BASIC ANALYTICAL CHEMISTRY TRAINING

ANALYTICAL CHEMISTRY

Analytical chemistry is the study of the separation, identification, and quantification of the
chemical components of natural and artificial materials. Qualitative analysis gives an
indication of the identity of the chemical species in the sample and quantitative analysis
determines the amount of one or more of these components. The separation of
components is often performed prior to analysis.

Analytical methods can be separated into classical and instrumental. Classical methods
(also known as wet chemistry methods) use separations such as precipitation, extraction,
and distillation and qualitative analysis by color, odor, or melting point. Quantitative
analysis is achieved by measurement of weight or volume. Instrumental methods use an
apparatus to measure physical quantities of the analyte such as light absorption,
fluorescence, or conductivity.

Identification of one or more constituents of a sample

Examination to determine how much of a particular species is present

analysis.

Information concerning the spatial arrangement of atoms in a molecule or crystalline

compound or confirmation of the presence or position of certain organic functional groups
structural analysis.

qualitative analysis.
quantitative

ANALYTICAL CHEMISTRY (Continued)

A general classification of important analytical techniques

SPECTROMETRIC

Molecular Spectrometry

UV, UV-Visible spectrometry

Quantitative determination of elements and compounds, mainly as trace and minor
constituents

Infra red (IR) spectrometry Infra red (IR) spectrometry

Identification and structural analysis of organic compounds

Nuclear Magnetic Resonance (NMR) spectrometry

Identification and structural analysis of organic compounds

Mass Spectrometry (MS) Mass Spectrometry (MS)

Identification and structural analysis of organic compounds
Identification and determinations of elements and isotopes at trace levels

Three techniques are important for analytical purposes :

1. Visible and ultraviolet spectrometry (electronic)
2. Infrared spectrometry (vibrational)
3. Nuclear magnetic resonance spectrometry (nuclear spin)

UV/VIS SPECTROMETRIC

The full electromagnetic radiation spectrum is continuous and each region merges slowly
into the next. For spectroscopy purposes, we choose to characterize light in the ultraviolet
and visible regions in terms of wavelength expressed in nanometers.

UV/VIS SPECTROMETRY (Continued)

Instrumentation
The purpose of a spectrophotometer is to provide a beam of monochromatic radiation to
illuminate a sample and so measure the ratio I/Io. Any spectrophotometer will consist of
the component parts of :

UV/VIS SPECTROMETRY (Continued)

The Beer-Lambert Law states that the concentration of a substance in solution is directly
proportional to the 'absorbance ', A, of the solution.
Absorbance A = constant x concentration x cell length

The law is only true for monochromatic light, that is light of a single wavelength or narrow
band of wavelengths, and provided that the physical or chemical state of the substance
does not change with concentration.

When monochromatic radiation passes through a homogeneous solution in a cell, the

intensity of the emitted radiation depends upon the thickness (l) and the concentration (C)
of the solution.

Io is the intensity of the incident radiation and I is the intensity of the transmitted radiation.
The ratio I/Io is called transmittance. This is sometimes expressed as a percentage and
referred to as %transmittance.

Mathematically, absorbance is related to percentage transmittance T by the expression:

A = log10(Io/I) = log10(100/T) = kcL
where L is the length of the radiation path through the sample, c is the concentration of
absorbing molecules in that path, and k is the extinction coefficient - a constant
dependent only on the nature of the molecule and the wavelength of the radiation.

UV/VIS SPECTROMETRY (Continued)

The most conveniently available source of visible radiation with which we are familiar is the
tungsten lamp. For the UV region, the most common source is the deuterium lamp and a
UV/Visible spectrometer will usually have both lamp types to cover the entire wavelength
range.

A molecule of any substance has an internal energy which can be considered as the sum
of the energy of its electrons, In complex molecules the energy levels are more closely
spaced and photons of near ultraviolet and visible light can effect the transition. These
substances, therefore, will absorb light in some areas of the near ultraviolet and visible
regions.

UV/VIS SPECTROMETRY (Continued)

In the expression A = kcl, c is expressed in molar-1 and l in m, then k is replaced by the

symbol and is called the molar absorption coefficient. The units of are mol-1m2. was
formerly called the molar extinction coefficient and concentrations were often expressed as
mol l-1, mol dm-3 or M and the cell length in cm to give units mol-1lcm-1, mol-1dm3cm-1
and M-1cm-1 respectively.

CHROMATOGRAPHIC

Chromatography is the collective term for a set of laboratory techniques for the separation
of mixtures. It involves passing a mixture dissolved in a "mobile phase" through a
stationary phase, which separates the analyte to be measured from other molecules in the
mixture based on differential partitioning between the mobile and stationary phases. Subtle
differences in a compound's partition coefficient result in differential retention on the
stationary phase and thus changing the separation.

Chromatography may be preparative or analytical. The purpose of preparative

chromatography is to separate the components of a mixture for further use (and is thus a
form of purification). Analytical chromatography is done normally with smaller amounts of
material and is for measuring the relative proportions of analytes in a mixture.

The mobile phase is the phase which moves in a definite direction. It may be a liquid (LC),
a gas (GC). The mobile phase consists of the sample being separated/analyzed and the
solvent that moves the sample through the column. In the case of HPLC the mobile phase
consists of a non-polar solvent(s) such as hexane in normal phase or polar solvents in
reverse phase chromotagraphy and the sample being separated. The mobile phase moves
through the chromatography column (the stationary phase) where the sample interacts
with the stationary phase and is separated.

The stationary phase is the substance which is fixed in place for the chromatography
procedure. Examples include the silica layer in thin layer chromatography.

CHROMATOGRAPHIC (Continued)

Some techniques of chromatographic are :

Chromatographic bed Shape

Column Chromatography

: The stationary bed is within a tube.

Planar Chromatography

: The stationary phase is on a plane.

- Paper Chromatography

: Involves placing a small dot or line of sample solution onto

a strip of chromatography paper.
- Thin Layer Chromatography : Similar to paper chromatography, stationary phase of a thin
layer of adsorbent like silica gel, alumina, or cellulose on a
flat, inert substrate.

Physical state of mobile phase

Gas Chromatography

: Mobile phase is a gas. It is always carried out in a column,

which is typically "packed" or "capillary"

Liquid Chromatography

: Mobile phase is a liquid. Liquid chromatography can be

carried out either in a column or a plane.

GAS CHROMATOGRAPHY

Is used to separate volatile components of a mixture. A small amount of the sample to be

analyzed is drawn up into a syringe. The syringe needle is placed into a hot injector port of
the gas chromatograph, and the sample is injected. The injector is set to a temperature
higher than the components boiling points. So, components of the mixture evaporate into
the gas phase inside the injector. A carrier gas, such as helium, flows through the injector
and pushes the gaseous components of the sample onto the GC column. It is within the
column that separation of the components takes place. Molecules partition between the
carrier gas (the mobile phase) and the high boiling liquid (the stationary phase) within the
GC column.

GAS CHROMATOGRAPHY (Continued)

Instrumentation

Carrier and Detector Gases

The carrier gas must be chemically inert. Commonly used gases include nitrogen, helium,
argon, and carbon dioxide. The choice of carrier gas is often dependant upon the type of
detector which is used. The carrier gas system also contains a molecular sieve to remove
water and other impurities. Hydrogen and air is commonly used for detector gases.

GAS CHROMATOGRAPHY (Continued)

Injection Port
For optimum column efficiency, the sample should not be too large. The most common
injection method is where a microsyringe is used to inject sample through a rubber septum
into a flash vapouriser port at the head of the column.
There are 2 type of inlet system :
- Packed column inlet :
Packed Column Injector

Septum Purged Injector

- Split/splitless inlet :

Split Injector
Splitless Injector
Cold on Column Injector

GAS CHROMATOGRAPHY (Continued)

Column
There are two general types of column :
Packed columns : contain a finely divided, inert, solid support material (commonly based
on diatomaceous earth) coated with liquid stationary phase. Most packed columns are 1.5
- 10m in length and have an internal diameter of 2 - 4mm.
Capillary columns : have an internal diameter of a few tenths of a millimeter. They can be
one of two types; wall-coated open tubular (WCOT) or support-coated open tubular
(SCOT). Wall-coated columns consist of a capillary tube whose walls are coated with liquid
stationary phase. In support-coated columns, the inner wall of the capillary is lined with a
thin layer of support material such as diatomaceous earth, onto which the stationary phase
has been adsorbed. SCOT columns are generally less efficient than WCOT columns.

GAS CHROMATOGRAPHY (Continued)

Detector
There are many detectors which can be used in gas chromatography. Different detectors
will give different types of selectivity. A non-selective detector responds to all compounds
except the carrier gas, a selective detector responds to a range of compounds with a
common physical or chemical property and a specific detector responds to a single
chemical compound.
Detector
Flame ionization (FID)
Thermal conductivity (TCD)
Electron capture (ECD)
Nitrogen-phosphorus
Flame photometric (FPD)
Photo-ionization (PID)
Hall electrolytic conductivity

Support gases
Hydrogen and air
Reference
Make-up
Hydrogen and air
Hydrogen, air possibly oxygen
Make-up
Hydrogen, oxygen

Selectivity
Most organic cpds.
Universal
Halides, nitrates, nitriles, peroxides, anhydrides, etc.
Nitrogen, phosphorus
Sulphur, phosphorus, tin, boron, arsenic, etc.
Aliphatics, aromatics, ketones, esters, aldehydes,
Halide, nitrogen, nitrosamine, sulphur

GAS CHROMATOGRAPHY (Continued)

Recorder
Two devices are used to record the GC traces/areas under peaks:
Integrating recorders
computer program
Each type of device records the messages sent to them by the detector as peaks,
calculates the retention time, and calculates the area under each peak; all of this
information is included in the printout.

CONCENTRATION

In chemistry, concentration is the measure of how much of a given substance there is

mixed with another substance. This can apply to any sort of chemical mixture, but most
frequently the concept is limited to homogeneous solutions, where it refers to the amount
of solute in the solvent.

To concentrate a solution, one must add more solute (e.g. alcohol), or reduce the amount
of solvent (e.g. water). By contrast, to dilute a solution, one must add more solvent, or
reduce the amount of solute.

For scientific or technical applications, a qualitative account of concentration is almost

never sufficient; therefore quantitative measures are needed to describe concentration.

There are a number of different ways to quantitatively express concentration. They are
based on mass, volume, or both. Depending on what they are based on it is not always
trivial to convert one measure to the other, because knowledge of the density might be
needed to do so. At times this information may not be available, particularly if the
temperature varies.

The most common used for unit of concentration are : Percentage, Molarity, Normality,
Molality, Part per million, Part per billion, etc.

CONCENTRATION (Continued)

Percentage (%)

There are a number of different ways to quantitatively express percentage concentration.

They are based on mass, volume, or both.

Mass Percentage
The mass of a substance in a mixture as a percentage of the mass of the entire mixture.
(Mass fraction Xm can be used instead of mass percentage by dividing mass percentage to
100.) Commercial concentrated aqueous reagents such as acids and bases are often
labeled in concentrations of weight percentage with the specific gravity also listed. In older
texts and references this is sometimes referred to as weight-weight percentage
(abbreviated as w/w% or wt%).

Mass Percentage (wt %)

Example :
If a bottle contains 40 grams of ethanol and 60 grams of water, then it contains 40%
ethanol by mass or 0.4 mass fraction ethanol.
Note that the total weight of the solution will be 100 grams, but the total volume of the
solution will be more than 100 milliliters because ethanol is less dense than water.

Weight of substance
Weight of mixture

x 100

CONCENTRATION (Continued)

Mass - Volume Percentage

Mass-volume percentage, (sometimes referred to as weight-volume percentage or percent
weight per volume and often abbreviated as % m/v or % w/v) describes the mass of the
solute in g per 100 mL of the resulting solution. Mass-volume percentage is often used for
solutions made from a solid solute dissolved in a liquid. For example, a 40% w/v sugar
solution contains 40 g of sugar per 100 mL of resulting solution.

Mass volume Percentage (% w/v)

Example :
A 40% w/v sugar solution contains 40 g of sugar per 100 mL of resulting solution.

Volume - Volume Percentage

Volume-volume percentage (sometimes referred to as percent volume per volume and
abbreviated as % v/v) describes the volume of the solute in mL per 100 mL of the resulting
solution. This is most useful when a liquid - liquid solution is being prepared.

Volume Percentage (% v/v)

Weight of substance
Volume of mixture

x 100

Volume of substance x 100

Volume of

mixture

Example :
For example, a 40% v/v ethanol solution contains 40 mL ethanol per 100 mL total volume.

CONCENTRATION (Continued)

Molarity (M)
Molarity (in units of mol/L, molar, or M) or molar concentration denotes the number of
moles of a given substance per liter of solution. A capital letter M is used to abbreviate
units of mol/L.

Example :

Normality (N)
The normality of a solution is the number of gram equivalent weight of a solute per liter of
its solution.The definition of normality depends on the exact reaction intended.

Example :
For example, hydrochloric acid (HCl) is a monoprotic acid and thus has 1 mol = 1 gram
equivalent. One liter of 1 M aqueous solution of HCl acid contains 36.5 grams HCl. It is
called 1 N (one normal) solution of HCl.

CONCENTRATION (Continued)

Molality (m)
The number of moles of solute per kilogram of solvent (not solution).

Example :
Adding 1.0 mole of solute to 2.0 kilograms of solvent constitutes a solution with a molality
of 0.50 mol/kg. Such a solution may be described as "0.50 molal". The term molal solution
is used as a shorthand for a "one molal solution", i.e. a solution which contains one mole
of the solute per 1000 grams of the solvent.

Equivalents (Eq)
Expression of concentration in equivalents per liter (or more commonly, milliequivalents
per liter) is based on the same principle as normality. A normal solution is one equivalent
per liter of solution (Eq/L).

Part per Million (ppm)

The amount of a given substance in a total amount of 1,000,000 regardless of the units of
measure used as long as they are the same. e.g. 1 milligram per kilogram. 1 part in 10 6.

Part per Billion (ppb)

Thank You.