1. Analytical methods Vladimíra Kvasnicová

2. 1.SPECTROPHOTOMETRY 2.CHROMATOGRAPHY 3.POTENTIOMETRY 4.VOLUMETRIC ANALYSIS

3. Spectrophotometry spectrophotometer

4. Material used for the analysis: SOLUTION

5. PRINCIPLE interaction between a compound of interest and a monochromatic radiation a part of the radiation is absorbed by the compound and a rest of the radiation is detected by a detector quantity of the absorbed radiation is directly proportional to the quantity of the compound

6. The spectrophotometry is a quantitative method: CONCENTRATION of a solution is analyzed  concentration  darker colour   absorption

7. Important terms sample = solution used for the analysis unknown sample = sample of unknown concentrat. standard = sample of known concentration blank = solution free of compound of interest chromophore = part of a structure of the compound related to the absorption of a radiation of certain wavelength

8. violet380 – 450 nm blue450 – 495 nm green495 – 570 nm yellow570 – 590 nm orange590 – 620 nm red620 – 750 nm see http://en.wikipedia.org/wiki/Electromagnetic_spectrum

9. The figure was found at http://en.wikipedia.org/wiki/Electromagnetic_spectrum (2006)http://en.wikipedia.org/wiki/Electromagnetic_spectrum

10. Used radiation colour sample: VIS light colourless sample: UV radiation

11. A / „absorption spectrum“

12. Complementary colours

13. SCHEME of the instrument

14. What quantity is measured? TRANSMITTANCE = the ratio of intenzity (I) of a radiation passed through the sample to the intenzity (I o ) of the radiation entering the sample T = I / I o T = 0 – 1 or it is expressed in % (0 – 100 %)

15. How the quantity of absorption is expressed? New quantity is defined: ABSORBANCE A = - log 10 T = - log 10 (I/I 0 ) = log 10 (I 0 /I) = log 10 (1/T) A = 0 – 1.0 (1.5 or more) the upper limit is determined by detector sensitivity

16. Tpassed (%)absorbed (%)A 110000 0.999910.004 0.9090100.05 0.5050 0.3 0.1010901.0 0.011992.0 0.0010.199.93.0 0.00010.0199.994.0

17. Tpassed (%)absorbed (%)A 110000 0.999910.004 0.9090100.05 0.5050 0.3 0.1010901.0 0.011992.0 0.0010.199.93.0 0.00010.0199.994.0  detector senzitivity

18. Calculation of concentration: 1.Beer-Lambert´s law 2.Calibration curve 3.Calculation based on values of standard solutions

19. Calculation of concentration: Beer-Lambert´s law A =  x l x c or T = 10 - (  x l x c) A = absorbance (A = -log T) T = transmittance (T = 10 -A )  = molar absorption coefficient l = thickness of cuvette (in cm), c = molar concentration

20. Calibration curve 3 or more standards processed by the same method linear calibration curve A =  x l x c y = kx + q

21. Calculation using standards A st = c st x l x  A us = c us x l x  A st / c st = l x  A us / c us = l x  l x  = l x  A st / c st = A us / c us c us = A us x (c st / A st ) c us = A us x f f = average of all (c st / A st ) used in the experiment

22. Exercises 1) A u = 0,25C u = ? A s = 0,40C s = 4mg / L 2) 1000mg/L glucose standard (C s ) reads T = 0,49. T of unknown sample is 0,55. What is glucose concentration of unknown sample? (in mg/L and mmol/L) MW = 180g 3) Protein standard: T = 0,33; patient’s sample: T = 0,44 Compare the patient’s protein concentration with the standard

23. Accuracy of the determination absorption by other substances found in the solution must be eliminated BLANK sample is used → its absorbance must be subtracted from the absorbance of unknown sample  final absorbance (= result) is related solely to the compound of interest

24. Spectrophotometry in the practical training „Determination of urine creatinine“ analysed sample: own urine 1.colorless creatinine is transformed to a colour compound by chemical reaction 2.absorbance of the compound is used to establish creatinine concentration using a calibration curve

25. Chromatography chromatograph

26. Not all chromatography techniques are instrumental... TLC chromatography = task of the practical training

27. PRINCIPLE Seperation of a mixture of solutes is based on a differential distribution of the solutes between two immiscible phases: stationary phase (solid or liquid) mobile phase (liquid or gase) The mobile phase carries solutes through the stationary phase with different velocities according to their mutual affinity.

28. if the „affinity“ of a substance to the mobile phase is high, the substance moves faster than a substance having lower affinity if the „affinity“ of a substance to the stationary phase is high, the substance is retarded in the phase and moves more slowly than a substance having lower affinity

29. The figure was found at http://www.chemistry.vt.edu/chem-ed/sep/lc/lc.html (November 2006)http://www.chemistry.vt.edu/chem-ed/sep/lc/lc.html

31. What is the aim of the analysis? 1.to separate solutes one from the other 2.to identify the solutes (= qualitative analysis) 3.to determine their concentrations (= quantitative analysis)

32. Classification of chromatographic techniques 1)by the mobile phase  Liquid Chromatography (LC)  Gas Chromatography (GC) 2)by the arrangement  Flat (Plane) Chromatography  Column Chromatography

33. Liquid Column „manual“ chromatography

34. Liquid Column „instrumental“ chromatography

35. Liquid Plane Chromatography example:

36. Gas Chromatography (GC) The figure was found at http://www.cofc.edu/~kinard/221LCHEM/ (November 2006)http://www.cofc.edu/~kinard/221LCHEM/

37. 3)by physicochemical interactions  Adsorption Chromatography  Partition Chromatography  Gel Permeation Chromatography (GPC)  Ion Exchange Chromatography (IONEX)  Affinity Chromatography

38. Physicochemical mechanisms of separation adsorptiondissolving sieving efect - gel permeation ion exchange complementary interactions „affinity“ Adopted from presentation: analyticke_metody / Petr Tůma

39. The figure was found at http://fig.cox.miami.edu/~cmallery/255/255tech/255techniques.htm (November 2006)http://fig.cox.miami.edu/~cmallery/255/255tech/255techniques.htm

40. The figure was found at http://fig.cox.miami.edu/~cmallery/255/255tech/255techniques.htm (November 2006)http://fig.cox.miami.edu/~cmallery/255/255tech/255techniques.htm

41. The figure was found at http://fig.cox.miami.edu/~cmallery/255/255tech/255techniques.htm (November 2006)http://fig.cox.miami.edu/~cmallery/255/255tech/255techniques.htm

42. Evaluation of chromatogram Spots are compared with standards: R f = a /b R f = retardation factor or „rate of flow“ a = start to center of spot b = start to solvent flow 1) Plane Chromatopgraphy (TLC) The figure was found at http://sms.kaist.ac.kr/~jhkwak/gc/catofp/chromato/tlc/tlc.htm (November 2006)http://sms.kaist.ac.kr/~jhkwak/gc/catofp/chromato/tlc/tlc.htm

43. 2) Column Chromatography (HPLC, GC) Peaks are compared with standards: t R = retention time  identification of solutes h = height of the peaks  concentration of solutes

44. Chromatography in the practical training „ TLC of fat-soluble dyes“ adsorption plane liquid chromatography mobile phase: toluene (nonpolar) stationary phase: plate of silica gel (polar) stadards of dyes → comparison of R f unknown sample: composed of 2 unknown dyes

45. „Show of HPLC and GC - a visit of the analytical laboratory“ HPLC = High Performance Liquid Chromatography (or High Pressure LC) normal or reversed phase HPLC GC = Gas Chromatography

46. Scheme of HPLC Mobile phase Degasser Pump Sample injection Column Detector Waste

47. Potentiometry potentiometer

48. PRINCIPLE Potentiometry is an electrochemical method based on the measurement of voltage of an electrochemical cell when no current flows. two electrodes: working (indicating) electrode reference electrode

49. Scheme:

50. The electrodes working electrode its potential is influenced by composition of a solution reference electrode its potential is stable (constant, known) It is impossible to measure one potential  potential difference (= voltage) is measured

51. working electrodes The figure was found at http://food.oregonstate.edu/images/ph/beck8.jpg (2006)http://food.oregonstate.edu/images/ph/beck8.jpg

52. Nernst equation E = E 0 + (RT/nF) ln a M E = electrode potential E 0 = standard electrode potential R = gas constant (8.314 J K -1 mol -1 ) F = Faraday´s constant (96 458 C mol -1 ) T = absolute temperature (25 0 C = 298 K) n = oxidative number of ion of interest (M) a = activity of ion of interest

53. E = E 0 + (RT/nF) ln a M ln a = 2.303 log a; R, T, and F values used  E = E 0 + (0.059/n) log a M ! REMEBER ! electrode potential is dependent on temperature, activity, and charge of a compound of interest! you will not calculate the potential: standards are used to calibrate potentiometer

54. General classification of electrodes 1)I. type (metal or gas electrodes) 2)II. type (metal + insoluble salt) → REFERENCE ELECTRODES 3)redox electrodes (Pt, Au) 4)membrane electrodes → ISE = Ion Selective Electrodes (determination of ions in medicine H +, Na +, K +, Cl -,...)

55. Standard hydrogen electrode (SHE) gas electrode its potential is used as a standard: E SHE = 0 under all conditions REFERENCE ELECTRODE but not in a practise

56. Reference electrodes calomel el.argent chloride el. SHE

57. „Glass electrode“ ISE (H + ) pH determination membrane electrode

58. Gass-sensing membrane electrode

59. analyzed sample gas permeable membrane glass electrode

60. Potentiometry in the practical training „ Measuring pH of phosphate buffer“ various solutions of phosphate buffer pH determination by pH-meter calibration of the instrument by standards glass combination electrode („twin“)

61. Glass combination electrode The figure was found at http://www.ph-meter.info/img/combination-electrode.png (October 2007)http://www.ph-meter.info/img/combination-electrode.png

63. Volumetric analysis (= titration)

64. The method is based on a chemical reaction between a solute of interest and a titrimetric reagent burette: titrimetric reagent titrimetric flask: diluted sample of a solute of interest titration = determination of exact concentration

65. PRINCIPLE A titrimetric reagent of known concentration is slowly added from the burette into the titrimetric flask containing a sample until a stoichiometric ratio of the reactans is reached (= point of equivalence) point of equivalence = reactants are present in a stoichiometric ratio given by the chemical equation describing the reaction used for the analyse

66. Titrimetric reagent (R) known, stable composition its concentration can be exactly determined by a primary stadard of known concentration it reacts quickly the reaction can be described by known chemical equation at the point of equivalence a detectable physico-chemical change proceeds

67. Determination of exact concentration of the titrimetric reagent (R) primary standard is used as a sample in the flask a theoretical (calculated) consumption of R is compared with an actual (analysed) consumption: V t / V a = f f = factor of a titrimetric reagent (0,900 – 1,100) actual concentration of R (= titr): c a = f x c t the factor is used for conversion of a theoretical value of R to its actual conc. used for analyses

68. Calculation of sample concentration based on knowledge of a stoichiometry of chemical reaction a A + b B → c C + d D a, b, c, d = stoichiometric coefficients = substance amounts (n) A = „titrimetric reagent“, B = analysed sample a / b = n(A) / n(B)

69. c = n / V→n = c x V c = molar concentration (mol/l) n = substance amount (mol) V = volume of a solution a, b = stoichiometric coefficients a x n(B) = b x n(A) a x c B x V B = b x c A x V A

70. stoichiometry of the reaction is known concentration and consumed volume of the titrimetric reagent at a point of equivalence is known sample volume used for the analyse is known the only unknown value is cBcB

71. Exercises 1) titrimetric reagent: 23,8 ml NaOH, (factor = 0,9685; C = 0,1M), sample = 10ml H 2 SO 4 ; C = ? 2) titrimetric reagent: 10ml KMnO 4 (0,1M), sample: 20ml FeSO 4 ; C = ? (mol/ L, % ), MW = 152g 3) H 3 PO 4 → Na 2 HPO 4 sample: 20ml H 3 PO 4 (C = 0,3M ), titrimetric solution: 0,2M NaOH V = ?

72. Titration is made by one person: „drop by drop addition of a titrimetric reagent under continual mixing of reactants in a flask“

74. Indication of point of equivalence 1)by an indicator  simple but subjective evaluation  equivalence point  total volume of R added when permanent colour change of a solution is observed  „the first excess“ of R is indicated  the solution is „overtitrated“ 2)by an instrument (e.g. by potentiometer)  objective  TITRATION CURVE is evaluated

75. Titration curve sample: acid / titrimetric reagent: base titrimetric reagent measured value indicators

76. sample: base / titrimetric reagent: acid titrimetric reagent

77. Classification of volumetric analyses 1)neutralization (acid-base titration)R: acid /base H + + OH - → H 2 O 2)oxidation-reduction (redox) R: ox./red. reagent oxidation: red → ox + e - reduction: ox → red + e - 3)precipitation titration R: e.g. AgNO 3 formation of an insoluble salt 4)complexometric titration R: e.g. EDTA formation of a stable complex

78. Titration in the practical training „Determination of acidity of gastric juice“ analyte: HCl found in gastric juice titrimetric reagent: NaOH → neutralization titration (= alcalimetry) indicator: phenolphtaleine (colourless → violet) c(HCl) → calculation of pH of gastric juice pH before and after a stimulation of the stomach is determinated

79. Instructions for the labs + theory of the methods: http://www.lf3.cuni.cz/chemie/ see Study/ Practical trainings