Analytical methods. Vladimíra Kvasnicová

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1 Analytical methods Vladimíra Kvasnicová

3 Laboratory analysis analyte = compound of interest 1. Qualitative analysis ~ WHAT IS IT? = determining the nature of a pure unknown compound(s) in a mixture 2. Quantitative analysis ~ WHAT IS ITS CONCENTRATION? = measuring the proportions of known components in a mixture

4 Biochemical laboratory: automatic analyzer (Oct 2007)

Classification of analytical methods Spectral methods absorption spectrometry (UV/VIS) mass spectrometry Separation methods chromatography LC

5 Classification of analytical methods Spectral methods absorption spectrometry (UV/VIS) mass spectrometry Separation methods chromatography LC Liquid Chromatography GC Gas Chromatography electrophoresis Gel Electrophoresis Capillary Electrophoresis Electrochemical methods potentiometry

6 Biochemical practicals (labs) WHERE? chemical laboratory (4 th floor, room 419) WHEN? teaching weeks: 2, 3, 4, 5! white laboratory coat is required! for instructions and all other you need for practicals go to see entries: 104, 105, 106, 107

7 Spectrophotometry spectrophotometer

8 Material used for the analysis: SOLUTION

9 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

10 The spectrophotometry is a quantitative method: CONCENTRATION of a solution is analyzed concentration darker colour absorption

11 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

12 violet blue green yellow orange red nm nm nm nm nm nm see

13 Used radiation colour sample: colourless sample: VIS light UV radiation

14 A / λ absorption spectrum

15 Complementary colours

16 SCHEME of the instrument

17 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 %)

18 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 = (1.5 or more) the upper limit is determined by detector sensitivity

19 T passed (%) absorbed (%) A detector senzitivity

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

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

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

23 Calculation using standards A st = c st x l x ε A st / c st = l x ε A us = c us x l x ε A us / c us = l x ε l x ε = l x ε 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

24 Exercises 1) A u = 0,25 C u =? A s = 0,40 C s = 4mg / L [2,5mg/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 [839mg/L = 4,7mM ] 3) Protein standard: T = 0,33; patient s sample: T = 0,44 Compare the patient s protein concentration with the standard [4/3]

25 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

Fluorescence physico-chemical effect 1. excitation of a molecule to higher energy state 2. emission of light after 10-9 to 10-6 sec 3. its wavelegth is higher, energy is lower fluorophores 1.

26 Fluorescence physico-chemical effect 1. excitation of a molecule to higher energy state 2. emission of light after 10-9 to 10-6 sec 3. its wavelegth is higher, energy is lower fluorophores 1. natural NADH, vitamin A, amino acids (Phe, Trp, Tyr) 2. fluorescence markers for proteins and DNA, e.g. fluorescein, rhodamine, ethidium bromide Energy levels Carcinom cells nucleus tubulin actin

27 Mass spectrometry measuring of masses of individual molecules molecule is ionized to molecular ion (M + ) by electron beam ionized particle turns in a magnetic field radius of its trajectory is related to mass / charge ratio

28 Mass spectrum molecular ion

29 Chromatography chromatograph

30 Not all chromatography techniques are instrumental... TLC chromatography (Thin Layer Chromatography)

31 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.

32 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

The figure was found at http://www.chemistry.

33 The figure was found at (November 2006)

35 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)

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

37 Liquid Column manual chromatography

38 Liquid Column instrumental chromatography

39 example: Liquid Plane Chromatography

40 Gas Chromatography (GC) The figure was found at (November 2006)

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

42 Physicochemical mechanisms of separation adsorption dissolving sieving efect - gel permeation ion exchange Adopted from presentation: analyticke_metody / Petr Tůma complementary interactions affinity

The figure was found at http://fig.cox.miami.

43 The figure was found at (November 2006)

44 The figure was found at (November 2006)

45 The figure was found at (November 2006)

46 Evaluation of chromatogram 1) Plane Chromatopgraphy (TLC) 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 The figure was found at (November 2006)

47 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

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

49 Electrophoresis

50 Electrophoresis = an analytical method based on movement of charged particles because of an external electric field velocity of a particle depends on the: a) size, shape and charge of the particle b) given applied voltage

51 Electrophoresis anion - negatively charged ion, it moves to the anode (+) cation - positively charged ion, it moves to the catode (-) amphoteric - a substance that can have a positive, zero, or negative charge, depending on conditions (e.g. proteins)

52 Classification of electrophoretic techniques 1. free-boundary electrophoresis separation is carried out entirely in a liquid phase, i.e. no support is used (capillary electrophoresis) 2. electrophoresis in a supporting medium paper, gel (agarose, polyacrylamide) it can be done horizontally or vertically

53 Capillary electrophoresis The figure was found at (Feb 2008)

54 Capillary electrophoresis - SCHEME The figure was found at (Feb 2008)

Gel electrophoresis - horizontal The figure was found at http://www.

55 Gel electrophoresis - horizontal The figure was found at (Feb 2008)

56 Gel electrophoresis - vertical SDS-PAGE animation The figure was found at (Feb 2008)

57 Effects of electrophoretic parameters on separation ph changes charge of analyte and hence its mobility, it can affect structure of analyte (denaturing, dissociating) ionic strength changes voltage or current: increased ion. str. usually reduces migration velocity and increases heating temperature: overheating can denaturate (precipitate) proteins; lower t. reduces diffusion but also reduces migration velocity, no effect on resolution current: too high current causes overheating voltage: migration velocity is proportional to voltage time: resolution (separation of bands) increases linearly with time, but dilution of bands (diffusion) increases with the square root of time medium: major factors are endosmosis and pore-size effects, which affect migration velocities

58 Process of electrophoresis 1. sample application 2. adjustment of voltage or current - DIRECT CURRENT! (gel-electrophoresis about volts, capillary electrophoresis about 20,000 volts) 3. separation time: minutes (e.g. gel-electrophoresis of serum proteins 30 min.) 4. electrophoresis in supporting medium: fixation, staining 5. evaluation: qualitative (standards) quantitative (densitometry)

59 Equipment used for the gel electrophoresis of serum proteins power suply (direct current) electrophoresis chamber containers for staining and destaining gel applicator

60 Electrophoresis examples from clinical medicine separation of serum proteins, isoenzymes, nucleic acids immunoelectrophoresi s (immunoglobulins) The figure was found at (Feb 2008)

61 Electrophoresis examples from clinical medicine separation of serum proteins, isoenzymes, nucleic acids immunoelectrophoresi s (immunoglobulins) The figure was found at (Feb 2008)

Evaluation by densitometry - peaks 60% 3% 9% 12% 16% The figures are from http://www.sebia-usa.

62 Evaluation by densitometry - peaks 60% 3% 9% 12% 16% The figures are from and respectivelly (Feb 2007)

63 AMPHOLYTES amino acids, proteins ph-dependent electrical charge H H H H2C C COOH H2C C COO Isoelectric focusing pk 1 = 2,34 pk 2 = 9,69 NH 3 + NH 3 + cation zwitterion anion isoelectric point: pi = (pk 1 +pk 2 )/2 proteins differing by pi 0,01 can be separated H C C COO 2 NH 2

64 Potentiometry potentiometer

65 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

66 Scheme:

67 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

68 working electrodes The figure was found at (2006)

69 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 ( 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

70 E = E 0 + (RT/nF) ln a M ln a = 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

71 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 -,...)

72 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

73 Reference electrodes calomel el. argent chloride el. SHE

74 Glass electrode ISE (H + ) membrane electrode ph determination

75 Gass-sensing membrane electrode

76 tělo sensoru vnitřní elektrolyt skleněná glass elektroda electrode ph-metr referentní elektroda skleněná glass elektroda electrode CO +H O HCO +H+ film elytu permeabilní membrána permeabilní gas membrána permeable membrane analyzed analyzované sample prostředí CO 2 (g) analyzované prostředí

77 Glass combination electrode The figure was found at (October 2007)

79 Biochemical practicals (labs) WHERE? chemical laboratory (4 th floor, room 419) WHEN? teaching weeks: 2, 3, 4, 5! white laboratory coat is required! for instructions and all other you need for practicals go to see entries: 104, 105, 106, 107

Introduction to biochemical practicals. Vladimíra Kvasnicová

Introduction to biochemical practicals Vladimíra Kvasnicová arrangement of practicals laboratory safety regulations laboratory equipment dealing with automatic pipette instructions: http://vyuka.lf3.cuni.cz/