Biological molecules (2.1.2)
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Water, , is a simple covalent molecule. The central oxygen shares a single covalent bond with each hydrogen.
Water is polar because the oxygen atom attracts the shared electrons more strongly, meaning that it has a partial negative charge, while the hydrogen atoms are partially positive. This polarity results in water molecules interacting with each other through hydrogen bonds.
Hydrogen bonds are strong electrostatic intermolecular interactions. They form between water molecules as the partially positively charged hydrogen atom is attracted to the lone electron pair of the electronegative oxygen atom.
Hydrogen bonds are responsible for some unique properties of water, such as cohesion, adhesion, heat capacity, and surface tension.
Water is a polar molecule, so it interacts with charged species, behaving as a solvent. This property facilitates numerous metabolic reactions that occur in aqueous environments.
In ionic compounds such as sodium chloride, the positive ions are attracted to the partially negative oxygen of the water molecules, while ions are attracted to the partially positive hydrogens.
As water freezes, it becomes less dense as hydrogen bonds lock the molecules into a fixed crystalline structure, where there is increased spacing between molecules. This reduced density causes ice to float.
A lot of energy is needed to break the hydrogen bonds between water molecules, which means that the structure of water also results in a high boiling point.
Most organisms (but particularly small ones) require a stable temperature range to survive.
The specific heat capacity of water is the amount of heat energy required to raise the temperature of 1 of water by 1 .
Water’s high specific heat capacity means it resists temperature changes, providing a stable environment. This stability makes large water bodies, such as oceans and lakes, thermally stable habitats, ideal for aquatic life.
Cohesion is the attraction between molecules of the same substance. In water, hydrogen bonding causes strong cohesion, allowing water molecules to form continuous columns for transport in the xylem during transpiration.
Cohesion also contributes to water’s high surface tension, enabling small organisms to move across the water surface.
Adhesion is the attraction between molecules of different substances. In plants, water molecules adhere to the walls of xylem vessels, helping maintain a continuous column of water and aiding its movement against gravity during transpiration. Adhesion also contributes to capillary action.
A monomer is a small, single molecule that can bind chemically to other monomers to form a larger structure.
A polymer is a large chain molecule composed of repeating units of monomers that are bonded together. Polymers play a crucial role in the formation of complex biological macromolecules.
Examples of monomers and polymers include:
- Amino acids link together to form proteins.
- Nucleotides join to create nucleic acids like DNA and RNA.
- Monosaccharides, such as glucose, bond to form polysaccharides as starch and cellulose.
Condensation reactions occur when two molecules combine to form a larger molecule, releasing one molecule of water ().
These reactions are essential for the synthesis of many biological polymers. For example, peptide bonds form between amino acids through condensation reactions to make polypeptides.
Hydrolysis reactions involve splitting a large molecule into smaller molecules by adding one molecule of water ().
These reactions are the reverse of condensation reactions and are essential for breaking down polymers into their monomer components. For example, the peptide bonds in a dipeptide can be broken by adding water in a hydrolysis reaction to produce two amino acid monomers.
There are four groups of biological macromolecules: carbohydrates, lipids, proteins, and nucleic acids consisting of the following chemical elements:
- carbohydrates –
- lipids –
- proteins –
- nucleic acids –
Carbohydrates are biological molecules made from sugars.
Monosaccharides are the simplest form of sugar, consisting of a single monomer. Examples include glucose, fructose, and galactose.
Two monosaccharides join via a condensation reaction to form a disaccharide. Many monosaccharides join via condensation reactions to form a polysaccharide.
Monosaccharides can be classified by the number of carbon atoms they contain:
- trioses ()
- pentoses ()
- hexoses ().
Monosaccharides are polar molecules and are therefore soluble in water. Pentose and hexose monosaccharides usually exist as ring structures in solution, with the carbon atoms numbered for identification.
Glucose () contains six carbon atoms and is therefore a hexose monosaccharide.
Glucose exists in two isomeric forms: -glucose and -glucose. They have the same chemical formula but different structures and properties.
In -glucose, the (hydroxyl) group on carbon 1 is on the opposite side of the ring from the group, whereas in -glucose it is on the same side.
Ribose and deoxyribose are pentose monosaccharides, meaning they have five carbon atoms.
They are found in the nucleotide monomers that form RNA (ribonucleic acid) and DNA (deoxyribonucleic acid).
Ribose and deoxyribose are very similar in their structure, but deoxy ribose lacks an oxygen atom at carbon 2.
Monosaccharides are linked by glycosidic bonds, which form in a condensation reaction between hydroxyl () groups on adjacent monosaccharides, releasing a molecule of water.
The name of the glycosidic bond indicates the carbon atoms involved in the linkage. For example, an 1,4 glycosidic bond forms between carbon 1 of one -glucose molecule and carbon 4 of another.
Two -glucose molecules combine to form the disaccharide maltose, joined by an 1,4 glycosidic bond.
Two monosaccharides bonded together via a glycosidic bond form disaccharides:
- Maltose = -glucose + -glucose
- Sucrose = -glucose + -fructose
- Lactose = -galactose + glucose.
The glycosidic bonds between monosaccharides can be broken down through hydrolysis reactions, where one molecule of water is added to break the bond.
Hydrolysis is crucial in biological processes, such as digestion and the breakdown of stored carbohydrates for cellular respiration.
Starch is the insoluble, primary storage polysaccharide in plants and is formed from a few hundred to a few thousand -glucose monosaccharides bonded together. It is made from a mixture of two polysaccharides.
The two polysaccharides which form starch are:
- Amylose constitutes 10–30 of starch and has an unbranched, helix-shaped chain of -glucose molecules linked by 1,4 glycosidic bonds.
- Amylopectin constitutes 70–90 of starch and has both 1,4 glycosidic bonds and 1,6 glycosidic bonds, creating a branched molecule. Enzymes involved in breaking down these molecules can simultaneously work at multiple branch points, leading to rapid release of glucose for energy. More accessible hydroxyl groups make amylopectin more soluble in water.
Glycogen is the primary storage polysaccharide in animals and fungi, characterised by a highly branched and uncoiled structure.
The highly branched structure of glycogen means there are numerous terminal glucose units leading to rapid release of glucose for energy. Branched polysaccharides with more accessible hydroxyl groups are also more soluble in water.
Glycogen’s structure is similar to amylopectin, but its branching is more extensive. This makes it more water-soluble and its terminal glucose molecules more accessible to enzymes.
Amylose is coiled, making it insoluble and compact which is ideal for storage.
Amylopectin and glycogen are both highly branched molecules, making them compact and allowing more energy to be stored. Their branches provide multiple free ends where glucose molecules can be rapidly added in condensation reactions or removed in hydrolysis reactions, enabling quick adaptation to cellular energy demands.
Glucose is stored in plants and animals until it’s needed for respiration. Storage molecules can be broken down by hydrolysis to provide glucose, which is converted from biochemical energy (oxidised) to usable energy (ATP) for cells.
Cellulose is a structural polysaccharide in plants. It is composed of long, linear chains of -glucose molecules joined by 1,4 glycosidic bonds.
Every other -glucose molecule in cellulose is inverted, producing a linear structure with extensive hydrogen bonding within and between polysaccharide chains.
Cellulose’s high tensile strength is a combined result of several key structural features:
- The strong 1,4 glycosidic bonds within a polysaccharide chain.
- There hydrogen bonding between polysaccharide chains within a microfibril.
- The alignment of microfibrils into macrofibrils.
- The alignment of macrofibrils into fibres.
Individual cellulose chains are held together by hydrogen bonds to form microfibrils.
Microfibrils are organised into larger macrofibrils, further stabilised by additional hydrogen bonds.
The alignment of macrofibrils can vary depending on their role within a plant.
Cellulose is the main structural component of plant cell walls. The cell wall matrix includes other molecules such as lignin, to further increase cell wall strength.
Its structure gives it the following properties:
- Its high tensile strength enables cell walls to withstand turgor pressure and maximise leaf surface area for light absorption.
- Spaces between macrofibrils make cellulose fibres permeable to water and solutes, facilitating diffusion into and out of plant cells.
- Cellulose is a source of dietary fibre (roughage). Few organisms have the enzyme cellulase, which hydrolyses cellulose, meaning that it is not absorbed during digestion.
The bonding and structure of the key polysaccharides in plants and animals are summarised below.
Lipids are non-polar macromolecules.
Unlike other biological macromolecules, lipids are not classed as polymers because they are not composed solely of monomer subunits.
Liquid lipids are classed as oils whereas solid lipids are fats.
Triglycerides are non-polar, hydrophobic molecules consisting of one glycerol molecule (an alcohol) and three fatty acid chains. They are the main components of fats and oils and are insoluble in water, but soluble in organic solvents, e.g., ethanol.
Fatty acids have a methyl group () at one end of a hydrocarbon chain (R group) and a carboxyl group () at the other end.
Triglycerides are formed through a condensation reaction where each hydroxyl group () of glycerol reacts with a carboxyl group () of a fatty acid to form an ester bond.
Three fatty acids are attached to one glycerol molecule, three water molecules are released per triglyceride formed.
Ester bonds are broken during the digestion of the triglyceride molecule through hydrolysis reactions consuming three water molecules per triglyceride hydrolysed.
Fatty acids can differ in hydrocarbon chain length (R group) and in their saturation.
Saturated fatty acids have no double bonds. They form a straighter structure and pack tightly together, resulting in higher melting points and being in a solid state at room temperature. They are primarily found in animal fats.
Unsaturated fatty acids contain one (monounsaturated) or more (polyunsaturated) double bonds, leading to ‘kinks’ in the hydrocarbon chains that prevent tight packing. This lowers the melting point, so they are usually liquids at room temperature. They are typically found in vegetable oils.
Phospholipids are essential components of cell membranes.
The structure of phospholipids is similar to triglycerides: glycerol plus two fatty acid chains and one phosphate. This results in a hydrophilic polar ‘head’ and two hydrophobic fatty acid ‘tails’.
Phospholipids act as surfactants (surface-active agents) that reduce surface tension between immiscible substances such as oil and water.
Their hydrophilic phosphate heads interact with water, while their hydrophobic fatty acid tails interact with non-polar substances.
Phospholipids are amphipathic. In aqueous environments, phospholipids spontaneously arrange into a bilayer with the hydrophilic heads facing outwards, facing the surrounding water and the hydrophobic tails facing inward. This arrangement forms the basic structure of cell-surface membranes.
Cholesterol is a sterol, a bulky organic molecule with an alcohol functional group, found within the phospholipid bilayer of cell membranes.
Cholesterol is amphipathic with a hydrophilic hydroxyl group and hydrophobic steroid rings. It fits between phospholipids in animal cell membranes, helping maintain membrane stability and fluidity by preventing excessive movement at high temperatures and tight packing at low temperatures.
Cholesterol is also a precursor of steroid hormones, vitamin D and bile salts.
Triglycerides are energy storage molecules with a high energy density due to their many carbon–hydrogen bonds.
They are insoluble, so can be stored without affecting water potential. Plants often store triglycerides as oils, while animals store them as fat in adipose tissue.
They also provide thermal insulation, protection and buoyancy. Oxidation of triglycerides releases metabolic water, which is important in some desert animals.
Amino acids are the monomers from which polypeptides and proteins are made.
Polypeptides are linear chains of more than two amino acids, whereas proteins consist of one or more polypeptides folded into a specific 3D functional structure.
Amino acids contain a central carbon atom, attached to a hydrogen atom, an amino group (), a carboxylic acid group () and a variable side chain group (R group).
There are 20 amino acids common to all organisms and they differ only by their R group.
Essential amino acids come from the diet and non-essential ones can be synthesized in the body.
Polypeptides are formed when amino acid monomers are linked to each other during condensation reactions by peptide bonds (strong covalent bonds).
The peptide bond is formed between the carboxyl group of one amino acid and the amino group of another amino acid.
The reaction is catalysed by peptidyl transferase.
Peptide bonds can be broken down by hydrolysis reactions. These reactions use one molecule of water to break the covalent peptide bond between two amino acid monomers.
This reaction is catalysed by proteases.
There are four levels of protein structure:
Primary structure refers to the specific sequence and number of amino acids in a polypeptide chain, determined by the DNA base sequence.
This linear sequence determines how the protein folds into its three-dimensional structure and therefore its function. A change in the amino acid sequence can alter the protein’s structure and affect its function.
Secondary structure refers to the localised folding of a polypeptide chain into -helices and -pleated sheets
In an -helix, hydrogen bonds form between atoms in the polypeptide backbone, causing the chain to coil into a spiral.
In a -pleated sheet, several polypeptide chains lie parallel to one another and are held together by hydrogen bonds. These hydrogen bonds stabilise the secondary structure of the protein.
Tertiary structure is the overall three-dimensional shape of a protein, determined by interactions between the R groups of amino acids. The shape of a protein is closely related to its function.
Tertiary structure is stabilised by:
- Hydrogen bonds between polar R groups.
- Ionic bonds between oppositely charged R groups (impacted by ).
- Disulfide bonds / bridges are covalent bonds between cysteine residues (impacted by ).
- Hydrophobic interactions, where non-polar R groups cluster together away from water.
- Hydrophilic interactions, where polar R groups are attracted to water and orientate towards the outside of the protein.
Quaternary structure occurs when two or more polypeptide chains (which may be identical or different) associate to form a functional protein.
The subunits are held together by the same interactions found in tertiary structure (hydrogen bonds, ionic bonds, disulfide bonds and hydrophobic / hydrophilic interactions), but these occur between polypeptides rather than within one polypeptide.
For example, haemoglobin consists of two -globin and two -globin subunits and is only functional when all four polypeptides are associated.
Globular proteins are compact, roughly spherical in shape, and soluble in water meaning that they can be transported in the circulatory system.
Examples include haemoglobin, enzymes and antibodies.
Insulin is a globular protein produced in the pancreas and is essential for regulating blood glucose levels. It is compact, soluble structure allows it to be transported in the blood, and its specific three-dimensional shape enables it to bind to complementary receptors on target cells.
Conjugated proteins are globular proteins which require a non-protein component, such as a cofactor or prosthetic group, to function.
Haemoglobin is a conjugated protein because it contains the prosthetic group haem: a ring shaped organic molecule which binds to iron.
Lipoproteins are conjugated proteins containing lipid and protein elements. Similarly, glycoproteins contain a carbohydrate element.
Haemoglobin is a conjugated protein with a quaternary structure. It is found in red blood cells and consists of two and two polypeptide chains. Each subunit contains a haem group. The haem groups give blood its distinctive red colour.
Haemoglobin’s function is to carry oxygen in the blood. Oxygen reversibly binds to the ion in the haem group. Each haem group bonds with one oxygen molecule, therefore haemoglobin can carry four oxygen molecules in total.
Catalase is a conjugated protein that functions as an intracellular enzyme which breaks down hydrogen peroxide (a toxic byproduct of cellular metabolism) into water and oxygen. It contains four haem groups.
Fibrous proteins are long, insoluble polypeptide chains with structural roles, such as in keratin and collagen.
They are not folded into compact shapes and have repetitive amino acid sequences. Their strength comes from extensive hydrogen bonding and additional cross-links, such as disulfide bonds, between chains.
These proteins form strong fibres and are well suited to support and protection.
Keratins are a group of fibrous proteins found in hair, nails, horns and feathers.
They have a high cysteine content, allowing many disulfide bonds to form between polypeptide chains, giving keratin its strength.
The greater the number of disulfide bonds, the tougher and less flexible the material; for example, nails contain more disulfide bonds than hair.
Elastin is a fibrous protein present in tissues such as blood vessels, skin and alveoli. The limited crosslinking between polypeptide chains in this protein allows for flexibility and elasticity; the tissues can stretch and recoil to their original shape.
Collagen is a fibrous protein found in connective tissues such as skin, tendons, ligaments, cartilage and the nervous system.
It consists of three polypeptide chains wound together in a triple helix, like a rope, making it strong yet flexible. The chains are held together by hydrogen bonds and covalent cross-links, and are staggered to avoid weak points.
Collagen molecules form fibrils and fibres and are insoluble in water.
An ion is an atom or group of atoms with an electrical charge. Ions with a positive charge are cations, while those with a negative charge are anions.
Inorganic ions are crucial in various cellular processes, existing in the cytoplasm and body fluids of organisms. Their concentrations can vary and each type of inorganic ion has a specific role based on its properties.
Hydrogen ions determine the of a solution. The concentration of is inversely related to ; more ions correspond to lower .
is crucial for enzyme activity, as changes can alter enzyme structure, particularly the shape of its active site.
Calcium ions () are vital for muscle contraction, nerve impulse transmission, and blood clotting. also regulates protein channels, affecting cell membrane permeability.
Iron ions () are essential for oxygen transport in haemoglobin and myoglobin. in haemoglobin binds oxygen, allowing red blood cells to carry it throughout the body.
Iron ions are also essential in electron transport during respiration and photosynthesis.
Sodium ions () are necessary for glucose and amino acid transport across cell membranes via co-transport. They also play a role in nerve impulse transmission.
Potassium ions () are essential for nerve transmission, particularly in repolarising the axon membrane after a nerve impulse. They also aid in water reabsorption in the kidneys and regulate the opening of stomata in plants.
Ammonium ions () are products of the oxidative deamination of proteins in the liver and kidneys and they are then converted to urea.
Ammonium ions are also involved in the process of nitrogen assimilation in plants, where they can be taken up by roots and converted into amino acids, contributing to plant growth and development.
Nitrate ions () are absorbed by plants from the soil and provide nitrogen for protein synthesis, essential for plant growth and repair.
In addition, nitrate ions play a role in the synthesis of nucleic acids (DNA and RNA) in plants, supporting cell division and growth.
Hydrogencarbonate ions () work with ions in transporting carbon dioxide in the blood.
Hydrogencarbonate ions also help regulate acid–base balance in the blood and act as a buffer to maintain the level.
Chloride ions () are involved in carbon dioxide transport and help maintain the balance in the blood.
Chloride ions are also required for the production of hydrochloric acid () in gastric juice, which aids digestion in the stomach.
Phosphate ions () form phosphate groups, essential components of DNA, RNA, ATP, and phospholipids.
In ATP, the phosphate bonds store energy, which is released during cellular processes. Phosphate ions are essential for energy transfer through ATP and play a critical role in signal transduction pathways, regulating various cellular processes.
Hydroxide ion () are important in the process of maintaining by neutralising excess ().
Sugars can be classified as reducing or non-reducing depending on their structure and their functional groups. Reducing sugars can act as reducing agents by donating electrons.
Glucose, fructose, lactose, and maltose are reducing sugars, while sucrose is non-reducing.
Benedict’s test detects reducing sugars in a sample.
Benedict’s reagent is a blue solution of () that contains copper(II) sulfate ions. When this reacts with reducing sugars, copper(II) is reduced to copper(I). The copper(I) oxide formed is insoluble in water and is detected as a brick-red precipitate.
Method:
- Add Benedict’s reagent (blue) to 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 fully react with the reducing sugars in the sample.
- Heat the test tube in boiling water or in a water bath for a few minutes.
Results:
A colour change from blue to green, then yellow, orange, and finally brick-red, indicates the presence of reducing sugars in increasing concentrations.
Hydrolysing paired with Benedict’s test can be used to test for non-reducing sugars.
Method:
- Follow the protocol for detecting reducing sugars, and if the results are negative, proceed.
- Boil the sample with dilute hydrochloric acid. This breaks the glycosidic bonds in the sample, breaking the non-reducing sugars down into reducing sugars.
- Neutralise the sample with sodium hydrogencarbonate.
- Add an excess of Benedict’s solution.
- Heat the test tube in boiling water or in a water bath for a few minutes.
Results:
A colour change from blue to green, then yellow, orange, and finally brick-red indicates the presence of reducing sugars in increasing concentrations — i.e. the non-reducing sugars that have been broken down by hydrolysis into monosaccharides and therefore turned into reducing sugars.
Quantitative analysis requires concentrations to be known. In colourimetry, solutions of known concentrations can be used as a ladder of standards to construct a calibration curve.
Serial dilution can be used to create a series of standard solutions. This involves progressively diluting a stock solution of known concentration to create a range of concentrations.
A colorimeter is a device that directs a specific wavelength of light through a sample and measures the amount of light absorbed. The absorbance value gives a quantitative measure of the colour intensity.
Before taking measurements, the colorimeter must be calibrated using a series of standard solutions to create a calibration (standard) curve, which can then be used to determine the concentration of unknown samples by comparing them to the curve.
Benedict’s solution can be used to perform a semi-quantitative, visual test to estimate the concentration of reducing sugars in an unknown sample:
- First prepare standard solutions with known concentrations of a reducing sugar, such as glucose, using simple or serial dilutions.
- Each standard solution should then be mixed with the Benedict’s solution and heated in a boiling water bath.
- The unknown solution must also be mixed with the Benedict’s solution and heated in a boiling water bath.
Compare the colours in the standard solutions with the colour of the unknown sample to estimate its sugar concentration.
For the comparisons to be accurate, the volume of Benedict’s added to the sample / standard and the heating method should be consistent.
The iodine test is a qualitative test that detects starch in a sample.
Method:
- Add a few drops of iodine (in potassium iodide solution, which is insoluble in water) to the sample.
Results:
A blue-black colour indicates the presence of starch.
The emulsion test is used to identify lipids in a sample.
Method:
- Add ethanol to the sample and shake to mix.
- Add the mixture to a test tube containing water.
Results:
A white emulsion (cloudy appearance) indicates the presence of lipids. The more lipid present, the more pronounced the milkiness/white colour.
The emulsion test is qualitative and cannot provide concentration values.
The biuret test is a qualitative test used to detect proteins in a sample.
Biuret solution is a premixed alkaline copper(II) sulfate solution which reacts in the presence of peptide bonds. The reagent requires at least two peptide bonds, so free amino acids or dipeptides give a negative result.
Method:
- Add sodium hydroxide to the food sample to make it alkaline.
- Add a few drops of blue copper(II) sulfate solution to the sample.
Results:
A colour change from blue to lilac / purple indicates the presence of proteins in the sample.
Biosensors detect specific substances in a sample by using a biological component, such as an enzyme, antibody, nucleic acid, or cellular organelle, which interacts with the target substance.
When the target substance is present in the sample, it interacts with the biological agent, generating a chemical signal. This signal is then detected by a transducer, which converts the chemical signal to an electrical signal. The strength of the electrical signal can then be measured to determine the concentration of the substance in the solution.
Thin layer chromatography is a technique used to separate a mixture into its individual components. It can be applied to mixtures containing proteins, carbohydrates, vitamins, and nucleic acids.
Chromatography separates substances using two phases:
- The mobile phase: this is the solvent that moves the components of the mixture.
- The stationary phase: this is the solid that remains fixed, over which the mobile phase flows. Typically a glass or plastic plate, coated with a thin layer of silica gel or alumina.
Components in the mixture separate based on their solubility in the mobile phase, and their affinity for the stationary phase. Components with higher solubility in the mobile phase travel further.
Paper chromatography uses chromatography paper as the stationary phase and a solvent as the mobile phase.
Method:
- A spot of the mixture is placed on the baseline of the chromatography paper and allowed to dry.
- The paper is suspended in the solvent, with the spot above the solvent level.
- As the solvent moves up the paper, the components of the mixture travel at different rates.
Substances that are more soluble in the solvent move further, while those with a greater attraction to the stationary phase move less. This separates the mixture into distinct spots, forming a chromatogram.
When carrying out thin-layer chromatography:
- Draw the origin line in pencil, as ink may dissolve in the solvent.
- Keep the solvent below the origin line so the samples do not dissolve directly into the solvent.
- Keep the paper / plate vertical to ensure even solvent movement by capillary action.
- Allow the solvent to travel near the top, but not reach it, so the solvent front can be measured accurately.
- Cover the container to prevent solvent evaporation and maintain a solvent-saturated atmosphere.
Paper chromatography can effectively separate a mixture of amino acids:
- Place a spot of the unknown amino acid mixture on the origin line.
- Place additional spots of amino acid standards on the origin line beside the unknown sample. Note that the spots must not touch.
- Put the chromatography paper in a solvent.
- Once the solvent front has travelled up most of the paper, remove the paper and mark on the solvent front.
- To visualise the amino acid spots, the paper may need to be dried and then treated with ninhydrin solution and heated, which reacts with the amino acids to produce a visible blue-violet colour.
The unknown mixture can be compared with the standards to identify the amino acid components.
Paper chromatography can be used to separate a mixture of monosaccharides:
- To prepare the sample, dissolve a solid or liquid mixture of monosaccharides in a suitable solvent.
- Place the sample spot on the origin line.
- Apply spots of known standard monosaccharide solutions next to, but not touching, the sample spot.
- Place the chromatography paper in the solvent, ensuring that the solvent does not cover the sample spots and leave until the solvent front nears the top of the paper.
Where the monosaccharide is colourless, you can visualise using a chemical stain within the sample or on the developed paper.
Compare the positions of the spots from the sample mixture to those of the known standard solutions.
stands for retention factor and represents how far through a stationary phase a component has moved compared to the distance moved by its solvent.
Compounds can be identified by comparing their values to those of known substances.
For the chromatogram shown above, the calculated value of reference A is:
The greater the value, the closer the distance moved by a sample is to the distance moved by the solvent. This indicates a sample is more strongly attracted to the solvent than to the stationary phase.
All values will be less than 1.
Note that values are specific to the stationary phase and solvent being used.