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3rd Year Fall > C3110 Midterm 2 > Flashcards

C3110 Midterm 2 Flashcards

1
Q

What are the 6 main components of an LC?

A

Autosampler, solvent delivery system, sample injection valve, high-pressure chromatography column, detector, computer to control system and display

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2
Q

What compounds are chosen for LC over GC?

A

Compounds that are not sufficiently volatile for GC

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3
Q

Why do smaller particles give better resolution? (2 reasons)

A
  1. More uniform flow reduces A term
  2. Less distance the solute must diffuse reduces C.
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4
Q

What is a consequence of smaller particle size?

A

Requires a higher pressure

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5
Q

What are the benefits of smaller particles? (3)

A

Higher plate number, shorter tun time, and lower detection limits

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6
Q

What contributes to extra column broadening in HPLC? (4)

A

Connecting tubing, extra column variance, column frits, and sample filtration

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7
Q

Describe the HPLC column

A

Filled with stationary phase; highly pure, spherical, microporous particles of silica

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8
Q

What is a guard column

A

When samples are filtered or centrifuged, the guard column protexts the main chromatographic phase

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9
Q

Most common bonded phase (sp)

A

C18 (ODS)

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10
Q

Describe reversed-phase chromatography

A

Most common for HPLC.
Sp is nonpolar and solvent is polar.
Analyte is nonpolar.
Less polar solvent is a stronger mobile phase.

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11
Q

Polarity Index

A

ranks solvents based on their ability to displace polar solutes; higher index=more polar solvent

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12
Q

Describe normal-phase chromatography

A

Polar sp and a nonpolar solvent
Analyte is polar
More polar solvent has a higher eluent strength

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13
Q

Eluent strength

A

measure of the solvent absorption energy on base silica. Pentane is defined as zero.

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14
Q

What is the acronym HILIC stand for?

A

Hydrophilic interaction chromatography

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15
Q

Describe HILIC

A

Separates compounds too polar for RPLC
Strongly polar sp (CH3CN with buffer)
Polar sp is coated with a layer of water, and the polar analytes partition

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16
Q

Why do analytical chemists dislike silica? (4)

A
  1. Variance
  2. Surface Complexity
  3. Not uniform
  4. Highly pH dependent
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17
Q

What are the problems with HPLC solvents?

A

Pure HPLC grade solvents are expensive.

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18
Q

What is a sparging system in HPLC

A

Removes dissolved gases by sweeping them out of solution by fine bubbles of an inert gas.

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19
Q

Why is sparging required?

A

Air bubbles create difficulties for pumps, columns, and detectors.
Dissolved oxygen can absorb UV.

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20
Q

How (3 steps)/when are HPLC columns cleaned?

A

When: before storage or after a series of runs.
How:
1. Replace buffer with water and wash with 5-10 mobile phase volumes (Vm)
2. Wash with 10-20 Vm of a strong eluent
3. Store the column with the solvent to inhibit bacterial growth.

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21
Q

What is the general elution problem?

A

One set of conditions is not suitable to separate a complex mixture of analytes in a reasonable amount of time.

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22
Q

What is gradient elution and why is it used?

A

Gradient elutions uses a continuous change of solvent composition to increase the mobile phase strength.
Used as a solution for the general elution problem.

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23
Q

What common detectors are used for HPLC?

A

UV, refractive index, evaporative light scattering, charged aerosol, electrochemical, fluorescence, nitrogen chemiluminescence, conductivity, mass spectrometry

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24
Q

Explain how spectrophotometric detectors work.

A

UV detectors – common because many solutes absorb UV light.
Photodiode array records spectrum of each solute as it elutes.
This is matched with library spectra to identify the analyte.

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25
Q

Describe fluorescence detectors.

A

The eluate is excited with a laser and fluorescence at long wavelengths is measured.
Up to 100 times more sensitive than UV.
Few analytes fluoresce.
Derivitization can be useful here.

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26
Q

Electrochemical detector

A

responds to analytes that can be reduced or oxidized.
Potential is controlled with respect to a reference electrode (usually Ag|AgCl). Current measured between working electrode and aux electrode.

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27
Q

Refractive index detector

A

Responds to almost every solute.
Poor detection limits.
When a solute with a different refractive index than the mp enters the cell, light is deflected and pixels of a diode array are irradiated.
Useless in gradient elution.
Useless in trace analysis.

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28
Q

Name the two most common LC-MS interfaces.

A
  1. Electrospray Ionization (ESI)
  2. APCI
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29
Q

How would we identify issues in an LC-MS experiment?

A
  1. If there is an issue but the internal standard performance check is normal, the issue is in the chromatography of the unknown.
  2. If the performance check fails, the issue is with the instrument.
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30
Q

What is retention based on in Ion-Exchange Chromatography?

A

the attraction between solute ions and charged sites bound to the stationary phase.

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31
Q

Anion exchangers

A

contain positively charged groups bonded to sp (covalent), attract solute anions

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32
Q

Cation exchangers

A

contain negative groups bonded to sp that attract solute cations

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33
Q

Resin

A

Amorphous particles of organic material

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34
Q

Polystyrene resins

A

co-polymerization of styrene and divinylbenzene

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35
Q

How does ion-exchange work to remove salts from water (use NaCl as an example salt)?

A

In a cationic resin, H+ ions.
Na+ displaces H+ and H+ is released.
In an anionic resin, OH- ions.
Cl- displaces OH- and this is released.
Can analyze the output solutions with titration.

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36
Q

Ion-exchange resin capcity

A

Values indicate how many ions of analyte are exchanged for 1 H+ (cationic resin) or 1 OH- (anionic resin).

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37
Q

When does a group lose their ion-exchange capacity?

A

If the pH extends beyond where the charge is present.

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38
Q

Selectivity coefficient

A

Describes the selectivity of a resin for H+ or the ion analyte.
K=[R-Na+][H+]/[R-H+][Na+]

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39
Q

Cross-linking

A

Used to form insoluble hydrocarbon polymers.

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40
Q

What increases with the extent of cross-linking?

A

Selectivity of polystyrene resins.
Pore size of resin shrinks as cross-linking increases.

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41
Q

What general trends are seen in ion-exchanger favoring?

A

Ion-exchangers favor binding of ions of higher charge, decreased hydrated radius, and increased polarizability.

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42
Q

Is Pu^4+ or Li+ bound more strongly?

A

Pu^4+ (higher selectivity)

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43
Q

Is La^3+ or Ag^+ more strongly bound?

A

La^3+

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44
Q

Which is bound weaker? Mg^2+ or Al^3+?

A

Mg^2+

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45
Q

What is the measure of the amount of charge exchanged?

A

Equivalents

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46
Q

Define equivalents

A

The amount of cation that will exchange with one mole of a monovalent ion

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47
Q

Ion-exchange capacity

A

The number of ionic sites on the resin that can participate in the exchange process.

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48
Q

What are the 3 classes of ion exchangers (note their relative sized molecules)?

A
  1. Resins (small molecules)
  2. Gels (large molecules)
  3. Inorganic (harsh chemical conditions)
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49
Q

How is gradient elution used in ion-exchange chromatography?

A
  1. Increasing ionic strength
  2. Changing pH
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50
Q

List 3 other applications of ion-exchange chromatography.

A
  1. Convert one salt into another
  2. Preconcentration of trace components to obtain enough for analysis
  3. Purify water
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51
Q

Reverse Osmosis

A

water is forced through a membrane containing pores through which ions do not pass

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52
Q

What are the 3 desired attributes of a new chromatographic method?

A
  1. Adequare resolution
  2. short run time
  3. Rugged - not affected by small variations in conditions.
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53
Q

What is the expected k for a good separation

A

0.5-20

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54
Q

What is the expected resolution of a good separation

A

greater than/equal to 2

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55
Q

What are the 4 initial steps of method development?

A
  1. Determine a goal
  2. Select method of sample prep
  3. Choose detector
  4. Test initial system
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56
Q

What are some things that should be optimized in method development?

A

Sp, column (particle size), flow rate, mp composition (or add gradient elution), sample size, temperature, operating pressure, resolution, k, alpha

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57
Q

What stays the same in method transfers (column change)?

A

Flow rate

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58
Q

What variable should be checked during method development, to ensure it is close to the maximum theoretical value?

A

N, number of theoretical plates

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59
Q

What are 4 ways to adjust the separation factor?

A
  1. Fine-tune solvent composition
  2. Vary column temperature
  3. Change solvent polarity
  4. Change sp (last resort)
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60
Q

What factors add to column optimization? (3)

A

Length, particle size, flow rate

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61
Q

Supercritical fluid

A

formed above the critical T/P of a substance. A phase where distinct liquid/gas phases do not exist. Properties: exhibit solvent properties of liquids, can effuse through solids like gases, small changes in pressure/temperature have great impacts on density.

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62
Q

What is a condition to make use of supercritical fluids in HPLC?

A

Critical temperatures must lie within the operating conditions of a normal HPLC instrument.

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63
Q

What additions would need to be added to normal HPLC equipment to undergo SCFE?

A

Temperature control of column
Flow restrictor (maintains T/P as required)

64
Q

What happens in SCE with an increase in P?

A

This increases the solvent effect and shortens elution times.

65
Q

What term is better in the van Deemter eqn for SCE than HPLC and why?

A

C term is better due to lower density and lower IM forces, which leads to faster partitioning

66
Q

What are some common SCFs used for SCE?

A

CO2, N2O, NH3, n-Butane

67
Q

What detectors are used in SCE?

A

Any of GC/LC detectors
FID used commonly

68
Q

What are the advantages of SCE compared to GC and HPLC? (5)

A
  1. SCE faster than LC
  2. SCE produces less band broadening than GC
  3. Plate height is lower at higher flow rates
  4. Faster separations with no loss in selectivity or resolution
  5. Variation in mp used to change selectivity factors
69
Q

What are advantages of supercritical solvents? (3)

A
  1. Cheap
  2. Easily recovered/vented
  3. Green
70
Q

What are liquid-liquid extractions used to extract?

A

Nonvolatile analytes from an aqueous solution into an organic solvent

71
Q

What equipment is used in liquid-liquid extractions?

A

Separatory funnels

72
Q

Describe continuous liquid-liquid extractions.

A

Solvent boils from the flask and condenses into the extraction vessel. Dense droplets of solvent fall through the liquid column and extract the analyte. When the liquid level reaches a certain point, the solvent is pushed through the return tube to the solvent reservoir.

73
Q

Describe dispersive liquid-liquid microextraction.

A

Syringe used to inject liquid to create emulsion.
Mix and vortex. Centrifuge to separate extract. Remove solvent and inject extract to chromatography.

74
Q

What is solid phase extraction used for?

A

Used to concentrate and purify samples for analysis

75
Q

Describe solid phase extraction.

A

Aqueous solution with analyte is passed through a small volume of sp. The analyte adheres to sp. Concentrated analyte is then eluted.

76
Q

What does the acronym QuEChERS stand for?

A

Quick
Easy
Cheap
Effective
Rugged
Sage

77
Q

Describe the 4 steps of QuEChERS

A
  1. Analytes extracted from solid samples with ACN and centrifugation.
  2. Aliquot of extract deposited into a QuEChERS tube (containing MgSO4, ion exchangers, and other sorbents)
  3. Agitate and centrifuge so solids sink
  4. Analyze aliquot of liquid extract by chromatography
78
Q

What are the advantages of QuEChERS? (2)

A
  1. Little organic solvent required
  2. Many samples handled at once
79
Q

Mass Spectrometry

A

Technique for studying the masses of atoms/molecules/fragments using the behaviour of their ions in the gas phase under the influence of electric or magnetic fields

80
Q

3 Essential Components of a mass spectrometer

A
  1. Ion Source
  2. Mass analyzer
  3. Detector
81
Q

Most common ionization source for gases

A

EI or CI

82
Q

Most common ionization source for liquids

A

ESI and APCI

83
Q

Most common ionization methods for solids

A

LDI, MALDI

84
Q

Describe how MS works

A

Ions are created in the ion source. Neutral molecules are converted into ions by reactions with electrons or other chemical rxns (proton transfer or adduct formation). Ions are accelerated by an electric field, and they separate by their m/z ratio in the mass analyzer. An electron multiplier detector converts each ion into a cascade of electrons that reach the anode, where current is measured.

85
Q

Molecular ion

A

m/z particle that represents the intact molecule with only one electron removed

86
Q

Protonated molecule

A

[M+H]+

87
Q

Base Peak

A

Has the highest intensity

88
Q

Nominal Mass

A

the integer mass of the most abundant naturally occuring stable isotope

89
Q

Exact Mass

A

The exact mass of the most abundant naturally occuring stable isotope

90
Q

Resolution in MS

A

smallest difference in m/z values that can be detected as separate peaks, dm.

91
Q

Resolving power

A

ability of a mass spectrometer to separate 2 peaks with simila mass.

92
Q

What compounds have significant molecular ions (base peak)

A

Compounds with benzene rings

93
Q

Describe the Nitrogen Rule

A

Odd nominal mass = odd number of N atoms.
Even nominal mass = even number of N atoms.

94
Q

Ratio of Two Isotopes

A

Intensity=nx1.08% + mx0.012%

95
Q

What are the types of mass spectrometers? (6)

A
  1. Magnetic sector
  2. Transmission quadrupole
  3. Time of flight
  4. Quadrupole ion trap
  5. Linear ion trap
  6. Orbitrap
96
Q

How does a magnetic sector MS work?

A

Separates gaseous ions by accelerating them in an electric field and deflecting ions of different m/z ratios through different arcs in a magnetic field.

97
Q

What tool ensures all ions produce a similar electrical response at the detector in magnetic sector MS?

A

Conversion dynode

98
Q

In what type of MS is it necessary to use an electric sector to reduce the spread of kinetic energy of ions?

A

Magnetic sector

99
Q

How is high resolution attained in double-focusing MS?

A

An electric sector is employed with the magnetic sector to select ions with a narrow range of KE. Resolution of 10^5 is possible.

100
Q

What are the 2 most common time-of-flight mass analyzers?

A
  1. Quadrupole
  2. Ion trap
101
Q

How does the quadrupole work?

A

The quadrupole only allows 1 m/z through with a stable trajectory at a time; the retention factors and DC potentials are scanned

102
Q

How does an ion trap work?

A

The ion trap ejects 1 m/z at a time, rather than filtering.

103
Q

What does MS require to prevent molecular collisions?

A

High vacuum

104
Q

Does gas from GC or LC need to be removed prior to ion separation?

A

LC

105
Q

Describe electron ionization

A

Electrons emitted from a hot filament are accelerated through 70V before interacting with molecules M. Resulting molecular ion has extra energy to break into fragments. The original and fragment ions are accelerated to the mass analyzer.

106
Q

Describe chemical ionization

A

Ion source is filled with reagent gas around 1mbar. Energetic electrons convert CH4 into a variety of reactive products, including CH5+, which reacts with analyte M to give MH+, the most abundant ion.

107
Q

Does EI or CI produce more fragmentation?

A

EI

108
Q

Describe APCI.

A

Uses a corona discharge to create a variety of gaseous ions from aerosol droplets. Unfragmented ions are produced.

109
Q

Describe electrospray (ESI)

A

ESI employs a high voltage at the exit of the column, with coaxial N2 gas flow around the capillary. A fine aerosol of charged droplets with ions is created. Analyte is often associated with other ions. Control pH ensures selected analytes are in proper ionic form.

110
Q

Reconstructed total ion chromatogram

A

Shows the signal from all ions above a chosen m/z emerging from chromatography as a function of time

111
Q

Extracted ion chromatogram

A

shows the signal for one ion taken from the complete mass spectrum

112
Q

Selected ion monitoring

A

takes one or a few values of m/z and spends all the time measuring just that one or few ions. Improves signal to noise.

113
Q

Selected reaction monitoring (MS/MS)

A

a precursor ion isolated by one mass filter passes into a collision cell in which it breaks into products.

114
Q

How many mass analyzers are in MS/MS?

A

2

115
Q

What is the advantage of MS/MS?

A

Extremely sensitive for just one analyte

116
Q

3 Types of MS/MS in triple quad

A
  1. Product ion scan
  2. Precursor ion scan
  3. Neutral loss scan
117
Q

What would the plot of the van Deemter eqn appear as without the B term?

A

Linear

118
Q

What would the plot of the van Deemter eqn appear as without the C term?

A

Rational function

119
Q

What would the van Deemter plot appear as without the A term?

A

Slightly shifted downwards

120
Q

Why does the curve of the van Deemter plot become nearly flat at high flow rates as the particle size decreases?

A

Once the particle size is small enough, the equilibration time between the mp and sp becomes very rapid, which reduces the C term

121
Q

Why is a superficially porous column with higher particle size similar in the van Deemter plot for a normal column with lower particle size?

A

The equilibration of the solute between mp and sp is on the same time scale

122
Q

What happens when particle size in a packed column is decreased? (2)

A
  1. Requires a higher operating pressure
  2. Increased resolution
123
Q

Are the following sp RPLC or NPLC?
Octadecyl, octyl, amino, cyano, diol, phenyl, pentafluorophenyl

A

RPLC: octadecyl, octyl, phenyl, pentafluorophenyl (weak polarity)
NPLC: amino, cyano, diol (high polarity)

124
Q

In RPLC, the sp is ____

A

Nonpolar

125
Q

In normal phase chromatography, the solute is ___

A

Polar

126
Q

How do we increase eluent strength of solvent in NPLC?

A

Increase polarity

127
Q

How do we increase the eluent strength of the solvent in RPLC?

A

Decrease polarity of solvent

128
Q

In what phase (RPLC or NPLC) is the solute more soluble in the mp as the polarity of the mobile phase decreases?

A

RPLC

129
Q

What defines a strong mp in LC? (3)

A
  1. Quickly elutes an analyte from the column
  2. Analyte is more soluble in the mp than the sp
  3. Analyte has limited interactions with the sp
130
Q

Describe Evaporative light-scattering detector

A

Eluate is nebulized, forming an aerosol that is then passed through a drying tube where the solvent is evaporated, leaving analyte aerosol particles. These particles scatter light from a diode laser, generating the signal.

131
Q

What LC detector has virtually no selectivity?

A

Refractive index

132
Q

What detector has an eluate that is first nebulized, before a positive charge is added to aerosol particles as they pass over a charged Pt needle?

A

Charged aerosol detector

133
Q

Which LC detectors are compatible with gradient elution?

A
  1. UV
  2. Evaporative light scattering
  3. Charged aerosol
134
Q

Which LC detectors are not compatible with gradient elution?

A
  1. Electrochemical
  2. Refractive index
135
Q

What is the purpose of solid phase extraction prior to chromatography?

A

To isolate the analyte from the sample matrix to simplify the analysis

136
Q

How does ion-exchange sorbent retain acrylamide?

A

The strong acid of the ion-exchange resin protonates acrylamide and retains it by ionic attractions

137
Q

Why are there many peaks for acrylamide in UV?

A

UV is not specific and many components in the acrylamide extract absorb UV

138
Q

What is the purpose of a buffer in QuEChERS?

A

To protect pH sensitive analytes

139
Q

What is the purpose of acetonitrile in the QuEChERS process?

A

To extract organic contaminants from the sample

140
Q

What is the purpose of solid sorbents in QuEChERS?

A

To remove nonpolar and aromatic substances

141
Q

What component of QuEChERS absorbs residual water?

A

MgSO4

142
Q

What component of QuEChERS measures the recovery of the analyte?

A

Internal Standard

143
Q

What is the role of NaCl and MgSO4 in QuEChERS?

A

to create aqueous and organic layers and drive organic components into the organic phase

144
Q

What do anion exchangers remove in QuEChERS?

A

Absorbed anions, such as fatty acids

145
Q

Why is it beneficial to use larger particles for solid phase extraction?

A

The use of larger particles in solid phase extraction allows for more efficient analyte separation

146
Q

What is the most likely m/z value for benzene?

A

78

147
Q

Which analytical technique(s) is destructive to the sample?

A

MS

148
Q

Collision Cell

A

uses N2 or Ar gas to fragment the precursor ion into fragments called product ions

149
Q

Mass Analyzer 1

A

selects one precursor ion from the mixture of original ions

150
Q

Detector in Tandem MS

A

typically ion transducer such as an electron multiplier

151
Q

Mass analyzer 2

A

analyzes the product ions and selects which one reaches the detector

152
Q

Why is selected reaction monitoring also called MS/MS?

A

It uses consecutive mass filters

153
Q

Why is there a low level of noise in selected reaction monitoring?

A

Selected reaction monitoring exhibits low levels of noise because it is unlikely that interfering species present in the collision cell will produce fragments of the same m/z as the product ion selected for by the second mass serparator.

154
Q

Describe the process of how a magnetic mass spectrometer works

A
  1. Molecules enter the ion source where they are ionized through collisions with electrons
  2. Ions are accelerated out of the ion source and into the analyzer tube, all with the same KE, by a voltage applied between the ion acceleration plates
  3. The ions encounter a magnetic field applied perpendicular to their direction of travel
  4. The magnetic field deflects the ions on a circular path, with radius dependent on m/z, towards the end of the analyzer tube
  5. ions deflected on a circular path with a radius equal to the radius of curvature will travel through the slit leading to the detector. Heavier ions are not deflected enough and lighter ions are deflected too much to reach detector.
  6. mass spectrum created by varying magnetic field strength
155
Q

Collision-induced dissociation

A

occurs when ions are accelerated through an electric field into a region of sufficient gas P which allows collisions of the ions with the gas molecules, which produces fragments

156
Q
A

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