December: Carbohydrates, Diffusion and Digestion

In this session, we continued looking at sugars and starches, and we saw how starches can be broken back down into simple sugars.  We used iodine to test for starch, and Benedict's Reagent to test for simple sugars. 

What are sugars and carbohydrates?

Carbohydrates are sugars, starches and cellulose, and as the name suggests, they contain only carbon, hydrogen and oxygen.

From the Royal Society of Chemistry's 'Chemistry for Biologists':

"Carbohydrates (also called saccharides) are molecular compounds made from just three elements: carbon, hydrogen and oxygen. Monosaccharides (e.g. glucose) and disaccharides (e.g. sucrose) are relatively small molecules. They are often called sugars. Other carbohydrate molecules are very large (polysaccharides such as starch and cellulose)."

Carbohydrates: rice, honey, sucrose, and glucose

We considered some different types of sugar and looked at molecular models.  Table sugar is sucrose, while the sugar made by photosynthesis is glucose.  Dextrose tablets sold in pharmacies are also glucose (dextrose is just another name for glucose from a natural source). Fructose or 'fruit sugar' is very similar to glucose in structure. 

Monosaccharides are simple sugars, which means they can't be broken down into simpler compounds.  They are single links in a chain.  Glucose, fructose and galactose are common simple sugars.

Disaccharides contain two simple sugars joined together, so they're a chain made of two links. Sucrose is a disaccharide -it contains made from one glucose molecule joined to one fructose molecule. Maltose is another disaccharide and one place this occurs is in the mouth, after we have chewed food and starches have started to break down.

Testing for Sugars - Benedict's Reagent

We introduced Benedict's Reagent (or Benedict's solution), which is used to test for reducing  sugars.  'Reducing sugars'  include all monosaccharides - eg glucose and fructose, and also some 'reducing' disaccharides, including maltose. It does not detect sucrose; sucrose is known as a 'non-reducing sugar'.  'Reducing sugar' just means that 

Angie made Benedict's reagent from copper sulphate, sodium citrate and sodium carbonate.  It is a transparent blue solution which, if heated with reducing sugars, produces a brick - red precipitate.  What this means in practice is that the solution will turn cloudy and anything from yellow to orange to brick - red. 

Honey, glucose and sugar solutions with Benedict's Reagent

We placed samples of glucose (dextrose tablets), honey and table sugar in test tubes with some Benedict's Reagent, and put them in a hot water bath for 5 minutes.  The dextrose tablets and honey produced an orange-yellow precipitate, while the sucrose solution remained transparent blue.

Below is a range of results from the Benedict's test for reducing sugars.  You can see one negative result, which is transparent blue, and colours from yellow to reddish-orange in the positive results.

Range of results from Benedict's test

Positive result appearing!

Aside: the colour change with Benedict's solution is caused by copper oxide precipitating out of the solution, so the blue copper (II) ions, which are soluble, change to red-brown copper (I) ions, which are insoluble.  Because they're insoluble, they form a precipitate.  At the end of the day, some of the weakly positive results looked distinctly coppery when the camera flash reflected off them.  For more on the chemistry of the Benedict's test, see Brilliant Biology Student.

Using Benedict's solution and heating is the classic test for the presence of glucose and other reducing sugars, but a quicker alternative is to use ready-made glucose test sticks.  These are sold so you can test blood or urine for the presence of glucose.  We added honey solution, glucose solution and sucrose solution to some of these, and saw that the colour change was similar to that seen with Benedict's solution.

Saliva breaks down starch!

Alison's initials in spit

Last month we modelled starches built from long chains of sugars.  This time, we investigated how these chains can be dismantled.

We each took a piece of filter paper which had been soaked in rice water - a starch solution.  The starchy filter paper was wrapped around a glass slide. We put saliva on a matchstick and wrote our initials on the filter paper in saliva.  Next, we added a few drops of iodine solution to the filter paper.  The areas containing starch immediately turned blue-black - this is the standard test for the presence of starch, Our initials appeared in white.  Angie had been very much looking forward to showing everyone this activity so was pleased that it was such a hit!  We had some creative patterns drawn in spit. We could have just stopped working at this point and left everybody happily experimenting with spit, starch and iodine!

So what happened here? Saliva contains the enzyme amylase, and this breaks starch down into maltose, which is two units of glucose joined together - so it's breaking down a long chain of glucose molecules into short couples. The starch in our initials was quickly broken down.  We tested that our saliva was working by spitting into a test tube and adding starch solution, then iodine. Initially the solution turned blue-black, but then it began to clear around the saliva. We found that if our test tubes were kept warm, the blue-black colour cleared faster.  This gave us evidence that our amylase could work to digest starch on a larger scale than the filter paper initials. Lachlan's saliva appeared to be very effective!

Visking Tubing as a Model Gut

Next, we tried to model one of the processes which happen during digestion. We made a model gut from Visking tubing, and added some cooked rice (with the cooking water) . The tubing was placed inside a large test tube, which was filled with water to the same level as the rice and water inside the tubing.  This was quite tricky to set up. 

We started by testing the water inside and outside the tubing for starch using iodine solution.  The water inside the tubing contained starch, but the water outside did not. Then we took samples of the water inside and outside and tested for reducing sugars using Benedict's solution. None contained reducing sugars at this stage.  

Testing for starch inside and outside the visking tubing.

Our scientists generously donated some amylase and added this to the visking tubing in the model gut. 

We kept our model guts warm by holding them in our hands or placing them in a warm water bath. At intervals (about 10 minutes) we tested again for glucose inside and outside the tubing.  We took care to keep one pipette for sampling from inside the tube, and one for outside the tube, to avoid contaminating our samples. 

This is what we hoped would be happening, at the molecular level:

Everyone had a positive result using Benedict's solution on the sample taken from inside the visking tubing, but it took a while to find sugars present outside the tubing. Angie only found out the real answer to to this later! See 'Digestion Discussion' below for the full explanation. We considered why this might have been. Why hadn't much glucose diffused through the partially-permeable Visking tubing?  Temperature was likely to be a factor, and also the liquid level had fallen so low in some of the tubes, after repeated sampling for tests, that there may not have been sufficient fluid left in there to allow diffusion to occur. Another possibility was that our rice and starch solution was so concentrated that the starch had formed a coating on the tubing and was blocking diffusion. If we wanted to investigate further, we would need to set up an experiment where we varied just one of these things at a time. The main reason which Angie only worked out later, though, is that salivary enzymes break starch down into maltose initially, and while maltose is a reducing sugar, it is too large to easily diffuse through the Visking tubing. It's great when you solve a mystery!

Starch present inside, but not outside, the tubing, at the start and end of the test.

Model guts in warm water bath, to help our amylase along.

At the end of the day, Angie re-tested the liquid outside the visking tubing and found reducing sugar present outside the membrane in all the models . It all makes sense now!

Hydrolysis of starches

Starch is broken down into sugars by a process called hydrolysis.

From BBC Bitesize Chemistry of Carbohydrates:

Breaking starch down
In animals, during digestion, starch molecules are broken down in the body into small glucose molecules, which can pass through the gut wall and into the bloodstream as an energy supply for the body’s cells. The enzyme amylase is the biological catalyst for this reaction.
Amylase is found in the mouth and gut of animals. The stomach also contains acid, which can also break down starch. The breakdown of starch can be carried out in the laboratory using acid or amylase. Enzymes such as amylase act as biological catalysts in the breakdown of complex food molecules into smaller ones in the digestive system.

Hydrolysis of starch

Starch molecules break down by reacting with water molecules. If any molecule reacts with water molecules to break apart, then this is called an hydrolysis reaction. 

Using acid to break the bonds between sugars

Another way of breaking down starches is by heating them with an acid.  This breaks the bonds between the long chains of sugars.  This process can also break the bonds between simple sugars in sucrose, producing glucose and fructose. 

Angie demonstrated that sucrose solution does not produce a colour change when heated with Benedict's solution, so does not contain free glucose.   However, when sucrose solution is heated with hydrochloric acid, then it tests positive for glucose. The sucrose molecule, which was one glucose molecule joined to a fructose molecule, had been broken down into the simple sugars.

Digestion Discussion

Our model gut represents some aspects of human digestion, but digestion occurs in several stages. We chew food, then saliva acts on it, then stomach acid and enzymes act on it, and finally the gut aborbs nutrients.   Digestion is a big topic and one which you'd need to study separately, but when you look at it, think about our activity and consider how it was similar.

Why did it take so long to find reducing sugars outside the Visking tubing?

We used saliva to help break down starches into components. One component of saliva is the enzyme amylase, which breaks starch down into maltose.  Benedict's test detects the presence of maltose - it is a reducing sugar.  However, maltose is a disaccharide; it is made from two glucose molecules joined together.  This means its molecule size is larger than a monosaccharide, so little, if any, will diffuse through Visking tubing.  Over time, and in the presence of water, and especially if there is an acid environment, the maltose will break down ('hydrolyse') into two glucose molecules, which can pass through the Viking tubing - and some maltose molecules will also diffuse through on their own.  They can 'get lucky', and fit through holes in the selectively permeable membrane if they hit it at the right angle! Think about our molecular models from last time and how a short chain might fit through the net sometimes.

Here's a nice summary of what happens in human digestion: 

Carbohydrase enzymes are secreted by the mouth, pancreas and small intestine.  The carbohydrase enzyme,amylase is secreted by the mouth and found in saliva. It starts to work as soon as we begin to chew our food.  Amylase digests long, complex starch (polysaccharide) molecules, into smaller, simpler maltose (disaccharide) molecules. As maltose is a disaccharide it still needs further digestion before it can be absorbed.  The enzyme maltase breaks it down into glucose.  

In our bodies, salivary enzymes start off the digestive process, along with chewing, but further digestion in the stomach breaks maltose down into glucose, which is the form that our bodies can absorb.  Digestion occurs at more than one site - so our model is very simple compared to what happens in the body.  Glucose does diffuse through the mucosa (mucous membranes inside the gut) in our bodies as it does in the Visking tubing, but as well as this 'passive transport', our bodies also use 'active transport' to take up all of the glucose.  Passive transport would just equalise the concentration either side of the membrane, but our bodies want to get all the available glucose from the food, so we also use something called Active transport, which you can read about in this BBC Bitesize section on movement across cell membranes .  

You can read a critique of the Visking tubing gut model  if you'd like to consider further how this model worked, and how it compares to what happens in our bodies.

Links

Carbohydrates and testing for them - BBC Bitesize

BBC Bitesize: Enzymes and Digestion

Investigating the effect of amylase on a starchy foodstuff | Nuffield Foundation 

Evaluating Visking tubing as a model gut - Nuffield Practical Science 

Starch and glucose - diffusion through semi-permeable membrane

https://www.youtube.com/watch?v=lgWE5m7xUJs

Chemistry for Biologists : Carbohydrates

November: Diffusion, Osmosis, Carbohydrates and Photosynthesis

Osmosis, Diffusion and Selective Permeability

Osmosis is the movement of pure water across a selectively permeable membrane. Diffusion is the movement of any substance from an area of high concentration (ie where there's lots of it) to an area of low concentration (where there is less of it). These are important definitions to know.

Selectively permeable membranes, also known as semi-permeable or partially-permeable membranes, have holes of a size which allow smaller molecules to pass through, but not larger ones. Cell membranes are selectively permeable. We used Visking tubing (also known as Dialysis tubing) as an example of such a membrane.

We demonstrated that water can travel through Visking tubing by osmosis. Visking tubing containing sucrose solution was placed in beaker of water. A narrow tube was fixed in the top of the tubing, to allow us to observe changes in the fluid level inside the tubing. The water level rose over time as water osmosised into the concentrated sucrose solution in the tube.

Carbohydrates: Sugars and Starches

Testing for Starch

Alison demonstrated that when iodine solution is added to starch, it turns a blue-black colour.  This colour change shows that starch is present and is the standard test for starch.

Can Starch and Iodine diffuse across Visking tubing?

We took lengths of soaked tubing and knotted them near one end to form a long bag.  Into this we put starch solution, then we closed the tube with a clip to form a sausage shape, and rinsed the outside to remove any deposits of starch which might affect our results.  After patting it dry with paper towels, we measured the mass and recorded it, and we noted the appearance of the tubing.  The starch solution looked milky white.

A beaker was filled with water and iodine solution was added until the liquid in the beaker was a pale golden yellow.  Our starch-filled sausage of Visking tubing was placed in the beaker.

Visking tubing containing starch solution, in beaker of iodine solution

We then observed carefully in a serious, scientific way.

After 5 minutes, some colour change was visible in some of the tubing.  The change happened at different rates and to different degrees.  After an hour, all tubes showed a colour change, ranging from lilac to blue-black, showing that iodine had diffused across the membrane to reach the starch.  However, the solution in the beakers remained pale golden-yellow, showing that no starch had diffused out of the tubing.

Photosynthesis and Molecular Modelling

Imagine if, just using sunlight, water and carbon dioxide, you could make sugar!  Oh hang on, that's what plants do....

We used lego bricks to model atoms and molecules.  A black brick represents carbon, white is hydrogen, and red is oxygen.  We discussed the difference between atoms, molecules, and mixtures.  We made oxygen molecules (O2) , carbon dioxide (CO2) and hydrogen (H), then we made water (H2O), and fizzy water.

Next, we worked as plants; we dismantled water and carbon dioxide molecules and, using sunlight energy provided by Lydia, we reassembled our elements as oxygen and glucose.  

Lydia is being the sun, while our 'plants' make glucose.

Making the molecular models was a very Serious Scientific Process.

Molecular Models and Partially Permeable Membranes

Molecular models help us to understand how molecules work together as mixtures or compounds. We looked at molecular models of iodine, water and starch. Iodine and water molecules are very small, simple sugars such as glucose are larger, other sugars such as sucrose are larger still, and starch molecules are enormous.

Alison showing two lego glucose molecules 

Starch contains many simple sugars joined together in a chain.  Usually over 300 glucose molecules are joined together to make one starch molecule.  We don't have enough lego to do this, so we made some simplified models.

Angie is holding a molecular model of sucrose

Angie built glucose, fructose and sucrose molecular models using a traditional chemistry modelling set, and we discussed the differences between this type of model and the lego models. Starch is harder to model as it contains more than 300 glucose molecules.

Selective Permeability and Passive Transport

To keep the models manageable, we used one small multi-coloured multilink block to represent glucose, and the chains of multilink blocks represent starches.  We demonstrated selective permeability by showing that small blocks representing glucose could pass through the holes in a net, but when several of these 'glucose' blocks were joined together to make a chain representing a starch, they couldn't fit through. In the same way, large starch molecules could not pass through our visking tubing to get out into the iodine solution, but the small iodine molecules could travel through the tubing to get into the sausage of starch solution.

Cell membranes are an example of selectively permeable membranes. There is another important mechanism for transporting substances into and out from cells - Active Transport - but diffusion and osmosis are types of 'passive transport'.  This means no energy input is required, and substances move from an area where they are concentrated, to one in which they are dilute - ie from where there is lots to where there is less of the substance.