CM2142 ANALYTICAL CHEMISTRY

Table of Contents Page

2.1 2.2 2.3 A 2.3 B

Gas Chromatography High Performance Liquid Chromatography Ion-Selective Electrodes I Ion-Selective Electrodes II

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Experiment 2.1

Gas Chromatography
Introduction Like all chromatographic methods, gas chromatography depends on the differential migration of the components of a mixture because of their distribution between two phases. In this case, the partition occurs between the gas phase (the carrier gas) and the liquid phase (on the column packing). In a mixture of substances with similar dipole moments but differing volatilities, the more volatile components will spend more time in the mobile gas phase, and hence move faster than the less volatile components which will lag behind in the stationary liquid phase. By analogy with distillation columns, chromatographic columns are often considered to be made up from a number of theoretical plates. The number of plates, N, is a measure of the efficiency of the column and is given by:
t N = 16 R W where W is the peak width at the base and t R is the retention time of the solute. Often it
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is easier to measure the width at half the peak height (width at half maximum), W1/2 :
t N = 5.54 R W 1/2
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For comparative purposes, a more convenient parameter is the Height Equivalent to a Theoretical Plate, or HETP (H) with H = L/N where L is the length of the column. The smaller the value of the HETP, the better the column. H is basically a measure of column efficiency which is concerned with the broadening of an initially compact band of solutes as it passes through the column. H is also a function of the carrier gas velocity and is given by the van Deemter equation: H = A + B/v + Cv where A, B and C are approximately constant for a given system, and v is the velocity of the carrier gas in cm/s.

The relative retention volume of a compound is invariant under a fixed set of experimental conditions. Retention volume is thus a useful parameter for the identification of an unknown band. However, it is possible for two different compounds to have identical relative retention volumes using a particular column. Thus for unequivocal identification at least two columns (one polar and the other nonpolar) should be used for the qualitative analysis of a mixture. For the quantitative analysis of components in a mixture, the necessary conditions depend on whether relative quantities or absolute quantities are to be determined. In the former case, measurement of the peak areas of a single chromatogram suffices whereas in the latter case, a calibration curve for each component must be obtained under the same conditions used for running the unknown sample. Peak area can be measured by triangulation, disc or electronic integrator, planimeter or weighing the cut out areas from the chromatogram.

Experimental

Determination of HETP
1. Ask the demonstrator or lecturer-in-charge to show you how to operate the chromatograph. Check the type of column being used in the instrument. The column, detector and injector temperatures have been preset and should not be altered. 2. Adjust the flow rate of the carrier gas according to the table given on the instrument. Allow two to three minutes for carrier gas flow to stabilize before injecting 1 L of the ethylbenzene by means of the micro-syringe. Wait for the elution peak to appear on the chart. 3. Repeat step 2 for different flow rates of carrier gas.

4. Calculate the HETP at each setting and plot a graph of HETP versus carrier gas flow rate for ethylbenzene. Choose any three points on the curve and calculate the A, B and C terms of the van Deemter equation. Comment on the results. Qualitative and Quantitative Analysis 1. Adjust the carrier gas flow rate according to the instructions.

2. Clean the microsyringe with acetone before each sample. Rinse with the sample at least three times before injecting. Inject 1 L of sample A provided which is made up of equal volumes of toluene and ethylbenzene. From the known volume and density of each component used, calculate the weight percentage and mole percentage of the components. Compare these results with the relative areas determined experimentally. 3. Repeat step (2) with sample B which is made up of 3 mL cyclohexane, 4 mL of npropanol and 3 mL o-xylene. 4. Repeat step (2) with sample C which contains equal volumes of ethanol, npropanol, n-butanol and n-pentanol. Plot the logarithm of retention volume against the number of carbon atoms in the molecule. Predict the retention volume for n-hexanol under the same experimental conditions. 5. Inject l L of sample D which contains three of the constituents present in any of the mixtures A, B and C. Determine the qualitative and quantitative composition (vol %) of this sample.

Effective Carbon Number The detector used in this experiment is a flame ionization detector (FID). The signal obtained depends directly on the number of ionizable compounds introduced into the detector. It is a useful general detector for the analysis of most organic compounds. The number of ions produced for organic compounds is roughly proportional to the number of reduced carbon atoms in the flame. Functional groups, such as carbonyl, alcohol, halogen and amine, yield fewer ions or none at all in a flame. In addition, the detector is insensitive toward noncombustible gases such as H2O, CO2, SO2 and NOx. The effective carbon number (ECN) for a compound is the sum of the contributions made by the individual carbon atoms in the molecule modified by the functional group contributions (Table 1). For example, butanol has four carbon atoms contributing a total of 4.0 to the ECN, but also one hydroxyl group that contributes -0.6, leaving 3.40 as the ECN for butanol. The ECN can be used to calculate relative response factors in cases where pure materials are not available for detector calibration.

(a) Calculate the expected ECN for toluene, ethylbenzene, cyclohexane, n-propanol and o-xylene, ethanol, n-butanol, and n-pentanol from the table. Find the relative ratio of the ECN by dividing with the smallest ECN. (b) Work out the experimentally determined ECN by normalizing the peak area to per mole of the compound. Determine the relative ratio as in (a). Compare both sets of relative ECN to see if they agree. Table 1: Individual contributions to the effective carbon number Atom C C C C C C O O O O Cl Cl N Type Aliphatic Aromatic Olefinic Acetylenic Carbonyl Nitrile Ether primary alcohol secondary alcohol tertiary alcohol, esters two or more on single aliphatic C on olefinic C in amines ECN 1.0 1.0 0.95 1.30 0.0 0.3 -1.0 -0.6 -0.75 -0.25 -0.12 each - 0.12 each Similar to O in corresponding alcohols

References

1.

G. D. Christian and J. E. O'Reil1y, Instrumental Analysis, 2nd ed., Allyn & Bacon, 1986 D. A. Skoog and J. J. Leary, Principles of Instrumental Analysis, 4th ed., Saunders. College Publishing, 1992.

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Experiment 2.2

High Performance Liquid Chromatography

Introduction Gas chromatography, because of its speed and sensitivity has been used much more widely since its development than the liquid column chromatographic techniques. The latter techniques, however, have potentially broader use because approximately 85% of all known compounds are not sufficiently volatile or stable to be separated by gas chromatography. The traditional approach in liquid chromatography has been to use relatively large diameter columns and small flow rates under gravity feed or low pressure pumping. Separation times are frequently on the order of hours and the collected fractions must usually be analyzed separately, adding more hours to the analysis. The wealth of chromatographic theory accumulated in recent years, primarily from gas chromatography, has led to the development of techniques and equipment for high performance liquid chromatography (HPLC). High performance liquid chromatography - as compared with the classical liquid chromatographic technique - is characterized by (1) small diameter (2-5 mm), reusable columns; (2) column packings with very small (5-50 m) particles and the development of new substances to be used as stationary phases; (3) relatively high inlet pressures and controlled flow of the mobile phase; (4) precise sample introduction without the need for larger samples (5) special continuous detectors capable of handling small flow rates and detecting very small amounts (6) automated standardized instruments (7) rapid analysis with high resolution. The basic chromatographic theory is applicable to HPLC. However, compared to gas chromatography, HPLC has one important difference. In chromatography, separation is based on specific interactions between the sample molecules and the stationary and mobile phase. In gas chromatography the mobile phase does not participate in these interactions. Being, essentially an inert gas, the only role of the mobile phase is to carry the sample molecules through the system. Thus, in gas chromatography, separation is based on the interactions between the sample and the stationary phase. The situation is, however, different in liquid chromatography (and HPLC) - here the mobile phase is not inert anymore as far as the interactions are concerned but has an active role in them.

Thus, in HPLC, we have an additional variable which can be properly selected to improve the separation power of the system.

HPLC apparatus consists of five principal parts (Fig. 1) : (1) the mobile phase supply system (2) the sample injection system (3) the column (4) a suitable detecting device (5) a recorder. The mobile phase supply system It contains a pump to provide the high pressure required and the solvent reservoirs which can be filled with a range of solvents of different polarities. The solvents must be pure and degassed. The selection of the solvents depends on a number of parameters. In adsorption and partition chromatography, the polarity plays the most important role, but the viscosity and characteristics which may influence the function of the detector are also important.

Fig.1 Schematic of an apparatus for HPLC

Sample injection system Often the limiting factor in the precision of liquid chromatographic measurement lies in the reproducibility with which samples can be introduced onto the column packing. The problem is exacerbated by band broadening, which accompanies overloading columns. Thus, the volumes used must be minuscule - a few tenths of a microliter to perhaps 500 L. Furthermore, it is convenient to be able to introduce the sample without depressurizing the system. The most widely used method of sample introduction in liquid chromatography is based upon sampling loops, such as shown in Figs. 2 and 3. Sampling loops provide a choice of sample sizes from 5 to 500 L. Such loops permit the introduction of samples at pressures up to 7000 psi with precisions of a few tenths percent relative. Micro sample injection valves, with sampling loops having volumes of 0.5 to 5 L, are also available.

Columns Typical HPLC columns are 25 - 50 cm long and are packed with extremely small diameter (5 - 50 m) particles. The column packings may be a porous solid such as silica or alumina for adsorption chromatography. For partition chromatography, the packings have the stationary phase chemically bonded to support particles. These are the so-called bonded phases which are very durable. Bonded phases are prepared by chemical reaction between the surface hydroxyl groups of silica particles and a linear organic molecule or an organosilane. The efficiency of an HPLC column should improve dramatically as the particle size is decreased. Fig. 4 shows the effect of particle size of packing on the plate height. It is seen that a reduction of particle size from 45 to 6 m results in a tenfold or more decrease in plate height. In liquid chromatography, the minimum in the plate height occurs at very small linear velocity (Fig. 5). Such flow rates are too low for most practical applications.

Experimental Rapid Separation & Analysis of Some Soft Drinks Ingredients

Instrumentation A reversed phase HPLC system with PC control is used to separate the ingredients of soft drinks samples. The system consists of a dual reciprocating plunger solvent delivery module connected to a degasser, a manual injector with a 5 L sample loop. A 250 mm x 4.6 mm stainless steel column packed with 5 m octylsilane is used. Detection is made using photodiode array as the photo-detector element. Simultaneous measurement of chromatograms in the wavelength range of 190nm-800nm is possible.

Preparation of eluting solvent Eluting solvent is prepared using HPLC grade methanol and distilled water. Solvents are filtered through 0.45 m filters, mixed then placed into a screw capped reagent bottle. Separation is achieved using 60% methanol and 40% water.

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Preparation of samples Dilute the degassed unknown 1:4 with distilled water to prevent overloading the column with solute and consequently reducing the observed chromatographic performance. Use a syringe with a Luer lock attached to a 0.45 m filter to remove any particulate matters. Preparation of individual standards Prepare 25 mL of individual standard of 20 ppm solution of caffeine from the 500 ppm standard caffeine solution provided. And from the 50 ppm standard tartrazine solution, prepare 5 ppm individual standard for tartrazine. Use distilled water for all preparations.

Preparation of combined standards Prepare 25 mL of each of the combined standards according to the table below. Concentration (ppm) Std 2 Std 3 20 30 3 6

Caffeine Tartrazine

Std 1 10 1

Std 4 50 10

Note: All solutions must be filtered with a 0.45 m filter prior to introduction into the column. Before filtering the solutions, flush the filter with 3 x 2 mL of water, followed by 3 x 2 mL of the standard, discard these. Collect a fresh lot of approximately 3mL of filtered solution into a clean test tube.

Procedure Ask the Demonstrator to show you how to operate the HPLC system. 1. To start chromatographic run, click on Control, Single Run. Input the File name and Data file and note down the drive and path in which the chromatograms are saved.

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2. Press the START button. At the status bar, a message Waiting for trigger appears. 3. Rinse the syringe with the sample at least 3 times. Fill the syringe with 60 L of sample; bubble-free. Turn the injector to INJECT position. Insert the syringe and turn the injector to LOAD position and inject the sample; the excess will drain off. Turn the injector to the INJECT position. Remove the syringe. 4. Wait for the peak to elute. Integrate the peak and note the retention time. 5. Repeat Step 3 for the other individual standard(s). 6. Each injection (standards and samples) MUST be done two times. 7. Inject the combined standards and create calibration curves for the two compounds. 8. Identify the components and determine the concentrations of those present in the soft drink. References 1. D. A. Skoog and J. J. Leary, Principles of Instrumental Analysis, 4th ed., Saunders College Publishing, 1992. 2. Sandy Lindsay, High Performance Liquid Chromatography , Analytical Chemistry by Open Learning, John Wiley & Sons, 1991.

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Experiment 2.3 A

Ion Selective Electrodes - I

The fluoride electrode is an example of ion selective membrane electrodes. It has become a particularly important analytical tool, as prior methods for determining fluoride ion concentration involve time consuming indirect methods of limited accuracy. A single crystal of doped lanthanum fluoride is such that only the F- ions can move about freely within an immobile framework of lanthanum ions. The electrode shows true Nernstian response from approximately 10 to 10-6 M fluoride activity: E = K RT F ln[F ]

The fluoride electrode is limited in extremely dilute solutions only by the finite solubility of the crystal. Moreover, since the measured potential is proportional to the activity of the free fluoride ions in solution, the sample must be treated to release the fluoride ion if complexes are present. For accurate measurements, the total ionic strength of samples and standards must also be held constant. If the complexing agent is hydrogen (pH < 5), the solution pH is adjusted to between 5 to 8; and when the complexing agent is ferric ion, aluminium ion, etc., these can form complexes with a polyvalent anion such as citrate to release the bound fluoride. Procedure (A) Determination of fluoride ion concentration in toothpaste Use of TISAB Since water may contain Al3+ or Fe3+ ions, it is necessary to release all F- ions into the free form. To maintain a constant ionic strength of the standards and the sample solution, a Total Ionic Strength Adjustment Buffer (TISAB) (see Appendix 1) is added 1:1 to the standards and the sample solution. This buffers the solution to pH 5 6 avoiding H+ interference, and preferentially complexes any Al3+ or Fe3+ ions present. TISAB also provides a high total ionic strength background, swamping out variations in the total ionic strength between samples and standards.

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Use of the fluoride electrode Place the fluoride ion selective electrode and the reference electrode in appropriate standardizing solutions (prepared in step 1). Use magnetic stirring and wait for a stable reading (about 2 minutes) before recording the electrode potential. Wipe the electrodes between solutions to prevent sample carry-over. Always blot water from the sensing element with a soft tissue. Never wipe the sensing element except to remove adherent deposits. Scratches on the sensing element will cause a slow response to changes in the ion level. 1. From a stock 50 ppm F- solution, prepare a series of standard F- ion solutions with concentrations of 0.5, 1, 2, 3, and 5 ppm in 50mL volumetric flasks. To each flask add 25 mL of TISAB before making up to volume with deionized water. Measure the corresponding electrode potential. Plot the potential vs. log[conc.(ppm)]. 2. Weigh out approximately 200 mg of a sample of toothpaste and place this in a 250 mL beaker containing 50 mL of TISAB. Boil the mixture for 2 min. After cooling, the solution is quantitatively transferred to a 100 mL volumetric flask and diluted to the mark with deionized water. To prevent sample carry-over, rinse and immerse the electrodes in deionized water, stir for two minutes, rinse and blot the electrodes. Measure the electrode potential of the toothpaste solution. Hence determine the fluoride ion concentration in ppm of the given toothpaste sample from the calibration graph. The unit of ppm is parts per million or mg of F- ions per kg of solution or sample. Suppose we found the concentration of the fluoride ions to be 4 ppm from the calibration curve. This is the same as 4 mg/liter or 4 x 10-3 mg/mL of solution. In the experiment, 200 mg of toothpaste was used to prepare 100 mL of solution. The total F- ion in the 100 mL solution is 4 x 10-3 mg/mL x 100 mL = 0.4 mg. The F- contained in 200 mg of toothpaste (or in 100 mL of solution) would be 0.4 mg / 200 mg = 0.002 mg/mg = 2000 mg/kg = 2000 ppm.

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(B)

Fluoride Titrations As the fluoride electrode makes a highly sensitive end point detector for titrations of fluoride with lanthanum nitrate, titrations are used to measure the fluoride ion concentration. They are slower but more precise than direct electrode measurement. In the following, you are asked to determine the concentration of a given fluoride solution by titration with a standard lanthanum nitrate solution, La(NO3)3.

1.

Pipette exactly 25.0 mL of the unknown fluoride solution into a beaker and add 50 mL of deionized water. Using a burette, add increments of about 0.50 to 1.00 mL of lanthanum nitrate in the beginning of the titration and about 0.10 mL in the region of the end point. Continue the titration to 3 4 mL past the end point. Stir gently throughout the titration. Locate the end point by plotting the observed potential readings versus milliliters of lanthanum nitrate solution added. The point of greatest inflection is taken as the end point. Calculate the unknown fluoride concentration.

2.

Appendix 1 TISAB (provided) TISAB may be purchased as a prepared solution (Orion 94-09-09), or it may be prepared as follows: Place approximately 500 mL of distilled water in a liter beaker. Add 57 mL of glacial acetic acid, 50 g of NaCl, and 0.30 g of sodium citrate. Stir to dissolve. Place the beaker in a water bath (for cooling), insert a calibrated pH electrode into the solution, and slowly add ~ 5M NaOH until the pH is between 5.0 and 5.5. Cool to room temperature. Pour into a 1 liter flask, and add distilled water to the mark. Reference G. Ewing, "Instrumental Methods of Chemical Analysis", McGraw-Hill, 5th ed., USA, 1985. 15

Experiment 2.3 B

Ion Selective Electrodes - II

In this experiment, the cupric ion selective electrode is used. This allows the cupric ions in aqueous solutions to be measured quickly and accurately. As for the fluoride ion electrode (Experiment 2.3A), the total ionic strength of the samples and standards must be held constant for accurate determination. Determination of Copper By Calibration Curve 1. Prepare a 1000ppm standard solution by pipetting the appropriate volume of the 0.1M standard Cu2+ solution into a 50mL volumetric flask. Add 1mL of ISA (5M NaNO3) and dilute to the mark with deionized water. 2. To prepare a 100ppm standard, transfer the 1000ppm solution to a 100mL beaker. Pipette 5mL of this 1000ppm standard into a 50mL volumetric flask. Add 0.9mL of ISA (to obtain a constant background ionic strength of 0.1M NaNO3) and dilute to the mark with deionized water. 3. Prepare the rest of the standards 10ppm, 1ppm and 0.1ppm by serial dilution in the same way, adding 0.9mL of ISA to each flask before diluting to the mark with deionized water. 4. Transfer the 0.1ppm standard into a 100mL beaker. Place the cupric ion electrode and the single junction reference electrode in the 0.1ppm standard. Use magnetic stirring and wait for a stable reading ( about 2 min. ) before recording the electrode potential. 5. Rinse the electrodes, blot dry and repeat for the other standards. 6. To prevent sample carry-over, rinse and immerse the electrodes in deionized water, stir for two minutes and blot dry.

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7. Measure the electrode potential of the unknown Cu2+ solution (in 0.1M NaNO3) 8. Plot a graph of the electrode potential against log(concentration) of the standards and hence determine the concentration of Cu2+ ions in the unknown given.

By Titration 1. 2. Prepare 100 mL of ~0.01 M EDTA solution. Calculate its molarity. Pipette 50.0 mL of the ~0.001 M Cu2+ solution into a 100 mL plastic beaker and add 1 mL of ISA to it. Rinse the electrodes clean, blot dry and place them in the solution. Add small increments of the EDTA solution from a 10 mL burette. Note the corresponding electrode potential. Use magnetic stirring throughout the titration. Plot a graph of electrode potential against the volume of EDTA solution added. From the graph determine the end point of the titration and calculate the exact molarity of the copper solution.

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