ENGI 9628 – Environmental Laboratory

Lecture 4: Environmental
Instrumental Analysis

Faculty of Engineering & Applied Science

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1. Introduction

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Classification of analytical methods
• Classical
– Also called wet-chemical methods
– Separation of component of interest (analyte) from
the sample by precipitation, extraction, or
distillation
– Followed by gravimetric or titrimetric
measurement for quantitative analysis
• Instrumental
– Use of new methods for quantitative analysis

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Instrumental analysis
• Converts information stored in the physical or chemical
characteristics of the analyte into useful information
• Four components for an instruments:
Energy source  Devices to stimulate measurable
response from analyte
Sensor Analytical device capable of monitoring specific
chemical species continuously and reversibly
Transducer  Devices that convert information in
nonelectrical domains to electrical domains and the converse
Detector  Device that indicates a change in one variable
in its environment (eg., pressure, temp, particles)  can be
mechanical, electrical, or chemical

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Selecting an instrumental method
• What accuracy is required
• How much sample is available
• What is the concentration range of the analyte
• What components of the sample will cause
interference
• What are the physical and chemical properties of the
sample matrix
• How many samples are to be analyzed

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Evaluation of an Instrumental Method
(1) Accuracy  describes the correctness of an
experimental result
− absolute error  Δx = xi – µ
− relative error  Δx/µ
Evaluation: Standard Reference Materials (SRM)
– provided by National Institute of Standards and
Technology (NIST)
– specifically prepared for validation of analytical
methods

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(2) Precision  describes the reproducibility of results
– degree of mutual agreement among data that have
been obtained in the same way
– a measure of the random, or indeterminate error of
an analysis
Evaluation: Standard Reference Materials (SRM)
– absolute standard deviation 

– relative standard deviation  RSD = S/mean

– coefficient of variation  RSD ×100%
– variance  = S2
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(3) Sensitivity  ability of an instrument or method to
discriminate between small differences in analyte
concentration

(4) Detection limit the minimum concentration or mass

of analyte that can be detected at a known confidence
level  the LOD (limit of detection) defines as
3 × standard deviation of the blank

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(5) Dynamic Range  extends from the lowest
concentration at which quantitative measurements can be
made (LOQ, limit of quantitation), to the concentration at
which the calibration curve departs from linearity (LOL,
limit of linearity)  An analytical method should have a
dynamic range of at least 2 orders of magnitude

(6) Selectivity  refers to the degree of an analytical

method to which the method is free from interference by
other species contained in the sample matrix  no
method is totally free from interference from other
species

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Calibration of an instrumental method

• Analytical methods require calibration

• Process that relates the measured analytical
signal to the concentration of analyte
• 3 common methods
– Calibration curve
– Standard addition method
– Internal standard method

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• Calibration curve
 standards containing known concentrations of the analyte are
introduced into the instrument  response is recorded
 response is corrected for instrument output obtained with a
blank
blank contains all of the components of the original sample
except for the analyte
 resulting data are then plotted to give a graph of corrected
instrument response vs. analyte concentration
 an equation is developed for the calibration curve by a least-
squares technique so that sample concentrations can be
computed directly
• Standard addition method  usually involves adding one or
more increments of a standard solution to sample aliquots of
the same size (spiking)
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 Schematic of a calibration curve plot
showing limit of detection (LOD),
limit of quantification (LOQ),
dynamic range, and limit of linearity
(LOL).

Standard addition

Add a known concentration directly to an analytical sample to aid in quantization 

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Internal standard method
Example: A multiple standard addition method was used for
determining Fe3+ in a natural water sample. Ten-milliliter
aliquots of the sample were pipetted into 50.00 mL volumetric
flasks. Exactly 0.00, 5.00, 10.00, 15.00, and 20.00 mL of a
standard solution containing 10 ppm of Fe3+ were added to
each followed by an excess of thiocyanate ion to give the red
complex Fe(SCN)+2. After dilution to 50 mL, absorbances of
the five solutions were measured in a 1.00 cm cell at 480 nm
and found to be 0.24, 0.43, 0.62, 0.81, and 1.00, respectively.
What is the concentration of Fe3+ in the original water sample?

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2. Spectroscopy

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Color spectrum
• An obvious difference between objects is their color.
The absorption of visible light is what makes things
colored.
• Sunlight (or white light) as uniform or homogeneous
in color, it is actually composed of a broad range of
radiation wavelengths in the ultraviolet (UV), visible
and infrared (IR) portions of the spectrum.
• The component colors of the visible portion can be
separated by passing sunlight through a prism, which
acts to bend the light in differing degrees according to
wavelength.

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• For example, a blue dye used in a pair of jeans appears blue
because the light at the red end of the spectrum is absorbed.
This leaves the blue light to be reflected to the observer’s eye.

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Wave length & Amplitude

 Wavelength is defined as the distance between adjacent peaks

(or troughs), and may be designated in meters, centimeters or
nanometers (10-9 meters).
 Frequency is the number of wave cycles that travel past a fixed
point per unit of time, and is usually given in cycles per
second, or hertz (Hz).
 The energy carried by a photon of a given wavelength of
radiation

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The electromagnetic spectrum

ES range from very short wavelengths (including gamma and x-rays)

to very long wavelengths (including microwaves and broadcast radio
waves).
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Spectroscopy
• Spectroscopy is the study of the interaction between
radiation (electromagnetic radiation, or light, as well
as particle radiation) and matter.
• Spectrometry is the measurement of these interactions
and an instrument
which performs such
measurements is a
spectrometer.

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UV/visible spectroscopy
• Ultraviolet-visible spectroscopy (UV/ VIS) involves the
spectroscopy of photons  uses light in the visible and adjacent
near ultraviolet (UV) and near infrared (NIR) ranges molecules
undergo electronic transitions in this region of energy space
• UV/VIS spectroscopy  routinely used in the quantitative
determination of solutions of transition metals and highly
conjugated organic compounds. It is possible to do so because
transition metals are often colored because of the possibility of
electronic transitions within the metal atoms. Organic molecules,
especially those with a high degree of conjugation also absorb light
in the UV or visible regions of the electromagnetic spectrum.

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How it works
• The absorbance of a sample is proportional to the number of
absorbing molecules in the spectrometer light beam (e.g. their molar
concentration in the sample tube), it is necessary to correct the
absorbance value for this and other operational factors if the spectra
of different compounds are to be compared in a meaningful way.

• The corrected absorption value is called "molar absorptivity", and is

particularly useful when comparing the spectra of different
compounds and determining the relative strength of light absorbing
functions (chromophores).

• Molar absorptivity (ε) is defined as:

ε = A/ c l
(where A= absorbance, c = sample concentration in moles/liter & l =
length of light path through the sample in cm.)

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• A reference cell containing only solvent is used.
• Light is passed simultaneously through the sample cell and reference
cell.
• The transmitted radiation is detected and the spectrometer records the
absorption spectrum by scanning the wavelength of the light passing
through the cells.
• The spectrometer compares the light passing through the sample with
that passing through the reference cell.
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Beer–Lambert law
ε
=A

• ε  used to identify a substance

• If ε and l are known for a compound  the concentration of the solution
can be calculated
• is the base for developing a spectrophotometer

A = Log10 (1/T) or A = 2 – Log10 (%T) T= I/I0  Transmission

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Example: A 3.96 ×10-4 M solution of a substance had an
absorbance of 0.534 at 350 nm in a 1 cm path-length cuvet.
A blank solution containing only solvent had an absorbance
of 0.025 at the same wavelength. What is the molar
absorption coefficient of the compound?

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3. Atomic Absorption
Spectroscopy (AAS)

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Principle of atomic absorption

• The atoms absorb light

and make transitions to
respective higher
electronic energy levels
 the analyte
concentration is
determined from the
amount of absorption

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Four components of an AAS
• Light source (usually
Hollow Cathode Lamp)
• Atomizing cell (Flame
or Furnace)
• Monochromator
• Detector and read out
device

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Schematic diagram of an AAS

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(1) Light source

• Hollow Cathode Lamps

(HCL).
• Tungsten (W) anode and a
hollow cylindrical cathode
made of the element
determined.
• Inert gas (neon or argon).

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How HCL works
• Applying a potential difference between the anode and
the cathode leads to the ionization of inert gas atoms.
• These gaseous ions bombard the cathode and eject metal
atoms from the cathode in a process called sputtering.
Some sputtered atoms are in excited states and emit
radiation characteristic of the metal as they come back to
the ground state.

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Inert gas molecules (gray)  become positive electrical charge (green)
Metal atoms (blue)  get energy from green inert gas molecules
from ground state to excited state (red)  go back to the ground
state and emit radiation characteristic of the metal
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(2) Atomizing cell
• Elements to be analyzed needs to be in atomic sate.
• Atomization is separation of particles into individual
molecules and breaking molecules into atoms .This
is done by exposing the analyte to high
temperatures in a flame or graphite furnace.

Elements detectable by atomic absorption are highlighted in pink in this

periodic table 32
A: Flame atomization

• Constant temperature with time

• Only analyze solutions
• Samples are usually introduced into nebuliser by
suction.
• In nebuliser, the sample is dispersed into tiny droplets,
which can be readily broken down into atoms by
flame.
• Need 0.5 -1mL sample per analysis

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Type of flame used

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B: Electro thermal atomization - graphite furnace

• Accept solutions, slurries, or solids

• Accept very small qualities of samples  Need 5-
100uL sample per analysis
• Higher energy provided to atomize the sample
particles into ground state might excite the atomized
particles to higher energy level and thus lowering the
detection limit

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• The furnace goes through several steps
– Drying (usually just above 110 ºC)
– Ashing (up to 1000 ºC)
– Atomization (Up to 2000-3000 ºC)
– Cleanout (quick ramp up to 3500 ºC). Waste is
blown out with a blast of Ar.
• The light from the source (HCL) passes through the
furnace and absorption during the atomization step is
recorded over several seconds.

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(3) Monochromator
• Used to separate out all of the thousands of lines.
• Selects the specific wavelength of light which is
absorbed by the sample, and to exclude other
wavelengths.
(4) Detector and Readout Device
• The light selected by the monochromator is directed
onto a detector that is typically a photomultiplier tube,
whose function is to convert the light signal into an
electrical signal proportional to the light intensity
the signal could be displayed for read out or further
fed into computers.
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3. Inductively Coupled Plasma
Optical Emission Spectroscopy
(ICP-OES)

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Inductively coupled plasma – optical emission
spectroscopy (ICP-OES)

• Uses principle of atomic emission

• Atomization unit  Plasma
• Mononchromator similar to atomic absorption
• Detector similar to atomic absorption

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Principle of atomic emission

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Atomization - plasma
• A plasma is an electrically neutral, highly ionized gas
that consists of ions, electrons, and atoms.
• Analytical plasma is derived from an electric or
magnetic field.
• Analytical plasmas operate with pure argon or helium
which makes combustion impossible.
• Analytical plasmas typically range in temperature
from 600 to 8,000K.

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ICP torch
• The ICP (inductively coupled plasma) is a
radiofrequency-(RF, 27.12 MHz, 40 MHz) induced
plasma that uses an induction coil to produce a
magnetic field (H).
• The ICP operates between 1 and 5 kilowats.
• The induction coil is wrapped two or three times
around the ICP torch and has water flowing through it
for cooling purposes.

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ICP torch
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Sample introduction- liquid

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Sample introduction- solid

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• Solids are introduced into the plasma either directly
or after electrothermal vaporization, arc vaporization,
or laser ablation.
• In a arc, a small amount of solid sample is mounted
on an electrode and vaporized by the an electric
current before being transported to the ICP.
• Direct laser ablation  utilizes a pulsed laser to
vaporize the solid sample. The plume is carried by
argon gas to the ICP torch for analysis by AES. The
laser plume is generated in an inert environment to
minimize combustion or metal oxide formation.

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Absorption vs Emission

Absorption Emission
Depends upon number of Depends upon the number
ground state atoms of excited state atoms
Presence of Hollow Absence of Hollow
Cathode Lamp Cathode Lamp
Temperature in the Temperature is high
atomizer is adjusted to enough to atomize and
atomize the analyte in the excite the analyte into
ground state higher levels

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4. Gas Chromatography (GC)

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Chromatography
• Chromatography basically involves the separation
of mixtures due to differences in the distribution
coefficient (equilibrium distribution) of sample
components between 2 different phases (a mobile
phase and a stationary phase)
• Distribution Coefficient =
Concentration of component A in stationary phase
Concentration of component A in mobile phase

• If the mobile phase is gas  Gas chromatography

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GC schematic
Detector (flame
Sample injection ionization
detector or FID)
Carrier gas
(nitrogen or Air
helium) Hydrogen

Long Column (30 m)

Sample separation

• Velocity of a compound through the column depends

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upon affinity for the stationary phase
 top view
Flame
Injection Port Ionization
Detector

Column

Oven

front view

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 Flame ionization detector (FID)
Teflon insulating ring Coaxial cable to
Analog to Digital
Gas outlet converter
Collector
Ions
Flame
Sintered disk
Platinum jet
Air

Hydrogen

Capillary tube (column) 52

• FID  responds to compounds that produce
ions when burned in an H2-air flame
– all organic compounds
• Little or no response to (use a Thermal
Conductivity Detector for these gases)
– CO, CO2, CS2, O2, H2O, NH3, inert gasses
• Linear from the minimum detectable limit
through concentrations 107 times the minimum
detectable limit

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 Other detectors
• Thermal Conductivity TCE

FID output
Detector (TCD)
– Difference in thermal
conductivity between methane
the carrier gas and
sample gas causes a time
voltage output

ECD output
– For measuring CO, CO2,
CS2, O2, H2O, NH3,
inert gasses
• Electron Capture Detector
(ECD)
time
– Specific for halogenated Mixture containing lots of methane and
organics a small amount of TCE 54
 MS as the detector: GC-MS
A mixture of compounds is separated by gas
chromatography, then identified by mass spectrometry.

Uses the difference in mass-to-charge ratio (m/e) of

ionized atoms or molecules to separate them from each55
Sample preparation: purge and trap
• Way to measure dilute samples by concentration of
constituents
• Trap constituents under low temperature
• Heat trap to release constituents and send to GC column

N2

Trap

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Sample qualification: retention time
Retention time  time required for the sample to travel from the
injection port through the column to the detector.

Response

X 2

X1

X0

1 3 6
Retention Time 57
Sample quantification: peak area

4
Response

1
3
h1 h2 h4
h3
W 1 W 2 W 3 W 4

V 3

V 4

Retention time (min)

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 Area under curve is
mass of compound
adsorbed to stationary
phase

Carrier gas
Gas phase concentration

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 Resolution
 A characteristic of the separation of two adjacent peaks

RAB = 2 |dR(B) - dR(A)|/|w(B) + w(A)|

dR(A) and dR(B)  retention distances (time or volume) of each
eluted component A and B
w(A) and w(B)  respective widths of each peak at its base 60
5. High Performance Liquid
Chromatography (HPLC)

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Liquid column chromatography

• A sample mixture is passed through a column

packed with solid particles which may or may not be
coated with another liquid.
• With the proper solvents, packing conditions, some
components in the sample will travel the column
more slowly than others resulting in the desired
separation.
• Mobile phase is liquid  liquid chromatography

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Diagram of LC DIAGRAM OF SI M PLE LI QUI D COLUM N CH ROM ATOGRAPHY

Solvent(mobil e or
movi ng phase)
OOOOOOOOOOO
A +B +C Sampl e OOOOOOOOOOO
(A +B+C) OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO OOOOOA OOOO
OOOOOOOOOO Column OOOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOOO Solid Particl es OOOOOOOOOO
OOOOOOOOOOO (packing material- OOOOOB OOOO
OOOOOOOOOO stationary phase) OOOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOO
OOOOOOOOOOO OOOOOC OOOO
OOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOO OOOOOOOOOOO

El uant (eluate)

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Schematic diagram of LC

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Four basic LC
(1) Liquid/Solid Chromatography (adsorption chromatography)
A.Normal Phase LSC (nonpolar mobile phase)
B.Reverse Phase LSC (nonpolar stationery phase)
(2) Liquid/Liquid Chromatography (partition chromatography)
A.Normal Phase LLC B.Reverse Phase LLC
(3) Ion Exchange Chromatography  Separation is based on the
competition of different ionic compounds of the sample on the
ion-exchange resin (column-packing)
(4) Gel Permeation Chromatography (exclusion chromatography)
 mechanical sorting of molecules based on the size of the
molecules in solution  Small molecules are able to permeate more
pores and are, therefore, retained longer than large molecules.
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MOBILE PHASE LIQUID

Liquid-Liquid Liquid-Solid
FORMAT Chromatography Chromatography
(Partition) (Adsorption)

STATIONARY Liquid Solid

PHASE

Normal Phase Reverse Phase Normal Phase Reverse Phase

Mobile Phase - Mobile Phase -

Nonpolar Polar
Stationary phase - Stationary phase -
Polar Nonpolar

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Selecting a LC operating mode
Sample Type LC Mode
Positional isomers (One of a set of
structural isomers which differ only LSC or LLC
in the point at which a side-chain group
is attached)
Moderate Polarity Molecules LSC or LLC
Compounds with Similar Functionality LSC or LLC
Ionizable Species IEC
Compounds with Differing SolubilityLLC
Mixture of Varying Sized Molecules GPC
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LC solvents
Polar Solvents: Water > Methanol > Acetonitrile >
Ethanol > Oxydipropionitrile

Non-polar Solvents : N-Decane > N-Hexane > N-

Pentane > Cyclohexane

LC detectors
Ultraviolet Detector  200-400nm/ 254 nm

Reflective Index Detector  Universal Detector

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