Question

Discuss the fundamental principles, instrumentation, and analytical capabilities of thefollowing techniques:

•Potentiometry

•Cyclic voltammetry

•Anodic Stripping voltammetry

(b) Which technique is most selective for the determination of Calcium? Explain.

(c) Using a typical example, discuss the mechanism of ionization used in mass spectrometry

Answer 1

Ans -(a) •Cyclic voltammetry- Cyclic Voltammetry (CV) is an electrochemical technique which measures the current that develops in an electrochemical cell under conditions where voltage is in excess of that predicted by the Nernst equation. CV is performed by cycling the potential of a working electrode, and measuring the resulting current

A CV system consists of an electrolysis cell, a potentiostat, a current-to-voltage converter, and a data acquisition system. The electrolysis cell consists of a working electrode, counter electrode, reference electrode, and electrolytic solution. The working electrode’s potential is varied linearly with time, while the reference electrode maintains a constant potential. The counter electrode conducts electricity from the signal source to the working electrode. The purpose of the electrolytic solution is to provide ions to the electrodes during oxidation and reduction. A potentiostat is an electronic device which uses a dc power source to produce a potential which can be maintained and accurately determined, while allowing small currents to be drawn into the system without changing the voltage. The current-to-voltage converter measures the resulting current, and the data acquisition system produces the resulting voltammogram.

The potential of the working electrode is measured against a reference electrode which maintains a constant potential, and the resulting applied potential produces an excitation signal such as that of figure 1.² In the forward scan of figure 1, the potential first scans negatively, starting from a greater potential (a) and ending at a lower potential (d). The potential extrema (d) is call the switching potential, and is the point where the voltage is sufficient enough to have caused an oxidation or reduction of an analyte. The reverse scan occurs from (d) to (g), and is where the potential scans positively. Figure 1 shows a typical reduction occurring from (a) to (d) and an oxidation occurring from (d) to (g). It is important to note that some analytes undergo oxidation first, in which case the potential would first scan positively. This cycle can be repeated, and the scan rate can be varied. The slope of the excitation signal gives the scan rate used.

Figure 1: CV Excitation Signal

A cyclic voltammogram is obtained by measuring the current at the working electrode during the potential scans.² Figure 2 shows a cyclic voltammogram resulting from a single electron reduction and oxidation. Consider the following reversible reaction:

M++e−⇌M

Figure 2: Voltammogram of a Single electron oxidation-reduction

In Figure 2, the reduction process occurs from (a) the initial potential to (d) the switching potential. In this region the potential is scanned negatively to cause a reduction. The resulting current is called cathodic current (ipc). The corresponding peak potential occurs at (c), and is called the cathodic peak potential (Epc). The Epc is reached when all of the substrate at the surface of the electrode has been reduced. After the switching potential has been reached (d), the potential scans positively from (d) to (g). This results in anodic current (Ipa) and oxidation to occur. The peak potential at (f) is called the anodic peak potential (Epa), and is reached when all of the substrate at the surface of the electrode has been oxidized.

•Potentiometry-

In potentiometry we measure the potential of an electrochemical cell under static conditions. Because no current—or only a negligible current—flows through the electrochemical cell, its composition remains unchanged. For this reason, potentiometry is a useful quantitative method. The first quantitative potentiometric applications appeared soon after the formulation, in 1889, of the Nernst equation, which relates an electrochemical cell’s potential to the concentration of electroactive species in the cell.1

Potentiometry initially was restricted to redox equilibria at metallic electrodes, limiting its application to a few ions. In 1906, Cremer discovered that the potential difference across a thin glass membrane is a function of pH when opposite sides of the membrane are in contact with solutions containing different concentrations of H3O+. This discovery led to the development of the glass pH electrode in 1909. Other types of membranes also yield useful potentials. For example, in 1937 Kolthoff and Sanders showed that a pellet of AgCl can be used to determine the concentration of Ag+. Electrodes based on membrane potentials are called ion-selective electrodes, and their continued development extends potentiometry to a diverse array of analytes.

use a potentiometer to determine the difference between the potential of two electrodes. The potential of one electrode—the working or indicator electrode—responds to the analyte’s activity, and the other electrode—the counter or reference electrode—has a known, fixed potential. In this section we introduce the conventions for describing potentiometric electrochemical cells, and the relationship between the measured potential and the analyte’s activity.

(c) mass spectrometry- Mass spectrometry is an analytical technique that involves the study in the gas phase of ionized molecules with the aim of one or more of the following:

• Molecular weight determination
• Structural characterization
• Gas phase reactivity study
• Qualitative and quantitative analysis of components in a mixture.

Mass spectrometry consists basically of weighing ions in the gas phase. The instrument used could be considered as a sophisticated balance which determines with high precision the masses of individual atoms and molecules. Depending on the samples chemical and physical properties, different ionization techniques can be used. One of the main factor in choosing which ionization technique to be used is thermolability. For samples that are not themolabile and relatively volatile, ionization such as Electron Impact and/or Chemical Ionization can be effectively used. For samples that are thermolabile such as peptides, proteins and other samples of biological interest, soft ionization techniques are to be considered. Among the most used soft ionization techniques are Electrospray (ESI) and Matrix Assisted Laser Desorption (MALDI). The name given to a particular mass spec technique is usually pointing to the ionization method being used.

Atomic and molecular masses are assigned relative to the mass of the carbon isotope, ¹²C, whose atomic weight is defined as exactly 12. The actual mass of ¹²C is 12 daltons, with one dalton is equal to 1.661 10^-24 g. The mass of a molecule or an ion can be presented in daltons (Da) or kilodaltons (kDa).

Chemical Ionization

For organic chemists, Chemical Ionization (CI) is especially useful technique when no molecular ion is observed in EI mass spectrum, and also in the case of confirming the mass to charge ratio of the molecular ion. Chemical ionization technique uses virtually the same ion source device as in electron impact, except, CI uses tight ion source, and reagent gas. Reagent gas (e.g. ammonia) is first subjected to electron impact. Sample ions are formed by the interaction of reagent gas ions and sample molecules. This phenomenon is called ion-molecule reactions. Reagent gas molecules are present in the ratio of about 100:1 with respect to sample molecules. Positive ions and negative ions are formed in the CI process. Depending on the setup of the instrument (source voltages, detector, etc...) only positive ions or only negative ions are recorded.

In CI, ion molecule reactions occur between ionized reagent gas molecules (G) and volatile analyte neutral molecules (M) to produce analyte ions. Pseudo-molecular ion MH+ (positive ion mode) or [M-H]- (negative ion mode) are often observed. Unlike molecular ions obtained in EI method, MH+ and [M-H]- detection occurs in high yield and less fragment ions are observed.

Positive ion mode:

GH+ + M ------> MH+ + G

Negative ion mode:

[G-H]- + M ------> [M-H]- + G

These simple proton transfer reactions are true gas-phase Acid-Base processes in the Bronsted-Lowrey sense. A"tight" ion source (pressure=0.1-2 torr) is used to maximize collisions which results in increasing sensitivity. To take place these ion molecule reactions must be exothermic. Proton transfer is one of the simple processes observed in positive CI:

RH+ + M -----> MH+ + R

One of the decisive parameter in this reaction is the proton affinity. For the reaction to occur, the proton affinity of the molecule M must be higher that the one of the gas molecule. The main reagent gases used in CI are: Ammonia, Methane, and Isobutane. The predominant reactant ions formed are given in the mechanisms shown below. Choice of reagent gas affects the extent of fragmentation of the quasi-molecular ion.

Methane (positive ion chemical ionization):

• CH4 + e -----> CH4+. + 2e ------> CH3+ + H.
• CH4+. + CH4 -----> CH5+ +CH3.
• CH4+. + CH4 -----> C2H5+ + H2 + H.

Isobutane (positive ion chemical ionization):

• i-C4H10 + e -----> i-C4H10+. + 2e
• i-C4H10+. + i-C4H10 ------> i-C4H9+ + C4H9 +H2

Ammonia (positive ion chemical ionization):

• NH3 + e -----> NH3+. + 2e
• NH3+. + NH3 ------> NH4+ + NH2.
• NH4+ + NH3 --------->N2H7+

In methane positive ion mode chemical ionization the relevant sample peaks observed are MH+, [M+CH5]+, and [M+C2H5]+; but mainly MH+. This corresponds to the masses M+1, M+29, and M+41.

In isobutane positive ion mode chemical ionization the main peak observed is MH+.

In ammonia positive ion mode chemical ionization the main peaks observed are MH+, and [M+NH4]+. If more than one protonation site is present, additional NH3 adducts might be seen corresponding to [M+NH3+NH4]+. This corresponds to the masses M+1, M+18, and M+35.

In some cases, protonated dimers or other adducts might be seen; loss of H2O followed by protonation or adduct ion formation is seen for some classes of compounds. If the spectrum you observe does not seem to show the proper adduct ions, or shows extensive fragmentation, be wary when you try to interpret the results. There is an abundance of data available in the literature discussing chemical ionization mechanisms applicable to specific classes of compounds.

Two factors determine the choice of the reagant gas to be used:

1. Proton affinity PA
2. Energy transfer

NH3 (ammonia) is the most used reagent gas in CI because of the low energy transfer of NH4+ compare to CH5+ for example. With NH3 as reagent gas, usually MH+ and MNH4+ (17 mass units difference) are observed.

Negative Ion Chemical Ionization

Three mechanisms can be underlined:

1. Electron capture reaction due to attainment of slow moving, low energy "thermalized" electrons which may be transfered more efficiently to sample molecules.
2. Electron transfer from ionized reagent gas (e.g. NH2- may transfer an electron to a molecule having a greater electron affinity than NH2).
3. Reagent gas ions participate in true CI reactions (e.g. proton abstraction, according to relative acidities).

Molecular ions observed in negative ion chemical ionization mass spectra are usually M- or [M-H]-.

•Anodic Stripping voltammetry-

This experiment is designed to introduce anodic stripping voltammetry (ASV), an electrochemical method for trace analyses of metals. Metal ions in solution are first reduced to metallic form and concentrated as mercury amalgam in a mercury film electrode. After concentration, they are re-oxidized into solution ("stripped") from the electrode. Any metal that forms a stable amalgam with mercury can be analyzed. The pre-concentration step permits analysis of very low levels of metal ions. The subsequent analysis step can be done in a number of ways; the linear sweep (DC) voltammetric method is employed here. The method is used to analyze trace levels of metals in a variety of environmental samples. Quantitation is acheived via the method of standard additions.

The ASV method is an example of an ultra-sensitive analysis, and as such will test your skills at careful, quantitative manipulations to the utmost. The most important principle to keep in mind as you do the experiment is that, since it is an ultra-sensitive analysis, all reagents and equipment must be "ultrapure" and you must also be "ultracareful". It is very easy in procedures such as this for contamination to occur at levels comparable to the analyte levels in the sample. All glassware must be carefully cleaned and protected from subsequent contamination. All reagents used must be "ultrapure" so that they do not add significant levels of the analyte to the sample.

(b) Methods of Determination a. Ionized Calcium – (Ca ) – ion-selective electrodes (not practical) 2+ b. Total Calcium 1) Titration (now obsolete) – Clark and Collip Ca is precipitated with oxalate and determined by redox titration with pe 4 rmangenate (MnO ) or cesium. Alternatively, complexometric titration with 2 EDTA using a fluorescent end-point indicator. 2) Colorimetric – Calcium in alkaline solution reacts with o-cresolphthalein complexone to form a purple complex proportional to the calcium concentration. Magnesium ions, which also react, are removed by 8-quinolinol or 8- hydroxyquinoline. 3) Atomic Absorption Spectrophotometry – Highly accurate, method of choice

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