part 2 chemical analysis

Question 1

What are the main steps of the analytical procedure?

Answer 1

1. The analyte must be extracted in a suitable solvent
2. Isolate the analyte from the matrix and the interferents by separation
3. Separate components by chromatography
4. Detection of the analytes (identification = qualitative ; quantitative)
5. Mass spectrometry for the selective detection, identification, and quantitation of analytes

Question 2

What is a solution?

Answer 2

A liquid phase containing more than one substance: the solvent and the solutes

Question 3

DEF solvent power

Answer 3

The ability of the solvent to solubilize a given compound

Question 4

How to choose a solvent?

Answer 4

1. Solvent power: like dissolve like. Polar solvents tend to dissolve polar (hydrophilic) compounds. The target compound needs to be very soluble in this solvent.
2. Boiling point/volatility: extracting solvent should be sufficiently volatile so that it can be removed easily from the extracted material by distillation
3. Health & safety: non-toxic and non-flammable (ex: choose toluene over benzene because far less toxic)

Question 5

DEF extraction

Answer 5

• The transfer of the analyte from a pahse into the other

- The process of transferring a substance from any matrix to an appropriate liquid phase

Question 6

Liq-liq extraction

Answer 6

It is a method to separate solutes when they exhibit different solubilities in two immiscible liquids

Question 7

DEF recovery

Answer 7

When the analyte is completely transferred into the solvent, the extraction is quantitative, that is 100% of the analyte was recovered. Recovery is generally less than 100%, extraction is rarely fully quantitative.
Recovery = analyte extracted into the solvent / analyte originally present in the sample

Question 8

How to increase the extraction efficiency?

Answer 8

1. increase the solubility (thermodynamic considerations)
   - solvent selection (solvent power)
   - increasing temperature: max temp solvent boiling point
   - increasing pressure
2. increase rate (kinetic considerations)
   - stirring
   - increase temperature
   - phase contact (grinding, fine powder instead of coarse particles, mixing)

Question 9

DEF chromatography

Answer 9

It is a physical method of separation in which the components to be separated are distributed between two phases: a stationary phase and a mobile phase

Question 10

What are the two main types of chromatography?

Answer 10

• gas chromatography: the mobile phase is a gas, carried out in a column
• liquid chromato: mobile phase is a liquid, can be carried out either in a column or on a plane. (HPLC = very small particles & high pressure)

Question 11

What is column chromatography?

Answer 11

It is a separation technique in which the separation bed is a tube. The particles of the solid stationary phase or support coated with a liquid stationary phase may fill the whole inside volume of the tube (packed column) or be concentrated on or along the inside tube wall leaving an open, unrestricted path for the mobile phase in the middle part of the tube (open-tubular column).

Question 12

What is an eluant?

Answer 12

The liquid or gas entering a chromatographic bed and used to perform a separation by elution

Question 13

DEF adsorption chromato

Answer 13

Chromatography in which separation is based mainly on differences between the adsorption affinities of the sample components for the surface of an active solid. An increase in the concentration of a dissolved substance at the interface of a
condensed and a liquid phase due to the operation of surface forces. Adsorption
can also occur at the interface of a condensed and a gaseous phase.

Question 14

DEF normal phase and reversed phase chromato

Answer 14

• normal phase chromato: an elution procedure in which the stationary phase
  is more polar then the mobile phase. (typically silica particles with silanol groups on surface)
• reversed phase: an elution procedure used in liquid
  chromatography in which the mobile phase is more polar then the stationary phase (typically reversing the polarity with hydrocarbon chains)

Question 15

Which interactions are involved in normal and reversed phase chromato?

Answer 15

In normal phase:
- interactions: hydrogen bonding or dipolar interactions
- the more polar is our component the higher is the affinity for the stationary phase
- the shorter will be the distance travelled by our component
ADSORPTION CHROMATO: silica gel polar due to Si-OH (silanol) groups on particle surface.

In reversed phase, the stationary phase is less polar than the mobile phase
PARTITIONING CHROMATO: silica gel functionalized with hydrocarbon chain. The liquid phase is bonded onto the surface of silica particles (bonded phase).
-> the order of elution is opposite compared to a normal phase chromato

Question 16

What is a chromatogram?

Answer 16

The chromatogram is a graph reporting the detector signal vs. analysis time

Question 17

DEF retention time

Answer 17

Retention time is a qualitative analysis. It is the time interval between sample injection and maximum detection. In other words, it is the time a component takes to travel through the column. Tr depends on the type of mobile phase and stationary phase, on the length of the column and other parameters such as the temperature & flow.

Question 18

What are two important parameters needed for the separation?

Answer 18

• Retention: affinity of the components for the stationary phase
• Selectivity: different affinities for each component. Selectivity is the ability of the analytical method to determine a given analyte without interferences by other components.

RETENTION + SELECTIVITY = SEPARATION

The separation depends on:

• the interaction with the stationary phase
• the volatility (boiling point, distribution in the gas phase)

Question 19

What are the main constituents of an HPLC?

Answer 19

1. mobile phase: solvents
2. pump pushing the solvent with a constant flow
3. injector
4. column
5. detector (containing a mass spec)
6. work station

Question 20

How to get a quantitative analysis with chromatography?

Answer 20

By measuring each peak area in the chromatogram. The peak area is determined by integration of the chromatographic peak. The relationship between peak area and concentration is determined through calibration. Calibration is performed by analysing solutions of the standard analyte at known
concentrations (calibration solutions) under the same conditions utilized for
sample analysis.

Question 21

What is the most powerful type of chromato?

Answer 21

The gas liquid chromatography! The column is a capillary column of approx 10-100m, which a liquid film as a stationary phase, and helium as a mobile phase. Temperature is applied, having an influence on the retention time. Indeed, the distribution of the compound into the mobile phase is increased by increasing the temperature of the oven.
The column is composed of a layer of polyamide coating, a layer of fused silica, and finally the stationary phase.
The liquid part of the stationary phase is made of polysiloxanes, which can have different polarities depending on the substitution of the side chains (can be determined by the proportion of chemical nature R1 and R2 groups).

Question 22

Which compounds can be analyzed by GC?

Answer 22

• without transformation: gases (CO2, H2, CH4, CO..), VOCs, semi-volatile compounds (volatile at high temps)
• after chemical transformation (derivatization). Non-analyzable as such due to low volatility (strong cohesive forces, ionic compounds, hydrogen bonding), but can be volatilized after chemical transformation. It is typically the case for polar compounds (-O-H –> -O-CH3 ; -O-SiMe3)
• after chemical or thermal degradation. Non-volatile high molecular weight compounds are fragmented into smaller compounds. Typically for macromolecules

CHEMICAL DEGRADATION: HYDROLYSIS

THERMAL DEGRADATION = PYROLYSIS

Question 23

What are the main elements composing a mass spectrometer?

Answer 23

1. GC
2. ion source
   Ions are accelerated
3. mass analyzer (ions are separated according to their mass to charge ratio m/z)
4. electromultiplier (ions collected and quantified)
5. work station & results

Mass spectrum: % abundance vs. m/z

Question 24

What are the main types of ions (for mass spec)?

Answer 24

• molecular ion M+• is a positive ion obtained by removing an electron from the molecule. The relative mass of the molecular ion corresponds to that of the neutral molecule. It is generally formed by “hard” ionization techniques (e.g. electron ionization). Common in gas chromato. (occurs at high energy)
• quasi-molecular ion MH+, which is formed by the addition of a proton to the molecule (M+H+ –> MH+). It is generally formed by “soft ionization techniques (e.g. electrospray ionization ESI). Common in liquid chromatography. (occurs at low energy)
• fragment ion A+ which is formed from the dissociation of a precursor ion. Those ions are produced in “hard” ionization techniques from the fragmentation of the molecular ion and derived ions. Their presence makes the mass spectrum informative and specific of the specific compound. (useful for the interpretation of the mass spectrum)

Question 25

How can masses be expressed in mass spectrometry?

Answer 25

- Monoisotopic mass: exact mass of an ion or molecule calculated using the mass of the most abundant isotope of each element. (most abundant isotope of chlorine: 35Cl so mass of methyl chloride is 49.992329u) - Nominal mass: mass of a molecular ion or molecule calculated using the isotopic mass of the most abundant contituent element isotope of each element rounded to the nearest integer value and multiplied by the number of atoms of each element. (for methyl chloride: 50u) - Accurate mass: experimentally determined mass of an ion of known charge. (accurate mass = measured mass ; exact mass = calculated)

Question 26

Electron ionization

Answer 26

Molecules M interact with a beam of electrons with high energy emitted from an electrically heated filament and accelerated by an electrical field. The interaction may cause the ionization of the molecule by electron abstraction. M + e-(primary) --> M+• + 2e-(secondary) The energy of primary electrons (70 eV) is higher than that of electron removal (≈ 10 eV). Because of the extra energy, the molecular ion can be fragmented into ions and neutral molecules/radicals (in turn fragmented to other ions)

Question 27

How is a mass analyzer working?

Answer 27

The ions are separated in a quadrupole. Ions are accelerated into the internal cavity of four metallic rods forming an oscillating electromagnetic field. Ions travel through the rods with trajectories depending on their mass to charge ratio m/z. By increasing the electric potentials, only ions with a given m/z value have a stable trajectory and can be transmitted through the quadrupole to be revealed by the detector. The scan enables the detection of all the ions within a given m/z interval. -> quadrupole works like a mass filter

Question 28

Main types of ions to look at in a mass spectrum

Answer 28

1. M+1: molecule with 13C for example 2. molecular ion M+•: molecule that lost one e-. Corresponds to the mass of the neutral molecule. 3. BASE PEAK: the most abundant ion (relative abundance of 100%), which corresponds to a loss of hydrogen atom 4. fragment ions: loss of H2, loss of OH, methyl ions... Comments on the molecular ion: - M+• is the most abundant: typical of aromatic hydrocarbons - M+• is barely detectable: aliphatic hydrocarbons (without side chains)

Question 29

Briefly, what is liq chromatography-mass spec?

Answer 29

An aerosol is formed at high temperature (200-500°C) in the presence of nitrogen gas. The mobile phase and solutes are evaporated. The vapor interacts with electrons emitted by a source of energy. Electron ionization forms primary ions from nitrogen ions (e.g. N2+•). Nitrogen ions react with water molecules (e.g. mobile phase) forming proton donor ions (e.g H2O+). The quasi molecular ions of the analyte are formed by proton transfer. A + H3O+ --> AH+ (protonized analyte) + H2O

Question 30

In a few words, electrospray ionization?

Answer 30

- Formation of charged droplets due to the high difference of potential (strong electrical field) - Solvent evaporates by the action of dry and hot nitrogen flow - Droplets are getting smaller until the cohesive forces of the liquid cannot sustain the repulsive electrostatic forces - Droplets explode forming even smaller droplets - Process continues until the molecules of the analyte become charged GOOD FOR POLAR ANALYTES and useful for the analysis of high molecular weight analytes (macromolecules)