This presentation contains HPLC column theory and details. You can find the complete structure of chromatographic column and its theory.

1. The first chromatography column was
developed by the Russian botanist
Mikhail Tsvet who, in 1901, washed
an organic solution of plant pigments
through a vertical glass column packed
with an adsorptive material.
He discovered that the pigments
separated into a series of discrete
coloured bands on the column, divided
by regions entirely free of colour.
CHROMATOGRAPHIC COLUMN
INVENTION

2. CHROMATOGRAPHIC COLUMN
Details of column

3. CHROMATOGRAPHIC COLUMN
Details of column
polyimid, 350 oC
Silica
Stationary phase
microbore: d < 150 m
standard capillary: 150 m < d < 500 m
widebore: d > 500 m
d

4. CHROMATOGRAPHIC COLUMN
Stationary Phase - Silica

5. CHROMATOGRAPHIC COLUMN
Stationary Phase - Silica

6. CHROMATOGRAPHIC COLUMN
Physical Characteristics of column
Default Column Optimum Column†
Column Bed Dimensions 150 x 4.6mm 250 x 4.6mm
Particle Size 5µm 3* or 5µm
Surface Area 200m2/g >200m2/g
Pore Size 100Å 100Å
Carbon Load 10% 16 - 20%
Bonding Type Monomeric Mono- or Polymeric
Base Material Silica Silica
Particle Shape Spherical Spherical
* mobile phase backpressure may be excessive
† Optimum Column: Alltima C18™, 5µm, 250 x 4.6mm (Part No. 88056)
*Note that the choice may represent a compromise. Here, the “optimum” column for resolution sacrifices speed.

7. CHROMATOGRAPHIC COLUMN
Physical Characteristics of column
Column Dimensions
Length and internal diameter of packing bed
• Effect on chromatography
Short (30-50mm) - short run times, low backpressure
Long (250-300mm) - higher resolution, long run times
Narrow ( 2.1mm) - higher detector sensitivity
Wide (10-22mm) - high sample loading

8. CHROMATOGRAPHIC COLUMN
Physical Characteristics of column
Particle Shape : Spherical or irregular
Effect on chromatography
Spherical particles offer reduced back pressures and longer
column life when using viscous mobile phases like 50:50
MeOH:H2O.

9. CHROMATOGRAPHIC COLUMN
Physical Characteristics of column
Particle Size
The average particle diameter, typically 3-20µm
Effect on chromatography
Smaller particles offer higher efficiency, but also cause higher
backpressure. Choose 3µm particles for resolving complex,
multi-component samples. Otherwise, choose 5 or 10µm
packings.

10. CHROMATOGRAPHIC COLUMN
Physical Characteristics of column
Surface Area
Sum of particle outer surface and interior pore
surface, in m2/gram
Effect on chromatography
High surface area generally provides greater retention,
capacity and resolution for separating complex, multi-
component samples. Low surface area packings generally
equilibrate quickly, especially important in gradient analyses.

11. CHROMATOGRAPHIC COLUMN
Physical Characteristics of column
Pore Size
Average size of pores or cavities in particles, ranging from
60-10,000Å
Effect on chromatography
Larger pores allow larger solute molecules to be retained
longer through maximum exposure to the surface area of the
particles.
Choose a pore size of 150Å or less for sample MW  2000.
Choose a pore size of 300Å or greater for sample MW >
2000.

12. CHROMATOGRAPHIC COLUMN
Physical Characteristics of column
Bonding Type
Monomeric - single-point attachment of bonded phase molecule
Si
R
R
(CH2)17CH3Si
CH3
CH3
(CH2)17CH3XOH +
monomeric
bonding
Si
CH3
X
(CH2)17CH3X+
polymeric
bonding
OH
OH O
O
Si
CH3
(CH2)17CH3
Polymeric - multi-point attachment of bonded phase molecule

13. CHROMATOGRAPHIC COLUMN
Physical Characteristics of column

14. CHROMATOGRAPHIC COLUMN
Physical Characteristics of column
Monomeric - single-point attachment of bonded phase molecule
Polymeric - multi-point attachment of bonded phase molecule
Effect on chromatography
This product presents a very high stability, batch-to-batch
reproducibility and good hydrophobic properties. The fact
that the silane possesses only one bond with the surface
makes this phase less stable at low pH, which may lead to
silane hydrolysis and consequently leaching.
Effect on chromatography
The silica surface is more hydrophobic, has greater stability in
strong acidic condition (pH 2, 3) and has a longer lifetime.

15. CHROMATOGRAPHIC COLUMN
Physical Characteristics of column
Carbon Load
Amount of bonded phase attached to base material,
expressed as %C
Effect on chromatography
Higher carbon loads generally offer greater resolution and
longer run times. Low carbon loads shorten run times and
many show a different selectivity.

16. CHROMATOGRAPHIC COLUMN
Physical Characteristics of column
End capping
“Capping” of exposed sialons with short hydrocarbon chains
after the primary bonding step
Effect on chromatography
End capping reduces peak-tailing of polar solutes that interact excessively with
the otherwise exposed, mostly acidic silanols. Non-end capped packings
provide a different selectivity than do end capped packings, especially for such
polar samples.

17. CHROMATOGRAPHIC COLUMN
Physical Characteristics of column
End capping
“Capping” of exposed sialons with short hydrocarbon chains
after the primary bonding step