This lesson is called the effective pH, on the rate of an enzyme reaction: practical, and is from the Unit: Biological molecules and enzymes.

Hi there, my name's Mrs. McCready, and I'm here to guide you through today's lesson, so thank you very much for joining me.

In our lesson today, we're going to carry out an investigation into the effect of changing pH on the rate of an enzyme reaction.

So I hope you're ready to get really stuck into this, and use lots of different equipment and processes in order to be able to complete this investigation.

So we're gonna come across a number of keywords in our lesson today, and they're here on the screen now.

You may wish to pause the video and make a note of them, but I will introduce them to you as we come across them.

So in our lesson today, we're going to, first of all, prepare the practical before we then complete it.

So are you ready to go? I hope so, let's get started.

So we know that enzymes are biological catalysts.

That means that they speed up the rate of a chemical reaction, and we can measure the rate of a chemical reaction by measuring how much change happens over a period of time.

So how many substrates disappear, or how many products are formed, and how long it takes for that to happen? So we are going to investigate the effect of changing the pH on the rate of that chemical reaction.

Specifically, we're going to look at the rate of the reaction of the enzyme amylase as it digests starch into maltose, which is a type of sugar.

So we're going to be looking at this reaction where starch is being digested into maltose by this enzyme called amylase, and what we're gonna do is change the pH to see what effect that has on the rate of the reaction.

Now, when we measure the rate of the reaction, we are going to use a solution called iodine.

Now, iodine is usually orange/brown, but in the presence of starch, it turns blue/black.

That's a very specific colour term that we're going to use, blue/black.

So iodine will turn from orange/brown to blue/black in the presence of starch, and that is how we're going to measure whether starch is present or whether it has all been digested down to maltose.

So we will see that as time passes, and therefore, the reaction has carried on, the amount of starch should decrease, assuming that the enzyme can function at the pH that it's sat in.

If it can't, well, we'll see what happens there.

So we're going to change the pH of each reaction and we're gonna measure the effect that has on the rate of the reaction.

Now, that means that our variables for this investigation are the pH as the independent variable.

So the pH is the variable that we're choosing to change, that's what the independent variable is.

Whereas, the dependent variable, the one we are choosing to measure, will be whether starch is present or not, and we'll be able to measure that by looking at the colour of iodine, whether it's brown, orange, or blue/black.

Okay, so let's just check our understanding then.

Who is most accurately describing the investigation we are completing today? So Andeep says, "We will observe the change from amylase to starch using iodine solutions at different pHs." Lucas says, "We will measure the time taken for amylase to digest starch to maltose at different pHs." And Aisha says, "We will measure the time taken for amylase to change the pH in the presence of starch." But who is most accurately describing our investigation? I'll give you five seconds to think about it.

Okay, so hopefully you've decided that Lucas is most accurately describing the investigation we are completing today, well done! Now for this experiment, we are going to need a range of different equipment.

So we're gonna need iodine solution, a spotting tile, a pasta or a plastic pipette.

We'll also need 5 centimetres cubed of starch in a test tube, and 5 centimetres cube of amylase in a test tube, along with 1 centimetre cube of pH7 buffer solution.

And then both of those test tubes need to be sat in a water bath at 21 degrees Centigrade.

Now iodine solution is an irritant, so you must wear safety goggles throughout this experiment, and obviously, follow other precautions as well.

Now as far as the starch and the amylase buffer solution is concerned, you will need to leave both of these in the water bath for about five minutes to get up to temperature.

So the 5 centimetres cubed of starch solution needs to be in one test tube, and in a separate test tube, you'll need 5 centimetres cubed of amylase solution, plus 1 centimetres cubed of the pH7 buffer solution in that separate test tube.

And then both of those need to be in the water bath for about five minutes.

Whilst you're waiting for the starch and for the amylase and buffer solutions to get up to temperature, you will need to prepare your spotting tile.

So you'll need to take the iodine solution and add a few drops into 10 wells of the spotting tile.

So add each of those in, and remember that iodine is an irritant, so please make sure you are wearing safety goggles.

Once you've done that, and whilst you are still waiting for the five minutes to pass for the solutions to get up to temperature in the water bath, you will also need to get a stopwatch and make sure that it is set to zero minutes and seconds so it's ready to go.

So you'll need to set up your equipment and materials neatly.

So in order to do that, you'll need your water bath at 21 degrees Centigrade and into the water bath, you'll need to place a test tube with 5 centimetres cubed of starch suspension, and a test tube with 5 centimetres cubed of amylase, and 1 centimetres cubed of pH buffer.

You will also need a spotting tile with drops of iodine solution on it, plus some other equipment including a stopwatch, some glass rods, a beaker of water, and a pipette.

So make sure all of your equipment and materials are set up and ready to go.

So what I'd like you to do is starting with the first, put these statements into the correct method order.

So Statement A is to measure the starch into one test tube and the amylase plus buffer solution into another test tube.

Statement B is to add 10 iodine solution spots into a spotting tile.

Statement C is to wear safety goggles, and Statement D is to place the solutions into the water bath.

So which order do these statements go in to reflect the method? I'll give you five seconds to think about it.

Okay, so hopefully, you've started off by wearing safety goggles.

Then, I hope you chose Statement A, to measure the starch into one test tube and the amylase and buffer solution into another test tube, and then Statement D, to put those solutions into a water bath.

And then whilst you're waiting, Statement B, to add the 10 iodine solution spots to a spotting tile, well done if you've got them in the correct order.

So what I'd like you to do now is to assemble the practical equipment required for this investigation, make sure that it's all ready to go.

So you firstly need to measure 5 centimetres cubed of starch suspension into one test tube, and then measure 5 centimetres cubed of amylase into another test tube, then to that same test tube where the amylase is add 1 centimetres cubed of pH7 buffer solution.

After that, you need to put both of those two test tubes into the water bath for about five minutes.

Meanwhile, you'll need to iodine solution to 10 spots on the spotting tile, and then get a stopwatch, and reset it to zero minutes and seconds.

Remembering, that iodine solution is an irritant and you must wear safety goggles throughout this process.

Off you go, come back to me when you're ready and set up.

Okay, so hopefully you're ready and set up, so let's just check that you've got everything sorted.

So you should have the starch solution in one test tube, and the amylase and buffer solution in a separate test tube, and both of those test tubes should be in the water bath at 21 degrees Centigrade.

You should also have 10 spots of iodine solution in a spotting tile, and you should also have a stopwatch reset to zero minutes and seconds.

So make sure that that is exactly as you have with your equipment, that you're all ready to go.

Okay, let's move on to completing this practical then.

So what is it that we're going to be doing now that we've got our equipment set up? So once the starch and the amylase plus buffer solutions have both been in the water bath for about five minutes, what you will need to do is combine the two solutions together into one test tube.

So you'll need to pour the starch solution into the amylase plus buffer solution test tube, pour them carefully, you don't want to waste any of the liquid, you don't want it to pour into the water bath or all over the worktop.

As soon as they are mixed, you must immediately start the stopwatch.

So what's going to happen is as soon as those solutions are combined, the amylase will start to digest the starch molecules down into maltose, and depending on the pH, will depend on how fast that reaction occurs.

Now we are going to measure the rate of that reaction by continuously sampling this reaction.

So in order to do this, we're going to take a sample of the solution every minute for up to 10 minutes.

So every minute, we're going to take a sample of the solution in the test tube out from the test tube, and then add it to one of the spotting tiles.

And I suggest you add them in sequence, so it's obvious where you've started, and obviously where you've got to.

You can see that the spotting dots on this tile are numbered sequentially, 1, 2, 3, 4 on the top row, and then 5, 6, 7, 8 on the second row, 9, 10 on the third row, reading left to right.

I suggest you use your spotting tile in an equally logical and methodical way.

So every minute you're going to take a sample from the test tube, add it to the spotting tile, and then return any leftover sample back into the test tube in the water bath.

So you're going to record the time at which iodine stops turning to blue/black and stays orange/brown.

Now, that might happen very quickly after a few minutes, for instance, or it might not happen at all.

The iodine may still continue to be changing to blue/black at 10 minutes.

Now if you get to 10 minutes and it's still turning blue/black, then you can stop the experiment at that point.

So you'll see that to start off with iodine will turn blue/black because there is starch present because there hasn't been enough time for the amylase to digest all of the starch down to maltose.

Now, if amylase is able to catalyse the reaction, then the amount of starch that is present within the test tube will over time reduce.

And at some point, there will eventually be no starch left, and therefore, no starch to turn the iodine solution blue/black, and so the iodine will stay orange/brown, and you are going to sample your solution every minute to see at what time that stopping of changing of colour occurs, if it occurs at all.

Once you've got the results for the pH7 buffer solution, then you will need to repeat the whole experiment again, setting it up as we have done, and then repeating it using buffer solutions of pH2 and 12 separately.

Now, pH2 is an acidic solution, whereas pH12 is an alkaline solution.

And remember that when you're using both of these solutions, because they are either acidic or alkaline, they are therefore, also irritants just like iodine is.

So again, you must continue to wear your safety goggles throughout this practical, please.

So just to review that before you get started properly, what I'd like you to do is just watch this video so you understand exactly what it is that you're going to be doing.

Okay, so let's have a quick check of understanding.

We should take a sample every minute and test it with iodine, true or false? I'll give you a few seconds to think about it.

So hopefully you've said that is true, but can you justify your answer? Use one of the two statements in order to do that.

I'll give you a few more seconds to think about it.

Okay, so hopefully, you've said that that is true because it means that we obtain results at accurately timed intervals, well done! So when you are ready, what I would like you to do is to combine the starch and the amylase and buffer solution, start the stopwatch, and then continuously sample the solution every minute for up to 10 minutes.

Remember, you must wear safety goggles throughout.

Then, I would like you to repeat the experiment using buffer solutions at pH2, and then separately at pH12, and record your results in a table.

So you'll need to record the pH buffer that you're using and the time taken in minutes of how long it takes for the iodine solution to stop turning blue/black, and to remain orange/brown.

Now I've got a third column up here called rate of reaction, for now, I would just like you to leave this blank, please.

So pause the video, carry on carefully with your experiment, and come back to me when you are ready.

Okay, let's see how you get on then.

So you should have recorded your results in a table, and your results may look a bit like mine, where at pH7, it took about 3 minutes, and at pHs2 and 10, it both took more than 10 minutes.

You will need to leave the rate of reaction column blank for now.

Now, well done for completing that experiment.

It was quite complicated and time-consuming, and I hope you did it carefully, and successfully, well done! So in our lesson today, we have conducted an experiment to investigate the effect of changing the pH on the rate of enzyme reaction, specifically, the enzyme amylase, which digests starch into maltose.

And we used a continuous sampling method to do this.

We've also demonstrated safe and appropriate use of apparatus, and we have collected data at accurately timed intervals.

We've also controlled the temperature of the reaction using a water bath, and we set it this time at 21 degrees Centigrade.

Now, I hope you enjoyed your practical, and all the complexities that that involved, well done for persevering and getting through it nice and safely.

Thank you very much for joining me in our lesson today, and I hope to see you again soon, bye!.