51LC F12 Kinetics Week 5

7/27/2019 51LC F12 Kinetics Week 5

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EXPERIMENT 5

DETERMINING ABSOLUTE CONFIGURATION OF SECONDARY ALCOHOLS

USING THIN-LAYER CHROMATOGRAPHY (TLC) AND YOUR SMARTPHONE

During this experiment, you will determine the absolute configuration of an optically pure

secondary alcohol using the chiral acyl-transfer catalyst HBTM. The configuration will bedetermined using thin-layer chromatography (TLC), your smartphone (or any other photo-takingdevice), and ImageJ software. Calibration curves will be provided for you.

REACTIONS: Esterification of a secondary alcohol using chiral acyl-transfer catalyst

TECHNIQUES: Microscale Experiment Set-up, Thin-Layer Chromatography, ImageJ Analysis

READING ASSIGNMENT

Technique 5.3: Measuring Volume and Transferring Liquids in Techniques inOrganic Chemistry 3

rdEd. pgs 4247.

Technique 17: Thin-Layer Chromatography in Techniques in Organic Chemistry 3rd

Ed. pgs 221235.

Working with ImageJ Handout (attached) Org. Lett. 2011, 13, 4470-4473. (found by going to pubs.acs.org)

PRE-LAB ASSIGNMENT

! In your lab notebook, as part of your purpose section, write a paragraph describing themethod used in the attached paper. Explain the rationale for determining absoluteconfiguration and how that applies to your laboratory experiment.

! In your lab notebook, make a list of chemicals that you will use in this experiment.Include molecular weight, molarity, and volume to be used.

! In your lab notebook, rewrite the procedure for thin-layer chromatography using yourown words.! In your lab notebook, rewrite the procedure for determining the absolute configuration of

an unknown secondary alcohol.

! In your lab notebook, rewrite the procedure for using ImageJ and the calibration curveswith your own words.

EXPERIMENTAL NOTES

Hexanes, ethyl acetate, and toluene are volatile organic solvents. They are also

flammable. Use them in the hood and wear gloves when working with them!

Phosphomolybdic acid is a stain used with thin-layer chromatography. It is a skinirritant! Gloves should be worn when working with it. The major component of the stainis ethanol, which is also flammable. Keep away from an open flame.

Triethylamine is a skin irritant and is quite pungent. Use only in the hood!

Benzyl alcohol, 2-phenylethanol, propionic anhydride, HBTM and 1-phenylethyl

propionate are irritants. Wear gloves and avoid all contact with skin, eyes, andclothing.


2/11

The Importance of Chirality and Absolute Configuration:

Understanding Molecular Reactions in 3-D

As you learned last quarter, molecules are not the flat, two-dimensional pictures that we

draw on paper, but are actually three-dimensional structures. This reality leads to a concept

called chirality, which you should already be familiar with from 51A lecture. Enantiomers canhave differences as minor as dissimilar odors, as in the case of carvone, or as major as havingdifferent biological effects, such as thalidomide, shown below.

For those of you more biology inclined, you should remember that enzymes are complexthree-dimensional structures, which is why they are incredibly efficient and incredibly role-

specific. In the same way that only certain keys can be used to unlock a door, only certaincompounds can be accepted by an enzyme. For drug development and enzyme recognition in

particular, chemists today realize the importance of being able to identify the absoluteconfiguration of chiral centers in compounds.

There are methods available to determine the absolute configuration of stereocenters in agiven molecule that contain a certain type of function group. This week, you will be using a

method that determines the absolute configuration of a chiral secondary alcohol adapted from apublished method in the scientific literature that was developed here at UC Irvine (Org. Lett.

2011,13, 4470-4473). Alcohols are the most common functional group found in isolated naturalproducts.

As described in the paper, a single enantiomer of a secondary alcohol, whose absoluteconfiguration is unknown, is reacted with propionic anhydride and an amine base, triethylamine

in this case, in the presence of one enantiomer of a catalyst called HBTM (drawn below).

What is the absolute configuration of the HBTM enantiomer drawn above?

(S)-ThalidomideResponsible for

birth defects(>20,000 victims)

N

SN

Ph

O

O O

N

SN

Ph

O

R1 R2

OH

NEt3 R1 R2

O

O

HBTM

HN

HN

O

O

O

O

HN

H N

O

O

O

O

(R)-ThalidomidePain killer and

cancer treatment


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The alcohol is reacted, in parallel reactions, with each enantiomer of HBTM. Thealcohol will react faster with one of the HBTM enantiomers (the matched case) than with the

other HBTM enantiomer (the mismatched case). After an experiment confirms which reactionprogresses faster, that information is compared to a mnemonic, shown below, which provides the

absolute configuration of the alcohol.

The original paper used

1

H NMR to discover which enantiomer of HBTM reacts fastest.You will be using TLC, with analysis via a free program called ImageJ, used by biologists to

determine quantities of biological compounds in gels that they run.As you read the Organic Letters paper and the procedure for this experiment, you should

think about the mechanism of the reaction and why the mechanism leads to the difference inreaction speed between the two HBTM enantiomers. In particular, think about the different in

the 3-D environment during each set of reaction conditions. Understanding this will be critical towriting a coherent lab discussion. What is the difference between the two enantiomers of

HBTM? Why might that difference have an effect on the reaction rate?

Part I: Reaction Set-up and TLC (work as a group)

A solution of optically pure 1-phenylethanol has been prepared for you. The absoluteconfiguration of the alcohol is unknown. Your goal is to determine which enantiomer of 1-

phenylethanol you possess using the method described in the attached Organic Letters paper.Instead of using

1H NMR to determine the absolute configuration, you will use TLC.

Four stock solutions have been prepared for you. Solution 1 is 1-phenylethanol of unknown

absolute configuration in toluene (0.30 M). Solution 2 is a mixture of the organocatalyst S-HBTM (0.015 M) and triethylamine (0.60 M) in toluene. Solution 3 is a mixture of the

organocatalystR-HBTM (0.015 M) and triethylamine (0.60 M) in toluene. Solution 4 ispropionic anhydride (0.60 M) in toluene.

Obtain two glass vials. Label one vial A and one vial B. Write the unknown number of stock

solution 1 in your lab notebook. Use graduated plastic pipets for the additions written below.

1. Add 0.5 mL of solution 1, 1-phenylethanol in toluene, to your vial A.

2. Add 0.5 mL of solution 2, S-HBTM + Et3N in toluene, to your vial A.

R

H OH

(S)-HBTMfaster

R

HO H

(R)-HBTMfaster

If...

Then...

(R) Configuration (S) Configuration


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3. Now, add 0.5 mL of solution 1, 1-phenylethanol in toluene, to your vial B.

4. Then, add 0.5 mL of solution 3,R-HBTM + Et3N in toluene, to your vial B.

5. Now, prepare your timer or watch the clock. Add 0.5 mL of solution 4, propionic anhydride in

toluene, to vial A. This is the start of the reaction for vial A (t = 0).

6. After 1 minute, add 0.5 mL of solution 4, propionic anhydride in toluene, to vial B. This is the

start of the reaction for vial B (t = 0).

7. After 30 minutes have passed for vial A, add 0.25 mL of methanol. Swirl the vial.

8. After 30 minutes have passed for vial B, add 0.25 mL of methanol. Swirl the vial.

9. Prepare a single TLC plate with three lanes. Spot the first lane with a provided standardsolution of benzyl alcohol (0.050 M). Spot the second lane with solution from vial A, and the

third lane with solution from vial B.

10. Run the TLC in a TLC chamber with a 40% ethyl acetate in hexanes solvent system.

11. Stain and visualize the TLC using the below instructions.

To Stain your TLC and Visualize Spots

After running your TLC plate in your TLC chamber, pull it out, mark the solvent frontline, and allow the plate to dry. Then, submerge your TLC plate in the jar with the PMA stain

and quickly remove it. Allow the excess liquid to drain in the jar or dab the plate excess liquid ona paper towel. The plate will come out light green. Heat the TLC plate in the oven, which has

been preheated to 160oC, for 45 seconds. When you pull the plate out, dark blue spots should

appear. If you are not seeing spots you may GENTLY heat the plate on a hot plate so as NOT TO

MELT the plastic. Note the Rf values for the starting material and the product.For each of the TLC plates, lane 1 should contain only 1 spot (benzyl alcohol spot).

Lanes 2 and 3 should each contain two spots with different Rf values (reactions A and B).

12. After removing the TLC plate from the oven, allow it to cool on the benchtop and take apicture of it with a tablet, smartphone, camera, or computer device. The TLC should look

something like the picture below.


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Part II: TLC Analysis to Determine Absolute Configuration (work individually)

Send the pictures you took of the TLC plate to your e-mail or plug in your camera and load thepictures onto your desktop. We will be using image density software called ImageJ, which was

developed by the National Institute of Health. You can download the ImageJ software for free at

http://rsbweb.nih.gov/ij/.

Analyze the TLC using ImageJ, according to the instructions below, to find the ratio of benzyl

alcohol standard to 1-phenylethanol and the ratio of benzyl alcohol standard to 1-phenylethylpropionate for both reaction A and reaction B. Plug in these values to your calibration curve

equations for each compound, provided at the end of this document, to determine theconcentration of starting material and the concentration of ester product for each reaction. Based

on these concentrations, find the reaction conversion for both reactions and determine whichenantiomer of the HBTM catalyst reacted faster. Determine the absolute configuration based on

the reaction conversions via the mnemonic developed in the cited Organic Letters paper.


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LAB WRITE-UP (work individually)

Introduction:

! Provide a logical introduction that discusses the purpose of this laboratory experiment.Theory:! Explain the method for determining absolute configuration. This explanation should

ALSO provide answers to the following questions: Why are you using both enantiomers

of HBTM? Why are you running two reactions? What is the purpose of looking atreaction conversion?

Mechanism:

! Provide the mechanism for esterification of 1-phenylethanol to form 1-phenylethylpropionate using only one enantiomer of HBTM. Reagents include 1-phenylethanol,

propionic anhydride, HBTM and triethylamine. Remember to form the activated acyltransfer intermediate.

Results:

You should have printed copies of the TLC plate after lanes are placed on it with ImageJ, theintegrated peaks from ImageJ, and the values that correspond to the integrated peaks. You should

also have a numerical analysis of how you determined reaction conversion from your TLC.! Write a short paragraph that summarizes but does not evaluate your results. This should

include the calibration curve equations given to you, the values obtained using thecalibration curves, and the conversion values determined for reactions A and B.

Discussion:

Your Discussion section should be a paragraph that comments on and evaluates the results ofthe experiment. Below you will find a list of pointers that should help you write a thorough

discussion section. Simply answering the questions or commenting on the issues would not beconsidered a good discussion section.

! Explain your results. Provide a conclusion on which enantiomer of 1-phenylethanol youhave been working with. Explain a thorough rationalization for how you determined the

absolute configuration based on your results.! Consider the calibration curve trend lines given to you and the concentration values you

found for compounds in reaction A and B during. Is this method reliable for aquantitative analysis of material on the TLC? Provide a rationalization for either answer

with supporting evidence.! Why did you receive two calibration curves? Provide a reasonable explanation.


7/11

Working With ImageJ Handout

ImageJ is a program that can be used to compare the density of bands or spots. It is mostcommonly used for biochemistry techniques. This handout assumes that your picture from your

TLC stain is a .tif, .jpg, or .png file.

1. Download ImageJ from http://rsbweb.nih.gov/ij/.2. Open the ImageJ program.

3. Open the image file usingFile > Open in ImageJ.4. Convert the image to grayscale usingImage > Type > 8-bit.

5. Make sure the rectangle selections tool is clicked.

6. Your TLC plate should be vertical in your image. Draw a rectangle around the first lane.7. After drawing the rectangle around your first lane, press 1. This will leave lane 1 in place,

while creating a second rectangle directly on top of it.8. Use your arrow keys to move the second rectangle over the second lane.

9. Press 2. This will leave lane 2 in place, while creating a third rectangle directly on top of it.10. Use your arrow keys to move the third rectangle over the third lane (See Figure 1).

11. Press 3. This will automatically bring up a new window that has integrated the lanes fordensity. This window represents the relative density of the contents of the rectangle over each

lane. The rectangles are arranged top to bottom on the profile plot (See Figure 2).12. Use theLine tool to draw a line across the baseline for each peak in the TLC lanes. It is

necessary to make sure to close off the peak in order to measure its size.

13. Use the Wandtool to click inside of each of the peaks in the TLC lanes. As you click on a

peak, it will be highlighted in yellow. There will also be a window that pops up to give areavalues for each of the peaks (Figure 3). You will need these values to calculate ratios. Keep track

of the order that you click on the peaks, as that is the order they will show up on the Resultswindow.


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Lane 1

Lane 2

Lane 3

Figure 1. Figure 2.

Figure 3. The labeling numbers in the figure have been added using an image program to aid

in visualization. IMAGEJ WILL NOT PROVIDE YOU WITH LABELING NUMBERS.Make sure you remember the order in which you select peaks and match those peaks to the

numbers in the popup window.


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14. Each plate should provide you will the benzyl alcohol standard intensity (lane 1), as well as

the integrated intensities for a specific concentration of 1-phenylethyl propionate and 1-phenylethanol (for reaction A and B separately).

15. For each plate, determine the ratio of benzyl alcohol standard to 1-phenylethanol (intensity ofbenzyl alcohol standard / intensity of 1-phenylethanol at a given concentration) and the ratio ofbenzyl alcohol standard to 1-phenylethyl propionate (intensity benzyl alcohol standard / intensity

of 1-phenylethyl propionate at a given concentration). Recording the data in a table such asbelow will help you organize the numbers. Is the bottom or top spot on the TLC the alcohol?

!! Intensity!

!! Lane!1! Lane!2! Lane!3!

Alcohol!Spot! !! !! !!

Ester!Spot! 7! !! !!

! ! ! !Ratio!Alcoholstd/AlcoholRxn! 7! !! !!

Ratio!Alcoholstd/Esterrxn! 7! !! !!

16. You will use the ratios calculated in step 15 and plug them into the calibration curvesprovided below to determine concentrations of starting material and product for reactionA and

reaction B.


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Calibration

Curvefor1-Phenyleth

anol y

=0.

0697x-0.

8749

R2

=0.

9743

0246810

12

14

16

0

0.

02

0.

04

0.

06

0.

08

0.

1

0.

12

Concentration(M)

RatioofBenzylAlcoholStandardto1-Phenylethanol


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CalibrationCur

vefor1-PhenylethylPropionate

y

=0.

096x-0.

7997

R2

=0.

9847

0246810

12

14

0

0.

02

0.

04

0.

06

0.

08

0.

1

0.

12

Concentration(M)

RatioofBenzylAlcoholStandardto1-Phenylethyl

Propionate

51LC F12 Kinetics Week 5

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