The document discusses various stationary phases and modes of interaction in chromatography. It describes different types of particles like monolith silica and core-shell technologies. It explains characteristics of common stationary phases like C18, their carbon load, surface area, endcapping, and interactions like hydrophobic bonding. The document provides examples of separations on different columns like C18 and C8. It discusses stationary phases like phenyl, biphenyl, pentafluorophenyl and their specific interactions. In conclusion, it encourages readers to contact the company for any chromatography needs.

1. Stationary phases
and
Respective mode of interaction
Kunal Kangane 1
Ad Hoc Scientific-
A company with a purpose

2. Ad Hoc Scientific Private limited
• We are over 3 years old now
• Over 14 years of business development experience.
• 24 years of column packing experience.
• Presence in India, Southeast Asia, UAE and USA.
• Silica from world’s largest silica manufacturers.
• Global standard column hardware.
• Similar to some of the recognized brands
• Office and stock in Pune
2

3. Outline
• Column technologies.
• Silica comparison and studies.
• Endcapping.
• Stationary Phase- mode of interactions
• Column Chemistry
3

4. Types of particles in chromatography media
• Monolith Silica
• Core-shell technology particles
• Traditional Fully porous particle
7

5. Onyx Monolith Columns
• Monoliths are rod-shaped continuous bed silica or polymeric materials,
which offer an alternative to conventional particle packed columns for
analytical and preparative liquid chromatography.
• Silica monoliths are more easily prepared in comparison with their
polymeric counterparts and provide larger surface areas for small
molecule separations.
• The main advantages of monoliths are their superior performance for
fast separations with low pressure at high flow rates and favorable
mass transfer efficiency
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6. The Stationary Phase: An Overview of
HPLC Media
• Silica Manufacturing
• Free Silanols
• Metal Content
• The reversibility of the reaction
• Ligand binding
• Endcapping (BDS)
• The negative charge

7. 7
Free Silanols
Column Packing Materials - Silica Surface
Si
OH
Si
OH
Si
OH
O
Si
OH
O
Hydroxylated
Si
O
H
Silanol groups with
physically adsorbed water
Si
OH OH
Geminal silanol
groups
Si
OH
Si
OH
O
Hydrogen Bonded
O
Siloxane Bonds

8. Traditional Silica Morphology
8
Conventional Silica Particle
2-8 pH range
• Sol gel silica is a polymer
• TEOS is the monomer
• The bonding is achieved with the
formation of Siloxane bridge.
• The reaction is reversible

9. Hybrid Silica Morphology
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• Polymer grafted alongside TEOS
• Hybrid silica
• X-terra, X-Bridge, Tri-art, Gemini
• The reaction is reversible
• pH 1-12 stable

10. 10
Users must be aware of the following:
• Surface characteristics of their particular stationary phases
• Analyte–surface interactions,
• For example, very hydrophobic sample matrices such as corn oil, highly
aromatic materials and waxes can stick to reversed-phase packing surfaces
and change their characteristics.
• Certain analyte–matrix combinations can affect stationary phases
detrimentally.
• Contamination results from the accumulation of strongly retained
substances from repeated injections
Surface Interactions

11. 11
Nature of the analyte
Mobile phase
Stationary Phase

12. 12
Brand Bonde
d
Phase
D50 D90/D1
0
Carbon
load (%)
Surface area
(m2sq./g)
Pore
size
pH range
ALPHA C18 4.3 1.23 17 450 100A 1.5-10.5
Luna C18 17.5 400 100A 2-10
Oyster C18 17.5 450 100A 2-10
YMC PRo C18 16 335 120A 2-8
Inertsil ODS3 C18 15 450 100A 2-7.5
Hypersil ODS C18 10 170 100 2-8
Sunfire C18 16 340 100A 2-8
C18- the first choice for reverse phase

13. 13
Si
O
Si
O
Si
O
Si
O
Si
O
Si
O
Si
O
Si
O
Si
O
Si
OH
C
H3
O
H OH O
H OH OH OH O
H OH OH OH
O O O
- OH
O O O
O
Si
Si Si
Si
Si
C
H3
CH3
C
H3
CH3
C
H3
CH3
C
H3 CH3
CH3
CH3 CH3
C
H3
CH3
CH3
CH3
OH
OH
N
CH3
CH3
CH3
C
H3
Hydrophobic
• Weak, transient interactions
between a non-polar stationary
phase and the molecules
• Hydrophobic & van Der
Waals interactions
• Retention will be predicted by
Log P values
Hydrophobic Interactions

14. Carbon Load
• Carbon load is the percent by weight of carbon on the
stationary phase.
• Reverse phase: more the carbon load, more is the
retention for non-polar compounds.
• Higher carbon loads will generally give higher column
capacities, greater resolution, and longer run times.
• Low carbon loads will shorten analysis times and may
provide selectivity differences because of the greater
exposure of the base material.
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15. Choosing right carbon load
• Choose high carbon loads for complex samples which
require the maximum degree of separation.
• Choose low carbon loads to give shorter analysis times for
simple sample mixes and for samples which require a high
water content for solubility or stability.
• More organic will be required in the mobile phase to
elute the same sample on columns with higher carbon
load.
1

16. Surface Area
• The adsorbent surface area has a major influence on the
stationary phase properties.
• HPLC retention is proportional to the total surface area of
the adsorbent in the column.
• The pore diameter influences the overall surface area and
with it the capacity of the column.
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17. Endcapping
• Bare silica is polar in nature.
• In a non-endcapped column, the bare silica is
exposed and available for attachment of polar
analytes.
• Thus, non-endcapped silica result into peak tailing for
polar solutes.
• Endcapping reduces peak-tailing of polar solutes that
interact excessively with the otherwise exposed,
mostly acidic silanols.
• Non-endcapped packings provide a different
selectivity than do endcapped packings, especially for
such polar samples.
1

18. • For functionalized silica, the only interaction that should
occur is with the grafted function and not a non-specific
binding with the surface.
• Also, in harsh conditions, the end-capping prevents the
surface from being attacked and destroyed.
• Alpha HPLC columns are packed with a double endcapped
silica which make them 1.5 to 10.5 pH stable.
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19. Fluconazole on Inertsil ODS 3V 150X4.6 3u C18
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20. 20
Fluconazole on Alpha 150X4.6 3u C18

21. Carbimazole and Thiamazole
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22. C8
• Nonpolar stationary phase: C8
• Rugged, general purpose, less retentive than C18
• Also provides steric Interaction
• Polar mobile phase: Water, ACN, Methanol
• The MOST polar compound comes out first
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23. Phenyl Hexyl
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• 6 Carbon chain provides a hydrophobic mode of interaction.
• Phenyl ring has alternate double bond.
• ∏-∏ interaction. (avoid ACN)
• Dipole interaction

24. Biphenyl
• Phenyl upon a phenyl
• Advanced ∏-∏ interactions
• Dipole interactions
• Good for polar aromatic compounds
• You can also find Diphenyl
• Phenyl adjacent to another phenyl
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25. Penta Flouro Phenyl
• Strong H-F bonding
• Hydrogen bond
• ∏-∏ interactions
• Dipole interactions
• Steric/ Electrostatic interactions
• Slight hydrophobic interactions
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26. HILIC
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• Acetonitrile-rich mobile phase and a water-enriched
layer adsorbed onto the hydrophilic stationary phase
• The more hydrophilic the analyte, the more the
partitioning equilibrium is shifted towards the
immobilized water layer on the stationary phase,
• The more the analyte is retained.
• In other words, a separation based on the polarities
of the compounds and
• the degree of solvation takes place.

27. Cyano
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28. THANK YOU
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Reach us at
• kunalk@adhocscientific.com
• technical@adhocscientific.com
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