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Chromatographic Techniques

• Gas Chromatography (GC)


• High Performance Liquid Chromatography (HPLC)
Hyphenated Techniques
• GC-MS
• LC-MS
• LC-NMR
• LC-NMR-MS
Natural product isolation, structure elucidation, pharmaceutical
analysis, impurity profiling, degradation studies, trace analysis
etc
Chromatography
• To separate individual
compounds from a sample
mixture.
• The separation of a sample
Mobile phase
mixture occurs through the
repeated distribution of the
components between the
stationary phase located in a Stationary phase
separation column or on a thin
layer (paper or thinlayer plate)
and a gaseous (only in the
column) or liquid mobile
phase. Separation of plant pigments
Chromatography
What is chromatography

AA C
AC C C AA
C AABC B B BB C CC
A A
B B A B B C CC
B ABB A B C
C
Sample separated components

Chemical and physical separation of components for


qualitative and quantitative analysis
Chromatography - Classification
General Classification Specific Method Stationary Phase Mechanism

Liquid/liquid Liquid adsorbed Partition


Liquid chromatography on solid (immiscible liquids)
(LC). Mobile phase: liquid
Liquid/bonded phase Organic species Partition (liquid/
bonded to solid surface bonded surface)

Liquid/solid Solid Adsorption

Size exclusion Liquid in polymeric Partition/sieving


solid
Ion exchange Ion-exchange resin Ion exchange

Gas chromatography Gas/liquid Liquid adsorbed on solid Partition


(GC). Mobile phase : gas (gas/liquid)
Gas/solid Solid Adsorption

Supercritical fluid Organic species Partition


chromatography bonded to solid (supercritical fluid/
Mobile phase: SF surface bonded surface)
Gas Chromatography
• Technique of choice for separating thermally stable and volatile compounds
• Basics – Distribution of sample between two phases, one stationary bed of large
surface area, second is mobile phase – a gas that percolates through the
stationary phase
• GLC: stationary phase is a liquid coated on a inert solid support
• GSC: solid adsorbent as a stationary phase
• Versatile and selective
• Large range of liquid phases with usable temp upto 450 degrees
• Can be used to analyze gaseous, liquid and solid samples
• Sample MUST be volatile at temperatures BELOW 3500C
• Advantages of GC
– Speed – very fast, minutes
– Resolution – high resolution of closely related compounds
– Qualitative analysis – retention time
– Quantitative analysis – from area under peak
– Sensitivity – high sensitivity, (ppm range)
– Simplicity – simple to operate and understand
Instrumentation for GC

• Carrier gas
– N2, He, H2
oven
• Injector
• Column
• Detector
• Computer

Mobile Phase Column Chromatogram


Column Types
• Capillary (open tubular)
– Inner wall modified with thin (1 m) film of liquid
– 0.3 - 0.5 mm ID; 10 - 50 m length
– Can load few g analyte only
– WCOT, SCOT, PLOT, FSOT

• Packed
– Solid particles either porous or non-porous coated with thin (1 m)
film of liquid
– 1 - 8 mm ID; 1 - 10 m length
– Can load upto few hundred g analyte
GC - Choice of Liquid Phase
• Solute classification: • Liquid phase classification:
• (Most Polar) • FFAP, 20M-TPA,Carbowaxes,
– Water, glycerol, amino-alcohols, Ucons, Versamid 900, Hallcomid,
hydroxy acids, polyphenols, Theed, Mannitol
dibasic acids etc.
• (Polar)
• Tetracyanoethylpentaerythritol,
– Alcohols, Fatty acids, phenols,
amines, oximes nitro compounds Zonyl E7, Ethofat, Amine 220 etc.
etc. • All polyesters, Tricresyl
• (Intermediate) phosphate, Benzyl cyanide,
– Ethers, ketones, aldehydes, Dimethyl sulfolane etc.
esters, etc. • Squalene, Hexadecane, Dow 11,
• (Low Polarity non polar) SE 30, SF 96 etc.
– Aromatic hydrocarbons,
saturated hydrocarbons,
mercaptans, sulfides, olefins etc.
GC - Columns, Theory and Technique
Solid Supports
•To support a thin uniform layer of liquid phase, large surface
area – 1 to 20 sq. mt./gram, uniform pore diameter of 10 m or
less, inert to sample, regular shaped particles, should have
high mechanical strength
•Diatomaceous silica (kieselguhr) – composed of skeletons of
diatoms – unicellular algae
•Diatomite surfaces are covered with silanol (SiOH) or
Sioloxane (Si-O-Si) groups capable of hydrogen bonding with
solvents or solutes
•Chromosorb A - preparative scale, holds 25% liquid phase,
surface is not highly adsorptive
•Chromosorb G – for separation of polar compounds, low
surface area
•Chromosorb P – calcined diatomite, pink color, hard, more
adsorptive than other chromosorb grades
GC – Temperature is important!
Injection Port Temperature
Hot enough to vaporize the sample rapidly, low enough to avoid
thermal decomposition or rearrangement of the sample
Column Temperature
High enough so that analysis is accomplished in reasonable time,
low enough that separation is achieved
Retention time approx. doubles for every thirty degrees decrease in
temperature
Lower the temperature, higher the ratio of partitioning coefficients
in SP, desirable to employ temperature programming
Detector temperature
Type of detector, hot enough to prevent condensation of sample,
peak broadening and loss of component peaks characteristic of
condensation, thermal conductivity detector more sensitive to
temperature than ionization-type detectors
GC – Headspace Sampling
• Volatile components in samples if matrix is solid or the sample is
liquid requiring extensive clean-up or if vapors above the liquid
are of interest
• Measured amount of sample in a vial sealed with septum and
crimped cap
• The sample vial is placed in a carousel and thermostatted with a
known volume of inert gas
• Internal pressure in closed headspace vial increases
• Can be sampled with gas-tight syringe or analyzed by automated
system
• Multiple headspace extraction – Stepwise sampling of headspace
from a single sealed sample, analyte concentration becomes
smaller after each extraction till all of analyte is removed
Gas Chromatography-Mass Spectrometry
(GC-MS)
Gas Chromatography-Mass Spectrometry
(GC-MS)
1. Dodecane
2. Biphenyl
1 2 3 4 3. Chlorobiphenyl
4. Hexadecanoic acid methyl ester
GC-MS Applications

• Detection of residual solvents in pharmaceuticals


• Organic volatile impurities can result during manufacture (used to
enhance yields, improve crystallization or solubility
• Class 1, class 2, and class 3 solvents (class 3 solvents pose a lower
risk to human health
• Manufacturers must ensure that residual solvents or contaminants are
not present in their products
• These are analyzed using headspace sampling GC (HS-GC) or GC-
MS
• Constiuents of essential oils are mainly studied by GC and GC-MS
• Fatty acids are normally determined by GC, GC-MS after making
their methyl esters
GC-MS Applications
Analysis of aldehydes in
tap water
Aldehydes give unpleasant
smell to tap water or bottled
mineral water
WHO limits formaldehyde
at 900 mg/L whereas in
Japan it is regulated at 80
mg/L
Pentafluorobenzylhydroxyl
amine forms oximes that
are volatile and analyzed by
headspace sampler GC-MS
High Performance Liquid Chromatography
(HPLC)
High Performance Liquid Chromatography
(HPLC)
Twin Piston Pump
The twin piston pumps with short
stroke are among the most commonly
used pumps for HPLC.
Both pump heads are switched in
series, whereby the piston in the first
pump head delivers a specific volume
per stroke.

An excentric disk presses piston 1 to the right and


displaces the solvent. The double ball saphire valves
ensure that the solvent stream can flow in only one
direction.
The second piston is used to produce a nearly
complete pulsation damping. With the twin piston
pump, a pressure of 40 MPa is achieved.
HPLC - Injector
Six-port Rheodyne valve in which the
sample fills an external loop
A clockwise rotation of the valve rotor
places the sample-filled loop into the
mobile-phase stream, with subsequent
injection of the sample onto the top of
the column
HPLC Column
Column dimension (size), particle size and pore size, stationary phase

Water/Methanol

C18 4.6 x 250 mm 5m 300oA

Dimension Pore Size


Stationary Phase Particle Size
Column – Stationary Phases
• Porous silica is the widely used normal phase stationary material. Its
chemical composition could be expressed as SiO2 x H2O
• The matrix of the primary silica gel particle consists of a core of
silicon atoms joined together with oxygen atoms by siloxane bonds
(silicon-oxygen-silicon bonds).
• Residual hydroxyl groups confer upon silica gel its polar properties
Column – Stationary Phases
• C18 silica is the most widely used reverse phase stationary material

• -Si-OH + Cl-Si(CH3)2-R → Si-O-Si(CH3)2-R; R = octadecyl, octyl

• Partitioning of solute between two immiscible liquids one which is


fixed (bonded to stationary phase) and the other is mobile phase
• The less polar the solute, greater attraction, longer retention,
• Least polar eluted last, napthalene being less polar is attracted
more strongly and eluted last
Column – Stationary Phases
The dependence of the retention time tR on the polarity of a substance
makes it possible to assemble a polarity scale for a number of compound
classes
This scale can be used to approximately predict the elution sequence of
the substances