Uv vis spectroscopy practical.


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Uv vis spectroscopy practical.

  1. 1. [SALUM MKATA B.PHARM 3] 2014 1 DATE: May 28, 2014. ULTRA-VIOLET VISIBLE SPECTROSCOPY PRACTICAL REPORT: Aim: To apply the Beer-Lambert relationship to an aqueous solution containing an absorbing substance and thus determine its respective concentrations. INTRODUCTION AND THEORY: Ultraviolet–visible spectroscopy or ultraviolet-visible spectrophotometry (UV- Vis or UV/Vis) refers to absorption spectroscopy or reflectance spectroscopy in the ultraviolet-visible spectral region (about 190-820nm).The Ultraviolet-visible absorption based on molecules containing π-electrons or non-bonding electrons (n- electrons) which can absorb the energy in the form of ultraviolet or visible light to excite these electrons to higher anti-bonding molecular orbitals. The more easily excited the electrons (i.e. lower energy gap between the HOMO and the LUMO), the longer the wavelength of light it can absorb. UV/Vis spectroscopy is routinely used in analytical chemistry for the quantitative and qualitative determination of different analytes, such as transition metal ions, highly conjugated organic compounds, and biological macromolecules. Spectroscopic analysis is commonly carried out in solutions but solids and gases may also be studied The ultraviolet region (about 400-190) is particularly important for the qualitative and quantitative determination of many Organic compounds, especially those with a high degree of conjugation, also absorb light in the UV or visible regions of the electromagnetic spectrum. The solvents for these determinations are often water for water-soluble compounds, or ethanol for organic-soluble compounds. While in the visible region (about 400-820nm), spectrophotometry methods are widely used for the quantitative determination of many trace substances, especially inorganic species. The basic principle of quantitative absorption spectroscopy lies in comparing the extent of absorption of a sample solution with that of a set of standards under radiation of a selected wavelength through the application of Beer-Lambert law. The Beer-Lambert law states that the absorbance of a solution is directly proportional to the concentration of the absorbing species in the solution and the path length. Thus, for a fixed path length, UV/Vis spectroscopy can be used to determine
  2. 2. [SALUM MKATA B.PHARM 3] 2014 2 the concentration of the absorber in a solution. It is necessary to know how quickly the absorbance changes with concentration. Equation: A = Where: A= absorbance P= power of transmitted PO =power of incident ϵ= extinction coefficient (mol-1 dm3 cm-1 ) b= path length of cell (cm) c= concentration of absorbing species. Note: In many cases, the sample compound does not absorb radiation appreciably in the wavelength provided, it’s then necessary to form an absorbing substance by reacting a compound in question with other reagents. What a special about Ultraviolet-visible spectrometer: The instrument used in ultraviolet-visible spectroscopy is called a UV/Vis spectrophotometer. It measures the intensity of light passing through a sample ( ), and compares it to the intensity of light before it passes through the sample ( ). The ratio is called the transmittance, and is usually expressed as a percentage (%T). The absorbance, , is based on the transmittance: The UV-visible spectrophotometer can also be configured to measure reflectance. In this case, the spectrophotometer measures the intensity of light reflected from a sample ( ), and compares it to the intensity of light reflected from a reference
  3. 3. [SALUM MKATA B.PHARM 3] 2014 3 material ( ) (such as a white tile). The ratio is called the reflectance, and is usually expressed as a percentage (%R). The basic parts of a spectrophotometer are; a light source, a holder for the sample, a diffraction grating in a monochromator or a prism to separate the different wavelengths of light, and a detector. The radiation source is often a Tungsten filament (300-2500 nm), a deuterium arc lamp, which is continuous over the ultraviolet region (190-400 nm), Xenon arc lamp, which is continuous from 160- 2,000 nm; or more recently, light emitting diodes (LED) for the visible wavelengths. The detector is typically a photomultiplier tube, a photodiode, a photodiode array or a charge-coupled device (CCD). Single photodiode detectors and photomultiplier tubes are used with scanning monochromators, which filter the light so that only light of a single wavelength reaches the detector at one time. The scanning monochromator moves the diffraction grating to "step-through" each wavelength so that its intensity may be measured as a function of wavelength. Fixed monochromators are used with CCDs and photodiode arrays. As both of these devices consist of many detectors grouped into one or two dimensional arrays, they are able to collect light of different wavelengths on different pixels or groups of pixels simultaneously. Figure.1
  4. 4. [SALUM MKATA B.PHARM 3] 2014 4 APPARATUS AND CHEMICAL USED Apparatus: 1) Spectrophotometer 2) Cuvettes 3) Measuring flask (10mls.) 4)1mls and 10mls capacity pipettes 5) Pipette filler 6) Storage bottle Figure.2 spectrophotometer. 7) Digital mass balance. 8) Test tube rack 9) Beaker. 10) Stopper 11) Stop watch Figure.3 Digital mass balance Chemical/ Reagents: 1) Salicylic acid (0.1%) 2) Acetate buffer (0.05M) 3) Distil water
  5. 5. [SALUM MKATA B.PHARM 3] 2014 5 PROCEDURES: 1) We turn on the spectrophotometer and allow it to warm up for at least 20 min. Then we determine the absorption spectrum using the standard acetate buffer. 2) We select one of the cuvettes for the blank solution (in this case acetate buffer) and we do not interchange it with the other cuvettes also we did not handle the lower portion of cuvettes through which the light passes. 3) We always rinse the cuvettes with several portions of solution by using acetate buffer before taking a measurement. 4) Then we wipe the outside of cuvettes with tissue paper. 5) Then we inserted the cuvette into the cell holder with the index line facing us to avoiding scratching. 6) Then we turn the wavelength control knob to 265nm with blank solution calibrate the spectrophotometer. A. Preparation of the solution: 1) We prepared a stock solution of 0.1% salicylic acid by: accurately measuring 100mg of salicylic acid by digital mass balance then we dissolved the obtained mass in 100ml of acetate buffer in beaker. 2) From that stock solution above we prepared serial dilution containing 0.05%, 0.025%, 0.01%, 0.005%, 0.0025, and 0.001%.(Note all percentages are in weight by volume ). We apply dilution law of same substance as: C1 V1=C2V2 Where: M1=concentration of concentrated (stock) solution. V1= volume of concentrated (stock) solution.
  6. 6. [SALUM MKATA B.PHARM 3] 2014 6 M2= concentration of diluted solution. V2= volume of diluted solution. Note: V2 will be equal to 10ml because we have to diluting the stock solution by adding sufficient amount of acetate buffer to make a solution of 10ml in measuring flask. Also C1 and C2 known then V1 obtained by following formula 3) Then we labeled test tube from 1 up to 6. After obtained required volume of stock solution to be taken from beaker containing stock solution of salicylic acid. Then we add sufficient amount of acetate buffer to make 10ml solution results summarized in following table. TABLE.1; S/N 1 2 3 4 5 6 Concentration (%w/v) 0.05 0.025 0.01 0.005 0.0025 0.001 Volume of stock solution need(ml) 5.00 2.50 1.00 0.50 0.25 0.10 Volume of diluted solution(ml) 10 10 10 10 10 10 4) Then we prepared (standard blank) in which 20ml of acetate buffer is substituted for the sample. B. Determination of absorbance 1) We set blank solution in the cuvette and calibrate at wavelength at wavelength control of 296 nm. 2) Then we inserted cuvette containing the sample- then we read and recorded the absorbance for all solutions in above section A. 3) Then we determine the absorbance of unknown sample.
  7. 7. [SALUM MKATA B.PHARM 3] 2014 7 Results obtained as follows NOTE: since we have to represents concentration in MOLARITY then: Concentration in Molarity= Concentration in g/dm3 Molar mass in g/mol Concentration in g/dm3 : Since Y g in 1000 cm3 X g in 100 cm3 Since X is mass of sample in above table then mass Y which is in 1000cm3 can be obtain. But molar mass of salicylic acid= 138g/mol. Then we change those concentrations in w/v% in Table 1 above into Molarity Then we obtain Table below. TABLE .2; TRIAL CONCENTRATION(MOL/LITRE) ABSORBANCE 1 7.24 x 10-5 0.007 2 1.81x 10-4 0.003 3 3.62x 10-4 0.007 4 7.24x10-4 0.011 5 1.81x10-3 0.015 6 3.62x10-3 0.018 Unknown Z 0.042
  8. 8. [SALUM MKATA B.PHARM 3] 2014 8 DISCUSSION: We plot the graph of Absorbance vs. concentration in Excel sheet as shown below: ANSWERS TO GIVEN QUESTIONS AND SOURCES OF ERROR: 1) Yes, it can, if an appropriate mathematical model can be fitted to the data obtained from standards. For example the older versions of the Sedex Evaporative Light-Scattering Detector (ELSD) (image below) gave quadratic response to most analytes, so one had to use a quadratic fit for the standards, and calculating the concentration of unknowns required solution of by the quadratic formula. Figure.4 y = 3.7129x + 0.006 R² = 0.8294 0 0.005 0.01 0.015 0.02 0.025 0 0.0005 0.001 0.0015 0.002 0.0025 0.003 0.0035 0.004 Absorbance concentration (mol/litre) ABSORBANCE VS. CONCENTRATION (MOLE/LITRE)
  9. 9. [SALUM MKATA B.PHARM 3] 2014 9 In NIR spectroscopy, non-linear fit methods such as Principal Component Analysis and Multiple Linear Regression are often used because the sample response may not strictly obey Beer's law, especially for reflectance spectra. 2) There no any added advantage of using choosing a wavelength at absorbance plateau other than the maximum since: It really depends on what is the largest source of error. Taking the readings at the peak maximum is best at low absorbances because it gives the best signal- to-noise ratio, which improves the precision of measurement. If the dominant source of noise is photon noise, the precision of absorbance measurement is theoretically best when the absorbance is near 1.0. So if the peak absorbance is below 1.0, then using the peak wavelength is best, but if the peak absorbance is well above 1.0, you might be better off using another wavelength where the absorbance is closer to 1. Another issue is calibration curve non-linearity, which can result in curve-fitting errors. The non-linearity caused by polychromatic light is minimized if you take readings at either a peak maximum or a minimum, because the absorbance change with wavelength is the smallest at those wavelengths. On the other hand, using the maximum increases the calibration curve non-linearity caused by stray light. But, Very high absorbances cause two problems:  The precision of measurement is poor because the transmitted intensity is so low, and the calibration curve linearity is poor due to stray light. The effect of stray light can be reduced by taking the readings at a wavelength where the absorbance is lower or by using a non-linear calibration curve fitting technique. Finally,  If spectral interferences are a problem, the best measurement wavelength may be the one that minimizes the relative contribution of spectral interferences (which may or may not be the peak maximum). In any case, don't forget: whatever wavelength you use, you have to use the exact same wavelength for all the standards and samples.
  10. 10. [SALUM MKATA B.PHARM 3] 2014 10 3) Cuvettes may be circular, square or rectangular (the latter being uncommon), and must be constructed of a material that will transmit both the incident and emitted light. Square cuvettes or cells will be found to be most precise since the parameters of path length and parallelism are easier to maintain during manufacture. However, round cuvettes are suitable for many more routine applications and have the advantage of being less expensive. Figure 5.Examples of sample cells for UV/Vis spectroscopy. From left to right (with path lengths in parentheses): rectangular plastic cuvette (10.0 mm), rectangular quartz cuvette (5.000 mm), rectangular quartz cuvette (1.000 mm), cylindrical quartz cuvette (10.00 mm), cylindrical quartz cuvette (100.0 mm). 4) The Beer-Lambert Law will not be obeyed if  The photons of light striking the detector do not all have an equal chance of absorption by the sample. This can happen if they have different absorption coefficients, different path lengths through the sample, or if they encounter different concentrations of sample molecules. Also  If anything else is present in the sample that absorbs light or causes light scattering, the measured absorbance will not be zero when the analyte's concentration is zero, contrary to Beer's Law.  If the absorber undergoes any type of chemical reaction or equilibrium that varies as a function of concentration, Beer's Law will not be obeyed with respect to the overall or total concentration, because the
  11. 11. [SALUM MKATA B.PHARM 3] 2014 11 concentration of the actual absorbing molecule is not proportional to the overall concentration of the solution. The "c" in Beer's Law refers to the concentration of just the absorber, not to the total concentration of all the compounds reacting with or in equilibrium with the absorber. Even if Beer's Law holds exactly for each individual compound, the total absorbance of the mixture will not follow Beer's Law with respect to the total concentration if the proportion of each compound changes with concentration (unless by chance the absorptivity of all those compounds happens to be exactly the same).  Deviations in absorptivity coefficients at high concentrations (> 0.01M) due to electrostatic interactions. Changes in refractive index at high analyte concentration.  Fluorescence or phosphorescence of the sample and Non- monochromatic radiation. 5) From the graph we obtain the equation below: Y = 3.7129X + 0.006 Hence the unknown concentration can be obtained since: Y=value from y-axis i.e. the absorbance X=value from x-axis i.e. the concentration in Mol/litre But absorbance (Y value) of unknown sample is obtain which is equal to 0.042 Then we can find the value of unknown concentration! Take 0.042=3.7129X+ 0.006 Then 0.042-0.006=3.7129X 0.036=3.7129X Then divide by 3.7129 both side to obtain X=9.696x 10-3 There fore Unknown concentration in mol/litre of solution is equal to 9.696x 10-3
  12. 12. [SALUM MKATA B.PHARM 3] 2014 12 6) The molar extinction coefficient of SA From below equation A Where: A= absorbance ϵ= extinction coefficient (mol-1 dm3 cm-1 ) b= path length of cell (cm) c= concentration of absorbing species. Take A1 =0.007 and A2=0.003 Also C1= 3.62x 10-4 and C2=1.81x 10-4 b =1cm A1=ϵ1c1b and A2=ϵ2C2b 0.007= ϵ1 x 3.62x 10-4 x1cm …………………………………….eqn.1 0.003= ϵ2x1.81x 10-4 x1cm…………………………………………eqn.2 Then ϵ1=19.33 ϵ2=16.57 Then molar absorbivity of SA Will be 19.33+16.57=35.9 35.9/2=17.95moll-1 cm-1
  13. 13. [SALUM MKATA B.PHARM 3] 2014 13 Source of errors: 1) Stray Light: A problem when working at limits of a spectrometer’s range. 2) Cells and Solvents: Everything else except the sample should be as transparent as possible. 3) Sample Preparation: If two samples are prepared so that one carries along a greater concentration of insoluble particulates, then additional scattering will lead to an apparent greater absorption. 4) Slit Width Affects Absorbance Measurements: If a significant variation in absorptivity occurs over the spectral bandwidth admitted by the slit, a non- linear variation (non-Beer’s Law) with concentration will be observed. This arises because the spectrometer measures the average transmissivity over the spectral bandwidth, but transmissivity and concentration are not linearly related. Keep slit width large to increase S/N ratio, but must keep it small enough to maintain a linear relationship with concentration changes. This effect is minimized if the absorptivity changes slowly with wavelength. Select a wavelength near a peak maximum. Use a slit width to provide a bandwidth about 1/10 the spectral feature width. 5) Wavelength error: In liquids, the extinction coefficient usually changes slowly with wavelength. A peak of the absorbance curve (a wavelength where the absorbance reaches a maximum) is where the rate of change in absorbance with wavelength is smallest. Measurements are usually made at a peak to minimize errors produced by errors in wavelength in the instrument, that is errors due to having a different extinction coefficient than assumed. CONCLUSION, ACKNOWLEDGEMENT AND REFERENCES: CONCLUSION: From above we can conclude that UV/Vis spectroscopy is best method which routinely used in analytical chemistry for the quantitative determination of different analytes, such as transition metal ions, highly conjugated organic compounds, and biological macromolecules.
  14. 14. [SALUM MKATA B.PHARM 3] 2014 14 Spectroscopic analysis is commonly carried out in solutions but solids and gases may also be studied. A UV/Vis spectrophotometer may be used as a detector for HPLC. The presence of an analyte gives a response assumed to be proportional to the concentration. For accurate results, the instrument's response to the analyte in the unknown should be compared with the response to a standard; this is very similar to the use of calibration curves. The response (e.g., peak height) for a particular concentration is known as the response factor. ACKNWOLEDGEMENT: 1. TO MR. EDSON-LAB. TECHNICIAN 2. TO Dr. E. KAALE. 3. TO Dr. SEMPOMBE-HEAD OF DEPARTMENT OF MEDCINAL CHEMISTRY 4. TO MY FELLOW GROUP MEMBER AND THE REST OF CLASS. REFERENCES: 1) http://en.wikipedia.org/wiki/Molar_extinction_coefficients 2) http://www.thestudentroom.co.uk/showthread.php?t=1424595 3) https://answers.yahoo.com/question/index?qid=20130721231123AABslPR 4) http://chemwiki.ucdavis.edu/Organic_Chemistry/Organic_Chemistry_Wit h_a_Biological_Emphasis/Chapter__4%3A_Structure_Determination_I/S ection_4.3%3A_Ultraviolet_and_visible_spectroscopy 5) http://en.wikipedia.org/wiki/Cuvette 6) Practical protocol by Department of Medicinal chemistry-School of Pharmacy, MUHAS.