5: Chromatography, Mass Spectrometry Flashcards
measure of analytes ability to dissolve in aqueous or organic phase in a biphasic system
Partition coefficient
In planar chromatography: the ratio of solute’s distance travelled to the solvent’s distance travelled
Rf value
Type of chromatography in which particles for SP have pores so small that only small molecules can infiltrate them; larger molecules will be excluded
Size Exclusion chromatography
Phenomenon observed in chromatography when molecules arrive at around the same time – some early, some on time, some late. Consider the multiple paths a molecule may take thru the column
Peak Broadening
diffusion of solute along the length of the column in the flowing mobile phase; molecules are travelling down column bc of MP flow, but they are also wanting to move in different directions – may cause peak broadening
Longitudinal diffusion
flow thru column not uniform in one direction due to packings – may cause peak broadening
Eddy Diffusion
pores in packing material in which analytes infiltrate; take analytes longer to pass thru column – may cause peak broadening
Mass transport broadening
Separate equilibrations of sample between stationary phase and mobile phase happen here:
theoretical plates
more plates = more efficient separation
H = A + B/µ + C*µ
Van Deemter Equation
H = HETP = height equivalent to theoretical plate; measure of
resolving power of a column (m)
A = eddy diffusion (particle size, bed uniformity)
B = longitudinal diffusion (flow rate, MP)
C = mass transfer (particle size, SP thickness)
µ = linear velocity (m/s)
migration rate of analyte on column
retention factor/capacity factor (k’)
k’ = RT-MT/MT
where RT = retention time
MT = time for mobile phase to travel thru column (represents compound that doesn’t interact wit stationary phase)
How to calculate Relative Retention Time (RRT)
(RT of analyte)/(RT of ISTD)
How to calculate Response Ratio
(response of analyte)/(response of ISTD)
Injection mode used for larger quantities; majority of sample split off to waste and only a fraction enters column
Split
Injection mode all sample introduced to column (good for trace)
Splitless
Chromatographic detector where everything eluted off column is consumed by flame; hydrogen flame; organic compounds degraded by flame to ions which are then collected by electrode and translated into an electrical current (peak)
Flame Ionization Detector (FID)
-He (carrier gas); H2 (fuel), Air, N2 (make-up)
-Sensitive; Large dynamic range
Chromatographic detector where H2 and O2 burn to form plasma; collector electrode measures charged particles that hit it; Thermionic bead (NP bead) coated with alkali metal promotes ionization of compounds
Nitrogen Phosphorous Detector
Chromatographic detector where radioactive nickel acts as source of beta particles (slow electrons) which pass along path of carrier gas; compounds pass thru in carrier gas an capture electrons; these compounds are typically halogens (i.e. Cl-)
Electron-Capture Detector (ECD)
-Selective for electronegative species
Type of ionization method where sample exposed to 70eV electrons; energy of e- interacting w/ molecule is much greater than the bonds so ionization occurs
Electron ionization (EI)
-Sometimes called electron impact (wrong)
In electron ionization, a bond breaks in such a way that each fragment gets one of the shared electrons. The result is uncharged free radicals that won’t be detected in a mass spec.
Homolytic Cleavage
In electron ionization, a bond breaks in such a way that one atom gets both shared electrons; more common in polar bonds
Heterolytic cleavage/ ionic fission
Describe how a mass spec works.
-Effluent dumped into ion source; filament in ion source; source of electrons causes fragmentation and production of charged particles; repeller has electrical potential applied to it so it directs charged particles then into a series of lenses
-Charged particles pass through lens stack to create a beam before entering quadrupole
-Quadrupole: opposite rods have the same charge
(+ or -); continually alternate polarity of poles to help filter specific masses of ions
-Reach detector (electron multiplier): each collision along the side results in ejection of multiple electrons which repeats down the length of the horn; this helps to take a small amount of charged particles and amplify them into a measurable signal
Collison cell
Second quadrupole in a triple quad; contains argon
-ions travel thru Q1 and filter –> reach Q2 and collide with Argon to create product ions which are then filtered in Q3
Ion Trap
Type of mass spec analyzer where you apply voltages to ring electrodes to repel masses and trap them into space to accumulate; you then change voltage to selectively eject them; higher sensitivity than quadrupole
Time of Flight
masses form and send into TOF tube; impart kinetic energy on ions, smaller ions will travel faster with the same amount of kinetic as larger ions; detector converts time spent in tube to specific mass
What is baseline resolution?
1.5
Ideally, will an ISTD elute before, after, or at the same time as the compound of interest
Before
Representation of data where one or more m/z values representing one or more analytes of interest are recovered from the entire data set for a chromatographic run
Extracted Ion Chromatogram
Quadrupoles use alternating:
radio frequencies and DC voltages
the point at which the droplets break apart and a charge is imparted to the molecules within
Rayleigh Instability Limit
What types of compounds will exhibit M+ as their base peak?
Those that readily fragment
Those that do not readily fragment
Those that do not readily fragment
Ionization technique that uses a fine spray of LC effluent that is vaporized in a high temperature tube
APCI
Ionization technique that uses photons as the energy source for the ionization process
APPI
Column efficiency must be calculated from an isothermal GC or isocratic LC run
True
False
True
a variant of normal phase chromatography that partly overlaps with other chromatographic applications such as ion chromatography and reverse phase LC
Hydrophilic interaction chromatography (HILIC)
Non-Polar Stationary Phase (think opposite)
-Polar matrix
-Non-polar analyte of interest
-Aqueous samples
-Elute using non-polar solvent to disrupt analytes attraction to
stationary phase
Reverse Phase SPE
Polar Stationary Phase (think opposite)
-Polar analytes in non-polar sample
-Not as useful in toxicology
Normal Phase SPE
Which ionization technique produces less fragmentation as compared to the other?
Chemical Ionization
Electron Ionization
Chemical Ionization
Sensitive detection of organic compounds over a wide linear dynamic range:
TCD
NPD
ECD
PFPD
FID
FID
Selectivity and sensitivity for trace-level detection and analysis of compounds with electronegative functional groups:
NPD
TCD
ECD
FID
PFPD
ECD
This for an analyte is a function of temperature, pressure, stationary phase and mobile phase:
The distribution/partition coefficient
Column efficiency
The resolution coefficient
Theoretical plates
The distribution/partition coefficient
In gas chromatography, the vaporization chamber in the injection port is typically heated:
Above the lowest boiling point of the sample
To be equivalent to the lowest boiling point of the sample
Below the lowest boiling point of the sample
Above the lowest boiling point of the sample
Put these solvents in the order of increasing elution strength for a polar molecule for reverse phase HPLC: acetonitrile, water, tetrahydrofuran, methanol
water methanol tetrahydrofuran acetonitrile
__1__ water
__2__ methanol
__4__ tetrahydrofuran
__3__ acetonitrile
Base peak
the ion with the highest abundance in the mass spectrum which is assigned a relative value of 100%
The incorporation of each deuterium adds _____ mass unit(s) to the fragment in the mass spectrometer.
1
The factors that can be modified to control or modify resolution are:
Temperature, Velocity and Capacity
Pressure, Solubility and Capacity
Selectivity, Velocity and Capacity
Selectivity, Capacity and Efficiency
Temperature, Pressure and Solubility
Selectivity, Capacity and Efficiency
Absorption in chromatography is a process of:
Partitioning
Attenuation
Bulk phenomenon
Surface property
None of the above
Partitioning
The Retention Factor describes:
The distribution of an analyte between the stationary and the mobile phase
The migration rate of an analyte through a column
The velocity of the mobile phase
a and b
b and c
The migration rate of an analyte through a column
The term used when several different precursor-product ion pairs are monitored
Multiple reaction monitoring
LC ionization methods differ from GC ionization methods in that:
Polar molecules poorly ionize
A greater amount of energy is required
LC ionization requires the analyte to be stable at high temperature and pressure
The length of time it takes to ionize an analyte is longer
LC ionization can occur at atmospheric pressures
LC ionization can occur at atmospheric pressures
In a triple quad instrument, the second quadrupole is where:
Masses are stored
The mass of the ion is determined
The mass axis is calibrated
Fragmentation takes place
Detection occurs
Fragmentation takes place
In TOF testing, the time it takes for ions generated and accelerated to reach the detector is used to determine the _________ of the ion.
Mass
Retention Time
Absorbance
m/z ratio
Structure
Mass
Describes the relative interaction of two analytes with the stationary phase:
Selectivity
Column Performance
Efficiency
Capacity
Effectivity
Selectivity
Chemical Ionization
Type of ionization method where a reagent gas (i.e. methane) is ionized in a simple electron impact ion source. The now charged gas collides w/ sample molecules to produce ions or adducts.
-Different mass spectra depending on the reagent gas used.
Types of Pumps used in a Mass Spec
-Turbomolecular pump: turns fast enough to direct air in a certain direction; directs remaining molecules
-Rough pump: draws out any gasses that are ejected by turbo
