laboratory technology instrument

1. Dr. Animikh Ray
Scientist-C
Father Muller Research Center
Assistant Professor
Dept. of Biochemistry
Father Muller Medical College

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3. ⦁ Atomic spectroscopy is used to determine the concentration of elements in a sample.
⦁ Atomic spectroscopy has played a major role in the development of our current database for
mineral nutrients and toxicants .
⦁ Atomic spectroscopic methods are used for the qualitative and quantitative determination of more
than 70 elements.
⦁ Typically, these methods can detect parts-per-million to parts-per-billion amounts, and in some
cases, even smaller concentrations.
⦁ Atomic spectroscopic methods are also rapid, convenient, and usually of high selectivity.
⦁ There are several types of atomic spectroscopy. Atomic absorption spectroscopy (AAS), atomic
emission spectroscopy (AES), and inductively coupled plasma-mass spectrometry (ICP-MS) are the
most common.
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4. AAS is a technique used for determining the concentration of a
particular metal element within a sample.
AAS is used to analyse the concentration of over 62 different
metals in a solution.
AAS is a method of analysis based on absorption of radiation by
atoms when a solution of metallic salt is aspirated (drawing) into
a flame.
•Only a drop of sample needed
•The metals need not be removed from other components.
•Sensitive in the ppm range
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5. AAS method is similar to that of spectrophotometer.
The only exception is the replacement of the sample cell by a flame.
In AAS, a monochromatic light for a particular element is produced by a
hollow cathode lamp utilizing that element as the cathode.
The monochromatic light produced by the lamp is beamed through a long
flame into which is aspirated the solution to be analysed.
The heat energy dissociates the molecules and converts the components
to atoms.
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6. ⦁ In theory, virtually all of the elements
in the periodic chart may be
determined by AAS or AES.
⦁ In practice, atomic spectroscopy is
used primarily for determining mineral
elements.
Essential Nutrient Toxicity Concern
Calcium
Phosphorous
Sodium
Potassium
Chlorine
Magnesium
Iron
Iodine
Zinc
Copper
Selenium
Chromium
Manganese
Arsenic
Boron
Molybdenum
Nickel
Silicon
Lead
Mercury
Cadmium
Nickel
Arsenic
Thallium
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Mineral elements in foods classified according to
nutritional essentiality and potential toxic risk

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9. ⦁ AAS quantifies the absorption of electromagnetic radiation by well-
separated neutral atoms in the gaseous state,
⦁ Atomic spectroscopy is particularly well suited for analytical
measurements because atomic spectra consist of discrete lines, and every
element has a unique spectrum.
⦁ Therefore, individual elements can be identified and quantified accurately
and precisely even in the presence of atoms of other elements.
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10. ⦁ Atomic spectroscopy requires that atoms of the element of interest be in the
atomic state (not combined with other elements in a compound) and that they be
well separated in space.
⦁ In food or plasma, virtually all elements are present as compounds or
complexes and, therefore, must be converted to neutral atoms (atomized) before
atomic absorption or emission measurements can be made.
⦁ Atomization involves separating particles into individual molecules (vaporization)
and breaking molecules into atoms. It is usually accomplished by exposing the
analyte (the substance being measured) to high temperatures in a flame or
plasma.
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11. A schematic representation of the atomization of
an element in a flame or plasma.
(The large circle at the bottom represents a tiny
droplet of a solution containing the element (M) as a
part of a compound).
⦁ A solution containing the analyte is introduced
into the flame or plasma as a fine mist.
⦁ The solvent quickly evaporates, leaving solid
particles of the analyte that vaporize and
decompose to atoms that may absorb radiation
(atomic absorption) or become excited and
subsequently emit radiation (atomic emission).
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12. ⦁ AAS is an analytical method based on the absorption of ultraviolet or visible radiation by
free atoms in the gaseous state.
⦁ The process of AAS involves two steps:
1) Atomization of the sample
2) The absorption of radiation from a light source by the free atoms
(The sample, either a liquid or a solid, is atomized in either a flame or a graphite furnace.
Upon the absorption of ultraviolet or visible light, the free atoms undergo electronic
transitions from the ground state to excited electronic states).
⦁ Two types of atomization are commonly used in AAS:
◦ Flame atomization
◦ Electrothermal (graphite furnace) atomization
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13. ⦁ The technique uses basically the principle that free atoms (gas) generated in an atomizer can
absorb radiation at specific frequency.
⦁ AAS quantifies the absorption of ground state atoms in the gaseous state.
⦁ The atoms absorb UV or visible light and make transitions to higher electronic energy levels.
⦁ The analyte concentration is determined from the amount of absorption.
⦁ Concentration measurements are usually determined from a working (calibration) curve after
calibrating the instrument with standards of known concentration.
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14. The amount of radiation absorbed by the sample is given by Beer’s law:
A = log(I0/I) = abc
where:
A = absorbance
I0 = intensity of radiation incident on the flame
I = intensity of radiation exiting the flame
a = molar absorptivity
b = path length through the flame
c = concentration of atoms in the flame
Absorbance is directly related to the concentration of atoms in the flame.
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15. Atomic absorption spectrometers consist of 5 main components:
1. Radiation source: a hollow cathode lamp (HCL) or an electrode-
less discharge lamp (EDL)
2. Atomizer: a nebulizer-burner system or an electrothermal
furnace
3. Monochromator: an ultraviolet-visible (UV-Vis) grating
monochromator
4. Detector: a photomultiplier tube (PMT) or a solid-state detector
(SSD)
5. Computer
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16. Schematic representation of a double-beam atomic absorption spectrophotometer
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17. ⦁ Sample preparation is relatively simple.
⦁ In principle, any food or clinical sample may be analyzed with any of the atomic spectrocopy
methods (AAS, ICP-AES). In most cases, it is necessary to ash the food the food to destroy organic
matter and to dissolve the ash in a suitable solvent (usually water or dilute acid) prior to analysis.
⦁ Sample preparation for AAS technique involves destruction of organic matter by ashing, followed
by dissolution of the ash in an aqueous solvent, usually a dilute acid.
⦁ Proper ashing is critical to accuracy. Some elements may be volatile at temperatures used in dry
ashing (heating the sample to about 500◦C to burn off the organicmatter) procedures. Volatilization
is less of a problem in wet ashing (heating the sample in nitric acid and perchloric acid), except for
the determination of boron, which is recovered better using a dry ashing method.
⦁ However, ashing reagents may be contaminated with the analyte. It is therefore wise to carry blanks
throughout the ashing procedure.
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18. The most widely used light source is the hollow cathode lamp.
These lamps are designed to emit the atomic spectrum of a
particular element, and specific lamps are selected for use
depending on the element to be determined.
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19. The cathode of the lamp is a hollow-out cylinder of
the metal whose spectrum is to be produced.
Each analyzed element requires a different lamp.
The anode and cathode are sealed in a glass cylinder
normally filled with either neon or argon at low
temperature.
At the end of the glass cylinder is window
transparent to the emitted radiation.
1. Cathode Lamp

20. The cathode lamps are stored in a
compartment inside the AA
spectrometer.
The specific lamp needed for a given
metal analysis is rotated into position
for a specific experiment.

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22. When an electrical potential is applied
between the anode and cathode, some of
the fill gas atoms are ionized.
The positively charged fill gas ions
accelerate through the electrical filed
(gather in a line) to collide with the
negatively charged cathode and dislodge
individual metal atoms in a process called
sputtering.
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23. Then the sputtered metal atoms are elevated to an
excited state.
When the atoms return to the ground state, the
characteristic line spectrum (light) of that atom is
emitted.
Then the emitted light is directed at the flame
where unexcited atoms of the same element
absorb the radiation and are themselves raised to
the excited state.
Then the absorbance is measured.
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25. In AAS, the hollow cathode lamp and flame are light emitting source.
The phototube responds to radiation from the hollow cathode lamp as
well as flame.
This will create interference in absorption measurement.
This problem is corrected by a beam chopper.
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2. Beam chopper

26. Beam chopper is a motor driven
device that has open and solid (or
mirrors in some cases) alternating
regions.
One half of their rotation, i.e., open
region, permits the beam obtained
from lamp to pass through.
During the other half of their
rotation, i.e., mirror region, the
beam is reflected and not allowed
to pass through.
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27. Function of the Chopper
When the incident beam hits the solid surface, the beam is blocked and the detector is
only read the emitted signal from the flame.
As the chopper rotates to the open surface and the beam emerges to the detector.
Now the detector signal is the sum of the transmitted light signal plus that emitted
from the flame.
The signal processor is able to subtract the first signal from the second-one, thus
excluding the signal from the emission in flames.
Signal – 1 (blocked beam) = Pe
Signal – 2 (transmitted beam) = P + Pe
Overall difference signal = (P + Pe) – Pe = P (corrected signal)
This correction method for background emission in flame is called source modulation.
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29. The third unique component in the system is the burner, through which
the sample is introduced. The burner consists of three parts, namely,
nebulizer,
premix chamber and
burner head.
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3. The flame / Burner system

30.  Suck up liquid sample at a
controlled rate
 Create a fine aerosol for
introduction into the flame
 Mix the aerosol and fuel and
oxidant thoroughly for
introduction into the flame.
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4.Nebulizer

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33. Sample solution is aspirated
through a nebulizer and sprayed as
a fine aerosol into the mixing
chamber.
The design of the nebulizer is to
limit the size of the atomized
sample introduced to the flame to a
very small size (~ 5 – 10nm).
Droplets larger than this are
stopped by spoilers and end up
flowing to waste.
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34. The nebulizer chamber / premix chamber
thoroughly mixes acetylene (the fuel) and
oxidant (air or nitrous oxide) and by doing
so, creates a negative pressure at the end
of the small diameter, plastic nebulizer
tube.
This negative pressure acts to suck (uptake)
liquid samples up the tube and into the
nebulizer chamber, a process called
aspiration.
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Premix Chamber

35. The mixture of gases and sample is
directed into the burner head and
flame.
The burner is a specially one for AAS.
It has a long, flat topped head
positioned directly below and parallel
to the beam of light from the lamp.
The gases flow through a 10cm long
slot at the top of the burner head so
that a long, thin curtain of flame is
produced.
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Laminar burner

36.  The two holes, left and right, are
where the light beam enters and
leaves after passing through the
flame.
 The dark place at the top is a stain
from the heat of the flame.
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37. Destroy any analyte ions and
breakdown complexes.
Create atoms (the elemental form)
of the element of interest. FeO,
CuO, ZnO etc.
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Job of the flame

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39. 3. Monochromotors
Monochromotors are the devices that can selectively provide radiation of
desired wavelength out of the range of wavelengths emitted by the source
(lamp) or emitted by the analyte sample.
In AAS, the monochromotor select a given emission line and isolate it from
other lines.
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40. Light from the source (i.e., flame) enters
the monochromotor at the entrance slit
and is directed to the grating where
dispersion takes place.
The diverging wavelength of the light are
directed toward the exit slit.
By adjusting the angle of the grating, a
selected emission line from the source
can be allowed to pass through the exit
slit and fall onto the detector.
All other lines are blocked from exiting.
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42. In photomultiplier tubes numerous
dynodes are aligned in a circular or in a
linear manner.
Here most of the electrodes act as both
an anode and a cathode with each
dynode (electrode pair) having a
potential difference of +90 V; thus, the
potential increases by 90 V as an
electron goes from one electrode to the
next.
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Detectors

43. When nine dynodes are used, a common feature in PMTs,
the net result is a yield of 106 to 107 electrons from a single
emitted photon.
This causes considerable amplification of a weak signal
compared to a photon tube (in old instruments) that does
not amplify the signal.
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44. •AA can be used to identify the presence of an element (qualitative
analysis), or the concentration of a metal (quantitative analysis)
•Quantitative analysis can be achieved by measuring the absorbance of a
series of solutions of known concentration.
•A calibration curve and the equation for the line can be used to
determine an unknown concentration based on its absorbance.
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Determine the concentration of a solution from a
calibration curve.

45. The Copper Flame
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46. The Potassium Flame
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47. The Manganese Flame
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48. Water analysis (Ca, Mg, Fe, Si, Al, Ba content).
Food analysis.
Analysis of animal feedstuffs (Mn, Fe, Cu, Cr, Se, Zn).
Analysis of additives in lubricating oils and greases (Ba, Ca, Na, Li, Zn and
Mg).
Analysis of soil.
Clinical analysis (blood samples, plasma serum – Ca, Mg, Li, Na, K and Fe).
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49. Overview of AA
spectrometer.
Light Source Detector
Sample
Compartment
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