Chromatography -- what does it mean?

To write with colors -- literally translated from its Greek roots chroma and graphein ,
chromatography was first developed by the Russian botanist Mikhail Tswett
in 1903 as he produced a colorful separation of plant pigments through a
column of calcium carbonate. Chromatography has since developed into
an invaluable laboratory tool for the separation and identification of compounds.
Although color usually no longer plays a role in the process, the same principles
of chromatography still apply.

Mikhail Tswett
First Chromatographic Separation

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 Chromatography -- what does it mean?

Tswett stated: Chromatography is a

method in which the components of
a mixture are separated on an
adsorbent column in a flowing
Chromatography: Method
system.
Chromatograph: Machine
Analytical Nomenclature of IUPAC: Chromatographer: Person
Chromatography is a physical
method of separation in which the Chromatogram: Data
components to be separated are
distributed between two phases, one
of which is stationary (stationary
phase) while the other (the mobile
phase) moves in a definite direction
(IUPAC, 1993).

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 Chromatographic Tree
Chromatography

Super Critical Fluid Liquid Chromatography

Gas Chromatography
Chromatography

Column
Planer Chromatography High Performance
Chromatography
Liquid Chromatography

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High Performance Liquid Chromatograph

Shimadzu Prominence

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Chromatogram

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 Separation Technique
• Compounds are separated due to the molecules
moves at different rates in the column.

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 Separation Technique
• Due to different interaction between stationary
phase and different sample, the molecules move
at different rate, therefore separation can be
done.

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 HPLC Phases
Normal Phase Mode
Reverse Phase Mode
Reverse Phase Ion Pairing Mode
Ion Exchange Mode
Chiral Separation Mode

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 Normal Phase
Stationary Phase is Polar
Mobile Phase is Non Polar

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Base material for Stationary Phase
SiO2

O=Si=O Si OH

Polar Group

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Silica Gel

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Silica gel

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Silica gel

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Silica gel

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Stationary Phase for Normal Phase

Si Si-OH Unmodified Silica (USP-L3)

Si Si-CH2-CH2-CH2-NH2 Amino (USP-L11)

Si Si-CH2-CH2-CH2-CN Cyano (USP-L10)

OH
Si Si-CH2-CH2-CH2-OCHCH2 Diol (USP-L)
OH

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What is Interaction

HO

Si Si-OH

A
OH U

Minutes

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Interaction

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 Reverse Phase

Normal Phase
Reverse Phase

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Reverse Phase

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Reverse Phase

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Column for Reverse Phase

Si Si-CH2-CH2CH2-----CH3 (C18) ODS (USP-L1)

Si Si-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3 C8 (USP-L7)

Si Si-CH2-CH2-CH2-CH3 C4 (USP-L26)

CH3
Si Si-CH3 C1 (USP-L-13)
CH3

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 Column for Reverse Phase

Si Si-CH2-CH2CH2NH2 Amino

Si Si-CH2-CH2CH2CN Cyano

Si Si-CH2-CH2CH2- Phenyl

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 Mobile Phase for RP
• Water (buffer) + Organic Solvents
-When buffer is used, the concentration
and pH are important factors
-Methanol, Acetonitrile or THF are
common organic solvents for RP HPLC
Optimization of water (buffer) and organic
solvents ratio is very important

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Interaction of RP

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Interaction of RP

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Interaction of RP

CH3

H3C CH3
7 Carbons
8 Carbons
6 Carbons

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Increase of Solvent Polarity

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Interaction between Phases

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 HPLC Instrumentation
• 1. Pump
• 2. Column
• 3. Injector
• 4. Detector
• 5. Data Processor

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 HPLC configuration by
eluting mode
• Isocratic
• Binary
• Quaternary

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Gradient VS Isocratic

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Isocratic

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Low Pressure Gradient

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High Pressure Gradient

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 Injector
• Manual Injector
• Auto Injector (Sampler)
• Advantage of Auto Injector
(Sampler)

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 Detectors

• 1. UV-Vis /Photodiode Array Detector

• 2. Refractive Index Detector
• 3. Fluorescence Detector
• 4. Conductivity Detector
• 5. Electro Chemical Detector
• 5. Evaporate Light Scattering Detector
• 6. Mass Detector

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 Data Processor
• Software or Integrator or Recorder

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 Column Particle Physical
Characteristics
•Column Dimensions
• • Length and internal diameter of packing bed

•Particle Shape
• • Spherical or irregular

•Particle Size
• • The average particle diameter, typically 3-20µm

•Surface Area
• • Sum of particle outer surface and interior pore
surface, in m2/gram
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 Column Particle Physical
Characteristics
•Pore Size
• • Average size of pores or cavities in particles, ranging from
60-10,000Å
•Bonding Type
• • Monomeric - single-point attachment of bonded phase
molecule
• • Polymeric - multi-point attachment of bonded phase
molecule
•Carbon Load
• • Amount of bonded phase attached to base material,
expressed as %C
•Endcapping
• • “Capping” of exposed silanols with short hydrocarbon
chains after the primary bonding step

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 Column length
•Effect on chromatography

• • Short (30-50mm) - short run times, low backpressure

• • Long (250-300mm) - higher resolution, long run times

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 Column ID
• Narrow ( 2.1mm) - higher detector sensitivity, Sharp peak
• Popular ID – 4.6 mm
• Wide (10-22mm) - high sample loading

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Base Material

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 Particle Shape
•Effect on chromatography
•Spherical particles offer reduced back pressures and longer
column life when using viscous mobile phases like 50:50
MeOH:H2O.
•Nice peak Shape

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 Particle Size
•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.

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 Carbon Load
•Effect on chromatography
•Higher carbon loads generally offer greater
resolution and longer run times. Low carbon loads
shorten run times.

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 Surface Area
•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.

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 Pore Size
•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.

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 Bonding Type
•Effect on chromatography
•Monomeric bonding offers increased mass transfer
rates, higher column efficiency, and faster column
equilibration.
CH3 R
monomeric
OH + X Si (CH2)17CH3 Si (CH2)17CH3
bonding
CH3 R

OH CH3 O CH3
polymeric
+ X Si (CH2)17CH3 Si
bonding
OH X O (CH2)17CH3

Polymeric bonding offers increased column stability,

particularly when highly aqueous mobile phases are used.
Polymeric bonding also enables the column to accept higher
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sample loading.
 System Suitability

Repeatability,%RSD (CV)
Capacity factor, K’
Selectivity/Relative Retention Time
Theoretical Plates, N (Efficiency)
 Resolution
Asymmetry/Tailing Factor

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 Repeatability, CV (%RSD)

X= (X1+X2……..Xn-1+Xn)/n
N=: Number of Analysis
X1..Xn : Retention time (or area or heights)
X : Average
C.V : Coefficient of Variation

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Capacity Factor, K’

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Selectivity/Relative Retention Time, 

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Theoretical Plate

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Resolution

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Tailing Factor,T

T=W0.05/2f

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