Porphyrin compounds

One class of chromophores are porphyrin compounds. Porphyrin compounds contain a porphin ring, an organic macromolecule containing four heterocyclic structures composed of carbon and nitrogen. When porphin rings are substituted with functional groups, they are called porphyrins. Porphyrin compounds consist of 26 electrons in a conjugated system, allowing for the absorption of visible light, therefore appearing as coloured species.

The two NH groups in the centre of the heterocyclic ring structure (Figure 1) are capable of losing the two hydrogen ions, acting as an acid. When these hydrogen ions are lost, the nitrogen atoms each have a lone pair of electrons that are capable of acting as a ligand to form a coordinate covalent bond to a metal ion. The presence of a transition metal ion can also influence the colour of the porphyrin compound.

Chlorophyll

One example of a porphyrin compound is chlorophyll, the green pigment in plants that is responsible for absorbing light from the sun during photosynthesis. Chlorophyll contains a porphin ring with many substitutions and a magnesium ion in the centre. The presence of the magnesium ion does not influence colour, since it is not a transition metal, but the porphyrin ring allows for the absorption of red light, resulting in the emission of green light.

Chlorophyll is essential to the life of a plant because it is capable of absorbing visible light, which is required to catalyse the reaction in photosynthesis. Chlorophyll is contained in organelles called chloroplasts that are within specialised cells near the upper surface of leaves. When chlorophyll absorbs visible light, the energy from the light is used to synthesise glucose by forming chemical bonds, an anabolic process that is endothermic. Although there are different types of chlorophyll, most chlorophyll compounds strongly absorb light in the red and blue visible range, resulting in the green colour of many plants.

Heme

Another example of porphyrin compounds are cytochromes. Cytochromes are porphyrin compounds that contain an iron ion, known as a heme group. Heme groups appear in different proteins, such as hemoglobin, which is responsible for transporting oxygen and carbon dioxide in the bloodstream to and from the lungs. Heme consists of the porphin ring with many substitutions and an iron ion in the centre. The presence of both the iron ion and the porphyrin ring in heme allows blood to appear red in colour.

Cytochromes, with the presence of the iron ion, are capable of undergoing redox reactions in biological processes. The iron ion can increase or decrease its oxidation number by converting between iron(II) and iron(III), allowing it to act as a catalyst in chemical reactions. The colour of blood varies from orange-red to blue-red depending on the oxidation state of iron.

Hemoglobin and myoglobin

Comparison of structures and functions

Hemoglobin

As discussed previously, hemoglobin (or haemoglobin) is an example of a cytochrome. Cytochromes contain a heme group – a porphyrin ring bonded to an iron(II) ion. Hemoglobin is responsible for transporting oxygen and carbon dioxide in the bloodstream between cells and the lungs. Hemoglobin is a protein consisting of four separate polypeptide chains with four heme groups. Hemoglobin is found in red blood cells, and the presence of both the porphyrin ring and the iron ion provide the red pigment in blood.

Myoglobin

Myoglobin is also an example of a cytochrome. It is related to hemoglobin and structurally similar, except that it contains only one heme group and is found in the muscle tissue of mammals, rather than in the blood. Myoglobin performs a similar function to hemoglobin, but instead of transporting oxygen, it stores oxygen, allowing it to be available for cellular respiration. The concentration of myoglobin is very high for mammals that hold their breath for extended periods of time underwater, such as whales, allowing the stored oxygen to be used when the mammal is not actively breathing.

The presence of myoglobin in muscle tissue provides the red pigment seen in meat. Red meats, such as beef, are deep red in colour due to the high concentration of myoglobin, whereas white meat, such as chicken, contains low concentrations of myoglobin. Meats that are 'cured' and preserved with nitrates, such as bacon or ham, appear pink, since the presence of the nitrate group alters the absorption of light by myoglobin. In raw meat, the colour of myoglobin is red, when the iron ion has an oxidation state of +2. When meat is cooked, the oxidation state of iron changes to +3, resulting in a colour change to brown.

Oxygen binding

The function of the heme group in hemoglobin and myoglobin is to bind to oxygen molecules. Hemoglobin must bind oxygen in order to transport it to cells for cellular respiration from the lungs, while myoglobin binds to oxygen to store it in muscle cells for later use. Oxygen binds to heme by forming a complex with the iron(II) ion. One lone pair of electrons on oxygen is donated to the iron ion to form a coordinate covalent bond. The oxygen is acting as a ligand, a Lewis base, while the iron(II) ion is acting as a Lewis acid. The binding of oxygen to iron(II) causes the iron ion to undergo temporary oxidation to iron(III). The iron(III) ion then undergoes reduction when the oxygen is released to the cell for cellular respiration.

Figure 3. The 'molecular machine' that is hemoglobin. Hemoglobin converts between the oxygenated and deoxygenated forms (labelled as oxy and deoxy in the diagram) when it binds to molecular oxygen (O2).

Measuring oxygen binding to heme

Recall that hemoglobin is a protein consisting of four polypeptide chains and four heme groups. The binding of oxygen to the four heme groups is cooperative; that is, there is a greater affinity for oxygen to bind to heme once one oxygen binds to one heme group. This makes hemoglobin an effective transporter of oxygen, since each of the four heme groups is capable of binding to one oxygen molecule for a total of four oxygen molecules per hemoglobin protein. When oxygen binds to heme, the protein's three-dimensional shape changes.

The concentration of oxygen bound to hemoglobin is measured in terms of oxygen saturation, compared to the partial pressure of oxygen gas, giving an oxygen dissociation curve. Oxygen saturation is measured as a percentage of oxygen bound to hemoglobin (plotted on the y-axis) compared to the partial pressure of oxygen present in the bloodstream, measured in kilopascals (plotted on the x-axis). The shape of the curve is sigmoidal, that is shaped like the letter S. The sigmoidal shape reflects the cooperative binding observed in hemoglobin.

In medicine, the saturation of hemoglobin when bound to two oxygen, and therefore 50% saturated, is the measurement used to indicate good health and is indicated by the notation P50. A healthy person generally has a P50 of 3.5 kPa. Any increase or decrease in this value can indicate that there is a problem with the binding of oxygen to hemoglobin or with the levels of oxygen in the bloodstream. The sigmoidal shape of the curve is maintained when factors increase or decrease the binding of oxygen to hemoglobin, but the entire curve will shift left or right.

Temperature

When the body has an active infection from a virus or bacteria, the system responds by increasing the internal body temperature, a condition known as a fever. The purpose of the fever is to slow the progression of the infection and allow the immune system to destroy the virus or bacteria. Temperature also causes a change in the binding of oxygen to hemoglobin. At higher temperatures, the bond between oxygen and hemoglobin is weakened, resulting in a lower saturation at the same partial pressure of oxygen in the blood.

pH

Another factor affecting the binding of oxygen to hemoglobin is the pH of the blood. Since many proteins and enzymes are present and active in the blood, the pH is strictly maintained to ensure that these biomolecules can function optimally. Blood pH is maintained by a buffer system at a pH of 7.4 and any deviation above or below can indicate a health problem.

When the pH of the blood decreases below 7.4, the ability for oxygen to bind to hemoglobin decreases. Proteins, including hemoglobin, can change shape when the pH changes, a process known as denaturing. Hemoglobin denatures slightly at lower pH, resulting in a decreased ability to bind to oxygen. At higher pH values, oxygen binding is increased.

Carbon dioxide

The function of hemoglobin is to carry oxygen to cells for cellular respiration and deposit it at the site where it is needed. Then, in addition to transporting oxygen from the lungs to the cells for respiration, hemoglobin also transports the carbon dioxide produced during respiration back to the lungs to be removed from the body by exhalation. The presence of carbon dioxide in the bloodstream causes the pH to decrease, since carbon dioxide dissolves in water forming carbonic acid.

The concentration of carbon dioxide in the blood is part of the buffer system that keeps the pH of the blood maintained at 7.4. The body can increase or decrease the rate of cellular respiration and the breathing rate in order to keep the blood pH buffered by adjusting the concentration of carbon dioxide. When the blood becomes too alkaline, the breathing rate decreases to slow the exhalation of carbon dioxide, keeping it in the bloodstream and lowering the pH. If the blood becomes too acidic, the breathing rate increases to remove carbon dioxide from the bloodstream faster, raising the pH.

As discussed above, a decrease in the pH causes a decrease in the ability of oxygen to bind to hemoglobin, since the shape of the protein becomes altered at low pH values. When the concentration of carbon dioxide is high, such as near the cells that are performing cellular respiration, a decrease in pH occurs and therefore a decrease in the saturation of oxygen bound to hemoglobin. When the concentration of carbon dioxide is low, the pH of the blood increases, resulting in a greater bonding of oxygen to hemoglobin.

Fetal hemoglobin

For a fetus developing inside of its mother's body, the hemoglobin is different. Since the fetus does not breathe, the transfer of oxygen only occurs through the bloodstream in the umbilical cord that connects the fetus and mother through the placenta. As a result, fetal hemoglobin has a higher ability to bind to oxygen than adult hemoglobin. All fetal hemoglobin is replaced by adult hemoglobin about six months after birth.

Carbon monoxide

Hemoglobin also has the ability to bind to carbon monoxide, CO, a product of incomplete combustion of hydrocarbons. When there is insufficient oxygen present, the products of combustion include carbon soot and carbon monoxide, rather than carbon dioxide.

Carbon monoxide binds very efficiently to hemoglobin, preventing oxygen from binding, making carbon monoxide a competitive inhibitor. The release of carbon monoxide from hemoglobin does not occur as quickly as the release of oxygen, so the carbon monoxide remains bonded longer to hemoglobin. As a result, exposure to carbon monoxide deprives cells of the oxygen required for cellular respiration and can lead to organ failure and eventually death, which means carbon monoxide is classified as a poison. Treatment for exposure to carbon monoxide includes exposure to high levels of oxygen, often in a hyperbaric chamber, to increase the uptake of oxygen in the bloodstream and encourage the release of carbon monoxide from hemoglobin.

Anthocyanins

Another group of pigments are the class of molecules known as anthocyanins. Anthocyanins are water-soluble pigments found in many plants. The word anthocyanin comes from the Greek anthos meaning flower and kyanos meaning blue. Anthocyanins are typically red, blue or purple in colour, depending on the pH. They also contribute to the taste of foods, giving a tart or sour flavour.

Structure of anthocyanins

Anthocyanins are part of a class of compounds known as flavonoids and contain a common fused ring structure, known as a flavan nucleus, with various functional group substitutions, including carbonyl, hydroxyl and ether groups, making anthocyanins polar and therefore water soluble. One part of the flavan nucleus consists of a double-fused ring structure and the other part consists of a single phenolic ring, the combination allowing for enough conjugation for visible light to be absorbed and reflected. There are over 500 identified anthocyanins and the colour exhibited depends on the functional groups present and the pH. Anthocyanins tend to absorb green, yellow or orange light, therefore reflecting the complementary colours of red, purple or blue. Note that the structures of anthocyanins can be found in section 35 of the IB data booklet.

The effect of pH on colour

The specific colour exhibited by an anthocyanin depends on the pH. Since many of the functional groups on the flavan nucleus have acid-base activity, the pH can change the functional group, which alters the wavelength of light absorbed and reflected. For example, the quinoidal base exhibits a blue colour. If the pH is decreased, one of the carbonyl groups becomes reduced to a hydroxyl group, which changes the colour to red.

The variety of colours that results from changes in pH make anthocyanins excellent chemical indicators for acid-base reactions. Anthocyanins tend to be red or pink in acidic pH environments, purple in neutral environments, green-yellow for mildly alkaline pH and colourless in strongly alkaline. The specific colours exhibited vary, however, depending on the specific anthocyanin compound present.

The effect of metal ions on colour

Like other pigments, metal ions can bind to anthocyanins, forming a metalloanthocyanin. The presence of a metal ion can alter the colour of the pigment. Magnesium ions, aluminium ions, and iron(III) ions are known to bind to anthocyanin compounds, changing the colour. The metal ions that are present in the soil are absorbed by the roots of the plant and travel to the sites where the anthocyanins are present in the plant, such as in flower petals. Anthocyanins can form coloured complexes with Fe3+ and Al3+ ions, typically found in metal cans. This can result in canned fruit losing its colour.

Stability of anthocyanins

Anthocyanins break down at high temperatures and extreme pH environments. At high temperatures, the bond between the double fused ring and the single phenolic ring in the flavan nucleus breaks. This cleavage of the molecule results in the formation of products that are not coloured, since there is too little conjugation for light absorption and reflection in the visible range. This is often observed when foods containing anthocyanins are cooked and lose some colour, appearing more dull.

Extreme pH environments can also degrade anthocyanin compounds. Excessive reduction or oxidation of the functional groups by the addition or removal of hydrogen ions can result in a change in the bonding that decreases the conjugation, leading to a colourless compound. Consumption of anthocyanins in foods often results in a degradation of the compound, since the pH of the stomach secretions is around pH 1.

Carotenoids and chromatography

Carotenoids

One final group of pigments are carotenoids. These compounds are naturally produced by plants and algae as well as some bacteria and fungi. When carotenoids are consumed by other organisms, they can be stored in the body. Carotenoids absorb violet, blue and green light, therefore reflecting the complementary colours of yellow, orange and red.

There are two classes of carotenoids - xanthophylls, which contain oxygen, and carotenes, which do not contain oxygen. Carotenes consist of long, highly conjugated hydrocarbon chains with one or more cyclic structures on either end, for a total of 40 carbons per molecule. Carotenes resemble polyunsaturated fatty acids, and their nonpolar nature makes them soluble in fat and fatty tissues, not in water. Alpha (α) and beta (β) forms for carotene are the most common, but there are more than 600 forms of carotenoids identified.

Most animals are unable to produce carotenoids, with the exception of two insects: spider mites and aphids. All other animals must obtain carotenoids from their diet. The nonpolar structures are then stored in body fat. Beta-carotene can be converted to vitamin A in the body.

The function of carotenoids in plants and algae is to aid in the process of photosynthesis. They allow for the absorption of light from different wavelengths than chlorophyll. Chlorophyll typically absorbs red light, therefore reflecting green, while carotenoids absorb violet, blue and green light, therefore reflecting yellow, orange and red light. Absorption of more wavelengths of light by carotenoids allows for more energy absorption for photosynthesis.

Carotenoids also can act as an antioxidant and protect chlorophyll from sun damage. Carotenoids are easily oxidised due to the high level of conjugation. The molecule can be cleaved or have oxygen atoms added when in the presence of sunlight, breaking down the carotenoid. This feature allows the carotenoid to take the damage that would otherwise be passed on the chlorophyll.

Chromatography

Chromatography is a method of separating mixtures and is often used for separating mixtures of colours. Since many plants and animals contain a mixture of different pigments, chromatography is a helpful technique to separate and identify the different pigments that are present. Simple paper chromatography separates mixtures on the basis of polarity. A sample of the mixture is applied to a strip of paper and placed in a solvent. Thin layer chromatography (TLC) is a similar process that uses plastic or glass plates with a coating of silica gel, aluminium oxide or cellulose.

The components of the mixture, referred to as analytes, then travel up the paper depending on their ability to bind to the solvent. When a polar solvent is used, such as water, the polar analytes will be able to bind to the solvent and travel with it, leaving the nonpolar analytes behind. When a nonpolar solvent is used, such as carbon tetrachloride, the nonpolar analytes will travel with the solvent, leaving the polar analytes behind. The distances travelled by the different analytes are then compared relative to the distance travelled by the solvent to give a calculation of the retention factor, Rf, of each analyte. The retention factor value can be used to identify the analyte.

When different samples are run together, the separation of the analytes can lead to identification. Further testing can be done on the individual analytes now that they have been separated from the mixture. The analyte spots on the paper can be cut out and dissolved in a solvent or scraped off a TLC plate for further testing or identification, such as spectroscopic methods.

The choice of solvent will affect the ability of the analytes to travel. For example, anthocyanins are water-soluble, so they would travel best with polar solvents, while carotenoids are lipid-soluble, travelling best with nonpolar solvents. In Figure 6, the use of a nonpolar solvent results in the carotenoids, the yellow analytes, travelling with the solvent, while the anthocyanins, the purple and blue analytes, travelled the least. If a polar solvent had been used instead, the separation of the analytes would have been in reverse with the anthocyanins travelling the most and the carotenoids travelling the least.