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Introduction

1.1 The Scope of Analytical Chemistry:

Analytical chemistry has bounds which are amongst the widest of any technological discipline.

An analyst must be able to design, carry out, and interpret his measurements within the context of the

fundamental technological problem with which he is presented. The selection and utilization of

suitable chemical procedures requires a wide knowledge of chemistry, whilst familiarity with and the

ability to operate a varied range of instruments is essential. Finally, an analyst must have a sound

knowledge of the statistical treatment of experimental data to enable him to gauge the meaning and

reliability of the results that he obtains.

When an examination is restricted to the identification of one or more constituents of sample, it is

known as qualitative analysis, while an examination to determine how much of a particular species

is present constitutes a quantitative analysis. Sometimes information concerning the spatial

arrangement of atoms in a molecule or crystalline compounds is required or confirmation of the

presence or position of certain organic functional groups is sought. Such examinations are described

as structural analysis and they may be considered as more detailed forms of analysis. Any species

that are the subjects of either qualitative or quantitative analysis are known as anlayte.

1.1.1 The Function of Analytical Chemistry:

Some of the major areas of application are listed below.

(a) Fundamental research:

The first steps in unraveling the details of an unknown system frequently involve the

identification of its constituents by qualitative chemical analysis. Follow up investigations usually

require structural information and quantitative measurements. This pattern appears in such diverse

areas as the formulation of new drugs, the examination of meteorites, and studies on the results of

heavy ion bombardment by nuclear physicists.


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(b) Product development:

The design and development of a new product will often depend upon establishing a link

between its chemical composition and its physical properties or performance. Typical examples are

the development of alloys and of polymer composites.

(c) Product quality control:

Most manufacturing industries require a uniform product quality. To ensure that this requirement

is met, both raw materials and finished products are subjected to extensive chemical analysis. On the

other hand, the necessary constituents must be kept at the optimum levels, while on the otherimpurities such as poisons in foodstuffs must be kept below the maximum allowed by law.

(d) Monitoring and control of pollutants:

Residual heavy metals and organo-chlorine pesticides represent two well known pollution

problems. Sensitive and accurate analysis is required to enable the distribution and level of a

pollutant in the environment to be assessed and routine chemical analysis is important in the control

of industrial effluents.

(e) Assay:

In commercial dealings with raw materials such as ores, the value of the ore is set by its metal

content. Large amounts of material are often involved, so that taken overall small differences in

concentration can be of considerable commercial significance. Accurate and reliable chemical

analysis is thus essential.

(f) Medical and Clinical Studies:

The level of various elements and compounds in body fluids are important indicators of

physiological disorders. A high sugar content in urine indicating a diabetic condition and lead in

blood are probably the most well-known examples.


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Table (1.1): A general classification of important analytical methods

Group Property measured

gravimetric weight of pure analyte or of a stoichiometric compound containing it

volumetric volume of standard reagent solution reacting with the analyte

spectrometric intensity of electromagnetic radiation emitted or absorbed by the

analyte

electrochemical electrical properties of analyte solutions

chromatographic physico-chemical properties of individual analytes after separation

1.1.2 Glossary of Terms

Accuracy:

The agreement between a measured value and the accepted true value.

Precision:

The degree of agreement between replicate measurements of the same quantity. That is, it is the

repeatability of result. Good precision does not assure good accuracy.

Analyte:

Constituent of the sample which is to be studied by quantitative measurements or identified

qualitatively.

Blank:

A measurement or observation in which the sample is replaced by simulated matrix, the conditions

otherwise being identical to those under which a sample would be analyzed. Thus, the blank can be

used to correct for background effects and to take account of analyte other than that present in thesample which may be introduced during the analysis, e.g. from reagents.


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Primary standard:

A substance whose purity and stability are particularly well-established and with which other

standards may be compared.

Requirements of titration:

1- The reaction must be stoichiometric. That is, there must be well-defined and known reaction

between the analyte and titrant. In the titration of acetic acid in vinegar with sodium hydroxide, for

example, a well defined reaction takes place:

CH3COOH + NaOH CH3COONa + H2O

2- The reaction should be rapid.

3- There should be no side reactions, and the reaction should be specific.

4- There should be a marked change in some property of the solution when the reaction is complete.

This may be change in the color of the solution or in some electrical or other physical property of the

solution. In the titration of acetic acid with sodium hydroxide, there is a marked increase in the pH of

the solution when the reaction is complete. A color change is usually brought about by addition of an

indicator whose color is dependent on the properties of the solution, for example, the pH.

5-The point at which an equivalent or stoichiometric amount of titrant is added is called the

equivalence point. The point at which the reaction is observed to be complete is called the end point,

that is, when a change in some property of the solution is detected. The end point should coincide

with the equivalence point.

6-The reaction should be quantitative. That is, the equilibrium of the reaction should be far to the right

so that a sufficiently sharp change will occur at the end point to obtain the desired accuracy.

Requirements of primary standard:

1- It should be 100% pure.

2- It should be stable to drying temperatures.

3- It should be readily available.

4- It should have a high formula weight (to minimize weighing error).


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5- It should posses the properties required for a titration (soluble and react rapidly ....).

1.3 Classification of volumetric methods:

There are four general classes of volumetric methods.

1- Acid-Base:

Many compounds, both inorganic and organic, are either acids or bases and can be titrated with astandard solution of a strong base or a strong acid. The reactions involve the combination of

hydrogen and hydroxide ions to form water. The end points of these titrations are easy to detect,

either by means of indicator or by following the change in pH with a pH meter. The acidity and

basicity of many organic acids and bases can be enhanced by titrating in nonaqueous solvent, so the

weaker acids and bases can be titrated.

2- Precipitation:

In the case of precipitation, the titrant forms an insoluble product with the analyte. An example is

the titration of chloride ion with silver nitrate solution.

3- Complexometric:

In complexometric titrations, the titrant is a complexing agent and forms a water-soluble complex

with the analyte, a metal ion. The titrant is often a chelating agent.

4- Reduction-oxidation:

The "redox" titrations involve the titration of an oxidizing agent with a reducing agent, or vice

versa. An oxidizing agent gains electrons and a reducing loses electrons in a reaction between them.

1.3.1 Expressions of concentration:

1- Concentration of solutions:

Standard solutions are expressed in terms of molar concentrations or molarity (M).

Molar solution is defined as one that contains one mole of substance in each liter of a solution.

Molarity of a solution is expressed as moles per liter or as millimoles per milliliters.

moles = (moles/liter) x liters = molarity x liters

millimoles = molarity x milliliters


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mmole = M x ml

Molarity (M) = (moles of solute) / (volume of solution in liters)

2- The equivalent weight:

The equivalent weight is that weight of a substance in grams that will furnish one mole of the

reacting unit. Thus, for HCl, the equivalent weight is equal to the formula weight:

eq wt HClFormula wt

. . . HCl (g / mol)

1 (eq / mol)

The milliequivalent weight is one thousandth of the eq.wt.

A normal solution contains one gram eq.wt of solute in one liter of solution:

NNo

. of gram- equivalents

No. of liters

No. milligram - equivalentsNo. of milliliters

By rearrangement of these equations we obtain the expression for calculating other quantities:

No of gram eq. = N x No. of liters

No. of milligram eq. = N x ml = N

3- Formality (F):

Is the number of gram formula weight of solute dissolved in liter of solution?

F = number FWs solute

liters solution

4- Normality:

It is often convenient to calculate the titer of the titrant.

The titer is the weight of analyte that is chemically equivalent to 1 ml of titrant, usually expressed in

milligrams. For example, if 1 ml of a hydrochloric acid solution will exactly neutralize 4 mg of

sodium hydroxide, the titer is 4 mg/ml.

Titer (T) can be easily converted to normality as seen from the following equations:


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Tmg

ml , N =

mg

ml x Eq.wt

Thus T= N x Eq.wt

In the example, if the titer of hydrochloric acid solution is 4 mg/ml of sodium hydroxide, the normality

is obtained upon dividing by 40 mg/meq (the equivalent weight of sodium hydroxide) giving a

normality of 0.1 meq/ml.

5- Weight, Volume, and Weight-to-Volume Ratios

Weight percent (% w/w), volume percent (% v/v) and weight-to-volume percent (% w/v)

express concentration as units of solute per 100 units of sample. A solution in which a solute has a

concentration of 23% w/v contains 23 g of solute per 100 mL of solution.

Parts per million (ppm) and parts per billion (ppb) are mass ratios of grams of solute to one million or

one billion grams of sample, respectively. For example, a steel

that is 450 ppm in Mn contains 450 µg of Mn for every gram of steel. If we approxi- mate the density

of an aqueous solution as 1.00 g/mL, then solution concentrations can be expressed in parts per

million or parts per billion using the following relationships.

PPm = mg = µg

Liter mL

PPb = µg = ng

Liter mL

For gases a part per million usually is a volume ratio. Thus, a helium concentration of 6.3 ppm

means that one liter of air contains 6.3 µL of He.

1.3.2 The pH scale:

Sorensen suggested a scale based on the exponent of the hydrogen ion concentration but with sign

reversed. The concentration of H+ or OH- in aqueous solutions can vary over wide range, from 1 M

Or greater to 10-14 M or less.

The pH of a solution is defined as:

pH = - log [H+

]

A similar definition is made for the hydroxyl ion concentration:

pOH = - log [OH-]


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The following summary indicates the relationship: Table 2

H 1 10 10 10 10-

10 10

-

10 10 10 10-

1

0

10-

1

1

10-

1

2

10-

1

3

10-

1

4

pH 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

pO

H

1 13 12 11 10 9 8 7 6 5 4 3 2 1 0

Acidic N. Alkaline

pH scale of water

Kw = [H+] [OH-]

- log Kw = - log [H+] [OH-] = - log [H+] - log [OH-]

pKw = pH + pOH

A t 25°C 14 = pH + pOH

The reaction of any aqueous solution at room temperature is defined:

pH < 7 < pOH acidic reaction

pH = 7 = pOH neutral reaction

pH > 7 > pOH basic reaction

Example:

i-Find the pH of a solution of which [H+] = 4.0x 10-5.

pH = - log [H+] - log (4x10-5) = 5 - log 4 = 5 - 0.602 = 4.398

ii- Calculate the pH of a 2.0 x 10-3 M solution of HCl ?

HCl is completely ionized, so

[H+] = 2.0 x 10-3 M

pH = - log (2.0 x 10-3) = 3 - log 2.0 = 3 - 0.30 = 2.70


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Calculation of pH:

1- pH of strong acid and bases:

Ionization of strong acids and strong bases is complete. The concentration of H+ or OH- is

determined readily from the concentration of the acid or base.

pH = - log [H+]

pOH = - log [OH-]

2- pH of weak acids:

Ka = acidity constant =[ ]

[ ]

H

HA

[A ]-........................................................ (5)

.. [H+] = [A-]

Eq (1) becomes:

Ka =[ ]

[ ]

H

HA

2

................................................................................................ (6)

If the molecular concentration of acid is Ca:

undissociated part = [Ca - [H+]]

Eq. (2) becomes:

Ka =[ ]

]

H

Ca

2

- [H+.......................................................................................... (7)

[H+]2 = Ca . Ka

[H+] Ca . Ka

pH = ½ pCa + ½ pKa

3- pH of weak bases:

HA H+

+ A-

weak acid

BOH B+

+ OH-

weak base


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Kb =[ ]

[ ]

B

BOH

[OH ]-......................................................................................... (8)

.. [B+] = [OH-]

Eq (1) becomes:

Kb =[ ]

[ ]

OH

BOH

2

.......................................................................................... (9)

If the molecular concentration of base is Cb and undissociated part is very small,

Eq. (9) becomes:

Kb =[ ]OH

Cb

2

[OH-]2 = Ca . Kb

[OH-] Cb . Kb

pOH = ½ pCb + ½ pKb

But since pOH = pKw - pH

pKw - pH = ½ pCb + ½ pKb

and pH = pKw - ½ PCb - ½ pKb

4- pH of salts solution:

When an acid in solution is exactly neutralized with base, the resulting solution corresponds

to a solution of the salt of the acid-base pair. This is a situation which frequently arises in analyticalprocedures and the calculation of the exact pH of such a solution may be of considerable importance.

The neutralization point or end point in an acid-base titration is a particular example.

Salts may in all cases be regarded as strong electrolytes so that a salt AB derived from acid AH

and base BOH will dissociate completely in solution. If the acid and base are strong, no further

reaction is likely to occur and the solution pH remains unaffected by the salt. However, if either or

both acid and base are weak a more complex situation will develop. It is convenient to consider three

separate cases: (a) weak acid - strong base, (b) strong acid - weak base and (c) weak acid - weak

base.


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(a) Weak acid - strong base solution:

The conjugate base A- will react with water and undergo hydrolysis,

............................................... (10)

Prodrug undissociated acid and hydroxyl ions with an accompanying rise in pH. The equilibrium

constant for this reaction is known as the hydrolysis constant Kh.

KAH

Ah

[ ]

[ ]

[OH ]

[H O]

-

2

............................................................................... (11)

(The solvent term [H2O] is by convention omitted. Kb is simply related to Kw and Ka by equation

(12) and as such is a redundant constant whose use should be discouraged).

K

K

AH

A

OH

AKw

a

h

[ ]

[ ]

[ ]

[ ]

[OH ] [H ]

[H ]

[AH]- +

+............................ (12)

Equation (10) shows that the amounts of AH and OH- generated in the hydrolysis are equal.

Furthermore, if it is assumed that only a small amount of the salt is hydrolyzed, the concentration C A-

of the salt dissolved is approximately the same as the concentration of A-. Then from (12):

[ ]OH

C

K

K KA

w

a

b

2

[OH-] = K

KC C K Kw

aA A w a

. / /

1 2

.................................................. (13)

pOH = ½ pKw - ½ pKa + ½ pCA- = pKw - pH

pH = ½ pKw + ½ pKa - ½ PCs

The pH of the solution will be dependent upon both pK a for the acid HA and on the

concentration of the salt dissolved in the solution. For example, the pH of solutions of sodium

cyanide may be calculated as follows:

A-

+ H2O AH + OH-


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pKa for HCN = 9.22

pH = 7.0 + 4.6 + ½ log CA-

= 11.6 + ½ log CA-

Thus when:

C= 1 mole dm-3 pH = 11.6

C= 0.1 mol dm-3

pH = 11.1C= 2 mol dm-3 pH = 11.8

(b) Strong acid - weak base solution:

Similar reasoning shows hydrolysis leading to the production of hydrogen ions and drop in pH,

and enables an analogous expression for pH to be derived, i.e.

pH = ½ pKw - ½ pKb + ½ pC ....

1.3.3 Buffers:

A buffer is defined as a solution that resists change in pH when a small amount of an acid or

base is added or when the solution is diluted. A buffer solution consists of a mixture of a weak acid

and its conjugate base or a weak base and its conjugate acid at predetermined concentrations and

ratios. Typical mixtures are acetic acid and sodium acetate, ammonia and ammonium chloride, boric

acid and borate ...etc. The pH values of such mixtures will lie at point on rather flat regions of the

titration graphs, pH vs. milliters, for the various weak acids or bases.

Consider an acetic acid - acetate buffer. The acid equilibrium that governs this system is:

......................................................... (21)

Since we have added a supply of acetate ions to the system (from sodium acetate for example), the

hydrogen ion concentration is no longer equal to the acetate ion concentration. The hydrogen ion

concentration is:

B-

+ H2O BOH + H+

HOAc H+

+ OAc-


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[ ][ ]

[ ]H K

HOAc

OAca

............................................................................... (22)

Taking the negative logarithm of each side of this equation we have

log[ ] log log[ ]

[ ]H K

HOAc

OAca

................................................... (23)

pH pKHOAc

OAca log

[ ]

[ ]

upon inverting the last log term, it becomes positive:

pH pKOAc

HOAca

log[ ]

[ ]

This terms of the ionization constant equation is called the Henderson-Hasselbach equation. It is

useful for calculating the pH of a weak acid solution containing its salt. A general form can be

written for a weak acid HA that ionizes to its salt, A -, and H+:

..................................................................... (24)

pH pKA

HAa

log[ ]

[ ]...................................................................... (25)

pH pKconjugate

acida log

[ ]

[ ]

base

The mixture of a weak acid and its salt may also be obtained by mixing an excess of weak acid

and some strong base to produce the salt by neutralization or by mixing an excess of salt with strong

acid to produce the weak acid component of the buffer.

Mechanism of buffer for a mixture of a weak acid and its salt can be explained as follows. The

pH is governed by the logarithm of the ratio of the salt and acid.

pH cons t tan + log

[A ]

[HA]

-

HA H+

+ A-


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If the solution is diluted, the ratio remains constant, and so the pH of the solution does not

change. If a small amount of a strong acid is added, it will combine with an equal amount of the A

to convert it to HA. That is, in the equilibrium HA H+ + A, Le Châtelier's principle indicates that

add H+ will combine with A to form HA, with equilibrium lying far to the left if there is an excess

of A. The change in the ratio [A]/[HA] is small and hence the change in pH is small. If a small

amount of a strong base is added, it combine with part of the HA to form an equivalent amount of

A. Again, the change in the ratio is small.

The amount of acid or base that can be added without causing a large change in pH is governed

by the buffering capacity of the solution. The buffering capacity increases with concentrations of the

buffering species. In addition to concentration, the buffering capacity is governed by the ratio of HA

to A. It is maximum when the ratio is unity, that is, when the pH = pKa.

pH pK pKa a log[ ]

[ ]

1

1

...................................................................... (26)

In general the buffering capacity is satisfactory over a pH range of pKa ± 1.

Examples:

Calculate the pH of the solution produced by adding 10.0 ml of N hydrochloric acid to 1 liter

of a solution which is 0.1 N in acetic acid and 0.1 N in sodium acetate (K a= 1.82 x 10-5).

pH pKSalt

Acida log

[ ]

[ ]

Neglecting the volume change from 1000 to 1010 ml, the hydrochloric acid reacts with acetate ion

forming practically undissociated acetic acid.

[CH3COO] = 0.1 - 0.01 = 0.09

[CH3COOH] = 0.1 - 0.01 = 0.11

pH = 4.74 + log0 09011..

= 4.71 - 0.09 = 4.65

H+

+ CH3COO-

CH3COOH


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Hence on adding the strong acid, the pH changes only by 4.74-4.65 = 0.09 pH unit, whereas, if

10 ml of N-hydrochloric acid were added to 1 liter of pure water (pH= 7), the pH would have

changed from 7 to

- log (0.01) = 2, i.e., by 5 pH units. This illustrates the action of the acetic acid sodium acetate buffer.

Similar calculation applies for mixtures of weak bases and its salt. We can consider the equilibrium

between the base B and its conjugate acid BH+ and write a pKa for the conjugate (Brnsted) acid:

BH+ = B + H+ ....................................................................................... (27)

KaB H

BH

K

K

w

b

[ ][ ]

[ ].................................................................................... (28)

The logarithmic Henderson-Hasselbach form exactly as above:

[ ] .[ ]

[ ]

.[ ]

[ ]

H KBH

B

K

K

BH

B

aw

b

log[ ] log log[ ]

[ ]log log

[ ]

[ ]H K

BH

B

K

K

BH

Ba

w

b

pH pKB

HBpK pK

B

BHa w b log

[ ]

[ ]( ) log

[ ]

[ ]

pH pKoton accep

oton donorpK pK

oton accep

oton donora w b log

[Pr ]

[Pr ]( ) log

[Pr ]

[Pr ]

tor tor

Since pOH = pKw - pH, we can also write:

pOH pKBH

BpK

oton donor

oton accepb b

log[ ]

[ ]log

[Pr ]

[Pr ]+

tor

The alkaline buffering capacity is maximum at pH = pKa = 14 - pKb or pOH = pKb with a usual

range of pKa ± 1.

We must select a buffer with pKa value near the desired pH i.e. a weak acid and its salt give the

best buffering in acid solution and a weak base and its salt give the best buffering in alkaline

solution. Table 3 shows some typical buffer solutions.


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Table 1.3.4.1: Some typical buffer solutions

Solutions pH range

Phthalic acid and potassium hydrogen phthalate

Citric acid and sodium citrate

Acetic acid and sodium acetate

Sodium dihydrogen phosphate and disodium hydrogen phosphate

Ammonia and ammonium chloride

Borax and sodium hydroxide

2.2-4.2

2.5-7.0

3.8-5.8

6.2-8.2

8.2-10.2

9.2-11.2

1.3.4 Acid-Base Indicators:

Acid-base indicators are substances whose presence during a titration renders the end-point

visible. Thus, at a certain pH very near, or at the equivalence point of the titration the indicator

produces in the system a change which is easily perceptible to the eye, and may consist of:

a- Sharp transformation from one color to another or to colorless.

b-Formation of a turbidity in a clear solution, or clearing up of a turbidity.

c- Development or disappearance of a fluorescence.

Most of the color acid base indicators of practical value are organic in nature. As the color

changes of these indicators depend on the change of the pH, they must themselves be acids or bases.

Table 1.3.5.1 : A range of visual indicators of acid-base titrations

Indicator pKIn

Low pH

color

High pH

color

Experimental

color change

range/pH

cresol red

thymol blue

bromo-phenol blue

methyl orange

ca. 1

1.7

4.0

3.7

red

red

yellow

red

yellow

yellow

blue

yellow

0.2-1.8

1.2-2.8

2.8-4.6

3.1-4.4


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methyl red

bromo-thymol blue

phenol red

phenolphthalein

5.1

7.0

7.9

9.6

red

yellow

yellow

colorless

yellow

blue

red

red

4.2-6.3

6.0-7.6

6.8-8.4

8.3-10.0

The well known indicator phenolphthalein (below) is a diprotic acid and it’s colorless. It

dissociates first to a colorless form and then, on losing the second hydrogen to an ion with a

conjugated system; a red color results.

O

O

HO

OH

+ H2O

COO

HO

OH

OH + H 3O

COO

O

OPhenolphthalein

colorless, H2In Phenolphthalein

colorless, HIn- Phenolphthalein

Red, In2-

Methyl orange, another widely used indicator, is a base and is yellow in the molecular form.

Addition of a hydrogen ion gives a cation which is pink in color.

Increasing the concentration of indicators has a serious effect on one-color indicators such as

phenolphthalein.

1.3.5Back titration:

Na+ -

O3SN N N(CH3)2 + H3O

+

In, Yellow

Na+ -

O3SN N N(CH3)2

H

+ H2O

In+, Pink

+


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In back-titration, a known number of millimoles of reactant are taken in excess of the analyte.

The unreacted portion is titrated for example, in the titration of antacid tablets with a strong acid such

as HCl.

1.4 Gravimetric Methods of Analysis:

Gravimetry encompasses all techniques in which we measure mass or a change in mass.

When you step on a scale after exercising you are making, in a sense, a gravimetric determination of

your mass. Measuring mass is the most fundamental of all analytical measurements, and gravimetry

is unquestionably the oldest analytical technique.

1.4.1 Types of Gravimetric Methods:

1- Precipitation Gravimetry

Precipitation gravimetry is based on the formation of an insoluble compound following

the addition of a precipitating reagent, or precipitant, to a solution of the analyte. In most methods

the precipitate is the product of a simple metathesis reaction between the analyte and precipitant;

however, any reaction generating a precipitate can potentially serve as a gravimetric method. Most

precipitation gravimetric methods were developed in the nineteenth century as a means for

analyzing ores. Many of these methods continue to serve as standard methods of analysis.The

indirect determination of PO3-2 by precipitating HgCl2 is representative example, as is the direct

determination of Cl – by precipitating AgCl

2-electrogravimetry:

In electrogravimetry the analyte is deposited as a solid film on one electrode in an

electrochemical cell. The oxidation of Pb+2 and its deposition as pbo2 on a Pt anode is one example

of electrogravimetry. Reduction also may be used in electrogravimetry. The electrodeposition of Cu

on a Pt cathode, for example, provides a direct analysis for Cu +2

3- volatilization Gravimetry :


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When thermal or chemical energy is used to remove a volatile species, we call the method

volatilization gravimetry. In determining the moisture content of food, thermal energy vaporizes

the H2 O. The amount of carbon in an organic compound may be determined by using the chemical

energy of combustion to convert C to CO2

4- particulate gravimetry:

In particulate gravimetry the analyte is determined following its re- moval from the

sample matrix by filtration or extraction. The determination of sus- pended solids is one example of

particulate gravimetry.

1.4.2 Important OF Gravimetry:

Gravimetry is one of only a small number of techniques whose measurements re-

quire only base SI units, such as mass and moles, and defined constants, such as Avogadro’s

number and the mass of 12C.* The result of an analysis must ultimately be traceable to methods,

such as gravimetry, that can be related to fundamental physical properties. Most analysts never

use gravimetry to validate their methods. Verifying a method by analyzing a standard reference

material, however, is common. Estimating the composition of these materials often involves

a gravimetric analysis.

1.5 Volumetric Precipitation Titrations (Precipitimetry)

Precipitation titrations are volumetric methods based on the formation of a slightly soluble

precipitate. They are in many ways simpler than gravimetric methods. The precipitate needs not be

separated, and needs not be pure, as long as the impurity does not consume titrant. The substance is

determined simply by converting it into an insoluble form of known composition by adding a

standard solution of the titrant. The equivalence point is reached when an equivalent amount of the

titrant has been added. From the volume of the latter, the amount of the substance is calculated. The


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precipitate must be sufficiently insoluble to ensure completion of the reaction and to ensure a marked

change in the concentration of the ions of precipitate at the equivalence point of the titration

.

Table:1.5.1 Substances determined by precipitation titrations with Ag+.

AsO43-, Br-, CNO-, CO3

2-, CrO42-, CN-, Cl-, C2O4

2-, I-, PO43-, SCN-, S2-, fatty

acids

Table 1.5.2: Miscellaneous precipitation titrations

Analyte Reagent Precipitate

Cl-, Br-

SO42-, MoO4

2-

Zn2+

PO34-, C2O4

2-

Hg2(NO3)2

Pb(NO3)2

K4Fe(CN)6

Pb(OAc)2

Hg2Cl2, Hg2Br2

PbSO4, PbMoO4

K2Zn3[Fe(CN)6]2

Pb3(PO4)2, PbC2O4

1.5.2 Limitations of volumetric precipitation titrations:

Volumetric precipitation reactions have several limitations combining those of the

titrimetric methods in general, and some of those of the gravimetric methods. In particular, few

points need here be stressed in comparing volumetric precipitation reactions with the other

volumetric reactions, where no precipitates form.

1- The rate of reaction:

Particularly in dilute solutions, is often slow so that a long wait is necessary for each addition

of titrant to react completely. As the equivalence point is approached, a high degree of

supersaturation will not exist, and the rates of precipitation and attainment of solubility equilibrium

become too slow for convenience of titration. The most suitable alternative, and often the only one, is

to take advantage of a more rapid precipitation in the reverse direction, i.e. to add a measured excess

of titrant and back-titrate.


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2- The lack of suitable indicators for many precipitation titrations imposes another limitations in

such titrations.

1.5.3 Limitations of argentometric titrations:

1- Reducing agents, such as, sulphur dioxide interfere by reducing the silver ions, and must be

removed by previous oxidation.

2- Coloured compounds of any sort obscure the end point, which is taken as the faintest ting of

colour detectable on the precipitated silver halide, or in solution, as the case may be.

3- Silver halides are sensitive to photodecomposition, and the titration should be carried out in

diffused daylight, or artifical light.

4- Most cations except the alkalies and alkaline earths interfere in several ways. (a) Some, such as

Fe3+ form insoluble coloured hydroxide in neutral or slightly acid medium; (b) Some, such as Al 3+,

hydrolyse to insoluble basic salts in neutral or slightly acid solution, showing a tendency to

coprecipitate chloride; (c) Hg2+ form soluble complexes with halides of the type [HgI4]2-.

1.5.4 Solubility product:

Ionic reactions are actually complete when any change occurs that lowers the concentrations of

ions to very small values. The factor governing the completeness of a precipitation reaction is the

solubility of the precipitate formed. The more insoluble the precipitate, the more complete is the

reaction at the equivalence point of the titration, and the larger is the change in concentration of the

reacting ions. The equilibrium constant expressing the solubility of a precipitate is the familiar

solubility product constant.

Let us consider what happens when the sparingly soluble salt AB comes in contact with water.

Some of the salt dissolves in water and, assuming this compound to be an ionic solid, dissociates into

its ions A+ and B-. This reaction can be represented in its simplest form (i.e. without considering

hydration of the ions) by the equation:

AB A+ + B- ................................................................................... (1)


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However, as the salt AB dissolves, more and more A+ and B- are in solution, with the net result

that the chance of their recombining to form AB increases; that is, the equilibrium represented

simply by equation (2) for the saturated solution is established.

AB (solid) A+ + B- (solution) (2)

The equilibrium constant for this reaction is:

KA B

AB

[ ][ ]

[ ]

Since the concentration of AB is constant as long as the temperature remains constant and there

is some solid AB in contact with the solution, this equilibrium expression becomes.

K X const. = [A+] [B-] = SAB = Solubility product constant.

Let us consider in the same way the saturated solution of the sparingly soluble salt X m Yn

which dissociates into m cation, Xn+

and n anions, Ym-

. The equilibrium for this saturated solution

can be represented by the equation:

[Xn+]m [Ym-]n = SXm Yn

1.6 spectrometric Methods of Analysis:

Electromagnetic radiation, or light, is a form of energy whose behavior is described by the

properties of both waves and particles. The optical properties of electromagnetic radiation, such as

diffraction, are explained best by describing light as a wave. Many of the interactions between

electromagnetic radiation and matter, such as absorption and emission, however, are better

described by treating light as a particle, or photon.


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1.6.1 Wave Properties of Electromagnetic Radiation :

Wave Properties of Electromagnetic Radiation Electromagnetic radiation consists of

oscillating electric and magnetic fields that propagate through space along a linear path and with a

constant velocity (Figure). In a vacuum, electromagnetic.

In a vacuum, electromagnetic radiation travel at the speed of lightc,which 2.99*108 m/s. .

Electromagneticradiation moves through a medium other than a vacuum with a velocity, v, less than

that of the speed of light in a vacuum.

The difference between v and c is small enough (0.1%) that the speed of light to three

significant figures ,3.00 108 m/s, is sufficiently accurate for most purposes.

Oscillations in the electric and magnetic fields are perpendicular to each other,and to the

direction of the wave’s propagation. Figure 10.1 shows an example of plane-polarized

electromagnetic radiation consisting of an oscillating electric field and an oscillating magnetic

field, each of which is constrained to a single plane. Normally, electromagnetic radiation is

unpolarized, with oscillating electric and magnetic fields in all possible planes oriented

perpendicular to the direction of propagation.

The interaction of electromagnetic radiation with matter can be explained using either

the electric field or the magnetic field. For this reason, only the electric field component is shown inFigure 10.2. The oscillating electric field is described by a sine wave of the form

E =Ae sin(2πνt + Φ)

where E is the magnitude of the electric field at time t, A e is the electric field’s maxi-mum

amplitude, ν is the frequency, or the number of oscillations in the electric field per unit time,

and Φ is a phase angle accounting for the fact that the electric field’s magnitude need not be zero at t

= 0. An identical equation can be written for the magnetic field, M

M= Am sin(2πνt + Φ)


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where Am is the magnetic field’s maximum amplitude.An electromagnetic wave, therefore,

is characterized by several fundamental properties, including its velocity, amplitude, frequency,

phase angle, polarization, and direction of propagation. Other properties, which are based on

these funda-mental properties, also are useful for characterizing the wave behavior of electro-

magnetic radiation. The wavelength of an electromagnetic wave, λ, is defined as the distance

between successive maxima, or successive minima (see Figure.1.6.1).

1.6.3 Particle Properties of Electromagnetic Radiation

When a sample absorbs electromagnetic radiation it undergoes a change in energy. The

interaction between the sample and the electromagnetic radiation is easiest to understand if we

assume that electromagnetic radiation consists of a beam of energetic particles called photons. When

a photon is absorbed by a sample, it is “destroyed,” and its energy acquired by the sample. The

energy of a photon, in joules, is related to its frequency, wavelength, or wavenumber by the

following equations


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NuclearCore-

level

electron

Valence

Molecular electrons

vibrations

Molecular

rotations;

electron

Nuclear

spin

-rayX-UV IRMicrowaveRadio wave

where h is Planck’s constant, which has a value of 6.62606896(33)×10−34 J·s

Wavelength(m)

10 –

1

4

10 –12 10 –10 10 –8 10 –6 10 –4 10 –2 100 102

Frequency (s – 1) 1022 1020 1018 1016 1014 1012 1010 108

Type of transition

Spectral region

Visible

Wavelength (nm) 380

480 580 680 780

Figure 1.6.2

The electromagnetic spectrum

showing the colors of the

visible spectrum.

Violet Blue Green Yellow Orange Red


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2.1Chromatography

The term "chromatography" is derived from the original use of this method for

separating yellow and green plant pigments. Chromatography has since evolved into

a very general separation method for many types of mixtures. Examples of the

application of chromatographic methods are (i) the purification of reaction mixtures

in chemical synthesis, (ii) the purification of bio-molecules such as proteins for

pharmaceutical research, (iii) the analysis of complex sample mixtures such as

those obtained in forensics (body fluids, paints etc) and (iv) the analysis of

environmental samples.

Key terms:

Adsorbtion: Interaction of solute molecules (or atoms or ions) with the

surface of the stationary phase (note that it is different from

absorption where the molecules fill the pores of a solid).

Eluent: The mobile phase (usually for solvents)

Elution: Motion of the mobile phase through the stationary phase

Elution time: The time taken for a solute to pass through the system. A

solute with a short elution time travels through the stationary

phase rapidly, i.e. it elutes fast.

Mobile phase: The part of the chromatography system that is mobile.

Commonly a solvent mixture (as in column chromatography or thin

layer chromatography or a gas (as in gas chromatography).

Normal phase: “Unmodified” stationary phase where polar solutes interact


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strongly and run slowly

Reverse phase: “Modified” stationary phase where polar solutes run fast i.e.reverse order

Resolution: Degree of separation of different solutes. In principle, resolution

can be improved by using a longer stationary phase, finer stationary

phase (e.g. column packing or TLC plate coating) or slower elution.

Stationary phase: The part of the chromatography system that is fixed in place. Most

commonly a solid e.g. the packing in column chromatography or gas

chromatography or the coating on a chromatographic plate.

Rf value distance travelled by solute

distance travelled by solvent

Rf = retardation factor. The Rf value has to be between 0 and 1 (by definition),

and it is typically reported to two decimal places.

2.1.1 Description

Chromatography is based on selective adsorption of compounds on a solid (or

liquid) with high surface area (the stationary phase). As the solute mixture passes

over the solid, the components are adsorbed and then released from the surface atdiffering rates. This means that the solutes are continuously partitioned between the

adsorbent and the mobile phase, either a gas or a solvent mixture.

The stronger the interaction of the solute with stationary phase, the slower the

solute will progress. The motion of the solute and solvent through the stationary

phase in called elution. The process is analogous to fractional distillation or


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extraction, in which different compounds are partitioned between liquid and vapour,

or between two immiscible liquids, respectively.

Chromatography can be carried out in many ways. In column chromatography

and in thin layer chromatography (TLC) a solution of the mixture flows over a solid

adsorbent. Separation occurs as molecules are adsorbed and desorbed while

passing over the surface. In paper chromatography applications, the mixture is

partitioned between water molecules adsorbed on the paper and a solvent that movesover the paper. In gas chromatography (GC, also called vapour phase

chromatography), a mixture of volatile compounds is separated by passing the vapour

over an adsorbent packing in a long, heated tube.

.

Chromatographic methods have high "resolving power", i.e. they are capable of

sharp separations of closely related compounds, particularly when very small samples

are used. Both GC and column chromatography can be carried out with instruments

that detect extremely small amounts of compounds in the gas or liquid stream, as it

leaves the chromatographic column. In GC, the detector responds to the thermal

conductivity of the gas stream or the ionisation of the gas as it passes through a

flame.

This gas stream can be introduced straight into a mass spectrometer (MS) to

give the very important technique of GCMS which facilitates the separation and

identification of samples within a mixture. In liquid (column) chromatography

instruments, the detector senses changes in the refractive index or uv-visible

absorption of the solution.

Signals from the detector corresponding to each component in the mixture


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and proportional to the amount of the compound are recorded automatically on a

chart. These instruments thus provide powerful methods for quantitative analysis.

There are four main types of chromatography

Gas Chromatography

Liquid Chromatography

Thin - layer Chromatography

Paper Chromatography

2.2 Analysis of a Mixture by Gas Chromatography:

GC is carried out using an instrument containing a long but very thin metal

tube filled with an inert support (usually silica) as the stationary phase and a stream

of helium gas as the mobile phase. The coiled tube, the "column" is heated in a

thermostatically controlled oven. A dilute solution of the liquidsample is typically prepared in a volatile solvent such as diethyl ether. A small

amount of this sample solution (maybe 1 L) is then injected onto the column and is

carried forward by the helium carrier gas. The oven temperature is typically

gradually increased up to about 270oC over a 10 to 20 minute period. In general, the

lower the boiling point of the liquid, the quicker it will be carried through the column


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(so it has a short retention time). When the sample exits the column, the liquid is

detected by the detector and the amount of liquid is measured, this information is

usually plotted as a "trace" with x-axis as time and the y-axis the response of the

detector as the abundance (i.e. the amount detected).

Therefore the area of the peak is directly proportional to the amount of material

it represents. A typical GC trace is shown below. This sample contained three

compounds. Based on the peak areas we see a 49 : 48 : 3 ratio. The minor impurity

(retention time 11.48 minutes) is a high boiling contaminant.

Figur 2.1


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2.2.1Use of Gas Chromatography :

Is used in airports to detect bombs and is used is forensics in many different

ways. It is used to analyze fibers on a persons body and also analyze blood found at a

crime scene. In gas chromatography helium is used to move a gaseous mixture through

a column of absorbent material.

2.3 Liquid Chromatography

2.3.1 Brife History and Deffintion:

Liquid chromatography was defined in the early 1900’s by the work of the

Russian botanist, Mikhail S. Tswett. His pioneering studies focused on separating

compounds (leaf pigments), extracted from plants using a solvent, and in a column

packed with particles

Tswett filled an open glass column with particles. Two specific materials that he

found useful were powdered chalk (calcium carbonate) and alumina. He poured his

sample (solvent extract of homogenized plant leaves) into the column and allowed it to

pass into the particle bed.This was followed by pure solvent.

As the sample passed down through the column by gravity, different colored

bands could be seen separating because some components were moving faster than

others.He related these separated, different-colored bands to the different compounds

that were originally contained in the sample.


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He had created an analytical separation of these compounds based on the

differing strength of each compound’s chemical attraction to the particles. The

compounds that were more strongly attracted to the particles slowed down, while other

compounds more strongly attracted to the solvent moved faster. This process can be

described as follows:

The compounds contained in the sample distribute, or partition differently

between the moving solvent, called the mobile phase, and the particles, called the

stationary phase. This causes each compound to move at a different speed, thus

creating a separation of the compounds.

Tswett coined the name chromatography (from the Greek words chroma,

meaning color, and graph, meaning writing — literally, color writing) to describe his

colorful experiment. (Curiously, the Russian name Tswett means color).

Today, liquid chromatography, in its various forms, has become one of the most

powerful tools in analytical chemistry

.

Figure (2.2) Tswett’s Experiment

Liquid Chromatography (LC) Techniques2.4


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Liquid chromatography can be performed using planar (Techniques 1 and 2) or

column techniques (Technique 3). Column liquid chromatography is the most powerful

and has the highest capacity for sample. In all cases, the sample first must be dissolved

in a liquid that is then transported either onto, or into, the chromatographic device

2.5Technique (1): Thin - layer Chromatography

TLC is carried out on glass plates or strips of plastic or metal coated on one

side with a thin layer of adsorbent. The adsorbent contains a small amount of gypsum

(CaSO4) which acts as a binder to give an adherent coating.

For routine work, small TLC plates can be prepared by dipping microscope

slides in a slurry of the adsorbent in chloroform..

More uniform plates are obtained by mixing the adsorbent with an equal weight

of water, spreading the mixture on a glass plate and allowing it to set dry.Precoated

TLC plates are commercially available with various adsorbents in very uniform

layers.A series of photographs are provided at the end of this document to show the

steps in running a TLC plate.

To "load" the plate, very small samples of the sample mixture in some volatile

solvent (e.g. diethyl ether or chloroform) are applied as spots near one end and the

volatile application solvent is allowed to evaporate. The plate is then placed, sample

end down, in a closed vessel containing a shallow pool of the "developing solvent".

The solvent rises on the plate by capillary action, passing over the sample and

causing the compounds to move at varying rates depending on their relative affinities

for the adsorbent and the solvent. When the solvent front has risen to just below the

top of the plate, the plate is removed and the solvent is allowed to evaporate. The

zones or spots containing the various components of the mixture are then detected at


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various points along the plate. If the compounds are colourless, they are made visible

by treating the plate with a reagent, such as iodine vapour, that causes colour to

develop.

2.5.1 Identification by TLC:

The main uses of TLC are for quick observation of the number of compounds

present in a sample, and for qualitative detection of a given compound in a mixture.

For the latter purpose, a sample of the compound in question is placed in one position

at the bottom of the plate and a sample of the mixture is placed in an adjacent position.

With a plate 3 to 4 cm wide, it is possible to place samples in as many as five or six

"lanes".

If identical compounds are present in two or more lanes, they should appear at

the same height after the plate has been developed with solvent. The position of the

spot relative to the solvent front, called the Rf value (distance travelled by spot /

distance travelled by solvent), depends on the thickness of the coating, the amount of

sample and the temperature, and may vary from one plate to the next. Comparison of

two samples on the same plate is therefore essential.

In addition to the position of the known sample and that of a spot in the mixture,

the appearance and / or colour of the compound on the developed plate

May greatly strengthen the identification if it is distinctive after visualisation. If

there is a reagent specific for the compound of interest, this is sprayed in a fine mistover the surface.

A general reagent such as iodine will cause brown or black spots with

practically all compounds; these may be slightly different shades, but the colour is

usually not very characteristic. Another method for visualising spots is illumination of

the plate with an ultraviolet lamp. Many substances, particularly aromatic compounds,


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will show a bright fluorescence, which may have a characteristic colour. A further

possibility is use of an adsorbent layer that contains a trace of fluorescent dye.

Compounds that are fluorescent still show up as bright spots on a light background;

any others appear as a dark spot since they quench the fluorescence of the background

dye.

2.5.2Preparing to run TLC

Capillaries or TLC applicators for applying the samples to the chromatographic

plate can be prepared by heating and drawing out a soft glass pipette or melting point

or capillary tubes. Soften a 1 cm section in the centre of the glass tube by heating in a

low Bunsen burner flame, then remove the tube from the flame and draw it out to a

thread-like thickness. Allow the glass to cool then carefully break the glass in the thin

portion gives you the capillaries.

The developing jar can be prepared from a beaker or jar with watch glass

as a lid. The developing jar is usuallylined with a piece of filter paper (cut to

shape and size) which helps create a solvent saturated environment.The solvent

system is added to the developing jar which is then carefully turned at an angle


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to saturate the filter paper The solvent depth should be less than 5mm (typically

2-5 mm)

origin

TLC plates or sheets should be handled by the edges, and you should avoid touchingthe coated surface otherwise fingerprints will ruin the experiment. Samples of the test

solutions should be spotted on the coated side about 1 cm from one end of the sheet,

and about 0.5 cm apart, with the outer two spots about 0.5 cm from the edge of the

sheet. It is common practice to draw a light pencil line about 1cm up from the bottom

edge of the plate (this line defines the origin) and then dividethis line at about 5mm

intervals to create the lanes.

Samples of unknowns are typically spotted in the middle lanes of the plate. It is

important to avoid applying too large an amount of sample. The spot after

application should be about 1 to 2 mm in diameter. It is a good idea first to practice

applying spots to a small scrap of the sheet. You can check to see how well you are

doing by examining the spots under the UV lamp. If the spots are too pale at this

stage, then you will need more applications to the plate, or you should concentrate

your samples.

To apply the samples, touch the end of a capillary tube to the solution and then

touch this gently to the plate at the proper place. When the samples have been applied,

place the sheet, spotted end (origin) down, in a developing jar containing a pool of


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solvent no more than 5 mm deep. On no account must spots dip below the surface of

the solvent pool when the sheet is placed in the developing jar.

The solvent system used depends on the nature of the samples. Many

experimental procedures will define the solvent system used, but at times a chemist

will need to develop their own system. It is important to handle the plates without

touching the front surface of the silica covered plate or fingerprints will result and the

plate may be spoiled. Use clean forceps for transferring the plates in and out of the

developing and visualising jars. Once the plate is in place, cap the jar securely and

develop the chromatogram until the solvent has risen to within 1 or 2 cm of the top of

the sheet.

When the solvent front is close to the top of the sheet remove the sheet from the

jar, and mark the solvent front with a small scratch (using a spatula or forceps) or a

pencil mark. Recap the developing jar and out the TLC sheet to one side and it allow

it to dry horizontally. In some cases the spots on the plate will be visible to the naked

eye. In other cases the plate will need to be visualised using other methods such as a

UV lamp or with a stain such as iodine.

It is a good idea to draw circles around the spots with a pencil as a permanent

record of thev isualisation. The Rf value (retardation factor) for a spot can now be

calculated and recorded along with the colour (if any) of the spot.

solvent front y = distance travelled by solvent

x = distance travelled by sample(measured to the point of

maximum colour intensity)y origin

x Rf = x/y


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When looking at a developed TLC plate it is good idea to review the “quality” of the

analysis or note if there are any problems with

the plate that means it might need

to be rerun. A poor quality plate would mean that it is difficult to obtain the

desired information from the plate. Some common problems are described below:

Problem Possible causes

Samples does not show up Not enough sample, differentvisualisation method required

Streaking This could be due to the nature of thesample, due to sample overload or a poor

choice of solvent mixture.

Diffuse / blurred spots The origin line was below the solventlevel or the plate was developed for too long sothe solvent front reached the top of the plate.

Sample “lanes” are not straight

/ parallel A problem with the way the solvent ran dueto the plate being crooked in the solvent,touching the filter paper in the developing jar orthe plate was chipped.


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Preparing a sample to use on TLC by dissolvinga small amount of the sample in a small

Adding the volatile solvent to dissolve thesample

Spotting the sample onto the TLC plate with amicrocapillary


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Figur(2.3)

Handling the TLC plate with forceps in order to place

it in the developing tank.

With the lid on, the TLC plate is developed by allowingthe solvent to run up the plate. Note that the solventlevel is below the pencil line on the plate that marks

the origin (i.e. the line where the samples wereapplied).

After development, the TLC plate is removed andvisualised. Here we see it under a UV lamp.


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Used of Thin-layer Chromatography

:2.5.3

uses an absorbent material on flat glass or plastic plates. This is a simple and rapid

method to check the purity of an organic compound. It is used to detect pesticide or

insecticide residuesin food. Thin-layer chromatography is also used in forensics to

analyze the dye composition of fibers.

Techniqe : Paper Chromatography2.6

In Figure(2.4), samples are spotted onto paper (stationary phase). Solvent

(mobilephase) is then added to the center of the spot to create an outward radial flow.

This is a form of paper . (Classic paper chromatography is performed in a manner

similar to that of TLC with linear flow.) In the upper image, the same black FD&C dye

sample is applied to the paper

Figure(2.4): Paper Chromatography

The difference in separation power for this particular paper when compared to

the TLC plate. The green ring indicates that the paper cannot separate the yellow and


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blue dyes from each other, but it could separate those dyes from the red dyes. In the

bottom image, a green sample, made up of the same yellow and blue dyes, is applied

to the paper. As you would predict, the paper cannot separate the two dyes. In the

middle, apurple sample, made up of red and blue dyes, was applied to the paper. They

are well separated.

Paper Chromatography is one of the most common types of chromatography. It

uses a strip of paper as the stationary phase. Capillary action is used to pull the

solvents up through the paper and separate the solutes.

Techniqe : Solid-Phase Extraction (SPE)2.7

In this, the most powerful approach, the sample passes through a column or a

cartridge device containing appropriate particles (stationary phase). These particles are

called the chromatographic packing material.

Solvent (mobile phase) flows through the device. In solid-phase extraction (SPE), the

sample is loaded on to the cartridge and the solvent stream carries the sample through

the device.

As in Tswett’s experiment, the compounds in the sample are then separated by

traveling at different individual speeds through the device.

Here the black sample is loaded onto a cartridge. Different solvents are used in

each step to create the separation


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Figure (2.5): Column Chromatography Solid-Phase Extraction (SPE)

When the cartridge format is utilized, there are several ways to achieve flow.

Gravity or vacuum can be used for columns that are not designed to withstandpressure. Typically, the particles in this case are larger in diameter (> 50 microns) so

that there is less resistance to flow.

Open glass columns (Tswett’s experiment) are an example of this. In addition,

small plastic columns, typically in the shape of syringe barrels, can be filled with

packing- material particles and used to perform sample preparation. This is called

solid-phase extraction (SPE). Here, the chromatographic device, called a cartridge, isused, usuallywith vacuum-assisted flow, to clean up a very complex sample before it

is analyzed further.

Smaller particle sizes (


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to withstand high pressure are necessary. When moderate to high pressure is used to

flow the solvent through the chromatographic column, the technique is called (HPLC).

High Preformance Liquid Chromatography (HPLC)2.8

The acronym HPLC, coined by the late (Prof. Csaba Horváth for his 1970 Pittcon

paper), originally indicated the fact that high pressure was used to generate the flow

required for liquid chromatography in packed columns. In the beginning, pumps only

had a pressure capability of (500 psi) (35 bars). This was called high pressure liquid

chromatography (HPLC).The early 1970s saw a tremendous leap in technology.

These new HPLC instruments could develop up to 6,000 psi (400 bars) of pressure,and incorporated improved injectors, detectors, and columns. HPLC really began to

take hold in the mid-to late-1970s. With continued advances in performance during this

time (smaller particles, even higher pressure), the acronym remained the same, but the

name was changed to high-performance liquid chromatography (HPLC)

High-performance liquid chromatography (HPLC) is now one of the most

powerful tools in analytical chemistr It has the ability to separate, identify, and

quantities the compounds that are present in any sample that can be dissolved in a

liquid. Today, compounds in trace concentrations as low as parts per trillion (ppt) may

easily be identified

HPLC can be, and has been, applied to just about any sample, such as

pharmaceuticals, food, nutraceuticals, cosmetics, environmental matrices, forensic

samples, and industrial chemicals.

Figure (2.9) HPLC Column

Ultra- High Preformance Liquid Chromatography2.9


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In 2004, further advances in instrumentation and column technology were made to

achieve very significant increases in resolution, speed, and sensitivity in liquid

chromatography.

Columns with smaller particles (1.7 micron) and instrumentation with

specialized capabilities designed to deliver mobile phase at( 15,000 psi) (1,000 bar)

were needed to achieve a new level of performance. A new system had to be holistically

created to perform ultra-performance liquid chromatography (UPLC technology).

Basic research is being conducted today by scientists working with columns containing

even smaller (1-micron-diameter) particles and instrumentation capable of performing

at (100,000 psi) (6,800 bars). This provides a glimpse of what we may expect in the

future

High-Performance Liquid Chromatograph Work:2.9.1

.1: The Components Of HPLC System:

The components of a basic high-performance liquid chromatography

(HPLC)

Figure (2.10): High-Performance Liquid Chromatography

“HPLC” System

2.2: A Resrvoir Holds The Solvent:

Called the mobile phase, because it moves.


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2.3: A High-PressurePump:

Solvent delivery system or solvent manager) is used to generate and meter a

specified flow rate of mobile phase, typically milliliters per minute.

2.4: An Injector:

Sample manager “auto sampler” is able to introduce (inject) the sample into the

continuously flowing mobile phase stream that carries the sample into the

HPLC column.

2.5: The Column:

Contains the chromatographic packing material needed to effect the separation.

This packing material is called the stationary phase because it is held in place by the

column hardware. Is needed to see the separated compound bands as they elute from

the HPLC column (most compounds have no color, so we cannot see them with our

eyes).

The mobile phase exits the detector and can be sent to waste, or collected, as

desired.

When the mobile phase contains a separated compound band, HPLC provides

the ability to collect this fraction of the eluate containing that purified compound for

further study. This is called preparative chromatography (discussed in the section on

HPLC Scale).

Notice that high- pressure tubing and fittings are used to interconnect the

pump, injector, column, and detector components to form the conduit for the mobile

phase, sample, and separated compound bands.

2.9.2 Detectors And Computer Date Station:


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The detector is wired to the computer data station, the HPLC system component

that records the electrical signal needed to generate the chromatogram on its display and

to identify and quantities the concentration of the sample constituents (see Figure 2.11).

Figure (2.11): A Typical HPLC System

2.7: Detectors Types:

Since sample compound characteristics can be very different, several types

of detectors have been developed.

For example: if a compound can absorb ultraviolet light, a UV-absorbance

detector is used. If the compound fluoresces, a fluorescence detector is used. If the

compound does not have either of these characteristics, a more universal type of

detector is used, such as an (evaporative-light-scattering detector) (ELSD). The most

powerful approach is the use multiple detectors in series.

For example: A (UV and or ELSD detector) may be used in combination with a

mass spectrometer (MS) to analyze the results of the chromatographic separation.

This provides, from a single injection, more comprehensive information

about an analyte. The practice of coupling a mass spectrometer to an HPLC system is

called (LC/MS).


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HPLC Operation

2.9.3 chromatographic Column Works – Bands:

A simple way to understand how we achieve the separation of the compounds

contained in a sample is to view the diagram in (Figure 8).

Mobile phase enters the column from the left, passes through the particle bed, and exits

at the right. Flow direction is represented by green arrows.

Figure (2.12): Understanding how a chromatographic column works – Bands

First, consider the top image; it represents the column at time zero (the moment

of injection), when the sample enters the column and begins to form a band.

The sample shown here, a mixture of yellow, red, and blue dyes, appears at the

inlet of the column as a single black band. (In reality, this sample could be anything that

can be dissolved in a solvent; typically the compounds would be colorless and the

column wall opaque, so we would need a detector to see the separated compounds as

they elute).

After a few minutes (lower image), during which mobile phase flows

continuously and steadily past the packing material particles, we can see that the

individual dyes have moved in separate bands at different speeds.


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This is because there is a competition between the mobile phase and the

stationary phase for attracting each of the dyes or analytes.

Notice: The yellow dye band moves the fastest and is about to exit the column. The

yellow dye likes [is attracted to] the mobile phase more than the other dyes. Therefore,

it moves at a faster speed, closer to that of the mobile phase. The blue dye band likes

the packing material more than the mobile phase. Its stronger attraction to the particles

causes it to move significantly slower. In other words, it is the most retained compound

in this sample mixture. The red dye band has an intermediate attraction for the mobile

phase and therefore moves at an intermediate speed through the column. Since each dye

band moves at different speed, we are able to separate it chromatographically.

Peaks AreCreated:

As the separated dye bands leave the column, they pass immediately into the

detector. The detector contains a flow cell that sees (detects) each separated compound

band against a background of mobile phase (see Figure 9).

Figure (2.13): How peaks are created


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In reality, solutions of many compounds at typical HPLC analytical

concentrations are colorless. An appropriate detector has the ability to sense the

presence of a compound and send its corresponding electrical signal to a computer data

station.

A choice is made among many different types of detectors, depending upon the

characteristics and concentrations of the compounds that need to be separated and

analyzed, as discussed earlier.

2.10 A chromatogram:

A chromatogram is a representation of the separation that has chemically

(chromatographically) occurred in the HPLC system. A series of peaks rising from a

baseline is drawn on a time axis. Each peak represents the detector response for a

different compound. The chromatogram is plotted by the computer data station (see

Figure H). In Figure (H), the yellow band has completely passed through the detector

flow cell; the electrical signal generated has been sent to the computer data

station. The resulting chromatogram has begun to appear on screen.

Notice: The chromatogram begins when the sample was first injected and starts as a

straight line set near the bottom of the screen. This is called the baseline; it represents

pure mobile phase passing through the flow cell over time. As the yellow analyte band

passes through the flow cell, a stronger signal is sent to the computer.

The line curves, first upward, and then downward, in proportion to the

concentration of the yellow dye in the sample band. This creates a peak in the

chromatogram. After the yellow band passes completely out of the detector cell, the

signal level returns to the baseline; the flow cell now has, once again, only pure m it is

the first peak drawn. A little while later, the red band reaches the flow cell.

The signal rises up from the baseline as the red band first enters the cell, and the

peak representing the red band begins to be drawn. In this diagram, the red band has not

fully passed through the flow cell. The diagram shows what the red band and red peak


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would look like if we stopped the process at this moment.

Since most of the red band has passed through the cell, most of the peak has

been drawn, as shown by the solid line. If we could restart, the red band would

completely pass through the flow cell and the red peak would be completed (dotted

line). The blue band, the most strongly retained, travels at the slowest rate and elutes

after the red band.

The dotted line shows you how the completed chromatogram would appear if we had let

the run continue to its conclusion. It is interesting to note that the width of the blue

peak will be the broadest because the width of the blue analyte band, while

narrowest on the column, becomes the widest as it elutes from the column.

This is because it moves more slowly through the chromatographic packing

material bed and requires more time (and mobile phase volume) to be eluted

completely.

Since mobile phase is continuously flowing at a fixed rate, this means that the

blue band widens and is more dilute. Since the detector responds in proportion to the

concentration of the band, the blue peak is lower in height, but larger in width.

2.11 Identifying And Quanttating Compounds

2.11.1 Identification:

In Figure (H), three dye compounds are represented by three peaks

separated in time in the chromatogram. Each elutes at a specific location, measured by

the elapsed time between the moment of injection (time zero) and the time when the

peak maximum elutes.

By comparing each peak’s retention time (tR) with that of injected reference

standards in the same chromatographic system (same mobile and stationary phase), a

chromatographer may be able to identify each compound.


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Figure (2.14) Identification

In the chromatogram shown in (Figure 2.14), the chromatographer know that,

under these LC system conditions, the analyte, acrylamide, would be separated and

elute from the column at (2.85) minutes (retention time).

2.11.2Identifying And Quanttating :

Whenever a new sample, which happened to contain acrylamide (name of

a compound under test), was injected into the LC system under the same conditions, apeak would be presen t at (2.85) minutes (see sample B in Figure I-2). For a better

understanding of why some compounds move more slowly (are better retained) than

others, once identity is established, the next piece of important information is how much

of each compound was present in the sample.

The chromatogram and the related data from the detector help us calculate the

concentration of each compound. The detector basically responds to the concentration

of the compound band as it passes through the flow cell. The more concentrated it is,

the stronger the signal; this is seen as a greater peak height above the baseline.

In (Figure I- 2), chromatograms for Samples A and B, on the same time scale, are

stacked one above the other. The same volume of sample was injected in both runs.


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Both chromatograms display a peak at a retention time (tR) of (2.85 minutes),

indicating that each sample contains acrylamide. However, Sample A displays a much

bigger peak for acrylamide

.

Figure (2.15): Identification and Quantitation

The area under a peak (peak area count) is a measure of the concentration of

the compound it represents. This area value is integrated and calculated automatically

by the computer data station.

In this example, the peak for acrylamide in Sample A has 10 times the area of

that for Sample B. Using reference standards, it can be determined that Sample A

contains ( 10 picograms) of acrylamide, which is ten times the amount in Sample B (1

picogram).

Notice: There is another peak (not identified) that elutes at (1.8 minutes)

in both samples. Since the area counts for this peak in both samples are about the same,


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this unknown compound may have the same concentration in both samples.

2.11.3 Factor s That Determine The OverallL Separtion Power OR Resolution

OF HPLC Column:

The degree to which two compounds are separated is called chromatographic

resolution (RS). Two principal factors that determine the overall separation power or

resolution that can be achieved by an HPLC column are (mechanical separation power),

(created by the column length, particle size, and packed-bed uniformity), (chemical

separation power) and (created by the physicochemical competition for compounds

between the packing material and the mobile phase).

Efficiency is a measure of mechanical separation power, while selectivity is a

measure of chemical separation power.

2.11.4: Mechanical Separation Power (Efificency ):

If a column bed is stable and uniformly packed, its mechanical separation power

is determined by the column length and the particle size.

Mechanical separation power, also called efficiency, is often measured and

compared by a plate number (symbol = N). Smaller-particle chromatographic beds have

higher efficiency and higher backpressure

For a given particle size, more mechanical separation power is gained by

increasing column length.

However, the trade-offs are longer chromatographic run times, greater solvent

consumption, and higher back pressure. Shorter column lengths minimize all these

variables also reduce mechanical separation power, as shown in


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(Figure 2.16 ) .

Figure (2.16): Column Length and Mechanical Separating Power (Same Particle Size)

Figure (2.17): Particle Size and Mechanical Separating Power (Same Column Length)

For example: For a given particle chemistry, mobile phase, and flow rate, as shown in

(Figure 13) , a column of the same length and (i.d.) , but with a smaller particle size, will

deliver more mechanical separation power in the same time. However, its back pressure

will be much higher.

2.11.5: Chemical Separation Power (Seectivity ):

The choice of a combination of particle chemistry (stationary phase) and (mobile-

phase composition), the separation system will determine the degree of chemical

separation power (how we change the speed of each analyte).


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Optimizing selectivity is the most powerful means of creating a separation; this

may obviate the need for the brute force of the highest possible mechanical efficiency.

To create a separation of any two specified compounds, a scientist may choose among a

multiplicity of phase combinations (stationary phase and mobile phase) and retention

mechanisms (modes of chromatography).

2.12 HPLC Sepration Modes

In general, three primary characteristics of chemical compounds can be used to

create HPLC separations. They are:

• Polarity

• Electrical Charge

• Molecular Size

First, let’s consider polarity and the two primary separation modes that exploit this

characteristic: normal phase and reversed-phase chromatography.

2.12.1 Seprations Based On Polarity:

A molecule’s structure, activity, and physicochemical characteristics are determined

by the arrangement of its constituent atoms and the bonds between them. Within a

molecule, a specific arrangement of certain atoms that is responsible for special

properties and predictable chemical reactions is called a functional group.

This structure often determines whether the molecule is polar or non-polar.

Organic molecules are sorted into classes according to the principal functional group(s)


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each contains.

Using a separation mode based on polarity, the relative chromatographic

retention of different kinds of molecules is largely determined by the nature and location

of these functional groups. As shown in (Figure 13), classes of molecules can be ordered

by their relative retention into a range or spectrum of chromatographic polarity from

highly polar to highly non-polar.

Figure (13): Chromatographic Polarity Spectrum by Analyte Functional

Group

Water (a small molecule with a high dipole moment) is a polar compound.

Benzene (an aromatic hydrocarbon) is a (non-polar compound). Molecules with

similar chromatographic polarity tend to be attracted to each other; those with dissimilar

polarity exhibit much weaker attraction, if any, and may even repel one another. This

becomes the basis for chromatographic separation modes based on polarity.

Another way to think of this is by the familiar analogy (oil) (non-polar) and water

(polar) don’t mix. Unlike in magnetism where opposite poles attract each other,

chromatographic separations based on polarity depend upon the stronger attractionbetween likes and the weaker attraction between opposites. Remember, ³Like attracts

like´ in polarity-based chromatography.

Figure (2.18): Proper Combination of Mobile and Stationary Phases


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2.12.2: Affects An Affect On Separtion Based On Polarity:

To design a chromatographic separation system (see Figure 14), we create

competition for the various compounds contained in the sample by choosing a mobile

phase and a stationary phase with different polarities. Then, compounds in the samplethat are similar in polarity to the stationary phase (column packing material) will be

delayed because they are more strongly attracted to the particles. Compounds whose

polarity is similar to that of the mobile phase will be preferentially attracted to it and

move faster.

In this way, based upon differences in the relative attraction of each compound

for each phase, a separation is created by changing the speeds of the analytes. In the

figures (R-1), (R-2), and(R-3) display typical chromatographic polarity ranges for

mobile phases, stationary phases, and sample analytes, respectively.

Let’s consider each in turn to see how a chromatographer chooses the appropriate

phases to develop the attraction competition needed to achieve a polarity - based HPLC

separation.

Figure (2.19) Mobile Phase Chromatographic Polarity Spectrum

2.12.3MOBILE PHASE CHROMATOGRAPHIC POLARITY SPECTRUM:

A scale, such as that shown in Figure (15), upon which some common solvents

are placed in order of relative chromatographic polarity is called an eluotropic series.

Mobile phase molecules that compete effectively with analyte molecules for the

attractive stationary phase sites displace these analytes, causing them to move faster

through the column (weakly retained).

Water is at the polar end of mobile - phase- solvent scale, while hexane, an


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aliphatic hydrocarbon, is at the non-polar end. In between, single solvents, as well as

miscible- solvent mixtures (b) ended in proportions appropriate to meet specific

separation requirements), can be placed in order of elution strength.

2.12.4: STATIONARY PHASE PARTICLE CHROMATOGRAPHIC POLARITY

SPECTRUM:

Which end of the scale represents the ‘strongest’ mobile phase depends upon the

nature of the stationary phase surface where the competition for the analyte molecu

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