ANALYTICAL INSTRUMENTS

(Student’s copy)

Begseng, John Mark

Novillero, Eunell
Flores, Rikki Anne
Gutierrez, Charmaine Ivy
Maregmen, Debhrey Whriz
Nadiahan, Michelle
Nangpuhan, Angelika
Olivar, Micaela Joy
Gas analyzer is an instrument which is capable of analyzing the species of chemical gases is
present in the sample. Not only it identifies the species but it also has capability to give
measurement value of the quantity which it displays either in numerical form or shows it
graphically.

TYPES OF GAS ANALYZER

 INFRARED GAS ANALYZER

It is used to measure the quantity of various gas. The amount of gas is determined by amount of a
particular frequency of light absorbed by the gas when the light is passed through the gas.
Different molecules in the air absorb different frequencies of light, measuring the absorbed
frequency clearly gives the relation to the amount of particular gas in the air.
Two types:
• Dispersion infrared analyzers - used in laboratories as spectrophotometers
• Non-dispersion infrared analyzers- used for continuous measurement in industrial
applications specifically for measuring the concentration of carbon oxides (CO & CO2).
Advantages:
• Gas molecule doesn’t interact directly with the gas.
• Non-destructive analysis.
• Standard detectors for the measurement of gas in any given environment.
• Monitors emission levels over longer periods of time.
Disadvantages:
• simple measurement becomes a complex measurement
• Higher cost of measuring gas normalization parameter is greater than the cost of primary
dust measurement

 ULTRAVIOLET (UV) GAS ANALYZER

It is a radiant energy (optical) analyzer that uses ultraviolet region of the electromagnetic
radiation spectrum. The UV radiation will pass through the gas inside the gas cell then to the
measurement detector. Thermopiles are used as detectors. The simplest style of non-dispersive
analyzer uses a single light source, shining continuously through a single gas cell, and eventually
falling on a small thermopile which converts the received UV light into heat, and then into a
voltage signal.

Advantages and Disadvantages:

Separate measurements of NO and NO2 eliminate any need for the traditional converters that
have seen frequent use. Using ultraviolet minimizes several maintenance issues. With ultraviolet,
there is no converter and no need to use a chemiluminescent type of analyzer. Using a UV
analyzer in real time gives the user a view of NO2 and NO without the converter. With the
higher levels of NO and NO2, infrared can be effective, but on lower levels, ultraviolet adds
accuracy. But in general, the values given by the IR are more specific than the UV gas analyzer.
There are two general approaches to monitoring emissions from stack gases: In – situ analysis or
extractive analysis.

1. IN – SITU GAS ANALYZER

In-situ analyzers have sensors to take measurements directly in the gas stream. This allows for a
reading without any time delay. In-situ analyzers contain infrared , ultraviolet , or
electrochemical sensors. The analyzer probe sits directly in the gas stream, and the probe sensors
detect the concentration of the species of interest. For this particular in-situ analyzer, the
concentration of the species is measured by using a spectrometer and a xenon flash light beam.
The analyzer units can be configured to relay this information to a variety of devices, such as
computers, strip charts, or modems. A purge air unit is included to keep the sensors from
overheating and prevent contamination.

Advantages and Disadvantages:

In situ gas analyzer is usually small. It is used for direct measurements which saves time and
gives greater accuracy but needs to be calibrated. This can be used in a lot of pipe sizes and flow
rates but the probes and sensor should be designed to function at gas stream temperature and
pressure.

2. EXTRACTIVE GAS ANALYZER:

In extractive measurement a gas sample is taken from the stream, prepared and then evaluated.
Composition of the gas stream sample is determined using one of three sensors: infrared ,
ultraviolet , or electrochemical.
Two ways of sample taking:
• Full Extractive Method- Measurements made can be done either with the moisture still
present in the sample called hot or wet basis or, with a 'dried' sample called cool or dry
basis.
• Dilution Extractive Method- It extracts a very small representative portion of a gas
stream and very accurately dilutes the sample with air before transportation to an
analyser.
Two ways of dilution:
In-stack dilution- dilution of gas sample takes place within the sample probe itself or
dilution mechanism is part of the probe.
Out of stack dilution- dilution takes place after the probe, right at it’s discharge
Advantages
• Can detect extremely low concentrations.
• Many applications possible.
• Can handle a variety of flow rates and pipe sizes.

Disadvantages
• Larger than in-situ analyzers.
• Delayed analysis of gas because it must pass from stream to sample unit to analyzing
unit.
• Analyzers must be calibrated to specific ranges.
 SPECTROMETERS
- Instruments that measure or analyse a range of a given characteristic or wavelength of
a substance

1. SPECTROPHOTOMETER
- An optical device that measures the concentration or particles in a solution.
- Light with a pre-selected wavelength is allowed to pass through the sample. The
amount of light absorbed will be determined. The amount of light absorbed increases
with the increase in amount of particles present in the sample.

 Beer’s Law – also referred to as Beer-Lambert law or the Bouguer-Beer law. It was
named after August Beer. It states that the quantity of light directly proportional to the
concentration of the substance and the path length of the light through the solution.
A=εcl
where A is the absorbance, c is the, l is the path length, and ε is a constant of
proportionality known as the molar extinction coefficient.
 Single-beam instrument - the absorbance of control is determined followed by the
sample
o Advantages: low cost, high throughput and high sensitivity
o Disadvantages: prone to drift, dilution is required

 Double-beam spectrophotometers - the light beam is split into two beams by means of
mirrors. One light path goes through the sample chamber and the other light beam passes
through what is referred to as the reference cell or chamber
o Advantages: less prone to drift
o Disadvantages: high cost, low sensitivity, dilution is required

 Components of a spectrophotometer
o Radiant Source
 Ultraviolet Radiation - hydrogen lamp, deuterium lamp
 Visible Radiation - Tungsten filament lamp, carbon arc
 Infrared Radiation - Nernst glower
o Monochromator -breaks polychromatic radiation into component wavelength. The
types of prisms usually employed in commercial instruments are a 60° cornu
quartz and a 30° Littrow prism.
 Diffraction Grating – used in pairs
 Ruled Gratings
 Holographic Gratings
 Prisms

o Transport Vessels (cuvettes) – sample containers

o Detection Devices
 High sensitivity to allow the detection of low levels of radiant energy
 Short response time
 Long-term stability
 An electric signal which is easily amplified for a typical readout apparatus.
 o Amplification and Read-out – amplifiers, ammeters, potentiometers and
potentiometric recorder
 Applications of a spectrophotometer
o Qualitative Analysis
o Quantitative Analysis
o Enzyme Assay
o Molecular Weight Determination
o Other Physiochemical Studies
 Heats of formation of molecular addition compound and complexes in
solution
 Determination of empirical formula
 Formation constants of complexes in solution
 Hydration equilibrium of carbonyl compounds
 Association constants of weak acids and bases in organic solvents
 Protein-dye interactions
 Chlorophyll-Protein complexes
 Vitamin-A aldehyde–Protein complex
 Determination of reaction rates
 Dissociation constants of acids and bases
 Association of cyanine dyes

2. Mass Spectrophotometer
- Developed by Francis William Aston
- Identifies the chemical composition of a sample based on the mass-to-charge ratio of
charged particles and identifies the isotopic composition of its constituents
- Determines the structure of the compound by observing its fragmentation
 Applications
o Isotope ratio MS: Isotope Dating and Tracking
o Trace Gas Analysis
o Atom Probe
o Pharmacokinetics
o Protein Characterization
o Space Exploration
 Advantages
o Sensitive
o Excellent toll for identifying unknown components in a sample or confirming
their presence
 Disadvantages
o Cannot identify hydrocarbons that produce similar ions
o Unable to tell optical and geometrical isomers apart
o Cannot distinguish components with the same molecular formula
o Cannot distinguish between isomers of a compound having the same charge-to-
mass ratio

3. Infrared (IR) Spectrophotometer

- Deals with the infrared region of the electromagnetic spectrum
 - Analyzes samples based on reflection, emission and absorption
- The two main components are
o Sources – Nernst glower –a rod of a sintered mixture of the oxides of
Zirconium, Ytterbium and Erbium.
o Prism - Sodium chloride or other alkali metal halides are the best material to
form prism and cell container
o Cell container
o Detector – converts IR energy to electrical energy and is amplified by an
amplifier
 Applications
o Identification of functional group and structure elucidation
o Identification of structure substances
o Study of the progress of a chemical reaction
o Detection of impurities via infrared
o Quantitative analysis
 Advantages
o Provides qualitative and quantitative analysis without destroying the sample
o The sample does not need any particular preparation
o Very sensitive, thus, it requires a minimum sample quantity
o Solid, liquid, gases, and semi-solid samples can be analyzed
o Peak intensities, peak positions, peak widths, shapes, and functional groups
provide all helpful information
 Disadvantages
o Difficult handling procedures and maintenance of the sample cells
o There are no infrared spectra in atoms or monoatomic ions
o Requires very sensitive and properly tuned devices
o Aqueous solutions and complex mixtures are complicated to analyze via IR
spectroscopy
 In early 1900s, Gas chromatography (GC) was discovered by Mikhail Semenovich
Tsvett as a separation technique to separate compounds. Gas chromatograph uses a flow-through
slender tube called the column, through that totally different chemical constituents of a sample
pass in an exceedingly gas stream (carrier gas, mobile phase) at totally different rates counting
on their varied chemical and physical properties and their interaction with a selected column
filling, referred to as the stationary part.
Instrumental components
Carrier gas
The carrier gas should be with chemicals inert. Ordinarily used gases embody atomic
number 7, helium, argon, and CO2. The selection of carrier gas is usually dependent upon the
kind of detector that is employed. The carrier facility additionally contains a molecular sieve to
get rid of water and alternative impurities.
Sample injection port
For optimum column potency, the sample mustn't be large, and may be introduced onto
the column as a "plug" of vapor - slow injection of enormous samples causes band broadening
and loss of resolution.
Column
There are a unit 2 general styles of column, packed and capillary (also referred to as
open tubular). Packed columns contain a finely divided, inert, solid support material (commonly
supported diatomaceous earth) coated with liquid stationary part. Most packed columns area unit
one 0.5 - 10m long and have an interior diameter of two - 4mm.
Column temperature
For precise work, column temperature should be controlled to at intervals tenths of a
degree. The optimum column temperature is dependent upon the boiling purpose of the sample.
As a rule of thumb, a temperature slightly on top of the typical boiling purpose of the sample
ends up in associate extraction time of two - half-hour.
Detectors
There are several detectors which may be employed in gas activity. Totally different
detectors can offer differing kinds of property. The response of a mass flow dependent detector is
unaffected by make-up gas. Have a glance at this tabular outline of common rate detectors

Applications
 Identification of the oil elements by GC/MS
 Skin samples analysis

Environmental monitoring
 Food, beverage, flavor and fragrance analysis
 Forensic and criminal cases
 Biological and pesticides detections
 Security and chemical warfare agent detection
 Astro chemistry and Geo chemical Research
 RNA isolation
Limitations
1. Not suitable for detecting semi-volatile compounds
2. Only indicates if volatile organic compounds are presents.
 3. High concentration so methane is required for higher performance.
4. Frequent calibrations are required.
5. Units of parts per million range
6. Environmental distraction, especially water vapour.
7. Strong electrical fields Rapid variation in temperature at the detector and naturally
occurring compounds may affect instrumental signal.

I. High performance liquid chromatography

High Performance Liquid Chromatography (HPLC) is a form of column
chromatography that pumps a sample mixture or analyte in a solvent (known as the mobile
phase) at high pressure through a column with chromatographic packing material (stationary
phase).
Instrumentation
Main components in an HPLC system include the solvent reservoir, or multiple
reservoirs, a high-pressure pump, a column, injector system and the detector.
The column and the solvent
Confusingly, there are two variants in use in HPLC depending on the relative polarity of
the solvent and the stationary phase.
Normal phase HPLC
It is described as "normal", it isn't the most commonly used form of HPLC. The column
is filled with tiny silica particles, and the solvent is non-polar - hexane, for example. A typical
column has an internal diameter of 4.6 mm (and may be less than that), and a length of 150 to
250 mm.
Reversed phase HPLC
In this case, the column size is the same, but the silica is modified to make it non-polar
by attaching long hydrocarbon chains to its surface - typically with either 8 or 18 carbon atoms
in them. A polar solvent is used - for example, a mixture of water and an alcohol such as
methanol. Reversed phase HPLC is the most commonly used form of HPLC.

A flow scheme for HPLC:

Injection of the sample
Injection of the sample is entirely automated; it is not the same as in gas
chromatography because of the pressures involved
Retention time
The time taken for a particular compound to travel through the column to the detector is known
as its retention time. This time is measured from the time at which the sample is injected to the
point at which the display shows a maximum peak height for that compound.
The detector
There are several ways of detecting when a substance has passed through the column. A
common method which is easy to explain uses ultra-violet absorption.
Interpreting the output from the detector
The output will be recorded as a series of peaks - each one representing a compound in
the mixture passing through the detector and absorbing UV light.
Peaks are also use a another way of measuring the quantities of the compounds present.
Let's suppose that you are interested in a particular compound, X.
 The area under the peak is proportional to the amount of X which has passed the
detector, and this area can be calculated automatically by the computer linked to the display. If
the solution of X was less concentrated, the area under the peak would be less - although the
retention time will still be the same.

Other HPLC types:

 Ultra high performance liquid chromatography (UHPLC):
Where standard HPLC typically uses column particles with sizes from 3 to 5µm and
pressures of around 400 bar, uHPLC use specially designed columns with particles down to
1.7µm in size, at pressures in excess of 1000 bar.
 Fast protein liquid chromatography (FPLC)
FPLC is a system similar to high-performance liquid chromatography that is used to
separate or purify proteins and other biomolecules from complex mixtures.

Advantages and Disadvantages

 Speed, efficiency and accuracy
Compared to other chromatographic techniques, such as TLC, HPLC is extremely quick
and efficient. It uses a pump, rather than gravity, to force a liquid solvent through a solid
adsorbent material, with different chemical components separating out as they move at different
speeds.
 Cost and complexity
Despite its advantages, HPLC can be costly, requiring large quantities of expensive
organics. Techniques such as solid phase extraction and capillary electrophoresis can be cheaper
and even quicker, especially for analysis under good manufacturing practice.
 Sensitivity and resolution
In general, HPLC is versatile and extremely precise when it comes to identifying and
quantifying chemical components. With many steps involved, the precision of HPLC is largely
down to the process being automated and therefore highly reproducible.

II. Thin layer chromatography

Thin layer chromatography (TLC) is used to separate the components of a mixture using
a thin stationary phase supported by an inert backing. TLC is an analytical tool widely used
because of its simplicity, relative low cost, high sensitivity, and speed of separation. The goal of
TLC is to obtain well defined, well separated spots.
 Plates (stationary phase)
Silica gel and alumina are among the most common stationary phases, but others are
available as well. Many plates incorporate a compound which fluoresces under short-wave UV
(254 nm). The properties of your sample should be considered when selecting the stationary
phase.
 Solvent (mobile phase)
Proper solvent selection is perhaps the most important aspect of TLC, and determining
the best solvent may require a degree of trial and error. As with plate selection, keep in mind the
chemical properties of the analytes. Varying the ratio can have a pronounced effect of Rf
(Retention factor)
Rf values:
Measurements are often taken from the plate in order to help identify the compounds
present. These measurements are the distance travelled by the solvent, and the distance travelled
by individual spots.
The Rf value for each dye is then worked out using the formula:

How does thin layer chromatography work?

The stationary phase - silica gel:

Silica gel is a form of silicon dioxide (silica). The silicon atoms are joined via oxygen
atoms in a giant covalent structure. However, at the surface of the silica gel, the silicon atoms are
attached to -OH groups. The surface of the silica gel is very polar and, because of the -OH
groups, can form hydrogen bonds with suitable compounds around it as well as van der Waals
dispersion forces and dipole-dipole attractions.

What separates the compounds as a chromatogram develops?

As the solvent begins to soak up the plate, it first dissolves the compounds in the spot
that you have put on the base line. The compounds present will then tend to get carried up the
chromatography plate as the solvent continues to move upwards.
How fast the compounds get carried up the plate depends on two things:
 How soluble the compound is in the solvent. This will depend on how much attraction
there is between the molecules of the compound and those of the solvent.
 How much the compound sticks to the stationary phase - the silica gel, for example. This
will depend on how much attraction there is between the molecules of the compound and
the silica gel.
Common Problems in
TLC:
There are common problems in TLC that should be avoided. Normally, these problems
can be solved or avoided if taught proper techniques.
 Over-large Spots: Spotting sizes of your sample should be not be larger than 1-2 mm in
diameter. The component spots will never be larger than or smaller than your sample
origin spot.
 Uneven Advance of Solvent Front: Uneven advance of the mobile phase is a common
problem encountered in TLC. Consequences would be inaccurate Rf values due to the
uneven advance of sample origin spots. This uneven advance can be caused by a few
factors listed below.
o No flat bottom. When placing the TLC plate into the chamber, place the bottom of
the plate on the edge of the chamber (normally glass container (e.g. beaker)) and
lean the top of the plate along the other side of the chamber. Also, make sure that
the TLC plate is placed in the chamber evenly. Do not tilt the plate or sit it at an
angle.
o Not enough solvent. There should be enough solvent (depends on size of
chamber) to travel up the length of the TLC plate.
 o Plate is not cut evenly. It is recommended that a ruler is used so that the plate is
cut evenly.

Advantages and Disadvantages

 The solvents for the TLC plate can be changed.
 TLC can be used to ensure purity of a compound.
 Identification of most compounds can be done simply by checking Rf literature values.
 TLC plates do not have long stationary phases.
 Detection limit is a lot higher.
 TLC operates as an open system.
Refractometers - measure the degree to which the light changes direction, called the angle of
refraction. A refractometer takes the refraction angles and correlates them to refractive index
(nD) values that have been established. Using these values, you can determine the concentrations
of solutions.
A refractometer is an optical devise used for measuring the extent to which light is bent,
or refracted, when it moves through a substance. It works because light travels at different
velocities in different mediums.

4 Main Types of Refractometer:

 Traditional handheld refractometers
 Digital handheld refractometers
 Laboratory refractometers
 Inline process refractometers

How a Refractometer Works?

When light enters a liquid it changes direction; this is
called refraction. Refractometers measure the degree to which the light changes direction, called
the angle of refraction. A refractometer takes the refraction angles and correlates them to
refractive index (nD) values that have been established. Using these values, you can determine
the concentrations of solutions. For example, solutions have different refractive indexes
depending on their concentration in water.
The prism in the refractometer has a
greater refractive index than the solution. Measurements are read at the point where the prism
and solution meet. With a low concentration solution, the refractive index of the prism is much
greater than that of the sample, creating a large refraction angle and a low reading ("A" on
diagram). The reverse would happen with a high concentration solution ("B" on diagram).
Applications
Refractometers are designed for specific operations, such as measuring liquids such as
oils, or water-based liquids, gases, and transparent or translucent solids, such as gemstones.
Refractometers are mostly used to determine the index of refraction of liquid samples, and to
measure fluid concentrations, such as blood protein concentration, sugar content, and salinity.
Other functions of the refractometer include:
 Helping to determine the identity of a sample by comparing its refractive index to
previously known values
 Assessing the purity of a sample by comparing its refractive index to the value for the
pure substance
 Determining the concentration of a solute in a solution by comparing the solution's
refractive index to a standard curve
Advantages
 Very portable
 Inexpensive
 Easy to operate
 Instant results
 Versatile
Disadvantages
 Difficult to analyze materials with lower refractive index than that of the prism.
 One must bring a possibly hazardous material close to the eyes to read hand held
refractometers.
COLORIMETER - is a light-sensitive device used for measuring the transmittance and
absorbance of light passing through a liquid sample. The device measures the intensity or
concentration of the color that develops upon introducing a specific reagent into a solution.
There are two types of colorimeters:
 color densitometers - which measure the density of primary colors.
 color photometers - which measure the color reflection and transmission.

How does the instrument works?

In a colorimeter, a beam of light with a specific wavelength is passed through a solution
via a series of lenses, which navigate the colored light to the measuring device. This analyzes the
color compared to an existing standard. A microprocessor then calculates the absorbance or
percent transmittance. If the concentration of the solution is greater, more light will be absorbed,
which can be identified by measuring the difference between the amount of light at its origin and
that after passing the solution.
Applications
 It is widely used to monitor the growth of a bacterial or yeast culture.
 It is widely used in hospital and laboratory for estimation of biochemical samples, like
plasma, serum, cerebrospinal fluid (csf), urine.
 It is used to measure and monitor the color in various foods and beverages, including
vegetable products and sugar.
 It used for testing water quality.
 It is used to determine the concentrations of plant nutrients.
 It is used in color printing, textile manufacturing and paint manufacturing.

Advantages:
 The colorimeter is a simple instrument and requires little maintenance. An example of the
simplest form of colorimetry is the use of litmus paper to determine acidity or basicity.
 The quantity of material used is generally very small, but if the material is costly it is
frequently possible to use dilutions in a solvent such as water. If a unit cell is utilized
through which light is passed, it is generally simple, little in size, and low in cost. Some
colorimeters used in non-critical applications don’t require the use of a standard for
comparison at all.
 Sample testing in the laboratory can take minutes to hours for sample preparation testing
to be run, but colorimetry using hand-held devices can take minutes or even seconds to
run. In such instances, colorimetry offers obvious advantages.
 Very well applicable to the quantitative analysis of colored compounds
Disadvantages:
 They are not able to identify metamerism or colorant strength, are not ideally suited for
color formulation, and cannot be used under variable illuminant/observer conditions.
 It does not work in Ultraviolet and Infrared regions.
 In using colorimeter similar colors from interfering substances can produce errors in
result.
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