Advanced Analytical Chemistry Experiments (c) DPSM UP MANILA

7/27/2019 Advanced Analytical Chemistry Experiments (c) DPSM UP MANILA

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Experiment o. 1

POTETIOMETRIC DETERMIATIO OF FLUORIDE I TOOTHPASTE

OBJECTIVE

To employ potentiometric techniques in the determination of fluoride content of

toothpaste samples by using direct calibration and standard addition methods.

MATERIALS

a) Equipment

Potentiometer (or pH meter)Combination fluoride ion-selective electrode

Magnetic stirrer with spin bar

b) ReagentsStock Standard Fluoride Solution (500 ppm)

Weigh exactly 27.62 mg dry NaF (AR, dried for 4 hours at 110 C) and dissolve indistilled water. Transfer the solution in 25-mL volumetric flask and dilute to volumewith distilled water. Store in a plastic container.

Total Ionic Strength Adjustment Buffer (TISAB)Place approximately 250 mL of distilled water in a 500-mL beaker and add 28.5 mL of

glacial acetic acid, 29 g NaCl, and 2.0 g 1,2-EDTA. Stir to dissolve. Place beaker in a

cool water bath and slowly add 6 M NaOH (about 62 mL) with stirring until pH isbetween 5.3 and 5.5. Transfer to a 500-mL volumetric flask and add distilled water to the

mark.

c) Sample toothpaste: Ask students to bring different brands of toothpaste.

PROCEDURE

A. Direct Calibration MethodSample Preparation

1. Weigh accurately 1.0 g toothpaste sample in a 50-mL beaker and dissolve in a small

amount of water. Quantitatively transfer the solution into a 50-mL volumetric flask anddilute it to volume with distilled water.

2. Take 5.00 mL of the toothpaste solution (for Colgate, Beam or Hapee) or 1.00 mL of the

toothpaste solution (for Exceed or Unique) and transfer to a 50-mL volumetric flask. Add

25.0 mL of the TISAB solution. Dilute to the mark with distilled water and mix well.

Measurement of Standard Solutions1. Accurately measure out 25 mL of TISAB and 25 mL of distilled water. Transfer into a

100-mL beaker. Immerse the combination fluoride-selective electrode into the solution

and measure the developed potential while stirring on a magnetic stirrer. Avoid stirring

before immersing electrodes because any bubble entrapped on the surface of the electrodes


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can cause erroneous readings. Let electrodes remain in solution for 2 minutes (or until

equilibrium is established) before taking a final millivolt reading. A layer of insulating

material (e.g. tissue paper) between stirrer and beaker minimizes solution heating.

2. Turn off the magnetic stirrer and using a micropipette, add 50 L of 500 ppm standardfluoride solution to the beaker. Stir the solution and take the potential reading once

equilibrium is established. Repeat the same procedure until a total of 250 L of fluoridestandard solution have been added.3. The readings obtained from these measurements will be used in the construction of the

calibration curve.

Determination of Analyte

1. Measure the potential of the previously prepared toothpaste solution (with TISAB).

2. Prepare a standard calibration curve by plotting the potentials obtained from the solutions

against the logarithm of the fluoride concentrations.3. Determine the level of fluoride in the toothpaste sample using the results from your

regression.

B. Standard Addition Method1. Take 5.00 mL of the toothpaste solution (for Colgate, Beam or Hapee) or 1.00 mL of the

toothpaste solution (for Exceed or Unique) and transfer to a 50-mL volumetric flask.2. Add 25 mL of TISAB solution. Mix thoroughly and dilute to the volume with distilled

water. Measure potential as follows:

Transfer the solution to a 100-mL beaker. Place the beaker on a magnetic stirrer plate.

Immerse electrode into the solution and while stirring at a constant rate, read the initialpotential.

3. After taking the initial potential reading, without disturbing the set-up, add 50 L of 500ppm fluoride standard solution to the sample solution in the beaker. Read the potential

once the equilibrium is established.4. Repeat the addition of 50 L of the standard solution as done above. After each addition,

take the stable potential reading. Ensure that the potential readings obtained fall within thepotential range of the standard solutions used in the direct calibration method.


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Experiment o. 2

POTETIOMETRIC DETERMIATIO OF HYDROGE IO

I FEMIIE WASH

OBJECTIVE

To employ potentiometric techniques in the determination of hydrogen ion concentration

of feminine wash samples by using direct calibration and standard addition methods.

MATERIALS

a) EquipmentPotentiometer (or pH meter)

Magnetic stirrer with spin bar

b) ReagentsStandard Hydrogen Ion SolutionPrepare the following standard hydrogen ion solutions: 1.00 x 10

-1M, 1.00 x 10

-2M, 1.00

x 10-3

M, 1.00 x 10-4

M, 1.00 x 10-5

M, 1.00 x 10-6

M, and 1.00 x 10-7

M using HCl and 0.5 M

KCl solution.

c) Samples: Ask students to bring different brands of feminine and/or masculine wash.

PROCEDURE

A. Standard Calibration MethodSample Preparation

Measure accurately 5.00 mL of feminine wash (e.g. pH Care, Lactacyd) sample in a 25-mLvolumetric flask and add 0.5 M KCl to the mark.

Measurement of Standard Solutions

1. Immerse the hydrogen ion electrode into the 10-7

M standard solution and measure thedeveloped potential while stirring on a magnetic stirrer. Avoid stirring before immersing

electrodes because any bubble entrapped on the surface of the electrodes can cause erroneous

readings. Let electrodes remain in solution for 2 minutes (or until equilibrium is established)before taking a final millivolt reading. A layer of insulating material (e.g. tissue paper)

between stirrer and beaker minimizes solution heating.

2. Repeat the same procedure using the other standard solutions. The measurement ofstandard should be in order of increasing concentration.

3. The readings obtained from these measurements will be used in the construction of the

calibration curve.

Determination of Analyte

1. Measure the potential of the previously prepared feminine wash solution. Prepare a

standard calibration curve by plotting the potentials obtained from the solutions against the


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logarithm of the hydrogen ion concentrations.

2. Determine the level of hydrogen ion in the sample using the results from your regression.

B. Standard Addition Method1. Take 10.00 mL of the sample solution and transfer to a 25-mL beaker.

2. Place the beaker on a magnetic stirrer plate. Immerse electrode into the solution and whilestirring at a constant rate, read the initial potential.

3. After taking the initial potential reading, without disturbing the set-up, add 0.10 mL of 10-1

M hydrogen ion standard solution to the sample solution in the beaker. Read the potentialonce the equilibrium is established.

4. Repeat the addition of 0.10 mL of the standard solution as done above. After each addition,

take the stable potential reading. Ensure that the potential readings obtained fall within the

potential range of the standard solutions used in the direct calibration method.


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Experiment o. 3

QUALITATIVE UV-VISIBLE SPECTROPHOTOMETRY

OBJECTIVES

To be able to interpret spectra of a series of compounds in terms of the type of structuralgroups in the molecules

To demonstrate the utility of UV-Visible absorption spectrophotometry as a means ofidentifying organic and inorganic compounds

PROCEDURE

A. Preparation of Solutions1. Organic compounds

Prepare 50 mL stock solution of the following analyte at concentration of approximately10-3

to 10-5

M using either ethanol or hexane as solvent: acetone (0.01 M), styrene,

benzaldehyde, anthracene, trichloroacetic acid, benzoic acid, and other organic compoundsavailable in the stockroom.

2. Inorganic compounds

Prepare 50 mL stock solution of the following inorganic compounds at concentration of

approximately 0.01M using distilled water: CoCl2, CuSO4, FeCl3, Ni(NO3)2, KMnO4, and

K2CrO4, and other inorganic compounds available in the stockroom.

B. Recording of Spectra

Take the spectrum of each solution between wavelengths 1000 and 190 nm. The reference

substance is the solvent used for each solution. Measure also the spectrum of the solvent

versus air as reference.


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Experiment o. 4

SPECTROPHOTOMETRIC AALYSIS OF CAFFEIE AD BEZOIC ACID I

SOFT DRIKS

ITRODUCTIO:

In this experiment ultraviolet absorbance is used to measure two major components found

in softdrinks, caffeine and benzoic acid. Caffeine is added as a stimulant and sodium benzoate isa preservative. The samples that will be analyzed are restricted to non-diet soft drinks only

because the sugar substitute aspartame in diet soda has some ultraviolet absorbance that slightly

interferes in the present experiment. Darkly colored drinks are not also recommended as samples

because the colorants have ultraviolet absorbance. Mountain Dew, Sprite, and, probably, otherlightly colored drinks are suitable for this experiment. There is undoubtedly some ultraviolet

absorbance from colorants in these beverages that contributes systematic error to this

experiment.

Reagents

Stock solutions:

100 mg benzoic acid/L in water

200 mg caffeine/L should be available.0.10 M HCl

Procedure

1. Calibration standards: Prepare benzoic acid solutions containing 2, 4, 6, 8 and 10 mg/L in

0.010 M HCl. To prepare a 2 mg/L solution, mix 2.00 mL of benzoic acid standard plus 10.0 mLof 0.10 M HCl in a 100-mL volumetric flask and dilute to the mark with water. Use 4, 6, 8 and

10 mL of benzoic acid to prepare the other standards. In a similar manner, prepare caffeine

standards containing 4, 8, 12, 16 and 20 mg/L in 0.010 M HCl.

2. Soft drink: Warm ~20 mL of soft drink in a beaker on a hot plate to expel CO2 and filter the

warm liquid through filter paper to remove any particles. After cooling to room temperature,

pipet 4.00 mL into a 100-mL volumetric flask. Add 10.0 mL of 0.10 M HCl and dilute to themark. Prepare a second sample containing 2.00 mL of soft drink instead of 4.00 mL.

3. Verifying Beer's law: Record an ultraviolet baseline from 350 to 210 nm with water in thesample and reference cuvets (1.000 cm pathlength). Record the ultraviolet spectrum of each of

the 10 standards with water in the reference cuvet. Note the wavelength of peak absorbance for

benzoic acid (') and the wavelength for the peak absorbance of caffeine ("). Measure theabsorbance of each standard at both wavelengths and subtract the baseline absorbance (if yourinstrument does not do this automatically). Prepare a calibration graph of absorbance versus

concentration (M) for each compound at each of the two wavelengths. Each graph should go

through 0. The least-squares slope of the graph is the molar absorptivity at that wavelength.


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4. Unknowns: Measure the ultraviolet absorption spectrum of the 2:100 and 4:100 dilutions of

the soft drink. With the absorbance at the wavelengths ' and ", find the concentrations ofbenzoic acid and caffeine in the original soft drink. Report results from both dilute solutions.


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Experiment No. 5

DETERMINATION OF pKa OF AN ACID-BASE INDICATOR BY UV-VIS

SPECTROPHOTOMETRY

OBJECTIVE

To demonstrate the utility of UV-Vis absorption spectrophotometry as a means of determining

the pKa of several acid-base indicators

MATERIALSa) Equipment

pH meter

UV-Vis SpectrophotometerMagnetic stirrer with spin bar

25-mL beakers

micropipette

b) Reagents

1% solution of phenolphthalein in isopropanol

0.1 M KCl solution0.05 M HCl

1.0 M NaOH

Aqueous solutions: 0.04% bromocresol green, 0.04% bromocresol purple, 0.04%bromophenol blue, 0.04% bromothymol blue, 0.10% methyl orange, 0.10% sodium salt of

methyl red, and 0.04% phenol red

PROCEDURE

1. Titration of Analyte SolutionPrepare the analyte solution by dissolving several drops of the 0.04% dye solution and 2-3

drops of 1.0 M NaOH in 15 mL of 0.10 M KCl solution. (Note: For better results, highest

absorbance value of indicator should be between 0.7 and 1.0. If precipitation occurs, filter the

solution prior to analysis.) Record the UV-Vis spectrum and pH of the resulting solution. Titrate

the analyte solution by adding 2-5 L of 0.05 M HCl. Record the UV-Vis spectrum and pH aftereach addition of HCl until the spectrum no longer changes.

2. Determining the pKa of the Acid-Base Indicator

Determine the pKa of an indicator using graphical method by plotting log[(A -AIn-)/(AHIn -A)]

versus pH:


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Experiment o. 6

DETERMIATIO OF TRACE LEVELS OF COPPER AD LEAD I VEGETABLE

SAMPLES USIG THE ATOMIC ABSORPTIO SPECTROPHOTOMETER

OBJECTIVE

To illustrate how trace amounts of several nutritionally important elements in vegetable

samples can be determined through atomic absorption spectrophotometry

INTRODUCTION AND THEORY

Atomic Absorption Spectroscopy (AAS) is used for the qualitative and quantitativeidentification and determination of trace levels of metals in different samples. In AAS,

measurement is made of the radiation absorbed by the nonexcited atoms in the vapor state. It is

similar to molecular absorption spectroscopy, the major difference being that unbound atoms

rather than molecules are the absorbing species. In terms of instrumentation, the monochromatorin an atomic absorption instrument is placed after the sample. This arrangement is necessary to

remove unwanted radiation created during the atomization process.

In most common instruments, the sample solution is introduced into the flame in an

aerosol form. Before the salt vaporizes and dissociates into free gaseous atoms, the solvent must

first evaporate. At certain temperatures of an air-acetylene flame, atoms of many elements existmostly in the ground state. When a beam of radiant energy that consists of the emission spectrum

for the element that is to be determined is passed through the flame, some of the ground state

atoms absorb energy of characteristic wavelengths and are elevated to a higher energy state. Theamount of energy as a function of concentration of an element in the flame is the basis of atomic

absorption spectroscopy.

REAGENTS AND MATERIALS

CuSO4 or Cu(NO3)2

Pb(NO3)2HNO3 (conc.)

Leaves of vegetable samples (kangkong, camote tops, chili, etc.)*

Volumetric flasks (25, 50, 100, 250 mL)250-mL Erlenmeyer flasks

Glass funnel

BeakersPipettes

Rubber aspirators

* Leaves should be air dried 2 weeks prior to this experiment


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PROCEDURE

A. Preparation of Stock SolutionsWeigh accurately to the nearest 0.001 g, 0.5 g of copper metal and 0.1 g of ferric chloride

hexahydrate, dissolve each in 20 mL of 1:1 nitric acid and dilute up to the mark in separate250-mL volumetric flasks. Then, get a 5 mL aliquot from both of the prepared 250 mL

solution and dilute separately to a 100-mL volumetric flask to obtain 100 g/mL (ppm) eachof Cu and Fe stock solutions.

B. Preparation of Standard SolutionsSecure five (5) clean, 50-mL volumetric flasks and label with numbers 1 to 5. Place 0.50,1.25, 2.50, 5.00 mL of the stock solution to flasks 1, 2, 3, and 4 respectively to prepare 1.00,

2.50, 5.00, 10.00 ppm of standard solutions. Dilute the solution up to the mark with distilled

water. Flask 5 will serve as the blank.

C. Preparation of SampleAt least one (1) kg of the vegetable sample is needed in this analysis. (OTE: Ask yourinstructor for the kind of vegetable sample to be analyzed). Collect only the leaves from the

vegetable sample. Rinse the leaves and dry in the oven, maintain the temperature at 100 to

150C for 30 to 45 minutes. Take the dried leaves out from the oven and weigh accuratelytwo (2) grams of the dried sample. Place it in a 250-mL Erlenmeyer flask and 17.5 mLconcentrated nitric acid. Prepare three samples. Boil slowly at low setting for 20 minutes and

cool the solution. Add 10 mL distilled water and filter to a 50-mL volumetric flask. Dilute

the filtrate to the mark with distilled water passing through the filter paper.

D. Analysis of the Vegetable SampleRecord the absorbance of the standard solutions and the sample using the required

instrumental parameters (for Cu and Fe) of the atomic absorption spectrophotometer. If the

absorbance in not within the range, get a 5-mL aliquot of the sample then dilute to the markin a 25-mL volumetric flask and take again its absorbance. Perform three trials.

E. Standard Addition Method1. Secure five (5) clean 50-mL volumetric flasks and label with numbers 1 to 5. To each flask,

add 10 mL of the digested sample.

2. Place 0.00, 0.50, 1.25, 2.50, 5.00 mL of the stock copper solution to flasks 1, 2, 3, 4, and 5respectively to prepare 0.00, 1.00, 2.50, 5.00, 10.00 ppm of added standards. Dilute the

solutions up to the mark with distilled water.

3. Record the absorbance of the solution using the required instrumental parameters for Cu.4. Repeat procedures 1 to 3 but this time use iron as the standard to be added and set the required

instrumental parameters for Fe.


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Experiment o. 7 (Dry Lab)

IFRARED SPECTROMETRY: SAMPLIG METHODS AD QUALITATIVE

AALYSIS

OBJECTIVES

To obtain and study the infrared spectra of a selection of compounds with a range of

common functional groups


Qualitative analysis using the infrared spectroscopy is possible because of the uniquecomplexity of the infrared spectra of different compounds. The vibrational modes of motion in a

molecule give rise to the bands in the spectrum, and no two compounds give exactly the same

spectrum. There are, however, similarities between the spectra of similar compounds with

similar functional groups.

The frequencies associated with certain functional groups and certain substitutionpatterns have been studied extensively. As a result, correlation charts have been developed.

These certain correlation charts give frequency ranges over which we can expect to find

vibrational bands for the molecular subgroups of interest; though the frequency ranges are not

all-inclusive. The vibrational frequencies of a molecule depend on the number, weight andgeometrical arrangement of the atoms and the force constant of each interatomic bond. A change

in any one of these factors will alter the infrared spectrum of the molecule.

The acquisition of a high quality spectrum is possible by the proper choice of sample

handling technique. It is also important to remember that the spectrum should have no peaks

which are bottomed up, that is, regions where transmittance is near zero.

SAMPLIG TECHIQUES

Techniques for mounting the sample in the beam of the infrared spectrometer depend onwhether the sample is a gas, liquid, or solid. Intermolecular forces vary considerably in passing

from solid to liquid to gas, and the infrared spectrum will normally display the effect of these

differences in the form of frequency shifts or additional bands, etc. It is, therefore, mostimportant to record on a spectrum the sampling technique used.

LIQUIDS and SOLUTIONS

The simplest technique of all consists of sampling a liquid as a thin film squeezed

between two infrared-transparent windows. The thickness of the film can be adjusted by varying

the pressure used to squeeze the flats together; the film thickness is 0.1-0.3 mm. Care must betaken to keep the windows from moisture.

Liquid samples can also be examined in solution. The sample can be dissolved ina

solvent and the spectrum of this solution recorded. The solution is placed in a solution or liquid


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cell (also known as cavity cell) consisting of transparent windows (e.g. NaCl or KBr) with a

spacer between them of known thickness; its thickness determines the path length of the cell

usually 0.1-1.0 mm. A second cell containing pure solvent is placed in the reference beam so thatthe solvent absorptions are cancelled out and the spectrum recorded is that of the solute alone.

Pure liquid samples or mixtures can also be injected in the liquid cell neat and highquality spectrum obtained by choosing a suitable path length (i.e. right spacer).

SOLIDS

There are three common techniques for recording solid spectra: KBr discs, mulls, and

deposited films. Solids can be examined in solution but the solution spectra may have different

appearances from solid spectra since intermolecular forces will be altered.

KBr discs are prepared by grinding the sample with dry KBr and compressing the whole

into a transparent wafer or disc.

Mulls or pastes are prepared by grinding the sample with a drop of oil, the mull is then

squeezed between transparent windows as for liquid samples. Liquid paraffin (Nujol) mull is themost widely utilized.

Solid films can be deposited onto NaCl or KBr windows by allowing a solution in a

volatile solvent to evaporate drop by drop on the surface of the window. Polymers and variouswaxy or fatty materials often give excellent spectra in this way.

PROCEDURE

1. Record the spectra of the samples provided using the different sampling techniques. Identifyand label the prominent bands in each spectrum.

2. Give the information about the chemical structure of a compound that can be deduced from

the IR spectra.

3. Compare the different sampling techniques. Comment on the kind of sample that can be mostappropriately prepared for each technique.


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DETERMIATIO OF CAFFEIE I BEVERAGES USIG HIGH PERFORMACE

LIQUID CHROMATOGRAPHY

OBJECTIVES

To determine the concentration of caffeine in coffee, tea and cola beverage drinks using

reversed phase HPLC


Food and pharmaceutical products are subjected to strict quality control (QC) proceduresto ensure consistency of the formulation within specified limits. Caffeine is a common

component of coffee and cola beverages.

High performance liquid chromatography (HPLC) is used for the separation and

quantitative analysis of a wide variety of mixtures, especially, those where the components areinsufficiently volatile and/or thermally stable to be separated by gas chromatography (GC). It is

used extensively in the analysis of pharmaceutical products, foodstuffs and beverages,agrochemicals, polymers and plastics and for monitoring drugs and their metabolites in the body

fluids. The components of a mixture are carried through a column by a mobile liquid phase

pumped under high pressure. The order of elution is determined by the chemical nature of

components, the mobile phase and the stationary phase. Stationary phases are silica or

chemically modified silica (bonded phases) of a very small particle size (3 m to 10 m). Theeluted components are detected by monitoring the UV absorbance or fluorescence, the current

generated by redox reaction (amperometry) or the refractive index. The eluted components arecharacterized by their retention times, tR, or their capacity factors, k

and quantitative analysis is

accomplished by comparing the areas of analyte peaks or heights with those of standards.


HPLC with UV-vis detector Caffeine standard (AR)Reversed phase column (C18) MethanolVolumetric flasks (100 mL and 25 mL) Phosphoric acid

Syringe (25 L) Distilled waterPipettes (1 mL and 10 mL)

Rubber aspiratorBeakers (100 mL)Ultrasonicator

Analytical balance

Filter membrane (0.45 m)Sample beverages (to be assigned by instructor)


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PROCEDURE

A. Preparation of Caffeine Standard4. Into five clean and dry 100-mL volumetric flasks, weigh accurately the following

quantities of caffeine: 2.5, 5.0, 7.5 and 10.0 mg.

5. Dilute to the mark with previously prepared methanol:water (2:8), adjusted toapproximately pH 3.50 with phosphoric acid. This is the same solvent to be used as the

mobile phase.

6. Shake the five caffeine solutions adequately to ensure dissolution and then degas each for

five minutes then filter using 0.45 m filter membrane before injection into the column.7. Turn the pump and detector on. Set the pump flow rate at 2.3 mL/min and the detector

sensitivity at 0.08 AUFS (absorbance unit full scale) Turn the recorder on and set at slowspeed rate. Prior to injection of the standards into the column, allow the mobile phase topass through the column for 5 to 10 minutes. Simultaneously record the detector response

to ensure that there are no substances left on the column from previous experiments.

8. With provided syringe, inject 25 L or more of caffeine standards starting with the least

concentrated. Take the duplicate chromatograms for each of the caffeine standard solution.

B. Determination of Caffeine in Tea and Coffee1. Into a clean, dry 25-mL volumetric flask, pipette about 0.5 mL coffee and into another

clean, dry volumetric flask, pipette 5 mL tea.

2. Dilute each flask to the mark with methanol:water (2:8) solvent.

3. Follow steps 3 to 5 in procedure A.

C. Determination of Caffeine in Cola Beverage1. Pour 10 to 15 mL of the cola beverage into a clean and dry beaker. Pour this into another

clean and dry beaker back and forth to the original beaker until the bubbling ceases.

Alternatively, the beaker can be placed in an ultrasonicator for about 5 minutes untilbubbling ceases. The soda is now adequately decarbonated.

2. Into a clean and dry 25-mL volumetric flask, pipette 10 mL of the cola beverage and diluteto the mark with methanol:water (2:8) solvent.

3. Follow steps 3 to 5 in procedure A.

4. After the last chromatogram, flush the column with 50 mL of solvent (not adjusted to pH3.50).


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CYCLIC VOLTAMMETRY

OBJECTIVES

To determine the E values of the [FeIII

(CN)6]-3

/[FeII(CN)6]

-4couple

To evaluate the effects of scan rate, concentration of electroactive species, and supportingelectrolyte

PROCEDURE

Record and analyze the electroanalytical data using the Power Lab 4SP-driven

Potentiostat. The electrochemical cell is made up of three electrodes namely: platinum working

electrode, platinum auxiliary electrode, and Ag/AgCl reference electrode.

A. Generating a Cyclic VoltammogramAssemble the cell and fill it with 1M KNO3. The volume of the electrolyte solution

should be enough for the tip of the electrode to immerse. Purge the solution with N2 for 5

min and blanket the solution with N2 during the experiment. Set the initial potential at 600

mV and scan limits at 600 mV and -600 mV. Initiate the scan in the negative direction with a

scan rate of 100 mV/s. After deoxygenation is completed, switch on the working electrode.Allow the current to flow (10 s) to attain a constant value, then initiate the potential scan.

After taking the background cyclic voltammogram of the supporting electrolyte, turn off the

working electrode and clean the cell. Refill the cell with 4 mM K3[Fe(CN)6] in 1 M KNO3.Repeat the above procedure (E range: 600 to -800 mV) to obtain the voltammogram of

[FeIII

(CN)6]-3

/[FeII(CN)6]

-4couple.

B. Effect of Scan Rate VariationObserve the effect of scan rate on the voltammogram using 4 mM K3[Fe(CN)6] in 1 M

KNO3. Record the CVs at the rates of 50, 80, 100, and 200 mV.

C. Effect of Electroactive Species Concentration VariationObtain the cylic voltammograms on 2, 6, 8, and 10 mM K3[Fe(CN)6] using a scan rate of

100 mV/s. Record also the voltammogram of the unknown K3[Fe(CN)6] solution.

D. Effect of Supporting Electrolyte VariationInvestigate the effect of supporting electrolyte. Record the voltammograms of 4 mM

K3[Fe(CN)6] in 1 M Na2SO4.


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Experiment o. 10(Dry Lab)

GAS CHROMATOGRAPHY

OBJECTIVES

To separate and determine the composition of a mixture of hydrocarbons and to identify

unknown members of hydrocarbons by gas chromatography

To determine some chromatographic parameters in the separation of hydrocarbon

samples


Gas Chromatography (GC) is used for the separation and quantitative analysis of

mixtures where the components are sufficiently volatile and thermally stable to pass through a

chromatographic column in the vapor state. This normally requires elevated temperature of100C to 400C. It is used in the analysis of petrochemicals and many products absed on them,solvents, volatile natural products, pesticide and herbicide residues, and paints and polymers

after pyrolysis. The component of a mixture are carried through the column by an inert carriergas, usually nitrogen, and are generally eluting in the order of increasing boiling points, although

differing affinities for the stationary phase may affect the order of elution. The elutedcompounds are detected by monitoring a physical property of the gas stream leaving the column,such as the degree of induced ionization, thermal conductivity or emission of characteristic

electromagnetic radiation. Eluted compounds are characterized by their retention times, tR, and

quantitative analysis is accomplished by comparing the areas or heights of analyte peaks withthose of standards. Most modern chromatographic apparatus are equipped with electronic

integrators that could provide measurements of relative peak areas. While analysis based on thepeak height can be performed using the triangulation method. The baselines of the two sides of a

chromatographic peak are connected by a straight line and the perpendicular distance from thisline to the peak is measured. The efficiency of the column can be measured by knowing the

number of theoretical plates and the resolution. The number of theoretical plates, N, can bedetermined using the equation:

=16tR

w

2

where w is the width of the peak at its base and t R is the retention time. While the resolution Rs isthe measure of the ability of the column to separate two analytes A and B.

Rs =2 tR( )B tR( )A[ ]

wA + wB


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The rate of migration of the solutes in the column can be described by the capacity factor

k. For a solute A,

k'A =tR tM

tM

where tM is the dead time which is the measure of the average rate of migration of the mobile

phase.


XyleneToluene

EthylbenzeneDiethyletherMicroliter syringe

Volumetric flasks (10 mL)Pipets (0.2 mL)

PROCEDURE

Run chromatograms of pure xylene, toluene and ethylbenzene dissolved in diethylether.

Take note of the retention time.

A. Preparation of Standard Solutions of Toluene, Ethylbenzene and XylenePrepare 10-mL standard solutions of toluene, ethylbenzene, and xylene in diethylether as

summarized in the table below. Run chromatograms of the standard solutions. Take note ofthe retention time. Note the important settings of the instrument (e.g. injection port

temperature, oven temperature, etc.)

Determine the area under each peak by triangulation, cut and weigh, and peak integrationmethods. Set-up the calibration curves.

Composition (%)

Std # Toluene Ethylbenzene Xylene Benzene*

1 0.10 0.20 0.30 0.30

2 0.20 0.30 0.10 0.303 0.30 0.10 0.20 0.30

* will serve as marker of unretained solute


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B. Determination of Amount of Components in Unknown MixturesObtain an unknown mixture of the above hydrocarbons. Run chromatograms using the

same settings as that used for the standard solutions. Identify each component.

Determine the amount of each component in the unknown mixture from the three

calibration curves. Using the chromatogram of your unknown mixture, determine thecapacity factor, resolution, and average number of theoretical plates of the column used.Remember to note the length of the column.


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STRUCTURE ELUCIDATIO BY MR SPECTROPHOTOMETRY

OBJECTIVE

1) To elucidate the structure of a compound using Nuclear Magnetic Resonance (NMR)Spectroscopy2) To process, analyze and interpret NMR data using a processing software

MATERIALS

ACD/NMR Processor Academic Edition (freeware downloaded from www.acdlabs.com)

NMR FID data files: (a) NMR fid1.1, (b) NMR fid2.1

PROCEDURE

1. Download and install NMR processing software from www. acdlabs.com upon registration(free).

2. Run 1D NMR Processor.

3. Load/Open a sample FID file in folder:ACDFREE/EXAMPLES/SPECMAN/1DNMR/CATECHIN.FID/FID

4. Study how to process, analyze and report NMR data by reading through the Quick Start Guide

(1D NMR Processor: Basic Training) and NMR Processor Blog available at the acdlabs website.5. Perform the following operations starting with the assigned FID files (jdf format):

A) Process FID with Interactive Fourier Transform (FT)

B) Pick peak signalsC) Integrate proton signals

D) Measure J coupling

E) Attach structure from ChemSketch (included) or from any compatible chem drawing

softwareF) Assign proton signals to structure

G) Prepare report

Note:

File Molecular

Formula

NMR fid1.1 C9H12O4S

NMR fid2.1 C16

H18

O6S

2

Documents

Advanced Analytical Chemistry Experiments (c) DPSM UP MANILA