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MINISTRY OF SCIENCE AND EDUCATION OF THE REPUBLIC OF KAZAKHSTANSTATE UNIVERSITY NAMED AFTER SHAKARIM, SEMEY

Document of 3 level by MQS EMCD

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EMCD Educational - methodical materials for discipline «Physical methods

of Chemical research»

« »__09__2014 № 1 edition

EDUCATIONAL-METHODICAL COMPLEX OF DISCIPLINE

«Physical methods of Chemical research»

For the specialty 5B011200– «Chemistry»

Educational - methodical materials

Semey2014

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CONTENT

1. Glossary of the discipline 2. Brief synopsis of the lectures 3. Laboratory work 4. Self-study of students 5. Test and Measurement tools

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1 GLOSSARY OF THE COURSE «PHYSICAL METHODS OF CHEMICAL RESEARCH»

Spectroscopy - the study of the interaction of matter and electromagnetic radiation, usually as a function of the radiation wavelength.A continuous spectrum is a spectrum of emitted light that contains all wavelengths of the colors that compose white light (red, orange, yellow, green, blue, indigo, violet, from long to short wavelength). Continuous spectra are emitted by incandescent solids, liquids, or compressed gases. If some discrete lines are missing from a spectrum, it is an absorption spectrum (indicating the presence of elements that absorb particular wavelengths).A spectral line is a bright or dark line found in the spectrum of some radiant source. Bright lines indicate emission, dark lines indicate absorption. A bright spectral line represents light emitted at a specific frequency by an atom or molecule. Each different element and molecule gives off light at a unique set of frequencies. Astronomers can determine the composition of gases in stars by looking for characteristic frequencies. Spectroscopy is a scientific technique in which the visible light coming from objects (like stars and nebulae) is examined to determine the object's composition, temperature, density, and velocity.The spectrum is the band of colors that white light is composed of, in the order: red, orange, yellow, green, blue, indigo, violet (from long to short wavelength). Newton first discovered that sunlight could be divided into the visible spectrum.Analyzer The analyzer is the section of the mass spectrometer in which ions (formed in the source) are differentiated on the basis of their mass-to-charge ratios. The detector of the instrument is located after the mass analyzer in a beam-type instrument. Array detector In MS, an array detector is an electronic device that detects ions arriving at different spots along the array. A film (photoplate) detector for a mass spectrograph was an early example of an array detector; the film has since been replaced by electronic devices. Advantages are derived from the fact that scanning to bring ions of different m/z values to a point detector may be avoided. Beam mass spectrometer In a beam mass spectrometer, an ion beam emanating from the source transits through a mass analyzer component through to the detector of the instrument. The total ion beam flight path in a mass spectrometer can be a few tens of centimeters to as long as several meters. Calibration Calibration is a process in which the operation of the mass spectrometer in a specified manner is adjusted and certified to produce the accurate and known ion masses in the spectrum of a standard compound. Chemical ionization (CI) Chemical ionization is a process of ionization that involves the reaction of a reagent ion and a neutral molecule to yield a charged ionic form of the molecule. The first step in chemical ionization is creation of the

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reagent ion through electron ionization of the reagent gas molecules present in great excess. A stable population of reagent ions is formed through ion/molecule reactions, and these ions will eventually react with the neutral gas-phase sample molecules. In a common case, methane gas is ionized to form predominantly CH5

+, which then reacts with the neutral sample molecule in a process of protonation to form (M + H)+. Delayed extraction (DE) Delayed extraction is an experimental technique in time-of-flight mass spectrometry in which improved mass resolution is obtained by using a controlled time delay between the initial pulse of ion formation and acceleration of the ions into the flight tube of the instrument. The technique is also called time-lag focusing. Desorption ionization (DI) This is a general term used to group various methods (secondary ion mass spectrometry, fast atom bombardment, californium fission fragment desorption, and plasma desorption) in which ions are generated directly from a sample by rapid energy input into the condensed phase sample. There may be no discrete process of desorption (in the thermal sense), but instead a transfer of usually nonvolatile sample molecules into the gas phase as ions that can be mass-analyzed subsequently. Detection limit The detection limit of an instrument or system is the smallest flow of sample into the source of the mass spectrometer (or the lowest partial pressure of sample gas) that gives a signal that can be distinguished from background noise. Often this is listed as the limit of detection, and specified at a signal-to-noise ratio of 3. The limit of quantitation is usually higher. The detection limit of a method is not the sensitivity of the method. The detection limit is a value. It is not "lower than" some value, a statement that is as meaningless as it is common. Direct electrospray ionization (DESI) Charged droplets from the needle in an electrospray source are used to bombard a surface. A fraction of the molecules on the surface are entrained within the solvent droplets, which are directed in a gas flow through conductance-limiting apertures into a mass spectrometer. Electron ionization (EI) Electron ionization is the process of molecular ionization initiated by interaction of the gas-phase molecule with an energetic electron. The beam of electrons is emitted from a heated metal filament in the source, and the electrons are accelerated through a potential difference of 70 V. The collision between the molecule and the electron causes the ejection of an electron from the molecule (M) and produces a radical molecular ion in which the unpaired electron is indicated by the superscripted dot (M–⋅). The overall process is: M + e– → M+⋅ + 2e–. An older term for electron ionization is electron impact. Electron multiplier (EM) An electron multiplier is a detection device inside the vacuum of the mass spectrometer that converts the arrivals of ions at its front dynodes into a detectable, amplified electron current at the back lead of the device. The overall gain (signal out/signal in) can be as high as 104 –108. Positive ions exiting from the mass analyzer impact the first dynode surface, and the impact causes the release of several electrons, which are then accelerated through a potential to the next electrode. There, each electron impact causes the release of

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several secondary electrons, which are accelerated into the next dynode for a repetition of the impact–release process. A cascade of electrons is produced, generating a current that is further amplified and then sampled by an analog-to-digital converter to be recorded by the data system. Ionization energy The ionization energy is the minimum energy required to remove an electron from an atom or molecule in order to produce a positive ion. Magnetic analyzer (B) A magnetic analyzer creates a magnetic field perpendicular to the ion path and, in conjunction with entrance and exit slits along the flight path, selects and focuses ions of a selected momentum (and, nominally, then, with the same mass-to-charge ratio) through to the detector. A magnetic analyzer is also called a magnetic sector. An instrument that includes magnetic or electric analyzers is called a sector mass spectrometer. Plasma - a state of matter in which electrons and ions move freely. Resolution Resolution is defined in several different ways relative to the commonly given formula of m/Δm, where m is the mass of the ion at which resolution is specified. For two adjacent, symmetric peaks of equal height in a mass spectrum, the instrumental (physical or electrical) parameters are adjusted such that the peaks at masses m and (m/Δm) are separated by a valley that, at its lowest point, is just 10% of the height of either peak. Then, the resolution (10% valley definition) is m/Δm. The definition also can be given for 50% valley or 5% valley separations. For a single peak, the resolution is still calculated as m/Δm, but now Δm is the width of the peak at a height that is a specified fraction of the maximum peak height. Sensitivity The proper definition of sensitivity is that of a system response measured per amount of sample placed in the system Clearly, sensitivity is a system parameter. Source (S) The source (more specifically, the ionization source) is the device within the mass spectrometer in which ionization of sample molecules occurs. The source can be under vacuum, or it can operate at atmospheric pressure. A chromatographic method can interface with the source, or samples might be introduced via a probe or an automated sample introduction system. Ions are accelerated out of the source into the mass analyzer of the instrument.

2. A BRIEF SYNOPSIS OF THE LECTURES

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Lecture #1. Introduction to physical and chemical methods of analysis Purpose: To familiarize with the basic group of physical methods of analysisKey questions:1. Classification of physical methods of analysis2. Features of physical methods of analysis

Summary:1. Classification of physical methods of analysis

Measurements of physical properties of analytes, such as conductivity, electrode potential, light absorption, or emission, mass to charge ratio, and fluorescence, began to be used for quantitative analysis of a variety of inorganic, organic, and biochemical analyte. Highly efficient chromatographic and electrophoretic techniques began to replace distillation, extraction, and precipitation for the separation of components of complex mixtures prior to their qualitative or quantitative determination. These newer methods for separating and determining chemical species are known collectively as instrumental methods of analysis. Instrumentation can be divided into two categories: detection and quantitation. Measurement of physical properties of analytes - such as conductivity, electrode potential, light absorption or emission, mass-to-charge ratio, and

fluorescence-began to be employed for quantitative analysis of inorganic, organic, and biochemical analytes. Efficient chromatographic separation techniques are used for the

separation of components of complex mixtures. Instrumental Methods of analysis (collective name for newer methods for separation and determination of chemical species.)

Instrumentation is necessary to decipher these values. The challenge for the instrumental scientist is to mimic the 5 senses. Substances have physical and chemical fingerprints with unique thresholds. The object is to detect a chemical substance within a matrix and selectively perturb the substance of interest. Signals must be readable (in a voltage or electrical signal).

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The table below lists the names of instrumental methods that are based upon various analytical signals.

Table 1 Signals Employed in Instrumental MethodsSignal Instrumental MethodsEmission of radiation Emission spectroscopy (X-ray, UV, visible, electron);

fluorescence, phosphorescence, and luminescence (X-ray, UV, and visible)

Absorption of radiation Spectrophotometry and photometry (X-ray, UV, visible, IR); photoacoustic spectroscopy; nuclear magnetic resonance and electron spin resonance spectroscopy

Scattering of radiation Turbidimetry; nephelometry; Raman spectroscopyRefraction of radiation Refractometry; interferometryDiffraction of radiation X-Ray and electron diffraction methodsRotation of radiation Polarimetry; optical rotary dispersion; circular

dichroismElectrical potential Potentiometry; chronopotentiometryElectrical charge CoulometryElectrical current Polarography; amperometryElectrical resistance ConductometryMass-to-charge ratio Mass spectrometryRate of reaction Kinetic methodsThermal properties Thermal conductivity and enthalpyRadioactivity Activation and isotope dilution methods 2. Features of physical methods of analysis

The table 2 lists quantitative performance criteria of instruments, criteria that can be used to decide whether or not a given instrumental method is suitable for attacking an analytical problem. These characteristics are expressed in numerical terms that are called figures of merit. Figures of merit permit the chemist to narrow the choice of instruments for a given analytical problem to a relatively few. Selection among these few can then be based upon qualitative performance criteria such as speed, ease of convenience, skill required of operator, cost and availability of equipment, per sample cost.

Table 2 The most used criteria of instrumentsCriteria Figure of Merit

1. Precision Absolute standard deviation,relative standard deviation,coefficient of variation,variance

2. Bias Absolute systematic error,relative systematic error

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3. Sensitivity Calibration sensitivity, analytical sensitivity

4. Detection limit Blank plus three times standard deviation of a blank5. Concentration Range Concentration limit of quantitation (LOQ) to concentration

limit of linearity (LOL)6. Selectivity Coefficient of selectivity

Questions for self-control:1. What analytical methods are the photometric?2. What is the difference between spectrophotometric analysis and colorimetric methods?3. What reactions is used for transferring the determined component in the colored compound? Give examples.

References:1. В.П. Васильев. Теоретические основы физико-химических методов анализа. М.:ВШ, 1979г.2. Ю.С. Ляликов. Физико-химические методы анализа М.: ВШ, 1968г.

Lecture #2-6 Optical methods of analysis Purpose: to learn the basics of photometric methods for analysisKey questions:1. Nature of Electromagnetic Radiation2. Molecular Absorption of Electromagnetic Radiation 3. Quantitative Law of Radiation Absorption4. Quantitative Analysis in the UV-VIS5. Atomic Absorption Spectrometry6. Atomic Emission Spectrometry

Summary:1. Nature of Electromagnetic Radiation

Electromagnetic radiation is a form of radiant energy which exhibits both wave and particle properties. Some phenomena such as: refraction, reflection and rotation of plane-polarized light are examples of wave properties. On the other hand photoelectric effect suggests particle properties of the electromagnetic radiation. The “particle-wave” duality explains the behavior and the nature of electromagnetic radiation.

Wave properties. As indicated in Figure an electromagnetic wave has an electric component and a magnetic component oscillating in planes perpendicular to each other. Only electric component is active in ordinary energy transfer interactions with matter.

In Figure, wavelength, λ, is the distance between two corresponding point on the wave. Another important property of an electromagnetic wave is its frequency,

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ν. The units of frequency are cycles per second or sec-1. The wavelength and frequency are related to the velocity of light by expression:

λν = c/n

where c = the velocity of light in a vacuum and n is the refractive index.

Particle properties. To describe how electromagnetic radiation interacts with matter, a light beam is considered as a train of photons. The energy of each photon is proportional to the frequency of the radiation and is given by the relationship:

E = hν = hc/nλwhere E = the energy of photon in ergs, ν = the frequency of the electromagnetic radiation in cycles per second and h = Planck’s constant, 6.624 x 10-27 erg.sec.

The individual photon energy is the basis for the phenomenon of light absorption. When the photon energy matches an allowed energy transition within the material through which the photon is passing, the energy (the photon) can be absorbed. Photons cannot lose just part of their energy through normal absorption. The photon energy must be equal to an allowed energy transition in the absorbing species.

The entire range of radiation is commonly referred to as the electromagnetic spectrum. The ultraviolet and visible part of the spectrum is a very small part of the total range of possible (and detectable) frequencies of electromagnetic radiation.

At longer wavelengths than visible light (about 800 nm) there is the infrared (IR) region, and at frequencies higher than blue light there is the ultraviolet (UV) region (10-400 nm). The UV region is most useful for analytical purpose is 200-400 nm. The 10-200 nm region is the vacuum ultraviolet region and need special instruments for measurements.

2. Molecular Absorption of Electromagnetic Radiation The total energy state of a molecule includes electronic, vibrational and

rotational components. All of these energy components are quantified. The absorption of UV or visible radiation generates a transition between electronic

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levels of the molecule. The energy difference between molecular electronic levels is much greater than that between vibrational states. Thus for each electronic level of a molecule, there are also superimposed vibrational and rotational states as illustrated in Figure.

Therefore, for an electronic transition we observe a “broad band” absorption spectrum and that is typical of most absorptions of ultraviolet and visible radiation. The wavelength of the maximum of “the broad band” correspond to the electronic transition and the width of it to the superimposed vibrational and rotational transitions

3. Quantitative Law of Radiation AbsorptionIn quantitative absorption studies, a beam of

radiation is directed at a sample and the intensity of the radiation that is transmitted is measured. If the photons that strike the sample posses an energy equal to that required to cause a quantized energy change, absorption may occur.

Let us consider a monochromatic radiation. Consider the changes in radiation intensity that occurs as monochromatic radiation passes through the absorption cell in Figure. We

first fill the cell with a “blank” solution, which normally consists of the solvent plus sample constituents other than the principal absorbing species. With this “blank” solution in the cell, the transmitted intensity of the radiation (It) represents the incident intensity of the radiation minus that lost by scattering, reflection and any absorption by the other constituents (normally quite small). We denote this radiation intensity as I0.

A = log I0/I = εcl; I = I010-εcl

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Equation has been referred to as the Lambert-Beer law. In equation A is the absorbance. The term I/I0 is defined as the transmittance (symbol T), which is the fraction of the incident radiation that is transmitted by the sample. The percent transmittance is defined as 100 x T. Therefore from equation:

log T = - εclor -log T = εcl = A

In the derivation of the Lambert-Beer law it is assumed that: (1) the incident radiation is monochromatic, (2) the absorbing species act independently of each other in the absorption process and (3) the absorption occurs in a volume of uniform cross section.

The relationships between absorbance, transmittance and concentration at a given wavelength are illustrated graphically in Figure.

Instrumentation Limitations. Indeterminate instrumental variations which cause apparent deviations include: (1) stray radiation reaching the detector (reflected within the instrument), (2) sensitivity changes in the detector, and (3) power fluctuations of radiation source and the detector amplification system. Double beam operation tends to cancel out most of the random causes of deviation.

4. Quantitative Analysis in the UV-VISThe spectral domain of the UV/VIS is well known because it includes the

visible part of the spectrum and is widely used in quantitative analysis. Measurements are based on the Lambert-Beer law.

It is not necessary that the compound contain a chromophore as long as derivatization is carried out before measurement to ensure absorption of the light. Through derivatization, it becomes possible to quantify a chemical species that has no significant absorption. The derivatization (a chemical transformation) that has to be specific, total, rapid, reproducible and has to yield a UV-VIS absorbing derivative that is stable in solution. This is the principle of photometric, spectrophotometric or colorimetric analysis. The first two methods of analysis use more or less narrow spectral bands obtained with filters or monochromators. In the case of colorimetric analysis the measurements were carried out with white light without any optical instrument. Visual comparison of the sample color with that of reference solution of known concentration was performed. Instrumentation for UV-VIS Spectrometry.

A UV-VIS spectrophotometer consists of three main components: the source, the dispersive system (combined in a monochromator) and a detector. The sample can be placed in the optical path before or after the dispersive system and

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the recorded spectra can be treated using a number of different computer algorithms. Light Sources

Two light sources are commonly used in the UV-VIS domain: an incandescent lamp made from a tungsten filament housed in a glass envelope is used for the visible portion of the spectrum, above 350 nm; a medium pressure deuterium arc lamp is used for the ultraviolet portion of the spectrum. Dispersive system. Light emitted by the source is dispersed by prisms or gratings, which are integrated into an assembly called a monochromator. The monochromator extracts a narrow spectral band of the spectrum. For simple devices as dispersive system is used a wideband, interchangeable color filter. Detectors. Two types of detectors exist: photomultiplier tubes and semiconductors (e.g. silicon photodiodes and charge transfer devices (CCD/CID)). The detector measures the light signal at a given wavelength. It converts the light intensity selected by the monochromator exit into an electrical signal.

Single Beam Spectrophotometers Many routine measurements are conducted at fixed wavelengths using

simple photocolorimeters equipped with wideband, interchangeable color filters. An analytical blank (containing the solvent and reagents for the analysis, without the sample to be measured) is first placed in the optical path, and then is replaced by the solution to be analyzed.

Two rotating mirrors, called choppers, which are synchronized with the displacement of the grating, allow the comparison of transmitted light at the detector of the two beams with the same wavelength. Amplification of the modulated signal allows the elimination of the stray light. Double beam spectrophotometers allow differential measurements to be made between the sample and the analytical blank. They are preferable to single beam instruments for measurements in problematic solutions. Atomic absorption and emission spectroscopy, perhaps the oldest instrumental techniques now widely used, are two methods of quantitative analysis that can be used to measure approximately 70 elements (metals, metalloids and non-metals) in environmental samples.

Since atoms cannot rotate or vibrate as molecules do, only electronic transitions can occur when energy is absorbed. Because the transitions are quantized, line spectra are observed. The principle behind these methods of elemental analysis depends on measurements made on an analyte that is transformed into free atoms. The sample solution is heated in the instrument to a temperature of between 2000 or 3000 degrees Celsius to break chemical bonds, liberate the elements and transform them into a gaseous atomic state. Thus, the total concentration of the element is measured without distinguishing the chemical structures present in the cold sample. There are various ways to obtain free atoms and to measure the absorption or emission of radiation by these:

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a. atomic absorption spectrometry (AAS), in which the amount of the radiation absorbed by ground state atoms in a flame or in a small electrical oven (graphite furnace) is measured;

b. atomic emission spectrometry (AES), in which atoms are excited in a flame, electrical arc, spark, inductively coupled plasma, and the radiation intensity emitted by a fraction of excited atoms that return to their ground-state is measured.

Many models of instruments allow measurements to be conducted by these techniques, which rely on different principles, and concentrations in the mg/L (ppm) range or lower can be accessed.

5. Atomic Absorption SpectrometryPrinciples The law that governs the absorption of light by the atoms is Kirchoff’s law

and it states that incandescent gases can absorb the same radiation that they can emit in certain conditions.

The main steps in AAS measurements are:1. aspiration of sample into the flame (or graphite furnace);2. conversion of the sample into free atoms and the gaseous atomic state formation

(atomic cloud);3. absorption of the monochromatic radiation by the ground state atoms from the

atomic cloud;4. measurement of the transmitted radiation intensity;5. extraction of the quantitative information from the registered signal.The monochromatic light is given off by hallow cathode lamp, the cathode made from the same element as that being determined. Therefore, the wavelengths of radiation given off by the source are the same as those absorbed by the atoms in the flame.AAS is identical in principle to absorption spectrophotometry and the Lambert-Beer’s law is followed in this technique, too:

A = k × Cwhere A is the absorbance , C is the concentration of the element and k is a coefficient unique to each element at a given wavelength. The absorption depends on the number of ground state atoms of atomic cloud in the flame and on the path length in the flame. Both of these variables are difficult to determine, but the path length can be held constant and the concentration of the atomic cloud is directly proportional to the concentration of the analyte in the solution being aspirated.

The instrument yields the absorbance by rationing the transmitted intensities in the presence (I) and absence (I0) of the sample:

A = - logT = log(I0/I)Measurements are made by comparing the unknown to standard solutions.

The procedure used involves classical protocols: prepare the calibration curve or standard additions, as long as the range of concentrations stays within the linear

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conditions of absorbance. The main disadvantage of making measurements in AAS is that a different source is required for each element.

InstrumentationSimilarly to absorption spectrophotometry, the basic components for AAS

are a light source, a cell (the flame), a monochromator and a detector. The line bandwidth emitted by the source is equal to the line bandwidth absorbed by the atoms. The beam-light emitted by the source (I0), which must be a very narrow band characteristic of the analyte metal, passes through the atomization device (flame or graphite furnace). In the atomization device, most metallic compounds are decomposed and the metal is reduced to the elemental state, forming a cloud of atoms. The atoms absorb a part of I0 that is proportional to the analyte concentration in the sample. The attenuated beam-light (I) is then focused on the entrance slit of the monochromator, located after the atomization device. The monochromator’s role is to select a very narrow band of wavelengths and to eliminate extraneous light resulted from the atomization device. The optical path ends at the entrance slit of the detector (photomultiplier tube). The detector measures the ratio I0/I and the logarithm of the ratio is displayed. I0 is the full intensity of the beam-light emitted by the source and perceived by the detector in the absence of the sample, and I is the attenuated intensity of the beam-light perceived by the detector when the sample is present.A. SourcesThe key element in AAS is the source, which must emit a sharp-line because the width of the absorption line is very narrow, around 10-3 nm. A sharp-line source that emits monochromatic and specific wavelengths and used almost exclusively in AAS is the hollow cathode lamp (HCL). The diagrams of a HCL and of the excitation of the cathode atoms are presented in Figure.

HCL is a glass tube with a borosilicate or quartz window, depending on the wavelengths emitted by the cathode (e.g. quartz is used for lines in the UV region). The tube is filled with an inert gas (argon or neon) at a reduced pressure. Inside of the lamp, a cylindrical hollow cathode made of the element to be determined (a cathode of lead is used for lead determination) and a tungsten or zirconium anode are enclosed. A higher voltage (300V) is applied between the electrodes, causing the inert gas atoms to be ionized at the anode. The positive ions are accelerated toward the negative cathode and some of them possess enough energy to strip atoms from the cathode, which form, at the surface, an atomic gas. The atomic gas

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is excited to higher electronic level by collisions with the high-energy inert gas atoms. When the excited cathode atoms return to their ground state, the characteristic emission spectrum of elements is obtained. The emission steps may be represented as follows:

where M(C) is the element in its metallic state in cathode, M(G)* is the element in its excite atomic state and M(G) is the element in its atomic state.

These lines are passed through the atomization device, and certain ones are absorbed by the analyte atoms because they possess the right energy to result in the discrete electronic transitions. The most strongly absorbed line is the one corresponding to the electronic transition from the ground state to the first excited state, known as the resonance line.

The width of the emitted lines of the cathode-excited atoms depends on the Doppler, Stark (ionization) and Lorentz (pressure) effects but even then it is narrower than the corresponding absorption band of the sample atoms, and therefore, the entire source line-width is absorbed.

There are more then 100 types of HCLs made in pure elements. HCLs cannot be used for mercury and sodium (their boiling points are too low), and then classical lamps using metallic vapor are used instead.

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For certain sample types multi-element hallow cathode lamps are attractive, the cathode is an alloy of several elements and the lines of all the elements are emitted. They may exhibit shorter lifetimes than the single-element lamps, due to the selective volatilization of one of the elements form the cathode and its condensation on the walls of the lamp.

In some instances, a brighter source such as microwave-excited or electrode-less discharge lamp composed of the element to be measured, may be preferred. Electrode-less lamps with very intense emission use a radio-frequency emitter to excite the metallic vapor and are especially used for elements such as As, Hg, Se or P.B. Devices for sample atomization

Flame atomization. The flame is a mixture of a fuel and an oxidant gas and it has a rectangular base of about 10 cm by 1 mm. It is aligned with the optical axis of the instrument. The sample solution is aspirated by the nebulizer and is introduced in the flame as a fine spray. In the flame, the analyte is transformed in an atomic cloud following the processes: solvent evaporation, dissociation of the salt into free ions, ions reduction to gaseous atoms. For sensitive measurements atomization should be as complete as possible. Collisions with high-energy flame gas atoms are principally responsible for atomization. Thus, the hotter the flame, the more effective is the process. In Table 3 are listed some analytically useful flames and their temperature. The most popular flames in AAS are acetylene–air and acetylene–N2O (for refractory elements). The hydrogen– argon–entrained air flame is preferred for wavelengths below 200 nm where the acetylene–air flame absorbs a large fraction of the radiation. It is used for arsenic and selenium when they are separated from the sample by volatilization as their hydrides (AsH3, H2Se) and passage of these gases into the flame.

Table 3 Recommended flame type and their temperature for analytical determinations by flame AAS.

Combustible mixture Tmax (°K)

H2 – air 2300H2 – O2 2950CH4 – O2 2950C2H2 – air 2450C3H8 – air 1998C3H8 – O2 3173C4H10 – air 2200C2H2 – O2 3400C2H2 – N2O 3200H2 – Ar – entrained air 1850

Flames must also be stable and something must be known about the background levels of chemical species derived from the flame gases as well as

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those derived from the sample. Not all the parts of a flame are valuable for atomization or for observation of the atomic cloud spectrometrically. Free radicals present in the flame have absorption and emission spectra in the near UV and they can interfere with the measurement of the element. Thus, the observation flame height must be adjusted for some elements.

Electrothermal atomization. For the lowest possible detection limit and greater sensitivity the electrothermal or resistive furnace atomizer (Figure) was the second development in the sample module for AAS. It is a flameless device without nebulization and consists of a hollow graphite rod or cup that can hold a precise volume/quantity of the sample (a few mL or mg deposited with an automated syringe). This rod of 8 mm inner diameter and 40 mm length is oriented parallel to the optical axis and it is surrounded by a double

sleeve containing an inert gas to protect it from oxidation and allow circulating water to cool the device. A small volume of sample (1–5 mL) is placed in the graphite rod through a hole in the top, and then the rod is heated resistively by passing an electrical current. To avoid splashing, the temperature is gradually increased according to a three-step cycle:1. the sample is dried at a low temperature for a few seconds (~100 to 200 °C) 2. pyrolysis at 500 to 1400 °C to destroy organic matter that produces smoke and scatters the light source during measurement; the smoke from pyrolysis in flushed out by flowing argon gas, and finally the sample is rapidly thermally atomized at a high temperature up to 3500 °C. 3. the absorption of the metal atoms in the hollow portion of the rod is measured and a sharp peak of absorbance versus time is recorded as the light path passes through the atomic cloud. The heating is done in an inert atmosphere to prevent oxidation of the graphite at high temperature and also to prevent the refractory metal oxides. Certain metals such as molybdenum, vanadium, nickel, and barium react with the graphite at high temperature forming carbides and to prevent this pyrolytically coated tubes are used.

Electrothermal atomizers have a conversion efficiency of 100 % compared to 0.1 % from flame atomization, therefore the detection limits are often 100 to 1000 times improved over those of flame aspiration, many metals being determined at concentrations of 1 mg/L. Other high melting, electrically conducting

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substances can also be adapted as atomizers. A coil-shaped tungsten filament (W-coil) serves for low-cost, compact and portable instrumentation for environmental and clinical analyses. High heating temperatures up to 3000 °C can be achieved by using a simple power supply such as a car battery. For over 20 elements that have been determined using W-coil AAS, the detection limits are even better than or within a factor of 10 of those of graphite furnace, while the linear dynamic ranges of concentrations remain unaffected.

Chemical vaporization. Other distinctive modes of atomization are the conversion of the compounds of metalloids and many low-activity metals to hydrides. For hydride preparation sodium boronhydride is added to an acidified sample. For instance, arsenic, bismuth, tin, lead, antimony or selenium, which are difficult to reduce in a flame when they are in higher oxidation states, will react with the reducing agent in a separate vessel to form the volatile hydride. The generator vessel must be flushed with Ar or N2 to remove oxygen. When hydride formation is complete, the hydride is carried into a quartz cell placed in the flame by a further flushing, decomposed by heat, and measured by atomic absorption. For mercury, the bound mercury is reduced by a reducing agent such as tin chloride (SnCl2) in acid solution. The elemental mercury is formed instead of hydride and it may be volatilized easily by bubbling an inert gas through the solution and passing the gas through a special cell, which does not need to be put into the flame. This is called “cold vapor” method, and requires specialized instruments. Because chemical atomization is a selective reaction, these metalloids and low-activity metals are also concentrated by removal from the original matrix. The determination of these elements in the presence of considerable amounts of organic matter often requires the organic material digestion, with a strong oxidizing agent, and the interfering substances masking.

Quantitative Determinations in Atomic Absorption SpectrometryAAS is widely applicable as a generally sensitive technique for quantitative determination of elements (about 70). Hollow cathode lamps are available for all and microwave electrode-less lamps for many. AAS is capable of a precision of ± 2 % and when a double-beam procedure and a background correction are employed, precision can be better then ±1 %.

Sample preparation. In the majority of cases samples are present in solution and for samples that requires dissolution, acid digestion or solvent extraction may be the first step of analysis.

Liquids with high salt content offer problems when the solution is evaporated. Encrustation of salts around the slot of the burner leads to unsteadiness of the sample flow and of fuel and oxidant gases and introduces serious errors. In electrothermal atomization salts rise the smoke and high background correction. A way to remove these difficulties may be the complexation of the analyte in the sample solution and then the extraction of the complexed species. The extraction also offers the advantage of concentrating the analyte, eliminating the interferences, and enchanting the sensitivity of the measurements if an organic extractant is used. Some common complexing agents for metals and solvents are:

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ammonium pyrrolidine dithiocarbamate used with methyl isobutyl ketone; 8-hydroxyquinoline with ethyl acetate or butanol and ethylenediaminotetraacetic acid. Many metals, metalloids and low-activity metals are most sensitively determined by chemical conversion to hydrides.

Calibration curves and working conditions. Analytical signal readings are quantified by reference to a working curve, a plot of absorbance versus concentration for each analyte. The standard solutions must be prepared to be nearly identical in composition with the samples as possible in order to minimize the errors. Ultra-pure reagents and solvents should be used, especially in the case of organic solvents. In spectrophotometric measurements the precision is best when the absorbance varies in the range of 0.15 to 1.0 units. If an internal standard is used in a dual-channel instrument, the absorbance range can be extended with a good precision. When the concentration range of an element in a sample exceeds the optimum absorbance values, several options are open: the variation of the optical path length through the flame by a rotating burner; the use of a less sensitive line of the element; the dilution of the sample.Standard addition method. Analyte determination using standard addition method is attractive only if the concentration of the analyte in the sample is very low. The method involves spiking an equal volume of standard solutions, at least three different concentrations, to equal volumes of blank solution and sample aliquots, respectively. The absorbance for all these solutions is measured and then it is plotted versus concentration. The linear curve obtained is extended through the concentration axis and the distance from the point of its intersection to the origin is equal to the concentration of the analyte in the sample.

6. Atomic Emission SpectrometryPrinciples

The main steps in AES measurement are:1. Conversion of a sample to free-atom gas.2. Excitation of the atoms and their ions to higher electronic states.3. Measurement of the emission wavelengths and intensities.4. Extraction of qualitative and quantitative information from these signals.AES is a well-established technique for determining inorganic constituents in various types of samples. Basically, the sample solution is introduced into the excitation device as a fine spray, where the solvent evaporates leaving the dehydrated salt. The salt is dissociated into free gaseous atoms in the ground state and then a certain fraction of these atoms can be raised to an excited electronic state, by the effect of high temperature. These excited atoms return to their ground state emitting photons (hn) of characteristic wavelength. The intensity of the emitted radiation is proportional to the concentration of analyte in the sample.In AES, one or several specific spectral lines are monitored for each analyte. Hence, the emission of radiation can occur from either excited or ionized atoms, thousands of different spectral lines can be observed. Some of these lines could be more intense than those of the analyte, which can be present at ultra-trace levels, and, for this reason, a high performance monochromator is required. Flame

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photometric analysis is much simpler and it is not always necessary to have a high-resolution monochromator, a simple interference filter may be sufficed. The monochromator can be replaced by a polychromator – a double module in which the exit slit of monochromator has been replaced by a multi-channel detector mounted in the focal plane. It should be noted that the next generation will be represented by the grating spectrometers; in recent years prism spectrometers have become relatively rare. Nowadays, spectrometers able to solve many interferences and problems related to the matrix effects are used, the instrument being programmed to identify and quantify the elements in a real sample.

InstrumentationThe atomic emission spectrometer consists of three principal components: the device responsible for bringing the sample to a sufficient temperature; the optics including a mono- or polychromator that represent the heart of the apparatus; and a microcomputer that controls the instrument.A. Excitation sources

The excitation source is the most critical module because it must volatilize and atomize the sample as uniformly as possible since the concentration of atoms in the atomic cloud should be representative of that in a sample. It should also excite atoms. The excitation sources must fulfill the following criteria:1. high energy flux;2. reproducibility in sample introduction and energy transfer;3. stability of excitation;4. high sensitivity;5. ease of use.The main excitation sources are flame; high-voltage arc or spark, inductively coupled plasma and glow discharge.Flame is a lower-energy source and the emission spectrum is much simpler, as there are fewer lines. Only the alkaline and alkaline earth metals are determined by flame emission spectrometry.High-voltage arc and spark are used for solid samples and they are mostly used in semi-quantitative analysis in industry. Inductively coupled plasma (ICP) emission spectrometry is used for rapid multi-element determinations in environmental samples. Different from AAS, the chemical interference in ICP-AES is very low. The plasma is an incandescent ionized gas (argon) heated inductively by radiofrequency energy at 4–50 MHz and 2–5 kW. Argon gas, which is ionized by a discharge of a Tesla coil or a pilot spark, flows upward through the quartz tubes, the external tube having at its superior part two water-cooled copper tubes. The copper tubes are connected to the radiofrequency generator creating a variable magnetic field in the flowing gas inside the coil. This induces a circulating eddy current in a gas, which in turn heats it. As this environment becomes more and more conductive, the temperature increases considerable, the temperature reaching 9000 to 10,000 K. The plasma is isolated from the tubes by a gaseous sheath of flowing, non-ionized argon injected

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through an external tube concentric to the first one. The sample aerosol generated in the nebulizer and spay chamber is introduced into the ICP via a third tube with a

diameter of 1–2 mm. At this high temperature, the sample atomizes, ionizes and excites producing emission spectra. Also, the high temperature of plasma eliminates many chemical interferences present in flame because molecules of compounds formed completely dissociate. The plasma is well suited for refractory elements (boron, phosphorus, uranium, etc.) and difficult to excited elements (zinc, cadmium). The advantages of using an

ICP include the possibility to identify and quantify all the elements excepting argon, in concentration from ultra-trace levels to major components; working curves covering 5 decades of concentrations (1 ppb–100 ppm) and the detection limits are mostly in ppb range; multi-element analysis can be accomplished in 30 seconds consuming 0.5 mL of sample solution, 30 elements can be determined simultaneously. Spectral interferences from ion–atom recombination, spectral line overlaps, molecular bad emission, or stray light can occur that may alter the net signal intensity. These can be avoided by selecting alternate analytical wavelengths and making background corrections.

Wavelength SeparatorThis is the device that separates specific emission lines of the analyte using planar, concave or echelle gratings. Predefined elements, characterized by precise spectral lines, are detected by as many photomultiplier tubes as there are secondary slits, each corresponding to an analytical line or measurement channel. A fixed optic arrangement using echelle grating in association with a focusing prism produces a double dispersion of the lines, in both the horizontal (due to the grating) and vertical (due to the prism) dimensions. This order-separating device allows simultaneous detection over the whole spectral range. There are also instruments with dispersion surfaces compatible with two-dimensional sensors. Their sensitivity and spectral response allows simultaneous measurements of lines. Wavelength scanning instruments (monochromator type) have the dispersive system movable in order to focus each wavelength on the fixed exit slit. Several

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units of this type can be placed in series, constituting double or triple monochromators of high performance.

Quantitative Determinations in Emission Absorption SpectrometryAES is described as a very sensitive (detection limits of few ppt) and rapid quantitative elemental analysis. The dynamic range of emission methods is so large that concentration levels are identified by terms major (>10 %), minor (»1 %), and trace (<0.01 %). However, each application may be considered as an individual case. Certain instruments better tolerate dominating matrices such as soil or mud, where there are high concentrations of elements such as Si, Fe and Al.The calibration curves are used for quantitative determinations. A well selected set of standards employed to obtain plots of intensity (Ia) of the analytical lines versus concentration (C):

Ia = m × Cwhere m is the slope of the curve. In every case, a background correction Ib should be made if background is greater than the order of magnitude of acceptable error. A more correct equation for the working curve than may be:

Ia – Ib = m × CAccuracy and precision are dependent on the regulation of excitation conditions, including the selection of an appropriate integration time. Many sources fluctuate and times of volatilization and excitation of elements will vary. The effectiveness of flame spectrometric methods for a particular element depends only in part on external variables such as fuel gas pressure and width of the burner slot. It is also determined by processes called interferences or inter-element (matrix) effects.

Questions for self-control:1. Formulate the law of Bouguer-Lambert-Beer.2. What does the molar absorption coefficient characterize?3. Give examples of methods for visual colorimetric analysis. What are their advantages and disadvantages?4. List the methods of quantitative photocolorimetric analysis.

References1. Gary D. Christian, Analytical chemistry (6th edition), Wiley, New York, 2004.2. M. Gore, ed., Spectrophotometry and spectrofluorimetry, 2nd Edition, Oxford

University Press, Oxford, 2000.3. R. Kellner, J, M, Mermet, M. Otto, H.M. Widmer Eds, Analytical Chemistry,

Wiley-VCH, Weinheim, 1998.4. Rouessac, F. and Rouessac, A., Chemical Analysis. Modern Instrumentation

Methods and Techniques, John Wiley and Sons, Ltd., Chicester, New York, 2000.

5. A. F. Dăneţ, Metode Instrumentale de Analiză Chimică, Editura Ştiinţifică, Bucureşti, 1995.

6. Encyclopedia of Analytical Science, Vol. 9, pp. 5297-5353, Academic Press, London, 1995.

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7. “Environmental Chemistry”, Seventh Edition 2000, Stanley E. Manahan, Boca Raton: CRC Press LLC.8. “Chemical Analysis – Modern Instrumental Methods and Techniques” 2003, Francis Rouessac, Annick Rouessac, John Wiley & Sons, New York.9. “Analytical Chemistry”, Fifth Edition 1994, Gary D. Christian, John Wiley & Sons, New York.10. K.W. Jackson, Anal. Chem., 2000, 72, 159R-167R.

Lecture #7-11 Electrochemical methods of analysis Goal: to learn the basics of potentiometry, volamperometrii, coulometry, conductometry, elektrogravimetriiKey questions:1. Potentiometry2. Electrodes used in potentiometry3. Typical electrode system for measuring pH4. Types of potentiometric determinations

Summary:1. PotentiometryPotentiometric Measurements based on measuring the potential of electrochemical cells without drawing appreciable currentIncluding: - a reference electrode, an indicator electrode, and a potential measuring deviceUsed to– Locate end points in titrations.– Determine ion concentrations with ion-selective membrane electrodes– Measure the pH– Determine thermodynamic equilibrium constants such as Ka, Kb, and Ksp.A typical cell for potentiometric analysis:

For most electroanalytical methods, the junction potential is small

enough to be neglected. Reference electrode

– A half-cell with an accurately known electrode potential, Eref, that is independent of the concentration of the analyte or any other ions in the solution– Always treated as the left-hand electrode

Indicator electrode– which is immersed in a solution of the analyte, develops a potential, Eind, that depends on the activity of the analyte.

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– Is selective in its response Salt bridge

– Preventing components of the analyte solution from mixing with those of the reference electrode– A potential develops across the liquid junctions at each end of the salt bridge.– Potassium chloride is a nearly ideal electrolyte for the salt bridge because the mobilities of the K+ ion and the Cl－ ion are nearly equal.

2. Electrodes used in potentiometryReference ElectrodesIdeal reference electrode has a potential that is accurately

known, constant and completely insensitive to the composition of the analyte solution.

• Calomel reference electrodes:

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– where x represents the molar concentration of potassium chloride in the solution.– Concentrations of potassium chloride that are commonly used in calomel reference electrodes are 0.1 M, 1 M, and saturated (about 4.6 M).– The saturated calomel electrode (SCE) is the most widely used because it is easily prepared. The potential is 0.2444 V at 25°C.

The electrode reaction in calomel half-cell:

• Silver/Silver Chloride Reference Electrode

• The half-reaction is

• The potential of this electrode is 0.199 V at 25°CLiquid-Junction Potentials– A liquid junction consisting of a 1 M HCl solution that is in contact with a solution that is 0.01 M HCl– Both H+ and Cl- ions tend to diffuse across the inert porous barrier– H+ ions diffuse more rapidly than Cl- ions, and a separation of charge results– The potential difference resulting from this

charge separation is the junction potentialThe magnitude of the liquid-junction potential can be minimized by placing

a salt bridge between the two solutions:– The mobilities of the negative and positive ions are nearly equal– A saturated solution of potassium chloride is good from both standpoints. The net junction potential with such a bridge is typically a few millivolts.

Indicator ElectrodesIdeal indicator electrode responds rapidly and reproducibly to changes in the concentration of an analyte ion (or groups of analyte ions).• Three types:– Metallic

electrodes of the first kind electrodes of the second kind inert redox electrodes

– Membrane– Ion-sensitive field effect transistor

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Electrodes of the First KindA pure metal electrode that is in direct equilibrium with its cation in the solution:

For which

where Eind is the electrode potential of the metal electrode and axn+ is the activity of

the ion (or, in dilute solution, approximately its molar concentration, [Xn+]). Electrode of the first kind is not very popular because metallic indicator

electrodes are not very selective and respond not only to their own cations but also to other more easily reduced cations. Many metal electrodes can be used only in neutral or basic solutions because they dissolve in the presence of acids. Easily oxidized, can be used only, when analyte solutions are deaerated to remove oxygen. Certain metals do not provide reproducible potentials.

Limited electrodes are:– Ag/Ag+ and Hg/Hg2+ in neutral solutions Cu/Cu2+, Zn/Zn2+, Cd/Cd2+, Bi/Bi3+, Tl/Tl+, and Pb/Pb2+ in deaerated solutions.Electrodes of the Second KindMetal electrode respond to the activities of anions that form sparingly soluble precipitates or stable complexes with such cations.

The Nernst expression for this process at 25ºC is

Inert Metallic Electrodes for Redox SystemsSeveral inert conductors can be used to monitor redox systems. Such as –

platinum, gold, palladium, and carbon:– Platinum electrode immersed in a solution containing cerium (III) and cerium (IV)Membrane ElectrodesFundamentally different from metal electrodes both in design and in principle. Sometimes called p-ion electrodes because the data obtained from them are usually presented as p-functions, such as pH, pCa, or pNO3. Liquid-membrane Electrode

The potential of liquid-membrane electrodes develops across the interfacebetween the solution containing the analyte and a liquid-ion exchanger that

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selectively bonds with the analyte ion. Liquid-membrane electrodes have been developed for the direct potentiometric measurement of numerous polyvalent cations as well as certain anions. For Calcium the membrane electrode consists of a conducting membrane that selectively binds calcium ion, an internal solutions containing a fixed concentration of calcium chloride, a silver electrode that is coated with silver chloride to form an internal reference electrode. Internal solution/analyte solution separation membrane:– the ion exchanger is dissolved in an immiscible organic liquid that is forced by gravity into the pores of a hydrophobic porous disk. This disk serves as the membrane.– the Ion exchanger is immobilized in a tough PVC gel cemented to the end of a tube that holds the internal solution and reference electrode.

The dissociation equilibrium develops at each membrane interface:

A potential develops across the membrane when the extent of the ion exchanger dissociation at one surface differs from that at the other surface. The relation between the membrane potential and the calcium ion activities:

– where a1 and a2 are the activities of calcium ion in the external analyte and internal standard solutions, respectively. Since a2 is a constant:

Crystalline-membrane ElectrodesSome solid membranes that are selective toward anions in the same way that some glasses respond to cations. Membranes prepared from cast pellets of silver halides are for the selective determination of chloride, bromide, and iodide ions. An electrode based on a polycrystalline Ag2S membrane is for the determination of sulfide ion. In both types, silver ions are sufficiently mobile to conduct electricity through the solid medium.3. Typical electrode system for measuring pH

pH meter - A thin glass membrane that separates two solutions with different hydrogen ion concentrations. The sensitivity and selectivity of glass membranestoward hydrogen ions are reasonably well understood. A glass electrode system contains two reference electrodes: the external calomel electrode (Immersed in a solution of unknown pH) and the internal silver/silver chloride electrode. Whenever there is a charge imbalance across any material, there is an electricalpotential across the material:

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– the concentration of protons inside the membrane is constant, and the concentration outside is determined by the concentration, or activity, of the protons in the analyte solution.– This concentration difference produces the potential difference that we measure with a pH meter.

4 potentials:– EAg,AgCl and ESCE, are reference electrode potentials that are constant.– A 3rd potential is the junction potential Ej across the salt bridge that separates the calomel electrode from the analyte solution.– The 4th and most important potential is the boundary potential, Eb, which varies with the pH of the analyte solution.

Glass composition affects the sensitivity of membranes to protons and other cations.

Cross-sectional view of a silicate glass structureThe hydration of a pH-sensitive glass membrane involves an ion-exchange

reaction between singly charged cations in the interstices of the glass lattice andprotons from the solution:

Glass compositions that permit the determination of cations other than hydrogen. Incorporation of Al2O3 or B2O3 into the glass has the desired effect. Glass electrodes that permit the direct

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potentiometric measurement of such singly charged species as Na+, K+, NH4 +, Rb+, Cs+, Li+, and Ag+ have been developed.

4 Types of potentiometric determinationsDirect PotentiometryDirect potentiometric measurements provide a rapid and convenient method to

determine the activity of a variety of cations and anions. A comparison of the potential developed in a cell containing the indicator electrode in the analyte solution with its potential when immersed in one or more standard solutions of known analyte concentration.The Standard-Addition Method

The standard-addition method involves determining the potential of the electrode system before and after a measured volume of a standard has been added to a known volume of the analyte solution.

Potentiometric Titrations A potentiometric titration

involves measurement of the potential of a suitable indicator electrode as a function of titrant volume. The measurement is base on the titrant volume that causes a rapid change in potential near the equivalence point. Potentiometric titrations provide data that are more reliable than data from titrations that use chemical indicators. They are particularly useful with colored or turbid solutions and for detecting the presence of unsuspected species. Potentiometric titrations are not dependent on measuring absolute values of Ecell.Potentiometric titration results depend most heavily on having a titrant of accurately known concentration.Detecting the End Point

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A direct plot of potential as a function of reagent volume – the inflection point in the steeply rising portion of the curve, and take it as the end point.

Calculate the change in potential per unit volume of titrant (i.e., ΔE/ΔV) – If the titration curve is symmetrical, the point of maximum slope coincides with the equivalence point.

Calculate the second derivative for the data changes sign at the point of inflection – This change is used as the analytical signal in some automatic titrators.

QUESTIONS FOR SELF-CONTROL:1. Give a classification of electrochemical methods of analysis2. On what is the analysis of potentiometric based?3. What types of electrodes is used in potentsmetrii?4. Why should the standard electrode potential be permanent?

REFERENCES1. http://memo.cgu.edu.tw/hsiu-po/Analytical%20Chem/Lecture%207.pdf 2. В.П. Васильев. Теоретические основы физико-химических методов

анализа. М.:ВШ 1979г.3. Ляликов Ю.С. Физико-химические методы анализа М.:ВШ, 1968г.

Lecture #12-15 Chromatographic methods of analysis Purpose: To familiarize with the main types of chromatographic methods Key questions:1. The bases of chromatography2. Types of Chromatography3. Components of Column Chromatography4. Chromatographic Theory.5. Ion Chromatography6. Gas Chromatography (GC)7. High Performance Liquid Chromatography (HPLC)

Summary 1. The bases of chromatography

In 1903, the Russian botanist Mikhail Tswett (1872–1920), who is currently held as the father of chromatography, conducted his pioneering experiment involving the passage of a plant extract through a column filled with a sorbent material. The isolated species formed colored bands in the column, hence the name of the new technique [from the Greek khroma (colour) and grafein (to write)] according to the much-repeated “historical” version. This inspired the following “historical definition” of chromatography: “a separation method based on the different velocity at which the components of a sample go through a stationary phase pushed or swept by a mobile phase.”

Chromatographic separation involves an interaction and the partitioning of the analyte between two immiscible phases for which it exhibits some affinity, namely: the mobile phase and the stationary phase. The stationary phase in a

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chromatographic column can be solid (packing the column) or liquid (anchored to a solid surface). The mobile phase, also called the “eluent”, can be a gas, liquid or supercritical fluid that sweeps the sample components as it goes through the stationary phase. The way an analyte distributes itself between the two phases depends on its partition coefficient, which dictates the velocity at which it will travel or migrate across the stationary phase. If the chromatographic process is allowed to proceed for a long enough time, differences in migration velocity between the sample components eventually result in their separation. Those components that are strongly retained by the stationary phase will move slowly in the mobile phase flow, whereas those that are weakly bound to such a phase will move faster. There will thus be a spatial separation: each sample component will move separately from the others. As a result, each sample component will reach a preset point (the detector) after a different time, so there will be both spatial and temporal separation between all. Once each component has been isolated, it can be identified and/or quantified individually.2. Types of Chromatography

Chromatographic methods can be classified according to various criteria. One is based on the way the stationary and mobile phases are brought into contact; thus, there is column (three-dimensional) chromatography and planar (two-dimensional) chromatography. In column chromatography, the stationary phase is held in a narrow tube (a column) through which the mobile phase is passed by gravity or under pressure. Columns can be of the packed and open-end types; the former are filled with particles containing the stationary phase (SP) and the latter consist of hollow capillaries the walls of which are coated with the SP. In planar chromatography, the stationary phase is placed on a flat plate and the mobile phase travels across it by gravity or capillarity. Both types of chromatography rely on identical equilibria, however.

One other classifying criterion is the type of stationary and mobile phases used. Thus, there is liquid, gas and supercritical fluid chromatography, which use a liquid, a gas and a supercritical fluid, respectively, as the mobile phase. Liquid chromatography can be implemented in a column or on a planar surface; on the other hand, gas and supercritical fluid chromatography can only be performed in a column.

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A third criterion is the underlying retention mechanism, i.e. the type of physical–chemical interaction between the stationary phase and the sample

components. Such a mechanism can be essentially of five different types – however, the overall phenomenon behind the separation usually involves more than one, namely:(a) Adsorption. This occurs when the stationary phase consists of particles of a solid sorbent the surface of which retains the sample components that will compete for the mobile phase molecules. In this way, the retention–elution cycle is successively completed.(b) Partitioning. This is a liquid–liquid extraction process by which the sample components are separated as a function of differences in polarity. The column is packed with a solid support coated with a liquid immiscible with the mobile phase or eluent. The mobile phase is usually non-polar (an organic solvent) and the stationary phase polar. These are the ingredients of so-called “normal chromatogra-phy” as opposed to “reversed phase chromatography”; the latter uses a polar (aqueous) mobile phase and a non-polar stationary phase.(c) Ion exchange. In ion-exchange chromatography, the stationary phase is a resin with negatively or positively charged covalently bound groups that attract ions of the opposite sign via electrostatic forces. This type of chromatography is dealt with in detail later on.(d) Exclusion. Exclusion chromatography, also known as “gel chromatography”, relies on a screening effect dependent on the size of ions and molecules. The column is filled with a porous stationary phase or a gel, so the packing particles possess inner channels. The larger molecules in the sample can only pass between the gel particles, whereas the smaller ones can also penetrate the gel via tortuous paths. As a result, the bulkier particles will leave the column before the smaller ones. In addition, adsorption of the analytes onto the gel surface can give rise to partition coefficients greater or less than unity.

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(e) Affinity. In affinity chromatography, the solute interacts in a specific manner with a functional group in a molecule covalently bound to the stationary phase (immobilized on it). For example, only the protein reacting with an antibody will bind to a column if the antibody is immobilized on the stationary phase. Selectivity here can be modulated by choosing an appropriate group to interact with the analyte and exploiting both chemical bonding and steric effects.3. Components of Column Chromatography

A column chromatography assembly essentially includes the following elements:

Whichever the type of analyte interaction is involved, the stationary phase is packed in the column and the sample is inserted at one end and swept by continuous (or, less often, intermittent) flow of the mobile phase. As noted earlier, because each analyte interacts differently with the two phases, it will travel at a different velocity through the column. If velocity differences between analytes are sufficiently large and the column sufficiently long, the analytes will be separated into more or less sharp bands. An appropriate detector can be placed at the end of the column in analytical chromatography to identify and/or quantify each species (viz. to record a chromatogram, where each band will show as a peak at a position dependent on the retention time of the analyte concerned); alternatively, each species can be collected in a separate vessel for subsequent measurement with an appropriate detector. The longer the column is, the more efficiently can two species be separated; however, an increased dispersion of the sample components in the bulk mobile phase reduces the separation efficiency of the column. The optimization process should be aimed at improving such efficiency; this entails minimizing band broadening and altering the relative migration rates in order to achieve complete separation of species at the column offset. This requires the prior identification of the variables influencing the analyte migration rates and the factors resulting in band broadening. The most influential experimental variables in addition to the nature of the stationary and mobile phases - and the temperature in gas chromatography - are the flow-rate of the mobile phase and various characteristics of the column including the size of the packing particles, the length and diameter of the column, and the degree of uniformity of the packing - and also the thickness of the liquid film when the stationary phase is liquid.

4 Chromatographic Theory.The analyte (i.e. the sample component to be isolated) partitions itself

between the stationary and mobile phase depending on its specific affinity for

Solvent/s container

Pumping Injector Column Detector Data collection and presentation

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each. In 1941, Martin and Synge developed the earliest theoretical description of chromatography, which earned them the Nobel Prize in 1952. The central element in their theoretical development is the upstream partitioning concept and the definition of “theoretical plate” - by analogy with fractional distillation -, which is the column section where the average concentrations of the analyte in the stationary and mobile phase are consistent with the above-described partition coefficient.

X M « X S

The number of theoretical plates of a column dictates its separation efficiency. The ratio of the column length to the number of theoretical plates it contains is called the “plate height”. Therefore, the efficiency of a separation

relies on the number of theoretical plates of the column. A straightforward description of the procedure used to determine the size (length) of the theoretical plate (and hence the number of plates in a column) based on the dynamics of chromatographic separation is provided below. The size of a

theoretical plate is defined as the height equivalent to a theoretical plate (HETP), which is a function of various column-related factors including the following:(a) the effect of mass transfer;(b) the eddy effect;(c) the diffusive effect; and(d) miscellaneous effects including interactions between solutes, the size and uniformity of the packing particles, the thickness of the mobile phase, and variations in the flow-rate of the mobile phase across the column (as a result of gaseous phases being compressible). In Table 4 are presented auxiliary parameters and equations in chromatography.

Table 4 Auxiliary parameters and equations in chromatographyParameter Parameter

nameDefinition

Distribution constant (coefficient)

Equilibrium constant Amobile«Astationary

tR Retention time Time interval from sample insertion to maximum peak appearance

tM Dead time Time interval required for a non-retained solute to be transported through the column.

HETP

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Capacity factor Describes the migration velocity of a solute into the column. Theoretically 1<k´A<5. (k´A<15). Depending on the T, mobile phase composition and filling in the column.

Selectivity factor Indicating the relative situation of two peaks: being B the most retained and A the most quickly eluted ® a>1. Depending from mobile and stationary phases composition.

H: plate height N: number of theoretical plates

H and N indicate the efficiency of the column.>N and <H ® >efficiency

Resolution Indicates the column ability to separate two different solutes.RS ³ 1.5 ® complete separation

CS: solute concentration in the stationary phase;CM: solute concentration in the mobile phase;W: base-wide peak. W = 4σ.

Mass Transfer EffectAn analyte that was very strongly retained by the stationary phase would stop

at the first plate and require a large volume of mobile phase for elution. On the other hand, one not retained at all by the stationary phase would migrate with the eluent front. In between these two extremes, actual analytes are retained to a variable degree and require the use of specific conditions for separation.

During the separation process, the analyte passes from the stationary phase to the mobile phase and back many times. The transfer is not instantaneous; rather, it depends on the rate of diffusion of the analyte in the two phases. The diffusion rate in turn depends on the prevailing concentration gradient. Equilibrium, which can never be fully reached in a dynamic system of this type, is favored by high diffusion coefficients, short diffusion distances and large interface areas between the two phases. Thus, if the mobile phase is thick, the solute will have to travel long distances to reach the stationary phase and vice versa.

In summary, the analyte will travel farther down the column than one would expect from its partition coefficient alone. As a result, the actual chromatographic peak will be broader than expected.

This effect is especially prominent in gaseous phases as a result of the increased transfer coefficient within a gas.

The transfer of an analyte from the mobile phase to the stationary phase and back is favored by the following:(a) a high diffusion coefficient;

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(b) a small distance between the two phases;(c) a large interface area; and(d) a low velocity of the mobile phase.

Eddy EffectThe velocity of the mobile phase is not the same at every point of a

theoretical plate. In fact, it is lower near the stationary phase than in the middle of the stream; the travel of the mobile phase through the stationary phase can be compared to that of a mountain stream flowing along an uneven bed splitting into several independent courses at different points. As a result, the velocity at which the analyte travels varies between two extreme values. Thus, some analyte molecules (or ions) will travel very rapidly, while others will be delayed and give rise to a new dispersion factor and hence to broadened chromatographic peaks. In some cases, column efficiency can be improved by using a stationary phase of low viscosity with an increased diffusion coefficient for the solutes.

Diffusive EffectA solute in a mobile phase can travel (diffuse) freely in any direction, not

only towards the stationary phase or away from it. Diffusion causes displacements from the regions of increased concentration by effect of a concentration gradient. The resulting dispersion will be higher for solutes with high diffusion coefficients and mobile phases flowing at a high rate. This phenomenon (marked with B/v) has the opposite effects of mass transfer. The mass transfer, eddy and diffusive effects in combination do not delay retention of the solute by the stationary phase, so they do not alter the mean velocity of the solute (i.e. the maximum of the chromatographic peak). However, they have a decisive influence on the solute dispersion within the chromatographic system - they result in broader peaks and hence is poorer analyte separation.

Miscellaneous EffectsThe three above-described effects are not the only ones influencing

chromatographic development. In fact, a few others exist that contribute to peak broadening and thus influence HETP. Thus,(a) Each solute molecule or ion can act independently of the others. In the presence of interactions between one another, solutes may be eluted sooner than expected (e.g. in non-linear chromatography). Such interactions are absent from linear chromatography, so partition coefficients are identical at any point in the chromatographic system.(b) The affinity of the stationary phase for the solutes varies across its surface. Thus, sorbent surfaces can be partially blocked by other polar substances such as water.(c) The thickness of a liquid stationary phase is not uniform throughout the chromatographic system.(d) Because gaseous mobile phases are compressible, their flow-rate changes with the distance traveled through the column.5 Ion Chromatography

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Ion chromatography peaked in popularity in the 1960s and 1970s. Although the subsequent inception of a new generation of chromatographic methods has lessened its significance, it continues to be widely used in routine applications.

Originally, ion chromatograph relied on two successive processes, namely: (a) the chromatographic separation of ionic compounds by interaction and/or exchange with charged sites on the stationary phase, and (b) the neutralization or suppression of the large amount of salt species produced in the previous step by formation of non-conducting species (water or carbonic acid) and the enhancement of the intrinsic conductivity of the analytes by conversion into strong acids or bases. The latter step was dictated by the type of detector used (a conductimeter). Ion ExchangeA. Stationary Phase

The stationary phase consists of a resin with a backbone bearing of a specific functional group capable of exchanging ions. The backbone of a typical ion exchanger consists of a styrene-divinylbenzene copolymer. This forms a three-dimensional hydrocarbon structure containing many instances of the following chemical sequence. This backbone can be easily obtained and is physically and chemically stable under certain conditions. For use as an ion exchanger, it is supplied with ionic groups.

Depending on the character of the ionic group introduced, the exchanger can be of various types the most prominent of which are acid, cationic and basic (anionic). Table 5 shows selected examples of each type of exchanger. One end of the functional group is covalently bound to the hydrocarbon backbone; the other (counterion), which bears charge of the opposite sign, binds to it via electrostatic forces. This latter portion can be exchanged with other ions present in the mobile phase.

Table 5 Types of exchangersIonic exchangers Type Chemical group

Cation exchange Strong acidic Sulfonic acid: -SO3H; -CH2CH2SO3HWeakly acidic Carboxylic acid: -COOH; -CH2COOH

CH=CH2

CH=CH2

CH=CH2

(I) (II)

CH CH2 CH CH2

CH

CH CH2 CH

CHCH2 CH2 CH2CHCH

CH CH2 CH CH2

CH

CH CH2 CH

CHCH2 CH2 CH2CHCHR R

RR

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Anion exchange Strongly basic Quaternary amine: -CH2N(CH3)3+OH-;

-CH2CH2N(CH2CH3)3+OH-

Weakly basic Amine: -NH3+OH-;

-CH2CH2NH(CH2CH3)2+OH-

B. Ion-exchange Processes A resin bearing sulfonic groups (R–SO3

–H+), for example, will establish the following ionic equilibrium:

n R-SO3-H+ + Mn+ « (R-SO3

-)nMn+ + n H+

solid solution solid solution

In an acid medium, all ionic sites in the resin will be occupied by hydrogen ions. However, such H+ ions can be displaced by other ions. As a rule, the affinity of the resin will be maximal for ions possessing a high charge and charge density, and a small size - size here means the actual size of the ion in solution, hydration sphere included. Once the target ions have been bound to and retained by the resin, they can be eluted by using a solution containing an anion with a higher affinity for the resin (a strong acid) in order to displace the equilibrium back to the left.

C. Mobile Phase In ion chromatography, the mobile phase is usually an aqueous solution

occasionally containing some miscible organic solvent and an ionic species with buffering properties. Any conventional buffer is theoretically useful for this purpose. Anion exchangers are used with buffers consisting of positively charged species and the opposite is true for cation exchangers - phosphate buffer can usually be used with both types of exchanger. Eluent power and elution selectivity depend on the particular type and concentration of the species added to the mobile phase. In some applications, the composition of the mobile phase is changed during the chromatographic process; this is known as “gradient elution”.

D. Detectors The ideal detectors for ion chromatography are those based on

conductivity, which are universal for charged species and can be highly sensitive in addition to simple, inexpensive, easily miniaturized, robust and long-lasting. Their lack of selectivity poses no problem here as the analytes are previously isolated. However, ensuring that all analytes will be eluted from the column within a reasonable time entails using a high electrolyte concentration; this decreases the sensitivity of the determination because of the conductivity of the eluent concealing that of the sample components. As stated above, this problem can be overcome by using appropriate suppressors behind the ion-exchange column.

E. Ion Suppression The earliest suppressors used in ion chromatography were ion-exchange

resins that converted the solvent ions into scarcely ionized molecular species

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without altering the analyte ions. Thus, with HCl as eluent, the suppressor column contained an anionic exchanger in hydroxide form, so the exchange process was as follows:

H+(aq) + Cl–

(aq) + Resin+OH–(s) → Resin+Cl–

(s) + H2O

For anions, the suppressor was the acid form of a cation-exchange resin and the eluent a carbonate or bicarbonate solution.

Suppressor columns must be regenerated on a regular basis (every 8–10 h) by converting the packing back to the original acid or basic form.

More recently, new types of suppressors such as the following have been developed:(a) Hollow fibers of polymeric ion-exchange material that can be regenerated by

passing an appropriate solution over their outer surfaces.(b) Micro-membrane suppressors based on ion-exchange membranes that are

described later on.(c) Electrolytic membrane-based suppressors, where ion transfer across the

membrane is favored by applying an electrical field.(d) Packed-column mini-suppressors, which use two suppressor cartridges

connected to a 10-port valve. In its starting position, the eluate is passed through one of the cartridges only. When the regeneration capacity of such a cartridge, which is transparent, is exhausted (viz. when the pH indicator it contains exhibits a color change that signals the need for regeneration), the valve is switched to have the eluent pass through the second cartridge and the first is replaced.

(e) Continuously regenerated packed-column suppressors, which rely on the ion reflux principle. This is an ion-exchange technique involving the passage of water over an electrically polarized resin bed and using an electrolytic reaction to produce the eluent and suppressor medium.

Applications The quality of environmental water (rain, lake, underground, river) is

usually assessed from analyses for inorganic ions such as sulfate, chloride, nitrate, sodium, potassium, ammonium, magnesium and calcium. Monitoring ion contents in water involves the simultaneous separation and determination of anions and cations by ion chromatography. A number of approaches have been explored for this purpose including the use of mixed beds of cation and anion exchangers or two individual columns and as many detectors.

A. Determination of anions (CrO42–, MoO4

2–, BrO3–, SeO3

2–, SeO42–, HAsO4

2–

and WO42–) and cations (Cu2+, Ni2+, Pb2+ and Cd2+)

This is a joint determination of several ions in river water samples. The analytes are all known to be toxic to humans, animals and plants. Toxicological analyses must not only be highly selective, but also allow the speciation of ions as their deleterious effects depend on their specific valence states. This determination involves the chromatographic separation of both metal ions (following chelation

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with Na2EDTA) and non-metal ions (by suppressed anion-exchange chromatography). The novelty here is that the analysis time is reduced by performing gradient elution under optimal conditions; in this way, each analysis takes less than 20 min. The use of a gradient introduces a gradually increasing competitive advantage in the elution process (i.e. the sweeping of retained ions); however, the use of a conductimetric detector can lead to baseline drift and substantially increased salt concentrations in the eluent as a result. In this particular determination, elution gradients were programmed by using a baseline balancing method.

B. Determination of Na2+, NH4+, K+, Mg2+, Ca2+, IO3

–, BrO3–, Br–,

NO3– and Cl– in various types of water (river, pond, tap)

The process here involved the use of two columns packed with and anion exchanger and a cation exchanger, respectively. The columns were placed one after the other or accommodated in the loops of two injection valves.

Figure shows the assembly with the two columns arranged in a serial manner. The sequence in which the two were placed was found to influence the elution profile. A high cation concentration can result in the formation of ion-pairs with the anions; therefore, the cation-exchange column should precede the anion-exchange column when the eluent contains many cations. However, the peaks for the cations deteriorate when these are passed through the anion-exchange column, which is not the case with those for the anions. Also, ions such as calcium, iodate, bromate and nitrite cannot be determined under these conditions owing to the resulting peak overlap. When the sample volume (20 μL) was injected, the two valves were in their injection positions, so the sample was allowed to reach the loops accommodating the two columns. Cations were retained by the cation-exchange column while anions were passed through it and reach the anion-exchange column. Within 1.45 min, all anions were retained by the latter column, the second valve then being switched to the next position.

Separating the cations took 10 min, after which the valves were switched and all analytes allowed to reach the detector.

For comparison purposes, the detection was done with a spectrophotometer and a conductimeter. Cations were detected by UV-VIS absorption spectrophotometry in an indirect manner and the mobile phase was supplied with an absorbing additive (viz. the aminoacid L-hystidine). The passage of each cation through the detector produced a negative peak. Absorbance measurements were made at 210 nm. 6 Gas Chromatography (GC)

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Gas chromatography can be directly applied to volatile compounds; however, it is more commonly used with liquid samples the components of which can be readily volatilized at the working temperature by derivatization.

The sample should be volatilized immediately upon injection into the chromatographic system. The elution is done using an inert gas as the mobile phase, as this is intended to carry the analyte through the column rather than interact with it - unlike most liquid chromatography applications.

Gas chromatography can be of two different types depending on the nature of the stationary phase, namely: gas-solid chromatography (GSC) and gas-liquid chromatography (GLC). Gas–solid chromatography uses a stationary phase that retains the analytes by adsorption. Its scope of application is highly restricted as a result of it usually giving tailed peaks - a consequence of non-linear adsorption - and of strongly polar molecules being retained almost permanently. For these reasons, it is usually applied to species of low molecular weight only. Gas-liquid chromatography, henceforward called simply “gas chromatography”, relies on the partitioning of the analyte between a gaseous mobile phase and a stationary phase consisting of a liquid immobilized onto the surface of an inert support.

Instrumentation for Gas ChromatographyThe basic components of a gas chromatograph are depicted in Figure, where - W, waste; D, detector; C, computer; is, injection sample; gc, control of the gas flow-rate; sc, separation column; and, rc, reference column (nor present in any commercially available model).

The carrier gas, which must be chemically inert, is usually helium, argon, nitrogen, carbon dioxide or hydrogen. The choice is frequently dictated by the type of detector used. Also, the gas container must be equipped with pressure control and measurement facilities, a flow meter and a molecular sieve (to remove water or other impurities). The sample introduction system should allow the insertion of a sample plug of appropriate size into the system. The most common

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insertion method involves using a micro-volumetric syringe to inject the liquid or gaseous sample via a silicone rubber septum into a vaporization chamber located at the column top. The chamber is about 50 ºC below the boiling point for the least volatile component of the sample. The mobile phase then sweeps the sample to the first plate in the chromatographic column, which minimizes dispersion.

Injected sample volumes usually range from 10–3 to 20 μL. For sub-microliter volumes, a stream splitter is used to circulate the sample through several channels only one of which leads to the column; in this way, only a small fraction of sample reaches the column while the rest is sent to waste. Quantitative work, where reproducible insertion of the sample is crucial, requires the use of rotary valves similar to those employed in HPLC and FIA. Solid samples can also be introduced into the chromatographic system, using glass vials of very thin walls that are broken from the outside once the vials have entered the injection chamber at the top of the column.

The earliest GC systems used packed columns where the stationary phase was a thin film of liquid coating the surface of an inert, finely divided solid support. A packed column consists of a glass, metal or Teflon tube 2–50 cm long and 1–4 mm in inner diameter. The tube is packed with a finely divided, homogeneous solid coated with a thin film (0.05–1 μm) of the stationary phase (a liquid). For easier accommodation into the chromatographic oven, the column is coiled to a diameter of about 15 cm.

The efficiency of the column increases markedly with decreasing size of the packing particles; however, this also increases pressure within the system, so particles are rarely smaller than 150 μm in diameter.

Packed columns afford larger injected volumes than do capillary columns; however, throughput and efficiency are better with unpacked columns of a very small inner diameter (a few tenths of a millimeter). These capillary columns are used with a stationary phase consisting of a uniform film of liquid a few tenths of a micrometer thick that is used to coat the inner walls of the capillary tube. This type of column is also known as open-end column.

The materials from which these columns are made, and their coiled configuration, coincide with those of packed columns. Their inner diameters typically range from 250 to 320 μm, but can be smaller (200 or even 150 μm). The sample volume is usually very small, so the detector must be highly sensitive. The thickness of the stationary phase usually ranges from 5 μm for highly volatile species to 0.1 μm for less volatile ones.

Although packed columns are more inexpensive and easy to use, capillary columns provide higher resolution.

One problem with GC in both packed and capillary columns arises from the physical adsorption of polar compounds onto the surface of the stationary phase, which usually contains silicates. Overcoming it entails pre-treating the columns to remove SiOH groups that form on the support surface through hydrolysis by existing moisture.

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The liquid stationary phase used should be scarcely volatile, thermally stable and chemically inert; also, it should provide capacity and selectivity factor (k′ and α) values within appropriate ranges for the analytes. The most suitable stationary phase must be determined on a case-by-case basis; in any case, the material should be similarly polar to the sample components.

The column temperature is very important here and should be strictly controlled by using a thermostated oven. The optimum temperature in each case depends on the boiling point of the particular sample and the degree of separation required. Usually, the working temperature is slightly above the mean boiling point (bp) for the sample components. If the bp range spanned by such components is too wide, a temperature program is used instead to change the column temperature during the separation process. Usually, resolution improves with decreasing temperature, at the expense of longer elution times; a compromise must therefore usually be made in this respect. As a rule, the retention time doubles with each rise in temperature of 30 ºC.

The chromatographic column is followed by the detector. The types of detectors used are rather different from those employed in liquid chromatography and can be classified as follows in terms of sensitivity:(a) Medium Sensitivity:

Thermal Conductivity Detector, TCD or catharometer Gas density balance (to check other detectors, not commercial

(b) High sensitivity:(b.1)- non-radioactive ionization:

flame Ionization Detector, FID thermo-ionic

(b.2)- radioactive ionization: electron capture detector, ECD ionization of argon

The most commonplace - even in portable equipment - are the thermal conductivity detector (TCD), the flame ionization detector (FID) and the electron capture detector (ECD). In addition, a mass, IR or NMR detector is frequently used to facilitate the identification of individual components in mixtures.

The thermal conductivity detector (TCD, see Figure), also called “catharometer”, was used in the earliest GC applications. It relies on a combination of the thermal and electrical properties of the sample components. This type of detector is very simple, affords wide linear ranges, responds to both inorganic and organic compounds, and is non-destructive. On the other hand, it is

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scarcely sensitive, so it cannot be used with capillary columns, where sample size is usually very small.

The flame ionization detector (FID, see Figure) is the best for organic compounds and one of the most commonplaces in current commercial instruments. The effluent from the column reaches a burner where it is mixed with hydrogen and air that is electrically ignited. Burning of most organic compounds under these conditions produces ions and electrons that make the flame space conductive. By applying a potential difference between an electrode that can be the burner end itself and the collector electrode, placed above the flame, an electrical current is produced that represents the detector response to a sample component concentration.

This type of detector is scarcely sensitive to flow-rate changes in the mobile phase and not responsive to water, CO2, SO2 or nitrogen oxides, so it is unaffected by the presence of moisture or oxide impurities in the sample; however, it is highly sensitive to the target species. Also, it exhibits a very broad linear response range and low background noise, and is robust and easy to use, but has the disadvantage of its destructive character.

The electron capture detector (ECD, see Figure) irradiates the effluent column with γ radiation to alter the electrical conductivity of the gas. The presence of electron-withdrawing organic molecules decreases the current. This type of detector is selective for organic molecules bearing electronegative functional groups (halides, peroxides, quinones and nitro compounds), but is insensitive to amines, alcohols and hydrocarbons. It exhibits a high analytical sensitivity and scarcely alters the sample. Its linear response range, however, is somewhat narrow.7 High Performance Liquid Chromatography (HPLC)

Based on the general theoretical principles exposed in introducing chromatography, the contact surface between the mobile phase and stationary phase should be as large as possible. This makes the size of the particles constituting the stationary phase (or its support) especially influential; in fact, the smaller such particles are, the greater will be the number of theoretical plates of the column and the higher its separation efficiency. Packing uniformity is also very important to avoid distorted, poorly resolved chromatographic peaks. The

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small particle sizes used now do not allow the mobile phase to progress through the column solely by gravity. This entails using an external force such as that provided by pumping at a high pressure. This in turn calls for a stronger chromatographic system consisting of materials capable of withstanding the high pressures to be used. The system is made even more complicated in relation to traditional liquid chromatography by the need to:(a) separate chemically similar components;(b) deal with complex samples by integrating some sample pretreatment

operations on-line;(c) improve sensitivity and detection limits by —usually post-column—

derivatization of the resolved components in order to enhance their detection characteristics; and

(d) use carefully programmed gradients of the mobile phase.Propulsion Unit and Elution Modes (isocratic and gradient-based)The propulsion system is intended to provide the pressure required to force

the mobile phase to pass through the column at as controlled and uniform flow-rate as possible (i.e. in the absence of a pulsating flow). The most widely used propulsion system in this context is the piston pump, which can provide high pressures but causes the flow to pulsate every time the piston is loaded or emptied. This shortcoming, however, can be greatly circumvented by using two pumps in opposite phases. Gradient elution requires accurate, programmed control of the mixing of the solvents making up the eluent. This allows such variables as the solvent polarity, ionic strength and pH to be altered during elution.

The outlets of several flasks containing the different solvents (or solutions) are connected to a valve allowing their flow-rates to be continuously controlled. In this way, mixing proportions can be changed as required at any time. The solvents used must be highly pure and degassed as bubbling results in distorted or even spurious peaks. The problem can be worsened by the use of mixed solvents (e.g. acetonitrile or methanol in water) as air is less readily soluble in solvent mixtures than it is in individual solvents. This entails the use of a de-bubbling system to reduce the air concentration below saturation levels. Helium can remove up to 80% and evacuation up to 60% of the air initially present in the system. Many operators, however, choose to use a helium pretreatment followed by evacuation.

ColumnsHPLC columns vary in length, diameter and content. They should be as

chemically inert as possible and capable of withstanding high pressures. Stainless steel tubes 3.9 or 4.6 mm in diameter are quite suitable for HPLC work. A column consisting of particles ca. 5 μm in diameter tightly packed in a stainless steel tube of 4.6 mm i.d. easily provides 60 000–90 000 theoretical plates per linear meter.

The use of a 3–10 cm long pre-column packed containing the same packing as the column is usually advisable. Its importance lies in the facts that (a) it can act as a filter by retaining solid particles (impurities) in the sample or solvents that might partially block the column and alter its efficiency and selectivity; and (b) its

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absence shortens the column lifetime. The pre-column should be regenerated or replaced on a regular basis.

Most HPLC work involves partitioning with liquid stationary phases chemically bonded to a support surface. Especially commonplace among the ligands bonded to silica gel are hydrocarbon chains of 8 or 18 carbon atoms (C8 and C18, respectively). Columns modified with octadecyl ligands are often referred to as “ODS columns”. There are ODS1 and ODS columns, the two types differing in the proportion active residual OH groups, which is variable in ODS1 (it contains a specific number of OH groups) and zero in ODS. However, manufacturers tend to give their columns a trade name not specifying the treatment they have received.

The problem with silica gel as a chromatographic support is its narrow operating pH range (3–7.5) and working temperature. However, its properties can be altered by deactivating residual silanol groups using the “end cropping” method.

The UV detector often uses a single interference filter, so it can only measure concentrations at a few different wavelengths. The more sophisticated models use a monochromator to select the most suitable wavelength in each situation; this enables qualitative identification by stopping the mobile phase flow. The UV detector is normally more sensitive than the refractive index detector; thus, the former can detect concentrations down to 0.01 ppm or even as low as a few nanograms if an appropriate chromophore is used to derivatize the analytes post-column.

Diode array detectors constitute powerful tools for qualitative analysis as they allow spectra to be directly recorded without the need to stop the mobile phase flow. The ability to resolve overlapped spectra by using spectral derivatives or an alternative chemometric technique results in further increased separation power.

Fluorescence detectors can be more sensitive and slightly more selective than UV detectors.

Finally, amperometric detectors are widely used for the detection of electroactive biochemical substances.

QUESTIONS FOR SELF-CONTROL:1. What is the basis chromatographic method of separating substances?2. How is chromatographic methods classified for the state of aggregation?3. On what is ion exchange chromatography foundation?

REFERENCES1. Gary D. Christian, Analytical chemistry (6th edition), Wiley 20042. Harvey D., Modern Analytical Chemistry, Mc Graw Hill Comp. Inc., 20003. D. Projean, T. M. Tu and J. Ducharme, J. Chrom. B, 2003 (787) 243-253.4. D. Wittintgton, E. D. Kharash, J. Chrom. B, 2003 (786) 95 - 103.5. T. C. R. Santos, J. C. Rocha and Damiá Barceló, J. Chrom. A, 2000 (879) 3 - 12.

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6. V. Kmetec and R. Roskar, J. Pharm. Biomed. Sc. 2003 (32), 1061- 1066.7. P. Ptacek, J. Macek and J. Klima, J. Chrom. B, 2003 (789) 405 - 410.

3 LABORATORY WORK

Methodical recommendations for conducting laboratory works

Laboratory studies promotes to strengthening of the knowledge by the physical methods of chemical research, develops of the student’s independence and instills the skills of the experiment. For successfully work in the laboratory you must first study the theoretical material from textbooks, lecture notes and the manuals on chemical workshops. This produces a conscious attitude to the implementation of experimental techniques and the work will be understood. In the chemistry lab workers should strictly follow by the safety rules and regulations of the working with chemical utensils and appliances. We must learn to use chemical agents, chemical equipment, which are listed in the guidelines for the work on the chemical workshop. Guidelines should not be deprive independence, and oppositely the following by the orders speeds up the work, prevents possible damage to equipment, glassware and reagents. The success of the experimental work not depends only from the correctness of the choice of working methods, the sequence of measurement, weight measurements, but also from the correct systematic recording of results. To the implementation of the laboratory work are allowed students with admission after verification by teacher the theoretical knowledge on the subject, knowledge by the methodic of laboratory work and verification of preparing lab journal to conduct entries. After completing the laboratory work the student must bring order to your workplace and deliver them on duty or technician. After processing the results in the lab book the student must the report to teacher.

Thematic plan of laboratory work

Name of the theme Number of hours

Manual

Laboratory work № 1. Rules of working on the fotoelektrokolorimetre

1 Methodical instructions

Laboratory work № 2-4 Spectrophotometric determination of manganese using a calibration curve


Laboratory work № 5-6 Photocolorimetric determination of iron using a calibration curve


Laboratory work №7-8Colorimetric determination of nitrite.


Laboratory work № 9-10 2 Methodical instructions

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Measuring the pH of the salt and aqueous extracts of soils by potentiometryLaboratory work № 11-12Preparation and measurement of pH buffer solutions by potentiometry


Laboratory work № 13-15Potentiometric titration of a strong acid with strong base


Laboratory work № 16-17Determination of the neutral salt in the solution by ion-exchange chromatography


Laboratory work № 18-20Determination of the dynamic exchange capacity of cation exchanger


Laboratory work № 21-22Separation of iron (III) and copper (II) by paper chromatography


Laboratory work № 23-24Separation of a amino acids mixture


Laboratory work № 25-26Determination of ions of iron (III), copper (III), nickel (II), cobalt (II) in a meat broth by ion exchange chromatography


Laboratory work № 27-28The separation of inorganic ions by precipitation chromatography.


Laboratory work № 29-30Quantitative determination of nickel by the size of precipitate zone


Methodical instructionsLaboratory work № 1. Rules of working on the fotoelektrokolorimetre

Purpose: To learn the basic principles and rules of operation on KFK - 2Objectives:

choose the most optimal wavelength and size of the cell to determine the optical density of the proposed solutions.

measure the optical density of the two colored solutions.Stages of the work: 1. Rules for selecting the filter

You should be noted that in fotoelektrokolorimetry is used a monochromatic light of different wavelengths. Color filters are used to convert a polychromatic light in a monochromatic. The KFK-2 has a set of 11 filters. The using of specific filter allows to pass the rays of a certain length through the solution, the absorption of which is most characteristic of the substance. Typically, the effective wavelength and the color filter is

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shown in the applicable method. If such a reference does not, you can use the table to select the filter:

The Color of sample solution

Necessary color filter The wavelength of the transmitted light, nm

YELLOW BLUE 420 – 450ORANGE BLUE 430 – 460RED GREEN 460 – 500PURPLE GREEN 490 – 530BLUE ORANGE 590BLUE-GREEN RED 600 – 650LIGHT BLUE RED 750THE BLUE-VIOLET RED 750

Comment. Some of the solutions with the same color can selectively absorb the rays of different wavelengths. Thus, the optimal wavelength filter will be the one which optical density in the case of passage through the fluid sample is maximum. 2. Selection of cells

It is known that at the thicker fluid’s layer, through which the beam of light passes, the absorption of the light beam is greater and the optical density of the sample solution is higher. Each Colorimeter included a set of cells from 1 to 50 mm., differing from each other with the distance between the working faces, through which the light beam passes. This distance (in mm) is shown in one of the working faces. On the side of the cells you can see a label, shown the limit to which you must pour a liquid. The cell must be closed with special caps, when you are working with the volatile solutions.

Selection of cells is carried out so that the optical density of the sample solution is not lower than 0.15 and above 0.7. Within these limits Bouguer Lambert Beer law is most closely satisfied. Consequently, at an intense coloration of the solution you must take a cell with a smaller distance between the working faces, and at weak coloring - cell with a greater distance.3. Rules for working on the KFK-3

GETTING STARTED 1. Connect the photometer to 220V, 50/60Hz, and turn on the tumbler 2. Press the START – on the digital display appears the symbol " Г ", the corresponding it’s value and the value of the wavelength. Withstand the photometer turned on condition 30 minutes at the opening lid!

ORDER OF PROCEDURE 1. At the closed sample compartment lid to press "Г". On the left bottom of the digital display will show a flashing point symbol "Г". 2. Press the "П" or "E". At the left of the flashing-point respectively displayed the symbol " П" or "E", and at the right of the flashing point - respectively the values «100.0 ± 0.2» or «0.000± 0.002», meaning that the initial sampling pass (100.0%) or optical density (0.000 ) installed correctly. 3. Then the handle set to the right until it stops, and in this case the light beam is introduced into the cell with the test solution. The data on the right of the flashing-point corresponds with the transmittance or absorbance of the sample solution. 4. Repeat the operations three times and calculate the average value

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COMPLETING WORK ON THE DEVICE1. To pour reagents from the cells.2. To rinse the cuvette with distilled water and to place in a Petri dish with bottomed up (to rinse the cell after full completion the work or technique, should not do this in the intervals between individual measurements!).3. Turn off the device is (tumbler on the rear wall in the left corner switch to "off.").4. Close the sample compartment lid Notice ! When working on a KFK-3 you must follow to these rules: To monitor for the purity of the device, not to spill reagents. Do not clap with the sample compartment lid. Especially to be careful with cuvettes - do not scrape, you must wipe with a soft, clean cloth (gauze). At the changing the filter to continue the work no earlier than 5 minutes.

4 Construction of calibration curve and determination coefficient of the factorization 1. Prepare a series of solutions with known concentrations covering the range of possible concentration of this substance in the test solution. 2. Measure the optical density of all solutions and build a calibration curve by plotting the known concentrations on the horizontal axis and the vertical - the corresponding values of optical density. You should be ensure that the graph is expressed by a straight line.3. The coefficient of factorization F is calculated by the graph. For this, necessary determine the concentration (C) in the middle of the graph and the corresponding of this concentration the optical density (A).

If at the construction calibration curve is established that the relation between optical density and concentration is not linear, the determining of the coefficient of factorization F not required. Determination of the concentration in this case carried out according to calibration curve4. The results of this research work should be maked out as a table: Color of the solution

Wavelength (nm)

Filter’s color The size of the cell (mm)

The optical density (E)

Findings:

Laboratory work №2-4. Spectrophotometric determination of manganese using a calibration curvePurpose: To determine the concentration of manganese in the test solution using a calibration curveObjectives:


measure the optical density of the standard solutions.

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construct a calibration curve on millimeter paper find a manganese concentration in the proposed solution

Reagents: sulfuric acid (ρ = 1,84), phosphoric acid (ρ = 1,72), silver nitrate (0.2% solution), ammonium persulfate (NH4)2S2O3.Proceedings. 1. The preparation of the standard solution. In volumetric flask for 100 ml place exactly with graduated pipette 9.1 mL of 0.1n potassium permanganate solution (pre-cooked), add distilled water to the mark and mix. The thus prepared solution contains 0.1 mg of manganese in 1ml. 2. Build a calibration curve. For this purpose, take 5-6 flasks for 50.0 ml and prepare standard solutions with desired concentration from the basic standard solution of potassium permanganate. For this, fill with pipette 2.0, 4.0, 6.0, 8.0, and 10.0 ml of potassium permanganate solution to each flask; after that add by 1 ml of sulfuric acid (1.84), lead up to the mark with distilled water and mix.Graph construct as in lab work #1. Take as zero solution the distilled water. 3. Take 10 ml analyte solution with pipette, place it in a 100 ml conical flask and poured to it 1 ml of sulfuric acid, 0.5 ml of phosphoric acid, 40 ml of distilled water and mix. Then add 10 drops of silver nitrate solution and 1.2 g of crystalline ammonium persulfate. Resulting solution is heated until the purple-red color and the discontinuation of the gas bubbles (heating leads on a sand bath at a temperature of 65-75 ° C). Then the mixture is cooled and quantitatively transferred to a 100 ml volumetric flask, add distilled water to the mark and mix. Then the mixture is put on 15 minutes in a dark cupboard and is determined the optical density (E) at 526nm wavelength, cell length 20 mm. Performance of the work similarly as described, but at the measurement of the optical density for the zero solution is used the first standard solution. 4. Make a conclusion with reaction equations according to the results of experiments.

Laboratory work № 5-6. Photocolorimetric determination of iron using a calibration curvePurpose: To determine the concentration of iron in the test solution using a calibration curveObjectives:


measure the optical density of the standard solutions. construct a calibration curve on millimeter paper find a iron concentration in the proposed solution

Reagents - nitric acid (1:1), 10% solution of ammonium thiocyanate. Proceedings. 1. Preparation of standard solution. A portion of 0.864 g (chemically pure) dodekahydrate ammonium iron (III) sulfate is transferred into a 1000 ml volumetric flask, acidified with 5 ml of sulfuric acid (ρ = 1,84) and dilute with water to 1 liter. The solution contains 0.1 mg iron in 1 ml.2. Construction of calibration curve. In the 50 ml volumetric flasks with a graduated pipette are placed consistently 0.5, 1.0, 2.0, 3.0, 4.0, 5.0 ml of standard solution of iron

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salts, then into flasks is poured 1 ml of diluted (1:1 ) nitric acid and 5 ml 10% solution of ammonium thiocyanate. Volume is adjusted by distilled water, mixed and photometer with blue filter and the 10 mm layer cuvette. The optical density is measured 3-4 times and by calculating the average value of it, build a calibration curve. 3. Determination of iron ion (III) in solution. The sample solution containing from 0.05 to 0.5 mg of iron, is placed in a 50ml volumetric flask, acidified with 1 ml of dilute nitric acid, add 5 ml 10% solution of ammonium thiocyanate, and reduce the volume of the flask to the mark with water, mix. Determine the absorbance of the sample solution (3-4 times) in a cell of the same thickness of 10mm and blue filter. For the zero solution is took distilled water. The iron content in the test solution is determined by the value of optical density on the calibration curve. Results of the analysis are entered into the table.4. Make a conclusion with reaction equations according to the results of experiments.

Laboratory work №7-8. Colorimetric determination of nitrite.The purpose of the work: Finding the optimum conditions for photocolorimetric determining of substance’s concentration in solution by the calibration curve.Objectives:


measure the optical density of the standard solutions. construct a calibration curve on millimeter paper find a nitrite concentration in the proposed solution

Reagents and Equipment.1. Standard solution of nitrite. Sample of sodium nitrite of qualification "chemically pure" with mass 0.I50 g is dissolved in I liter of distilled water and is added I ml of chloroform. Before using the standard solution students should be diluted 5.0 ml of this solution with water to 50.0 mL in volumetric flask. The diluted solution contain 0.0I mg NO2 in 1 ml. 2. A mixture of sulfanilic acid and phenol. Sulfanilic acid in a sample of 1 g is dissolved by heating in I00 ml of saturated ammonium chloride, to the resulting liquid is added 1.5 g phenol and I00 ml of 2n hydrochloric acid solution. 3. Sulfuric acid (r = I, 7 g/cm3) 4. 1:1 solution of ammonia5. Volumetric flask of 50 ml. 6. Volumetric pipettes capacity of 1 and 5 ml. 7. Photocolorimeter KFK-3.

Proceedings. For construct a calibration graph prepare with diluting standard a series of colored

solutions containing nitrite from 0.0I to 0.07 mg in 50 mL (0.0I, 0.02, 0.03, etc.). For this "n" ml is placed in a 50 ml volumetric flask, is diluted to 25 ml with water, add 0.25 ml of sulfuric acid, 1 ml of sulfanilic acid and phenol and allowed to stand for I5 minutes. After that, add 6 ml of ammonia solution; adjust water to the mark and mix. Use these solutions for determine the optimal wavelength for measurements (filter) and choice the cell. At the chosen conditions measure the color intensity of the resulting solutions. For the whole of this series build calibration graph according to the obtained values of the

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optical density. Received from a teacher monitoring task analyze as described above, and on graph calculate the of concentration of NO2.

Make a conclusion with reaction equations according to the results of experiments.

Laboratory work № 9-10. Measuring the pH of the salt and aqueous extracts of soils by potentiometry The goal: to get acquainted with the methodology of determining the pH of salt extracts from soils.Objectives:

get salt and aqueous extraction of different types of soil (at least 2 samples) prepare a potentiometer to work, learn to calibrate the instrument with standard

buffer solutions determine the pH of the resulting salt and aqueous extracts of soils compare these values with literature data and health regulations and make

conclusions about the physical-chemical composition of the investigated soils by obtained values of pH

Equipment.Potentiometer, standard solutions with pH 1.00, 4.00, 4.01, 7.00, 10.00, 12.43, 1n potassium chloride, flasks (100ml) - 4 pieces, ground plugs to the flask - 4pcs.Proceedings.The pH of of salt extracts only determine for the soils with acidic and neutral reaction of an aqueous suspension at the interaction between the soil and the solution of potassium chloride. In this process absorbed cations imprint from the soil. pH of salt extraction indicates the level of potential (exchange) acidity of the soil. pH of aqueous extracts indicates the level of actual acidity of the soil1. Arrangements for the working on potentiometer. Connect to the device appropriate electrodes - ion-selective and standard, according to the description of the instrument. Immerse the electrodes into distilled water Turn on and let it warm the instrument. Ion-selective electrode for installing the mode require 15 minutes. Ionometric measurements suppose preliminary calibration of the electrode system. Graduation - is putting into the device's memory parameters, obtained in standard solutions. The device will fix the response of the electrode, which is putting in the solution of known concentration of the determining ions. Sufficient number of standards, ensuring the measurement is 2. Perform device graduation for working in the pH measurement mode. For this purpose, follow these steps:1) Prepare the standard solutions for pH measurement (at least 3 with intervals of possible measured values2) Remove the electrodes from distilled water and wipe the filter paper and place it in the first standard solution. After equilibration (2minuty) proceed to the measurement and instrument calibration by the first standard solution.3) On the keyboard of the user interface, press "graduation." The device offers you a list of the pH standard solutions, which consists of six standards. With cursor to select from the list a value of pH equal to your existing solution, and then press "Enter." The device displays the settings for the selected standard.

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4) Install the cursor on the position "Grad" and press "Enter." The device go to graduation and displays the current values of the calibration parameters. Then press "Enter" if the values do not cause you to doubt and you want to lead values in memory (otherwise press "Cancel" and repeat the operation). The device automatically detects standard solution.5) Remove the electrode from the first standard solution, gently rinse them with distilled water, wipe with filter paper and place it in the second standard solution. After establishment of equilibrium (2minuty) proceed to the measurement and calibration of the device according to the second standard solution. For this purpose, perform the same operations as described in Section 2-4. Then make the same operations with the third standard solution.6) After the calibration of the device by the third standard solution remove the electrode from the solution, gently rinse them with distilled water, wipe with filter paper and place it in a glass with pure distilled water.7) On the keyboard of the user interface, press "Install". The device is ready for the measurement of pH analyzed media.2. Methodology for work.

For prepare salt extracts use the 1n potassium chloride. The ratio of soil:solution is taken equal 1:2.5. Weighed portion 10 g of soil is placed in a 100 ml conical flask, pour 25 ml of 1n potassium chloride solution, cover the contents of the flask with cork and shaken for 10 minutes, at the same time turn on the pH meter and prepare it for using. After shaken, suspension is left standing. In salt solution suspension particles settle quickly, and clarified supernatant liquid decant into glass of potentiometer.

For prepare aqueous extracts use the ratio of soil: distilled water of 1:2.5. Weighed portion 10 g of soil is placed in a 100 ml conical flask, pour 25 ml of distilled water, cover the contents of the flask with cork and shaken for 10 minutes, at the same time turn on the pH meter and prepare it for using. After shaken, suspension is left standing. In salt solution suspension particles settle quickly, and clarified supernatant liquid decant into glass of potentiometer.

pH measurement is performed by the potentiometer on the following procedure:1. Remove the electrodes from distilled water, wipe with filter paper and place into analyzed solution.2. On the keyboard user interface press "Measure". The device offers you a list of measurements. With cursor select the pH position in the list of measurements of pH (measured value) and press "Enter." The device automatically starts measuring the pH of the medium.3. To allow recording of the measured pH values of the medium, place the cursor in the fourth position of the functional line. In this case will be launch automatically record of the readings with intervals noted in the position (for example, A: 30, readings recording interval 30sec). Fixing the results can also be done manually. To do this, after a certain period of time (determined by the investigator), press "Enter" key. In memory can be recorded 60 readings. Measuring the pH of the medium spend at least 2-3 minutes. Then press the "Install".4. Remove the electrode from the test solution by gently rinse of distilled water, wipe with filter paper and place it in the second test solution5. Perform the same operations as described in clauses 1-4. Then make the same operations with all test solutions

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6. After the measurement electrodes remove from the last solution, gently rinse them with distilled water, wipe with filter paper and place it in a glass of pure distilled water.7. Turn off the unit by pressing the "On / Off". The instrument automatically stores all the calibration parameters and the current settings, the recorded results.

Make a conclusion about comparing experimental data with literature and health regulations and about the physical-chemical composition of the investigated soils by obtained values of pH

Laboratory work № 11-12. Preparation and measurement of pH buffer solutions by potentiometry The goal: to prepare buffers and experimentally determine their pHObjectives:

prepare phosphate-citrate buffers determine the pH of the resulting phosphate-citrate buffers compare these values with literature data and calculate the absolute and relative

error

Equipment. Citric acid (dry), Na2HPO4 * 2H2O (solid), potentiometer, volumetric flask 1L - 2pcs, pipette 5ml - 7units.Proceedings. 1. Prepare phosphate-citrate buffer mixture. Phosphate-citrate mixture composed of two solutions: 1) 0.1 M solution containing 21.018 g per liter of citric acid (with one molecule of water), and 2) a 0.2 M solution, contained in a liter of 35.598 g Na2HPO4 * 2H2O. Mixing different amounts of the two solutions are got various pH values.0.1 M solution Citric acid (ml)

0.2 M Na2HPO4 * 2H2O solution (ml)

рН

17,82 2,18 2,610,14 9,86 4,86,78 13,22 6,214,30 5,70 3,48,40 11,6 5,62,61 17,39 7,20,85 19,15 7,81,83 18,17 7,415,89 4,11 3,07,37 12,63 6,0

pH measurement is performed by the potentiometer on the following procedure:1. Remove the electrodes from distilled water, wipe with filter paper and place into analyzed solution.2. On the keyboard user interface press "Measure". The device offers you a list of measurements. With cursor select the pH position in the list of measurements of pH (measured value) and press "Enter." The device automatically starts measuring the pH of the medium.

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3. To allow recording of the measured pH values of the medium, place the cursor in the fourth position of the functional line. In this case will be launch automatically record of the readings with intervals noted in the position (for example, A: 30, readings recording interval 30sec). Fixing the results can also be done manually. To do this, after a certain period of time (determined by the investigator), press "Enter" key. In memory can be recorded 60 readings. Measuring the pH of the medium spend at least 2-3 minutes. Then press the "Install".4. Remove the electrode from the test solution by gently rinse of distilled water, wipe with filter paper and place it in the second test solution5. Perform the same operations as described in clauses 1-4. Then make the same operations with all test solutions6. After the measurement electrodes remove from the last solution, gently rinse them with distilled water, wipe with filter paper and place it in a glass of pure distilled water.7. Turn off the unit by pressing the "On / Off". The instrument automatically stores all the calibration parameters and the current settings, the recorded results.

Find the absolute and relative error of the experiment.Make a conclusion about comparing experimental data with literature and accuracy of the method

Laboratory work № 13-15 Potentiometric titration of a strong acid with strong base The goal: to learn to identify the equivalence point without indicatorObjectives:

collect installation for the titration determine the pH during the entire titration process using the potentiometer construct two graphs pH = f (VNaOH) and ΔpH / Δ VNaOH = f (VNaOH) and

determine the equivalence point

Equipment. 0.1 M hydrochloric acid, 0.1 M sodium hydroxide solution, potentiometer, burette for 25ml - 7units. Proceedings. 1. Collect 3 samples of 0.1 M hydrochloric acid by 10 ml in the tank for titration2. Gather installation for titration. Prior the titration the electrode must be in containers of distilled water.3. Electrode dip in the test solution. Fix the pH of the solution4. Conduct titration, gradually adding 0.1-0.2 ml of 0.1 M sodium hydroxide solution. In this case, record the pH of the solution in the table. Titration lead untill pH = 11 and get several constant pH by adding new portions of alkali. Record the data in table:

VNaOH, мл рН ∆рН/ ∆ VNaOH

Based on these data to construct two graphs pH = f (VNaOH) and ΔpH / Δ VNaOH = f (VNaOH) and determine the equivalence point by the usual and derivative curve.

Compare the data with theoretical calculations (give the theoretical calculations of pH at each point). Find the absolute and relative error of the experiment. Conclude about the accuracy of the method of potentiometric titration.

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Laboratory work № 16-17. Determination of the neutral salt in the solution by ion-exchange chromatography

The goal: To determine the presence of neutral salts in the solution using cation exchanger.Objective: Prepare a cation exchanging column in the H+- form. Passing the test solution through prepared column of cation exchanger Calculation of the salt concentration

Proceedings1. Preparing a cation exchanging column in the H+- form. For this, 5g of dry cation exchanger must be weighed. Then transfer it to a large beaker, pour with a fivefold volume of saturated sodium chloride solution and remain for a day for swelling. Thereafter, the liquid is decanted and poured with 1n solution of sodium hydroxide and left for 3 hours at occasional stirring. Then, the liquid is decanted and cation exchanger is washed abundantly with water. Swollen cation exchanger is placed in a column: for this burette is filled with water and then grain of cation exchanger from above is brought. Excess water is released from the burette. The layer of cation exchanger must have a height of about 10cm. Then 500 ml of 2n solution of hydrochloric acid is passed through the column. Thereafter cation exchanger is washed with water until neutral reaction at the presence of methyl orange indicator, or until complete removal of chlorine - test with silver nitrate.

For this effluent is taken up in a small tube from the column and methyl orange indicator is added. If the color of the solution in the test tube turns yellow, it is considered that the eluate has a neutral reaction, and the column with the cation exchanger is ready for further work. If methyl orange in the eluate colored to pink or orange, a column with the cation exchanger must be washed by a small, an amount of 10-15 ml, portions of distilled water until the eluate medium will be neutral.

2. The sample of neutral salt solution is placed in a 100ml volumetric flask, brought to the mark with distilled water and mixed thoroughly. Take with a pipette 10 ml of the test solution and pass through prepared column of cation exchanger with velocity at approximately 2 drops of 1 second. The effluent from the column is collected in a conical flask.

For completely washing out the released acid, distilled water in small portions of 10-15 ml is rinsed through a column, collecting the washings in the same conical flask, until the medium in the eluate will be neutral (check by methyl orange, taking away smaller portions of the eluate in a test tube). Then the contents of the conical flask is titrated with 0.01n solution of KOH at the presence of methyl orange. Determination of neutral salt is conducted as long as obtaining 3-reproducible results.

The salt content, m, mg is calculated by the formula:

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where C - equivalent molar concentration of working solution, - the equivalent

molar mass of analyzed salt, g / mol; - total volume of the sample solution in ml, - aliquot of the sample solution, ml.

Note: at the working should be remembered that liquid at all times must be over a bed of cation exchanger. In case of formation of air bubbles in the column cation exchanger should be fluffing the glass rod.

Laboratory work № 18-20. Determination of the dynamic exchange capacity (DEC) of cation exchanger

The goal: determine the dynamic exchange capacity of cation exchanger

Objectives:

Proceedings1. Determination of water hardness

In a 250 ml of volume conical flask 10 ml of test water is poured with pipette, then 90 mL of distilled water, 5 ml of ammonia buffer and pinch of Chrome Dark Blue indicator are added there. The flask content is titrated with Trilon B by adding it dropwise as long as color will change from violet-red to blue. The titration is repeated until 3-reproducible results obtaining.

Calculation of the water hardness according to the formula is carried out:

, mEq / L (2.19),

where V - the average amount of Trilon B spent on titration, ml; CH - equivalent molar concentration of Trilon B, mol-eq / l; 10 - the volume of water (ml) taken for titration; 1000 - factor for the transition units from mole-equivalent to mmol- equivalent (mEq)2. Determination of the DEC of cation exchanger

Through the ion exchange column with the cation exchanger in the Na+-form test water is passed, which had previously been determined stiffness trilonometric . Set point slip calcium and magnesium ions in the eluate . For this purpose, a small test tube in the drop of indicator solution and ammonium buffer was added several drops of the resulting ion exchange column water. If the blue color of the sample , the breakthrough has not happened yet . If there solution is purplish tinge , the eluate from the column effluent had already appeared calcium or magnesium ions . At the same fixed amount of water to breakthrough missed . Ie Softened eluate. Cation volume is calculated by multiplying the cross-sectional area at the height of the column of cation layers . ( With a diameter of 2 cm column in its cross-sectional area of 3.14 cm2). Через ионообменную колонку с катионитом в Na+ -форме пропускают воду, жесткость которой предварительно определялась трилонометрически. Устанавливают момент проскока ионов кальция и магния в элюате. Для этого в маленькую пробирку вносят каплю раствора

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индикатора в аммонийном буфере и добавляют несколько капель вытекающей из ионообменной колонки воды. Если цвет содержимого пробирки голубой, проскока еще не произошло. Если у раствора наблюдается сиреневатый оттенок, то в элюате, вытекающем из колонки,уже появились ионы кальция или магния. При этом фиксируется объем пропущенной воды до проскока .т.е. объем умягченного элюата. Объем катионита вычисляют путем умножения площади сечения колонки на высоту слоя катионита. (При диаметре колонки в 2 см площадь сечения ее составляет 3.14 см2).

Расчет динамической обменной емкости катионообменника проводят по формуле:

, мэкв/м3 (2.20),

где V1 – объем умягченного элюата,мл; V2 – объем катионита, см3; Ж (H2O) – жесткость пропускаемой через колонки воды, мэкв/л; 1000 – множитель для перевода единиц измерения от мэкв/л к мэкв/м3.

БУМАЖНАЯ ХРОМАТОГРАФИЯ

Работа 1. Разделение железа (III) и меди (II) методом бумажной хроматографии Separation of iron (III) and copper (II) by paper chromatography

Цель работы: разделить и идентифицировать ионы железа и меди методом круговой бумажной хроматографии.

Сущность работы. Хроматография на бумаге – разновидность метода распределительной хроматографии. Носителем для неподвижного растворителя служит при этом фильтровальная бумага.

Анализ смеси веществ проводят по следующей схеме: на круглый обеззоленный фильтр в центр наносят каплю разделяемой смеси, фильтр подсушивают и помещают в хроматографическую камеру с ПФ. ПФ под действием капиллярных сил поднимается по «фитилю», достигает стартового пятна с разделяемой смесью, вместе с ней перемещаются с различной скоростью определяемые вещества.

Анализируемый раствор наносят на стартовую линию с помощью стеклянного капилляра в объеме не более 5–10 мкл. Чем меньше площадь стартового пятна, тем менее размытой будет зона вещества после хроматографирования. Поэтому пробу наносят в одну и ту же точку в несколько приемов, каждый раз подсушивая пятно.

Зоны разделяемых веществ имеют вид концентрических колец, которые могут быть видимыми и невидимыми; в последнем случае хроматограмму проявляют – опрыскивают раствором специфического реагента, либо подвергают воздействию УФ-излучения (см. рис.2.2).

Скорость перемещения компонентов определяется соответствующими коэффициентами распределения: чем меньше коэффициент распределения,

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тем быстрее вещество передвигается по сорбенту. В качестве характеристики удерживания используется величина Rf – подвижность, определяемая как отношение расстояния фронтов компонента и ПФ:

,

где l – расстояние, пройденное зоной компонента от старта пятна, см;L – расстояние, пройденное подвижной фазой, см. Под фронтом растворителя понимают видимую границу распространения растворителя по бумаге.

Рис.2.2. Круговая хроматограмма

1 – круглый фильтр; 2 – «фитиль», погружаемый в растворитель;.А – место нанесения анализируемого раствора

Величина Rf каждого катиона не зависит от концентрации определяемого катиона, температуры, присутствия других катионов и природы аниона, с которым связан изучаемый катион, но зависит от состава и свойств используемой ПФ, а также сорта хроматографической бумаги. У катионов железа (III) и меди (II) значения Rf значительно отличаются по величине. Поэтому удается их четкое разделение на бумаге.

Растворы, реактивы, аппаратура.1. Стандартный раствор соли Fe 3+, 1 мг/мл2. Стандартный раствор соли Cu 2+, 1 мг/мл3. Раствор K4[Fe(CN)6], 10% -ный4. Подвижная фаза – смесь этанола с 5М HCl (9:1) по объему5. Обеззоленная фильтровальная бумага «синяя лента»6. Капилляры стеклянные7. Хроматографическая камераВыполнение работы1. На круглом обеззоленном фильтре «синяя лента» диаметром 12,5 см

простым карандашом намечают контуры «фитиля» длиной 40 мм и шириной 4 мм (см. рис.2).

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2. На центр фильтра с помощью капилляра наносят каплю раствора разделяемой смеси. Раствор наносят в несколько приемов, чтобы впитывание происходило за счет капиллярных сил бумаги. Образовавшееся пятно осторожно обводят простым карандашом, т.е. фиксируют его положение на бумаге. Бумагу высушивают, вырезают «фитиль», как показано на схеме.

3. В хроматографическую камеру помещают кристаллизатор и тигель с 10 мл подвижной фазы. Кислоту добавляют к органическому растворителю, чтобы предотвратить адсорбцию ионов бумагой. На кристаллизатор сверху помещают фильтр, следя за тем, чтобы «фитиль» был погружен в растворитель, и закрывают камеру крышкой. Во время разделения не рекомендуется открывать крышку камеры, перемещать камеру.

3. Когда произойдет размывание первичного пятна растворителем, и фронт ПФ пройдет заданное расстояние, бумагу вынимают, отмечают карандашом границы фронта растворителя, высушивают в токе теплого воздуха и приступают к проявлению зон.

4. Для проявления зон локализации ионов Fe 3+ и Cu 2+ фильтр опрыскивают раствором K4[Fe(CN)6] из стеклянного пульверизатора (металлический непригоден!). В результате на хроматограмме проявляется синяя зона Fe4[Fe(CN)6]3 и коричневая зона Cu2[Fe(CN)6].

5.Рассчитывают для обоих катионов значения Rf , считая началом их пути наружную границу первоначального пятна, отмеченную карандашом, а концом пути – наружные границы появившихся после проявления кольцевых зон локализации. Расстояние же, пройденное фронтом растворителя, мм, отсчитывают от центра хроматограммы (центра бумажного круга).

6. Рассчитывают коээфициент разделения a как отношение подвижностей Rf и оценивают степень разделения катионов.

Работа 2. Разделение смеси аминокислот Separation of a amino acids mixture Цель работы: разделить и идентифицировать смесь простейших

аминокислот – a-аланина и аспарагиновой кислоты методом круговой бумажной хроматографии.

Сущность работы. Хроматография на бумаге – разновидность метода распределительной хроматографии. Носителем для неподвижного растворителя служит при этом хроматографическая бумага.

Разделению смеси аминокислот мешают следы металлов в бумаге для хроматографии, которые вымывают раствором 8-оксихинолина или комплексона III. Для этого из хроматографической бумаги № 1 или № 2 вырезают круглые листки диаметром 10–12 см и обрабатывают 0,1%-ным раствором 8-оксихинолина, приготовленным на смеси н-бутанола, ледяной уксусной кислоты и воды в соотношении по объему (8:1:1). Бумагу погружают на 1–2 мин в раствор 8-оксихинолина, затем подсушивают, помещают в хроматографическую камеру и пропускают ПФ до полного обесцвечивания темноокрашенных соединений 8-оксихинолина с катионами

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металлов. Затем бумагу многократно промывают дистиллированной водой и сушат на воздухе. Эта подготовка выполняется заблаговременно.

Анализ смеси веществ проводят по следующей схеме: на круглый обеззоленный фильтр в центр наносят каплю разделяемой смеси, фильтр подсушивают и помещают в хроматографическую камеру с ПФ. ПФ под действием капиллярных сил поднимается по «фитилю», достигает стартового пятна с разделяемой смесью, вместе с ней перемещаются с различной скоростью определяемые вещества.

Анализируемый раствор наносят на стартовую линию с помощью стеклянного капилляра в объеме не более 5–10 мкл. Чем меньше площадь стартового пятна, тем менее размытой будет зона вещества после хроматографирования. Поэтому пробу наносят в одну и ту же точку в несколько приемов, каждый раз подсушивая пятно.

Зоны разделяемых веществ имеют вид концентрических колец, которые могут быть видимыми и невидимыми; в последнем случае хроматограмму проявляют – опрыскивают раствором специфического реагента, либо подвергают воздействию УФ-излучения (см. рис.2.2., предыдущая работа).

Скорость перемещения компонентов определяется соответствующими коэффициентами распределения: чем меньше коэффициент распределения, тем быстрее вещество передвигается по сорбенту. В качестве характеристики удерживания используется величина Rf – подвижность, определяемая как отношение расстояния фронтов компонента и ПФ:

,

где l – расстояние, пройденное зоной компонента от старта пятна, см;L – расстояние, пройденное подвижной фазой, см. Под фронтом растворителя понимают видимую границу распространения растворителя по бумаге.

При этом в качестве подвижной фазы используют смесь н-бутанола, ледяной уксусной кислоты и воды в объемном соотношении (4:1:5), смесь тщательно взбалтывают и после расслоения берут верхний слой. Проявителем служит раствор с массовой долей нингидрина 0,25% в водонасыщенном н-бутиловом спирте. Нингидрин дает с аминокислотами оранжево-коричневое окрашивание бумаги.

Растворы, реактивы, аппаратура.1. Стандартный раствор a-аланина, 0,5 мг/мл2. Стандартный раствор аспарагиновой кислоты, 0,5 мг/мл3. Раствор нингидрина 0,25% в водонасыщенном н-бутиловом спирте4. Подвижная фаза – н-бутанола, ледяной уксусной кислоты и воды в объемном соотношении (4:1:5)5. Хроматографическая бумага № 1 или № 26. Капилляры стеклянные7. Хроматографическая камераВыполнение работы

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1. На предварительно подготовленной хроматографической бумаге простым карандашом намечают контуры «фитиля» длиной 40 мм и шириной 4 мм (см. рис.2).

2. На центр бумаги с помощью капилляра наносят каплю раствора разделяемой смеси. Раствор наносят в несколько приемов, чтобы впитывание происходило за счет капиллярных сил бумаги. Образовавшееся пятно осторожно обводят простым карандашом, т.е. фиксируют его положение на бумаге. Бумагу высушивают, вырезают «фитиль», как показано на схеме.

3. В хроматографическую камеру помещают кристаллизатор и тигель с 10 мл подвижной фазы. На кристаллизатор сверху помещают круг бумаги, следя за тем, чтобы «фитиль» был погружен в растворитель, и закрывают камеру крышкой. Во время разделения не рекомендуется открывать крышку камеры, перемещать камеру.

3. Когда произойдет размывание первичного пятна растворителем, и фронт ПФ пройдет расстояние, не доходя до края бумаги, бумагу вынимают, отмечают карандашом границы фронта растворителя, высушивают в токе теплого воздуха и приступают к проявлению зон.

4. Для проявления зон локализации аспарагиновой кислоты и a-аланина бумагу опрыскивают проявителем из стеклянного пульверизатора. Из появляющихся двух кольцевых окрашенных зон локализации первая принадлежит аспарагиновой кислоте, вторая – a-аланину.

5. Рассчитывают для обеих аминокислот значения Rf , считая началом их пути наружную границу первоначального пятна, отмеченную карандашом, а концом пути – наружные границы появившихся после проявления кольцевых зон локализации. Расстояние же, пройденное фронтом растворителя, мм, отсчитывают от центра хроматограммы (центра бумажного круга).

6. Рассчитывают коээфициент разделения a как отношение подвижностей Rf и оценивают степень разделения аминокислот.

Практическая работа №5Тема: Определение ионов железа (ІІІ), меди (ІІІ), никеля (ІІ), кобальта (ІІ) в мясном бульоне ионобиенной хроматографией. Determination of ions of iron (III), copper (III), nickel (II), cobalt (II) in a meat broth by ion exchange chromatography.Цель: качественное определение ионов железа (ІІІ), никеля (ІІ), кобальта (ІІ) в мясном бульоне ионобиенной хроматографией.Реактивы и оборудование: фарфоровая ступка, колонки, мерные колбы, пипетки, сорбент – оксид алюминия.Ход работы: подготовить 5 хроматографических колонок, исслед.р-р (молоко или мясной бульон) объемом 1 мм залить в каждую хроматографическую колонку, наполненную сорбентом – оксидом алюминия.

Обнаружение ионов кобальта (ІІ) и никеля (ІІ)

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Колонку №1 с сорбентом промывают водой, проявляют 1%-ным спиртовым раствором рубеановодородной кислоты, образуется фиолетово-красная зона. Разделение не происходит.Колонку №2 промывают водой, проявляют концентрированным раствором аммиака (2-3 капли). Вверху колонки появляется розовая зона, постепенно приобретающая бурую окраску свойственную аммиакату кобальта, ниже наблюдается голубая зона аммиаката никеля.

При проведении опыта в бульоне «роллтон» были обнаружены ионы кобальта (ІІ) и никеля (ІІ).CO2++4NH3 [Co(NH3)4]2+

Ni2++4NH3 [Ni(NH3)4]2+

Обнаружение ионов железа (ІІІ) меди (ІІІ), кобальта (ІІ).

Колонку №3 промывают водой (3-5 капель). Вверху образуется бурая окраска характерная для ионов железа, затем голубая зона ионов меди, внизу розоавя зона ионов кобальта.

Колонку №4 промывают с помощью 1-5%-ного раствора пексациано-ІІ-феррата калия, дающего характерную окраску с ионами железа (ІІІ).

При проведении опыта в бульоне «роллтон» не были обнаружены ионы железа (ІІІ), меди (ІІ) кобальта (ІІ).

Обнаружение ионов свинца (ІІ)Колонку №5 промывают водой (3-5) капель и проявляют 7-8 каплями 2N р-ра хромата калия. Образуется желтая зона соединений свинца. Зарисовать колонки с полученными р-ми. Сделать вывод о возможности обнаружения и разделения неогранических ионов в молоке и мясном бульоне методом колоночной хроматографии.

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В бульоне «роллтон» не были обнаружены ионы свинца (ІІ)

Наблюдение: Для проведения опыта был взят мясной бульон «роллтон» для обнаружения в нем ионов железа (ІІІ) меди (ІІ), никеля (ІІ) и кобальта (ІІ). При проведении опыта было выяснено что в бульоне «роллтон» находятся ионы никеля (ІІ) и кобальта (ІІ). Ионы никеля (ІІ) и кобальта (ІІ) были определены концентрированным раствором аммиака.Вывод: методом колоночной хроматографии обнаружили ионы никеля (ІІ) и кобальта (ІІ) в бульоне «роллтон».

Практическая работа №6Тема: Разделение ионов методом осадочной хроматографии. The separation of inorganic ions by precipitation chromatography.Цель: Изучение разделение ионов методом осадочной хроматографии, на примере разделение катионов в виде иодидов. Окись алюминия, которая является носителем, смешанная с иодидом натрия-осадителем, образует осадочно-хроматографирующую смесь. В качестве хроматографируемых растворов можно использовать искусственные смеси солей серебра, висмута, ртути, свинца. Яркоокрашенные осадки этих этих металлов образует на хроматографической колонке разноцветные зоны. Реактивы и оборудование:

1. нитрат висмута, 0,1 н раствор2. нитрат серебра, 0,1 н раствор3. нитрат ртути, 0,1 н раствор4. нитрат свинца, 0,1 н раствор5. окись алюминия, х.ч.6. иодид натрия, х.ч. 7. хроматографическая колонка.(d=0,5 см, h=8-10 см)Ход определения:

Хроматографическую смесь готовят следующим образом: 1 г иодида натрия, не содержащего свободного иода, и 3 г окиси алюминия тщательно перемешивают в фарфоровой ступке. Приготовленную смесь помещают в хроматографические колонки так, чтобы слой смеси занимал ½ объема

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трубки. Расчитывают по значениям произведений растворимости иодидов, какой должен быть порядок расположения зон, и готовят смеси растворов солей в любых сочетаниях и соотношениях. Например: 1,0 мл Hg(NO3)2:0,5 мл, Pb(NO3)2 :1,5 мл AgNO3 и.т.п. 0,5 мл Ві (NO3)3 приготовленной смеси заливают в хроматографическую колонку. При протекании раствора через хроматографирующую смесь образуется хроматограмма из нескольких зон. Описывают полученную хроматограмму непосредственно после ее получения и на следующий день, так ткак многие осадочные хроматограммы изменяются во времени.ПР(AgУ)=83*10-17 ПР(PbУ2)= 1,1*10-9

ПР(HgI2)=4,5*10-29 ПР (Bi I3)=8,1*10-19

1) 0,1 н раствора Bi(NO3)2 на 100 мл V=0,1л√экв = Сэкв*V; √экв*Mэкв = m Mэкв = *395=131,7√экв =0,1*0,1=0,01 m=0,01*131,7=1,317 г

f экв= = =

2) 0,1 н р-ра AgNO3

√экв = Сэкв*V=0,1*0,1=0,01 на 100 мл V=0,1лMэкв =1*170 г=170m=0,01*170=1,7 г

2) 0,1 н раствора Hg(NO3)2 на 100 мл V=0,1л√экв = Сэкв*V=0,1*0,1=0,01Mэкв = *325=162,5m=0,01*162,5=1,625 г

4) 0,1 н р-ра Pb(NO3)2 на 100 мл V=0,1л√экв = Сэкв*V=0,1*0,1=0,01

Mэкв = *331=165,5m=0,01*165,5=1,655 г

a) Bi(NO3)3+3NaI BiI3 +3NaNO3

Bi3+ +3I- BiI3

б) AgNO3+NaI AgI +NaNO3

Ag++I- AgIв) Hg(NO3)2 +2NaI HgI2 +2NaNO3

Hg2++2I- HgI2

г) Pb(NO3)2+2NaI 2NaNO3+PbI2

Pb2++2I- PbI2

Цвета:

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Bi(NO3)3 -0,5 мл красно-коричневыйAgNO3 -1 мл желтыйHg(NO3)2 -1,5 мл красный

Pb(NO3)2 1 мл желтый

Наблюдение:

Для проведения опыта были взяты такие соли как Hg(NO3)2, AgNO3, Bi(NO3)3, Pb(NO3)2 и окись алюминия которая является носителем, смешанная и с иодом натрия – осадителем, образуют осадочно-хроматографическую смесь. В колонке образуются хроматограмма по следующей последовательности начиная с Pb(NO3)2

Hg(NO3)2, Bi(NO3)3, AgNO3. чем ниже произведение растворимости, тем выше он находится в колонке.Вывод: изучили разделение ионов методом осадочной хроматографии.

Практическая работа №7Тема: Количественное определение никеля по величине зоны осадочной хроматографии. Quantitative determination of nickel by the size of precipitate zone Цель: Изучение количественного определения никеля по величине зоны осадочной хроматографии.Реактивы и оборудование: колонки, пробирки, оксид алюминия с диметилглиоксимом, соли никеля, дистил.вода, нитрат никеля. В основу количественного определения вещества положена главная особенность осадочных хроматограмм, связанная с равномерными распределением веществ в зоне, следствием чего является наличие пропорциональной зависимости между размерами зон осадочных хроматограмм и концентрацией исследуемого растворе. Сущность указаного метода состоит в том, что для каждого конкретного случая предварительно строят калибровочную кривую зависимости величины зон хроматограмм от концентрации растворе, а затем при тех же условиях получают осадочную хроматограмму того же вещества, но неизвестной концентрации, которую и определяют по калибровочной кривой.

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Методика определения: Готовят серию колонок единственного внутренного диаметра и заполняют смесью оксида алюминия с диметилглиоксимом. Колонки заполняют смесью на 2/3 мл высоты, уплотняют постукиванием о твердую поверхность. Для построения калибровочной кривой готовят серию стандартных растворов соли никеля путем последовательного разбавления дистиллированной водой раствора нитрата никеля данной концентрации.Техника разбавления раствора.

Берут 10 пробирок, вносят в каждую по 1 мл исследуемого раствора известной концентрации, например 1 М, затем в пробирку вносят определенное количествоводы, указанное в таблице. Концентрация разбавленного раствора в пробирках вычисляется по формуле:C1-10= (3.30)Где П-величина разбавления С-исходная известноя концентрация раствора, 1 М.

№ V исходного р-ра мл

V добавлен.воды

мл

Разб.р-ра, П

С разб.р-ра, моль/л

H зоны, мм

1 1 1 2 0,5 172 1 2 3 0,33 153 1 3 4 0,25 144 1 4 5 0,2 12,55 1 5 6 0,17 126 1 6 7 0,14 11,57 1 7 8 0,125 108 1 8 9 0,11 99 1 9 10 0,1 810 1 10 11 0,09 7,5

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Результаты вносят в соответствующую графу таблицы. В колонки со смесью вносят по 0,5 мл раствора известной концентрации. Через 3-5 мин после полного впитывания раствора проводят измерения высоты ало-розовой зоны диметилглиоксимата никеля и строят график в координатах по оси абсцисс – концентрация (моль). Для определения концентрации ионов никеля в исследуемом растворе готовят колонку с вышеуказанной смесью и тщательно уплотняют. Вносят в колонку 0,2 см 3 исследуемого р-ра никеля, не подвергая разбавлению. В течении 3-5 минут проводят измерения высоты хроматограммы. По предварительно построенному калибровочному графику определяют концентрацию исследуемого вещества в растворе.

Расчеты: 1 М Ni(NO3)2 на 100 мл =0,1 лC = ; √=C*V; √=1*0,1=0,1

√= =m=M*√=183 * 0,1=18,3

C1= = =0,5 моль

C2= = =0,33 моль

C3= = =0,25 моль

C4= = =0,2 моль

C5= = =0,17 моль

C6= = =0,14 моль

C7= = =0,125 моль

C8= = =0,11 моль

C9= = =0,1 моль

C10= = =0,09 моль

Вывод: Для проведения опыта, был взят сорбент оксида алюминия с метиллиоксимом, и раствор нитрата никеля. Заполнили колонки сорбентом, приготовили 1 М раствор Ni(NO3)2 затем взяли 10 колонок и 10 пробирок. В каждую пробирку вносили по 1 мл Ni(NO3)2 в определенное количество воды как указано в таблице. Затем в каждую колонку вливали с каждой пробирки раствор, при получении ало-розовой зоны, измерили высоту этой зоны и вносили в таблицу. Затем построили калибровочную кривую. Для

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определения концентрации ионов никеля в исследуемом растворе приготовила колонку со смесью и тщательно уплотнила. Затем вносила в колонку 0,2 мл исследуемого раствора никеля, не подвергая разбавлению. В течении 3-5 минут проводят измерение высоты хроматограммы. Высота Һ=8 мм, по предварительно построенному калибровочному графику определила концентрацию которая =0,1 моль/л.

Практическая работа №8

Тема: Определение содержания цинка в растворе методом ионообменной хроматографии.Цель: Определение содержания цинка в растворе методом ионообменной хроматографии.Реактивы и оборудование: цинк, катионит КУ-2, дисстилированная вода, мерная колба HCl, K4[Fe(CN)6], колонки, индикатор метилоранжевый, пипетки, вата.

Сущность метода: Определение основано на реакции обмена. Между ионами цинка в растворе и ионами водорода сильнокислотного катионита КУ-2:

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2 R – SO3H + Zn2+=[R – SO3]2Zn + 2H+(3.22)Через колонку пропустить раствор соли цинка сорбируется, а в растворе образуется эквивалентное количество кислоты, которое можно оттитровать щелочью.

Подготовка катионита:В стакан помещают 10 г воздушно-сухого сильнокислотного катионита, заливают дистиллированной водой и оставляют на 30 минут для набухания зерен. Затем катионит отмывают от пыли декантацией дистиллированной водой. Набухший катионит переносят в стеклянную колонку (d=12-20мм, h=300мм), в которую предварительно на ½ высоту наливают воду, а на дно помещают стеклянную вату. Над слоем ионита, все время должна находится жидкость. В случае попадания пузырьков воздуха в колонку катионит надо взрыклить стеклянной полачкой. Для переведения катионита в Н-форму через колонку пропускают 2N раствор соляной кислоты. Если в колонке 10 г катионита, то достаточно пропустить 200 мм раствора HCl. Катионит обычно содержат ионы железа (ІІІ), поэтому оканчание промывания колонки устанавливают по отсутсвую Fe3+ в элюанте. Для этого отбирают в пробирку или на часовое стекло 2 капли раствора K4[Fe(CN)6], в присутствии Fe3+ выпадает синий осадок K4[Fe(CN)6], кислоту пропускают через катионит со скоростью 10 мл/мин примерно 2 капли в секунду. После окончания пропускания кислоты жидкость в колонке опускают до верхнего слоя катионита и промывают катионит дистиллированной водой до значения РН, которое имеет воды. Приготовленный таким образом катионит готов к работе.

Методика определения:Анализируемый раствор соли помещают в мерную колбу на 100 мл и доводят водой до метки. Отбирают пипеткой 10 мл раствра и помещают в колонку. Раствор пропускают через катионит со скоростью 1,5-2,5 мл/мин. Вытекающий из колонки раствор собирают в коническую колбу для титрования емкостью 250 мл. Затем через катионит с этой же скоростью пропускают 20-30 мл дистиллированной воды, наливая ее в колонку из промывалки отдельными порциями по 5-10 мл. Новую порцию воды наливают тогда, когда жидкость в колонке достигает поверхности катионита. Затем пропускают воду со скоростью 8-10 мл/мин. Всего пропускают 100 мл воды. Полноту вымывая проверяют по метилову оранжевому. Для этого отбирают в пробирку несколько капель вытекающего из колонки раствора и прибавляют индикатор. Промывные воды собирают в ту же коническую колбочку. Все содержимое конической колбы оттитровывают 0,05N раствором NaOH с индикатором метилоранжем определение проводят 3 раза. Содержание цинка в мл вычисляют по формуле:

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m = ЭZn *VNaOH*NNaOH*

где ЭZn –эквивалентная масса цинка, равная ½ его атомной массы г/мольVк-общий объем исследуемого раствора, млVА –объем анализированного раствора, мл

После насыщения колонки катионит регенируют. Через колонку с катионитом пропускают 200 мл 2л раствора HCl для извлечения поглашенных катионов, затем отмывают катионит от кислоты дистиллированной водой и до следующего определенная сохраняют в колонке, заполненой водой.

Расчеты: 1) С(HCl) =2М√экв=M*V; C экв= ; m =Mэкв*√экв

√экв(HCl) =2*0,200=0,4моль* эквm(HCl) =0,4*36,5=14,6

2) масса NaOH для приготовления 100 мл 0,05N раствора

√экв= C экв*V=0.05*0,1=0,005 моль/эквMэкв=1*40=40m =√экв* Mэкв=0,005*40=0,2 г3) массы сухого ZnSO4 для приготовления 100 мл 0,1 н раствора√экв= C экв*V√экв= 0,1*0,1=0,1 моль/эквm =√экв* Mэкв

m = (ZnSO4) = 0,01*80,5=0,805г4) расчет содержания цинка в раствореm = ЭZn* VNaOH* VNaOH*

ЭZn-эквивалентная масса цинка, ЭZ = ArVк-общий объем исследуемого раствора, млVА –объем анализированного раствора, мл

V1(NaOH) =19 млV2(NaOH) =19,3 млV3(NaOH) =19,1 млрасчитаем Vср (NaOH)Vср (NaOH) = =19,133 мл

расчитаем ЭZn = Ar = *65=32,5

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Vк=100 млVА=10 млNNaOH*0,05 нVNaOH*19,133 млподставляем в формулу:m =32,5*19,133*0,05* =311 мгВывод: Определили содержания цинка в растворе методом ионообменной хроматографии, в растворе ZnSO4 содержится Zn=311 мл

Практическая работа №9

Тема: Качественное разделение ионов меди и кадмия на катиоите методом распределительной хроматографии.

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Цель: Качественное разделение ионов меди и кадмия на катиоите методом распределительной хроматографии.Реактивы и оборудование: соляная кислота, дисстилированная вода, метилоранж, колонка, колба, катионит КУ-2, штатив для крепления колонки.Разделение веществ методом распределительной хроматографии основано на различии в коэфициентах распредеоения хроматографируемых веществ между двумя несмешивающимся фазами-растворителями. Методика распределительного хроматографического разделения позволяетразделять смеси неорганических ионов и органических кислот качественно разделяют смесь неорганических ионов Ca2+ и Cu2+ на катионите КУ-2. в колонку с катионитом КУ-2 в Н-форме внести 25 мл смеси ионов меди и кадмия с концентрацией 0,01 N каждого иона. Смесь пропускают со скоростью 2-3 мл в минуту. Вытекающий из колонки раствор выбрасывают. Под колонку помещают чистую колбу емкостью 250 мл, в колонку вливают небольшое количество раствора глицерино-щелочной смеси порциями по 3-4 см3 (5 см3

глицерина и 5 г гидроксида натрия в 100 г воды)Повторяют эту операцию до момента полного извлечения меди из колонки, что определяется везуально по голубой окраске фильтрата и по качественной реакции рубеановодородной кислоты. Для этого на фильтровальную бумагу наносят каплю рубеановодородной кислоты и каплю фильтрата наличие серого пятна на бумаге свидетельствует о присутствии меди в расторе. Ионы кадмия вымывают из колонки 5% соляной кислоты и определяют качественно раствором K4[Fe(CN)6] по наличию белого осадка. Изобразите схему полученной хроматограммы и объясните результаты.Регенерация сорбента.Регенерацию сорбента проводят пропуская через колонку 50 мл 5% соляной кислоты. Затем сорбент отмывают от кислоты дистиллированной водой, проверяя пропускаемую через колону дистиллированную воду метилоранжем. Окраска не должна изменяться и должна соответствовать заготовленному стандарту. Стандарт готовится в стаканчике с тремя каплями метилоранжа в 25 см2 дистиллированной воды. Расчеты:

1) для приготовления 5% р-ра HCl объемом 100 мл, из 37 %( HCl) 37 %=1,183 г/мл( HCl) 5 %=1,024 г/мл

V (HCl) =100 млm = * V=1,024 г/мл100 мл=102,4 гсоответственно m(HCl) в 37 % р-ра =5,12 г

m(р-ра)= = 13,83 г

V= = 11,7 млVводы=100 – 11,7=88,32) расчет на приготовление 0,01 Н р-ра CuSO4*5H2O и р-ра Cd (CH3COO)2

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Сэкв= ; √экв=Сэкв* V*0,01*0,1=0,001 г/моль/эквm Cd (CH3COO)2 =Мэкв*√экв=115*0,001=0,115 гМэкв Cd (CH3COO)2=М*fэкв=230* =115 г/моль/эквб) массу CuSO4*5H2O приготовления 100 мл 0,01 Н р-ра√экв=Сэкв* V√экв=0,01*0,1=0,001 моль*эквm=Сн*Мэкв* V=0,01*125*0,1=0,125 гm (CuSO4*5H2O) =0,125 г

Вывод: Провели качественное разделение ионов меди и кадмия на катионите методом распределительной хроматографии.

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ЛИТЕРАТУРА

1. Гиндуллина Т.М. Хроматографические методы анализа: учебно-методическое пособие /Т.М. Гиндуллина, Н.М. Дубова – Томск: Изд-во Томского политехнического университета, 2010. – 80 с.

2. Васильев В. П. Аналитическая химия. Ч. 2– М.: Дрофа, 2007. 384 с.3. Основы аналитической химии. 2 кн. /Под ред. Ю. А. Золотова.– М.:

Высшая школа, 2004. –359 с. (кн.1), 503 с. (кн.2)4. Васильев В. П. Аналитическая химия: лабораторный практикум / В. П.

Васильев, Р. П. Морозова, Л. А. Кочергина.– 3-е изд., стер.– М. : Дрофа, 2006.– 414 с.

5. Аналитическая химия и физико-химические методы анализа/Под ред. О.М.Петрухина. – М.: Химия, 2001. – 496 с.

6. Основы аналитической химии.Практическое руководство. Под ред.Ю.А.Золотова. – М.: Химия, 2001.– 463 с.

7. Аналитическая хроматография / К. И. Сакодынский, В. В. Бражников, С. А. Волков и др.– М.: Химия, 1993.– 463 с.:

8. Айвазов Б.В. Введение в хроматографию. – М.: Высшая школа, 1983. – 250с.

9. Вяхирев Д.А., Шушунова А.Ф. Руководство по газовой хроматографии. – М.: Высшая школа, 1987. – 335с.

10.Дорохова Е. Н., Прохорова Т.В. Аналитическая химия. Физико- химические методы анализа. – М.: Высшая школа, 1991. – 256с.

4 Methodical recommendations for conducting Students, Self-Study and Office Hours

The kind of work

The contents References

Fulfillment period

Quantity of hours

Form of control

The work with lectures

Consolidate the obtained knowledge by participating in office hour, studying the scientific and journalistic periodic press

1, 3, 4 1-14 weeks 17,5 orally

The preparation to laboratory

Activate self study Consolidate the obtained

1, 3, 4 1-14weeks

17,5 Written work

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works knowledge by participating in office Hour. Mastering by the elementary techniques of researches of objects

Colloquium Check the obtained knowledge through examinations and tests

7, 14 week 10 orally

SCHEDULE OF THE SURRENDER TASKS OF SSS, OH

№ Name of the theme

The contents References

Mark

Fulfillment period

Form of control

1 Introduction to physical and chemical methods of analysis

Classification of physical methods of analysis. Features of physical methods of analysis

1, 2, 3 100 1week orally

2 Optical methods of analysis

Photometric methods. Visual methods of quantitative analysis. Photocolorimetric methods. Bouguer-Lambert - Beer law.To pass a colloquium #1

1, 2, 3, 4 200 2-6 week orally

3 Electrochemical methods of analysis

Potentiometry. Electrodes used in potentiometry. Types of potentiometric determinations. Methods for determining the equivalence point.

4 100 7-11 week orally

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Coulometric method. Voltammetry, polarography, amperometric titration. Electro-gravimeter.

4 Chromatographic methods of analysis

The bases of chromatography. The characteristics of chromatography method. The laboratory equipment. The Thin-layer chromatography. The gas-liquid chromatographyTo pass a colloquium #2

1, 2, 3, 4 200 12-15 week orally

THE TOPICS OF COLLOQUIUMS ON COURSE "PHYSICAL METHODS OF CHEMICAL RESEARCH"

1. Introduction to physical and chemical methods of analysis. Optical methods of analysis- 7 week2. Electrochemical methods of analysis. Chromatographic methods of analysis. - 14 week

POINT-RATING SYSTEM OF STUDENT ACTIVITIES

Lecture - 15, laboratory work – 15, OH – 15, SSS -45№ Name of parts Number

of partitions

The maximum mark for

one

Sum

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partition1 Visiting lectures 15 100 1500

2 Perform laboratory work 6 200 1400

3. Implementation of the OH plan

15 100 1500

4. Implementation of SSS schedule

15 100 1500

5. Colloquium 2 100 200In total 6100

Note: Maximum Mark exhibited at 100% performance of all requirements for this section.

Estimation Colloquium«excellent» – 100 - 90. (100%-90%)

«good» - 89 -75 ( 89%-75%)«satisfactory» – 74 – 50 ( 74%-50 %)«unsatisfactory» – 0-49

POLICY AND PROCEDURE OF THE COURSEAdministrative requirements to the students in the process of studying- In late for class are removed: 50 points for the lecture 30 points for the laboratory work- Untimely perform tasks leads to a lowering of the rating 10 points.- Attendance at laboratories is permitted only in white coats.- Compliance with the safety rules in the chemical laboratory.

Grading of the total control

Literal equivalent

Traditional marking

system

Points Percent (%)

А excellent 4, 00 95-100А - 3, 67 90-94В+

good3, 33 85-89

В 3, 00 80-84В - 2, 67 75-79С+

satisfactory

2, 33 70-74С 2, 00 65-69

С - 1, 67 60-64D+ 1, 33 55-59D 1, 00 50-54

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F unsatisfactory 0, 00 0-49

BASIC LITERATURE: 1. Modern analytical chemistry. D. Harvey. De Pauw University, 20092. Fundamentals of analytical chemistry. Skoog West, Holler. 8 edition3. В.П. Васильев. Теоретические основы физико-химических методов

анализа. М.:ВШ 1979г.4. Ляликов Ю.С. Физико-химические методы анализа М.:ВШ, 1968г.5. Иоффе Б.В., Костиков Р.Р., Разин В.В.. Физические методы

определения строения органических соединений. М.: Высшая школа, 1984, 335с.

6. Филимонов В.Д., Добычина Н.С., Тигнибидина Л.Г. Физико-химические методы исследования структуры и чистоты биологически активных соединений. Томск: 1987, 57с.

ADDITIONAL LITERATURE:1. Беллами Л. Инфракрасные спектры сложных молекул. М.: ИИЛ, 1963,

444с.2. Прикладная инфракрасная спектроскопия. Под ред. Кендалла Д. М.:

Мир, 1970, 376с.3. Джонстон Р. Руководство по масс-спектрометрии для химиков-

органиков. М.: Мир, 1975, 236с.

5 MEASUREMENT TOOLSEXAM QUESTIONS

1. Properties of electromagnetic radiation2. The dual models of radiation’s behavior3. Structure of electromagnetic radiation4. Regions of electromagnetic radiation5. Spectroscopy classes6. Principles of absorption spectroscopy7. Principles of emission spectroscopy8. Sources of energy9. Flame as source of energy10. Plasma as source of energy11. Wavelength selector12. The main properties of wavelength selector

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13. Absorption filters14. Interference filters15. Construction of a typical monochromator16. modern detectors17. Semiconductor as the photosensitive surface18. Thermal transducer19. Signal processor20. General requirements at absorbing radiation21. Conditions of appearing fundamental absorption lines22. The most important transitions in the molecular spectra23. Characteristic of width of absorption lines24. Transmittance of radiation25. Absorbance of radiation26. Beer-lambert low27. Atomization in atomic absorption28. Flame atomization as general method29. Electro thermal atomization as general method30. Fuel-oxidant mixture in flame atomization31. Three stages of atomization32. Various ways of relaxation33. Fluorescence as a type of emission34. Using a plasma in atomic emission35. Structure of simplest electrochemical cell36. Ohm’s low37. Two half-cells of electrochemical cell38. Shorthand notation as a useful representation of structure of cell39. Determining of electrochemical cell’s potential40. Redox reactions41. Nernst equation42. Standard electrodes43. Standard hydrogen electrode44. Saturated calomel electrodes45. Silver chloride electrodes46. Electrodes of second kind47. Indicator electrodes48. electrodes of first kind49. Ion-selective electrodes50. Determination of membrane potential51. Glass electrodes52. Solid-state ion-selective electrodes53. Principles of chromatographic separations54. Classification of chromatographic methods by states of mobile and

stationary phases55. Approaches using to contact mobile and stationary phases

EMCD042-18-34.1.23 /03-2014

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from 71

56. Three mechanisms of separation in chromatography57. Columns in column chromatography58. Important parameters of chromatographic peak59. Resolution of separation in column chromatography60. Selectivity factor in column chromatography61. Theoretical plates in column chromatography62. Gas chromatography63. Packed columns and it’s containing64. Capillary columns65. Solute’s boiling point as factor of good elution66. Process of gas chromatography

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