chroma Flashcards
are multistage separation
methods in which the components of
a sample are distributed between
two phases, of which one is stationary
and the other is mobile.
Chromatographic separation
techniques
separate different components present in a sample since samples contain a mixture
polar and nonpolar, ionic or non ionic compounds
Chromatographic separation
techniques
may be a solid or a liquid
supported on a solid or a gel
STATIONARY PHASE
may be packed in a column,
spread as a layer, distributed as
a film, or applied by other
techniques
STATIONARY PHASE
may be in a gaseous or liquid
form, or a supercritical fluid
MOBILE PHASE
any substance at a temperature
and pressure above its critical point, where distinct
liquid and gas phases do not exist, but below the
pressure required to compress it into a solid.
supercritical fluid -
supercritical fluid -
Border of being a liquid
and being a gas.
SP of Ion exchange
negatively charged
beads
Separated by size by
virtue of density
Size Exclusion
pressure needed when the column is very
packed
high pressure
T/F: Column is a very porous material but need
help of pressure when it is very packed
TRUE
Gas chrom SP:
liquid or solid
Liquid Chromatography SP:
solid or liquid
Gas chrom MP:
gas
Liquid Chromatography MP
liquid
a graphical representation of the
detector response,
concentration of analyte in the
eluent, or other quantity used as
a measure of eluent
concentration versus eluent
volume or time
CHROMATOGRAM
volume between the point at
which the eluents meet and the
top of the column
DWELL VOLUME/GRADIENT DELAY VOLUME
T/F:dwell volume: mobile phase
T
Air can cause a __________ on the chromatogram
peak
the time required for elution of an unretained component
HOLD-UP TIME (TM)
shown as an
air or unretained solvent peak, with the baseline scale in minutes
HOLD-UP TIME (TM)
the volume of mobile phase required for elution of an unretained
component
HOLD-UP VOLUME (VM)
time for the unretained component to be eluted
Tm
Volume required to push the unretained component =
Vm
Faster flow rate - ________ components are removed
more components are removed
formula for Vm
Vm = Tm (hold-up time) x F
a measure of column efficiency
NUMBER OF THEORETICAL PLATES (N)
depends upon the substance being chromatographed as well as the
operating conditions, such as the flow rate and temperature of the
mobile phase or carrier gas, the quality of the packing, the uniformity of
the packing within the column, and, for capillary columns, the thickness
of the stationary phase film and the internal diameter and length of the
column
NUMBER OF THEORETICAL PLATES (N)
Bell curve
Gaussian peaks
Used in industry
w/ electronic integrators
the portion of the chromatographic recording of the detector response
when a single component is eluted from the column
PEAK
if separation is incomplete, two or more components may be eluted as
one unresolved peak
PEAK
employed as a system suitability criterion in a test
for related substances when baseline separation
between two peaks is not achieved
PEAK-TO-VALLEY RATIO (p/v)
ratio of the adjusted retention time of a component relative to that of
another used as a reference, obtained under identical conditions
RELATIVE RETENTION (r)
“unadjusted relative retention”
RELATIVE RETENTION TIME (RRT)
the separation of two components in a mixture
RESOLUTION (RS)
the time elapsed between the injection of the sample and the
appearance of the maximum peak response of the eluted sample
zone
RETENTION TIME (tR
may be used as a parameter for identification
RETENTION TIME (tR)
characteristic of the compounds they represent but are not unique
RETENTION TIME (tR)
Not used to characterize compounds
since it is not unique but it is a parameter
for identification. L
RETENTION TIME (tR)
volume of mobile phase required for elution of a component
RETENTION VOLUME (VR)
relative retention calculated for two adjacent peaks (by convention,
the value of the separation factor is always >1)
SEPARATION FACTOR (⍺)
“tailing factor”, of a peak
SYMMETRY FACTOR (AS
system suitability tests is done at the
start
integral part of GC and LC methods
SYSTEM SUITABILITY TESTS
used to verify that the chromatographic system is adequate for the
intended analysis
SYSTEM SUITABILITY TESTS
pH of the mobile phase (HPLC):
based on the concept that the equipment, electronics, analytical
operations, and samples analyzed constitute an integral system that
can be evaluated as such
SYSTEM SUITABILITY TESTS
. concentration of salts in buffer (HPLC)
ratio of components in mobile phase (HPLC): amount of the minor
component (≤50%) can be adjusted ________ but cannot exceed ______-
in relation to the total mobile phase
± 30%; ± 10%
wavelength of UV-Vis detector (HPLC):
± 3 nm
stationary phase column length (GC):
stationary phase column inner diameter (GC):
: ± 50%
. particle size (HPLC): -
-25% to 50% of the prescribes column length and
particle size ratio
flow rate (GC):
± 50%
flow rate (HPLC):
± 50% (isocratic)
column temperature (HPLC):
± 10°
oven temperature (GC):
± 10%
oven temperature program (GC):
± 20%
A gaseous mobile phase flows under pressure through a ___________
either coated with a liquid stationary phase or packed with liquid
stationary phase coated onto a solid support
heated tube
Principle of Gas Chrom:
The analyte is loaded onto the
head of the column via a ______ , where it
evaporates. It then condenses at
the head of the column, which is
at a lower temperature.
heated
injection port
liquid stationary phase is deposited on a
finely divided, inert solid support, such as
diatomaceous earth, porous polymer, or
graphitized carbon, which is packed into a
column that is typically 2–4 mm in internal
diameter and 1–3 m in length
PACKED COLUMN
T/F: Packed Column causes less problem in sample introduction
T
T/F: Packed Column do not produce high-resolution
chromatography
T
contain no packed solid support, the liquid
stationary phase is deposited on the inner
surface of the column and may be
chemically bonded to it
CAPILLARY COLUMN
higher efficiency
larger sample volume neede
most commonly used carrier gas in
capillary GC is helium
CAPILLARY COLUMN
A liquid mobile phase is pumped under pressure through a stainless
steel column containing particles of stationary phase with a diameter
of 3–10 mm (1.7 mm in ultra- high-performance liquid
chromatography (UPLC))
HPLC
The analyte is loaded onto the
head of the column via a loop
valve and separation of a
mixture occurs according to the
relative lengths of time spent by
its components in the stationary
phase.
HPLC
most commonly used stationary phases are modified silica or
polymeric beads
HPLC
beads are modified by the addition of
ong-chain
hydrocarbons
(HPLC) Mobile phase in Normal Phase
NP
(HPLC) Mobile phase in Reverse Phase
Polar
includes stainless steel, lined stainless steel, and polymeric
columns, packed with a stationary phase
CHROMATOGRAPHIC COLUMN
the chromatographic technique which has seen the
most intensive development in recent years, leading to
improved columns, detectors and software
control.
HPLC
(HPLC) The variety of columns and detectors means that the selectivity of the method can be .
readily adjusted
less risk of
sample degradation Compared to gas chromatography (GC
HPLC
