Analytical methods. Vladimíra Kvasnicová

Analytical methods Vladimíra Kvasnicová

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

Biochemical laboratory: automatic analyzer http://success.shoreline.edu/mlt/debhitachi9172.jpg (Oct 2007)

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

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 http://vyuka.lf3.cuni.cz/ see entries: 104, 105, 106, 107

Spectrophotometry spectrophotometer

Material used for the analysis: SOLUTION

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

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

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

violet blue green yellow orange red 380 450 nm 450 495 nm 495 570 nm 570 590 nm 590 620 nm 620 750 nm see http://en.wikipedia.org/wiki/electromagnetic_spectrum

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

A / λ absorption spectrum

Complementary colours

SCHEME of the instrument

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 %)

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

T passed (%) absorbed (%) A 1 100 0 0 0.99 99 1 0.004 0.90 90 10 0.05 0.50 50 50 0.3 0.10 10 90 1.0 0.01 1 99 2.0 0.001 0.1 99.9 3.0 0.0001 0.01 99.99 4.0 detector senzitivity

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

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

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

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

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]

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. 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

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

Mass spectrum molecular ion

Chromatography chromatograph

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

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.

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.vt.edu/chem-ed/sep/lc/lc.html (November 2006)

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)

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

Liquid Column manual chromatography

Liquid Column instrumental chromatography

example: Liquid Plane Chromatography

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

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

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.edu/~cmallery/255/255tech/255techniques.htm (November 2006)

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 http://sms.kaist.ac.kr/~jhkwak/gc/catofp/chromato/tlc/tlc.htm (November 2006)

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

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

Electrophoresis

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

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)

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

Capillary electrophoresis The figure was found at http://www.hood.edu/images/content/academics/instruments/agilent_capillary_electrophoresis_system.jpg (Feb 2008)

Capillary electrophoresis - SCHEME The figure was found at http://en.wikipedia.org/wiki/capillary_electrophoresis (Feb 2008)

Gel electrophoresis - horizontal The figure was found at http://www.mun.ca/biology/desmid/brian/biol2250/week_three/electro4.jpg (Feb 2008)

Gel electrophoresis - vertical SDS-PAGE animation The figure was found at http://fig.cox.miami.edu/~cmallery/150/protein/page.jpg (Feb 2008)

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

Process of electrophoresis 1. sample application 2. adjustment of voltage or current - DIRECT CURRENT! (gel-electrophoresis about 70-100 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)

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

Electrophoresis examples from clinical medicine separation of serum proteins, isoenzymes, nucleic acids immunoelectrophoresi s (immunoglobulins) The figure was found at http://www.sebia-usa.com/images/controlgel1.jpg (Feb 2008)

Evaluation by densitometry - peaks 60% 3% 9% 12% 16% The figures are from http://www.sebia-usa.com/products/hyrys2.html and http://erl.pathology.iupui.edu/labmed/gener27.htm respectivelly (Feb 2007)

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

Potentiometry potentiometer

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

Scheme:

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

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

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

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

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 -,...)

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

Reference electrodes calomel el. argent chloride el. SHE

Glass electrode ISE (H + ) membrane electrode ph determination

Gass-sensing membrane electrode

tělo sensoru vnitřní elektrolyt skleněná glass elektroda electrode ph-metr referentní elektroda skleněná glass elektroda electrode CO +H O HCO +H+ film elytu 2 2 3 - permeabilní membrána permeabilní gas membrána permeable membrane analyzed analyzované sample prostředí CO 2 (g) analyzované prostředí

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