Analytical chemistry : Part A

deals with separating, identifying, and quantifying the relative amounts of the components of an analyte.Analyte = the thing to analyzed; the component(s) of a sample that are to be determined There are several different areas of analytical chemistryClinical analysis - blood, urine, feces, cellular fluids, etc., for use in diagnosis. Pharmaceutical analysis - establish the physical properties, toxicity, metabolites, quality control, etc Environmental analysis - pollutants, soil and water analysis, pesticides Forensic analysis - analysis related to criminology; DNA finger printing, finger print detection; blood analysis . Industrial quality control - required by most companies to control product quality. Bioanalytical chemistry and analysis - detection and/or analysis of biological components (i.e., proteins, DNA, RNA, carbohydrates, metabolites, etc.)

Chemical analysis may be qualitative or quantitative.

Qualitative analysis involves attempting to identify what materials are present in a sample. Answering the question: ‘What is itQuantitative analysis involves determining how much of a material is present in a sample. Answering the question: ‘How much is present

Qualitative analysis:Deals with the identification of elements, ions, or compounds present in the sample.Qualitative testes may be performed by selective chemical reaction or with use of instrumentation. The white ppt of AgCL Functional group of organic compound in IR Quantitative analysis Deals with the determination of how much of one or more constituents is present.

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A history of sample composition should be known. (Its known that blood contain glucose) A qualitative test should be done before the more difficult quantitative analysisFundamentals of chemical analysis”Electrolytes:Substances that give ions when dissolved in water are called electrolytes.

They can be divided into acids, bases, and salts, because they all give ions when dissolved in water. These solutions conduct electricity due to the mobility of the positive and negative ions, which are called cations and anions respectively.

Strong and Week Electrolytes: Strong electrolytes completely ionize when dissolved, and no neutral molecules are formed in solution.

For example, NaCl, HNO3, CaCl2 etc are strong electrolytes. An ionization can be represented by, NaCl (s) = Na+ (aq) + Cl- (aq)

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Since NaCl consists of cations Na+ and anions Cl-. No molecules of NaCl are present in NaCl solid or NaCl solution. The ionization is said to be complete. The solute is one hundred percent (100%) ionized. weak electrolytes Small fractions of weak electrolytes molecules ionize when dissolve in water. Some neutral molecules are present in their solutions. For example, ammonia, NH4OH, carbonic acid, H2CO3, acetic acid, CH3COOH, and most organic acids and bases are weak electrolytes. The following ionization is not complete, H2CO3(aq) = H+(aq) + HCO3

-(aq)

Atomic and molecular weights:

Atomic MassThe units in which the mass of an atom are expressed are atomic mass units. At one time, the lightest atom was assigned a mass of 1 amu and the mass of any other atom was expressed in terms of this standard.

Today atomic mass units are defined in terms of the 12C isotope, which is assigned a mass of exactly 12.000... amu.

IsotopesIsotopes are atoms of the same element with different numbers of neutrons, such as the 20Ne and 22Ne isotopes of neon or the 35Cl and 37Cl isotopes of chlorine

Atomic WeightThe atomic weight of an element is the weighted average of the atomic masses of the different isotopes of an element.Ex. Naturally occurring carbon is a mixture of two isotopes, 12C (98.89%) and 13C (1.11 %). Individual carbon atoms therefore have a mass of either 12.000 or 13.03354 amu. But the average mass of the different isotopes of carbon is 12.011 amu.

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Molecular Weight:The molecular weight of a compound is the sum of the atomic weights of the atoms in the molecules that form these compounds.

Ex: The molecular weight of the sugar molecule found in sugar isthe sum of the atomic weights of the 12 carbon atoms, 22 hydrogen atoms, and 11 oxygen atoms in a C12H22O11 molecule.

342.299 amu

175.993 amu11 O atoms = 11(15.9994) amu =

22.174 amu22 H atoms = 22(1.0079) amu=

144.132 amu12 C atoms = 12(12.011) amu=

C12H22O11 has a molecular weight of 342.299 amu.

A mole of C12H22O11 would have a mass of 342.299 grams.

This quantity is known as the molar mass, a term that is often used in place of the terms atomic weight or molecular weight.

Concentrationconcentration, is the measure of the relative proportions of two or more quantities in a mixture. The concentration of a solute is very important in studying chemical reactions because it determines how often molecules in solution.

The simplest statement of the concentrations of the components of a mixture is in terms of their percentages by weight or volume. Mixtures of solids or liquids are frequently specified by weight percentage concentrations, such as alloys of metals, whereas mixtures of gases are usually specified by volume percentages.

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Very low concentrations may be expressed in parts per million (ppm), as in specifying the relative presence of various substances in the atmosphere.

Concentration may be expressed in a number of ways :1.Percent Composition by Mass (%) ;

This is the mass of the solute divided by the mass of the solution (mass of solute plus mass of solvent), multiplied by 100 %.

Ex. Determine the percent composition by mass of a 100 g salt solution which contains 20 g salt. Solution:20 g NaCl / 100 g solution x 100 = 20 % NaCl solution

2.Mole Fraction ( X ) ;This is the number of moles of a compound divided by the total number of moles of all chemical species in the solution. Keep in mind, the sum of all mole fractions in a solution always equals 1.00.

Ex. What are the mole fractions of the components of the solution formed when 92 g glycerol is mixed with 90 g water? (molecular weight water = 18; molecular weight of glycerol = 92) .number of moles = weight (g) / molecular weight (g / mol)

90 g water = 90 g x 1 mol / 18 g = 5 mol water92 g glycerol = 92 g x 1 mol / 92 g = 1 mol glycerolTotal mol = 5 + 1 = 6 molX for water = 5 mol / 6 mol = 0.833X for glycerol = 1 mol / 6 mol = 0.167It's a good idea to check your math by making sure the mole fractions add up to 1:X water + X glycerol = .833 + 0.167 = 1.00

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3.Molarity (M) ;

Molarity is probably the most commonly used unit of concentration. It is the number of moles of solute per liter of solution (not necessarily the same as the volume of solvent!).

Molarity = moles of solute (mol) / volume of solution (Liter) .

Ex. What is the Molarity of a solution made when water is added to 11 g CaCl2 to make 100 mL of solution?

11 g CaCl2 / (110 g CaCl2 / mol CaCl2) = 0.10 mol CaCl2100 mL x 1 L / 1000 mL = 0.10 LMolarity = 0.10 mol / 0.10 LMolarity = 1.0 M or 1.0 mol / L

4. Molality (m); Molality is the number of moles of solute per kilogram of solvent. Because the density of water at 25°C is about 1 kilogram per liter, molality is approximately equal to molarity for dilute aqueous solutions at this temperature. Molality = moles of solute (mol) / Kilogram of solvent (Kg) .

Ex. What is the molality of a solution of 10 g NaOH in 500 g water? 10 g NaOH / (4 g NaOH / 1 mol NaOH) = 0.25 mol NaOH500 g water x 1 kg / 1000 g = 0.50 kg watermolality = 0.25 mol / 0.50 kgmolality = 0.05 mol / kgmolality = 0.50 m or 0.50 mol / Kg5. Normality (N) ;Normality is equal to the gram equivalent weight of a solute per liter of solution. A gram equivalent weight or equivalent is a measure of the reactive capacity of a given molecule. Normality is the only concentration unit that is reaction dependent.

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Ex. 1 M sulfuric acid (H2SO4) is 2 N for acid-base reactions because each mole of sulfuric acid provides 2 moles of H+ ions. On the other hand, 1 M sulfuric acid is 1 N for sulfate precipitation, since 1 mole of sulfuric acid provides 1 mole of sulfate ions.

Dilutions , You dilute a solution whenever you add solvent to a solution. Adding solvent results in a solution of lower concentration. You can calculate the concentration of a solution following a dilution by applying this equation: Mi Vi = Mf Vf where M is molarity, V is volume, and the subscripts i and f refer to the initial and final values. Ex. How many millilieters of 5.5 M NaOH are needed to prepare 300 mL of 1.2 M NaOH? Solution:(5.5 M ) (Vi) = (1.2 M) (0.3 L) Vi = 1.2 M x 0.3 L / 5.5 M Vi = 0.065 L Vi = 65 mL So, to prepare the 1.2 M NaOH solution, you pour 65 mL of 5.5 M NaOH into your container and add water to get 300 mL final volume.

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Hydrogen Ion concentration (H+):

The concentration of H+ in a solution is a measure of the solution acidity and represented as pH.

pH is generally expressed without units, the number arises from a definition based on the activity of hydrogen ions in the solution. The pH scale is a reverse logarithmic representation of relative hydrogen proton (H+) concentration.

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In aqueous systems, the hydrogen ion activity is dictated by thedissociation constant of water (Kw)

In pure water

Due to this dissociation constant, a neutral solution (hydrogen ion activity equals hydroxide ion activity) has a pH of approximately 7. Aqueous solutions with pH values lower than 7 are considered acidic, while pH values higher than 7 are considered basic

Q1: What is the pH of a 0.001 M solution of the strong acid HCl ?

Q2: What is the pH of a 0.0001 M solution of the strong base NaOH ?

Q3: A sample of Orange juice was found to have a pH of 4.0 what were the H+ and the OH- concentrations in the juice ?

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BUFFER SOLUTIONSBuffers help maintain a constant pH, when small quantities of an acid or an alkali are added to it.1.Acidic buffer solutionsAn acidic buffer solution is simply one which has a pH less than 7. Acidic buffer solutions are commonly made from a weak acid and one of its salts - often a sodium salt.You can change the pH of the buffer solution by changing the ratio of acid to salt, or by choosing a different acid and one of its salts.2.Alkaline buffer solutionsAn alkaline buffer solution has a pH greater than 7. Alkaline buffer solutions are commonly made from a weak base and one of its salts.

Buffer CapacityA buffer solution has to contain things which will remove any hydrogen ions or hydroxide ions that you might add to it - otherwise the pH will change. Acidic and alkaline buffer solutions achieve this in different ways.

Acidic buffer solutions:

By Adding an acid to this buffer solution. The buffer solution must remove most of the new hydrogen ions otherwise the pH would drop markedly.

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Hydrogen ions combine with the ethanoate ions to make ethanoicacid. Although the reaction is reversible, since the ethanoic acid is a weak acid, most of the new hydrogen ions are removed in this way.

Because of the equilibria involved, the pH will fall a little bit.

Adding an alkali to the acid buffer solutionAlkaline solutions contain hydroxide ions and the buffer solution removes most of these. hydroxide ion is Removed by reacting with ethanoic acidto form ethanoate ions and water.

Alkaline buffer solutions

By adding an acid to this buffer solution. The hydrogen ions are removed by reacting with ammonia to form ammonium ions .

Adding an alkali to this buffer solutionThe hydroxide ions from the alkali are removed by a simple reaction with ammonium ions.

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Q: Describe in equilibrium equations how the alkaline hydrazine buffer will maintain a constant pH after adding :

1. a small amount of acid to the buffer ?

2.A small amount of base to the buffer ?

Acid-Base Titration An acid-base titration is a method in chemistry that allows quantitative analysis of the concentration of an unknown acid or base solution. It makes use of the neutralization reaction that occurs between acids and bases, and that we know how acids and bases will react if we know their formula.

The key equipment used in a titration are:1.Burette 2.Pipette 3.Acid/Base Indicator (the one used varies depending on the reactants) 4.Conical Flask 5.Standard Solution (a solution of known concentration, a common one is aqueous Na2CO3) 6.Solution of unknown concentration

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Beaker Conical Flask Burette

ACID-BASE INDICATORSIndicators : A substances that generally is added to a solution in the receiving vessel and which undergoes some sort of color change when the reaction is over.

The color change of the indicator marks the endpoint in the titration, so named because it is at this point that the delivery of the titrant is stopped and the volume of the titrant used in the reaction recorded.

Types of Indicators1. Litmus Litmus is a weak acid, which will simplify to HLit. The "H" is

the proton which can be given away to something else. The "Lit" is the rest of the weak acid molecule.

There will be an equilibrium established when this acid dissolves in water. Taking the simplified version of this equilibrium:

The un-ionised litmus is red, whereas the ion is blue.

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what would happen if you added hydroxide ions or some more hydrogen ions to this equilibrium .

Adding hydroxide ions:

Adding hydrogen ions :

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2.Methyl orange :is one of the indicators commonly used in titrations. Its structure is simple enough to be able to see what is happening as it loses and gains hydrogen ions.

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3.PhenolphthaleinPhenolphthalein is another commonly used indicator for titrations, and is another weak acid.

In this case, the weak acid is colourless and its ion is bright pink. Adding extra hydrogen ions shifts the position of equilibrium to the left, and turns the indicator colourless. Adding hydroxide ions removes the hydrogen ions from the equilibrium which tips to the right to replace them - turning the indicator pink.

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The pH range of indicators

8.3 - 10.0 phenolphthalein

3.1 - 4.4 methyl orange

5 - 8litmus

pH rangeindicator

The litmus colour change happens over an unusually wide range, but it is useful for detecting acids and alkalis in the lab because it changes colour around pH 7. Methyl orange or phenolphthalein would be less useful.

For example, methyl orange would be yellow in any solution with a pH greater than 4.4. It couldn't distinguish between a weak acid with a pH of 5 or a strong alkali with a pH of 14.

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Acid-Base NeutralizationA salt is any compound which can be derived from the neutralization of an acid and a base.

The word "neutralization" is used because the acid and base properties of H+ and OH- are destroyed or neutralized.

In the reaction, H+ and OH- combine to form HOH or H2O or water molecules.

A neutralization is a type of double replacement reaction.

The following are some examples of neutralization reactions to form salts .

Problem:Calculate the molarity of an acetic acid solution if 34.57 mL of this solution are needed to neutralize 25.19 mL of 0.1025 M sodium hydroxide

Strategy: Figure out how many moles of the titrant (in this case, the base) were needed.

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Use the balanced chemical equation to calculate the moles of analyte (in this case, the acid) present.

Use the volume of analyte to find the concentration of the analyte.

Example:Calculate the molarity of an Carbonic acid solution if 20 mL of this solution are needed to neutralize 40 mL of 0.02 M sodium hydroxide

Oxidation Reduction Reactions (Redox reactions)

Redox reactions are a family of reactions that are concerned with the transfer of electrons between species.

You don't have an oxidation reaction without a reduction reaction happening at the same time.

Oxidation refers to the loss of electrons, while reduction refers to the gain of electrons.

Each reaction by itself is called a "half-reaction", simply because we need two (2) half-reactions to form a whole reaction.

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Solid copper (with no charge) being oxidized (losing electrons) to form a copper ion with a plus-2 charge. Silver ion (silver with a positive charge) is being reduced through the addition of two (2) electrons to form solid silver

We can now combine the two (2) half-reactions to form a redox equation

Oxidizing and Reducing agents Oxidizing agent: a chemical substances that causes another substance to be oxidized. (it is reduced in the process(. Reducing agent: a chemical substances that causes another substance to be reduced.)it is oxidized in the process(.Since oxidation and reduction are symmetric processes, always occurring together, there is always an oxidizing agent and a reducing agent in the reaction.

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SOLUBILITY and PRECIPITATION

SolubilitySolubility is the amount of a solute that will dissolve in a specific solvent under given conditions.

The dissolved substance is called the solute and the dissolving fluid (usually present in excess) is called the solvent, which together form a solution.

The process of dissolving is called solvation, or hydration if the solvent is water.

A solution at equilibrium that cannot hold any more solute is said to be saturated. The equilibrium of a solution is mainly dependent on temperature. The maximum equilibrium amount of solute which can normally dissolve per amount of solvent is the solubility of that solute in that solvent.

It is often expressed as a maximum concentration of a saturated solution. These maximum concentrations are often expressed as grams of solute per 100 milliters of solvent.

The solubility of one substance dissolving in another is determined by 1. Intermolecular forces between the solvent and solute.2. Temperature.3.Entropy change that accompanies the solvation.4.The presence and amount of other substances.5. Pressure or partial pressure of a solute gas.

Solubility constants are used to describe saturated solutions of ionic

compounds of relatively low solubility .

For salts, solubility in aqueous solutions or the maximum amount of salt that can be dissolved is the solubility constant.

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The solubility constant is a special case of an equilibrium constant. It describes the balance between dissolved salt and undissolved salt. The solubility constant is also "applicable" to precipitation, the reverse of the dissolving reaction. Solutions may, under special conditions, hold more solute than the solvent can normally dissolve. This is called supersaturation

Solvents are normally characterized as polar or nonpolar. The general rule is "Like Dissolves Like." This means that polar solvents will dissolve ionic compounds and covalent compounds which ionize, while nonpolar solvents will dissolve nonpolar covalent compounds.

For example, Table salt, an ionic compound, will dissolve in water, but not in ethanol.Water and nonpolar solvents are immiscible; they do not form homogeneous mixtures but separate into two distinct phases or form milky emulsions.

Precipitation

Precipitation is the formation of a solid in a solution during a chemical reaction.

When the chemical reaction occurs the solid formed is called the precipitate.

This can occur when an insoluble substance, the precipitate, is formed in the solution due to a reaction or when the solution has been supersaturated by a compound. The formation of a precipitate is a sign of a chemical change. In most situations, the solid forms ("falls") out of the solute phase, and sinks to the bottom of the solution (though it will float if it is less dense than the solvent, or form a suspension(

This effect is useful in many industrial and scientific applications whereby a chemical reaction may produce a solid that can be collected from the solution by various methods (e.g. filtration, decanting, centrifuging).

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An example of a precipitation reaction:Aqueous silver nitrate (AgNO3) is added to a solution containing potassium chloride (KCl) and the precipitation of a white solid, silver chloride is observed.

The silver chloride(AgCl) has formed a solid, which is observed as a precipitate.This reaction can be written emphasizing the dissociated ions in a combined solution

A final way to represent a precipitate reaction is known as a net ionic reaction. Ag+(aq) + Cl-(aq) → AgCl(s)

Question / Aqueous Baruim sulphate (BaSO4) is added to a solution containing Soduim chloride (NaCl) and the precipitation of a solid, Baruim chloride( BaCl2) is observed.

What is the ionic and the net precipitation equations ?

Solubility ProductThe most insoluble salts dissolve in water to at least some degree, and their saturated solutions have dynamic equilibria .

Not all salts are completely dissolved in water, but we will assume that in a saturated solution an equilibrium exist between the solid saltand its dissolved ions.For Example:In a saturated solution of silver chloride we have an equilibrium

The equilibrium constant can write as:

The concentration of the solid is a constant and therefore can included with the equilibrium constant the result is the solubility constant

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In the case of an insoluble solid such as Mg(OH)2, the coefficients in the equilibrium are not all equal to one.

Q1/ The molar solubility of PbCl2 is 1.7 x 10-3 moles in a liter. What is the Ksp of PbCl2?

Q2/ What is the molar concentration of [Ag+] in AgCl solution

(Ksp = 1.8 x 10-10)

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Spectrophotometric and photometric techniques

Principles of Spectrophotometry:A spectrophotometer consists of two instruments:

1.Spectrometer : for producing light of any selected color (wavelength) ( light Source).

2.Photometer for measuring the intensity of light (detector).

The instruments are arranged so that liquid in a cuvette can be placed between the spectrometer beam and the photometer.

A spectrophotometer is employed to measure the amount of light that a sample absorbs. The instrument operates by passing a beam of light through a sample and measuring the intensity of light reaching a detector. The photometer delivers a voltage signal to a display device, normally a galvanometer.

Nature of light The beam of light consists of a stream of photons. When a photonencounters an analyte molecule there is a chance the analyte will absorb the photon. This absorption reduces the number of photons in the beam of light, thereby reducing the intensity of the light beam. Reaching the detector

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Beer's law ;The decrease in light intensity because of sample absorption is proportional to: Cell path length and the Analyte concentration. When the light of a specific wavelength passes through a solution there is usually a quantitative relationship (Beer's law) between the solute concentration and the intensity of the transmitted light.

(I0) the intensity of light passing through a blank . The blank is a solution that is identical to the sample solution except that the blank does not contain the solute that absorbs light (I) the intensity of light passing through the sample solution is measured. (T) the transmittance and the (A). absorbance If A = 0, then no photons are absorbed. If A = 1.00, then 90% of the photons are absorbed; only 10% reach the detector

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SpectrophotometryThere are two major classes of spectrophotometers; single beam and double beam. double beam spectrophotometer measures the ratio of the light intensity on two different light paths, and a single beam spectrophotometer measures the absolute light intensity. Although ratio measurements are easier, and generally stabler, single beam instruments have advantages; for instance, they can have a larger dynamic range. Types of spectrophotometers

# Visible-region spectrophotometers

Visible region 400-700nm spectrophotometry is used extensively in colorimetry science.

components: 1. The light source shines through the sample. 2. The sample absorbs light. 3. The detector detects how much light the sample has absorbed. 4. The detector then converts how much light the sample absorbed into a number.

Schematic of a single beam uv-vis spectrophotometer

# UV and IR spectrophotometers The most common spectrophotometers are used in the UV and visible regions of the spectrum, and some of these instruments also operate into the near-infrared region as well.

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Spectrophotometers designed for the main infrared region are quite different because of the technical requirements of measurement in that region. One major factor is the type of photosensors that are available for different spectral regions, but infrared measurement is also challenging because virtually everything emits IR light as thermal radiation, especially at wavelengths beyond about 5 μm.

Flame Emission Photometry (FEP)

1. Sample solution sprayed or aspirated as fine mist into flame.

Conversion of sample solution into an aerosol by atomiser (scent spray) principle.

No chemical change in the sample in this stage. [atomiser does not convert anything into atoms].

2. Heat of the flame vaporizes sample constituents. Still no chemical change.

3. By heat of the flame + action of the reducing gas (fuel), molecules & ions of the sample species are decomposed and reduced to give ATOMS.

4. Heat of the flame causes excitation of some atoms into higher electronic states.

5. Excited atoms revert to ground state by emission of light energy, hυ , of characteristic wavelength; measured by detector.

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Flame Photometer

FluorescenceFluorescence is a luminescence that is mostly found as an optical phenomenon in cold bodies, in which the molecular absorption of a photon triggers the emission of another photon with a longer wavelength.

The energy difference between the absorbed and emitted photons ends up as molecular vibrations or heat.

Usually the absorbed photon is in the ultraviolet range, and the emitted light is in the visible range, but this depends on the type of the particular fluorophore.

Fluorescence is named after the mineral fluorite, composed of calcium fluoride, which often exhibits this phenomenon.

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Physical ProcessFluorescence occurs when a molecule or quantum dot relaxes to its ground state after being electronically excited.

Excitation:

Fluorescence (emission):

= hν is a generic term for photon energy where: h = Planck's constantand ν = frequency of light. (The specific frequencies of exciting and emitted light are dependent on the particular system.)

= State S0 is called the ground state of the fluorophore (fluorescent molecule) and S1 is its first (electronically) excited state.

Applications in Lighting The common fluorescent tube relies on fluorescence. Inside the glass tube is a partial vacuum and a small amount of mercury.

An electric discharge in the tube causes the mercury atoms to emit light.

The emitted light is in the ultraviolet )UV) range and is invisible, and also harmful to living organisms, so the tube is lined with a coating of a fluorescent material, called the phosphor, which absorbs the ultraviolet and re-emits visible light.

Chromatography

Chromatography is a separations method that relies on differences in partitioning behavior between a flowing mobile phase and a stationary phase to separate the components in a mixture.

A column (or other support for TLC) holds the stationary phase and the mobile phase carries the sample through it.

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Sample components that partition strongly into the stationary phase spend a greater amount of time in the column and are separated from components that stay predominantly in the mobile phase and pass through the column faster.

Components of a mixture may be interacting with the stationary phase based on charge, relative solubility or adsorption.

Chromatography terms

The analyte is the substance which is to be purified or isolated during chromatography A chromatogram is the visual output of the chromatograph. Different peaks or patterns on the chromatogram correspond to different components of the separated mixture.

A chromatograph takes a chemical mixture carried by liquid or gas and separates it into its component parts as a result of differential distributions of the solutes as they flow around or over the stationary phase

The mobile phase is the analyte and solvent mixture which travels through the stationary phase. The retention time is the characteristic time it takes for a particular molecule to pass through the system under set conditions.

The stationary phase is the substance which is fixed in place for the chromatography procedure and is the phase to which solvents and the analyte travels through or binds to.

Examples include the silica layer in thin layer chromatography.

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Capillary-action chromatography

1. Paper chromatographyThis is an older technique which involves placing a small spot of sample solution onto a strip of chromatography paper.

The paper is placed into a jar containing a shallow layer of solvent and sealed. As the solvent rises through the paper it meets the sample mixture which starts to travel up the paper with the solvent.

Different compounds in the sample mixture travel different distances according to how strongly they interact with the paper. This allows the calculation of an retention factor ( Rf )value and can be compared to standard compounds to aid in the identification of an unknown substance.

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2.Thin layer chromatographyThin layer chromatography (TLC) is a widely-employed laboratory technique and is similar to paper chromatography. However, instead of using a stationary phase of paper, it involves a stationary phase of a thin layer of adsorbent like silica gel, alumina, or cellulose on a flat, inert substrate. Compared to paper, it has the advantage of faster runs, better separations, and the choice between different adsorbents.

Different compounds in the sample mixture travel different distances according to how strongly they interact with the adsorbent. This allows the calculation of an Rf value and can be compared to standard compounds to aid in the identification of an unknown substance.

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Column chromatography

Column chromatography based on utilizing a vertical glass column filled with some form of solid support (stationary phase), with the sample to be separated placed on top of this support.

The rest of the column is filled with a solvent (mobile phase) which, under the influence of gravity (pressure), moves the sample through the column.

Similarly to other forms of chromatography, differences in rates of movement through the solid medium are depend on the solubility of the sample (Analyte) in either the stationary or mobile phase.

1.Liquid Chromatography

The mobile phase in liquid chromatography is a liquid of low viscosity which flows through the stationary phase bed.

This bed may be comprised of an immiscible liquid coated onto a porous support, a thin film of liquid phase bonded to the surface of a sorbent, or a sorbent of controlled pore size.

High performance liquid chromatographyHigh performance liquid chromatography (HPLC) is a form of column chromatography used frequently in biochemistry and analytical chemistry.

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The analyte is forced through a column (stationary phase) by a liquid (mobile phase) at high pressure, which decreases the time the separated components remain on the stationary phase and thus the time they have to diffuse within the column.

Gas Chromatography

Gas chromatography makes use of a pressurized gas cylinder and a carrier gas, such as helium, to carry the solute through the column.

There are three types of gas chromatography :

1.Gas adsorption chromatography involves a packed bed comprised of an adsorbent used as the stationary phase. Common adsorbents are zeolite, silica gel and activated alumina. This method is commonly used to separate mixtures of gases. 2.Gas-liquid chromatography is a more common type of analytical gas chromatography. In this type of column, an inert porous solid is coated with a viscous liquid which acts as the stationary phase. Diatomaceous earth is the most common solid used. Solutes in the feed stream dissolve into the liquid phase and eventually vaporize. The separation is thus based on relative volatilities.

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3.Capillary gas chromatography is the most common analytical method. Glass or fused silica comprise the capillary walls which are coated with an absorbent or other solvent. Because of the small amount of stationary phase, the column can contain only a limited capacity. However, this method also yields rapid separation of mixtures.

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