ANALYTICAL METHODS

Question 1

WHAT ARE SEPARATION METHODS?

Answer 1

-gas chromatography, liquid chromatography, thin-layer chromatography, column or planar chromatography

Question 2

WHAT ARE SPECTRAL METHODS?

Answer 2

-absorption (UV / VIS) or mass spectrometry

Question 3

WHAT ARE VOLUMETRIC ANALYSIS METHODS?

Answer 3

-titrations

Question 4

WHAT ARE ELECTROCHEMICAL METHODS?

Answer 4

-potentiometry

Question 5

WHAT IS CHROMATOGRAPHY?

Answer 5

-based on weak bond interactions between the studied substance and the different polar
phases
-Polar substances travel slower because they are attracted to stationary phase
-The mobile phase carries solutes through the stationary phase with different velocities according to their mutual affinity.

Question 6

WHAT IS THE MOBILE PHASE?

Answer 6

-liquid or gas
USUALLY NONP-POLAR

Question 7

WHAT IS THE STATIONARY PHASE?

Answer 7

-solid or liquid
USUALLY POLAR

Question 8

WHAT IS THIN-LAYER CHROMATOGRAPHY (adsorption / planar)?

Answer 8

-stationary phase = liquid fixed on the supporting material / solid adsorbent in the form of a thin layer
EG: AZO DYE (benzene nuclei separated by an azo group)

-thin layer contains the stationary phase (monolayer of silica gel)
-determination of the starting line (right above the mobile phase) and end point
-apply unknown sample onto the starting line of silica gel and next to it 3 standard samples (known azo dyes)
-thin layer is immersed into mobile phase (non-polar solvent e.g. toluene)
-mobile phase rises through the thin layer and it transports individual compounds with it
-when mobile phase reaches the end point, we compare how far the standards travelled to our sample through a so-called retardation factor = ratio of distance travelled by sample/mobile phase travelled
-each azo dye has its own specific retardation factor

Question 9

WHAT IS GAS CHROMATOGRAPHY?

Answer 9

-mobile phase is inert gas (helium, N2 or argon)
-stationary phase is a liquid or solid and mainly volatile substances like lipids
-capillary column (heated and coiled tube to separate components)
-Inner wall of the column is covered with stationary phase – fixed film of suitable liquid
-compounds that interact better with the gas phase tend to have lower boiling points (are volatile) and low molecular weights, while compounds that prefer the stationary phase tend to have higher boiling points or are heavier
-flame ionizing detector
EXAMPLES = ANALYSIS OF FATTY ACIDS, ALDEHYDES, KETONES IN BLOOD

Question 10

WHAT IS HIGH PERFORMANCE LIQUID CHROMATOGRAPHY?

Answer 10

-stationary phase – very small granules (2-5 μm) of silica gel, hydrated silica, which is then modified and silicon groups bind to hydrocarbon chains, forming octadecyl silicone gel
- mobile phase = liquid, solvent - acetonitrile, methane
-reservoir of mobile phase (solvent) -> solvent pump -> sample injector -> column -> detector
EXAMPLES: Water / fat soluble vitamins, corticoides, corticosteroides, antidepressants

Question 11

WHAT IS LIQUID CHROMATOGRAPHY?

Answer 11

-based on weak bond interactions between the studied substance and the different polar
phases
-mobile phase is a nonpolar solvent (acetonitrile, methanol)
-stationary phase is a polar molecules (silica gel, octadecyl silica)
-polar substances will attach to stationary phase and non-polar will travel faster
-if reversed: stationary phase is nonpolar and mobile phase is polar - the nonpolar substances will travel slower and polar faster
-detector records for an chromatogram (narrow and high peaks = good) = QUANTITATIVE

Question 12

WHAT DOES THE CHROMATOGRAPHIC WAVE INDICATE?

Answer 12

-the passage of the substance through the detector

Question 13

WHAT IS CHROMATOGRAPHIC RETENTION TIME?

Answer 13

-time taken to pass through the column
-specific for each substance
QUALITATIVE

Question 14

WHAT IS A SPECTROPHOTOMETER?

Answer 14

photometer measures the absorption of waves– emits a ray of monochromatic light or UV (colorless) radiation and measures how much radiation was absorbed by the analyzed substance

Question 15

WHAT IS AN ABSORPTION SPECTRA?

Answer 15

-a spectrum of electromagnetic radiation transmitted trough a substance, showing
dark lines or bands due to absorption at specific wavelengths

Question 16

WHAT IS SPECTROPHOTOMETRY AND ITS PRINCIPLES?

Answer 16

-measures the amount of light that is absorbed or transmitted by a chemical compound
-different substances will absorb / transmit different frequencies of light
-compounds absorbs light radiation of a specific wavelength
-a part of the radiation is absorbed by the compound and the rest of the radiation is detected by the detector
-the light absorbed by the sample is directly proportional of the concentration of sample in the solution
-as concentration increases, absorption increases

Question 17

WHAT DOES THE CALIBRATION CURVE SHOW?

Answer 17

-plots absorbance vs concentration, which is then used to calculate the value of an unknown quantity
-standard solutions help find the relationship between absorbance and concentration
-beer lambert law used

Question 18

WHAT IS THE BEER LAMBERT LAW?

Answer 18

-linear / directly proportional relationship between absorbance and concentration of an analyte

-exponential relationship between transmittance and concentration of an analyte

A = e x l x c

Question 19

WHAT IS ELECTROPHORESIS?

Answer 19

-fast & effective technique - movement of charged particles because of an external electric field
-free boundary (no support = capillary electrophoresis) or supporting medium (gel / paper)
-speed is constant, depends on size of charger (bigger the charge the faster the molecule) and the size of the molecule ( the bigger the molecule the slower it moves)
-electrodes made of inert metal so that it will not react with surrounding solution
-ions put into electric field - cations move to negative cathode electrode (REDUCTION HERE), anions to positive anode electrode (OXIDATION HERE) - velocity is measured
EG: ANALYSIS OF BLOOD PLASMA PROTEINS

Question 20

WHAT IS THE ISOELECTRIC POINT?

Answer 20

-value of pH in solution where a zwitterion stops moving in an electric field (e.g. proteins)

Question 21

WHAT IS POTENTIOMETRY?

Answer 21

-an electrochemical analytical methods, that is based on the measurement of voltage of
electrochemical cell, when no current flows
-reduction at cathode
-oxidation at anode

Question 22

WHAT IS ISOELECTRIC FOCUSING?

Answer 22

• separation according to pH gradient between two electrodes
• separated substances travel under electric current until the point they reach a region of pH equal to its isoelectric point - proteins can be separated using this method

Question 23

WHAT IS ELECTRODE POTENTIAL?

Answer 23

-value of potential of the glass membrane electrode Is not always 0
-the electrode potential of the SHE is defined to be 0 volts under each temperature
-the measured voltage of the electrochemical cell is equal to the electrode potential of electrode of interest
-Nernst equation (relationship between the electrode potential and the activity of any ion of
interest)
-Voltmeter measures the cell potential

Question 24

WHAT IS AN INDICATING ELECTRODE?

Answer 24

-potential depends on the amount of analysed
substance, SHE (standard hydrogen electrode)
-responds to analyte
EG: METALLIC, MEMBRANE INDICATOR, MOLECULAR SELECTIVE

Question 25

WHAT IS A REFERENCE ELECTRODE?

Answer 25

-one electrode of system to act as a reference against which potential measurements can be made
-it has a fixed potential, constant response, obeys Nernst equation, reversible, intensive to composition of solution
EG: STANDARD HYDROGEN, CALOMEL, SILVER CHLORIDE

Question 26

WHAT IS AN ELECTROCHEMICAL CELL?

Answer 26

-any metal which has the ability to be reduced or oxidized

Question 27

WHAT IS ELECTRODE POTENTIAL DEPENDENT ON?

Answer 27

-temperature, activity and charge

Question 28

HOW CAN YOU MEASURE pH BY POTENTIOMETRY?

Answer 28

-measuring system consists of a pH measuring electrode and a reference electrode
-potential difference between both electrodes is a function of the pH value
-based on use of glass membrane that binds H+
-analyte must be conductive and is part of the electrical circuit

Question 29

WHAT ARE MEMBRANE INDICATOR ELECTRODES / SELECTIVE ELECTRODES?

Answer 29

-electrodes based on determination of cations or anions by the selective adsorption of these ions to a membrane surface
-selectively bind ions of interest, need electrical conductivity, minimal solubility so that membrane will not dissolve in solution during measurement

Question 30

WHAT IS A TITRATION?

Answer 30

-an analytical procedure that allows us to measure the volume of solution, which is
needed to react exactly with the another solution of unknown concentration

Question 31

WHAT IS THE EQUIVALENCE POINT?

Answer 31

-endpoint of titration

Question 32

WHAT ARE THE TITRATION TYPES?

Answer 32

ACID-BASE:
acids = HCl, H2SO4, HNO3, HClO4
bases = NaOH, KOH, Ba(OH)2
indicators = methyl violet (yellow-violet), methyl orange (pink-yellow), phenolphthalein (colorless-pink)

REDOX REACTIONS:
oxidation: Red ⇒ Ox + e
reduction: Ox + e ⇒ Red
oxidizing agents:
MnO4- (in acid solution: MnO4- + 8H+ + 5e ⇔ Mn2+ + 4H2O) = determines Fe2+, H2O2, As3+,I,NO2-
Cr2O72- (Cr2O72- + 14H+ + 6e ⇔ 2Cr3+ + 7H2O)
I2 (I2 + 2e ⇔ 2I) = determines SO3(2-), S2O3(2-)
reducing agents: Na2S2O3 (thiosulfate ion to tetrathionate ion + 2e-) = determines I2

PRECIPITATION: common agent is AgNO3 = determines Cl-, Br-, I-, SCN-

COMPLEX ION: EDTA = determines a lot of metal ions (Ca2+, Mg2+)

Question 33

WHAT IS THE RELATIONSHIP BETWEEN ABSORBANCE AND TRANSMITTANCE?

Answer 33

A = log10 T

Question 34

ABSORBANCE TO TRANSMITTANCE

Answer 34

0 - 100%
1 - 10%
2 - 1%
3 - 0.1%
4 - 0.01%
5 - 0.001%

Question 35

WHAT IS THE DIAMETER OF THE CAPILLARY AND WHAT IS IT FILLED WITH?

Answer 35

-0.3mm
-buffer solution