146
800-356-1688or 814-353-1300
HPLCCOLUMNS
Column Selection
System volume, or extra column volume, also affects efficiency. As extra column vol-
ume increases, lower efficiency is experienced as bandbroadening increases. Typically,
column IDs less than 3.0mm, considerednarrow bore columns, require systems with
minimizedextracolumnvolume.Table IIIdefines theclassificationof columnsaccord-
ing to internal diameter or bore. Another contributor to overall system volume and
column ID choice is the system delay volume. Delay volume is the volume contained
between thepumps and thecolumn,often including themixingchamber and injection
valve. Delay volume is especially significant during gradient analysis. Narrow bore
columns often require lower flow rates, and these lower flow rates will not sweep the
delayvolume inhighvolume systemsquickly.This extends analysis timeand creates an
increasedgradient lag time.For fastgradientanalysisandLC/MS,narrowborecolumns
and systemswith low extra column volume are recommended.
PhysicalCharacteristics
SilicaType
Thephysical characteristicsof the supportmaterial canbe selected to control retention
andpeak shape.Thebase silica, commonlyporous spherical particles,used in theman-
ufacturing of the column can first be selectedby type, namelyTypeA, TypeB, or Base
Deactivated.TypeB silica is typicallyhigher inpurityandprovides limited silanol activ-
ity.Whenanalyzingbasiccompounds, especiallywithout theuseofmobilephasemod-
ifiers, TypeB silica is recommended formore symmetric peak shape. TypeA andBase
Deactivated silica are recommended for acidic, neutral, and slightlybasic compounds.
Another criterion for choosinga column line is theporosityof the silica.Thepore size,
or pore diameter, which is commonly expressed in Å, is the average diameter of the
silicapores.This relates inversely to available surface area. Smaller pore volumes create
a larger surfacearea inagivenparticleand, therefore,canbeused tocontrol theamount
of stationaryphase bonded to the particle.
The carbon load, or% carbon in the packingmaterial, is themeasure of the amount,
or load,of stationaryphase.Carbon loaddirectlyaffects retention.Highercarbon loads
typically result inhigher retention characteristics. Figure1 illustrates the relative reten-
tion capacities of commercially available columns for hydrophobic compounds.
Allure® columns were designed formaximum retention of smallmolecules by utiliz-
ing high carbon load, surface area, and ligand density. In contrast, Viva columns,
considered wide pore, have a large pore diameter and are used for the analysis of
largermolecules as commonly seen inbiological separations.Table IV summarizes the
physical characteristics and recommendeduses forRestek column lines.
Silicacolumnscommonlyhavea temperature limitof 80 °C. Increased temperaturecan
be used todecreasemobile phase viscosity and, therefore, lower the backpressure of a
Table IV
Physical characteristicsand recommendeduses forRestekcolumns,
basedon silica lines.
Table III
Commonclassifications
for LCcolumnsby internal diameter.
Classification
Internal Diameter
Capillary
<1.0mm ID
Micro bore
1.0mm ID
Narrow bore
2.1-3.0mm ID
Standard bore
3.2-4.6mm ID
Semi-prep
10 to 21.2mm ID
Prep
30 to 50mm ID
Column
Pore Surface
Carbon
Line
Size
Area Load Range*
(Å)
(m
2
/g)
(%)
Usage
Allure
60
450 12–27 Very high retention (highest retention available)
High purity
5µm particle size only
Ultra II
100
300 11–19 High retention
High purity
Full range particle size - 1.9, 2.2, 3 and 5µm for UHPLC andHPLC
Ultra
100
300
2–20 High retention
High purity
3 and 5µm particle size only
Pinnacle II
110
180
2–13 Moderate retention
AcidicType A (not for RP analyses of bases)
3 and 5µm particle size only
PinnacleDB
140
150
4–11 Moderate retention
Base deactivated silica
1.9, 3 and 5µm particle sizes
Viva
300
100
3.5–9 Low retention
Wide-pore silica for biological separations
Table II
Optimal flow rates for
variousparticlediametersand
column internal diameters.
Column ID
Optimal flow rate
(mm)
(mL/min.)
1.9µm dp 2.2µm dp 3µm dp 5µm dp
4.6
1.50
1.00
3.2
0.73
0.50
3.0
1.12
1.00
0.65
0.40
2.1
0.55
0.47
0.31
0.20
1.0
0.07
0.05
*Ranges are based on phases available for each silica line. See column product listings formore specific
information.
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Mar 2011
