FLAME ATOMIC ABSORPTION SPECTROSCOPY Introduction Flame atomic absorption is a very common technique for detecting metals and

metalloids in environmental samples. It is very reliable and simple to use. Figure 1 shows which elements are commonly detected through atomic absorption. The technique is based on the fact that ground state metals absorb light at specific wavelengths. Metal ions in a solution are converted to atomic state by means of a flame. Light of the appropriate wavelength is supplied and the amount of light absorbed can be measured against a standard curve.

Figure 1. Elements detectable by atomic absorption are highlighted in pin in this periodic table. History The history of spectroscopy starts with the use of the lens by !ristophanes about "#$ %.&.' and the studies of mirrors by Euclid ($)) %.&.* and +ero (1)) %.&.*. ,eneca (") !.-.* observed the light scattering properties of prisms. and in 1)) !.-. /tolemy studied incidence and refraction. !lha0en in 1)$1 studied reflection and refraction of light. and in 1#2) 3oger %acon determined the focal points of concave mirrors. !round 14)). the telescope was developed in +olland and by 141). 5alileo had made improvements on the telescope design. ,ir Isaac 6ewton (14"#718#8* performed many e9periments on the separation of light to obtain a spectrum and the indices of refraction of different colors of light' he applied those principles to the telescope. Fraunhofer. about 111"712. observed diffraction phenomena and was able to measure wavelength instead of angles of refraction. +erschel (11#$* and Talbot (11#2* discovered atomic emission when certain atoms were placed in a flame. :heatstone concluded in 11$2 that metals could be distinguished from one another on basis on the wavelengths of this emission. In 11"1.

Foucault observed atomic emission from sodium and discovered that the element would absorb the same rays from an electric arc. In the later 11)). scientists such as ;irchoff. %unsen. Michelson and %almer studied the composition of the sun based on their emissions at different wavelengths. ;irchoff summari0ed the law which states that. <Matter absorbs light at the same wavelength at which it emits light<. It is under this law that atomic absorption spectroscopy wor s. :oodson was one of the first to apply this principle to the detection of mercury. In 1=22. :alsh suggested the use of cathode lamps to provide an emission of appropriate wavelength' and the use of a flame to produce neutral atoms that would absorb the emission as they crossed its path. Instrumentation and applications for atomic absorption greatly e9panded after the 1=2)s. Principle The technique of flame atomic absorption spectroscopy (F!!,* requires a liquid sample to be aspirated. aerosoli0ed. and mi9ed with combustible gases. such as acetylene and air or acetylene and nitrous o9ide. The mi9ture is ignited in a flame whose temperature ranges from #1)) to #1)) >&. -uring combustion. atoms of the element of interest in the sample are reduced to free. une9cited ground state atoms. which absorb light at characteristic wavelengths. as shown in figure #. The characteristic wavelengths are element specific and accurate to ).)17).1nm. To provide element specific wavelengths. a light beam from a lamp whose cathode is made of the element being determined is passed through the flame. ! device such as photon multiplier can detect the amount of reduction of the light intensity due to absorption by the analyte. and this can be directly related to the amount of the element in the sample.

Figure #. ?peration principle of an atomic absorption spectrometer. The %eer7Lambert law (also called the %eer7Lambert7%ouguer law or simply %eer@s law* is the linear relationship between absorbance and concentration of an absorber of electromagnetic radiation. The general %eer7Lambert law is usually written asA

where ! is the measured absorbance. a is a wavelength7dependent absorptivity coefficient. b is the path length. and c is the analyte concentration. :hen wor ing in concentration units of molarity. the %eer7Lambert law is written asA

where B is the wavelength7dependent molar absorptivity coefficient with units of M71 cm71. The C subscript is often dropped with the understanding that a value for is for a specific wavelength. If multiple species that absorb light at a given wavelength are present in a sample. the total absorbance at that wavelength is the sum due to all absorbersA

where the subscripts refer to the molar absorptivity and concentration of the different absorbing species that are present Parts and Functions Figure " shows an atomic absorption spectrometer. This instrument in particular is designed to operate either with a flame or with a graphite furnace. The graphite furnace is additionally equipped with an auto sampler.

Flame atomic absorption hardware is divided into si9 fundamental groups that have two maDor functionsA generating atomic signals and signal processing. ,ignal processing is a growing additional feature to be integrated or e9ternally fitted to the instrument. The instrument parts are shown in figure ".

Figure ". schematic of basic instrumental parts of atomic absorption spectrometer ! cathode lamp (1* is a stable light source. which is necessary to emit the sharp characteristic spectrum of the element to be determined. ! different cathode lamp is needed for each element. although there are some lamps that can be used to determine three or four different elements if the cathode contains all of them. Each time a lamp is changed. proper alignment is needed in order to get as much light as possible through the flame. where the analyte is being atomi0ed. and into the monochromator. The atom cell (#*. shown in figure 4. is the part with two maDor functionsA nebuli0ation of sample solution into a fine aerosol solution. and dissociation of the analyte elements into free gaseous ground state form. 6ot all the analyte goes through the flame. part of it is disposed as shown in the figure. !s the sample passes through the flame. the beam of light passes through it into the monochromator ($*. The monochromator isolates the specific spectrum line emitted by the light source through spectral dispersion. and focuses it upon a photomultiplier detector ("*. whose function is to convert the light signal into an electrical signal. The processing of electrical signal is fulfilled by a signal amplifier (2*. The signal could be displayed for readout (4*. or further fed into a data station (8* for printout by the requested format. Operations o Fla!e !* Types of flame -ifferent flames can be achieved using different mi9tures of gases. depending on the desired temperature and burning velocity. ,ome elements can only be converted to atoms at high temperatures. Even at high temperatures. if e9cess o9ygen is present. some metals form o9ides that do not redissociate into atoms. To inhibit their formation. conditions of the flame may be modified to achieve a reducing. nono9idi0ing flame. Table 1 shows the characteristics of various flames.

Table 1. &haracteristics of different flames ,ource 3eynolds et al.. 1=8). %* Eltrasonic 6ebuli0ation /roper nebuli0ation is required to brea up an aqueous sample into a fine mist of uniform droplet si0e that can be readily burned in the flame. Most instruments utili0e the direct aspiration. -uring aspiration. the gas flow brea s down the liquid sample into droplets. and the nebuli0ation performance depends on the physical characteristics of the liquid. ?nly about 1)F of the sample gets into the flame. !nother option for nebuli0ation is the use of an ultrasonic wave beam. which generates high frequency waves in the liquid sample. This causes very small liquid particles to be eDected into a gas current forming a dense fog. &* ,lotted Tube !tom Trap This device is a heated quart0 tube that can be placed in a conventional flame. !s the dissociated ground state atoms pass into the tube. they are delayed and stay longer in the optical path. increasing the sensitivity of the instrument. Cali"ration !s with other analytical techniques. atomic absorption spectrometry requires careful calibration. E/! G!HG& demands calibration through several steps. including interference chec sample. calibration verification. calibration standards. bland control. and linear dynamic range. The ideali0ed calibration or standard curve is stated by %eer@s law that the absorbance of an absorbing analyte is proportional to its concentration.

Figure 8. Ideali0edHdeviation response curve Enfortunately. deviations from linearity usually occur. especially as the concentration of metallic analyte increases due to various reasons. such as unabsorbed radiation. stray light. or disproportionate decomposition of molecules at high concentrations. Figure 8 shows an ideali0ed and deviation of response curve. The curvature could be minimi0ed. although it is impossible to be avoided completely. It is desirable to wor in the linearity response range. The rule of thumb is that a minimum of five standards and a blan should be prepared in order to have sufficient information to fit the standard curve appropriately. Manufacturers should be consulted if a manual curvature correction function is available for a specific instrument. If the sample concentration is too high to permit accurate analysis in linearity response range. there are three alternatives that may help bring the absorbance into the optimum wor ing rangeA 1* sample dilution #* using an alternative wavelength having a lower absorptivity $* reducing the path length by rotating the burner hand.

HI#H PERFORMANCE LI$%I& CHROMATO#RAPHY Introduction +igh7performance liquid chromatography (or +igh pressure liquid chromatography. +/L&* is a form of column chromatography used frequently in biochemistry and analytical chemistry to separate. identify. and quantify compounds. +/L& utili0es a column that holds chromatographic pac ing material (stationary phase*. a pump that moves the mobile phase(s* through the column. and a detector that shows the retention times of the molecules. 3etention time varies depending on the interactions between the stationary phase. the molecules being analy0ed. and the solvent(s* used. History Liquid chromatography was defined in the early 1=))Is by the wor of the 3ussian botanist. Mi hail ,. Tswett. +is pioneering studies focused on separating compounds Jleaf pigmentsK. e9tracted from plants using a solvent. in a column pac ed with particles. Tswett filled an open glass column with particles. Two specific materials that he found useful were powdered chal Jcalcium carbonateK and alumina. +e poured his sample Jsolvent e9tract of homogeni0ed plant leavesK into the column and allowed it to pass into the particle bed. This was followed by pure solvent. !s the sample passed down through the column by gravity. different colored bands could be seen separating because some components were moving faster than others. +e related these separated. different7colored bands to the different compounds that were originally contained in the sample. +e had created an analytical separation of these compounds based on the differing strength of each compoundIs chemical attraction to the particles. The compounds that were more strongly attracted to the particles slowed down. while other compounds more strongly attracted to the solvent moved faster. This process can be described as followsA the compounds contained in the sample distribute. or partition differently between the moving solvent. called the mobile phase. and the particles. called the stationary phase. This causes each compound to move at a different speed. thus creating a separation of the compounds. Tswett coined the name chromatography Jfrom the 5ree words chroma. meaning color. and graph. meaning writingLliterally. color writingK to describe his colorful e9periment. J&uriously. the 3ussian name Tswett means color.K Today. liquid chromatography. in its various forms. has become one of the most powerful tools in analytical chemistry. Principle +/L& separates mi9ture of compounds on the basis of polarity. M/olarity refers toA the greater the difference in electron affinity i.e. electronegativity between atoms in a covalent bond. the more polar the bond. partial negative charges are found on the most electronegative atoms. the others are partially positive. The molecular electrostatic potential energy of a hydrogen ion at a particular location near a molecule. 6egative electrostatic

potential corresponds to A partial negative charges . /ositive electrostatic potential corresponds to A partial positive charges.N It is used to analy0e. identify. purify and quantify compounds. It has a mobile phase. a stationary phase. and detector. The mobile phase is continuously pumped at a fi9ed flow rate through the system and mi9ed by the pump. The inDector is used to introduce a plug of a sample into the mobile phase without having to stop the mobile phase flow. O without introducing air into the system. The mi9ture of components is carried in a narrow band to the top of the column. ,ome compounds in the sample mi9ture will have greater preference for stationary phase than the mobile phase and will be retained in the column longer. Those components that are not retained as strongly as are carried by the mobile phase down the column. The detector is then used to respond to a physico7chemical property of analyte. this response is digitally amplified and sent to a data system where it is recorded as a @chromatogram@. Operation The sample to be analy0ed is introduced in small volume to the stream of mobile phase. The analyte@s motion through the column is slowed by specific chemical or physical interactions with the stationary phase as it traverses the length of the column. The amount of retardation depends on the nature of the analyte. stationary phase and mobile phase composition. The time at which a specific analyte elutes (comes out of the end of the column* is called the retention time' the retention time under particular conditions is considered a reasonably unique identifying characteristic of a given analyte. The use of smaller particle si0e column pac ing (which creates higher bac pressure* increases the linear velocity giving the components less time to diffuse within the column. leading to improved resolution in the resulting chromatogram. &ommon solvents used include any miscible combination of water or various organic liquids (the most common are methanol and acetonitrile*. :ater may contain buffers or salts to assist in the separation of the analyte components. or compounds such as trifluoroacetic acid which acts as an ion pairing agent. ! further refinement to +/L& has been to vary the mobile phase composition during the analysis' this is nown as gradient elution. ! normal gradient for reversed phase chromatography might start at 2F methanol and progress linearly to 2)F methanol over #2 minutes' the gradient chosen depends on how hydrophobic the analyte is. The gradient separates the analyte mi9tures as a function of the affinity of the analyte for the current mobile phase composition relative to the stationary phase. This partitioning process is similar to that which occurs during a liquid7 liquid e9traction but is continuous. not step7wise. In this e9ample. using a waterHmethanol gradient. the more hydrophobic components will elute (come off the column* when the mobile phase consists mostly of methanol (giving a relatively hydrophobic mobile phase*. The more hydrophilic compounds will elute under conditions of relatively low methanolHhigh water. The choice of solvents. additives and gradient depend on the nature of the stationary phase and the analyte. ?ften a series of tests are performed on the analyte and a number of trial runs may be processed in order to find the +/L& method which gives the best separation of pea s.

Co!ponents The basic components of an +/L& system include a solvent reservoir. pump. inDector. analytical column. detector. recorder and waste reservoir. ?ther important elements are an inlet solvent filter. post7pump inline filter. sample filter. precolumn filter. guard column. bac 7 pressure regulator andHor solvent sparging system. The function of each of these components is briefly described below.

!n +/L& system begins with the solvent reservoir. which contains the solvent used to carry the sample through the system. The solvent should be filtered with an inlet solvent filter to remove any particles that could potentially damage the system@s sensitive components. ,olvent is propelled through the system by the pump. This often includes internal pump seals. which slowly brea down over time. !s these seals brea down and release particles into the flow path. an inline solvent filter prevents any post7pump component damage. The ne9t component in the system is the sample inDector. also nown as the inDection valve. This valve. equipped with a sample loop of the appropriate si0e for the analysis being performed. allows for the reproducible introduction of sample into the flow path. %ecause the sample often contains particulate matter. it is important to utili0e either a sample filter or a precolumn filter to prevent valve and column damage. Following the inDector. an analytical column allows the primary sample separation to occur. This is based on the differential attraction of the sample components for the solvent and the pac ing material within the column. +owever. a sacrificial guard column is often included Dust prior to the analytical column to chemically remove components of the sample that would otherwise foul the main column.

Following the analytical column. the separated components pass through a detector flow cell before they pass into the waste reservoir. The sample components@ presence in the flow cell prompts an electrical response from the detector. which is digiti0ed and sent to a recorder. The recorder helps analy0e and interpret the data. !s a final system enhancement. a bac pressure regulator is often installed immediately after the detector. This device prevents solvent bubble formation until the solvent is completely through the detector. This is important because bubbles in a flow cell can interfere with the detection of sample components. !lternatively. an inert gas sparging system may be installed to force dissolved gasses out of the solvent being stored in the solvent reservoir. Trou"les'ootin( Lea s

/ressure

,ensitivity

6oise

Para!eters Internal diameter The internal diameter (I-* of an +/L& column is an important parameter that influences the detection sensitivity and separation selectivity in gradient elution. It also

determines the quantity of analyte that can be loaded onto the column. Larger columns are usually seen in industrial applications. such as the purification of a drug product for later use. Low7I- columns have improved sensitivity and lower solvent consumption at the e9pense of loading capacity. Larger I- columns (over 1) mm* are used to purify usable amounts of material because of their large loading capacity. !nalytical scale columns (".4 mm* have been the most common type of columns. though smaller columns are rapidly gaining in popularity. They are used in traditional quantitative analysis of samples and often use a EP7Pis absorbance detector. 6arrow7bore columns (17# mm* are used for applications when more sensitivity is desired either with special EP7vis detectors. fluorescence detection or with other detection methods li e liquid chromatography7mass spectrometry &apillary columns (under ).$ mm* are used almost e9clusively with alternative detection means such as mass spectrometry. They are usually made from fused silica capillaries. rather than the stainless steel tubing that larger columns employ.

/article si0e Most traditional +/L& is performed with the stationary phase attached to the outside of small spherical silica particles (very small beads*. These particles come in a variety of si0es with 2 Qm beads being the most common. ,maller particles generally provide more surface area and better separations. but the pressure required for optimum linear velocity increases by the inverse of the particle diameter squared. This means that changing to particles that are half as big. eeping the si0e of the column the same. will double the performance. but increase the required pressure by a factor of four. Larger particles are used in preparative +/L& (column diameters 2 cm up to R$) cm* and for non7+/L& applications such as solid7phase e9traction. /ore si0e Many stationary phases are porous to provide greater surface area. ,mall pores provide greater surface area while larger pore si0e has better inetics. especially for larger analyte. For e9ample. a protein which is only slightly smaller than a pore might enter the pore but does not easily leave once inside. /ump pressure /umps vary in pressure capacity. but their performance is measured on their ability to yield a consistent and reproducible flow rate. /ressure may reach as high as ") M/a (4))) lbfHin#*. or about ")) atmospheres. Modern +/L& systems have been improved to wor at much higher pressures. and therefore are able to use much smaller particle si0es in the columns (S# Qm*. These <Eltra +igh /erformance Liquid &hromatography< systems or E+/L&s can wor at up to 1)) M/a (12.))) lbfHinT*. or about 1))) atmospheres. The term <E/L&<. though sometimes used is a trademar of :aters &orporation and not the name for the technique in general.

BATAN#AS STATE %NI)ERSITY &ollege of Engineering. !rchitecture. Fine !rts and &omputing ,ciences &epart!ent o Electronics * Co!!unications+ Instru!entation * Control and Mec'atronics En(ineerin(

I&E "2# (!nalytical Instrumentation*

Hi(' Per or!ance Li,uid C'ro!ato(rap'y Fla!e Ato!ic A"sorption Spectroscopy

,ubmitted byA 53?E/ " Alday+ -e rey Al!eron+ Ar.in -ed Baylosis+ Allen Mic'ael &alan(in+ -o!ar -arlos+ /edric0 Mendo1a+ El.is %,I&E U "1)1

,ubmitted toA En(r2 Aries A2 Arce(a Instructor