HPLC COLUMNS
Column Selection
2
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Figure1
Stationaryphase selection.
SampleSolubility
ReversedPhase
NormalPhase
Neutral
Ionic
Acidic
Aqueous
C18
IBD
Organic
Acids
Basic
Amino
Cyano
Basix
IBD
PFP
PFPPropyl
Hydrophilic
Aqueous
C18
IBD
Organic
Acids
C1
C4
C8
Hydrophobic
C18
Phenyl
Biphenyl
Mixed
Basix
IBD
Silica
Cyano
Amino
IBD
Nonpolar
Organics
Wateror
PolarOrganics
SelectinganHPLCColumn
Choosing the best column for your application requires consideration of stationary phase chemistry,
retention capacity, particle size, and column dimensions. Identifying the best stationary phase for your
separation is the most critical step of column selection, and your decision should be based on sample
solubility and the chemical differences among the compounds of interest. Figure 1 is a handy tool for
stationaryphase selection.
Reversedphase columns (e.g. alkyl,phenyl, cyano)workwell forwater-solublehydrophobic compounds.
Some stationary phases incorporate both polar and nonpolar functionality and can be used in either
reversed phase or normal phasemodes (e.g. Ultra IBD, Allure® Basix, andAllure® PFP Propyl).While
straight chain alkyl (e.g. C18) stationary phases are historically themost commonly used, many newer
phases provide better separations. Alkyl phases are best suited for analyzing neutral compounds with a
high ratio of carbon:heteroatoms where the major distinction among analytes is their hydrophobicity.
However, for analyzing compounds that are highly polar, aromatic, or halogenated, nonalkyl stationary
phases oftenprovide significantlybetter selectivity (Figure 2).
Retention capacity is another important consideration and is influencedby surface area and carbon load
(% carbon in thepackingmaterial).Allure® columnsweredesigned formaximum retentionusingahigh
densityof ligandsbonded toa large surface area silica.Ultra,Kromasil®,Pinnacle™ II andPinnacle™DB
columns have the same high liganddensity, but aremoremoderately retentive due to their lower surface
areas. Surface area is inversely proportional to pore size; thus, larger pore sizes result in less retention.
However, wide pore (e.g. 300Å) packings, such as Viva, are ideal when analyzing larger molecules, as a
larger pore size is necessary to allow the analytes to ‘fit’ into the pores.
Particle sizeandcolumndimensionsalso influencecolumnchoice. In selectingaparticle size, theprimary
consideration is efficiency (plates/meter) versus columnpressure.A3µm columnwill have approximate-
ly 50%more efficiency than a 5µm column, if all other conditions are constant for both columns. As
particle size is further decreased (e.g.<2µm), theoretically, efficiencieswill increaseproportionally, based
on theVanDeemter equation (and the usable flow rate range ismuchwider). Please note that column
backpressure also increases as particle size decreases. Columndimensions include internal diameter and
length, where themost commonly used internal diameter (ID) forHPLC columns is 4.6mm. In theory,
resolution andpressure shouldbe independent of column ID as long as flow rate is adjusted tomaintain
the samemobile phase linear velocity (flow rate is proportional to column cross-sectional area). Table I
shows the approximate optimum flow rates for four column IDs.
Table I
Approximateoptimum flow rates for variousanalytical column IDs.
5µmParticles
3µmParticles
ID (mm)
Optimum FlowRate (mL/min.)
Optimum FlowRate (mL/min.)
4.6
1.00
1.5
3.2
0.50
0.73
2.1
0.20
0.31
1.0
0.05
0.07
HPLCTechTipsWall Chart
Almost everything you need to
remember about HPLC, con-
densed into 3 feet by 2 feet:
mobile phase basics, buffers
(types, pK a values, pH ranges,
formulamasses, more), misci-
bility and solubility chart
(invaluable!), system setup and
optimization, detector tips,
pressure conversion factors,
most-used chromatographic
equations, column storage
essentials.
(lit. cat.# 59894A)
Free
Literature
ordering
note
For assistance in selecting
anHPLC column, please
contact RestekTechnical
Service at 814-353-1300 or
800-356-1688 (ext. 4) or
.
