CHEM 241L Final Exam

key components of the SDS

molecular weight (ID), hazard identification, first aid, ingredient composition, handling/exposure, physical/chemical properties

how to use a micropipette

setting the right volume, attaching a sterile tip, drawing up liquid to the first stop, and dispensing to the second stop

how to use a seperatory funnel

Fill the closed, secured funnel up to three-quarters full, stopper it, and repeatedly invert, gently shake, and vent gas through the stopcock until pressure stops building. Return it to the stand, remove the stopper, let the layers settle, drain the denser bottom layer out the stopcock, and pour the top layer out the top.

how to fill graduated cylindar and read meniscus

place the graduated cylinder on a flat surface, pour slowly, and read the bottom of the curved liquid surface (meniscus) directly at eye level

Experiment BUC and BUS: What were the lab objectives, and how did we achieve them?

We calibrated a micropipette by weighing dispensed water and plotting volume corrections. We calibrated a pH probe, ran unbuffered control titrations, made a pH 4.50 buffer by two methods, and measured its resistance to added HCl and NaOH

Experiment BUC and BUS — How did we use gravimetric analysis to calibrate the micropipette?

We weighed the beaker before and after each water delivery. The difference gave the mass of water delivered. Divide that mass by water’s density at the measured temperature to get the actual volume. Correction = actual volume − pipette setting.

Experiment BUC and BUS — How do you pick an acid/base pair for a buffer?

Choose a weak acid and its conjugate base with a \(pK_a\) close to the target pH, within about ±1 pH unit. Acetic acid and acetate are suitable for pH 4.50 because acetic acid has a \(pK_a\) of about 4.76.

Experiment BUC and BUS — How do you calculate the amounts needed to make a theoretical buffer?

For 100.0 mL of 0.050 F buffer at pH 4.50, calculate the total moles, \(0.050(0.1000)=0.00500\) mol. Henderson–Hasselbalch gives the acetate-to-acetic-acid ratio, \(10^{4.50-4.76}=0.550\). Split the total using that ratio to get about 0.00323 mol acetic acid and 0.00177 mol acetate. That’s 6.45 mL of 0.50 M acetic acid and 0.241 g sodium acetate trihydrate.

Experiment BUC and BUS — What is the practical buffer method?

Start with one member of the conjugate pair and create the other by partial neutralization. Add strong base to a weak acid to make some conjugate base, or add strong acid to a weak base to make some conjugate acid.

Experiment BUC and BUS — How do you make the practical buffer?

Add 10.0 mL of 0.50 M acetic acid to about 80 mL DI water. Stir and monitor with a calibrated pH probe while gradually adding 0.50 M NaOH until the pH reaches 4.50. Transfer to a 100.00 mL volumetric flask, add the beaker rinses, dilute to the mark, and mix.

Experiment BUC and BUS — What are the benefits and drawbacks of the practical method?

You can adjust directly to the measured target pH and start with one member of the conjugate pair. You need a calibrated pH probe and careful additions near the target because you can overshoot.

Experiment BUC and BUS — What is the theoretical buffer method?

Start with both members of the conjugate pair. Use Henderson–Hasselbalch to find their ratio and the desired formal concentration and final volume to find the amount of each.

Experiment BUC and BUS — How do you make the theoretical buffer?

Measure the calculated volume of acetic acid and weigh the calculated mass of sodium acetate trihydrate. Combine them in a 100 mL volumetric flask, dissolve the salt, dilute to the mark, and mix. Check the pH with the calibrated probe.

Experiment BUC and BUS — What are the benefits and drawbacks of the theoretical method?

The calculated amounts give you a reproducible recipe. The measured pH can differ from the prediction because Henderson–Hasselbalch is an approximation and the result depends on accurate reagent measurements and stock concentrations.

Experiment BUC and BUS — What is the Henderson–Hasselbalch equation?

pH=pKa+log([A-]/[HA]) Put conjugate base on top and weak acid on the bottom. You can use moles because the two species share the same volume. If you add strong acid or base, use the moles after neutralization.

Experiment BUC and BUS — What is buffer capacity, and how do you calculate it?

Buffer capacity is the number of moles of strong acid or base per liter needed to change the pH by one unit.

1. Calculate C_added = (titrant molarity × corrected cumulative volume added) / total solution volume.

2. Plot C_added on the y-axis and pH on the x-axis.

3. Buffer capacity is the absolute value of the local slope: |change in C_added / change in pH|.

Example: 0.00244 M / 0.10 pH unit = 0.0244 mol/L per pH unit.

Manual: BUC.5–7, Equation BUC.4 and Figure BUC.1; BUS Part 2.4.

Experiment BUC and BUS — Practice Q1: How did you choose reagents, prepare the buffer, and measure its capacity?

Choose acetic acid and acetate because pKa 4.76 is close to pH 4.50. Mix calculated amounts for the theoretical buffer, or add NaOH to acetic acid until pH 4.50 for the practical buffer. Titrate separate 20 mL samples with HCl and NaOH in 0.50 mL steps. Capacity = |change in titrant concentration / change in pH|.

Experiment BUC and BUS— Practice Q2: Which buffer preparation method is best in a lab, and why?

The practical method lets you monitor the pH as you add acid or base and stop at the target.

Experiment BUC and BUS — Practice Q3: What is gravimetric analysis, and how did we use it?

Gravimetric analysis uses mass to determine a quantity. We divided the mass of dispensed water by its density to find the actual volume.

Experiment BUC and BUS — Practice Q4: How do you apply a micropipette correction between two calibration points?

Interpolate between the two surrounding points, then add the signed correction to the pipette setting.

Experiment BUC and BUS — Practice Q5: Can a pH 6.0 buffer resist added HCl, NaOH, or both?

Both, in small amounts. The conjugate base consumes H+ from HCl, and the weak acid consumes OH- from NaOH.

Experiment AM — What were the lab objectives, and how did we achieve them?

We used UV-Vis spectra and Beer’s law to track the two forms of bromothymol blue (BB) and estimate its pKa. We used solvent-solvent extraction to compare how the forms partitioned between water and ethyl acetate. (AM.1, AM.13–16)

Experiment AM — What are the pH and pKa equations?

pH = -log10[H3O+]

pKa = -log10(Ka)

pH = pKa + log10([A-]/[HA])

When [A-] = [HA], pH = pKa

Experiment AM — Which indicator did we use, and what is its useful pH range?

Bromothymol blue (BB). Its color transition is about pH 6.0–7.6. In lab, we measured spectra over the wider range of pH 5–10. (AM.5, Part 2)

Experiment AM — What color is bromothymol blue in its acidic and basic forms?

Acidic HIn is yellow. Basic In- is blue. A mixture of both looks green. (AM.5–6)

Experiment AM — What does a UV-Vis spectrophotometer measure?

It measures how much light a sample absorbs at different wavelengths. A spectrum plots absorbance versus wavelength. (AM.3)

Experiment AM — How does a UV-Vis spectrophotometer work?

Light source → sample cuvette → wavelength separator → detector. The instrument compares transmitted light to the blank at each wavelength to calculate absorbance. (AM.3, Part 2)

Experiment AM — Why did we use UV-Vis for bromothymol blue?

The acidic and basic forms absorb most strongly at different wavelengths. Measuring absorbance at each form’s peak lets us track how its concentration changes with pH. (AM.5, AM.13–14)

Experiment AM — How is absorbance related to concentration?

Beer’s law: A = εbc. At a fixed wavelength and path length, absorbance is directly proportional to concentration. (AM.4)

Experiment AM — What was the purpose of solvent-solvent extraction?

To see how the two forms of BB partition between immiscible water and ethyl acetate. Their different charges give them different solubilities. (AM.7)

Experiment AM — How does bromothymol blue’s structure change as pH rises?

HA loses a proton, the sulfur-containing five-membered ring opens, and the negative charge spreads through the rearranged structure. This changes the color from yellow to blue. (AM.6–7)

Experiment AM — How are absorbance and transmittance related?

T = I/I0, %T = 100 × T, and A = -log10(T). Use fractional T in the log. More transmitted light means lower absorbance. (AM.3, Equations AM.2–3)

Experiment AM — What are the organic and aqueous layers?

The organic layer is ethyl acetate. The aqueous layer is the water-based BB solution. The liquids form separate layers because they are largely immiscible. (AM.7, Part 1)

Experiment AM — Which layer is on top, and why?

Ethyl acetate is the top layer because it is less dense than water. The aqueous layer is on the bottom. (AM Part 1)

Experiment AM — Which layer did we analyze after extraction?

We collected UV-Vis spectra of the bottom aqueous layers from both the pH 5 and pH 10 extractions. (AM Part 2, Subpart C)

Experiment AM — Practice Q1. Which form of BB prefers each layer, and why?

Basic In- prefers the aqueous layer because it is negatively charged. Acidic HIn prefers the ethyl acetate layer because it is neutral. (AM.7)

Experiment AM — Practice Q2. What is the point where the pH 5–10 spectra cross, and why does it matter?

It’s the isosbestic point, near 500 nm in the practice graph. Both forms absorb equally at that wavelength, so the absorbance stays constant as their ratio changes at fixed total BB concentration.

Experiment AM — Practice Q3. How did we determine the pKa of bromothymol blue?

Plot absorbance versus pH at each form’s peak wavelength. Plot change in absorbance / change in pH against average pH. The derivative peak or trough gives the pKa, where [HIn] = [In-]. (AM.14–16)

Experiment AM — Practice Q4. What does pKa tell you about a reagent?

It tells you how readily an acid gives up a proton. Lower pKa means a stronger acid. At pH = pKa, the protonated and deprotonated forms are equally abundant. (AM.4–5)

Experiment AM — Practice Q5. Draw a UV-Vis spectrophotometer and explain how it works.

Draw: light source → cuvette → wavelength separator → detector. The detector measures how much light passes through at each wavelength. Comparing that light to the blank gives absorbance. (AM.3, Part 2)

Experiment AM — Practice Q6. Why did we use a glass cuvette after extraction?

Residual ethyl acetate can dissolve plastic and interfere with the reading. Glass resists ethyl acetate. (AM Parts 1–2)

Experiment AM — What were the lab objectives, and how did we achieve them?

We extracted ethanol into pentanol and measured its GC peak area. In GCC, we used external standards to find the partition coefficient. In GCS, we used standard addition to find the percent ethanol in three beers. (GCC.1, GCS.1)

Experiment GCC and GCS — How do you make and use a standard calibration curve?

Plot average ethanol peak area (y) versus known % ethanol in pentanol (x). Fit a line and use the unknown extract’s peak area to solve for its ethanol concentration. (GCC.7, GCC.21)

Experiment GCC and GCS — How do you make and use a standard addition curve?

Spike equal beer aliquots with different amounts of ethanol and include an unspiked sample. Extract them all the same way. Plot average peak area (y) versus % ethanol added (x). The magnitude of the x-intercept gives the original % ethanol in the beer. (GCS.4–6, GCS.12)

Experiment GCC and GCS — What is solvent-solvent extraction?

An analyte distributes between two immiscible liquids. Here, some ethanol moves from the aqueous sample into pentanol. (GCC.8–9)

Experiment GCC and GCS — How did we perform the extraction?

Add NaCl to 1.0 mL of beer, then add 1.0 mL of pentanol or ethanol-in-pentanol standard. Cap, invert for 1–2 minutes, let the layers separate, and sample the top pentanol layer. (GCC.18–19, GCS.5–6)

Experiment GCC and GCS — What were the roles of salt and pentanol?

NaCl salts out ethanol, pushing more into the organic layer and making extraction more reproducible. Pentanol is the organic solvent that extracts ethanol and forms the top layer. (GCC.18–19)

Experiment GCC and GCS — Why extract ethanol before running the beer samples on the GC?

Extraction moves ethanol into pentanol while leaving much of the water, sugars, and proteins behind. This gives the GC a simpler sample to analyze. (GCS.3–4)

Experiment GCC and GCS — What is gas chromatography?

GC separates vaporized compounds as a carrier gas moves them through a column. The detector records a chromatogram of signal versus time. (GCC.3–6)

Experiment GCC and GCS — How does the GC instrument work?

Hot injector vaporizes the sample → carrier gas moves it through the column → compounds separate based on volatility and interactions with the stationary phase → thermal conductivity detector records the peaks. (GCC.3–6, GCC.17)

Experiment GCC and GCS — Why was GC suitable for our samples?

Ethanol and pentanol are volatile and can be separated by GC. Ethanol has a lower boiling point and elutes first under our lab conditions. (GCC.7)

Experiment GCC and GCS — How did we use the chromatogram qualitatively and quantitatively?

Qualitatively, we matched retention times to identify ethanol. Quantitatively, we integrated the ethanol peak and used its area to determine concentration. (GCC.7, GCC.20–21)

Experiment GCC and GCS — What is retention time?

The time from injection to the top of a compound’s peak. We compared it with the ethanol reference under the same GC conditions to identify ethanol. (GCC.7)

Experiment GCC and GCS — What is a peak on a chromatogram?

A peak is a rise in detector signal as a component leaves the column. The earlier of the two reference peaks was ethanol. (GCC.7, Figure GCC.6)

Experiment GCC and GCS — What is peak area, and how did we use it?

Peak area is the integrated detector signal above the baseline. We used average ethanol peak area from replicate injections as the y-value on our calibration plots. (GCC.20–21, GCS.11–12)

Experiment GCC and GCS — What assumptions did we make about the beer matrix, and what limited the results?

We treated each beer as mostly water and assumed native and spiked ethanol extracted to the same extent within that beer. Different beer compositions can affect extraction, and inconsistent injections or overlapping peaks can affect the measured area. (GCS.3–4)

Experiment GCC and GCS — What is baseline correction, and how did we do it?

Estimate the average detector voltage before the ethanol peak and subtract that signed value from the whole trace. This shifts the baseline to zero. (GCC.20, GCS.11)

Experiment GCC and GCS — Why is baseline correction necessary for quantitative data?

A baseline offset makes the integrated peak area too high or too low. That biases the ethanol concentration calculated from the area. (GCC.20–21, GCS.11–12)

Experiment GCC and GCS — Practice Q1. What is the difference between a standard calibration curve and a standard addition curve?

External calibration uses separate standards and the unknown’s signal to find its concentration. Standard addition spikes the sample itself and uses the x-intercept to find its original concentration. (GCC.7, GCS.4, GCS.12)

Experiment GCC and GCS — Practice Q2. Why use standard addition to measure ethanol in beer?

Each spiked aliquot contains the same beer matrix, so the standards experience the same matrix effects as the original ethanol. The method works without knowing K if extraction is consistent across the aliquots. (GCS.3–4)

Experiment GCC and GCS — Practice Q3. If ethanol’s partition coefficient is 1.33, which layer contains more ethanol?

With equal layer volumes, the organic pentanol layer contains more. K = [ethanol]organic / [ethanol]aqueous = 1.33, so the organic concentration is 1.33 times higher. If volumes differ, compare concentration × volume. (GCC.8–9)

Experiment GCC and GCS — Practice Q4. What chemical property helps predict GC elution order?

Boiling point. More volatile compounds generally elute earlier, so ethanol elutes before pentanol. Polarity and interactions with the stationary phase can also affect the order. (GCC.4–7)

Experiment GCC and GCS — Practice Q5. How do GC and LC phases differ, and what makes a sample suitable for GC?

GC uses a gas mobile phase and, in our lab, a liquid stationary coating. LC uses a liquid mobile phase and commonly a packed solid stationary phase or bonded coating. GC analytes should vaporize without decomposing. (GCC.3–4, GCC.17)

Experiment GCC and GCS — Practice Q6. Why must the sample be injected rapidly into the GC?

A quick injection lets the sample flash-vaporize and enter the column as a narrow band. Slow injection can broaden peaks and reduce separation. Start recording at the moment of injection. (GCC.17, GCS.9)

Experiment LCS — What were the lab objectives, and how did we achieve them?

We extracted spinach pigments, separated them by reversed-phase LC, and detected them at 450 nm. We used retention times and peak widths to calculate relative retention and resolution for chlorophyll a and b. (LCS.1, 22–23)

Experiment LCS — What is relative retention, and how do you calculate it?

Relative retention (alpha) compares the adjusted retention times of two peaks. Alpha = (tR2 - tM) / (tR1 - tM). Peak 2 elutes later, and tM is the unretained peak time. (LCS.8, 22–23)

Experiment LCS — What is resolution, and how do you calculate it?

Resolution measures how well two peaks are separated relative to their widths. Rs = 2(tR2 - tR1) / (w1 + w2). Use baseline widths and the same time units throughout. (LCS.8, 22–23)

Experiment LCS — How do you find retention time and peak width?

Read retention time at the peak apex, measured from injection. Estimate baseline width by extending the two steep sides to the baseline and subtracting the left intercept time from the right. (LCS.8, 22)

Experiment LCS — Which extraction layer contains the pigments we analyze?

The top, dark green, acetone-rich organic layer. Drain the bottom aqueous brine layer first. (LCS.16–17)

Experiment LCS — What is the basic diagram of how LC works?

Solvent reservoir → pump → sample injector → column → UV-Vis detector → waste. The detector records absorbance versus time as pigments elute. (LCS, Instrumentation)

Experiment LCS — In what order do pigments elute in reversed-phase LC, and why?

More polar pigments generally elute first because they interact less with C18. Chlorophyll b elutes before chlorophyll a because it is more polar. Nonpolar carotenes elute late. (LCS.5–7)

Experiment LCS — What were the stationary and mobile phases?

The stationary phase was nonpolar C18 bonded to silica. The mobile phase was a changing mixture of ethyl acetate, acetonitrile, and water. (LCS, Instrumentation)

Experiment LCS — What is the salting-out effect?

Saturated NaCl helps separate the acetone-rich organic layer from the aqueous layer and favors keeping the pigments in the organic layer. (LCS.5, 16–17)

Experiment LCS — How did we load and run the LC?

Equilibrate with mobile phase 1, load the filtered extract into the injection loop, then switch to INJECT and start recording. Switch mobile phases as the designated peaks elute. (LCS, Experimental Procedure)

Experiment LCS — How did we use the separatory funnel?

Mix spinach extract with saturated NaCl, shake and vent, then let the layers separate. Drain the bottom aqueous layer and retain the top organic layer. Repeat the brine wash. (LCS.16–17)

Experiment LCS — How did we use UV-Vis in the LC setup?

The column effluent passed through a flow cell. We recorded absorbance at 450 nm versus time to detect the eluting pigments. (LCS, Instrumentation and Experimental Procedure)

Experiment LCS — What does good versus poor resolution look like?

Good resolution gives distinct peaks with a valley near the baseline. Poor resolution gives overlapping peaks. Rs of about 1.5 or higher generally indicates baseline separation. (LCS.8, 22–23)

Experiment LCS — What mobile phases did we use, and why did we switch them?

Ethyl acetate / water / acetonitrile by volume: MP1 = 5/10/85, MP2 = 35/10/55, MP3 = 70/0/30. The step gradient becomes less polar so it can elute increasingly nonpolar, strongly retained pigments. (LCS.7, Mobile Phase Composition)

Experiment LCS — Practice Q1. Why measure LC flow rate, and how did we do it?

Flow rate affects retention times and separation. Collect effluent for 5 minutes with mobile phase 1, then divide volume by time. The target was 0.7–0.8 mL/min. (LCS, Determination of Mobile Phase Flow Rate)

Experiment LCS — Practice Q2. What type of column did we use, and which pigments elute first?

A reversed-phase C18 column. More polar pigments elute first because they interact less with the nonpolar stationary phase. (LCS.7, Instrumentation)

Experiment LCS — Practice Q3. What happens if you use a normal-phase column with the original mobile phases?

The original polar solvents would compete strongly for the polar stationary phase, especially at the start. Pigments could elute early with poor separation, and water can change the behavior of bare silica. (LCS.6–7)

Experiment LCS — Practice Q4. What happens with a normal-phase column and a nonpolar mobile phase?

The order would generally reverse. Nonpolar pigments elute first, and polar pigments are retained longer by the polar stationary phase. (LCS.6–7)

Experiment LCS — Practice Q5. What is salting out, and why did we need it?

Adding saturated NaCl helps split the extract into two layers and keeps more pigment in the acetone-rich layer we inject. (LCS.16–17)

Experiment LCS — Practice Q6. What are two ways to improve poor peak resolution?

Adjust the mobile-phase composition or switch timing to spread the peaks apart. Optimize the flow rate to reduce band broadening. (LCS.7–9, Experimental Procedure)

Experiment LCS — Practice Q7. How would you sketch and measure a pigment’s peak width?

Draw a peak above a baseline. Extend both steep sides down to the baseline, label the two intercept times, and calculate width = right time - left time. (LCS.8, 22)

Experiment VIS — What were the lab objectives, and how did we achieve them?

We measured glucose indirectly using an enzyme assay and a 420 nm calibration curve, then corrected for dilution. We also compared how temperature, reaction time, and enzyme amount affected the signal. (VIS.1, 4, 10–12)

Experiment VIS — What is the difference between monosaccharides and disaccharides?

A monosaccharide is one sugar unit, like glucose or fructose. A disaccharide is two linked units. Sucrose contains glucose and fructose. (VIS.2–3)

Experiment VIS — How does digestion break a disaccharide into monosaccharides?

Hydrolysis breaks the glycosidic bond. We used acid and heat to split sucrose into glucose and fructose, then adjusted the pH before the assay. (VIS.2, 12)

Experiment VIS — Why did we need an enzymatic assay to quantify glucose?

Glucose gives no useful visible absorbance at 420 nm. The assay selectively converts its presence into a measurable signal from yellow ferricyanide. (VIS.3–4)

Experiment VIS — How do enzymes work?

An enzyme binds its substrate at an active site and speeds up the reaction by lowering the activation energy. The enzyme can be reused. (VIS.3)

Experiment VIS — How do time, temperature, and enzyme concentration affect the assay?

At a fixed reading time, cold or less enzyme generally gives less product. A shorter reaction gives less time for product to form. Very high heat can denature enzymes. (VIS, Experimental Procedure)

Experiment VIS — Why are enzymes highly specific?

The substrate fits the enzyme’s active site like a key fits a lock. The site’s shape and chemistry determine which substrates bind and react. (VIS.3)

Experiment VIS — What two reactions let us measure glucose by spectroscopy?

1. Glucose oxidase converts beta-D-glucose to gluconolactone and produces H2O2.

2. Horseradish peroxidase uses H2O2 to oxidize ferrocyanide to yellow ferricyanide, measured at 420 nm. (VIS.4)

Experiment VIS — How do you find the original glucose concentration from a diluted sample?

Use C = (A - b)/m from the calibration line to find the assay concentration. Then multiply by every dilution factor. For the manual’s Gatorade prep: 50 × (5.0/0.25) = 1000. (VIS.10–12)

Experiment VIS — Practice Q1. What species is measured at 420 nm?

Yellow ferricyanide, [Fe(CN)6]3-. Its absorbance measures glucose indirectly. (VIS.4)

Experiment VIS — Practice Q2. The assay reads 0.150 g/L. It contains 0.50 mL of a 20× diluted Gatorade stock in 5.0 mL total. What was the original concentration?

0.150 × (5.0/0.50) × 20 = 30.0 g/L glucose. (VIS.11–12, using the practice question’s volumes)

Experiment VIS — Practice Q3. Which temperature gives the highest ferricyanide absorbance: 0, 40, or 80 °C?

40 °C. The enzymes work faster than at 0 °C, where the reaction is slow. At 80 °C, heat can denature the enzymes, so less ferricyanide forms. (VIS, Experimental Procedure)

Experiment VIS — Practice Q4. How do mono- and disaccharides differ, and how do you split a disaccharide?

A monosaccharide is one sugar unit. A disaccharide is two units joined by a glycosidic bond. Hydrolysis breaks that bond, such as when acid and heat split sucrose into glucose and fructose. (VIS.2–3, 12)

Experiment VIS — Practice Q5. What sugars are in Gatorade, what does the assay detect, and how could you measure the glucose bound in sucrose?

Check the bottle label for sucrose and sources of free glucose, such as dextrose. The assay detects beta-D-glucose. Hydrolyze sucrose and adjust the pH before running the assay to include the glucose released from sucrose. Fructose still goes unmeasured. (VIS.2–4, 12)