Analytical methods

Vladimíra Kvasnicová

1. SPECTROPHOTOMETRY

2. CHROMATOGRAPHY

3. POTENTIOMETRY

4. VOLUMETRIC ANALYSIS

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 380 – 450 nm

blue 450 – 495 nm

green 495 – 570 nm

yellow 570 – 590 nm

orange 590 – 620 nm

red 620 – 750 nm

see http://en.wikipedia.org/wiki/Electromagnetic_spectrum

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

Used radiation

• colour sample: VIS light

• colourless sample: UV radiation

A /

„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 (Io) of the

radiation entering the sample

T = I / Io

T = 0 – 1 or it is expressed in % (0 – 100 %)

How the quantity of absorption is expressed?

New quantity is defined: ABSORBANCE

A = - log10 T

= - log10 (I/I0) = log10 (I0/I) = log10 (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

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 = x l x cor

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

Calibration curve

3 or more standards

processed by the

same method

linear calibration curve

A = x l x c

y = kx + q

Calculation using standards

Ast = cst x l x Aus = cus x l x

Ast / cst = l x Aus / cus = l x

l x = l x

Ast / cst = Aus / cus

cus = Aus x (cst / Ast)

cus = Aus x f

f = average of all (cst / Ast) used in the experiment

Exercises

1) Au = 0,25 Cu = ?

As = 0,40 Cs = 4mg / L

2) 1000mg/L glucose standard (Cs ) 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

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

Spectrophotometry in thepractical 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

Chromatography

chromatograph

Not all

chromatography

techniques are

instrumental...

TLC chromatography = task of the practical training

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

Liquid Plane

Chromatography

example:

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 dissolvingsieving efect

- gel permeation

ion exchangecomplementary interactions

„affinity“ Adopted from presentation: analyticke_metody / Petr Tůma

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

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

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

Evaluation of chromatogram

Spots are compared with

standards:

Rf = a /b

Rf = 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)

2) Column Chromatography (HPLC, GC)

Peaks are compared with

standards:

tR = retention time

identification of solutes

h = height of the peaks

concentration of solutes

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 Rf

• unknown sample: composed of 2 unknown dyes

„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

Scheme of HPLC

Mobile phase

Degasser

Pump

Sample injection

Column

Detector

Waste

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

Nernst equation

E = E0 + (RT/nF) ln aM

E = electrode potential

E0 = 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 0C = 298 K)

n = oxidative number of ion of interest (M)

a = activity of ion of interest

E = E0 + (RT/nF) ln aM

ln a = 2.303 log a; R, T, and F values used

E = E0 + (0.059/n) log aM

! 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: ESHE = 0

under all conditions

REFERENCE ELECTRODE but not in a practise

Reference electrodes

calomel el. argent chloride el.

SHE

„Glass electrode“

ISE (H+)

pH determination

membrane electrode

Gass-sensing membrane electrode

skleněnáelektroda

referentníe lektroda

tě losensoru

vnitřníe lektro lyt

perm eabilním em brána

analyzovanéprostředí

skleněnáelektroda

CO (g)2

perm eabilním em brána

analyzovanéprostředí

film e lytu

pH-m etr

CO +H O HCO +H+2 2 3-

analyzed sample

gas permeable membrane

glass electrode

glass electrode

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“)

Glass combination

electrode

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

Volumetric analysis (= titration)

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

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

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

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:

Vt / Va = f

• f = factor of a titrimetric reagent (0,900 – 1,100)

• actual concentration of R (= titr): ca = f x ct

• the factor is used for conversion of a theoretical value of R to its actual conc. used for analyses

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)

a / b = n(A) / n(B)

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 cB x VB = b x cA x VA

a x cB x VB = b x cA x VA

• stoichiometry of the reaction is known • concentration and consumed volume of the

titrimetric reagent at a point of equivalenceis known

• sample volume used for the analyse is known

the only unknown value is

cB

Exercises

1) titrimetric reagent: 23,8 ml NaOH, (factor = 0,9685; C = 0,1M), sample = 10ml H2SO4; C = ?

2) titrimetric reagent: 10ml KMnO4 (0,1M), sample: 20ml FeSO4 ; C = ? (mol/ L, % ), MW = 152g

3) H3PO4 → Na2HPO4

sample: 20ml H3PO4 (C = 0,3M ), titrimetric solution: 0,2M NaOH V = ?

Titration is made by one person:„drop by drop addition of a titrimetric reagentunder continual mixing of reactants in a flask“

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

Titration curvesample: acid / titrimetric reagent: base

titrimetric reagent

measured value

indicators

sample: base / titrimetric reagent: acid

titrimetric reagent

Classification of volumetric analyses

1) neutralization (acid-base titration)R: acid /base• H+ + OH- → H2O

2) oxidation-reduction (redox) R: ox./red. reagent

• oxidation: red → ox + e-

• reduction: ox → red + e-

3) precipitation titration R: e.g. AgNO3

• formation of an insoluble salt

4) complexometric titration R: e.g. EDTA• formation of a stable complex

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

Instructions for the labs+ theory of the methods:

http://www.lf3.cuni.cz/chemie/

see Study/ Practical trainings