‫جلیلی‬
‫به نام خداوند بخشنده ی مهربان‬
)1
Atomic Absorption Spectroscopy (AAS)
Atomic Absorption Spectroscopy in analytical chemistry is a technique for determining the
concentration of a particular metal element within a sample. Atomic absorption spectroscopy
can be used to analyze the concentration of over 62 different metals in a solution.
Although atomic absorption spectroscopy dates to the nineteenth century, the modern form
was largely developed during the 1950s by a team of Australian chemists, lead by Alan
Walsh, working at the CSIRO (Commonwealth Science and Industry Research Organization)
Division of Chemical Physics, in Melbourne Australia. Typically, the technique makes use of
a flame to atomize the sample, but other atomizers such as a graphite furnace are also used.
Three steps are involved in turning a liquid sample into an atomic gas:
1. Desolvation – the liquid solvent is evaporated, and the dry sample remains
2. Vaporizations – the solid sample vaporizes to a gas
3. Volatilization – the compounds making up the sample are broken into free atoms.
The flame is arranged such that it is laterally long (usually 10cm) and not deep. The height of
the flame must also be controlled by controlling the flow of the fuel mixture. A beam of light
is focused through this flame at its longest axis (the lateral axis) onto a detector past the
flame.
The light that is focused into the flame is produced by a hollow cathode lamp. Inside the lamp
is a cylindrical metal cathode containing the metal for excitation, and an anode. When a high
voltage is applied across the anode and cathode, the metal atoms in the cathode are excited
into producing light with a certain emission spectra. The type of hollow cathode tube depends
on the metal being analyzed. For analyzing the concentration of copper in an ore, a copper
cathode tube would be used, and likewise for any other metal being analyzed. The electrons
of the atoms in the flame can be promoted to higher orbitals for an instant by absorbing a set
quantity of energy (a quantum). This amount of energy is specific to a particular electron
transition in a particular element. As the quantity of energy put into the flame is known, and
the quantity remaining at the other side (at the detector) can be measured, it is possible to
calculate how many of these transitions took place, and thus get a signal that is proportional
to the concentration of the element being measured.
[Courtesy of http://www.chemistry.nmsu.edu/Instrumentation/ICP_MS.html]
Cold Vapor Atomic Absorption (CVAA)
Since atoms for most AA elements cannot exist in the free, ground state at room temperature,
heat must be applied to the sample to break the bonds combining atoms into molecules. The
only notable exception to this is mercury. Free mercury atoms can exist at room temperature
and, therefore, mercury can be measured by atomic absorption without a heated sample cell.
In the cold vapor mercury technique, mercury is chemically reduced to the free atomic state
by reacting the sample with a strong reducing agent like stannous chloride or sodium
borohydride in a closed reaction system. The volatile free mercury is then driven from the
reaction flask by bubbling air or argon through the solution. Mercury atoms are carried in the
gas stream through tubing connected to an absorption cell, which is placed in the light path of
the AA spectrometer. Sometimes the cell is heated slightly to avoid water condensation but
otherwise the cell is completely unheated.
As the mercury atoms pass into the sampling cell, measured absorbance rises indicating the
increasing concentration of mercury atoms in the light path. Some systems allow the mercury
vapor to pass from the absorption tube to waste, in which case the absorbance peaks and then
falls as the mercury is depleted. The highest absorbance observed during the measurement
will be taken as the analytical signal. In other systems, the mercury vapor is rerouted back
through the solution and the sample cell in a closed loop. The absorbance will rise until an
equilibrium concentration of mercury is attained in the system. The absorbance will then
level off, and the equilibrium absorbance is used for quantitation.
The entire cold vapor mercury process can be automated using flow injection techniques.
Samples can be analyzed in duplicate at the rate of about 1 sample per minute with no
operator intervention. Detection limits are comparable to those obtained using manual batch
processes. The use of flow injection systems also minimizes the quantity of reagents required
for the determinations, further reducing analysis costs.
The sensitivity of the cold vapor technique is far greater than can be achieved by
conventional flame AA. This improved sensitivity is achieved, first of all, through a 100%
sampling efficiency. All of the mercury in the sample solution placed in the reaction flask is
chemically atomized and transported to the sample cell for measurement.
The sensitivity can be further increased by using very large sample volumes. Since all of the
mercury contained in the sample is released for measurement, increasing the sample volume
means that more mercury atoms are available to be transported to the sample cell and
measured. The detection limit for mercury by this cold vapor technique is approximately 0.02
µg/L. Although flow injection techniques use much smaller sample sizes. They provide
similar performance capabilities, as the entire mercury signal generated is condensed into a
much smaller time period relative to manual batch-type procedures.
Flame Atomic Absorption (FLAA)
Flame Atomic Absorption Spectroscopy is a fast and easy technique with an extremely high
sensitivity (especially for elements like Pb, Cd, Cu and Cr), although problems can arise as a
result of chemical (a much worse situation than with ICP-AES) and spectral interferences.
The sample is atomized in the flame, through which radiation of a chosen wavelength (using
a hollow cathode lamp) is sent. The amount of absorbed radiation is a quantitative measure
for the concentration of the element to be analyzed. The most current gas mixtures used are
air/acetylene and nitrous-oxide/acetylene. The latter resulting in higher atomization
efficiencies and thus better detection limits for elements like Si, Al, Sc, Ti, V and Zr. The
air/acetylene flame can be used for easy atomizable elements (e.g. As and Se). Background
correction can be achieved with a deuterium lamp although several disadvantages
subsequently occur.
A disadvantage of the AAS technique is the non linearity of the calibration curves when
absorbance becomes higher than 0.5 to 1. The relative standard deviations are between 0.3
and 1% for absorbances of 0.1 to 0.2. Detection limits for flame AAS vary enormously: from
1 - 5 ppb (e.g. Ca, Cd, Cr, Cu) to more than 1000 ppb (e.g. P). Some elements (e.g. B, C, Br)
cannot be measured at all.
[Courtesy of http://www.mtm.kuleuven.ac.be/Research/Equipment/Chemical/FAAS.html]
Overview of Technique
In flame atomic absorption spectroscopy a liquid sample is aspirated and mixed as an aerosol
with combustible gasses (acetylene and air or acetylene and nitrous oxide.) The mixture is
ignited in a flame of temperature ranging from 2100 to 2800 degrees C (depending on the
fuel gas used.) During combustion, atoms of the element of interest in the sample are reduced
to the atomic state. A light beam from a lamp whose cathode is made of the element being
determined is passed through the flame into a monochronometer and detector. Free, unexcited
ground state atoms of the element absorb light at characteristic wavelengths; this reduction of
the light energy at the analytical wavelength is a measure of the amount of the element in the
sample.
Specific Sample Considerations
Plant: solid samples must be in liquid form to be aspirated by the instrument. Therefore, solid
material must be liquefied by means of some form of extract or digest protocol. Procedures
have been devised that make the total amount of an element in the sample available for assay
or that use some particular property to extract that portion of the element which exists in
some chemical forms but not in others. The [plant dry ash/double acid] extraction method
determines the total element content of the sample.
Soil: for ecological purposes there is more interest in measures of extractable or labile soil
constituents than in total element content. Certain partitions of the total soil content of a given
element are operationally defined by an extraction procedure, and arguments are usually
offered that these partitions, so defined, correspond to different levels of biological
availability or activity. The [HCl/H2SO4 double acid] extraction method, also referred to as
North Carolina and Mehlich-1, is widely used to determine bioavailable Ca, K, Mg, Mn, P,
and Zn in sandy acid soils characteristic of the eastern and southeastern United States.
Water: aquatic samples of course need no liquefaction step, but researchers must still decide
which analyte partition (dissolved, suspended, total) is of interest. Differing treatments of
each sample partition are detailed in the U.S. EPA's discussion of [Content partitioning] of
water samples.
[Courtesy of http://www.uga.edu/~sisbl/aaspec.html]
Graphite Furnace Atomic Absorption (GFAA)
Graphite furnace atomic absorption spectrometry is a highly sensitive spectroscopic
technique that provides excellent detection limits for measuring concentrations of metals in
aqueous and solid samples.
GFAA has been used primarily in the field for the analysis of metals in water. GFAA could
be used to determine metals in soil, but the sample preparation for metals in soil is extensive
and is not practical for field applications. GFAA cannot be described as a truly field portable
instrument. GFAA instruments are extremely sensitive and therefore, must be operated in a
clean, climate controlled environment. This can be difficult but not impossible to achieve in a
field environment. In addition, the 220-volt electrical power requirement often precludes
remote operation. However, GFAA is an example of “taking the laboratory to the field.”
Miniaturization of electronics has significantly reduced instrument size and weight, making it
easier to use the instrument in a field laboratory.
In atomic absorption (AA) spectrometry, light of a specific wavelength is passed through the
atomic vapor of an element of interest, and measurement is made of the attenuation of the
intensity of the light as a result of absorption. Quantitative analysis by AA depends on: (1)
accurate measurement of the intensity of the light and (2) the assumption that the radiation
absorbed is proportional to atomic concentration.
Samples to be analyzed by AA must be vaporized or atomized, typically by using a flame or
graphite furnace. The graphite furnace is an electrothermal atomizer system that can produce
temperatures as high as 3,000°C. The heated graphite furnace provides the thermal energy to
break chemical bonds within the sample and produce free ground-state atoms. Ground-state
atoms then are capable of absorbing energy, in the form of light, and are elevated to an
excited state. The amount of light energy absorbed increases as the concentration of the
selected element increases.
GFAA has been used primarily for analysis of low concentrations of metals in samples of
water. GFAA can be used to determine concentrations of metals in soil, but the sample
preparation for metals in soil is somewhat extensive and may require the use of a mobile
laboratory. The more sophisticated GFAAs have a number of lamps and therefore are capable
of simultaneous and automatic determinations for more than one element.
Logistical needs include reagents for preparation and analysis of samples, matrix modifiers, a
cooling system, and a 220-volt source of electricity. In addition, many analytical components
of the GFAA system require significant space, which typically is provided by a mobile
laboratory.
The advantages of GFAA spectrometry include:




Greater sensitivity and detection limits than other methods
Direct analysis of some types of liquid samples
Low spectral interference
Very small sample size
[Courtesy of http://www.clu-in.org/char/technologies/graphite.cfm]
Inductively Coupled Plasma – Mass Spectroscopy (ICP-MS)
The Inductively Coupled Plasma coupled with a mass spectrograph give very high sensitivity
for the determination of elements and even isotopes. This technique has the ability to detect
very low levels (parts per billion) of most elements in a sample. The dynamic range is
typically ten orders of magnitude and data reduction is relatively simple. Rapid data
acquisition and data reduction enable the measurement of large numbers of samples in a short
period of time. ICP-MS is the technique of choice for trace element analysis of natural
waters, minerals, and rocks. High precision is achieved by using multiple internal standards.
[Courtesy of http://www.chemistry.nmsu.edu/Instrumentation/ICP_MS.html]
Inductively Coupled Plasma – Optical Emission
Spectroscopy (ICP-OES)
Inductively coupled plasma optical emission spectroscopy is a major technique for elemental
analysis. The sample to be analyzed, if solid, is normally first dissolved and then mixed with
water before being fed into the plasma.
Atoms in the plasma emit light (photons) with characteristic wavelengths for each element.
This light is recorded by one or more optical spectrometers and when calibrated against
standards the technique provides a quantitative analysis of the original sample.
ICP instruments comprise various optical spectrometers, nebulizers (including glass
concentric, parallel flow, JY pneumatic, cross flow, V groove, micro concentric, ultrasonic,
CMA), spray chambers, ICP torch, and RF generators.
[Courtesy of http://icp-oes.net/]
Fourier Transform Infrared (FTIR)
FTIR spectrometers record the interaction of IR radiation with a sample, measuring the
frequencies at which the sample absorbs the radiation and the intensities of the absorption.
Determining these frequencies allows identification of the sample's chemical make-up, since
chemical functional groups are known to absorb radiation at specific frequencies. The
intensity of the absorption is related to the concentration of the component. Intensity and
frequency of sample absorption are depicted in a two-dimensional plot called a spectrum.
Intensity is generally reported in terms of percent transmittance, the amount of light that
passes through it. In the interferometer the light passes through a beam splitter, which sends
the light in two directions at right angles. One beam goes to a stationary mirror then back to
the beam splitter. The other goes to a moving mirror. The motion of the mirror makes the
total path length variable versus that taken by the stationary-mirror beam. When the two meet
up again at the beam splitter, they recombine, but the difference in path lengths creates
constructive and destructive interference pattern called an interferogram.
The recombined beam passes through the sample. The sample absorbs all the different
wavelengths characteristic of its spectrum, and this subtracts specific wavelengths from the
interferogram. The detector now reports variation in energy versus time for all wavelengths
simultaneously. A laser beam is superimposed to provide a reference for the instrument
operation.
[Courtesy of http://www.chemistry.nmsu.edu/Instrumentation/PE_Spec1.html]
Description
Infrared spectroscopy is an established analytical technique that identifies compounds by
fingerprint light absorption spectra. A sample's molecular constituents are revealed through
their characteristic frequency-dependent absorption bands. Click here for a typical IR
spectrum. Laboratory infrared (IR) instruments have been used extensively for decades in
fixed laboratory settings. Recently, manufacturers of the instruments have significantly
reduced their overall size and power requirements to perform field analysis, while increasing
their durability. Types of portable instrumentation include Multiple Internal Reflectance
Infrared Spectrometers, Long Range Gas Monitors, Open Path Infrared Flammable Gas
Detectors and Infrared Ambient Air Monitors, and Open Path Fourier Transform Infrared
(FTIR) systems.
Typical Uses
In the environmental field, IR's primary use is air monitoring for volatile organic chemicals
(VOCs) by FTIR. FTIR is the preferred method because interferometry (specific to FTIR)
provides multiple, rapid scanning capability which allows near real-time analysis.
Applications for which FTIR is suitable include fence-line or site perimeter monitoring,
worker exposure monitoring, emission rate assessment, air impact measurement during
emergency removals, air impact evaluation during remedial actions, vapor suppression
technique evaluation, accidental release early warning systems, and industrial facility
monitoring. Open-path FTIR was first employed at waste sites in 1990. Its use has grown as
the technology has improved and matured. It has been employed at numerous sites over the
last few years for applications in fugitive industrial emissions, industrial health and safety
monitoring, and indoor air assessments. Recently, manufacturers of FTIR have reconfigured
the systems into high-powered, durable projectors and mirrors for use in field applications. In
October 1996, EPA issued Toxic Compendium Method TO-16 recognizing open-path FTIR
as an ambient air monitoring method.
‫‪)2‬‬
‫دستگاه طيف سنجي جذب اتمي‬
‫‪Atomic Absorption Spectrophotometery‬‬
‫تصاویری از دستگاه ‪AAS‬‬
‫شرح مختصر روش (اساس فيزیکی)‬
‫اساس این روش‪ ،‬میزان جذب پرتو توسط اتمهای عنصر مورد نظر میباشد و میزان پرتوی‬
‫جذب شده متناسب با غلظت عنصر مورد نظر است‪ .‬در این فرآیند ابتدا توسط المپهای‬
‫کاتدی توخالی یا تخلیه الکتریکی تولید پرتو تک رنگ میشود‪.‬از طرفی نمونه مورد‬
‫نظر نیز در حالل خاصی بصورت محلول در آمده و توسط وسیله ای به نام مهپاش به داخل‬
‫شعله تزریق شده و در آنجا بصورت اتم آزاد در می آید‪ ،‬پس از عبور پرتوی تک رنگ‬
‫مقداری از این پرتو توسط این اتم های آزاد جذب میشود و از شدت آن کاسته میگردد‪.‬‬
‫سپس با محاسبه مقدار پرتوی جذب شده توسط آشکارساز و به وسیله منحنی های‬
‫کالیبراسیون میتوان غلظت عنصر مجهول را در محلول محاسبه کرد‪.‬‬
‫کاربردها‪:‬‬
‫کاربرد اصلی این دستگاه آنالیز عنصری میباشد‪ .‬از جمله کاربردهای دیگر آن‬
‫عبارتند از ‪ :‬تعیین سرب در حد ‪PPM‬تراشه فوالدی‪ ،‬سنجش سرب یا كادمیوم در یك قطره‬
‫خون ‪ ،‬سنجش نقره در آب باران مصنوعي‪ ،‬جستجوي ناخالصي ها در آلیاژها و فعال كردن‬
‫واكنشگرها ‪ ،‬آنالیز آب ‪ ،‬آنالیز مستقیم هوا ‪ ،‬آنالیز مستقیم سنگ معدن فلزات و‬
‫فلزهاي تصفیه شده ‪ ،‬سنجش عناصر آلیاژي در فوالد همانند منگنز‪ ،‬منیزیم‪ ،‬كروم‪ ،‬مس‪،‬‬
‫نیكل‪ ،‬مولیبدن‪ ،‬وانادیم‪ ،‬كبالت‪ ،‬تیتانیوم‪ ،‬قلع‪ ،‬آلومینیوم و سرب‪.‬‬
‫اشعه ورودی ‪:‬‬
‫پرتوی ورودی پرتویی تک طول موج با شدت ‪ I0‬میباشد‪.‬‬
‫اشعه خروجی ‪:‬‬
‫پرتویی است که شدت آن کاهش یافته و مقداری از آن توسط اتم های آزاد جذب گردیده‬
‫است‪.‬‬
‫نوع پردازش خروجی ‪:‬‬
‫نوع پردازش خروجی نمودار بوده که میزان نور جذب شده را نشان میدهد‪.‬‬
‫تنوع دستگاهی ‪:‬‬
‫انواع مختلفی از این دستگاه در کشور وجود دارد ولی کال دارای ‪2‬نوع سیستم حرارتی‬
‫بوده که اولی بصورت شعله و دومی بصورت کوره است‪.‬‬
‫نوع ماده ‪:‬‬
‫حدود ‪ 75‬عنصر فلزی و شبه فلزی را دارا‬
‫این نوع آنالیز توانایی آنالیز‬
‫میباشد‪.‬ولی توانایی آنالیز مواد غیر فلزی را بصورت مناسب ندارد‪ .‬مواد به صورت‬
‫محلول مورد آنالیز قرار میگیرند‪.‬‬
‫شکل‪ ،‬مقدار‪/‬اندازه ماده ‪:‬‬
‫بستگي به روش استفاده شده دارد‪ ،‬از یك میلي گرم(جامدهایي كه با طیف سنج جذب‬
‫اتمي گرافیت كوره اي بررسي مي شوند)‪ ،‬تا ‪ 10‬میلي لیتر براي محلول هایي كه با‬
‫كار تابشي معمولي بررسي مي شوند‪.‬‬
‫مدت زمان آزمایش ‪:‬‬
‫وابسته به نوع اتمایزر و روش مورد استفاده از ‪ 5‬دقیقه تا ‪ 4‬یا ‪ 8‬ساعت‪.‬‬
‫دقت ‪:‬‬
‫دقت این روش در حد ‪ ppm‬میباشد و برای برخی عناصر تا غلظت‬
‫‪ 1ppm‬قابل تشخیص است‪.‬‬
‫هزینه دستگاه ‪:‬‬
‫هزینه دستگاه ‪ 250,000,000‬لاير میباشد‪.‬‬
‫هزینه آزمایش ‪:‬‬
‫هر عنصر به وسیله شعله ‪60000‬لاير ‪،‬هر عنصر به وسیله کوره ‪150000‬لاير‪.‬‬
‫آزمایشگاههای دارنده این دستگاه ‪:‬‬
‫پژوهشکده مواد و انرژی‪ ،‬مرکز فرآوری موادمعدنی ایران‪ ،‬سازمان زمین شناسی و‬
‫اکتشافات معدنی ایران ‪ ،‬دانشگاه آزاد میبد‪ ،‬دانشگاه آزاد شهرضا ‪ ،‬دانشگاه صنعتی‬
‫شریف ‪ ،‬دانشگاه باهنر کرمان ‪.‬‬
‫‪)3‬‬
‫اسپکتروفتومتری‬
‫اسپكتروفتومترها‪ ،‬تجهيزاتي است كه جذب يا عبور طول موجهاي مشخصي از انرژي تابشي (نور) از يك آناليت را در‬
‫يك محلول تعيين ميكنند‪ .‬به دليل تفاوت در تعداد و آرايش گروهها‪ ،‬پيوندهاي دوگانه اتمهاي كربن در هر مولكول نور‬
‫را در طول موجهاي خاص با الگوي طيف مشخص‪ ،‬جذب ميكند‪ .‬بر اساس قانون بير ‪ -‬المبرت )‪(Beer - Lambert‬‬
‫‪ ،‬مقدار نوري كه در اين طول موج مشخص جذب ميشود مستقيما ً با غلظت آن نمونه شيميايي متناسب است‪.‬‬
‫اسپكتروفتومترهاي مرئي و فرابنفش‪ ،‬رايجترين دستگاههاي جذب سنجي در مراكز تشخيصي و آزمايشگاهي است‪.‬‬
‫(طیفسنجی نوری يا اسپکتروفتومتری (به انگليسی )‪:( Spectrophotometry‬در شيمی‪ ،‬روشی است برای سنجش و‬
‫مطالعه طيف الکترومغناطيسی ‪.‬در اين روش با استفاده از ميزان اندازه جذب نور نمونهها‪ ،‬غلظت آنها را تعيين میکند‪.‬‬
‫همچنين از آن میتوان برای تجزيه و تحليل نمونههای دیانای و آرانای استفاده نمود ‪.‬‬
‫اين شيوه در دستگاه طيفسنج نوری با نام اسپکتروفوتومتر مورد استفاده قرار میگيرد‪.‬‬
‫اسپكتروفتومتر نور مرئي‬
‫در آزمايشگاهها‪ ،‬بخش گسترده اي از اندازه گيريها بر اساس واكنشهاي جذب سنجي صورت ميپذيرد‪ .‬فعاليت اكثر‬
‫آنزيمها‪ ،‬تري گليسيريد‪ ،‬كلسترول‪ ،‬ليپو پروتئينها‪ ،‬قند‪ ،‬كراتينين‪ ،‬اوره و ‪ . . .‬طيف وسيعي از آناليتها با كاربردهاي‬
‫باليني و تحقيقاتي‪ ،‬طيف وسيعي از داروها و بخش گستردهاي از متابوليتها با اسپكتروفتومتري قابل سنجش است‪.‬‬
‫بررسي ساختمان مولكولي‪ ،‬شناسائي تركيبات‪ ،‬مقايسه ساختمانها‪ ،‬يافتن طول موج ماكزيمم جذب و ‪ . . .‬از ديگر‬
‫كاربردهاي اسپكتروفتومتري در مسائل تحقيقاتي است‬
‫در روشهاي اسپکتروفتومتری (طيف سنجي)‪ ،‬تاثير محلولها بر امواج الكترومغناطيسي مورد مطالعه قرار ميگيرد‪.‬‬
‫محدوده طيف الكترومغناطيس ميتواند از اشعه ماوراء بنفش تا امواج راديويي باشد‪.‬‬
‫محاسبه مي شود‪ .‬طبق ‪ A=e lc‬است و از رابطه ‪ Lambert‬و‪Beer‬مقدار نور جذب شده توسط محلول‪ ،‬تابع قوانين‬
‫قانون بير‪ ،‬هر گاه يک اشعه نور تک رنگ از درون محلولی با رنگ مکمل عبور کند‪ ،‬مقدار نور جذب شده توسط‬
‫محلول‪ ،‬با غلظت آن نسبت مستقيم دارد‪ .‬طبق قانون المبرت‪ ،‬مقدار نور جذب شده توسط اليه های مختلف محلول‬
‫همواره ثابت بوده و با شدت نور تابيده شده بستگی ندارد‪ .‬بر اساس قوانين بير و المبرت رابطه بين غلظت محلول و‬
‫نور جذب شده به صورت خطی است و معموال در محدوده اي كه جذب با غلظت رابطه خطي دارد‪ ،‬تعيين غلظت مواد‬
‫انجام مي شود‪.‬اگر غلظت نمونه و استاندارد به هم نزديك باشد و غلظتها هم در محدوده خطي باشند‪ ،‬مي توان با استفاده‬
‫از تناسب محاسبات را انجام داد‪.‬‬
‫‪:‬معرفی دستگاه‬
‫اجزاء دستگاه‬
‫‪ 6:‬قسمت اصلي در ساختمان اسپكتروفتومترها وجود دارد كه عبارت است از‬
‫)‪(Light Source‬منبع نور‬
‫)‪(Monochromator‬تكفام ساز‬
‫)‪(Focusing Device‬متمركز كننده پرتو‬
‫)‪(Cuvet‬محل نمونه‬
‫)‪(Detector‬آشكارساز‬
‫)‪(Display Device‬دستگاه نمايش خروجي‬
‫)‪(Light Source‬منبع نور‬
‫منبع نور در اثر افزايش حرارت به كمك الكتريسيته در يك المپ تأمين ميشود‪ .‬شرايط اصلي اين منبع‪ ،‬شدت كافي‪،‬‬
‫پايداري و پيوستگي اجزاي آن است‪ .‬براي تامين نور مرئي به منظور اندازهگيري‪ ،‬محلولهاي نسبتا رقيقي كه تغيير در‬
‫‪nm-900‬شدت رنگشان متناسب با تغيير در غلظتشان است‪ ،‬معموال از المپهاي تنگستن (با طول موج توليدي بين‬
‫‪ )330.‬استفاده ميشود‬
‫اين قسمت از دستگاه پرتو چند فام را به پرتو تكفام تبديل ميكند‪ .‬اين عمل ممكن )‪(Monochromator‬تكفام ساز‬
‫است توسط منشور يا سيستم گريتينگ انجام شود‪ .‬فيلترها شيشههاي رنگي است كه بخش وسيعي از پرتوها را جذب‬
‫‪.‬كرده و فقط طول موجهاي محدودي را عبور ميدهد‬
‫)‪(Focusing Device‬متمركز كننده پرتو‬
‫با تركيبي از عدسيها‪ ،‬شكاف بين دو تيغه باريك فلزي و آئينهها در مسير پرتو تابش‪ ،‬پرتوها موازي ميشود و با تنظيم‬
‫عرض شكاف ميتوان عرض پرتو را تنظيم كرد‪ .‬هر چقدر عرض شكاف نور به كار رفته كمتر باشد‪ ،‬كيفيت پرتوها‬
‫‪.‬بهتر خواهد بود‬
‫)‪(Cuvet‬محل نمونه‬
‫كووتها قسمتي از دستگاه است كه نمونه مورد نظر يا بالنك در آن قرار ميگيرد‪ .‬اين بخش معموال به صورت استوانه‬
‫يا مستطيل بوده و از شيشه‪ ،‬كوارتز يا پالستيك ساخته ميشود‪ .‬كووتهاي پالستيكي و شيشهاي براي محدوده مرئي به‬
‫‪.‬كار ميرون‬
‫)‪(Detectors‬آشكارسازها‬
‫دستگاههايي است كه يك نوع از انرژي را به نوع ديگري تبديل ميكند و معموال به سه گروه اصلي تقسيم ميشود ‪-1 :‬‬
‫فتوالكتريك‪ - 2 ،‬فتوشيميايي و ‪ - 3‬حرارتي‪ .‬در دستگاههاي اسپكتروفتومتر از آشكارسازهاي فتوالكتريك استفاده‬
‫ميشود‪ .‬فتوسل و فتو تيوب از سادهترين آشكارسازها است‪ .‬فتو ترانزيستورها و فتوديودها نيز براي اين منظور استفاده‬
‫‪.‬ميشود‬
‫)‪(Display Device‬دستگاه نمايش خروجي‬
‫اين قسمت‪ ،‬ميتواند يك گالوانومتر‪ ،‬صفحه ثبات‪ ،‬اسيلسكوپ يا صفحه نمايشگركامپيوتر با نرم افزارهاي متنوع باشد‪.‬‬
‫)‪ -(Ultraviolet‬اسپكتروفتومتر فرابنفش‬
‫ساختماني همانند اسپكتروفتومتر نور مرئي داشته و به طول موجهاي نور فرابنفش حساس است‪.‬‬
‫)‪(Flame‬اسپكتروفتومتر نشر شعله‬
‫ساختمان اين دستگاه شبيه اسپكتروفتومتر يا فتومتر ساده است‬
‫با اين تفاوت كه در فتومتر‪ ،‬المپ الكتريكي و در اين دستگاه نور حاصل از سوختن ماده مورد آزمايش در درون شعله‬
‫به عنوان منبع نوري در نظر گرفته ميشود‪ .‬در طيف سنجي نشر شعله‪ ،‬نور حاصل مستقيما اندازهگيري ميشود‪.‬‬
‫اسپكتروفتومتر جذب اتمي )‪(Atomic Absorption‬‬
‫اسپكتروفتومترهاي جذب اتمي )‪ (AAS‬غلظت عناصر فلزي كه از نظر پزشكي براي حفظ سالمتي مهم است را اندازه‬
‫گيري ميكند‪ .‬در خصوص اين عناصر ميتوان به كلسيم‪ ،‬منيزيم‪ ،‬مس‪ ،‬روي و آهن اشاره نمود‪.‬‬
‫اسپكتروفتومترهاي جذب اتمي همچنين براي تعيين اينكه آيا سطح درماني داروهايي نظير ليتيم در خون‪ ،‬تامين شده است‬
‫يا خير و همچنين براي آشكارسازي و تعيين كميت سموم فلزي مورد استفاده قرار ميگيرد‪.‬‬
‫نمونه در اثر تابش نور برانگيخته شده و در برگشت به حالت پايه از خود نور نشر می کند که اين خاصيت را‬
‫لومينسانس گويند ‪.‬‬
‫کارايی دستگاه‪:‬‬
‫اين دستگاه جهت شناسايی کيفی و کمی ترکيباتی که قابليت لومينسانس را دارد می باشند به کار می رود‪ .‬از جمله‬
‫ترکيباتی که با اين دستگاه آشکارساز می شوند می توان به پروتئين ها‪ ،‬دلروها‪ ،‬سموم کشاورزی و ‪ ...‬نمونه های‬
‫بيولوژيکی مانند خون و فلزات و ‪ ...‬اشاره کرد‪.‬‬
‫‪)4‬‬
‫عنوان آزمایش ‪ :‬اسپکتروفتومتری‬
‫هدف آزمایش ‪:‬‬
‫‪-1‬‬
‫‪-2‬‬
‫‪-3‬‬
‫آشنایی با دستگاه اسپکتروفتومتر‬
‫تعیین ‪ λmax‬برای کرومات پتاسیم ‪1000ppm‬‬
‫تعیین ضریب جذب مولی‬
‫روش کار ‪ :‬برای انجام این آزمایش محلول هایی را‬
‫با غلظت های مختلف برای کرومات پتاسیم در ‪1000ppm‬‬
‫درست می کنیم و ضریب جذب مولی را بدست می آوریم‪.‬‬
‫ابتدا ‪ 7‬ارلن آماده کردیم و داخل ارلن یک ‪ 1‬سی سی‬
‫و در ارلن شماره دو ‪ 2‬سی سی و به این ترتیب در‬
‫ارلن های شماره سه چهار پنج شش و هفت ‪7-6-5-4-3‬‬
‫سی سی کرومات پتاسیم ریختیم سپس داخل هر کدام از‬
‫ارلن ها ‪ 5‬سی سی سود اضافه کردیم‬
‫و دستگاه اسپکتروفتومتررا با آب مقطر صفر کردیم‪.‬‬
‫وبعد از ارلن شماره یک مقداری را داخل سل ریختیم‬
‫( البته باید محلول را هم بزنیم و سعی کنیم از‬
‫قسمت غلیظتر نمونه را داخل سل بریزیم) و بعد از‬
‫اینکه دستگاه را صفر کردیم سل را داخل دستگاه‬
‫گذاشتیم و دستگاه را روی طول موج ‪ 350‬تنظیم کردیم‬
‫و همین کار را ادامه دادیم به این صورت که تا ‪370‬‬
‫پنج تا پنج تا طول موج را تنظیم میکردیم و از ‪370‬‬
‫تا ‪380‬دوتا دوتا و بعد از این طول موج‪ ,‬تا ‪400‬‬
‫دوباره پنج تا پنج تا پیش رفتیم و‪ λ‬های بدست آمده‬
‫را یادداشت کردیم‪.‬‬
‫‪Α‬‬
‫‪0/101‬‬
‫‪0/142‬‬
‫‪0/175‬‬
‫‪0/207‬‬
‫‪0/223‬‬
‫‪0/228‬‬
‫‪0/232‬‬
‫‪0/237‬‬
‫‪0/229‬‬
‫‪0/226‬‬
‫‪0/206‬‬
‫‪0/187‬‬
‫‪0/144‬‬
‫‪0/111‬‬
‫‪λ‬‬
‫‪350‬‬
‫‪355‬‬
‫‪360‬‬
‫‪365‬‬
‫‪370‬‬
‫‪372‬‬
‫‪374‬‬
‫‪376‬‬
‫‪378‬‬
‫‪380‬‬
‫‪385‬‬
‫‪390‬‬
‫‪395‬‬
‫‪400‬‬
‫روی جدول‬
‫بعد از‬
‫‪ λmax‬را که در طول موج ‪ 376‬است و پررنگتر نشون‬
‫دادم را بدست آوردیم‪.‬‬
‫ تنظیم کرده و‬376 ‫و بقیه محلول ها را در طول موج‬
.‫جذبشان را بدست می آوریم‬
376λ
‫ارلن شماره‬
‫دو‬
‫ارلن شماره‬
‫سه‬
‫ارلن شماره‬
‫چهار‬
‫ارلن شماره‬
‫پنج‬
‫ارلن شماره‬
‫شش‬
‫ارلن شماره‬
‫هفت‬
Α
0/439
0/614
0/788
0/956
1/099
1/174
‫بعد از بدست آوردن این جذب ها می توانیم ضریب جذب‬
‫ بدست آوریم و‬A=ЄbC ‫مولی را بوسیله فرمول‬
. ‫نمودارهای مربوطه را رسم کنیم‬
)5
Spectrophotometry
Objectives:
1. To measure the absorbance of the sample at different wavelengths.
2. To find out the unknown concentration of the sample.
3. Verification of Beer-Lambert's Law.
Theory:
A spectrophotometer is a photometer that can measure the intensity of light as a function of
its wavelength. Single beam and double beam are the two major classes of
spectrophotometers. Linear range of absorption and spectral bandwidth measurement are the
important features of spectrophotometers.
In Single Beam Spectrophotometers, all the light passes through the sample. To measure the
intensity of the incident light the sample must be removed so that all the light can pass
through. This type of spectrometer is usually less expensive and less complicated. The single
beam instruments are optically simpler and more compact, znc can also have a larger
dynamic range.
In a Double Beam Spectrophotometer, before it reaches the sample, the light source is split
into two separate beams. One beam passes through the sample and the second one is used for
reference. This gives an advantage because the reference reading and sample reading can take
place at the same time.
In transmission measurements, the spectrophotometer quantitatively compares the amount of
light passing through the reference and test sample. For reflectance, it compares the amount
of light reflecting from the test and reference sample solutions.
Many spectrophotometers must be calibrated before they start to analyse the sample and the
procedure for calibrating spectrophotometer is known as "zeroing." Calibration is done by
using the reference substance, and the absorbencies of all other substances are measured
relative to the reference substance. % transmissivity (the amount of light transmitted through
the substance relative to the initial substance) is displayed on the spectrophotometer.
The major sequence of events in spectrophotometry is as follows:
1. The light source shines through a monochromator.
2. An output wavelength is selected and beamed at the sample.
3. A fraction of the monochromatic light is transmitted through the sample and to the
photo-detector.
Single Beam Spectrophotometer:
Spectrophotometry deals with visible light, near UV and near IR. To acquire the spectral
information quicker in IR spectrophotometers, which use a Fourier transform technique and
is called Fourier Transform Infrared (FTIR).
Different Types of Spectrophotometers:
A. Single Beam: In this type, all the light passes through the sample .To measure the
intensity of the incident light the sample must be removed so that all the light can pass
through. This type of spectrometer is usually less expensive and less complicated.
B. Double Beam: In this type, before it reaches the sample, the light source is split into two
separate beams. From these one passes through the sample and second one is used for
reference. This gives an advantage because the reference reading and sample reading can take
place at the same time..
C. Visible Light (400-700 nm): Visible spectrophotometers can use incandescent, halogen,
LED, or a combination of these sources and these spectrophotometers vary in accuracy.
Plastic and glass cuvettes can be used for visible light spectroscopy.
D. Ultraviolet Light: UV spectroscopy is used for fluids, and even solids. Cuvettes, only
made of quartz, are used for placing the samples.
E. Infrared Light: IR spectroscopy helps to study different structures of molecules and their
vibrations. Different chemical structures vibrate in different ways due to variation of energy
associated with each wave length. For example, mid-range and near infrared (higher energy)
infrared tends to cause rotational vibrations and harmonic vibrations respectively.
Beer-Lamberts L+9iaw:
Diagram of Beer-Lambert absorption of a beam of light as it travels through a cuvette of
width .
Beer-Lambert’s law is the linear relationship between the absorbance and concentration of
the absorbing sample, i.e. a logarithmic relation exist between the transmission of light
through a substance ( ) and the product of absorption coefficient of a substance (
)
and distance travelled by the light through the material(path length ) The absorption
coefficient is the product of molar absorptivity, the concentration the material, or an
absorption cross section,
, and the (number) density,
Where,
is the molar absorptivity of the absorber,
the material and
is the concentration of the absorbing species in
is the density (number) of adsorbers.
For liquids, these relations are usually written as:
Whereas for gases, these relations are written as:
where,
=intensity of the incident light
=intensity of the transmitted light
=cross section of light absorption by a single particle
=density of absorbing particles
The transmission (or transmissivity) for liquids in terms of absorbance, is defined as:
The relationship between absorbance ( ) and percent transmittance (
and this can be written as:
) is also quantitative
Percent transmittance is
Whereas, for gases, it is usually defined as:
The above equation shows that the absorbance becomes linear relationship with the
concentration according to:
and
Thus, the absorbance is measured, if the path length and the molar absorptivity are known
and the concentration of the substance can be deduced.
According to the Beer-Lambert Law, absorbance is proportional to concentration, so that at
dilute solutions a plot of concentration vs. absorbance would be straight line, but the Law
breaks down for solutions of higher concentration, and so you might get a curve under those
circumstances.
Applications of a Spectrophotometer:
1. It is directly used to measure light intensity at different wavelengths.
2. It is used to determine the unknown concentration of solution.
3. Spectrometers can be used to determine the equilibrium constant of a reaction involving
ions.
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