Peoples notes flash cards

Question 1

Explain MS

Answer 1

Mass spectrometry is an analytical technique that measures the mass-to charge ratio of
ions. The results are typically presented as a mass spectrum, a plot of intensity as a function
of the mass-to-charge ratio. Mass spectrometry is used in many different fields and is
applied to pure samples as well as complex mixtures.
A mass spectrometer generates ions from the sample under investigation, it then separates
them according to their specific mass-to charge ratio (m/z) and then record the relative
abundance of each ion type.

Question 2

What is soft ionization vs strong ionization

Answer 2

Strong ionisation: M+ e- → M+ + 2 e- (fragment ions)
Soft ionisation: M → [M+H]+ (molecular ions)

Question 3

What are the components of a mass spectrometer and what do they do?

Answer 3

Inlet with sample induction, source with gas phase ions, analyzer with vacuum pumps that do the ion sorting and ion detector for ion detection. Then a data system will analyze this and give an output in the form of a mass spectrum.

Question 4

MS with regard to hard ionisation, Important ion sources:

Answer 4

Electron impact ionization (EI): In EI, a high-energy beam of electrons is directed at the sample, resulting in the ejection of one or more electrons from the analyte and the formation of highly energetic molecular ions. This method is commonly used in gas chromatography-mass spectrometry (GC-MS) for the analysis of volatile and semi-volatile organic compounds.

Fast atom bombardment (FAB): In FAB, a high-energy beam of atoms, usually from an inert gas such as argon or xenon, is directed at a solid sample or a solution of the analyte in a suitable matrix. The high-energy atoms cause ionization of the analyte, which is then extracted and analyzed by mass spectrometry.

Matrix-assisted laser desorption/ionization (MALDI): In MALDI, a sample is mixed with a suitable matrix material and dried on a target plate. A laser is then used to vaporize and ionize the sample and matrix, resulting in the formation of ions that can be analyzed by mass spectrometry.

Question 5

MS with regard to soft ionisation, Important ion sources:

Answer 5

ESI, Electrospray ionization (ESI) is a technique used in mass spectrometry to produce ions using an electrospray in which a high voltage is applied to a liquid to create an aerosol. MALDI In mass spectrometry, matrix-assisted laser desorption/ionization (MALDI) is an ionization technique that uses a laser energy absorbing matrix to create ions from large molecules with minimal fragmentation. Fast atom bombardment (FAB) is an ionization technique used in mass spectrometry in which a beam of high energy atoms strikes a surface to create ions.

Question 6

When do you use soft vs hard ionization?

Answer 6

Soft ionization is a useful technique when considering biological molecules of large molecular mass, such as the aformetioned, because this process does not fragment the macromolecules into smaller charged particles, rather it turns the macromolecule being ionized into small droplets.

EI which is a hard method is also the method that is most commonly used for GC-MS.

Question 7

Important analyzers: Quadrupole,TOF

Answer 7

The quadrupole mass analyzer consists of four parallel conducting rods of hyperbolic cross section spaced about a central axis along which ions are conducted. Depending on their mass and charge they will interact differently with the rod, large molecules or very charged molecules will interact more and smaller less charged will interact less.

Time-of-Flight (TOF) is a mass analyser that utilises an electric field to accelerate generated ions through the same electrical potential, and then measures the time each ion takes to reach the detector.

Question 8

Describe molecular spectrophotometry

Answer 8

1. Molecular spectrophotometry
   - We can measure the amount of colour and then relate it to the
   concentration
   - Speed of light: C , frequency: U, wavelength: λ, planck's
   constant: h
   Energy: E= h.u = h / cλ
   - M+ hu → M * ( Absorption)
   M* → M+ heat (relaxation)
   M* → M+ hu (emission , fluorescence)
   - The difference between the absorbed wavelength and the
   emitted is called stocks shift
   - Absorption: the sample is more concentrated than in emission

Question 9

Describe atomic spectrophotometry

Answer 9

Atomic spectrophotometry
- The flame is the source of energy that is given to these atoms,
they get excited.
- AES (Atomic emission spectroscopy): the energy in the flame
can be enough to excite the atom so they start to emit a light
that is detected
- AAS (Atomic absorption spectrometry): the sample is atomised
in the flame, we send in light after that corresponds to the

analytes. That light is absorbed by the atoms so we can
measure the absorbance.
- AFS (Atomic fluorescence spectroscopy); the external light
source is the laser.

Question 10

• If we measure the absorbance at one wavelength we can perform
  quantitative measurements
• Single beam spectrophotometer: describe what it is made of and their functions

Answer 10

light source, wavelenght selector (monocrhomator) sample and light detector

Question 11

Double beam spectrophotometer: describe what it is made of and their functions

Answer 11

There is a light source, scanning monocrhomator(wave lenght selector) motor with a roatting mirror (beam chopper) sample cuvet, derector, amplifier and display

Question 12

Describe what an optical filters do

Answer 12

Optical filters: absorption filter , interference filter. They are
usually colored glasses. Since one colour comes out of the
glass they have visually selected some of the visible lights.

Question 13

Describe what monocrhomators do and how they are bult and alternatives to them

Answer 13

Monochromators: prisma, gratings. It disperses light into its
components and selects a narrow band of wavelength to pass
onto the sample detector.
→ Grating Czerny-Turner monochromator:
They consist of a cathode made from the element of interest, an
anode and an inert filler gas contained in a glass envelope.
(the light source goes into the entrance slit of the
monochromator and hits a concave mirror that is reflective so
that all the light is parallel and hit the grating. Depending on the
wavelength from the grating the light is reflected to this concave
mirror and then reflected again towards the exit slit. So
depending on the wavelength the diffraction angle is different.)

Question 14

What is important to keep in mind with the cuvette?

Answer 14

Cuvette /sample container
What is important for the cuvette is that it has to be transparent for the
light that we use.

Question 15

What is a detector, what does it do?

Answer 15

Detectors
It is a device that identifies / records or registers a stimulus such as an
environmental charge in pressure or temperature , an electric signal or
radiation from that radioactive material.
The photodetector principle is that when photons hit a metal surface,
electrons are ejected creating an electric current. The response of a
photodetector changes depending on the wavelength.

Question 16

What is a phototube and what does it do?

Answer 16

Phototube: it has a negatively charged cathode. When photons hit a cathode
electrons are emitted from the surface and are accelerated to the anode that
gives rise to the electric current. The more electrons are emitted the stringer
the current will be.

Question 17

What is a photo multiplier and what does it do?

Answer 17

b) Photomultiplier: it is a sensitive device in which electrons emitted from the
photosensitive surface strikes a second surface called a diode which is a
positive part with respect to the photosensitive emission. Electrons are
accelerated and strike the dybode with more than their original kinetic energy.
Each energetic electron knocks more than one electron from the dynode.
These new electrons are accelerated toward a second diode which is more
positive than the first. This process is repeated several times so that more
than up to 6.10 e- are collected to each photon striking the first surface.

Question 18

What is a photodioide and what does it do

Answer 18

The photodiode detects the absorption or fluorescence of the sample component and generates a signal that is proportional to its concentration.

In spectrophotometry, photodiodes are used to measure the absorbance or transmittance of a sample at a specific wavelength. The photodiode detects the light that passes through the sample and generates a signal that is proportional to its intensity. This signal can then be used to calculate the absorbance or transmittance of the sample at the given wavelength.

In fluorescence detection, photodiodes are used to detect the fluorescence emitted by a sample when it is excited by light at a specific wavelength.

Question 19

What affects band broadeing?

Answer 19

1. Multiple flow paths
2. Longitudinal diffusion
3. Mass transfer

Question 20

What is the mobile, stationary and analyte og GC?

Answer 20

mobile phase:carrier gas
stationary phase: usually a nonvolatile liquid, but sometimes a solid
analyte: gas or volatile liquid

Question 21

1. Qualitative analysis : how can you determine the identity of a peak?

Answer 21

• Retention time
• NPD ECD (provides some degree of specificity)
• Mass spectrometry (can identify the compound by its mass spectrum)

Question 22

2. Quantitative analysis: how can you determine the concentration of a
   compound?

Answer 22

• Calibration curve (external standard)
  Y axis , peak area
  X axis , concentration
• Internal standard
  The same amount of the IS is added to all standards and samples
  Y axis : analyt area / IS area
  Corrects for variations of the injected sample volume

Question 23

Describe the basics of capillary electrophoresis

Answer 23

Capillary electrophoresis (CE) is an analytical separation technique that uses a capillary as the separation column. It is based on the principles of electrophoresis, which involves the separation of charged analytes in an electric field.

In CE, a capillary with an inner diameter of typically 25-100 μm is filled with a buffer solution that serves as the electrolyte. The capillary is then placed in an electric field generated by a high voltage power supply, which causes the charged analytes to migrate through the capillary at different rates based on their charge, size, and shape.

The separated analytes are detected as they reach the detection point at the end of the capillary, using a suitable detector such as UV-Vis spectrophotometer, fluorescence detector or mass spectrometer. The detection is based on the absorbance, fluorescence, or mass-to-charge ratio of the analytes, depending on the type of detector used.

Highly charged and small molecules migrate faster.

Question 24

What is Electrophoretic mobility:

Answer 24

μep : It is the constant of proportionality between the speed of the ion
and the electric field strength
- Describes how fast an ion will migrate in an electric field
- Higher charge → μep increases
- Smaller hydrodynamic radius → μep increases
- Different ions have different electrophoretic mobilities, which enables
separation
- Mobilities are tabulated ( in a table), therefor migration order of
analytes can be foreseen.

E= electric field strength (V/m) = V/Lt = applied voltage/ total
capillary length.
- Separation is achieved when the analytes have different electrophoretic
mobilities μep.
- Mobilities increase with charge. Ex from slow to fast: charge -1 →
charge -2 → charge -3.

Question 25

2. Electroosmotic flow (EOF), electroosmotic mobility (μeo)

Answer 25

In capillary electrophoresis (CE) that uses silica, electroosmotic flow (EOF) occurs due to the interaction between the negatively charged silica surface and the buffer solution used as the electrolyte.

hen a high voltage is applied across the capillary, the negatively charged silica surface attracts the positively charged ions of the buffer solution, creating a diffuse layer of counterions that neutralizes the surface charge.

This diffuse layer of counterions creates a net flow of the buffer solution towards the cathode, which is opposite to the direction of the electrophoretic migration of the analyte. The resulting EOF effectively drives the analyte towards the detection end of the capillary, improving the separation and detection of the analytes.

The magnitude and direction of the EOF in CE depend on various factors, such as the pH and ionic strength of the buffer solution, the surface charge density of the silica, and the applied electric field strength. The EOF can be manipulated by altering these factors to optimize the separation and detection of different analytes in CE.

Electroosmotic mobility (µeo) is a parameter in capillary electrophoresis (CE) that describes the velocity of the electroosmotic flow (EOF) in a capillary under a given set of conditions. It is defined as the ratio of the electroosmotic flow velocity to the electric field strength.

• Increased electric field strength (E) → increase the electroosmotic velocity
• μeo is a constant.

Question 26

How can EOF be affected?

Answer 26

a) EOF ↑ with ↑ pH:
As pH is increased , additional silanol groups will be deprotonated, which
leads to an increased negative charge at the capillary surface.
A larger number of cations in the carrier electrolyte will be attracted to the
negatively charged capillary wall and can contribute to the creation of EOF.
b) EOF ↓ when adding an organic modifier to the electrolyte:
The organic modifier breaks the ordered structure of the water, which means
that the electrolyte is dragged forward less efficiently (EOF decreases)
c) EOF ↑ with ↑ temperature:
↑ temperature leads to a ↓ viscosity of the carrier electrolyte. Due to the ↓
viscosity the electrolyte can flow more easily through the capillary (EOF ↑)
d) EOF ↓ with ↑ electrolyte concentration.

Question 27

In what order do analytes migrate?

Answer 27

1. Cations – electrophoretic mobility of cations and EOF are both directed
   towards the cathode at capillary outlet (coelectroosmotic conditions)
2. Neutral analytes – Have no electrophoretic mobility. Just follow the EOF.
3. Anions – electrophoretic mobilities of anions are directed towards the capillary
   inlet (anode), while EOF is directed towards the cathode at capillary outlet
   (counter electroosmotic conditions)

Question 28

Will the anions reach the detector?

Answer 28

Yes – if the electroosmotic velocity (directed towards the outlet) exceeds the electrophoretic
velocity of the anion in question (directed towards the inlet).
No – if it is the other way around

Question 29

How the EOF is reversed by adding a cationic surfactant:

Answer 29

When the cationic surfactant is added in a sufficiently high concentration, a bi-layer is
formed.
The cationic surfactant turns with its positive part towards the negative capillary
surface and a second layer of the surfactant turns the positive part toward the
electrolyte due to hydrophobic interaction between the tails.
The effect of this is that the charge on the surface of the capillary changes from
negative (the silanol groups) to positive. Anions in the electrolyte will now be
attracted to the positive surface and drag along the electrolyte in an electroosmotic
flow directed toward the anode (+)/
The direction of EOF has been reversed.

Question 30

Counterelectroosmotic conditions for analytes that are anions:

Answer 30

In what order do analytes migrate?
- Anions – electrophoretic mobility of anions and EOF are both directed
towards the anode at capillary outlet (coelectroosmotic conditions)
- Neutral analytes – Have no electrophoretic mobility. Just follow the EOF.
- Cations – electrophoretic mobilities of cations are directed towards the
capillary inlet (cathode), while EOF is directed towards the anode at the
capillary outlet (counter electroosmotic conditions)

Question 31

Will the cations reach the detector?

Answer 31

Yes – if the electroosmotic velocity (directed towards the outlet) exceeds the electrophoretic
velocity of the anion in question (directed towards the inlet).
No – if it is the other way around