SelectinganLCColumn
ColumnDimensions
ParticleSizeandColumnLength
When choosing a column, the first two
parameters that should be considered are
the particle diameter and column length.
These two parameters are themajor con-
tributors to separation efficiency (N), also
known as theoretical plates. The number
of theoreticalplates isdirectlyproportion-
al to the length of the column over the
diameter of the particle.
ParticleDiameter
Particle diameter (dp), is commonly
expressed in micrometers (µm), and has
an inverse relationship to the efficiency of
the separation. As the particle diameter
decreases, the efficiency of the separation
increases proportionately. If all other
parameters remain equal, a 3 µm particle
diameter offers an approximate 60%
increase in efficiency over a 5µmparticle,
and a 1.9 µm particle diameter offers an
additional 60% over a 3 µm particle.
System backpressure also increases pro-
portionally as particle size decreases.
Selecting the proper particle diameter is a
way of controlling separation efficiency,
and even analysis speed, but is limited by
the pressure capabilities of the system.
Often, particle diameters are determined
by instrumentation. Table I is a guideline
for selecting the optimal particle size,
based upon pressure capability for com-
monmobile phases.
When choosing aparticlediameter, it isnot recommended tooperate significantlybelow theoptimal linear velocity, as losses in effi-
ciencycanbeobserveddue toaxial dispersion.As aquickestimateof particlediameterusability, check theoptimal linear velocity for
the organic solvent used and ensuremaximumpressures observed arewithin the pressure specifications of your instrument. Please
note that thesearemaximumpressuresobservedduringgradient analyses. Isocraticmobilephasesof lesserviscositywilloperatewith
less backpressure.
ColumnLength
Column length (L) directly relates to efficiency. Increasing column length increases efficiency. It is important to note that column
length is not an ideal way to increase resolution. Doubling the column length yields only a 1.4x gain in resolution (efficiency is a
square root term in the resolutionequation),whiledoublingbothanalysis timeand systembackpressure.Shorter column lengths are
suitable for fast gradients andhigher sample throughput,while longer column lengths aremore suitable forhigherpeakcapacityand
shallow gradients.
Column InternalDiameter
Column internal diameter (ID) is the inner diameter of the column hardware holding the packing material, and is commonly
expressed inmillimeters (mm).Column ID is ultimately related to efficiency and flow rate through the vanDeemter equation.This
chromatographic concept relates column efficiency (often called band broadening) to linear velocity. Linear velocity is the distance
mobile phase travels per unit time,while flow rate is the volume of mobile phase per unit time.A specific linear velocity has a flow
rate that is dependent upon the internal diameter of the column. As column ID is lowered, a lower flow rate is needed to
maintain the same linear velocity. Flow rate is the volumeofmobilephaseneeded to create thedesired liner velocity. It is important
tonote that as particle size decreases, optimal linear velocity increases.Columnswith smaller particle sizes, namely 1.9 and 2.2µm,
are capableof runningmuchhigher flow rates and therefore creatinghigher sample throughput.Table II (next page) canbeused to
find the optimal flow rate, as it relates toparticle size and internal diameter, and is a good startingpoint formethoddevelopment.
800-356-1688or 814-353-1300
145
HPLCCOLUMNS
Column Selection
Table I
Empericallydeterminedmaximumpressuresexhibited for
acetonitrileandmethanol gradients for variousparticle sizesand flow rates
Flow rate
Pressure (psi)
Flow rate
Pressure (psi)
(mL/min.)
Acetonitrile@25°C
(mL/min.)
Methanol@25°C
1.9µm 2.2µm 3µm
1.9µm 2.2µm 3µm
0.2
2436
1755
1045
0.2
3198
2304
1371
0.3
3655
2633
1567
0.3
4797
3455
2057
0.4
4873
3510
2090
0.4
6395
4607
2743
0.5
6091
4388
2612
0.5
7994
5759
3429
0.55
6700
4826
2873
0.55
8794
6335
3771
0.6
7309
5265
3135
0.6
9593
6911
4114
0.7
8527
6143
3657
0.7
11192
8062
4800
0.8
9745
7020
4180
0.8
12791
9214
5486
0.9
10964
7898
4702
0.9
14390
10366
6171
1
12182
8775
5224
1
15989
11518
6857
Data are for 2.1 x 50mm columns using a gradient of 5%B to 95%B (A: water, B: organic solvent).
SeeTable II for optimal flow rates for alternate column internal diameters.
Bold blue numbers represent optimal linear velocity for the given particle size and ID. For longer
column lengths, the approximate pressure corresponds to the increase in column length. A 2-fold
increase in column length yields a 2-fold increase in back pressure.
Equation1
The resolutionequationdefinesvariablesaffecting separations.
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Mar 2011
