, details of quantitative preparation
VARIABLES e.g. independent, dependent, units used, control variables
REPEATABILITY e.g. repeats for each change in independent variable, quantitative processing of data e.g. means
RISK ASSESSMENT e.g. potential chemical hazards and control, potential electrical hazards and control
