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HPLC Column Dimensions.

HPLC columns are manufactured in a variety of different internal diameter and length combinations, as well as having an assortment of particle sizes.  HPLC column dimensions will affect the efficiency sensitivity, and speed of an analysis. The choice of column dimensions will depend on the chromatographic application; analytical, semi-preparative, preparative, number of analytes in the mixture etc.  However, the dimensions can also be altered to improve chromatography by achieving more efficient, sensitive, and faster analyses.  The primary column dimensions of particle size, length, and internal diameter will be discussed and their effect on chromatographic separations demonstrated, providing some practical insight into how to select the best column dimensions for a particular application.

 
: HPLC column dimensions Figure 1: HPLC column dimensions and the chromatographic parameters which they influence.
 

 
Particle Size
The particle size of the support material is of primary importance when selecting a stationary phase.  By reducing the particle size diameter, not only is the efficiency of the column increased (plate height (H) is reduced), but the optimum linear velocity at which the minimum plate height is achieved is also increased.  More efficient peaks can be achieved at elevated flow rates, leading to a reduction in analysis time accompanied, potentially, by an increase in resolution.  Shorter diffusion paths and, hence, increased mass transfer kinetics (C term from the Van Deemter equation) are one reason behind this reduction in plate height.  Figure 2 shows the relationship between particle size, flow rate (linear velocity), and plate height for columns packed with various silica particle diameters.
 
Van Deemter curves for various particle diameters

Figure 2: Van Deemter curves
for various particle diameters.

 

It may seem that the smallest diameter particles will be the obvious choice for high efficiency separations; however, this benefit does come at a substantial price, that of increased back pressure.  The pressure increase is inversely proportional to the square of the particle diameter as shown in Equation 1.

Equation 1
 

Where any of the numerators increase, flow (F) or column length (L) for instance, a resultant increase in back pressure will be observed.  Where any of the denominators decrease, column radius (r) or particle size (dp ) for instance, an increase in back pressure is also observed.  Note that the radius and particle size functions are raised to the second power, thus causing their impact on back pressure to be much more dramatic.

When selecting the appropriate particle size for an application some useful guidelines are as follows:

  • Smaller diameter particles - complex mixtures with similar components and high throughput separations
  • Large particles - routine analyses where analytes have greater structural differences
  • Particles 10+ µm - preparative HPLC

Particle size particularly affects the efficiency term of the fundamental/Purnell resolution equation (Equation 2).  Efficiency is inversely proportional to particle size (Equation 3); hence, a decrease in particle size will result in an increase in efficiency.  This increase in efficiency will allow the use of shorter columns and/or faster flow rates which will ultimately result in faster analyses without loss of resolution, as is demonstrated in Figure 3.  Note the increase in system back pressure caused by reducing the particle size.

Equation 2   Equation 3
 
Effect of column length  

Figure 3: Effect of column length on efficiency and resolution.1-2

 

 

Column: Hypersil GOLD 200 x 2.1 mm
Mobile phase: A -H2O; B - MeCN
Gradient: 65-95 %B in 1.5 min, hold for 1.5 min
Flow rate: 600 µL/min
Temperature: 40 °C
Detection: 247 nm
Injection volume: 0.2 µL

  1. Acetophenone
  2. Propiophenone
  3. Butyrophenone
  4. Valerophenone
  5. Hexanophenone
  6. Heptanophenone
  7. Octanophenone
 

 

Particle Size Distribution
The particle size distribution is another key parameter that should be considered; however, as this is not a user controlled variable, little practical attention is given to it (Figure 4).

Any particle size stated for an HPLC column (e.g. 5 µm) will actually be the mean value from a distribution of particle sizes, achieved practically by the manufacturer using automated sub-sieve sizing techniques.  In reality a wide distribution of particles will lead to heterogeneity within the column and, as such, an increase in variable path lengths the analyte molecules can take through the column (Van Deemter A term, often called the column packing term) which will give rise to band broadening.

D10/D90 ratios are often quoted for packing materials as a relative measure of the particle diameter distribution.  D10 = particle diameter at 10% of the total size distribution and D90 = particle diameter at 90% of the total size distribution.  The closer this value is to unity, the more homogeneous the particle diameter distribution.

This phenomenon has contributed to the sudden upsurge in the commercial availability of superficially porous silica (SPS) or core-shell particles with small pore volumes (80 Å - 120 Å) which exhibit increased efficiency/reduced plate height for the analysis of low molecular weight analytes.  SPS particles have been available in their 300 Å guise for the analysis of high molecular weight analytes for a number of years because of the improved mass transfer kinetics (C term) which were debilitating for conventional porous microspheres.  These particles consist of a solid silica core with a layer of porous silica deposited around the outside, effectively shortening the path length for diffusion within the internal pore structure and improving mass transfer kinetics.

However, for lower molecular weight analytes (< 600 Da) the effects of increased mass transfer are less important and the increase in efficiency/reduction in plate height attributed, in the main part, to the very narrow particle size distribution that can be achieved from creating a particle with a solid core and the reduction of multiple path effects (A term).

: HPLC column dimensions Figure 4: Particle size distribution
 

 

Length

Efficiency is directly proportional to column length (Equation 3).  Increasing column length will increase efficiency.  Doubling column length increases resolution but only by a factor of 1.4.  Short column lengths (30-50 mm) will give short run times and low backpressures and are ideal for gradient analyses.  Longer columns (250-300 mm) will give greater resolution but with longer analysis times and at a greater cost. 
images/column-dimensions-EQ03.jpg
The separation of four paraben compounds detailed in Figure 5 demonstrates the effect of column length.  As can be seen decreasing the column length reduces analysis time, however, resolution is also decreased.
Effect of column length  

 

Figure 5: Effect of column length.3

 

 

Column: Hypersil GOLD
Mobile phase: H2O/MeCN (50:50) + 0.1% formic acid
Temperature: 30 °C
Flow rate: 0.2 mL/min
Detection: UV 254 nm
Injection volume: 1 µL

  1. Methyl paraben
  2. Ethyl paraben
  3. Propyl paraben
  4. Butyl paraben

 


Internal Diameter
As was shown previously (Equation 1) a decrease in column internal diameter will result in an increase in back pressure.

column dimensions EQ01

Larger diameter HPLC columns require higher flow rates; therefore, larger volumes of mobile phase will be used.  Changing from a 4.6 mm (1) to a 3.2 mm (2) ID column can reduce the flow rate and solvent volume required to reach the same optimum linear velocity without increasing the run time.  The new flow rate can be calculated using Equation 4.

column dimensions EQ04
 

Where:
F = flow rate (mL/min)
dc = column diameter (mm)
dp = particle diameter (µm)

Note: For columns with the same particle size the last term becomes obsolete.

When column internal diameter is decreased an increase in sensitivity (2-3 fold) can be expected when injecting the same analyte mass.  This is due to there being an increased analyte concentration in the mobile phase.

HPLC instrumentation may need to be adapted for columns with very narrow internal diameters to minimize band broadening effects which result from extra column effects i.e. mixing volumes out with the column; these can be reduced by reducing tubing length and diameter, using a microvolume detector flow cell etc.  Instrument manufacturers will be able to aid in recommendations for these types of alterations.

Generally columns with an internal diameter of 2-5 mm are used for analytical applications. 
For preparative applications the column internal diameter is much larger at 10-25.4 mm. 

A change in column diameter will affect the loading capacity, both the volume of sample and the mass of analyte which can be injected onto the column, along with the flow rates which can be used.  Some guidelines for these parameters are listed in Table 1.

 
Internal Diameter (mm) Typical Injection Volume (µL) Loading Capacity (mg) Typical Flow Rates
4.6 15 1 0.5-2
10 100 4.7 4-15
21.2 400 19.5 10-50
30 1000 42.5 40-100
50 2000 118 100-300
100 10000 473 400-1000

Table 1:
HPLC column dimensions, capacities, and flow rates.

 


Effect of Column Dimensions on Isocratic and Gradient Elution
It’s interesting to draw a quick but effective analogy between isocratic and gradient HPLC.

In isocratic HPLC, the analytes enter the HPLC column and, depending upon the partition co-efficient of the analyte between the mobile and stationary phase material (governed by many factors including the hydrophobicity of the analyte and its shape), moves at a constant pace along the column, undergoing successive partitioning events into the stationary phase, which controls analyte retention. Retention in isocratic HPLC is measured using the retention factor (k) which is defined as (Equation 5):
 
column dimensions EQ05
 

Where:
t0 = retention time of a compound which does not interact with the stationary phase surface (typically estimated using the injection solvent peak or a compound such as Uracil in reversed phase HPLC or hexane for normal phase HPLC).

In gradient HPLC, things are somewhat different.  The gradient is formed by increasing the percentage of organic solvent.  Consequently; at the beginning of the analysis, when the mobile phase strength is low, the analyte will be partitioned wholly into the stationary phase (or ‘focused’) at the head of the column and will not be moving through the column at all.  As the mobile phase strength increases, the analyte will begin to partition into the mobile phase and move along the column.  As the mobile phase strength increases continuously, the rate at which the analyte moves along the column subsequently increases and the analyte ‘accelerates’ through the column.  At some point within the column, the analyte may be wholly partitioned into the mobile phase, and will be moving with the same linear velocity as the mobile phase.  The point at which this occurs depends upon the nature of the analyte and its interaction with the stationary phase material.  As the rate at which the analyte elution changes during gradient HPLC, the retention factor (k) used above for isocratic separations is not applicable. Instead the use of an ‘average’ retention value, k*, or the retention factor of the analyte as it passes the mid-point of the column (Equation 6).  Most analytes will be moving at the same rate as they pass the mid-point of the column and hence the retention factors for all analytes in gradient HPLC are very similar.

 
column dimensions EQ06
 

Where:
tg = gradient time (min.)
F = flow rate
S = constant determined by strong solvent and sample compound (for small molecules < 500   Da the value is between 2 and 5; a value of 4 is used by convention when the value is not accurately known.  Proteins have much larger values; typically between 50 and 100 and need longer gradient times for separation).  S can be estimated from Equation 7:

 

column dimensions EQ07

 

ΔΦ = change in volume fraction of organic (final %B – initial %B)
VM = column void volume (Equation 8)

 
column dimensions EQ08
 

Increasing column length in isocratic separations can increase efficiency (N), increase retention (k) and, therefore, affect resolution (Figure 6).  In gradient HPLC it is possible to encounter peak reversals depending upon the shape of the gradient profile and the length of the column used.  In the example shown (Figure 7), because of the relationship between %B and gradient retention factor, using a 10 cm column will result in Compound A eluting first.

However, when a 25 cm column is used, the retention factors dictate that compound B has enough distance to ‘overtake’ compound A and elutes first.  It is also important to note that although the column length is more than doubled, the retention times of the peaks has not increased by this factor as the analytes are travelling at the velocity of the mobile phase.

 

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Figure 6: Effect of column length on
efficiency and resolution in isocratic separations.
Figure 7: Effect of column length on
analyte elution order in gradient separations.
 
 

References

  1. //www.analiticaweb.com.br/newsletter/04/Poster_Particulas-sub-2.pdf
  2. //www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma-Aldrich/Datasheet/t308183.pdf
  3. //www.analiticaweb.com.br/newsletter/13/HPLC_Columns.pdf
 

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