INTRODUCTION Spectroscopy is based on the absorption of photons by the analyte while ultraviolet spectroscopy is about a method to quantify the

light that is absorbed and scattered by a sample. The spectrometric method shows the absorption of electromagnetic radiation by the sample solution from an appropriate source. It occurs when the sample is placed between the photo detector and the light source. The intensity of a beam of light is measured before and after passing through the sample and the amount of the absorption of electromagnetic radiation can be related to the concentration of the analyte in the solution. Radiation is one of the type of energy transfers that is considered in terms of a wave motion and its presence can be detected by the ability to feel radian heat and also by the sense of the sight. The figure below shows the wave motion resulted from radiation:

Figure 1.1: the wavelength of electromagnetic radiation

The full electromagnetic radiation spectrum is continuous and it commonly treated as a wave phenomenon, characterized by frequency or wavelength. The range of this electromagnetic spectrum is based on the wavelength whereas from very short wavelengths, gamma and x-rays to very long wavelengths, microwaves (Reusch, 2013). One of the examples of electromagnetic radiation is visible light.

The wavelength is defined as the distance between two adjacent crests while the frequency is the number of the crest (peak) passing in a given period of time and it is given in cycles per second or hertz (Hz). The wavelength of the light in the ultraviolet and visible regions is expressed in nanometers, nm (10-9 meters). The working ranges of the UV-VIS are 200-380 nm for UV and 380-780 nm for Vis.

Figure 1.2: visible spectrum in electromagnetic spectrum

From the figure 1.2, it shows that the energy is directly proportional to the frequency but inversely proportional to the wavelength. The energy associated with the various kinds of radiation increases as the frequency increases and vice versa for the wavelength. There are many instrument designs for molecular UV/Vis absorption such as filter photometer, single-beam spectrophotometer, double-beam spectrophotometer and many others. Filter photometer is a simplest instrument for molecular UV/Vis absorption. It uses an absorption or interference filter to isolate a band of radiation. The filter is placed between the source and the sample to prevent the sample from decomposing when exposed to higher energy radiation. The single-beam spectrophotometer is a simplest spectrophotometer equipped with a fixed-wavelength monochromatic for wavelength selection. The limitations of single-beam spectrophotometers such as fixed-wavelength are minimized by using a doublebeam spectrophotometer (Davis).

THEORY Radiation which is a form of energy and considered in terms of wave motion could be felt by our sense and ability to feel radiant. It shows the relationship between the wavelength, frequency and velocity of light as below: Equation 2.1 Where: = wavelength in cm C = velocity of light (31010 cm/s) V = frequency in s-1 of Hz

Equation 2.1 shows that the wavelength is inversely proportional to the frequency. As the frequency becomes higher, the wavelength becomes shorter and causes the increase of energy level of the photon. The amount of energy called photon is possessed by the electromagnetic radiation and it also related to the frequency and wavelength. Equation 2.2 as below show the relationship between the amount of energy, frequency and wavelength.

Equation 2.2 Where: E = energy of photon h = Planks constant, 6.62 10-34 Joules-sec

Hence, light with the higher frequency should be absorbed to obtain the higher energy because of the higher the frequency, the higher the energy (Clark, 2007). The matters or organic compounds absorb radiation to raise the molecule to the higher energy level in the three basic processes. There are rotational transition, vibrational transition and electron transition. Rotational transition is about the molecules rotates about various axes and cause the energy of rotational become a definite energy level. The molecules can be raised to a higher energy rotational energy level as the molecules absorb the radiation. While the vibrational transition shows the definite energy level is contributed as the atoms or groups of atoms within the molecules vibrate relative to each other. These molecules absorb a discrete amount of energy and cause the rise of the vibrational energy level. Besides, the electron of a molecule may be raised to higher electron energy level is known as electron transition. Electrons in a molecule can be divided into four groups that are closed shell electron, covalent single bond electron, pair nonbonding outer shell electron and electron in orbitals. The closed shell electron not involved in the bonding while the pair nonbonding outer shell electron such as halogens are less tightly held and the electron can be excited by visible or UV radiation. The covalent single bond electron is called sigma bond, and the covalent double and triple bonds are called pi bond, The electrons in double and triple bonds are most readily excited and responsible for majority of electronic spectra in UV-VIS regions. A molecule also possesses unoccupied orbital called antibonding orbitals that correspond to excited-state energy levels. The absorption of radiation will contribute to the electronic transition to antibonding orbitals. The absorption of UV or visible radiation involves the excitation of the outer or valence electron. There are three types of electronic transition that are transitions involving , and n electrons, transition involving charge-transfer electrons and transitions involving d and f electrons (UV-Vis Absorption Spectroscopy). The electrons are promoted to an excited state from their ground state when an atom or molecule absorbs energy. These atoms will rotate and vibrate each other and cause the contribution of discrete energy levels.

Figure 2.1: Possible electronic transitions of , , and n electrons * transitions require large amount of energy for an electron in a bonding s orbital to be excited to the corresponding antibonding orbital. For

n* transitions that needs less

energy than * transitions is capable for saturated compounds containing lone pairs. n* and * transitions are influenced in the most absorption spectroscopy of organic compounds. These transitions need an unsaturated group in the molecule to provide electrons. While for the transition involving charge-transfer electrons, it shows the absorption of radiation that involving the transfer of an electron from the donor to an orbital associated with the acceptor (UV-Vis Absorption Spectroscopy). The absorbing groups in a molecule are called chromophores which is a functional group while the molecules containing a chromophore are named as chromogen. The chromophore exhibits a characteristic absorption spectrum in the ultraviolet or visible region. The presence of the auxochrome will enhance the absorption by a chromophore or shift the wavelength of absorption. The actual effects of an auxochrome on a chromophore depend on the polarity of the auxochrome. Besides, the spectra changes can be divides into four groups. The first group is bathochromic shift (red shift) and the second group is hypsochromic shift (blue shift) is both resulted from the removal of conjugation. The maximum wavelength shifts to longer wavelength for bathochromic shift and shorter wavelength for hypsochromic. The third and fourth group are hyperchromic effect that increase the absorption intensity and hypochromic that decrease the absorption intensity respectively.

Many molecules absorb visible light or it is called ultraviolet. Different molecules absorb radiation with different wavelengths. The absorbance of a solution increases as attenuation of the beam increases. Absorbance is directly proportional to the path length, b, and the concentration, c, of the absorbing species (Beer's Law). Beer's Law states as below: Equation 2.3

Equation 2.4

Equation 2.5

Where: A = absorbance = molar absorbtivity (L mol-1 cm-1) b = path length of sample c = concentration of compound in solution (mol L-1) k = constant T = transmittance I = intensity of light Besides, the other important thing is choice of solvent. The considerations that should be taken are cheap, good solvent transparent down to about 210 nm and the solvent that most commonly used is ethanol.

REFERENCES Beer's Law. (n.d.). Retrieved from http://teaching.shu.ac.uk/hwb/chemistry/tutorials/molspec/beers1.htm Clark, J. (2007). uv-visible absorption spectra. Retrieved from http://www.chemguide.co.uk/analysis/uvvisible/theory.html Davis. 10C UV/Vis and IR Spectroscopy. UCDAVIS CHEMWiki. Retrieved from: http://chemwiki.ucdavis.edu/Analytical_Chemistry/Analytical_Chemistry_2.0/10_Spectroscopic _Methods/10C_UV%2F%2FVis_and_IR_Spectroscopy Reusch, W. (2013). Visible and Ultraviolet Spectroscopy. Retrieved from: http://www2.chemistry.msu.edu/faculty/reusch/VirtTxtJml/Spectrpy/UV-Vis/spectrum.htm Theoretical principles. UV-Vis Absorption Spectroscopy. (n.d.). Retrieved from: http://teaching.shu.ac.uk/hwb/chemistry/tutorials/molspec/uvvisab1.htm Official Methods of Analysis of AOAC International, 17th edition, Current through Revision No. 2, 2003.

SCHEMATIC DIAGRAM

Diagram 1

A diagram of the components of a typical spectrometer is shown in the following diagram 1. Instruments for measuring the absorption of U.V. or visible radiation are made up of the following components; 1. Sources (UV and visible) 2. Wavelength selector (monochromatic) 3. Sample containers 4. Detector Sources of visible radiation The tungsten filament lamp is commonly employed as a source of visible light. This type of lamp is used in the wavelength range of 350 - 2500 nm. The energy emitted by a tungsten filament lamp is proportional to the fourth power of the operating voltage. This means that for the energy output to be stable, the voltage to the lamp must be very stable indeed. Electronic voltage regulators or constant-voltage transformers are used to ensure this stability.

Wavelength selector All wavelength selectors contain the following component parts;

An entrance slit A dispersing device (usually a prism or a grating) An exit slit

Cuvettes The containers for the sample and reference solution must be transparent to the radiation which will pass through them. Quartz or fused silica cuvettes are required for spectroscopy in the UV region. These cells are also transparent in the visible region. Silicate glasses can be used for the manufacture of cuvettes for use between 350 and 2000 nm. Detectors The photomultiplier tube is a commonly used detector in UV-Vis spectroscopy. It consists of a photo emissive cathode (a cathode which emits electrons when struck by photons of radiation), several dynodes (which emit several electrons for each electron striking them) and an anode. A photon of radiation entering the tube strikes the cathode, causing the emission of several electrons. These electrons are accelerated towards the first dynode (which is 90V more positive than the cathode). The electrons strike the first dynode, causing the emission of several electrons for each incident electron. These electrons are then accelerated towards the second dynode, to produce more electrons which are accelerated towards dynode three and so on. Eventually, the electrons are collected at the anode. By this time, each original photon has produced 106 - 107 electrons. The resulting current is amplified and measured. Photomultipliers are very sensitive to UV and visible radiation. They have fast response times. Intense light damages photomultipliers, they are limited to measuring low power radiation.

SAMPLE OF ANALYSIS NITRATE DETERMINATION IN ABSORBANCE MEASUREMENTS Apparatus UV-visible absorption spectra were obtained with a UV-VIS spectrophotometer Model 530, using 1.00 cm quartz cells. For all samples, the spectra were recorded between 190 and 500 nm with an interval of 1 nm (311 data points per spectrum), saved in ASCII format and transferred to PC Pentium 4 for subsequent manipulation. The reference procedure for nitrate determination in Caliche samples was adapted from the AOAC Official Method 892.01 for nitrogen determination in fertilizers. These analyses were carried out on a Gerhardt distillation system model Vapodest 6 with automated analysis of 12 samples. The nitrogen concentrations are reported as percentage of sodium nitrate (% NaNO3). Reagents All experiments were performed with analytical-reagent. The solutions of boric acid and sodium hydroxide, both 0.1 mol L-1, were prepared on a daily basis for nitrate quantitation. CHILEAN CALICHE SAMPLES BY UV-VISIBLE

EXPERIMENTAL The nitrate ion presents two UV absorption bands in water: a broad and very intense band at 220 nm, and a less intense one at 301 nm. Figure 1a shows the spectra for the entire set of 70 Caliche samples, where nitrate signals are identified. From the inspection of this Figure and the zoom window Figure 1b, between 250 and 350 nm, it is apparent that some samples present a strong overlapping of the band ascribed to nitrate ion and at least one spectral interference, whose absorption maximum is near 294 nm. According to our knowledge of the samples, this spectral interference appears to correspond to either sulfur- or iodine-containing species. Principal component analysis of these UV spectra (in the 250-350 nm regions) shows that two components explain. The corresponding Principal component analysis loadings and scores are present in Figures 2a and 2b respectively. As can be seen, most samples are grouped along the dashed line which is shown in the score-score plot of Figure 2b. For these particular samples, the spectral interference is minimal, and the univariate UV method provides nitrate concentrations which are comparable to those obtained by the reference Devarda method.

A group of samples, however, appear outside this region, some of them presenting significant contributions from the first PC score. On the other hand, the first PC loading shows a maximum at 294 nm Figure 2b, a wavelength corresponding to the spectral interference described above. For these samples, the univariatc method yields nitrate concentrations which are significantly higher than the reference methodology.