GenChem

3rd Class Chemical Separations

Today’s Agenda:

Develop the problem of the day.

Perform first challenge: extract pigments.

Discuss the challenge of chemical separations.

Perform second challenge: determine the best solvent for the problem.

Develop a consensus on the best answer.

Learn how to write an introduction.

Discuss writing the report.

GenChemOne of the central goals of Chemistry is to analyze chemical substances.

Detection: Does the sample contain substance X?Identification: What is the identity of the substance in this sample?

Central Goals and Questions

Separation: How can the substances present in the sample be separated?Quantitation: How much of substance X is in the sample?

Today we will investigate the SEPARATION of chemical substances

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Chemists use their knowledge and understanding of the properties of substances to separate the different components of materials.

Why is there a need to separate substances?

Identification Characterization

Elimination Quantitation

Application Transformation

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Separations D2L-graded pre-lab assignment

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In particular, biological materials tend to be

mixtures of hundreds of different substances.

What do you know about the properties of substances that cause leaves and flowers

to be colored?

They absorb visible light of certain colors and reflect others.. Chlorophyll, for example, absorbs red and blue light. Carotene absorbs green and

blue light.

GenChemLet′s think!

.

In your groups:

Discuss what properties of pigments in a leaf or a flower you could use to extract them;

Propose an experimental strategy to extract the different pigments in a plant;

ChlorophyllCarotene

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The solubility of one substance in another depends on the relative strength of the attractive forces between their particles

AA , BB >> AB Insoluble, Immiscible

AA, BB <= AB Soluble, Miscible

What is going on?

Could we use water or acetone or hexane to

extract the pigments from spinach leaves?

Demo

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Why does simply sticking a leaf in a beaker of solvent not work?

What do you know about the structure of leaves? Where are

the pigments located? How can you get them out?

Pigments are located in chloroplast walls inside cells. Solvent can’t get to them.

Need to break down cell walls.

Let′s think!

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Natural Product ExtractionNatural Product Extraction

Let′s think!

In your groups review your responses to the following pre-lab question and come up with a consensus answer.

Take the six-point "Procedure for BIological Sample Extraction" given in section 5-1 of your technique book and rewrite it as a procedure you can use to

extract the photosynthetic pigments from spinach leaves. This procedure

will need to contain the specific equipment and the amounts of chemicals you expect to use.

GenChemExtraction Tips

Make sure your procedure accounts for the following.• A description of how to make the 1:1 mixture of hexane and acetone including how much to make.

• Separation of the organic layer from the water layer. The recommended procedure is to use a transfer pipet to remove the dark green organic layer (top layer) into a small test tube, taking care to avoid collecting any of the milky, aqueous layer at the bottom of the mortar.

Don’t Don’t rinse the rinse the mortar mortar

with with acetone!acetone!

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First Challenge

Extract the pigments from spinach leaves.

Available resources:

Acetone, hexane, spinach leaves & sand;

Mortar and pestle;

Glassware.

Implement your

procedure.

Let′s explore!

You have 15 minutes

GenChemLet′s think!

Compare the color and depth of color of your pigment extract with those from other groups.

How do you explain the differences?

How dark should your extract be?

How can you make it darker?

Concentration of the extract by reducing the solvent volume. Place it in a small beaker and set the

beaker in a hood. Leave alone until the volume is reduced to about 1 ml. If it evaporates to dryness,

add 1 ml of additional solvent.

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Separating a Mixture

What do you think is in the extract?

What do you need to do next to determine how many pigments are in there?

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ChromatographySeparation based on unequalunequal attractions

between the different components of a moving mixture an a stationary phase.

Mobile phaseStationary

Support

Animation: http://www.youtube.com/watch?v=SdYb6GgBQ7s

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Imagine we inject a substance composed of molecules that have weak attractive forces with the STATIONARY PHASE

MOBILE PHASE or ELUTANT

BASED ON MOLECULAR INTERACTIONSPREDICT HOW WILL MOVE THROUGH THE TUBE

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What if we injected molecules with strong attractive forces with the stationary phase?

BASED ON MOLECULAR INTERACTIONSPREDICT HOW WILL MOVE THROUGH THE TUBE

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Suppose we put a mixture of two solutes and one solvent (mobile phase) into our coated tube.

What do you expect to happen?

What will happen if more solvent is added?

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With the proper selection of stationary and mobile phases you can separate materials based on

intermolecular forces.

Gas chromatography

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Thin Layer Chromatography (TLC)

TLC Plate

Support and Stationary Phase

Developing jar

What is it?

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Thin Layer Chromatography (TLC)

Spotting

Running

what’s the mobile phase?

what’s the stationary phase?

what makes the mobile phase move?

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Thin Layer ChromatographyThin Layer Chromatography

Let′s think!

In your groups review your responses to the following pre-lab question and come up with a

consensus answer.

Consider the 12-step procedure for performing a TLC that is given in chapter 6-1 of the technique book.  Describe how you will use this procedure to determine the best

solvent system to separate the components of a spinach leaf.  Do not repeat any of the 12 steps.  Instead,

describe what additional steps you will need to take to solve the problem. 

GenChemTLC TipsMake sure your procedure accounts for the following.•Don’t waste chemicals! •Don’t let dust, dirt, liquids (like water) and/or your fingerprints get on the TLC .•Make sure they fit.•Keep the spot small.•Mark original spot location.•Different strips may develop at different rates.•Mark solvent front.•Capillary tubes are GLASS WASTE.

Put them in your notebook!Put them in your notebook!

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Second Challenge

Determine the solvent mixture that, using TLC, best separates the various pigments present in

the spinach leaf extract. .

Available resources:

Solvents;

TLC strips;

Glassware.

Now that you have a feasible procedure, just

do it!

Let′s explore!

You have 60 minutes

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Compare the TLC plates developed with different solvent mixtures .

Begin by drawing pictures of the TLC plates in your notebook

What mixture is more efficient in separating the components of the mixture?

How many different components seem to be present in the original pigment mixture?

Identify the chlorophylls and carotenoids. What property can you use to do this?

Let′s think!

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Consider the spots on the various TLC plates.

How could you identify each individual

component? Propose an experimental strategy.

By performing the TLC with known compounds and comparing the results.

Let′s think!

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What properties of the spots allows us to use them

to identify the spots?

Color and distance the spot moves.

Let′s think!

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The distance the spot moves depends on how far the solvent

moves. It is the RATIO of the distance the spot moves to the

distance the solvent moves that is constant for any given component

and solvent.

This ratio is known as the Rf value.

Let′s think!

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Rf

retention factor

1. Create a formula for calculating Rf values.

2. For each strip, calculate a Rf value for each spot on the strip.

3. Make a table that lists, for each strip, the spot colors and the calculated Rf values.

Let’s apply

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Report your results for the identity of the best

solvent mixture.

In your group:

Discuss how the results obtained by the different groups compare with each other? Which are most credible? Which are least?

What were the strengths and weaknesses of the method you used to identify the chromophores? How could you avoid or reduce the difficulties that you encountered?

Let′s think!

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You’ll be looking at the behavior of

gases and the kinds of measurements that can be made.

Looking Ahead to Next WeekLooking Ahead to Next Week

Let′s think!

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Discussion

From your Technique Book: Discussion. Analysis of the data and presentation of conclusions regarding original hypothesis. This should include a defense of the conclusions citing the results and a discussion of the reliability of the data used.

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Discussion OutlineWhat were the major goals of today’s lab?

1. Determine the best solvent to use to separate the various components.

2. Determine how many components can be identified.

For each goal, what were the results?

1. The experimentally determined mixture of

Let′s think!

GenChemDiscussion of a Goal

For Each Goal: Interpret/analyze result. What is the result? (Cite data/give examples).What does it mean? Does it make sense? Explain the chemistry.Is it what was expected? Is it consistent with your observations?Comparisons to literature or peers.If something went wrong: What were you expecting and why? What went wrong? How do you know? How could you fix it next time?

Let’s apply

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Discussion: Goal 1The best separation was achieved with a mixture of acetone

and hexane consisting of one part acetone and three parts hexane. With pure acetone none of the pigments traveled very far and they did not separate. With pure hexane the pigments all traveled far, but also did not separate. The 1 to 3 mixture caused the pigments to end up further apart than for any other mixture with the chlorophylls traveling the farthest and the carotenoids the least.

Based on these results it can be said that all the pigments had strong affinity for the hexane and limited affinity for the acetone. It also appears that the chlorophylls have a greater affinity for the hexane than the carotenoids. That the two chlorophylls tracked closely together implies they have very similar affinities for the hexane which also implies they have very similar structures. The same is true for the carotenoids.

GenChemAdditional Goals

• If the experiment has more than one goal, you will discuss the results of each like we did for Goal 1.

• In addition to discussing the results of each goal, you also need to discuss data quality…

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Discussion: Data qualityIn a paragraph, discuss the quality and reliability of your data:

Discuss pros/cons/accuracy/precision of tools/techniques.Use technique book! Be specific and give examples.Suggest fixes for the problems you identify and additional experiments to improve findings. Don’t use generic “human error”. Any discussion of human error should focus on specific, known errors and their effect on the results.

Let’s apply

GenChemDiscussion: Data quality

The ability to determine separation depended on the size of the spots on the TLC strips. For some of the strips the spots were so big that they ran together. This made it difficult to determine how well they were separated. This problem could be addressed by repeating the TLC and applying less of the leaf extract to the plate.

It should also be noted that, based on the data presented in the results table, both the 1:3 acetone to hexane and 1:2 acetone to hexane solvent mixtures produced reasonable separations. While the 1:3 appeared to do a better job it is quite possible that a mixture between these two would be even better. More trials with mixtures between 1:2 and 1:3 would help resolve this uncertainty.

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• See Technique book pp. 68-69 for more help with writing a Discussion.

GenChemThe Worksheet

You will now complete a

worksheet for this experiment.

To be submitted BEFORE leaving class: Raw data. A copy of all measurements and observations you collected in lab. Calculations. Examples of the Rf calculation. Results. An organized presentation of the results obtained.

Discussion. A discussion of the results obtained, their credibility and their meaning.

If you don’t finish the worksheet in class you are to submit it tomorrow to the wooden dropbox outside the TSO

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Pointers1. Organize all your measurements into tables. Include all

calculated results along with measured quantities.

2. Give labeled drawings of the TLC strips including colors of the spots and any other pertinent observations.

3. Organize your table to “flow”. Your presentation should lead the reader to your conclusion.

4. You will be graded on credibility. Your presentation must be credible. Proper use of significant figures and labeling (units) strongly impacts credibility.

Let’s apply