Chapter 3 - HPLC columns and trends Flashcards
Column support
Spherical/irregular, porous particles used as column packing material.
Bonded phases
Stationary phase; support material modified with functional groups.
Packing material
A term that can refer to both the support or the bonded phase.
IBW
Instrumental bandwidth or system dispersion.
Hybrid phases
Support synthesized from both inorganic and organic materials.
SPP
Superficially porous particle.
Dwell volume
Volume measured from the point of the solvents mixing in the HPLC system to the head of the column.
Most common column length
150 mm
Most common inner diameter
4.6 mm
Most common particle size
3 μm C18
Most common flow rate
1 ml/min
Most common sample loading size
1 μg
Most common sample loading size
1 μg
Most common operating pressure
3000 psi or 200 bar
Common column lifetime
2000 injections
Void volume, sample capacity, and operating flow rate are proportional to
dc2
The limit of detection for minimum detectable mass is inversely proportional to
dc2
Longer columns have _______ plate counts and yield _______ resolution.
higher; better
Since plate numbers are additive, columns can be connected in series to produce
higher efficiency and resolution at the expense of analysis time and mass sensitivity.
Silica (SiO2) is the dominant support material because of its
mechanical strength, modifiable surface chemistry, and high efficiency.
Silica based bonded phases have a pH range of
2-8
Most chromatographic supports are porous to
provide a high surface area to maximize the interaction of solutes with the bonded phase.
Traditional problem areas of silica-based RPC columns.
- Batch to batch inconsistency
- Peak tailing of basic analytes
- Short column lifetime
- pH limitations
Silanol group formula
Si-OH
Hydrolysis
The reaction of an organic chemical with water to form two or more substances.
Bulky groups are incorporated within the silane to
sterically protect the acid-labile Si-O-Si bonds against acid hydrolysis.
Polar-embedded phases are not prone to phase collapse due to the
hydrophilicity of the polar-embedded group.
An AQ designation of the bonded phase means
that the stationary phase is compatible with 100% aqueous mobile phases.
Micro LC and nano LC benefits
- Increased mass sensitivity due to lower sample dilution
- Better compatibility with LC/MS
- Less solvent used
Superficially porous particles (SPP) are made of
a solid, nonporous core surrounded by a porous shell layer.
A guard column is a
small packed cartridge/column placed before an analytical column to protect it from contaminants.
The standard determination of enantiomers is HPLC using _______ stationary phases.
chiral
General guidelines for column selection.
- Select columns packed with 3- or sub 3 μm high-purity silica-bonded phases. Hybrids and SPP are highly recommended.
- For resolution of coeluting analytes on C18 columns, explore selectivity differences of polar-embedded, phenyl, PFP, or cyano phases.
PFP stands for
Pentafluorophenyl
SPP stands for
Superficially porous particles
