Milo_Revised

Correct. (set:$clicks = $clicks + 1) [[Click to Continue -> Question 11]]

Settling is impractical; it’s time-consuming and won’t fully clarify the extract. (set:$clicks = $clicks + 1) [[Retry | Question 10]]

What type of chromatography column would you use? [[A: Agilent Zorbax Silica column -> Q11 Chrom+UV A1]] [[B: Waters XBridge C18 column ->Q11 Chrom+UV A2 Correct]] [[C: Superdex 200 Increase 10/300 GL column -> Q11 Chrom+UV A1]] [[D: Thermo Fisher Dionex IonPac AS11-HC column -> Q11 Chrom+UV A1]] (set:$clicks = $clicks + 1)

Incorrect. Let's think this through. What type of chromatography should you use? [[A: Normal phase HPLC -> Q11 Chrom+UV]] [[B: Reversed phase HPLC -> Q11 Chrom+UV]] [[C: Anion exchange chromatography -> Q11 Chrom+UV]] [[D: Size exclusion chromatography | Q11 Chrom+UV]] [[D: Affinity chromatography | Q11 Chrom+UV]] Now think about which of the listed columns enable the kind of chromatography you want to use. (set:$clicks = $clicks + 1)

Caffeine and theobromine will have extremely similar oxidation and reduction potentials. While it is possible to use it for quantification it would be extremely difficult without extremely sensitive instruments. There are much easier ways to quantify caffeine. [[Retry | Question 11]] (set:$clicks = $clicks + 1)

FAAS is typically used to quantify metals in a solution. Since caffeine doesn't have any metals in it you will not be able to directly quantify caffeine using this method. [[Retry | Question 11]] (set:$clicks = $clicks + 1)

You measure the UV–Vis spectrum of your 10 mL caffeine/theobromine extract using a standard 1 cm pathlength cuvette. The absorbance at 272 nm is 0.8. For comparison, the UV–Vis spectra of pure caffeine and pure theobromine in water are shown below, along with a calibration curve for caffeine in water prepared using the same instrument and cuvette.

Based on the data, what is the caffeine concentration in your sample? [[A: 0.083 mM -> Q11 UV Vis A1]] [[B: 0.127 mM -> Q11 UV Vis A1]] [[C: 0.035 mM -> Q11 UV Vis A1]] [[D: None of the above -> Q11 UV Vis A1]] (set:$clicks = $clicks + 1)

The actual concentration of caffeine is 0.01 mM. What is your error? [[A: 730% -> Q11 UV Vis A2]] [[B: 1170% -> Q11 UV Vis A2]] [[C: 250% -> Q11 UV Vis A2]] [[D: None of the above -> Q11 UV Vis A2]]

Why might your error be so high? [[A: Theobromine and caffeine overlap on the UV-Spectra -> Q11 UV Vis A3]] [[B: You made a mistake calculating the concentration | Q11 UV Vis]] [[C: Random error - UV Vis has a lot of variability from sample to sample -> Q11 UV Vis A4]]

Correct. Because theobromine and caffeine overlap significantly on the UV spectra you cannot use UV-vis and Beers law to determine the concentration of caffeine. (set:$clicks = $clicks + 1) [[Choose a different quantification method | Question 11]]

While random error is always present, you measure everything in triplicate and get similar values everytime. Thus, you are confident in your measurements. (set:$clicks = $clicks + 1) [[Retry | Q11 UV Vis A2]]

Incorrect. [[Retry | Question 12]] (set:$clicks = $clicks + 1)

Correct. The 4.6 mm × 150 mm column is a standard analytical-scale HPLC column, ideal for routine separations and quantification of small molecules like caffeine and theobromine. It provides high resolution, moderate backpressure, and compatibility with typical HPLC flow rates (around 1 mL/min). [[Click to Continue -> Question 13]] (set:$clicks = $clicks + 1)

Incorrect. [[Retry | Question 13]] (set:$clicks = $clicks + 1)

Correct! This option balances the resolution with the overall run time. [[Click to Continue -> Question 14]] (set:$clicks = $clicks + 1)

Do you really think you can accurately quantify the amount of caffeine/theobromine based on these noisy peaks? [[Retry | Question 14]] (set:$clicks = $clicks + 1)

Correct! This option balances the signal to noise ratios while not saturating the detector. [[Click to Continue -> Question 15]] (set:$clicks = $clicks + 1)

Do you really think you can accurately quantify the amount of caffeine/theobromine considering it seems that the detector is saturated for one of the peaks in your sample? [[Retry | Question 14]] (set:$clicks = $clicks + 1)

Correct. [[Click to Continue ->Question 16]] (set:$clicks = $clicks + 1)

Looks like your math is off. Please retry. - Make sure you identified the peaks for caffeine and theobromine correctly. - Make sure you used the right equation for the right analyte [[Retry | Question 15]] (set:$clicks = $clicks + 1)

Correct. [[Click to Continue ->Question 17]] (set:$clicks = $clicks + 1)

Looks like your math is off. Please retry. [[Retry |Question 16]] (set:$clicks = $clicks + 1)

Correct. Even small quantities of chocolate can be harmful depending on the dog’s size and the type of chocolate. [[Click to Continue ->Question 18]] (set:$clicks = $clicks + 1)

Looks like your math is off. Please retry. [[Retry |Question 17]] (set:$clicks = $clicks + 1)

Incorrect. This is a qualitative question and knowing "yes" or "no" will not help determine if caffeine/theobromine can cause toxicity. (set: $clicks to $clicks + 1) [[Retry | Question 2]]

Incorrect. This is an important research question, however this will depend on the amount of caffeine/theobromine consumed. (set: $clicks to $clicks + 1) [[Retry | Question 2]]

Incorrect. The flavor profile should not affect toxicity. (set: $clicks to $clicks + 1) [[Retry | Question 2]]

Well done. This question not only is quantitative but also helps us to define some research boundaries. (set: $clicks to $clicks + 1) [[Click to Continue -> Question 3]]

Correct! Having replicate measurements is an essential part of any experiment. (set: $clicks to $clicks + 1) [[Click to Continue -> Question 4]]

Incorrect. Chocolate color reflects differences in composition, which may affect caffeine/theobromine levels — you cannot assume one type represents all others. [[Retry | Question 3]] (set:$clicks = $clicks + 1)

Incorrect: One sample per type doesn’t account for variability; color alone doesn’t guarantee consistent caffeine/theobromine levels. [[Retry | Question 3]] (set:$clicks = $clicks + 1)

Correct: Cocoa solids contain caffeine/theobromine in relatively high concentrations. Thus this variable is most likely to affect caffeine/theobromine content. (set:$clicks = $clicks + 1) [[Click to Continue -> Question 5]]

Incorrect: While caffeine/theobromine levels might vary from brand to brand, this won't be a significant change unless you change what type of chocolate you measure (i.e. white vs dark). (set:$clicks = $clicks + 1) [[Retry | Question 4]]

Incorrect: Sugar doesn't have caffeine/theobromine in it so this should not affect the caffeine/theobromine levels in a chocolate bar. (set:$clicks = $clicks + 1) [[Retry | Question 4]]

Incorrect: The shape of chocolate is independant of the ingredients (including the caffeine/theobromine amount). (set:$clicks = $clicks + 1) [[Retry | Question 4]]

Incorrect: Analyzing one sample won't reflect any differences across different bars. (set:$clicks = $clicks + 1) [[Retry | Question 5]]

Correct: To get representative sampling testing multiple samples from different batches and brands is always important. (set:$clicks = $clicks + 1) [[Click to Continue -> Question 6]]

Incorrect: While it's important to think through the cost of sampling you always want to have a variety of samples to test from (including different expense points). (set:$clicks = $clicks + 1) [[Retry | Question 5]]

Incorrect: Most common nuts do not contain any caffeine/theobromine. (set:$clicks = $clicks + 1) [[Retry | Question 6]]

Incorrect: Caramel does not contain caffeine/theobromine. (set:$clicks = $clicks + 1) [[Retry | Question 6]]

Incorrect: Fats do not contain caffeine/theobromine. (set:$clicks = $clicks + 1) [[Retry | Question 6]]

Correct: In order to get a representative sample you need to have homogenous samples or be able to homogenize the samples. Otherwise you risk getting drastically different results each time you measure. (set:$clicks = $clicks + 1) [[Click to Continue -> Question 7]]

This is correct. But this is not the only reason. (set:$clicks = $clicks + 1) [[Retry | Question 7]]

Correct. (set:$clicks = $clicks + 1) [[Click to Continue ->Question 8]]

Try again. (set:$clicks = $clicks + 1) [[Retry | Question 7]]

You have successfully digested the fats with an enzyme, thus removing the interference with your measurment. However, the enzyme you used also causes interference. How would you seperate the enzyme? [[A: Add a reagent that leads to aggregation of the enzymes. Then centrifuge the mixture to precipitate out the aggregated enzymes -> Q8 A1 A1]] (link:"B: Restart and try a different method")[(set:$clicks = $clicks + 1)(go-to:"Question 8")] [[C: Boil your mixture to kill the enzymes -> Q8 A1 A2]] (set:$clicks = $clicks + 1)

You successfully precipitated the enzymes removing the interference from both the fats and the enzymes. Well done. (set:$clicks = $clicks + 1) [[Click to Continue -> Question 9]] Thought question: Do you think the digested fats could potentially interfere with the analyses?

You boil the reaction mixture successfully killing the enzymes. However, you realize that the interference is not caused by the enzyme's activity but the presence of the enzyme itself. (set:$clicks = $clicks + 1) [[Try Again | Q8 A1]]

Well done! You have successfully removed fats from your chocolate. (set:$clicks = $clicks + 1) [[Click to Continue -> Question 9]]

Incorrect. Heating might melt fats but won’t chemically separate them; they can still co-extract or interfere with detectors. (set:$clicks = $clicks + 1) [[Retry |Question 8]]

Incorrect. Diluting doesn’t remove fats — they’re immiscible with water and can still form emulsions or residues. (set:$clicks = $clicks + 1) [[Retry |Question 8]]

This is always something you should check. Depending on the instrument and method you use the compatability will change. However many instruments do not like fats. (set:$clicks = $clicks + 1) [[Click to Continue -> Question 10]]

If the detector becomes saturated or your sample concentration is too high, dilute the sample and reanalyze. (set:$clicks = $clicks + 1) [[Retry | Question 9]]

It is important to look for similarities and find techniques to minimize interference coming from it. However most fats are long hydrocarbon chains while caffeine/theobromine is not. (set:$clicks = $clicks + 1) [[Retry | Question 9]]

While sometimes it is not neccesary to seperate your target analyte of interest, in this case, the fats do indeed cause interference. (set:$clicks = $clicks + 1) [[Retry | Question 9]]

You add 10 mL of water to extract caffeine/theobromine from chocolate and gently heat the mixture. How can you remove the solid chocolate particulates before analysis?

[[A: Centrifuge to precipitate the solid residue using a benchtop centrifuge -> Q10 A1 Correct]] [[B: Allow the solids to settle overnight, then pour off the top layer carefully. -> Q10 A2]]

At this point, you have 10 mL of water with caffeine/theobromine in it. Which method would you choose for quantifying caffeine? [[A: UV-Vis Spectroscopy -> Q11 UV Vis]] [[B: Chromatography with UV -> Q11 Chrom+UV]] [[C: Flame Atomic Absorption Spectrometry (FAAS) -> Q11 FAA]] [[D: Electrochemistry -> Q11 Electro]]

What size column should you use? [[A: 30 mm x 150 mm (preparative column)-> Q12 A1]] [[B: 4.6 mm x 150 mm (analytical column) -> Q12 A2 Correct]] (set:$clicks = $clicks + 1)

Based on literature, you decide to use a gradient elution where mobile phase A is water and B is acetonitrile. You prepare a 10 µM 1:1 mixture of caffeine and theobromine standard and record the following chromatograms for different gradients of A → B.

Which gradient program gives the best separation within a reasonable analysis time? [[A: Program A-> Q13 A1]] [[B: Program B -> Q13 A1]] [[C: Program C -> Q13 A2 Correct]] [[D: Program D -> Q13 A1]]

Next, you inject different volumes of your 10 mL caffeine/theobromine extract into the HPLC to monitor the chromatogram. Which of the following injection volumes would you choose?

[[A: 1 µL -> Q14 A1]] [[B: 10 µL -> Q14 A2 Correct]] [[C: 20 µL -> Q14 A3]]

You decide to proceed with a 10 µL injection volume for your 10 mL chocolate extract. For three replicate samples, you obtain the following data:

As a reminder, when you developed the HPLC method using a 10 µM 1:1 mixture of caffeine and theobromine standard, the retention times were: 4.3 min and 5.2 min. The calibration equations for each analyte are given below: Peak Area (Caffeine) = Molar Concentration of Caffeine x 23,400 Peak Area (Theobromine) = Molar Concentration of Theobromine x 26,600 What is the approximate average concentration of caffeine and theobromine in your 10 mL chocolate extract? [[A: Caffeine: 10 µM Theobromine: 80 µM -> Q15 Correct ]] [[B: Caffeine: 80 µM Theobromine: 10 µM -> Q15 Incorrect]] [[C: Caffeine: 10 µM Theobromine: 90 µM -> Q15 Incorrect]] [[D: Caffeine: 90 µM Theobromine: 10 µM -> Q15 Incorrect]]

What is the best "chemical analysis" question? [[A: Is there caffeine/theobromine in chocolate? -> Q2 A1]] [[B: How much caffeine/theobromine is in a 50-g chocolate bar? -> Q2 Correct Choice]] [[C: How quickly does caffeine/theobromine absorb into a dog's bloodstream after ingestion? -> Q2 A3]] [[D: How does the concentration of caffeine/theobromine influence the flavor profile of dark chocolate? -> Q2 A4]]

You don't remember the color of the chocolate bar Milo consumed. You want to compare the caffeine/theobromine levels in milk vs. dark vs. white chocolate. What would you need?

[[A: Replicate bars for each type -> Q3 A1 Correct]] [[B: The color of the bar should not affect its caffeine/theobromine content. Thus, we can take one type of chocolate and assume that it will be representative of the others. -> Q3 A2]] [[C: Only one bar of each type, since chocolate color alone determines caffeine/theobromine content. -> Q3 A3]]

What variable would most affect caffeine/theobromine content in a chocolate bar?

[[A: Cocoa percentage -> Q4 A1 Correct]] [[B: Brand of chocolate (Lindt, Hershey, Ghiradelli, etc.) -> Q4 A2]] [[C: Sugar content -> Q4 A3]] [[D: Shape of the chocolate bar -> Q4 A4]]

How should you collect a sample of chocolate to get a representative measurement? [[A: Analyze one piece from one bar -> Q5 A1]] [[B: Take multiple samples across bars and brands -> Q5 A2 Correct]] [[C: Only test the cheapest chocolate -> Q5 A3]]

What is the risk of testing only one piece of a heterogeneous bar (e.g. a bar with nuts or caramel)?

[[A: Nuts contain caffeine/theobromine -> Q6 A1]] [[B: Caramel has same caffeine/theobromine as cocoa -> Q6 A2]] [[C: Fats increase caffeine/theobromine concentration -> Q6 A3]] [[D: Composition varies across the bar -> Q6 A4 Correct]]

Because chocolates with higher cocoa percentages generally contain more caffeine and theobromine — the compounds responsible for chocolate toxicity in dogs — you choose to analyze dark chocolate, which typically contains the highest levels. After collecting appropriate chocolate samples, you record their weight and grind them into a fine powder.

Why are these two steps important in the analysis? [[A: To increase the surface area of the chocolate for efficient extraction of caffeine/theobromine for further analysis. -> Q7 A1]] [[B: To ensure each sample is homogeneous and that caffeine/theobromine concentrations can be normalized to the total mass analyzed. -> Q7 A2]] [[C: Both A and B are correct -> Q7 A3 Correct]] [[D: Neither A and B are correct -> Q7 A4]]

You removed the fats because they can interfere with your measurements. In what ways could the fats have caused interference? [[A: The fats are incompatible with the instrument and could foul or damage the detector -> Q9 A1 Correct]] [[B: The fats are highly concentrated resulting in a signal that is too high to measure -> Q9 A2]] [[C: The fats absorb at similar wavelengths or co-elute, producing overlapping signals with caffeine/theobromine. -> Q9 A3]] [[D: Removing the fats is an unneccasary step and was just to practice our lab techniques -> Q9 A4]]

What is your name? (input-box: bind $name) What is your EID? (input-box: bind $studentId) Once you have input your name and EID please [[click to continue |Question 1]]

Congratulations $name! You successfully designed and reasoned through a full quantitative analysis of caffeine and theobromine in chocolate! [[Click to Restart |Question 1]] (set:$clicks = $clicks + 1) You finished this on (current-time:) (current-date:) <script> var sendData = { 'name': harlowe.State.variables.name, 'studentId': harlowe.State.variables.studentId, 'clicks' : harlowe.State.variables.clicks }; $.ajax({ url: 'https://script.google.com/macros/s/AKfycbxl1yMMC3mrrLA5HvfhqgjNfqzMosjeGUSFcIwCz_QyYSv-3M01fUIxMPRr7G1G6L57/exec', // <--- Ensure this is in single quotes method: 'POST', dataType: 'json', data: sendData }).done(function() { console.log('Data sent successfully!'); }).fail(function(jqXHR, textStatus, errorThrown) { console.error('Error sending data:', textStatus, errorThrown); }); </script>

Incorrect. The chemical equation is not helpful in this case. (set: $clicks to $clicks + 1) [[Retry |Question 1]]

Incorrect. While this is important because metabolism rates determine the effect, it’s important to first know the quantity of caffeine/theobromine consumed; otherwise, you wouldn’t know how much caffeine is being metabolized. (set: $clicks to $clicks + 1) [[Retry |Question 1]]

Correct. It's important to ask yourself what you are trying to analyze before jumping straight into the analysis. (set: $clicks to $clicks + 1) [[Click to Continue -> Question 2]]

Correct. A C18 column would be the right choice for RP-HPLC. [[Click to Continue -> Question 12]] (set:$clicks = $clicks + 1)

[[Try Again |Question 18]]

Chocolate can be toxic to dogs as it contains both caffeine and theobromine — two related stimulants that can be dangerous even in small amounts.

One afternoon, Milo, a curious 10.0-kg golden retriever, found a 50-g bar of chocolate left on the counter and ate the entire thing. You want to know if it's safe for Milo, or if the amount of caffeine/theobromine he consumed could be dangerous. Where should you begin? [[A: Formulate the problem as a "chemical analysis" question->Q1 Correct Choice]] [[B: Determine the chemical equation that is pertinent to this problem->Q1 A2]] [[C: Research dog metabolism rates for caffeine and theobromine ->Q1 A3]] (set: $clicks to 0)

Now that you’ve determined the concentrations of caffeine and theobromine in your 10 mL chocolate extract, return to your notebook to find the mass of the chocolate sample you used. The recorded sample masses were 36.1 mg, 36.2 mg, and 36.5 mg. Using these data, calculate the amount (in grams) of caffeine and theobromine per 50 g bar of chocolate. As a reminder, your calculated values are: Caffeine = 10 µM and Theobromine = 80 µM [[A: Caffeine: 27 mg; Theobromine: 198 mg -> Q16 Correct ]] [[B: Caffeine: 43 mg;Theobromine: 252 mg -> Q16 Incorrect]] [[C: Caffeine: 52 mg; Theobromine: 363 mg -> Q16 Incorrect]] [[D: Caffeine: 38 mg; Theobromine: 477 mg -> Q16 Incorrect]]

Mild signs of toxicosis in dogs may appear after ingestion of approximately 20 mg/kg body weight of caffeine and/or theobromine. Based on the amounts of caffeine and theobromine you calculated in a 50 g chocolate bar, determine whether Milo, a 10 kg dog, would be at risk if he ate the entire bar. [[A: Yes, Milo's at risk. Call a veterinarian immediately. -> Q17 Correct]] [[B: Milo should be fine. -> Q17 Incorrect]]

You’ve successfully measured the amount of caffeine in chocolate and explored how your findings apply to a real-world context. You are now writing a report for your awesome TA's to grade. What all should you include in your reports? Type x for all that apply. Leave other boxes blank. [Exact concentration (mg caffeine per g chocolate)] (input-box: bind $a1) [Calibration curve & R squared value] (input-box: bind $a2) [Sources of error] (input-box: bind $a3) [Only "yes/no caffeine present"] (input-box: bind $a4) [Final conclusion about toxicity] (input-box: bind $a5) (link: "Submit")[ (if: ($a1 is "X" or $a1 is "x") and ($a2 is "X" or $a2 is "x") and ($a3 is "X" or $a3 is "x") and ($a5 is "X" or $a5 is "x") and $a4 is "" )[ (goto: "Post Data") ] (else:)[ (goto: "Q18 Incorrect") ] ]

Chocolate bars can contain up to 50 g of fat per 100 g. These fats can interfere with downstream analyses. How can you solve this problem? [[A: Digest the fats with an enzyme. -> Q8 A1]] [[B: Remove the fats using a liquid–liquid extraction with a nonpolar solvent such as petroleum ether. -> Q8 A2]] [[C: Heat the sample to melt and separate the fats from the chocolate before analysis. -> Q8 A3]] [[D: Dilute the chocolate extract with water to reduce the concentration of fats. -> Q8 A4]]