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Objective: The purpose of this experiment is to gain a deeper understanding of chemical analysis tools such as mass spec, NMR, and IR and techniques such as melting point, TLC, chromic acid test, and bromine decolorization test through the identification of an unknown compound. The goal of this experiment will be to come up with the identity of an unknown chemical through reasoning and analysis.
Pre-Lab:
For a compound to absorb the UV light used for TLC, a compound must have a “chromophore”. A chromophore is a structural characteristic that allows a chemical to absorb light in the UV range. The chemical must have two or more conjugated double bonds. What is a “conjugated” double bond?
Double bonds alternate with double and single bonds, i.e. double, single, double. Alternating single and double bonds allow for the delocalization of pi electrons across multiple atoms.
We can add a drop of our unknown to a TLC plate and then irradiate with the UV light to see if it absorbs that light. If it does, we know that we do have conjugated double bonds. If we conduct a TLC analysis and our unknown has an Rf value of 0.8, what additional piece of information will we learn about our unknown compound? Briefly explain what we would learn if the Rf value is 0.8 and what we would learn instead if our unknown had an Rf value of 0.2.
An RF value of 0.8 indicates that the compound travels far up the TLC plate and has low polarity. An RF value of 0.2 indicates that the compound stays close to the base of the plate, suggesting it has high polarity, interacting more with the polar stationary phase.
If our unknown does not have a chromophore, we can still conduct TLC analysis, afterwards, we have to visualize our compound with different chemicals. Iodine will be available in the lab. Read about TLC visualization from the website linked below and then briefly report on when we use chemical visualization, how we would choose the visualization technique, about the destructive (or not) characteristics of this kind of visualization and some specifics about iodine visualization. Iodine will be available in the lab.
Chemical visualization is utilized when compounds are colorless and do not glow under UV light, making them invisible on the TLC plate. Looking at chemical spots under UV light does not alter the compounds, making this technique non-destructive and allowing for further analysis.
Iodine is a useful chemical for analysis as it forms colored complexes with many organic compounds, making it a versatile reagent for TLC visualization. Iodine-spot complex formation is considered semi-destructive as spots may fade over time as iodine sublimes away, potentially allowing for subsequent visualization methods.
Analysis can also be performed by spraying TLC with reagents such as ninhydrin or PMA, but this process is destructive and often involves chemical reactions that permanently alter the compounds.
We can get some ideas about the size of an atom from melting point as well as mass spec. Generally, if you have a high melting point, 100 oC or higher, do you expect this to be a larger unknown compound or a small one? Give your answer and explain what interaction leads to a high melting point in large compounds.
While there are always exceptions, as a general rule, larger compounds tend to have higher boiling points due to increased Van der Waal forces that increase with molecular size.
What are the details of the chromic acid qualitative test? What functional group(s) does it test for and what are the results from a positive test and a negative test? If you are unsure of this chemistry, you can review the chromic acid test in the alcohol reactions chapter of Wade.
The chromic acid qualitative test is used to identify primary and secondary alcohols based on their oxidation reactions. If the compound contains a primary or secondary alcohol, the solution changes from orange to green. If the compound is a tertiary alcohol, then no color change will occur.
What are the details of the bromine decolorization test? What functional group(s) does it test for, and what are the results from a positive test and a negative test? If you are unsure of this chemistry, you can review the bromine decolorizing test in the alkene reactions chapter of Wade.
The bromine decolorization test is used to identify the presence of alkenes and alkynes based on an electrophilic addition reaction with bromine. The rapid disappearance of the reddish-brown color of bromine to a colorless solution indicates the presence of an alkene or alkyne, while no color change indicates no pi bond character.
Fill in the details of all of the peaks that you would expect to see and their general characteristics in each of the IR regions shown below. You should do as much as you can from memory and then referring to your notes, the textbook and/or the IR worksheet, double check the details in your table.
3300 cm-1 Amine (3300 Strong & Spike each NH), Terminal Alkyne sp C-H 3330 (Moderate & Narrow)
3300-3000 cm-1 Carboxylic Acid (3000-3300 Strong & Very Broad), Alkene (3000-3100 Strong & Narrow)
3000-2700 cm-1 Alkane (2900-3000 Strong, Narrow, Overlap)
2300-2200 cm-1 Terminal Alkyne C=C (2300 Weak & Narrow), Nitrile (2300 Strong & Narrow)
1740-1680 cm-1 Ester (1740, Moderate & Narrow), Amide (1690, Very Strong & Narrow),
1660-1640 cm-1 Alkene (1660 Moderate & Narrow)
1610-1590 cm-1 Aromatic (1600 Moderate & Narrow)
The DART-TOF mass spec that we own primarily gives only the molecular ion for molecules. It has a “soft” ionization technique. It does not knock off an electron but instead, it adds a proton to add a positive charge. Since one proton is added, how does that affect the mass that we will observe for our molecular ion? Briefly explain.
The Westminster Mass spec gives an [M+1] charge as it is designed to analyze impure compounds. Therefore, we would expect the mass of all molecules analyzed by mass space to have an amu that is 1 point higher than the expected literature value.
Once you determine the molecular ion, what are the steps to determine the formula? Outline the steps that you would take, doing as much from your memory as possible and then referring to your notes, the textbook and/or the Mass Spec worksheet to double check your steps.
Hyde 5 Step Program:
1.) Determine the M+
2.) Identify Other Molecules Present (Br, Cl, S, N, C13, O, I)
3.) Solve for Possible H and C to determine the chemical formula of the unknown
4.) Make an Initial Prediction
5.) Refine the Structure
The following can be answered in your lab notebook late, after NMR has been introduced in class. You would add the answers as “corrections,” keeping in mind that all other answers need to be in your notebook before the lab begins.
A quick look at an NMR spectrum helps us know about the presence of some functional groups. If we see peaks from 0-3, what do we know? If we see peaks from 4-6, what do we know? If we see peaks from 7-8, what do we know? If we see peaks from 9-10, what do we know?
Peaks from 0-3 indicate alkenes, alkanes, alkynes, and molecules that are 2 away from electronegative atoms.
Peaks from 4-6 indicate atoms that are neighbors to electronegative atoms or bonded to electronegative atoms.
Peaks from 7-8 indicate atoms that are part of aromatics. They can be Typical CH, 2 away from electronegative atoms, neighbors to electronegative atoms, or neighbors to two electronegative atoms.
Peaks from 9-10 indicate the presence of an aldehyde or a carboxylic acid.
A detailed analysis of NMR helps us completely solve for structure. If we observe a quadruplet, a triplet, and a doublet on our NMR, what information does that tell us about neighboring protons?
A quadruplet indicates 3 neighboring protons, a triplet indicates 2 neighboring protons, and a doublet indicates 1 neighboring proton.
Methods: Will be analyzing chemical: 304 Unknown 3
Selected chemical: 304 Unknown 3 for analysis. Unknown 3 was noted to be a fine white powder with no distinct aroma.
Prepared a sample for analysis by TLC by dissolving a small amount of the sample in reagent alcohol per lab handout. This solvent was not particularly effective as a large amount of the powder remained in the solution even when more reagent alcohol was added.
Prepared TLC plates and tested for the appearance of spots under UV light. Spots were vibrant under UV Light, and it was determined that no iodine chamber was needed for analysis to be completed.
2 TLC tests were conducted using a solvent of 2:1 hexane/ethyl acetate and 9:1 dichloromethane/methanol. The plates were removed from the mobile phase after approximately 5 minutes, and their RF values were recorded.\
Conducted a chromic acid test of the sample by dropping a small amount of chromic acid from a pipette onto a small amount of sample placed in a watchglass. An immediate color change of the chromic acid solution from orange to blue was noted.
Conducted a bromine decolorization test by dropping a small amount of Br2 from a pipette onto a small amount of the sample placed in a watch glass. No obvious decolorization occurred, even when more Br2 was added to the sample.
Prepared a small sample of unknown 3 for analysis by mass spec by loading the sample into a cuvette. Loaded into mass spectrometer and exported data for later analysis.
Prepared a small amount of unknown 3 for analysis by IR. Loaded solid dry sample into infrared spectrometer and exported data for later analysis.
Conducted analysis of the sample by melting point by loading the sample into a cuvette and "packing" it via a dropping tube.
Ran a "quick and dirty" test to determine ball park ranges of analysis, melting point range set to 40°C and 250°C. It was noted that the sample began melting at around 190°C.
Ran a second, more accurate melting point test with ranges of 165°C-200°C. End result of the melting point of 190.5°C-193.0°C.
A small amount of the sample was dissolved in both methanol and water to determine an effective solvent for NMR analysis. Barely any dissolution of the sample in methanol, but the sample readily dissolved in water.
Prepared sample for NMR analysis by placing in an Eppendorf tube and dissolving the sample with deuterated water spiked with TMS. Loaded sample into NMR and ran analysis. NMR data was run a second time with a sample dissolved in methanol spiked with TMS, as the data appeared inconclusive. Determined the first sample data to be most closely aligned with character-predicted unknown (see results section) and exported data for later analysis.
Performed a pH test to determine the acidity of the sample by dissolving a small amount of the product in water. The pH value was determined to be 3.0.
Results:
Chromic Acid Test: Color Change from orange to blue/green, indicating the presence of a primary or secondary alcohol or aldehyde (see image below labeled "Chromic Acid Test").
Bromine Decolorization Test: No decolorization - no straight alkene character (see image below labeled "Bromine Decolorization Test").
Melting Point: 190.5°C-193.0°C, melting point consistent with literature value for ascorbic acid. Literature value (190°C).
pH Test: pH of ~3.0. Tested pH of sample by dissolving a small amount of unknown in DI water and confirmed acidic character.
Bromine Decolorization Test
Negative
Chromic Acid Test
Positive
TLC:
TLC: High Rf values. Indicates largely non-polar, or slightly polar character.
Left TLC:
Solvent: 2:1 hexane/ethyl acetate
Spot 1: 4.5 cm
Solvent Front: 4.5 cm
Rf: 4.5 cm /4.5 cm = 1.0
The spot and solvent front ran all the way up to the top of solvent front so a second plate was made for comparison.
Right TLC:
Solvent: 9:1 Dichloromethane/methanol, this solvent appeared to work better than the prior solvent as the sample appeared to interact better with the stationary phase and not run to the top of the solvent front.
Spot 1: 3.5 cm
Solvent Front: 4.5 cm
Rf Value: 3.5 cm /3.5 cm = 0.77
Mass Spec:
Mass Spec: Asked technician to focus on m/z 50-125 for unknown 3 per laboratory handout instructions.
The Westminster mass spec machine adds one AMU to all samples, leaving us with an [M+2] value of 177 with another strong peak at 141. This peak likely indicates a molecule that easily breaks into fragments, such as a molecule with several polar bonds.
IR:
Strong narrow peak from about 1650 cm^ - 1750cm^-1: Consistent with ester character
Slightly smaller, narrow peak from 1750 cm^-1 - 1790 cm^-1: Also likely consistent with ester character
Strong, very broad peak from 2900 cm^-1 - 3200 cm^-1: Consistent with carboxylic acid character
Several strong and broad peaks of varying height from 3190cm^-1-3600 cm^-1: This could be indicative of alcohol, alkane, and alkene character and overlap of all of these peaks.
Several peaks within the finger print region: Typically ignored when running IR.
NMR:
NMR sample prepared by dissolving the unknown in deuterated water and spiked with TMS.
Strong peak at 0: Based on TMS and used as standard for comparison.
Quintuplet just below and around 4: Molecules neighboring an electronegative atom (2-4 ppm).
Singlet just below 5: A molecule bonded to an electronegative atom (4-6 ppm).
Singlet just above 5: A molecule bonded to an electronegative atom (4-6 ppm).
With several molecules bonded to and around other electronegative atoms, there is likely a lot of polar character within the molecule.
Attempted to dissolve the sample in deuterated methanol to minimal effectiveness.
Discussion:
TLC: The unknown compound reacted more strongly with the stationary phase with an Rf value of 0.77 when placed in the 9:1 Dichloromethane/methanol solvent as opposed to the 2:1 hexane/ethyl acetate solvent with an Rf value of 1.0, indicating that the compound has more polar character. Only a single spot was obtained on both plates, indicating that the compound is pure.
Chromic Acid Test and Bromine Decolorization Test: The unknown had a positive chromic acid test and a negative bromine decolorization test, indicating that there is primary or secondary alcohol character, but no straight chain alkene character within the molecule.
Melting Point: The second melting point analysis revealed a melting point range of 190.5°C-193.0°C. This range is within the margin of error for the literature value of ascorbic acid, 190°C, one of the possible unknowns for our experiment. This, combined with the results of the chromic acid and bromine decolorization test, provides reassurance that our unknown is ascorbic acid as the melting point matches as known literature value, and ascorbic acid has primary alcohol and cyclic alkene character, which would not react with the bromine decolorization test.
Mass Spectroscopy: The [M+2] value for our unknown was 177, which is consistent with the reading we would expect for ascorbic acid, which has an amu of 176. The second, notably large peak on the mass spec at 141 is likely representative of a fragment at one of the many polar bonds found within the molecule. This is highly likely as ascorbic acid has several polar bonds due to its alcohol, ester, and carboxylic acid groups that would make fragmentation via mass spec possible.
IR: The IR peaks for the unknown are consistent with the expected ranges for alkane, alkene, alcohol, ester, and carboxylic acid character. All of these peaks are consistent with the functional groups expected for ascorbic acid.
NMR: There were three prominent areas where peaks were observed, all located in areas that indicate bonds directly to or neighboring electronegative atoms, which is consistent with the structure of ascorbic acid.
There were no prominent peaks around or above 7 ppm indicative of aromatic character or carboxylic acid, which is consistent with previous results with ascorbic acid character, but with additional testing, such as the bromine decolorization test, TLC, and mass spec, to help confirm compound identity, these tests can be used in conjunction with the NMR to solidify the identity of the unknown.
pH: A final pH test was done to confirm the unknown compound's identity. A pH of approximately 3.0 was obtained, which is consistent with the acidic character of ascorbic acid.
Conclusion:
Based on the results from multiple analytical techniques, the unknown compound is most likely ascorbic acid. The TLC analysis confirmed the compound's polarity and purity. The chromic acid and bromine decolorization tests provided evidence of primary or secondary alcohol character without alkene presence, which aligns with ascorbic acid's structure. The melting point analysis closely matched the literature value for ascorbic acid, further supporting this identification. Mass spectrometry data, including the [M+2] peak at 177 amu, corresponded with ascorbic acid’s molecular weight, and the IR spectroscopy confirmed the presence of expected functional groups. NMR analysis showed peaks indicative of bonds to electronegative atoms, consistent with ascorbic acid’s structure. Lastly, the pH test confirmed the compound’s acidic nature. Collectively, these results provide strong evidence that the unknown compound is ascorbic acid.
Ascorbic Acid Structure
Ascorbic Acid Structure
Molecular Weight: 176.12 g/mol
Melting Point: 190
Polarity: Polar
Image Source: PubChem
Reflection: In this lab I gained a deeper understanding of how various chemical analysis techniques can be used to identify unknown compounds. I also learned how techniques build off of each other to create further confidence in chemical identification. I continued to practice techniques such as TLC, mass spec, pH testing, IR, and melting point while learning new skills such as NMR analysis, bromine decolorization test, and the chromic acid test. If I were to repeat this lab I would run the NMR again to see if I could generate and NMR scan that shows the cyclic or carboxylic acid character of ascorbic acid by dissolving in another solvent.
Post-Lab: None.
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