Liquid Chromatography Flashcards
n HPLC, we inject the sample, which is in solution form, into a LIQUID MOBILE PHASE.
The mobile phase carries the sample through a PACKED or CAPILLARY COLUMN that separates the sample’s components based on their ability to partition between the mobile phase and the stationary
phase
In a typical HPLC instrument, which has several key components:
– ___ that store the MOBILE phase;
– a ___ for pushing the MOBILE phase through the system;
– an ___ for INTRODUCING the sample;
– a ____ for SEPARATING the sample into its component parts; and
– a ___ for monitoring the ELUENT as it comes off the column
reservoirs
pump
injector
column
detector
The most common type of HPLC column is a STAINLESS STEEL TUBE with an internal diameter between ___ and a length between ___.
The column is packed with ___ particles with either an irregular or a spherical shape.
Typical column efficiencies are 40000–
60000 theoretical plates/m. Assuming a V /V of approximately 50, a 25-cm column with 50 000 plates/m has 12 500 theoretical plates and a peak
capacity of 110.
Analytical Columns
2.1 mm and 4.6 mm
30 mm and 300 mm.
3–10 nm porous silica
use LESS SOLVENT and, because the sample is CONCENTRATED, produce larger signals at the detector.
These columns are made from fused silica capillaries with internal diameters from ___ and lengths of ____
Capillary columns packed with ___ particles have been prepared
with column efficiencies of up to 250 000 theoretical plates
Capillary columns
44–200 μm
50–
250 mm.
3–5 μm
Two problems tend to shorten the lifetime of an analytical column.
Its solution is a ___
– (1) SOLUTES BIND IRREVERSIBLY to the stationary phase and degrade the column’s performance by decreasing the amount of stationary phase available for effecting a separation.
– (2) particulate material injected with the sample may CLOG the analytical column.
Guard Column
A Guard column usually contains the SAME PARTICULATE PACKING MATERIAL and stationary phase as the analytical column, but is significantly shorter and less expensive—a length of ___
7.5 mm
The properties of a stationary phase depend on the organosilane’s alkyl group.
If R is polar, the stationary phase is POLAr, Mobile is NONPOLAR
Example:
Normal-phase chromatography
where R contains cyano (C2H4CN)
a diol
or an amino(-C3H6NH2)
Most common form of HPLC.
The stationary phase is ___ and the mobile phase is ___.
Common examples:
Most reverse-phase separations carried out with polar mobile phase such as ___ or ___
pH must be less than 7.5 because hydrolysis attacks silica in basic conditions
Reversed-Phase Chromatography
Nonpolar, Polar
R group has C8 or C18
Methanol, Acetonitrile
For a normal-phase separation,
a solute of lower polarity spends
proportionally less time in the polar stationary phase and elutes before a
solute that is more polar.
retention times in normal-phase HPLC are controlled by adjusting the mobile phase’s properties. (make it even more nonpolar or less polar)
While in Reversed-phase HPLC, just keep Increasing polarity IN MOBILE PHASE for longer retention times (this is because the mobile phase is non-polar now compared to the polar stationary)
Changing the mobile phase’s composition as the separation progresses is one solution to this problem.
noted
A separation using a mobile phase that has a fixed composition is an ___.
For a reversed-phase separation we use an initial mobile phase that is more polar.
As the separation progresses, we adjust the composition of mobile phase so that it becomes less polar). Such separations are called
____.
isocratic elution
gradient elutions
Cant use syringe because of the pressure
Instead, we inject the sample using a ___. In the load position a
sample loop—which is available in a variety of sizes ranging from 0.5 μL to
5 mL—is isolated from the mobile phase and open to the atmosphere.
loop injector.
Ionisation methods being used in LC-MS (2)
Electrospray Ionisation (EI)
Atmospheric Pressure Chemical Ionisation (APCI)
For HPLC analysis, ANALYTE must be soluble in the ___ phase
Analyse samples over a wide polarity range
No upper molecular weight limit so large proteins can be analysed
mobile
The combination of two MS experiments.
THe only is to get structural information by fragmenting ions and to get better slectivity and sensitivity for quantitative analysis
MS/MS or TANDEM MASS spectometry
Sample prep:
Liquid
Clean liquid sample, remove particulate materials and matrix interferents
Solid samples
Dissolved in a suitable solvent or extraction
Gas samples
Bubbling them in a trap with a suitable solvent to prevent degradation
Noted
Hplc is easier than quantitative GC analysis because fo the fixed volume sample its more precise
It also does need an internal standard, just a calibration curve
noted
