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Homework answers / question archive / Experiment 2: Quantitative Analysis of Caffeine in Energy Drinks by External Standard Method using UV Spectroscopy Objectives In this experiment, the student will determine the concentration of caffeine (mg/bottle) in commercial energy drink samples using the Agilent Cary 100 UV-Visible spectrophotometer

Experiment 2: Quantitative Analysis of Caffeine in Energy Drinks by External Standard Method using UV Spectroscopy Objectives In this experiment, the student will determine the concentration of caffeine (mg/bottle) in commercial energy drink samples using the Agilent Cary 100 UV-Visible spectrophotometer

Chemistry

Experiment 2: Quantitative Analysis of Caffeine in Energy Drinks by External Standard Method using UV Spectroscopy

Objectives

In this experiment, the student will determine the concentration of caffeine (mg/bottle) in commercial energy drink samples using the Agilent Cary 100 UV-Visible spectrophotometer.  A UV absorption spectrum of a standard caffeine solution will be recorded to establish the appropriate wavelength for quantitative analysis. Method of external standard following Beer’s Law will be used to calibrate the UV spectrophotometer. Both multipoint and single point data handling techniques will be used to evaluate the concentration of caffeine in energy drinks. Statistical data handling methods will be employed to evaluate the quality of the final results. The student’s experimental results will then be compared to the manufacturer’s specifications in order to determine the identity of the unknown.

Introduction

I-Energy Drinks

An energy drink is a beverage containing stimulant drugs, usually including caffeine, which provide mental and physical stimulation. These drinks may or may not be carbonated, and many types of energy drinks also contain sugar or other sweeteners, herbal extracts, taurine, and amino acids. Energy drinks are often marketed to provide the benefits of caffeine. Caffeine helps muscle cells to contract more forcefully, producing more power and cutting down on lactic acid buildup. In a recent study, caffeine was shown to slow down the rate of breakdown of glycogen to glucose in the muscles. Glucose is the molecule that the muscles use for energy.  In another study it was found that caffeine acts to quicken the rate of glucose absorption by the body. These findings suggest that caffeine not only slows down the rate at which your muscles use up their energy, it speeds up the rate at which the body delivers new energy to them.

 

The chemical structure of caffeine is shown in Figure 1. It is a xanthine alkaloid found in seeds, leaves, and fruit of some plants. It is commonly consumed by people in the form of coffee or tea derived from the koala nut.

 

 

 

 

 

 

Figure

1

:

 Chemical Structure

of Caffeine

 
 
   
 

 

 

II- Ultraviolet Spectroscopy

 

The total energy of a molecule is the sum of its binding or electronic energy, vibrational energy, rotational energy and other types of energies.

Emolecule = Eelec + Evib + Erot + other types

Absorption of a photon by a molecule can increase molecular energy in a quantized way as illustrated in Figure 1. When a photon is absorbed, the energy of the absorbing species is increased by an amount proportional to the frequency of the photon.

????????? = ????????" − ????????′ =  ????????????????     

where    ????????" − ????????′ =   energy of absorption  

                        h =   Planck’s constant                                  

 =   frequency of the photon in = c/λ

               

All organic molecules absorb light in the ultraviolet and visible region of the electromagnetic spectrum. Energy absorbed in the UV region by the molecule produces changes in the electronic energy of the molecule where the energy differences between electronic levels in most molecules vary from 30 to 150 kcal/mol.  These changes in the electronic energy result from transitions of the bonding and nonbonding electrons in the ground state of a molecule to higher

energy levels (excited states) as illustrated in Figure

2

.

 

 

Figure

2

:

 Energy

L

evel

D

iagram of

P

ossible

M

olecular

T

ransitions

 
 

 

For most organic molecules, the lowest energy occupied molecular orbitals are the sigma orbitals (σ); single bonds. The pi orbitals (π) in unsaturated bonds lie somewhat higher than the sigma whereas n-orbitals or non-bonding, unshared electron pairs occupy the highest energy levels. A typical representation of the electronic energy levels is illustrated in Figure 3.

 

 

 

 

 

 

 

 

Figure

3

:

 Electronic Energy Levels and Transitions

 

Absorption of light by the electrons of a single sigma (σ) bond occurs in the vacuum-ultraviolet region (below 180 nm) where atmospheric components also absorb strongly. Obtaining spectra for these transitions is experimentally difficult. In general, spectra are limited to absorption by various functional groups or chromophores which contain lower energy pi (π) or non-bonding (n) electrons. In the near ultraviolet, ultraviolet and visible region, most of the observed absorption bands are due to transitions of the pi (π) and nonbonding (n) electrons to pi antibonding (π*) or sigma antibonding (σ*) orbitals respectively as depicted in Figure 3.  Absorption of UV or visible light by a molecule can occur only when the energy of incident radiation is the same as that of the possible electronic transition in the molecule (i.e. energy is quantized).  The absorbed energy excites the electrons in the molecule from the ground energy state to an energetically higher excited state.  This is accomplished by the promotion of a single π or n electron from an occupied molecular orbital to an unoccupied antibonding molecular orbital such as a (π*) or (σ*).  Figure 4 displays a molecular orbital diagram showing σ, π and n electrons for

 

formaldehyde

.

 

 

 

 

Figure

4

:

 

Molecular

O

rbital

D

iagram of

F

ormaldehyde

 

The molecular orbitals describe the distribution of electrons in a molecule, just as atomic orbitals describe the distribution of electrons in an atom. 

In formaldehyde, the carbonyl double bond (C=O) in the ground state consists of a pair of bonding sigma (σ) electrons and a pair of bonding pi (π) electrons as well as a pair of non-bonded (n) electrons on the oxygen.  Upon absorption of UV radiation of the correct energy (i.e. wavelength), one of the pi (π) bonding electrons or non-bonded (n) electrons is raised to the next higher energy orbital; an antibonding pi (π*) orbital. Figure 5 illustrates the possible excited electronic states for

 

the formaldehyde molecule

 

and the corresponding UV spectrum

.

 

 

Figure

5

:

 

E

lectronic

T

ransitions

for F

ormaldehyde

and Corresponding UV S

pectrum

 

300

 

 

It is important to note that the antibonding orbital consists of unpaired single electrons while bonding orbitals consists of two paired electrons.  As the electrons in the antibonding orbital are not paired, the electronic state represented by this configuration is unstable and of higher energy.  This is why the term antibonding is used and also why the molecule spontaneously returns to the more stable bonding ground state with the emission of energy in the form of radiation or nonradiant decay (heat).  

 

A UV-visible spectrum represents the absorption of light as a plot the intensity of absorption (i.e. absorbance) versus wavelength of energy in nanometres. In general, when a molecule absorbs light having sufficient energy to cause an electronic transition, vibrational and rotational transitions occur as well. A collection of molecules will have its members in many states of vibrational and rotational excitation. The energy levels for these states are quite closely spaced, corresponding to considerably smaller energy differences than for electronic levels. A molecule may therefore undergo electronic and vibrational/rotational excitation simultaneously. Since there is so many possible transitions each differing from the others by only a slight amount, a continuous broadband spectrum results with a maximum absorbance centred on the wavelength representing the major transition. For the formaldehyde molecule, Figure 5 displays a maximum absorbance at 200 nm resulting from the π à π* transition with a minor absorbance at 300 nm corresponding to the n à π* transition.

 

 

A variety of energy absorptions is possible depending on the nature of the bonds within a molecule.  If a molecule contains a carbonyl bond (C=O) and carbon-carbon double bonds (C=C), it will have energy absorptions characteristic of these two types of bonds. Electrons in organic molecules may be in strong sigma (σ) bonds or in weaker pi (π) bonds or in nonbonding (n) orbitals on electronegative atoms such as oxygen, sulphur or nitrogen.  When energy of the correct frequency is absorbed the electrons in covalent bonds or nonbonding orbitals can be excited to higher energy levels.  The possible transitions in organic molecules are σ à σ*, πà π*, n à π* or n à σ* transitions as illustrated in Figure 2. Most σ à σ* absorptions take place below 200 nm beyond the conventional range of most UV spectrometers.  As all organic molecules contain sigma bonds of nearly equal energy this transition is not particularity useful for quantitative or qualitative analysis. 

 

The type of transition that is used extensively in UV spectroscopy is the π à π * transition.  As discussed, molecules that contain double or triple bonds or aromatic rings can undergo π à π * transitions.  An important feature of UV spectroscopy is to define the presence, nature and extent of conjugation.  Increasing conjugation moves the absorption to longer wavelengths and finally to the visible region. A table of common absorption characteristics of some common chromophores is given in Table 1. For more comprehensive tables, one must refer to reference textbooks.

Table 1: Partial List of Electronic Transitions of Some Common Chromophores

 

Chromophore

Example

Excitation

λmax, 

nm

ε

Solvent

 

 

C=C

Ethylene

π  →  π*

171

15,000

hexane

C≡C

1-Hexyne

π  →  π*

180

10,000

hexane

C=O

Ethanol

n →  π* π  →  π*

290

180

 

hexane hexane

N=O

Nitromethane

n  →  π* π  →  π*

275

200

17

5,000

ethanol ethanol

C-X   X=Br

              X=I

Methyl bromide Methyl iodide

n  →  σ* n  →  σ*

205

255

200

360

hexane hexane

 

 

UV qualitative spectral analysis for the identification of organic molecules can be performed similar to infrared spectroscopy relying on the fact that every molecule has a unique UV-visible spectrum or fingerprint.  By comparing the spectrum of an unknown molecule with spectra of known compounds found in UV spectral libraries, an analyst can determine the identity of the molecule in question.

The UV absorption spectrum of caffeine in HCl presented in Figure 6 for the wavelength range of 200 to 320 nm shows two major absorption peaks at 206 and 272 nm.  The peak at 206 nm is associated with the  π à π* transition. The peak at 272 nm is associated with the n à π* transition. 

 

Figure

6

:

  UV Absorption Spectrum of

Caffeine in 0.01 M HCl

 

 

 

  1. Beer Lambert Law

Measurement of absorption of ultraviolet-visible radiation by organic molecules in solution provides a means for both qualitative and quantitative analysis. Quantitative analysis depends upon the fact that the Beer-Lambert law must be obeyed, which states that absorbance is directly proportional to analyte concentration at a particular analysis wavelength (A α C):

                                                            ???????? = ???????? ???????? ????????

where  A – absorbance                          b – pathlength of cuvette (cm)

                        c – analyte concentration (mg/L)

a – Absorptivity coefficient (L/(mg*cm))

 

As a first step, a UV spectrum of the analyte of interest must be recorded in order to determine the absorbance maximum (λmax) or analysis wavelength for quantification. Figure 6 displays the UV spectrum of caffeine and the absorbance maximum centred at 206 nm. Next, the absorbance values for a series of external calibration standards of analyte of known concentrations must be measured at the analysis wavelength. By plotting absorbance as a function of increasing analyte concentration, a Beer Lambert law linear calibration graph as seen in Figure 7 can be constructed.                                                              ???????? = ???????????????? + ????????                           

where                 A = absorbance 

C = concentration of analyte  m = slope  b = y-intercept

The final step in the analysis is to measure the absorbance values of the analyte in the unknown sample at the analysis wavelength. By rearranging the equation above, the concentration of analyte in an unknown sample can be determined:

 

 

 

 

 

 

 

 

 

 

 

Figure

7

:

 

Calibration

 

Plot of Absorbance versus Concentration

 

of

Caffeine

 

(

ppm

)

 

 

 

                                                             

It is important to note that the analyte of interest must be dissolved in a solvent that does not absorb UV radiation at the analysis wavelength. Table 2 lists the cut-off wavelength for UV absorption for various solvents in the UV spectral range.

Table 2: Short Wavelength Cut-off Limits of Various Solvents

 

Solvent

Cut-off Wavelength(nm)

 

Water

190

Acetonitrile UV

190

Hexane UV

195

Isopropyl Alcohol

205

Methanol

205

Ethyl Alcohol

210

 

  1. Double Beam UV-Visible Spectrometer 

All commercial instruments are capable of operating over the UV and visible portions of the electromagnetic spectrum.  This is necessary as the electronic transitions can occur, depending on the type of molecule, in either of these two regions.  There are five basic components for the spectrophotometer and these are:

 

  1. A source of radiation covering the required wavelength range. Two sources of radiation are used in UV-visible spectrometers which cover the necessary range of 200 to 1100 nm.  For the visible range measurements above 350 nm use a compact tungsten-halogen source housed in a quartz envelope and below 350 nm to 200 nm, a deuterium arc source is used. 
  2. A means for selecting a narrow band of wavelength. The function of the monochromator is to select a narrow band of wavelengths to pass through the sample cell.  In most modern spectrophotometers this is accomplished using a holographic reflection grating.
  3. A cuvette (cell) contains the solution to be analyzed. Cuvettes are made of quartz for analysis in the UV range, or optical glass for analysis in the visible range. It is important to use a cuvette made of material that is transparent in the region of analysis. The most commonly used cell is 1.0 cm in pathlength with a capacity of 3 - 5 mL of solution.
  4. A detector capable of measuring the intensity of the radiation once it has passed through the sample solution. Maximum sensitivity is achieved by the use of photomultiplier tubes or silicon diode arrays that are sensitive over the entire range of 200 nm to 1100 nm.

5)         A means of recording the spectrum as displayed on a computer screen.

A double beam ratio recording UV-visible spectrophotometer shown in Figure 8 allows for the comparison of the intensity of light that has passed through a sample and solvent reference.  A rapidly rotating sector mirror, the beam chopper, is used to alternate the beam of polychromatic radiation that comes from the source between the sample and the reference cell compartments. The reference beam passes through the blank solution (Po) while the sample beam passes through sample (P). The two radiant beams, “reference" and "sample" are received and recombined alternatively at the junction of the half silvered mirror (grid mirror). Alternately, half of the radiation of each beam is focused onto the detector while half of the radiation is lost through the transparent segments of the mirror. Each beam Po/2 and P/2 is received as pulses by the photomultiplier tube (detector) and the signal is averaged and ratio by electronics [T= (Po/2)/(P/2]). The function of the double beam spectrophotometer is to subtract out or remove the blank spectrum such that the detector sees only the absorbance of radiation by the UV absorbing analyte in the sample.   

 

 

 

 

 

 

V- Matched Cuvettes in Spectrometry

 

Figure

8

:

 

Double Beam Cary 100

 

UV

-

visible

S

pectrophotometer

 

Optical Diagram

 

 

 

Matched cuvettes are necessary when collecting a qualitative spectrum of an absorbing analyte solution or performing quantitative analysis. For dual beam UV-visible spectrophotometers, one cuvette holds the blank solution while the other cuvette contains the absorbing analyte solution. In order to make accurate measurements that reflect analyte absorption only and not contributions due to the blank (analytical matrix), it is imperative that the two cuvettes are optically matched. 

Matched cuvettes or cells are identical with respect to pathlength, reflective and refractive properties in the area where the light beam passes. Regardless of whether or not the cuvettes are made of optical glass or quartz the windows must be parallel to ensure a constant pathlength and must be as flat as possible to prevent reflection or refraction. In addition, they must be highly polished to keep light dispersion to a minimum. If the pathlengths were different, or if the wall of one cuvette reflects more or less light than another cuvette, then the absorbance measurement could be different for that reason and not because the solution concentration is different.

A general guideline with respect to matching cuvettes is that a solution which transmits 50% of the incident light should not have a reading differing by more than ±1 %T in any cuvette. Cuvettes are matched by placing analyte solution of intermediate absorbance in each cuvette and comparing absorbance readings at the wavelength where the solution absorbs maximally. Two cuvettes that give the same percent transmittance reading within ±1 %T are optically matched. In this experiment, you will be provided with two cuvettes that are optically matched. The cuvettes used in this experiment have a pathlength of 1.00 cm. 

It is important that cuvettes be placed in the instrument exactly the same way each time, since pathlength and reflective or refractive properties can change by rotating the cuvette. A vertical line on the cuvette, lined up with a similar line on the cuvette holder of the spectrophotometer is essential. Obviously cuvettes that have scratches should be avoided. Any liquid or fingerprints adhering to the outside wall must be removed with a soft cloth or Kimwipe and an appropriate solvent.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Prelab Questions

 

  1. State what is the diluent for this experiment. 

 

  1. State which solutions must be used to analytically rinse the volumetric flasks and volumetric pipets.

 

  1. Draw a diagram of the preparation of the caffeine stock and the external standard calibration solutions.

 

  1. Calculate the concentration of caffeine stock solution in ppm if the analyst weighed 0.0250 g of caffeine on the semi-micro analytical balance and dissolved it in a 200.0 mL volumetric flask and made to the mark.

 

  1. Calculate the concentration in ppm of the five external standard caffeine solutions if 1.00, 2.00, 3.00 4.00 and 00 mL aliquots of the stock solution are added to five separate 50.00 mL volumetric flasks, and made up to the mark with diluent.

 

  1. Draw a diagram of the preparation of the unknown energy drink samples.

 

  1. State the absorbance maximum wavelength for caffeine that will be used in quantitative analysis.

 

  1. Explain why it is necessary to use quartz cuvettes instead of glass. What is the pathlength of the cuvettes used in this experiment?

 

  1. Explain why it is necessary to ensure that the cuvette contains no air bubbles and must be free of fingerprints for analysis.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Procedure

 

NOTE: Use RO WATER throughout the entire procedure to make all of your solutions.

 

A - Preparation of Caffeine Stock Solution

  

  1. Using the semi-micro analytical balance, weigh 0.020 – 0.025 g of pure caffeine into a clean dry 150 mL beaker and record the mass to ±0.00001 g in your lab notebook.

  

  1. Add 70 mL of RO water to the beaker to dissolve the caffeine powder. Swirl the beaker. If caffeine does not dissolve readily, gently heat the caffeine solution on a hot plate at low heat for five minutes until it dissolves. 

 

NOTE: DO NOT boil the solution.  

  1. Analytically transfer the caffeine solution into a 200.0 mL volumetric flask that has been prerinsed with RO water. Rinse the 150 mL beaker three times with small volumes of RO water and add each rinse to the volumetric flask. Make the caffeine stock solution to the mark with RO water.  Stopper and invert 15 times.

B- Preparation of External Standard Caffeine Solutions

4.       Prepare five standards by pipetting the caffeine stock solution (prepared in step 3) the exact volumes indicated in the table below using 5.00 mL micropipet into 50.00 mL pre-rinsed volumetric flasks. Dilute each volumetric flask to the mark with RO water. Stopper and invert 15 times.

Table 3: External Standard Calibration Solution

 

Preparation

 

Standard

Number

 

Stock Caffeine

 

Volumes

pipet

t

ed (mL)

 

Calculated

Caffeine

Concentration (ppm

)

 

1

 

1.00

 

 

2

 

2.00

 

 

 

3

 

3.00

 

 

 

4

 

4

.00

 

 

 

5

 

5

.

0

0

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

C - Unknown Energy Drink Sample Preparation

5.     Obtain an unknown energy drink sample from your professor. This sample has already been degassed and filtered. 

In TRIPLICATE, using a 5.00 mL micropipet pipet and new tip, transfer 1.00 mL of the unknown energy drink sample into three separate 50.00 mL analytically rinsed volumetric flasks. Make each flask up to the mark with RO water. Stopper and invert 15 times.

NOTE: The Agilent Cary 100 UV-Vis Spectrometer (see Appendix C for operating procedure) will be used to complete Parts D and E of the procedure. 

D – Scan Package: Qualitative Absorption Spectrum 

  1. The spectrometer should already be turned on and warmed up. Obtain a pair of matched QUARTZ cuvettes from the professor or technologist.

NOTE: See the professor or technologist for tips on cuvette handling. Prior to each measurement, the cuvette must be rinsed 3X with the solution to be analyzed and wiped with a Kimwipe. Ensure that there are no air bubbles in the solution. Do not scratch the cuvettes, and do not touch the transparent faces of the cuvette with your fingers.  

  1. Set the baseline response of the instrument to zero using the blank solution. 
  2. Using the highest concentrated external standard solution (solution #5), record the qualitative absorption spectrum. Follow Appendix C - Scan Application to obtain the qualitative spectrum, and consult the professor or technologist for assistance.
  3. From the caffeine spectrum, select the wavelength for quantitative analysis corresponding to the absorbance maximum. 
  4. Print your results from the qualitative analysis.

E – Concentration Package: Quantitative Analysis 

  1. Zero the absorbance on the instrument using the diluent at the absorbance maximum wavelength determined in step 9 - procedure. 
  2. Using the absorbance maximum wavelength determined from step 9 - procedure, measure the absorbance values of each external standard calibration solution and energy drink sample. Follow Appendix C – Concentration Application to perform the quantitative analysis, and consult the professor or technologist for assistance. 

NOTE: The blank solution will be placed in the “reference” cuvette. The other cuvette will   be used for the external standards and samples. Always measure the standards in ascending order from lowest to highest concentration. 

  1. After all measurements have been recorded, rinse the sample and reference cuvettes with RO water, and store in the original cuvette box. 
  2. Download and save your instrumental output as a PDF file. Before leaving the lab, email the PDF file including the calibration plot and caffeine spectrum to yourself and your professor. Your professor will show you how to do this.

 

 

 

Laboratory Report Questions

A - Operating Parameters and Caffeine Stock, Substock and External Standard Solutions

  1. Record in a table the UV spectrometer instrumental settings (e.g. λ, bandwidth, scan speed, etc) that were used for analysis of caffeine. Settings can be found in the instrument printouts. Include your unknown sample number.

 

  1. Calculate the actual concentration (ppm) of caffeine in the stock solution prepared in steps 1-3 - procedure. Show the calculation. 

 

  1. Calculate the actual concentration (ppm) of caffeine in each external calibration standard solution prepared in step 4 - procedure. Show a sample calculation for external standard solution # 1 only. Record the actual concentration and corresponding absorbance value for each external standard solution in a table.  

 

  1. Construct a computer generated diagram illustrating the preparation of the caffeine stock and external calibration standard solutions. Be certain to include the actual mass of caffeine weighed, volumes pipetted, volumetric flask sizes, diluent used and actual (calculated) concentrations of all solutions. 

 

  1. Record the absorbance values for the replicate unknown energy drink samples in the same table as step 3- report.

 

  1. Construct a computer generated diagram illustrating the preparation of the unknown energy drink samples. Include the unknown number, volumes pipetted, volumetric flask size and diluent used. 

B - Multipoint Method

  1. Examine the output obtained from the UV spectrophotometer, and state the equation of the line and correlation coefficient in a table 
  2. Calculate the caffeine concentration in ppm in the diluted unknown energy drink sample for each replicate using the equation of the line. Show one sample calculation for replicate one only. Report your calculated results for each diluted energy drink sample replicate in a new table. 
  3. Back calculate the caffeine concentration in mg in the original energy drink bottle assuming the bottle size is 710 mL (mg/bottle) for each replicate sample. Show one sample calculation for replicate one only. Record your calculated results in the same table as step 8 - report. 
  4. Calculate the mean caffeine concentration (mg/bottle), absolute standard deviation, relative standard deviation and true value (µ) at 95% confidence interval in the original energy drink sample.  Show the calculation for each statistic, and include a statement about the meaning of your µ value. Record your final statistical results in a new summary table. 

 

NOTE: Before calculating any statistics, Q-test, at 95% confidence, any suspect datum (show your work, if performed). Perform the Q-test only if there is a true outlier in your replicate data set, otherwise omit this calculation!

 

C - Single Point Method

  1. Compare the external standard absorbance values to those for the replicate unknown samples in table format. Select and state the name of the external standard solution whose absorbance value is closest to that of the unknown replicate samples. Using the selected standard and the following equation, calculate the caffeine concentration in ppm in the diluted unknown energy drink samples.

                                                           

 

 

Show only one sample calculation. Record the calculated single point method results for each replicate sample in a new table. 

  1. Back calculate the caffeine concentration in mg in the original energy drink bottle assuming the bottle size is 710 mL (mg/bottle) for each replicate sample. Show one sample calculation for replicate one only. Record your calculated results in the same table as step 11 – report.

 

  1. Calculate the mean caffeine concentration (mg/bottle), absolute standard deviation, relative standard deviation and true value (µ) at 95% confidence interval in the original energy drink sample.  Record your final statistical results in a new summary table. 

NOTE: DO NOT SHOW STATISTICAL CALCULATIONS!

 

D – Results Summary and Unknown Identification            

  1. In a new final summary table, caffeine concentration (mg/ bottle), absolute standard deviation, relative standard deviation and true value (µ) at 95% confidence interval in the original energy drink sample for both multipoint and single point methods.  Results must be summarized in ONE table. 
  2. Using the multipoint method results only, create a table to compare your experimental caffeine concentration (mg/bottle) to ALL of the true values posted on SLATE. State the identity of your unknown energy drink sample. 

 

  1. Calculate the relative error for caffeine concentration (mg/bottle) in the energy drink sample for both multipoint and single point methods. Show one sample calculation for multipoint results only. Record your relative error values in a table. 

 

 

 

E – Qualitative UV Spectrum

 

  1. Using the qualitative UV absorption spectrum of (obtained using caffeine external standard #5), calculate the absorptivity coefficient in L/(cm*mg) at the analysis wavelength used in this experiment. Show the calculation. In a new table, list the absorptivity coefficient and the corresponding slope value for caffeine. 
  2. Using ChemDraw software or some other software, draw the caffeine molecule, and circle and label all of the non-bonding (n) and π electrons in caffeine.  

 

  1. The qualitative UV absorption spectrum shows two peaks at 206 nm and 272 nm. State what type of electron transition is responsible for each of these peaks.  

 

F – Discussion  (14 marks)   

 

  1. In step 7- of the procedure, in order to prepare the unknown soft drink solution, the sample had to be degassed. (7)
    1. Explain the purpose of degassing? 

 

    1. Suppose that the technician forgot to degas the sample. Explain how the accuracy of the final result in multipoint method would be affected? 

 

    1. Explain in detail how the precision of your final result in multipoint would be affected. 

 

  1. Discuss whether or not your multipoint final results agree with your single point final results. Support your answer with numerical results from the table prepared step-14 results. (3)

 

21. Suppose your partner prepared the external standards from your substock and that you prepared the diluted soft drink unknowns. Your preparation was perfect. Your partner was using an old version of the lab manual from Winter 2019. Instead of preparing the external standards using 1.00, 2.00, 3.00 and 4.00 mL and 5.00 mL, your partner pipetted 2.00, 4.00, 6.00, 8.00 and 10.00 mL. Your partner correctly calculated each external standard’s concentration before plotting the graph on Excel. (4)

 

  1. Explain how the appearance of this calibration plot compared to the calibration curve that you actually obtained in this lab which was made according to the F2020 procedure. 

 

  1. Explain how the slope and intercept of this calibration plot compared to the slope and intercept values that you obtained in this lab which was prepared according to the F2020 procedure. 

 

  1. Explain how your partner’s mistake would affect the accuracy and precision of your final result obtained with his/her’s calibration plot.

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