Principle of Atomic Absorption Spectrophotometry

Principle of

Atomic Absorption Spectrophotometry

Mr. Charnchai Suracheep

Introduction

Atomic Absorption Spectrophotometry,

which are standard instruments for

the determination of metal elements,

are widely applied of samples, such as

agriculture chemical, clinical and

biochemistry, minerals, food and

drugs, environmental and other.

Principle of Atomic Absorption Spectrophotometer

Principle of the Atomic Absorption Method

Atomized elements each absorb energy of a wavelength that is peculiar to that element. The

atomic absorption method uses as its light source a hollow cathode lamp which emits light of a wavelength that is peculiar to each element.

Elements within a solution are heated in a flame or electrically (2000K to 3000K) and subsequently

determined using the fact that the degree of absorption will vary with its concentration.

Light absorption process of atoms

Atomic Absorption Spectroscopy, AAS

Atomic Emission Spectroscopy, AES

Principle of Atomic Absorption Spectrophotometer

Ground state E0

Excited state E1

e

Absorption

Ground state E0

Excited state E1

e

Emission

ee

Sodium (Na) energy states

Ground state 0.0 eV

Excited state (I) 2.2 eV

Excited state (II) 3.6 eV

589.0 nm

330.3 nm

Electronic Transition

Relation between light absorption and density

• When light of a certain intensity is given to many atom in ground state, part of this light is absorbed by atoms.

Density C

l

I0 I

Lambert-beer’s LawDensity C

l

I0 II = I0 e-k .l .C

Abs = -logI/I0 = k .l. C

k : proportional constantl : path lengthC : density (concentration)


Calibration curve

• Graph show the relation between absorbance and concentration

Abs

orba

nce

Concentration (ppm)

Absorbance of unknown sample

Concentration of unknown sample


Atomization method

• Atomic absorption spectrometry measures absorption of free atom.

• “Free atom” means an atom not combined with other atoms.

• Elements in the sample to be analyzed are not in the free state, and are combined with other elements invariably to make a so-called molecule.

• The combination must be cut off by some means to free the atoms.

• This is called “atomization”• 2 types:

- Flame method

- Flameless method

Atomization method

Flame Method

Flame Atomization Method

With the Flame Method, the sample solution is converted into mist-form using a nebulizer, and then introduced into the flame. It is atomized by the temperature of the flame.

Measurement time: A few dozen seconds

Optical diagram of Flame Atomic Absorption Spectrometers

Flame selection• These flames vary in temperature, reducibility and

transmission characteristics.• Selected according to the element being analyzed,

and properties of the sample.

Flame Method

• Argon-hydrogen : Max. temp. 1,577 0C• Air-hydrogen : Max. temp. 2,045 0C• Air-acetylene : Max. temp. 2,300 0C•Nitrous oxide-acetylene : Max. temp. 2,955 0C

(For elements are hard to combine with oxygen (Al, Si, V, Ti, etc.))

Flame selection

Flame Method

Flameless Method (Graphite Furnace)

シール

Graphite cap Graphite holder

Cooling block

Aperture plate socket

Sampleinlet

Seal Graphite tube

Eject arm Spring Fixing knobGraphite tube

• Sample is injected in the formed graphite tube.

• An electric current of 300 ampere (maximum) is applied to the tube.



• In an actual measurement heating is done in 3 stage.

- Ashing stage (400-1000oC)

- Atomizing stage (1400-3000oC)

- Drying stage (100oC)

Other atomic absorption methods

• Methods having higher sensitivity than normal flame atomic absorption or electro-thermal atomic absorption

• Used for special elements including arsenic, selenium and mercury.

• Use chemical reactions in the process of atomization to vaporize in the form of an atom or simple molecule.

Structural Diagram of Hydride Vapor Generator

Absorption Cell

NaBH4

Gas Liquid Separator

Peristaltic Pump

Manifold

Reaction Coil

Sample

Carrier Gas Ar

HCl

Drain

Burner Head of AAS

• As, Se, Sb, Sn, Te, Bi, Hg and other metals produce a metal hydride by this method

6BH4- +As3++ 3H+ 3B2H6+3H2 +AsH3 (gas)

Hydride Vapor Generation Technique

Elements Concentration (ppb)

As 5~20

Sb 5~20

Te 5~20

Bi 5~20

Se 10~40

Hg 20~80

Sn 30~90

Cold Vapor Technique

SnCl2 + Hg2+ Hgo(gas)

reduce

5%KMnO4

5%H2SO4

SnCl2

253.6 nm

HHoo

Limit of Quantitative

Element Detection Limit

Flame (ppm) Furnace (ppb)

Ag 0.04 0.01

Al 0.5 0.03

As 0.02 ppb (HVG) 0.2

As 0.4 -

Cd 0.012 0.003

Cr 0.08 0.015

Cu 0.04 0.008

Hg 0.01 ppb (cold vapor) -

Hg 0.2 ppb (HVG) -

Mg 0.0035 0.003

Mn 0.025 0.01

Ni 0.08 0.13

Pb 0.2 0.06

Se 0.3 ppb (HVG) 0.2

Sn 2 N2O-C2H2 2

Zn 0.01 0.01

Interference effects

• Physical interference

• Spectral interference

• Chemical interference

Physical interference

• Flame– Spray efficiency fluctuations due to difference in

viscosity and surface tension between the standard and sample.

• Furnace– Sample dispersion ; Measurement value fluctuations due to tube temperature distribution

– Viscosity within the graphite furnace ; Adherence to sample tip causing errors in collection quantity.

• Example: samples, such as blood or juice, containing numerous organic components.

Spectral interference

• Spectral absorption line overlapping with the absorption line of the target element.

• Absorption and scattering by molecules

Target element Spectral line(nm)

Interferingelement

Spectral line(nm)

Al V Ca Ge Cd As Co In Cu Eu Fe Pt Ga Mn Hg Co Mn Ga Sb Pb Si V Zn Fe


Spectral absorption line overlapping with the absorption line of the target element.


• Absorption and scattering by molecules– Molecules absorption

• Alkaline metals + Halogens = Alkali halides

(Na, K)+(F, Cl, Br, I) = (Ex: NaCl, KI)

Chemical interference

• Generation of non-separable compounds by coexisting matrices

–Example : influence of PO4-, SO4

-, SiO2 relative to Ca, Mg in flame analysis

• (generation of Ca2PO4)

• Generation of low boiling point compounds by coexisting matrices

–Example: influence of chloride ions relative to Cd in furnace analyses

• (generation of CdCl2)

Matrix modifier effect

• Masking of obstructing matrices• Influence of phosphate on Ca is masked by La

• Conversion of obstructing matrices to compounds that easily undergo sublimation or evaporation– Sublimation agent

• Example: removal of chloride ion by ammonium salt of nitric acid or phosphoric acid

• Conversion of measured elements to stable oxides or metallic intermediary compounds– Stabilizing agent:

• Example: creation of measured element alloy using white metals (Pd, Pt, Rh)

Application examples of the matrix modifier method

Standard Addition Method

Mg concentrationafter filled up

X X+0.1 X+0.2 X+0.3

100 ml

Solvent

No.1 No.2 No.4No.3

10 ml Unknown sample

10 ml 10 ml 10 ml 10 ml

1.0 ppm X Standard solution (ppm : mg/1000ml)

20 ml30 ml

10 ml

Standard Addition Method

Calibration Curve of Standard Addition Method

Concentration of unknown sample

　　 2-Way Background Correction is Standard

•D2 lamp method ( 190-430 nm) – Molecular absorption

Background Correction

•Self-Reversal (SR) method – Spectra interference

Elements/ wavelengths where spectral interference becomes problematic

Target element Spectral line(nm)

Interferingelement

Spectral line(nm)

Al V Ca Ge Cd As Co In Cu Eu Fe Pt Ga Mn Hg Co Mn Ga Sb Pb Si V Zn Fe



Self-Reversal Method


Self-Reversal Method

10 mA

100 mA


Signal

Background

AA-6300

Atomic Absorption Spectrophotometer

Optical diagram of Double Beam System

High Performance Optical System

Easy Switching between Flame and Furnace

Flame -> Furnace: All that is involved is to remove the burner head, place the furnace unit, and fix it with the screw. No tools are required.

Remove the burner head.

Fit the furnace.

Remove the furnace.

Fit the burner head.

New Flame AtomizerNew Flame Atomizer

For chemical resistanceFor chemical resistance

• Neburizer w/ Ceramic Neburizer w/ Ceramic made Impact Beadmade Impact Bead

• Polypropylene-made Polypropylene-made ChamberChamber

• Solid Titanium-made Solid Titanium-made Burner HeadBurner Head

High Productivity

• Full Auto ASCFull Auto ASC- - Auto measurement up to 60 samplesAuto measurement up to 60 samples- Reagent addition 8 position- Reagent addition 8 position - Automatic dilution - Automatic dilution

• Optimize Flame analysisOptimize Flame analysis- - Automatic search the best fuel gas flow rateAutomatic search the best fuel gas flow rate-- Automatic search the Optimize Flame Automatic search the Optimize Flame analysis best burner heightanalysis best burner height

Enhanced Safety

High Temp. Burner

• Auto Gas Leak Check• Gas pressure monitoring to prevent flashback• Automatic flame monitoring• Automatic flame extinguish when power failure• Safety interlock for burner misuse• Auto Air/N2O flame changeover• Drain level sensor

Drain level sensor

Wizard Software System

* Select elements

*Set the calibration curve and samples condition

*Connect to PC

*Set the spectrophotometer

* Set the atomizer

Automated/ Optimized

Effectiveness of the automatic Line Search/Beam Balance

Effectiveness of the automatic burner height

(Cr : 4ppm standard solution used)


Burner height & Sensitivity (Cr)

Search for the optimal fuel flow rate

(Cu : 4ppm standard solution used)


Calibration curve

Display of saved signal

The 4 newest signals

Signals in real-time

Screen during measurement

•　 The Login ID and password need to be entered when the software is started up.

•　 Records of who logged in at what time are preserved in the “Event Log”.

User Management

User Management

Authority can be set in detail for each user

Initial Validation Screen

Summary Validation Report

Applicationof

Atomic Absorption Spectrophotometry

Application of AAS

Pretreatment (dissolution) is required for solid samples.

AASAAS

Precautions for pretreatment: Dissolve all the elements into the same solution evenly.

(Check with certified reference material.)

Ensure that elements are not lost in the solution. i.e., due to vaporization

or sedimentation (Check with recovery test.)

Contamination : Purified water, reagent (e.g., acid), container,

environment. (Check with blank operation.)

Ensure that the solution to be analyzed is stable for a long time (i.e., no

hydrolysis or sedimentation).

Consider the interference effect of the reagent on the analysis values.

Pretreatment

DilutionDilute the sample with purified water, dilute acid, or organic solvents.

Examples: food products (e.g., dairy products), pharmaceuticals, and biological samples (e.g., blood, urine).

Types of Pretreatment

Dry DecompositionHeat the sample to a high temperature (400 to 500C), Decomposition is possible in a short time (a few hours) and operation is simple.Elements with low boiling points (e.g., Hg, As, Se, Te, and Sb) will vaporize

Wet DecompositionHeat the sample together with acid to a low temperature (approx. 300C). Suitable for volatile elements.A long time is required for the decomposition of organic substances.

Microwave DecompositionDecompose the sample at high pressure by heating it together with acid to a temperature in the range 100 to 200C in a sealed Teflon container.The decomposition process is sealed; there is little vaporization of elements with low boiling points; the decomposition time is short; there is little contamination from the operating environment and the reagent; and only a small amount of acid is required.Examples: Sediment, soil, dust, ceramics, living organisms, food products, etc.

Wet Decomposition Method

Sample+Sulfuric acid

Nitric acid

Heating

Cooling tube

Waste gas

Simple method

(no cooling)

Kjeldahl flask wet decomposition method

Decompose the sample together with an acid in a sealed container.

Decomposition possible in a short time with little vaporization or contamination.- Ideal for the pretreatment of trace elements and trace samples.

- Food products, living organisms, pharmaceuticals, airborne dust, soil, etc.

Pretreatment Microwave Decomposition

High-pressure Decomposition Container

Microwave Digestion

TemperaturemeasurementTemperaturemeasurement

internal PC or Controller

internal PC or Controller

Control by Tmax and Pmax

Control by Tmax and Pmax

Microwavepower

Digestion Vessels 1 - 12

Pressure measurement

Pressure measurement

Real-Time Display

Sample Preparation using Pressure Digestionwith Microwave heating

Pretreatment Solubility of Elements in Samples

Total Content

Inorganic compoundswith low solubility

Sulfides, oxides,silicates, etc.

Simple soluble metals& compounds

Carbonates, oxides, etc.

Organiccompounds

Simplewater-soluble

ions

Pretreatment Methods

Dilution, Elution

Purified water,

solvents, etc.

Wet Decomposition

Hydrochloric acid, nitric acid, etc.

Dry/Wet Decomposition

Microwave Decomposition

Nitric acid,

sulfuric acid, etc.

Wet/High-pressure Decomposition

Hydrofluoric acid, nitric acid, etc.

Example Example

Application of AAS

EU Regulation for Hazardous Substances

IEC Recommendation for RoHSRoHS : Restriction of Hazardous Substance in Electrical and Electronic equipment.

Substances Polymers Metals Electronics

PBB/PBDE :

1000 ppm

GC-MS NA GC-MS

Cr6+ : 1000 ppm Colorimetric Method (Spectrophotometer)

Spot-test procedure/boiling=water-

Extraction procedure

(Clause8)

Colorimetric Method

(Spectrophotometer)

Hg : 1000 ppm Cold Vapor-AAS, ICP

Pb : 1000 ppm

Cd : 100 ppm

AAS, ICP AAS, ICP AAS, ICP

EU Regulation for Hazardous Substances

Preparation of circuit boards

Vibratory Disc Mill RS 100

Pre-Cutting with the Heavy Duty Cutting Mill

, bottom sieve 6 mm

Heavy Duty Cutting Mill SM 2000

after a grinding time of 2 min. endfineness 90 % < 125 µm

Sample Preparation

Target Ele

ment

Pretreatment Methods

Polymer MetalsElectronic

s

Hg

Microwave digestion (HNO3

+ HBF4

+ H2 O2

)

Cd

Pb

Microwave digestion (HNO

3+H2 O

2 )

(If contain ing Si,

Ti add HF)

a)Commonmethod (HCl : HN

O3 : water ; 2 : 1 : 2 ) b)IfcontainingZr,Hf,Ti,Ta,Nb ,W (HNO3 : HF ;

1 3: ) c) I f contai ni ngSn (H

: ; : )

Microwave di gesti onStepA

(HNO3+HBF

4+

H2O2

) Mic rowave digestio

n Step B (addHCl)

Pretreatment method, which follow by IEC 62321

Analyzing Cadmium (Cd) in RiceAnalyzing Cadmium (Cd) in Rice

Pretreatment Using Wet Decomposition

Put 5 g of the sample in a beaker.

Add 30 mL of nitric acid (1+1) and 0.5 mL of sulfuric acid.

Warm on a hot plate until the violent reaction subsides.

↓

Perform thermal decomposition until the contents approach a hardened and dried state.

When the contents turn dark brown, add 1 mL of nitric acid. Repeat this process.

When the contents turn light yellow or become transparent, expel the white smoke of the sulfuric acid and leave to cool.

Add nitric acid.

Heat on the hot plate to dissolve the salt content.

Leave to cool.

Dilute for measurement.

Level suggested by FAO/WHO Codex Committee ; 0.2 ppm max. in polished rice (propose

d)

Polished rice:0.118 ppm

Unpolished rice:0.070 ppm

0.1 ppm

Furnace method

Injected amount: 10 µL

Interference inhibitor: Pd 50ppm 5 µL

Ashing: 400C; Atomization: 1,800C

Results of Quantitative Analysis of Cd in Rice

Flame method

Air-C2H2

0.5 ppm

Polished rice : 0.118 ppm

Unpolished rice :0.073 ppm

The following 2 methods can be used to analyze unpolished and polished rice decomposed using acid:

Summary of Methods for Analyzing Cd in Rice

Comparison of Pretreatment Methods Wet oxidation: 3 to 5 hours; Dry ashing: 5 to 10 hours;

Microwave: 1 hour; Acid extraction: 2 hours

Expected Lower Limits for Quantitative Measurement Flame method : 0.100 ppm Furnace method : 0.001 ppm

Comparison of Measurement Times for Each Measurement

Method (n=3) 　

Flame method : 30 s

Furnace method : 360 s

of Atomic Absorption Spectrophotometry

Preventive Maintenance / Calibration

Maintenance

1. Cleaning the Burner head

Preventive Maintenance/Calibration

Weekly check

Clogged(by carbide or salt etc.)

Normal

Maintenance

2. Cleaning the Chamber with diluted water or alcohol

Weekly check


Burner Head

O-ring Chamber

Disperser

O-ring Fixing Plate

Nebulizer

Drain

Sample Suction

Nebulizer Construction

Sample Solution

Air

Maintenance

3. Cleaning the Nebulizer

Weekly check


Nebulizer

Cleaning wire

Do not apply the ultrasonic cleaner to the nebulizer

Hardware Validation

1. Wavelength Accuracy

- Using Hg hollow cathode lamp set at Emission mode

- Measure peaks should be within + 0.7 nm

(253.6nm 365.0nm 435.8nm 546..1nm 585.2 640.2nm)

(12 Month) Calibration


3. Baseline Drift

- Using Cu hollow cathode lamp ( 324.8 nm)

- Measuring time 1800 sec

- Measured value less than 0.006 Abs

2. Noise Level

- Using Se hollow cathode lamp ( 196 nm)

- NON-BGC Noise level should be < 0.015 Abs.

- BGC-D2 Noise level should be < 0.035 Abs.

5. Repeatability - Using Cu hollow cathode lamp - Standard Cu 2 ppm - Measure 5 time and CV value < 2%



4. Absorption - Using Cu hollow cathode lamp - Standard Cu 2 ppm - Measured value more than 0.23 Abs



6. Detection Limit

- Using Cu hollow cathode lamp

- Standard Cu 2 ppm

- Measure Standard is 3-5 time and calculate the mean value (A)

- Measure Blank solution is 3-5 time and calculate the standard deviation (S)

- Take the obtained value as the detection limit < 0.004 Abs.

Detection limit = (2.0 x 3 x S) / A



7. Stability

- Using Se and Cu hollow cathode lamp

- Standard Cu 2 ppm

- Measure std. Cu around 5 sec (B)

- Measure std. Cu continuous around 30 sec and measure amplitude of Abs. value (W)

- Take the ratio of W to B

Stability = W/B < 6.0 %

Principle of Atomic Absorption Spectrophotometry

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