HPLC Flashcards
HPLC can be divided into the following categories:
- adsorption
- ion exchange
- size exclusion
- partition chromatography
> reverse
> normal
the composition of mobile phase
- solvents
- buffers
- mobile phase modifiers
> ex: triethylamine (can dramatically influence retention time)
the choice of solvent affects ________ and ________
selectivity and retention
solvents often used in the lab
methanol*
DMSO
ethanol
acetonitrile*
tetrahydrofuran
dioxane
isopropanol
what are buffers used for in HPLC?
- control pH
- reduce peak tailing
- give well-shaped narrow peaks
pH affects selectivity (separation of compounds relative to one another)
criteria for choosing a detector
sensitivity
detectability
linearity
reproducibility
peak shape
flow and temp
a resolution of _____ or greater is considered necessary for good chromatographic analyses
1.25
k’
capacity factor
measure of degree of retention
how is retention time easily adjusted?
by changing the amount of organic solvent
rule of thumb: __% change in fraction of organic solvent in water will cause ___ or ____-fold change in k’
10%; two-three
isocratic mode
mobile phase composition remains constant throughout the chromatographic run
gradient mode
the mobile phase composition is either changed in a stepwise or continuous fashion throughout the run
___________ __________ of the analytes between the mobile phase and the stationary phase of the column results in their separation
differential eqm
what is HPLC?
separation technique
mixtures of compounds can be resolved by exploiting differences in their chemical and physical properties
stationary = column
mobile phase = solvent/buffers
a partition system in which the stationary phase is more polar than the mobile phase
normal phase
T or F. Normal phase more popular
F! Reverse phase; more popular in biological applications b/c of polar nature of many bio compounds
the choice of solvent affectes these (2)
retention and selectivity
chosen based on their polarity
solvents usually used
methanol and acetonitrile low UV cutoff; won’t contribute to background noise
DMSO, ethanol, tetrahydrofuran, etc.
For LC-MS/MS how does acetonitrile compare to methanol?
increases ionization efficiency
- lower sampel viscosity means fine droplets produced
what are buffers used for in HPLC?
control pH = pH affects selectivity or separation of compounds
reduce peak tailing
give well-shaped narrow peaks
The _________ of an analyte changes rapidly when changes in pH are within +/- pH unit of the pKa of the analyte
retention
ion suppressed analytes = better retention than analyzed analytes
which buffers are preferred in LC-MS/MS
volatile buffers
- minimizes ion suppression and maintains sensitivity
non-volatile buffers
phosphate
popular in HPLC but can lread to contam of ion source so not used in LC-MS/MS
what is back pressure?
the force necessary to push a liquid mobile phase through a tightly packed bed of tiny particles
- even at low flow rates
frequently the source of elevated back pressure
particulates
- sample
- mobile phase
- instrument wear and tear
how to troubleshoot back pressure
start at detector and work backwards up the flow path using systematic approach
what are detectors responsible for?
detecting compounds as they elute from column
criteria for choosing detector
sensitivity
detectability
linearity
reproducibility
peak shape
flow and temp
examples of detectors
UV
fluorescence
electrochemical
MS
etc.
fluorescence detector theory
ability of molecule to emit light after excited by light radiation
since many molecules do not fluoresce, numerous methods for deriving compounds have been developed
amperometric detector theory
- electrochemical detector
- column effluent flows past an electrode to which voltage is applied
- voltage = large enough => analyte molecules at interface between electrode and solution can either accept e- and be reduced or give up e- to be oxidized
- net movement of e- = current flow; current is proportional to conctn of analytes
t0
void time
time required to elute non-retained substances
the time that has elapsed from injection of sample into chrom. system to recording of peak max of the component of chromatogram
retention time (tR)
resolution
R
degree of separation between two components by chromatography
a resolution of _____ or greater is considered necessary for good chromatographic analyses
1.25
resolution is controlled by factors that affect:
- peak retention
> capacity factor [k]
> selectivity [alpha] - peak width
> efficiency [N]
the most cost- and time-effective approach approach to improvement of resolution
- first adjust the capacity factor (k), then selectivity factor (alpha) and then the efficiency factor
k’
(tR-t0)/t0
measure of retention time
alpha
k’2/k1
- measure of tretention time of two peaks relative to each other
N
measure of peak width
16(tR/W)^2
measure of the degree of retention
k’
capacity factor
how do we adjust retention time?
by changing the amount of organic solvent
T or F. A 10% change in the fraction of organic solvent in water will cause a two- or threefold change in k’
T
this is the most important factor in terms of resolution
selectivity factor
because selectivity is a function of the column packing, the __________ phase and the solute chemistry can be manipulated by changing: (4)
mobile
- composition of mobile phase (organic solvent, buffer and pH)
- stationary phase
- sample chemistry through derivatization
- separation temp
obtained by calculating the number of theoretical plates for a column
efficiency of a column (N)
what is a theoretical plate?
microscopic segment of a column where a perfect eqm is assumed to exist between the solute in the mobile and stationary phase
what does a large number of theoretical plates mean?
indicates relatively narrow peaks and thus, an efficient column
T or F. The larger the N, the more efficient
T
T or F. To maximize column efficiency, band broadening must be increased
F! DECREASED
- accomplished by using a well-packed column that contains a stationary phase packing that is small and uniform with respect to size distribution of particles
how to maximize column efficiency (4)
- decrease band broadening; well-packed column
- increase column length
- increasing mobile phase flow rate will also help but only to a certain pt
- less is better with vol of injection
reasons for extraction of samples
to prolong life of column
to see only peaks of interest
to improve sensitvity
why do people prefer LC-MS/MS?
short run time but sensitive and specific
