biological molecules Flashcards
1
Q
water molecules
A
polar molecule- has regions of negativity and regions of positivity.
oxygen and hydrogen do not share electrons equally in a covalent bond, o2 has a greater share of electrons in an o-h bond
This separation of charge due to the electrons in the covalent bonds being unevenly shared is called a dipole. When a molecule has one end that is negatively charged and one end that is positively charged it is also a polar molecule
Water is a polar molecule
2
Q
hydrogen bonds
A
polar molecules interact with each other as the + and - regions of the molecule attract each other and form H bonds
Hydrogen bonds are weak, when there are few, so they are constantly breaking and reforming. However when there are large numbers present they form a strong structure
3
Q
characteristics of water
A
small molecule, lighter than co2 or o2
liquid at room temp due to h+ bonds
takes a lot of energy to increase the temp of water to become gaseous
high boiling point
water becomes less dense when frozen- at below 4 degrees the h+ bonds fix the position of the polar molecules slightly further apart than the average distance in liquid state
produces a giant, rigid, open structure
o2 at the centre of tetrahedral arrangement of h2 atoms, this is why ice floats
cohesive properties- water moves as one mass because the molecules are attracted the each other (cohesion)
adhesive properties- water molecules are attracted to other minerals
water molecules are more strongly cohesive to each other than they are to air, results in water having a ‘skin’ of surface tension
4
Q
water as a solvent
A
As water is a polar molecule many ions (e.g. sodium chloride) and covalently bonded polar substances (e.g. glucose) will dissolve in it
This allows chemical reactions to occur within cells (as the dissolved solutes are more chemically reactive when they are free to move about)
Metabolites can be transported efficiently (except non-polar molecules which are hydrophobic)
5
Q
water as a transport medium
A
cohesion between water molecules means that when water is transported through the body, molecules will stick together. adhesion occurs between water molecules and other polar molecules and surfaces. the effects of adhesion and cohesion results in water exhibiting capillary action. this is the process by which water can rise up a narrow tube against the force of gravity
hydrogen bonds between water molecules allows for strong cohesion between water molecules
This allows columns of water to move through the xylem of plants and through blood vessels in animals
This also enables surface tension where a body of water meets the air, these hydrogen bonds occur between the top layer of water molecules to create a sort of film on the body of water (this is what allows insects such as pond skaters to float)
Water is also able to hydrogen bond to other molecules, such as cellulose, which is known as adhesion
This also enables water to move up the xylem due to transpiration
6
Q
water as a coolant
A
buffers temperature changes during chemical reactions in prokaryotic and eukaryotic cells because of the large amounts of energy required to overcome hydrogen bonding. maintaining constant temperatures in cellular environments is important as enzymes are often only active in a narrow temperature range
in order to change state (from liquid to gas) a large amount of thermal energy must be absorbed by water to break the hydrogen bonds and evaporate
This is an advantage for living organisms as only a little water is required to evaporate for the organism to lose a great amount of heat
This provides a cooling effect for living organisms, for example the transpiration from leaves or evaporation of water in sweat on the skin
7
Q
water being stable
A
The specific heat capacity of a substance is the amount of thermal energy required to raise the temperature of 1kg of that substance by 1°C
Water’s specific heat capacity is 4200 J/kg°C meaning a relatively large amount of energy is required to raise its temperature
The high specific heat capacity is due to the many hydrogen bonds present in water. It takes a lot of thermal energy to break these bonds and a lot of energy to build them, thus the temperature of water does not fluctuate greatly
The advantage for living organisms is that it:
Provides suitable habitats
Is able to maintain a constant temperature as water is able to absorb a lot of heat without big temperature fluctuations
This is vital in maintaining temperatures that are optimal for enzyme activity
Water in blood plasma is also vital in transferring heat around the body, helping to maintain a fairly constant temperature
As blood passes through more active (‘warmer’) regions of the body, heat energy is absorbed but the temperature remains fairly constant
Water in tissue fluid also plays an important regulatory role in maintaining a constant body temperature
8
Q
polymers and monomers
A
Monomers are the smaller units from which larger molecules are made
Polymers are molecules made from a large number of monomers joined together in a chain
Carbon compounds can form small single subunits (monomers) that bond with many repeating subunits to form large molecules (polymers) by a process called polymerisation
Macromolecules are very large molecules
They contain 1000 or more atoms and so have a high molecular mass
Polymers can be macromolecules, however, not all macromolecules are polymers as the subunits of polymers have to be the same repeating units
9
Q
covalent bonding
A
A covalent bond is the sharing of two or more electrons between two atoms
The electrons can be shared equally forming a nonpolar covalent bond or unequally (where an atom can be more electronegative δ-) to form a polar covalent bond
Generally, each atom will form a certain number of covalent bonds due to the number of free electrons in the outer orbital e.g. H = 1 bond, C = 4 bonds
Covalent bonds are very stable as high energies are required to break the bonds
Multiple pairs of electrons can be shared forming double bonds (e.g. unsaturated fats C=C) or triple bonds
When two monomers are close enough that their outer orbitals overlap this results in their electrons being shared and a covalent bond forming. If more monomers are added then polymerisation occurs (and / or a macromolecule forms)
10
Q
condensation reaction
A
Also known as dehydration synthesis (‘to put together while losing water’)
A condensation reaction occurs when monomers combine together by covalent bonds to form polymers (polymerisation) or macromolecules (lipids) and water is removed
11
Q
hydrolysis
A
Hydrolysis means ‘lyse’ (to break) and ‘hydro’ (with water)
In the hydrolysis of polymers, covalent bonds are broken when water is added
12
Q
chemical elements in biological molecules
A
Carbohydrates, lipids, proteins and nucleic acids contain the chemical elements carbon (C) and hydrogen (H) making them organic compounds
Carbon atoms are key to organic compounds because:
Each carbon atom can form four covalent bonds – this makes the compounds very stable (as covalent bonds are so strong they require a large input of energy to break them)
Carbon atoms can form covalent bonds with oxygen, nitrogen and sulfur
Carbon atoms can form straight chains, branched chains or rings
13
Q
carbohydrates
A
All carbohydrates contain the chemical elements C, H and O
As H and O atoms are always present in the ratio of 2:1 (eg. water H2O, which is where ‘hydrate’ comes from in ‘carbohydrate’) they can be represented by the formula Cx (H2O)y
The three types of carbohydrates are monosaccharides, disaccharides and polysaccharides
Carbohydrates have many different functions:
Source of energy e.g. glucose is used for energy-release during cellular respiration
Store of energy e.g. glycogen is stored in the muscles and liver of animals
Structurally important e.g. cellulose in the cell walls of plants
14
Q
lipids
A
All lipids contain the chemical elements C, H and O
However, the proportion of O in lipids is low compared to carbohydrates
There are many types of lipids, including triglycerides (fats and oils), phospholipids, waxes, and steroids (such as cholesterol)
Lipids have many different functions:
Source of energy that can be respired (lipids have a high energy yield)
Store of energy e.g. lipids are stored in animals as fats in adipose tissue and in plants as lipid droplets
Insulating layer e.g. thermal insulation under the skin of mammals and electrical insulation around nerve cells
An essential component of biological membranes
15
Q
proteins
A
Like carbohydrates and lipids, all proteins contain the chemical elements C, H and O
However, all proteins also contain N (nitrogen) and some proteins contain S (sulphur)
Proteins have many different functions:
Required for cell growth, cell repair and the replacement of biological materials
Structurally important e.g. in muscles, collagen and elastin in the skin, collagen in bone and keratin in hair
Proteins can also act as carrier molecules in cell membranes, antibodies, enzymes or hormones
16
Q
nucleic acid
A
Like carbohydrates, lipids and proteins, all nucleic acids contain the chemical elements C, H and O
However, all nucleic acids also contain N (nitrogen) in their bases and P (phosphorous) in the form of phosphate groups
Nucleic acids (DNA and RNA) have one function:
Carrying the genetic code in all living organisms
Nucleic acids are essential in the control of all cellular processes including protein synthesis
17
Q
monosaccharides- reducing sugars
A
Sugars can be classified as reducing or non-reducing; this classification is dependent on their ability to donate electrons
Reducing sugars can donate electrons (the carbonyl group becomes oxidised), the sugars become the reducing agent
Thus reducing sugars can be detected using Benedict’s test as they reduce the soluble copper sulphate to insoluble brick-red copper oxide
Examples of reducing sugars include: glucose, fructose and galactose
Fructose and galactose have the same molecular formula as glucose however they have a different structural formula
The different arrangement of atoms in these monosaccharides gives them slightly different properties
18
Q
monosaccharides- non reducing sugars
A
Non-reducing sugars cannot donate electrons, therefore they cannot be oxidised
To be detected non-reducing sugars must first be hydrolysed to break the disaccharide into its two monosaccharides before a Benedict’s test can be carried out
Example: sucrose
19
Q
different types of monosaccrides
A
There are different types of monosaccharide formed from molecules with varying numbers of carbon atoms, for example:
Trioses (3C) eg. glyceraldehyde
Pentoses (5C) eg. ribose
Hexoses (6C) eg. glucose
20
Q
glucose
A
The most well-known carbohydrate monomer is glucose
Glucose has the molecular formula C6H12O6
Glucose is the most common monosaccharide and is of central importance to most forms of life
The main function of glucose is as an energy source
It is the main substrate used in respiration, releasing energy for the production of ATP
Glucose is soluble and so can be transported in water
21
Q
alpha and beta glucose
A
Glucose exists in two structurally different forms – alpha (α) glucose and beta (β) glucose and is therefore known as an isomer
This structural variety results in different functions between carbohydrates- alpha bottom beta top
Different polysaccharides are formed from the two isomers of glucose
starch- alpha no beta
glycogen- alpha no beta
cellulose- no alpha alpha
22
Q
ribose and deoxyribose
A
Sugars that contain five carbon molecules are described as pentose sugars
Ribose and deoxyribose are important pentose sugars found in the nucleotides that make up RNA and DNA
Ribose and deoxyribose are very similar in terms of structure
Deoxyribose has lost one oxygen atom at carbon number 2
23
Q
forming the glycosidic bond
A
To make monosaccharides more suitable for transport, storage and to have less influence on a cell’s osmolarity, they are bonded together to form disaccharides and polysaccharides
Disaccharides and polysaccharides are formed when two hydroxyl (-OH) groups (on different saccharides) interact to form a strong covalent bond called the glycosidic bond (the oxygen link that holds the two molecules together)
Every glycosidic bond results in one water molecule being removed, thus glycosidic bonds are formed by condensation
24
Q
different types of glycosidic bonds
A
Each glycosidic bond is catalysed by enzymes specific to which OH groups are interacting
As there are many different monosaccharides this results in different types of glycosidic bonds
forming (e.g maltose has a α-1,4 glycosidic bond and sucrose has a α-1,2 glycosidic bond)
maltose (disaccharide) alpha 1,4
sucrose (disaccharide) alpha 1,2
cellulose (polysaccharide) beta 1,4
amylose (polysaccharide ) alpha 1,4
amylopectin (polysaccharide) alpha 1,4 and alpha 1,6
25
breaking the glycosidic bond
The glycosidic bond is broken when water is added in a hydrolysis (meaning ‘hydro’ - with water and ‘lyse’ - to break) reaction
Disaccharides and polysaccharides are broken down in hydrolysis reactions
Hydrolytic reactions are catalysed by enzymes, these are different to those present in condensation reactions
Examples of hydrolytic reactions include the digestion of food in the alimentary tract and the breakdown of stored carbohydrates in muscle and liver cells for use in cellular respiration
26
when sucrose is added
Sucrose is a non-reducing sugar which gives a negative result in a Benedict’s test. When sucrose is heated with hydrochloric acid this provides the water that hydrolyses the glycosidic bond resulting in two monosaccharides that will produce a positive Benedict's test
27
common disaccharides
Monosaccharides can join together via condensation reactions to form disaccharides
A condensation reaction is one in which two molecules join together via the formation of a new chemical bond, with a molecule of water being released in the process
The new chemical bond that forms between two monosaccharides is known as a glycosidic bond
To calculate the chemical formula of a disaccharide, you add all the carbons, hydrogens and oxygens in both monomers then subtract 2x H and 1x O (for the water molecule lost)
Common examples of disaccharides include:
Maltose (the sugar formed in the production and breakdown of starch)
Sucrose (the main sugar produced in plants)
Lactose (a sugar found only in milk)
All three of the common examples above have the formula C12H22O11
28
latent heat of vaporisation
In order to change state (from liquid to gas) a large amount of thermal energy must be absorbed by water to break the hydrogen bonds and evaporate
This is an advantage for living organisms as only a little water is required to evaporate for the organism to lose a great amount of heat
This provides a cooling effect for living organisms, for example the transpiration from leaves or evaporation of water in sweat on the skin
29
polysaccharide structure
Starch, glycogen and cellulose are polysaccharides
Polysaccharides are macromolecules (polymers) that are formed by many monosaccharides joined by glycosidic bonds in a condensation reaction to form chains
These chains may be:
Branched or unbranched
Folded (making the molecule compact which is ideal for storage eg. starch and glycogen)
Straight (making the molecules suitable to construct cellular structures e.g. cellulose) or coiled
30
starch structure
Starch is constructed from two different polysaccharides:
Amylose (10 - 30% of starch)
Unbranched helix-shaped chain with 1,4 glycosidic bonds between α-glucose molecules
The helix shape enables it to be more compact and thus it is more resistant to digestion
Amylopectin (70 - 90% of starch)
1,4 glycosidic bonds between α-glucose molecules but also 1,6 glycosidic bonds form between glucose molecules creating a branched molecule
31
glycogen structure
Glycogen is a polysaccharide found in animals
It is made up of α-glucose molecules
There are 1,4 glycosidic bonds between α-glucose molecules and also 1,6 glycosidic bonds between glucose molecules creating a branched molecule
Glycogen has a similar structure to amylopectin but it has more branches
32
starch summary
amylose:
monomer- alpha glucose
branched- no
helix (coiled)- yes
glycosidic bonds present- 1,4
source- plant
amylopectin:
monomer- alpha glucose
branched- yes every 20 monomers
helix (coiled)- no
glycosidic bonds present- 1,4 and 1,6
source- plant
glycogen:
monomer- alpha glucose
branched- yes every 10 monomers
helix (coiled)- no
glycosidic bonds present- 1,4 and 1,6
source- animal
33
cellulose structure
Cellulose is a polysaccharide found in plants
It consists of long chains of β-glucose joined together by 1,4 glycosidic bonds
β-glucose is an isomer of α-glucose, so in order to form the 1,4 glycosidic bonds consecutive β-glucose molecules must be rotated 180° to each other
Due to the inversion of the β-glucose molecules, many hydrogen bonds form between the long chains giving cellulose its strength
34
starch function
storage polysaccharide:
Compact
So large quantities can be stored
Insoluble
So they will have no osmotic effect, unlike glucose which would lower the water potential of a cell causing water to move into cells
Starch is the storage polysaccharide of plants. It is stored as granules in plastids such as amyloplasts and chloroplasts
Plastids are membrane-bound organelles that can be found in plant cells. They have a specialised function eg. amyloplasts store starch grains
Due to the many monomers in a starch molecule, it takes longer to digest than glucose
The amylopectin in starch has branches that result in many terminal glucose molecules that can be easily hydrolysed for use during cellular respiration or added for storage
35
glycogen function
storage polysaccharide:
Compact
So large quantities can be stored
Insoluble
So they will have no osmotic effect, unlike glucose which would lower the water potential of a cell causing water to move into cells
Glycogen is the storage polysaccharide of animals and fungi, it is highly branched and not coiled
Liver and muscles cells have a high concentration of glycogen, present as visible granules, as the cellular respiration rate is high in these cells (due to animals being mobile)
Glycogen is more branched than amylopectin making it more compact which helps animals store more
The branching enables more free ends where glucose molecules can either be added or removed allowing for condensation and hydrolysis reactions to occur more rapidly – thus the storage or release of glucose can suit the demands of the cell
36
cellulose function
Cellulose is the main structural component of cell walls due to its strength which is a result of the many hydrogen bonds found between the parallel chains of microfibrils
The high tensile strength of cellulose allows it to be stretched without breaking which makes it possible for cell walls to withstand turgor pressure
The cellulose fibres and other molecules (eg. lignin) found in the cell wall forms a matrix which increases the strength of the cell walls
The strengthened cell walls provide support to the plant
Cellulose fibres are freely permeable which allows water and solutes to leave or reach the cell surface membrane
As few organisms have the enzyme (cellulase) to hydrolyse cellulose it is a source of fibre
37
biochemical tests
There are a number of tests that can be carried out quickly and easily in a lab to determine if a sample contains a certain type of sugar
The following tests are qualitative - they do not give a quantitative value as to how much of each type of molecule may be present in a sample
Sugars can be classified as reducing or non-reducing; this classification is dependent on their ability to donate electrons (a reducing sugar that is able to donate electrons is itself oxidised)
OILRIG in Chemistry
38
benedict’s test
The Benedict’s test for reducing sugars
Benedict’s reagent is a blue solution that contains copper (II) sulfate ions (CuSO4 ); in the presence of a reducing sugar copper (I) oxide forms
Copper (I) oxide is not soluble in water, so it forms a precipitate
39
benedict’s test method
Add Benedict's reagent (which is blue as it contains copper (II) sulfate ions) to a sample solution in a test tube
Heat the test tube in a water bath or beaker of water that has been brought to a boil for a few minutes
If a reducing sugar is present, a coloured precipitate will form as copper (II) sulfate is reduced to copper (I) oxide which is insoluble in water
It is important that an excess of Benedict’s solution is used so that there is more than enough copper (II) sulfate present to react with any sugar present
A positive test result is a colour change somewhere along a colour scale from blue (no reducing sugar), through green, yellow and orange (low to medium concentration of reducing sugar) to brown/brick-red (a high concentration of reducing sugar)
This test is semi-quantitative as the degree of the colour change can give an indication of how much (the concentration of) reducing sugar present
40
test for non reducing sugars
Add dilute hydrochloric acid to the sample and heat in a water bath that has been brought to the boil
Neutralise the solution with sodium hydrogencarbonate
Use a suitable indicator (such as red litmus paper) to identify when the solution has been neutralised, and then add a little more sodium hydrogencarbonate as the conditions need to be slightly alkaline for the Benedict’s test to work
Then carry out Benedict’s test as normal
Add Benedict’s reagent to the sample and heat in a water bath that has been boiled – if a colour change occurs, a reducing sugar is present
Explanation:
The addition of acid will hydrolyse any glycosidic bonds present in any carbohydrate molecules
The resulting monosaccharides left will have an aldehyde or ketone functional group that can donate electrons to copper (II) sulfate (reducing the copper), allowing a precipitate to form
41
iodine test for starch
To test for the presence of starch in a sample, add a few drops of orange/brown iodine in potassium iodide solution to the sample
The iodine is in potassium iodide solution as iodine is insoluble in water
If starch is present, iodide ions in the solution interact with the centre of starch molecules, producing a complex with a distinctive blue-black colour
This test is useful in experiments for showing that starch in a sample has been digested by enzymes
42
lipids structure
Lipids are macromolecules that contain carbon, hydrogen and oxygen atoms. Unlike carbohydrates, lipids contain a lower proportion of oxygen
Lipids are non-polar and hydrophobic (insoluble in water)
There are two groups of lipid that you need to know:
Triglycerides (the main component of fats and oils)
Phospholipids
Lipids play an important role in energy yield, energy storage, insulation and hormonal communication
43
triglycerides
Triglycerides are non-polar, hydrophobic molecules
The monomers that make up triglycerides are glycerol and fatty acids
Glycerol is an alcohol (an organic molecule that contains a hydroxyl group bonded to a carbon atom)
Fatty acids contain a methyl group at one end of a hydrocarbon chain known as the R group (chains of hydrogens bonded to carbon atoms, typically 4 to 24 carbons long) and at the other is a carboxyl group
The shorthand chemical formula for a fatty acid is RCOOH
44
variation of fatty acids
Fatty acids can vary in two ways:
- Length of the hydrocarbon chain (R group)
- The fatty acid chain (R group) may be saturated (mainly in animal fat) or unsaturated (mainly vegetable oils, although there are exceptions e.g. coconut and palm oil)
Unsaturated fatty acids can be mono or poly-unsaturated
If H atoms are on the same side of the double bond they are cis-fatty acids and are metabolised by enzymes
If H atoms are on opposite sides of the double bond they are trans-fatty acids and cannot form enzyme-substrate complexes, and therefore are not metabolised. 'Trans-fat' is linked with coronary heart disease
45
phospholipids
Phospholipids are a type of lipid, therefore they are formed from the monomers glycerol and fatty acids
Unlike triglycerides, there are only two fatty acids bonded to a glycerol molecule in a phospholipid as one has been replaced by a phosphate ion (PO43-)
As the phosphate is polar it is soluble in water (hydrophilic)
The fatty acid ‘tails’ are non-polar and therefore insoluble in water (hydrophobic)
Phospholipids are amphipathic (they have both hydrophobic and hydrophilic parts)
As a result of having hydrophobic and hydrophilic parts, phospholipid molecules form monolayers or bilayers in water
46
phospholipids vs triglycerides
phospholipids:
no of fatty acid tails- 2
presence of phosphate- yes
polar/non polar- polar phosphate head
no of water molecules released during formation- 3
function- cell membrane component
triglycerides:
no of fatty acid tails- 3
presence of phosphate- no
polar- non polar
no of water molecules released during formation- 3
function- energy storage
47
the ester bond
Triglycerides are formed by esterification
An ester bond forms when a hydroxyl (-OH) group from the glycerol bonds with the carboxyl (-COOH) group of the fatty acid
An H from glycerol combines with an OH from the fatty acid to make water
The formation of an ester bond is a condensation reaction
For each ester bond formed a water molecule is released
Three fatty acids join to one glycerol molecule to form a triglyceride
Therefore for one triglyceride to form, three water molecules are released
48
lipids structure
Triglycerides are fats and oils
Fatty acid and glycerol molecules are the components that make up triglycerides
Fats and oils have a number of important functions in organisms: energy storage, insulation, buoyancy, and protection
49
energy storage
The long hydrocarbon chains in triglycerides contain many carbon-hydrogen bonds with little oxygen (triglycerides are highly reduced)
So when triglycerides are oxidised during cellular respiration this causes these bonds to break releasing energy used to produce ATP
Triglycerides, therefore, store more energy per gram than carbohydrates and proteins (37kJ compared to 17kJ)
As triglycerides are hydrophobic they do not cause osmotic water uptake in cells so more can be stored
Plants store triglycerides, in the form of oils, in their seeds and fruits. If extracted from seeds and fruits these are generally liquid at room temperature due to the presence of double bonds which add kinks to the fatty acid chains altering their properties
Mammals store triglycerides as oil droplets in adipose tissue to help them survive when food is scarce (e.g. hibernating bears)
The oxidation of the carbon-hydrogen bonds releases large numbers of water molecules (metabolic water) during cellular respiration
Desert animals retain this water if there is no liquid water to drink
Bird and reptile embryos in their shells also use this water
50
insulation
Triglycerides are part of the composition of the myelin sheath that surrounds nerve fibres
The myelin sheath provides insulation which increases the speed of transmission of nerve impulses
Triglycerides compose part of the adipose tissue layer below the skin which acts as insulation against heat loss (eg. blubber of whales)
51
buoyancy
The low density of fat tissue increases the ability of animals to float more easily
52
protection
The adipose tissue in mammals contains stored triglycerides and this tissue helps protect organs from the risk of damage
53
phospholipids
Phospholipids are a type of lipid, therefore they are formed from the monomer glycerol and fatty acids
Unlike triglycerides, there are only two fatty acids bonded to a glycerol molecule in a phospholipid as one has been replaced by a phosphate ion (PO43-)
As the phosphate is polar it is soluble in water (hydrophilic)
The fatty acid ‘tails’ are non-polar and therefore insoluble in water (hydrophobic)
Phospholipids are amphipathic (they have both hydrophobic and hydrophilic parts)
As a result of having hydrophobic and hydrophilic parts phospholipid molecules form monolayers or bilayers in water
54
phospholipids within the cell membrane
Phospholipids are the main component (building block) of cell membranes
Due to the presence of hydrophobic fatty acid tails, a hydrophobic core is created when a phospholipid bilayer forms
The core acts as a barrier to water-soluble molecules
The hydrophilic phosphate heads form H-bonds with water allowing the cell membrane to be used to compartmentalise
Compartmentalisation enables cells to organise specific roles into organelles, helping with efficiency
The composition of phospholipids contributes to the fluidity of the cell membrane
If there are mainly saturated fatty acid tails then the membrane will be less fluid
If there are mainly unsaturated fatty acid tails then the membrane will be more fluid
Phospholipids control membrane protein orientation
Weak hydrophobic interactions between the phospholipids and membrane proteins hold the proteins within the membrane but still allow movement within the layer
55
cholesterol
Another important lipid molecule found in the cell membrane of eukaryotic cells is cholesterol
Just like phospholipid molecules, cholesterol molecules have hydrophobic and hydrophilic regions
Their chemical structure allows them to exist in the bilayer of the membrane
Molecules of cholesterol are synthesised in the liver and transported via the blood
Cholesterol affects the fluidity and permeability of the cell membrane
It disrupts the close-packing of phospholipids, increasing the rigidity of the membrane (makes the membrane less flexible)
It acts as a barrier, fitting in the spaces between phospholipids. This prevents water-soluble substances from diffusing across the membrane
Molecules of cholesterol are used to produce steroid-based hormones such as oestrogen, testosterone and progesterone
56
biochemical tests- lipids
The emulsion test can be carried out quickly and easily in a lab to determine if a sample contains lipids
Lipids are nonpolar molecules that do not dissolve in water but will dissolve in organic solvents such as ethanol
Apparatus
Test tubes
Test tube rack
Ethanol
Pipettes
Food sample
Mortar and pestle (if food sample is solid)
Water
Gloves
Method
Add ethanol to the sample to be tested
Shake to mix
Add the mixture to a test tube of water
Results
If lipids are present, a milky emulsion will form (the solution appears ‘cloudy’); the more lipid present, the more obvious the milky colour of the solution
If no lipid is present, the solution remains clear
Limitations
This test is qualitative - it does not give a quantitative value as to how much lipid may be present in a sample
57
amino acid proteins
Proteins are polypeptides (and macromolecules) made up of monomers called amino acids
The sequence, type and number of the amino acids within a protein determines its shape and therefore its function
Proteins are extremely important for cell growth, cell repair and structure
They form all of the following:
Enzymes
Cell membrane proteins (eg. carrier)
Hormones
Immunoproteins (eg. immunoglobulins)
Transport proteins (eg. haemoglobin)
Structural proteins (eg. keratin, collagen)
Contractile proteins (eg. myosin)
58
amino acids
Amino acids are the monomers of polypeptides
There are 20 amino acids found in proteins common to all living organisms
The general structure of all amino acids is a central carbon atom bonded to:
An amine group -NH2
A carboxylic acid group -COOH
A hydrogen atom
An R group (which is how each amino acid differs and why amino acid properties differ e.g. whether they are acidic or basic or whether they are polar or non-polar)
59
the peptide bond
Peptide bonds form between amino acids
Peptide bonds are covalent bonds and so involve the sharing of electrons
In order to form a peptide bond a hydroxyl (-OH) is lost from the carboxylic group of one amino acid and a hydrogen atom is lost from the amine group of another amino acid
The remaining carbon atom (with the double-bonded oxygen) from the first amino acid bonds to the nitrogen atom of the second amino acid
This is a condensation reaction so water is released
Dipeptides are formed by the condensation of two amino acids
Polypeptides are formed by the condensation of many (3 or more) amino acids
A protein may have only one polypeptide chain or it may have multiple chains interacting with each other
During hydrolysis reactions, the addition of water breaks the peptide bonds resulting in polypeptides being broken down to amino acids
60
levels of protein structure
There are four levels of structure in proteins, three are related to a single polypeptide chain and the fourth level relates to a protein that has two or more polypeptide chains
Polypeptide or protein molecules can have anywhere from 3 amino acids (Glutathione) to more than 34,000 amino acids (Titan) bonded together in chains
61
primary structure
The sequence of amino acids bonded by covalent peptide bonds is the primary structure of a protein
The DNA of a cell determines the primary structure of a protein by instructing the cell to add certain amino acids in specific quantities in a certain sequence. This affects the shape and therefore the function of the protein
The primary structure is specific for each protein (one alteration in the sequence of amino acids can affect the function of the protein)
62
secondary structure
The secondary structure of a protein occurs when the weak negatively charged nitrogen and oxygen atoms interact with the weak positively charged hydrogen atoms to form hydrogen bonds
There are two shapes that can form within proteins due to the hydrogen bonds:
α-helix
β-pleated sheet
The α-helix shape occurs when the hydrogen bonds form between every fourth peptide bond (between the oxygen of the carboxyl group and the hydrogen of the amine group)
The β-pleated sheet shape forms when the protein folds so that two parts of the polypeptide chain are parallel to each other enabling hydrogen bonds to form between parallel peptide bonds
Most fibrous proteins have secondary structures (e.g. collagen and keratin)
The secondary structure only relates to hydrogen bonds forming between the amino group and the carboxyl group (the ‘protein backbone’)
The hydrogen bonds can be broken by high temperatures and pH changes
63
tertiary structure
Further conformational change of the secondary structure leads to additional bonds forming between the R groups (side chains)
The additional bonds are:
Hydrogen (these are between R groups)
Disulphide (only occurs between cysteine amino acids)
Ionic (occurs between charged R groups)
Weak hydrophobic interactions (between non-polar R groups)
This structure is common in globular proteins
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disulphide bonds
Disulphide bonds are strong covalent bonds that form between two cysteine R groups (as this is the only amino acid with a sulphur atom)
These bonds are the strongest within a protein but occur less frequently, and help stabilise the proteins
These are also known as disulphide bridges
Disulphide bonds can be broken by oxidation
This type of bond is common in proteins secreted from cells eg. insulin
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ionic bonds
Ionic bonds form between positively charged (amine group -NH3+) and negatively charged (carboxylic acid -COO-) R groups
Ionic bonds are stronger than hydrogen bonds but they are not common
These bonds are broken by pH changes
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hydrogen
Hydrogen bonds form between strongly polar R groups. These are the weakest bonds that form but the most common as they form between a wide variety of R groups
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hydrophobic interaction
Hydrophobic interactions form between the non-polar (hydrophobic) R groups within the interior of proteins
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tertiary structure determines function
A polypeptide chain will fold differently due to the interactions (and hence the bonds that form) between R groups
Each of the twenty amino acids that make up proteins has a unique R group and therefore many different interactions can occur creating a vast range of protein configurations and therefore functions
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quaternary structure
Quarternary structure exists in proteins that have more than one polypeptide chain working together as a functional macromolecule, for example, haemoglobin
Each polypeptide chain in the quaternary structure is referred to as a subunit of the protein
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globular protein structure
Globular proteins are compact, roughly spherical (circular) in shape and soluble in water
Globular proteins form a spherical shape when folding into their tertiary structure because:
their non-polar hydrophobic R groups are orientated towards the centre of the protein away from the aqueous surroundings and
their polar hydrophilic R groups orientate themselves on the outside of the protein
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how does globular proteins benefit from their orientation
This orientation enables globular proteins to be (generally) soluble in water as the water molecules can surround the polar hydrophilic R groups
The solubility of globular proteins in water means they play important physiological roles as they can be easily transported around organisms and be involved in metabolic reactions
The folding of the protein due to the interactions between the R groups results in globular proteins having specific shapes. This also enables globular proteins to play physiological roles, for example, enzymes can catalyse specific reactions and immunoglobulins (antibodies) can respond to specific antigens
Some globular proteins are conjugated proteins that contain a prosthetic group eg. haemoglobin which contains the prosthetic group called haem
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haemoglobin structure
Haemoglobin is a globular protein which is an oxygen-carrying pigment found in vast quantities in red blood cells
It has a quaternary structure as there are four polypeptide chains. These chains or subunits are globin proteins (two α–globins and two β–globins) and each subunit has a prosthetic haem group
The four globin subunits are held together by disulphide bonds and arranged so that their hydrophobic R groups are facing inwards (helping preserve the three-dimensional spherical shape) and the hydrophilic R groups are facing outwards (helping maintain its solubility)
The arrangements of the R groups is important to the functioning of haemoglobin. If changes occur to the sequence of amino acids in the subunits this can result in the properties of haemoglobin changing. This is what happens to cause sickle cell anaemia (where base substitution results in the amino acid valine (non-polar) replacing glutamic acid (polar) making haemoglobin less soluble)
The prosthetic haem group contains an iron II ion (Fe2+) which is able to reversibly combine with an oxygen molecule forming oxyhaemoglobin and results in the haemoglobin appearing bright red
Each haemoglobin with the four haem groups can therefore carry four oxygen molecules (eight oxygen atoms)
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haemoglobin function
Haemoglobin is responsible for binding oxygen in the lung and transporting the oxygen to tissue to be used in aerobic metabolic pathways
As oxygen is not very soluble in water and haemoglobin is, oxygen can be carried more efficiently around the body when bound to the haemoglobin
The presence of the haem group (and Fe2+) enables small molecules like oxygen to be bound more easily because as each oxygen molecule binds it alters the quaternary structure (due to alterations in the tertiary structure) of the protein which causes haemoglobin to have a higher affinity for the subsequent oxygen molecules and they bind more easily
The existence of the iron II ion (Fe2+) in the prosthetic haem group also allows oxygen to reversibly bind as none of the amino acids that make up the polypeptide chains in haemoglobin are well suited to binding with oxygen
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enzymes
Enzymes are biological catalysts
‘Biological’ because they function in living systems
‘Catalysts’ because they speed up the rate of chemical reactions without being used up or changed
Enzymes are also globular proteins
Critical to the enzyme's function is the active site where the substrate binds
Metabolic pathways are controlled by enzymes in a biochemical cascade of reactions
Virtually every metabolic reaction within living organisms is catalysed by an enzyme – enzymes are therefore essential for life to exist
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insulin
The first protein to have its sequence determined by scientists was the hormone insulin
Insulin is a globular protein produced in the pancreas. It plays an important role in the control of blood glucose concentration
It consists of two polypeptide chains
Polypeptide A has 21 amino acid residues
Polypeptide B has 30 amino acid residues
The two polypeptide chains are held together by three disulfide bridges
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fibrous proteins
Fibrous proteins are long strands of polypeptide chains that have cross-linkages due to hydrogen bonds
These proteins have little or no tertiary structure
Due to a large number of hydrophobic R groups, fibrous proteins are insoluble in water
Fibrous proteins have a limited number of amino acids with the sequence usually being highly repetitive
The highly repetitive sequence creates very organised structures that are strong and this along with their insolubility property, makes fibrous proteins very suitable for structural roles
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examples of fibrous proteins
Keratin makes up hair, nails, horns and feathers (it is a very tough fibrous protein)
Elastin is found in connective tissue, tendons, skin and bone (it can stretch and then return to its original shape)
Collagen is a connective tissue found in skin, tendons and ligaments
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collagen
Collagen is the most common structural protein found in vertebrates
It provides structural support
In vertebrates it is the component of connective tissue which forms:
Tendons
Cartilage
Ligaments
Bones
Teeth
Skin
Walls of blood vessels
Cornea of the eye
Collagen is an insoluble fibrous protein
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structure of collagen
Collagen is formed from three polypeptide chains closely held together by hydrogen bonds to form a triple helix (known as tropocollagen)
Each polypeptide chain is a helix shape (but not α-helix as the chain is not as tightly wound) and contains about 1000 amino acids with glycine, proline and hydroxyproline being the most common
In the primary structure of collagen almost every third amino acid is glycine
This is the smallest amino acid with a R group that contains a single hydrogen atom
Glycine tends to be found on the inside of the polypeptide chains allowing the three chains to be arranged closely together forming a tight triple helix structure
Along with hydrogen bonds forming between the three chains there are also covalent bonds present
Covalent bonds also form cross-links between R groups of amino acids in interacting triple helices when they are arranged parallel to each other. The cross-links hold the collagen molecules together to form fibrils
The collagen molecules are positioned in the fibrils so that there are staggered ends (this gives the striated effect seen in electron micrographs)
When many fibrils are arranged together they form collagen fibres
Collagen fibres are positioned so that they are lined up with the forces they are withstanding
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function of collagen
Collagen is a flexible structural protein forming connective tissues
The presence of the many hydrogen bonds within the triple helix structure of collagen results in great tensile strength. This enables collagen to be able to withstand large pulling forces without stretching or breaking
The staggered ends of the collagen molecules within the fibrils provide strength
Collagen is a stable protein due to the high proportion of proline and hydroxyproline amino acids present. These amino acids increase stability as their R groups repel each other
The length of collagen molecules means they take too long to dissolve in water (making it insoluble in water)
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inorganic ions
An ion is an atom (or sometimes a group of atoms) that has an electrical charge
An ion that has a +ve charge is known as a cation
An ion that has a -ve charge is known as an anion
An inorganic ion is an ion that does not contain carbon
Inorganic ions play an important role in many essential cellular processes
Inorganic ions occur in solution in the cytoplasm and body fluids of organisms
Some occur in high concentrations and others in very low concentrations
The concentration of certain ions can fluctuate and can be used in cell signalling and neuronal transmission
Each type of inorganic ion has a specific role, depending on its properties
Some inorganic ions act as cofactors
Cofactors are non-protein chemical compounds that are required for a protein to function
You should know the following inorganic ions, as well as their properties and roles in the body:
Hydrogen ions (H+)
Calcium ions (Ca2+)
Iron ions (Fe2+/Fe3+)
Sodium ions (Na+)
Potassium ions (K+)
Ammonium ions (NH4+)
Nitrate ions (NO3-)
Hydrogen carbonate ions (HCO3-)
Chloride ions (Cl-)
Phosphate ions (PO43-)
Hydroxide ions (OH-)
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hydrogen ions
Hydrogen ions are protons
The concentration of H+ in a solution determines the pH
There is an inverse relationship between the pH value and the hydrogen ion concentration
The more H+ ions present, the lower the pH (the more acidic the solution)
The fewer H+ ions present, the higher the pH (the more alkaline the solution)
The concentration of H+ is therefore very important for enzyme-controlled reactions, which are all affected by pH
The fluids in the body normally have a pH value of approximately 7.4
The maintenance of this normal pH is essential for many of the metabolic processes that take place within cells
Changes in pH can affect enzyme structure
For example, abnormal levels of hydrogen ions can interact with the side-chains of amino acids and change the secondary and tertiary structures of the proteins that make up enzymes
This can cause denaturation of enzymes
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calcium ions
Ca2+ is essential in the movement of organisms:
In synapses, calcium ions regulate the transmission of impulses from neurone to neurone
Ca2+ also stimulates muscle contraction
When an impulse reaches a muscle fibre, Ca2+ is released from the sarcoplasmic reticulum
This Ca2+ binds to troponin, removing the tropomyosin from myosin-binding sites on actin
This allows actin-myosin cross-bridges to form when the muscle fibre contracts
Ca2+ can also help to regulate protein channels, which affects the permeability of cell membranes
Many enzymes are activated by Ca2+, making these ions key regulators in many biological reactions
The presence of Ca2+ is also necessary for the formation of blood clots (it is known as a clotting factor or a cofactor)
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iron ions
There are actually two versions of iron ions (known as oxidation states)
Iron (II) ions, also known as ferrous ions (Fe2+)
Iron (III) ions, also known as ferric ions (Fe3+)
Iron ions are essential as they can bind oxygen
Haemoglobin is the large protein in red blood cells that is responsible for transporting oxygen around the body
Haemoglobin is made up of four polypeptide chains that each contain one Fe2+
This Fe2+ is a key component in haemoglobin as it binds to oxygen
Myoglobin in muscles functions in a similar way (it is an oxygen-binding protein) but is only made up of one polypeptide chain (containing one Fe2+)
Iron ions are also essential as they are involved in the transfer of electrons during respiration and photosynthesis, so they are key to the biological generation of energy
Iron ions are an essential component of cytochromes (that are themselves a component of electron transport chains)
Cytochrome c contains an iron ion that is essential to its function
During the electron transport process, this iron ion switches between the Fe3+ and Fe2+ oxidation states, which allows for electrons to be accepted and donated
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sodium ions
Na+ is required for the transport of glucose and amino acids across cell-surface membranes (e.g. in the small intestine)
Glucose and amino acid molecules can only enter cells (through carrier proteins) alongside Na+
This process is known as co-transport
First, Na+ is actively transported out of the epithelial cells that line the villi
The Na+ concentration inside the epithelial cells is now lower than the Na+ concentration in the lumen of the small intestine
Na+ now re-enters the cells (moving down the concentration gradient) through co-transport proteins on the surface membrane of the epithelial cells, allowing glucose and amino acids to enter at the same time
Na+ is also required for the transmission of nerve impulses
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potassium ions
K+ is essential for nerve transmission
Potassium ion voltage-gated channel proteins in a section of axon membrane open, allowing the diffusion of potassium ions out of the axon, down their concentration gradient. This returns the potential difference across the axon membrane back to normal (about -70mV) – a process known as
K+ allows for the reabsorption of water in the kidneys
Potassium ions also play an important role in guard cells and the opening of the stomata
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ammonium ions
NH4+ is the intermediate ion that forms during the deamination of proteins in the liver and kidneys
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nitrate ions
NO3- ions are present in the soil and are taken up by plants
NO3- provides an essential source of nitrogen for protein synthesis
Proteins are required for growth and repair in plant
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hydrogen carbonate ions
HCO3- ions work alongside hydrogen ions in the transport of carbon dioxide in the blood
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chloride ions
Cl- ions are also involved in the transport of carbon dioxide in the blood
These ions move in and out of red blood cells and help to maintain pH balance
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phosphate ions
PO43- attaches to other molecules to form phosphate groups, which are an essential component of DNA, RNA and ATP
In DNA and RNA, the phosphate groups allow individual nucleotides to join up (to form polynucleotides)
In ATP, the bonds between phosphate groups store energy
These phosphate groups can be easily attached or detached
When the bonds between phosphate groups are broken, they release a large amount of energy, which can be used for cellular processes
Phosphates are also found in phospholipids, which are key components of the phospholipid bilayer of cell membranes
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hydroxide ions
OH- ions play a vital role in bonding between biochemical molecules
One oxygen atom is covalently bonded to one hydrogen atom
The electronegative charge of the oxygen allows for the formation of hydrogen bonds
Hydrogen bonds are important in many molecules
DNA base pairing
Secondary, tertiary and quaternary protein structure
Water
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cations and anions
cations- h+, ca+,fe2+,na+,k+
anions- no3-, hco3-, cl-, oh-
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biochemical tests- proteins
The Biuret test can be carried out quickly and easily in a lab to determine if a sample contains proteins
Biuret ‘reagent’ contains an alkali and copper (II) sulfate which react in the presence of peptide bonds
Apparatus
Test tubes
Test tube rack
Food solution
Control solution (containing no proteins e.g. distilled water)
Sodium hydroxide
Copper (II) sulfate solution
Pipette
White tile
Gloves
Method
Add sodium hydroxide to the food solution sample to make the solution alkaline
Add a few drops of copper (II) sulfate solution (which is blue) to the sample
Biuret ‘reagent’ contains an alkali and copper (II) sulfate
Repeat steps 1 and 2 using the control solution
Compare the colours of the control solution and the food sample solution
Results
If a colour change is observed from blue to lilac/mauve, then protein is present.
The colour change can be very subtle, it’s wise to hold the test tubes up against a white tile when making observations
If no colour change is observed, no protein is present
For this test to work, there must be at least two peptide bonds present in any protein molecules, so if the sample contains amino acids or dipeptides, the result will be negative
Limitations
The Biuret test is qualitative - it does not give a quantitative value as to the amount of protein present in a sample
If the sample contains amino acids or dipeptides, the result will be negative (due to lack of peptide bonds)
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finding the concentration of a substance - semi quantitative test
Benedict’s solution can be used to carry out a semi-quantitative test on a reducing sugar solution to determine the concentration of reducing sugar present in the sample
It is important that an excess of Benedict’s solution is used so that there is more than enough copper (II) sulfate present to react with any sugar present
The intensity of any colour change seen relates to the concentration of reducing sugar present in the sample
A positive test is indicated along a spectrum of colour from green (low concentration) to brick-red (high concentration of reducing sugar present)
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serial dilutions
A semi-quantitative test can be carried out by setting up standard solutions with known concentrations of reducing sugar (such as glucose)
These solutions should be set up using a serial dilution of an existing stock solution
Each solution is then treated in the same way: add the same volume of Benedict’s solution to each sample and heat in a water bath that has been boiled (ideally at the same temperature each time) for a set time (5 minutes or so) to allow colour changes to occur
It is important to ensure that an excess of Benedict’s solution is used
Any colour change observed for each solution of a known concentration in that time can be attributed to the concentration of reducing sugar present in that solution
The same procedure is carried out on a sample with an unknown concentration of reducing sugar which is then compared to the stock solution colours to estimate the concentration of reducing sugar present
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producing serial dilations
Serial dilutions are created by taking a series of dilutions of a stock solution. The concentration decreases by the same quantity between each test tube
They can either be ‘doubling dilutions’ (where the concentration is halved between each test tube) or a desired range (e.g. 0, 2, 4, 6, 8, 10 mmol dm-3)
Serial dilutions are completed to create a standard to compare unknown concentrations against
The comparison can be:
Visual
Measured through a calibration/standard curve
Measured using a colorimeter
They can be used when:
Counting bacteria or yeast populations
Determining unknown glucose, starch, protein concentrations
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alterations
It is also possible to standardise this test but instead of waiting a fixed amount of time for a range of colours to be observed, time how long it takes for the first colour change to occur (blue to green)
The higher the concentration of reducing sugar in a sample, the less time it would take for a colour change to be observed
To avoid issues with human interpretation of colour, a colorimeter could be used to measure the absorbance or transmission of light through the sugar solutions of known concentration to establish a range of values that an unknown sample can be compared against a calibration curve
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colorimeters
A colorimeter is an instrument that beams a specific wavelength (colour) of light through a sample and measures how much of this light is absorbed (arbitrary units)
They provide a quantitative measurement
They contain different wavelengths or colour filters (depends on the model of colorimeter), so that a suitable colour can be shone through the sample and will not get absorbed. This colour will be the contrasting colour (eg. a red sample should have green light shone through)
Remember that a sample will look red as that wavelength of light is being reflected but the other wavelengths will be absorbed
Colorimeters must be calibrated before taking measurements
This is completed by placing a blank into the colorimeter and taking a reference, it should read 0 (that is, no light is being absorbed)
This step should be repeated periodically whilst taking measurements to ensure that the absorbance is still 0
The results can then be used to plot a calibration or standard curve
Absorbance/transmission of light against the known concentrations can be used
Unknown concentrations can then be determined from this graph
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biosensors
Advances in technology mean there are now other methods of finding the concentration of a substance
Biosensors are highly accurate analytical devices
They use a catalyst to turn a biological response into an electrical signal
The catalyst used is a very specific and stable enzyme
Blood glucose biosensors are often used by diabetics so they can monitor and regulate their blood glucose levels
Glucose oxidase is the enzyme used in these types of biosensors. It breaks down the glucose in the blood sample:
-Glucose oxidase uses FAD to oxidise glucose, forming FADH2
-FADH2 is then oxidised by the electrode in the device and this produces a current
-The current is a measurement of the glucose concentration
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blood glucose biosensor
Using a blood glucose biosensor
An individual produces a drop of blood on their finger using a pricker or a sterile lancet
The blood is transferred onto a test strip
The enzyme is located on the test strip
The test strip is inserted into the biosensor meter
A blood glucose reading is displayed as a digital figure
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chromatography
Chromatography is a technique that can be used to separate a mixture into its individual components
Chromatography relies on differences in the solubility of the different chemicals (called ‘solutes’) within a mixture
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chromatography phases
All chromatography techniques use two phases:
The mobile phase
The stationary phase
The components in the mixture separate as the mobile phase travels over the stationary phase
Differences in the solubility of each component in the mobile phase affects how far each component can travel
Those components with higher solubility will travel further than the others
This is because they spend more time in the mobile phase and are thus carried further up the paper than the less soluble components
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paper chromatography
Paper chromatography is one specific form of chromatography
In paper chromatography:
The mobile phase is the solvent in which the sample molecules can move, which in paper chromatography is a liquid e.g. water or ethanol
The stationary phase in paper chromatography is the chromatography paper
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paper chromatography
A spot of the mixture (that you want to separate) is placed on chromatography paper and left to dry
The chromatography paper is then suspended in a solvent
As the solvent travels up through the chromatography paper, the different components within the mixture begin to move up the paper at different speeds
Larger molecules move slower than smaller ones
This causes the original mixture to separate out into different spots or bands on the chromatography paper
This produces what is known as a chromatogram
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using chromatography to separate a mixture of monosaccharides
Paper chromatography can be used to separate a mixture of monosaccharides
Mixtures containing coloured molecules, such as ink or chlorophyll, do not have to be stained as they are already coloured
Mixtures of colourless molecules, such as a mixture of monosaccharides, have to be stained first
A spot of the stained monosaccharide sample mixture is placed on a line at the bottom of the chromatography paper
Spots of known standard solutions of different monosaccharides are then placed on the line beside the sample spot
The chromatography paper is then suspended in a solvent
As the solvent travels up through the chromatography paper, the different monosaccharides within the mixture separate out at different distances from the line
The unknown monosaccharides can then be identified by comparing and matching them with the chromatograms of the known standard solutions of different monosaccharides
If a spot from the monosaccharide sample mixture is at the same distance from the line as a spot from one of the known standard solutions, then the mixture must contain this monosaccharide
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using chromatography to separate a mixture of amino acids
Paper chromatography can be used to separate a mixture of amino acids
A spot of the unknown amino acid sample mixture is placed on a line at the bottom of the chromatography paper
Spots of known standard solutions of different amino acids are then placed on the line beside the unknown sample spot
The chromatography paper is then suspended in a solvent
Each amino acid will be more or less soluble in the mobile phase than others and will therefore separate out of the mixture travelling with the solvent at different times/distances from the line, depending on their:
Charge
Size
The unknown amino acid(s) can then be identified by comparing and matching them with the chromatograms of the known standard solutions of different amino acids
If a spot from the amino acid sample mixture is at the same distance from the line as a spot from one the known standard solutions, then the mixture must contain this amino acid
In order to view the spots from the different amino acids, it may be necessary to first dry the chromatography paper and then spray it with ninhydrin solution (this chemical reacts with amino acids, producing an easily visible blue-violet colour)
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calculating the Rf value
After a chromatogram has been obtained the molecules present in the sample mixture can be identified by calculating their retardation factor (Rf)
In order to calculate Rf values, a line must be drawn across the chromatogram to show how far the solvent travelled
This line is known as the solvent front
The distance between the origin line and the solvent front is the distance moved by the solvent
The origin line is the line at the bottom of the paper on which the samples were placed at the beginning of the experiment
The Rf value demonstrates how far a dissolved molecule travels during the mobile phase
A smaller Rf value indicates the molecule is less soluble and larger in size
The Rf value of each solute (each spot on the chromatogram) is calculated and then compared to the Rf values of known molecules/substances
The equation is:
Rf = distance moved by solute ÷ distance moved by solvent
The Rf value is a ratio so it is always lower than one
It has no units
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using Rfvalues to identify chloroplast pigments
Chromatography can be used to separate and identify chloroplast pigments that have been extracted from a leaf as each pigment will have a unique Rf value
Although specific Rf values depend on the solvent that is being used, in general:
Carotenoids have the highest Rf values (usually close to 1)
Chlorophyll B has a much lower Rf value
Chlorophyll A has an Rf value somewhere between those of carotenoids and chlorophyll B
Small Rf values indicate the pigment is less soluble and larger in size
