CHROMATOGRAPHY

 Method in identifying substances however the difference with spectroscopy is

that there is separation on the different components.
 A physical process where the components of a sample mixture are separated as
a result of their differential distribution between stationary and mobile phase.
o Differential distribution- depends on the solubility, polarity, volatility of our
molecules either on the stationary phase or mobile phase.
 It separates compounds from one another by passing a mixture through a
column which represents the stationary phase and retains some compounds
longer than the others.
2 COMPONENTS
1. Mobile Phase- solvent that moves through the column or through the stationary
phase.
 Mixture of different solvents
2. Stationary Phase- substances that stays fixed inside the column.

CHROMATOGRAM- graphical representation of detector response or movement of

components within the column.
 Peaks represent the eluting, exiting solutes or the substances being separated in
the column.
o Every peak represents separated substances.
o Separated substances have different distances to each other depending
on the polarity and solubility.
 Data represented by the chromatogram are usually used to identify
and quantify solute.
o The longer the time means it has the same polarity with the column.
o Eluent- fluid entering the column during stationary phase.
o Eluate- fluid exiting the column
o Concentration of analyte in the effluent.
 Effluent- liquid that discharge or flowing out the column/stationary
phase.
 Other quantity used as a measure of effluent concentration.
 Chromatographic peaks- represents the eluting solutes.

A. VOID VOLUME AND CAPACITY FACTOR

 If there is no partition that happened, then it will travel with mobile phase at the
same rate.
 Void Volume- The faster the solvents exits, the faster the particular substance
exits with mobile phase.
o If the compound travels together with mobile phase at the same rate.
o Length of time it takes an unretarted molecule to flow through the
column.
 Unretarted molecule- molecules that did not separate into the
stationary phase.
o Flowing rate- 1 mL per minute, it takes 1.8 minutes to pass through the
void volume.
 Capacity Factor- length of time it takes for retarded compound or compound that
has been separated in the column.
o It measures the debris to which it partitions into the stationary phase.
o When it separates, the components of compounds will also separate at
the different rate thus the retention time.
B. RETENTION TIME/VOLYME
- When a solute exits the injector or injector introduce the sample and it
passes through the column and the detector.
- The time from the injection to the detection is measured either in minutes or
seconds.
- The time between the point of injection and the time of emergence of separation
of component from the column.
- The more longer the retention time, the compound has the same
polarity/solubility with the column.
C. SEPARATION FACTOR
- The ratio of partition coefficient of two components to be separated.
- Relative solubility of the analyte into two phases.
- A result with more than 1.1 means there is a good separation.
- If peaks are far apart there is more difference in their partition coefficient between
a compound hence more separation factor.
- If increase then increase, if decrease then decrease din separation factor.
D. COLUMN EFFICIENCY
- The degree to which a column and/or other system components can physically
and chemically affect the separation of analytes.
- How well the column can separate molecules or compounds.
- Expressed in term of theoretical plates per meter.
- If peak is more broader, less efficient the column compared to a narrower or
sharper peak.
o As column efficiency increases, the analyte components will also elute/exit
in a smaller volume
o Narrower peaks are preferred since column has increased efficiency
- Increases column efficiency: small particle size; thin stationary-phase coating;
regularly shaped particles of stationary phase; high temperature; even stationary-
 phase coating; uniform stationary-phase particle size; high diffusion coefficient in
the mobile phase; high diffusion coefficient in the stationary phase.
- Decreases column efficiency: very low flow rate; large particle size of
stationary phase; thick stationary-phase coating; irregularly shaped particles of
stationary phase; low temperature; uneven stationary-phase coating; non-uniform
stationary-phase particle size; low diffusion coefficient in the mobile phase; low
diffusion coefficient in the stationary phase.
E. RESOLUTION FACTOR
- To quantify the separation between two peaks.
- How well two peaks are separated from each other.
- The more efficient a column, a greater degree of resolution it will produce
between a closely ending peaks.
- The greater resolution factor, more efficient a column.

CLASSIFICATIONS:
1. Based on shape of chromatographic beds.
 Planar- stationary phase is present in a plane.
o Example: Paper chromatography and Thin layer chromatography.
o Thin Layer Chromatography- they use a whattman filter paper or thin
layer glass.
 Column- used for Glass Chromatography or High Performance Liquid
Chromatography.
o Sometimes embedded with silica gels and other solid compounds
which interacts with components to be separated.
2. Based on the physical state of mobile and stationary phase.
 Solid, liquid or gas
 Mobile phase is solvent for HPLC, Paper chromatography and TLC. In gas
chromatography is a carrier gas.
3. Based on mechanism of separation.
 Adsorption chromatography- solute/components that is separated is adsorbed
on the surface of stationary phase.
 Partition chromatography- solute dissolved on the liquid phase coated on the
surface of a solid support.
 Ion exchange chromatography- mobile anions is held near cations that are
covenantly attached to a stationary phase.
o Interaction between anion and cation compounds.
 Molecular chromatography- small molecules present in solute will penetrate
the poorse(?) of particles while the large molecules are executed.
 Affinity chromatography- one kind of molecule in a complex mixture becomes
attach to a molecule that covanently bond to a stationary phase while all other
molecules are washed through or removed.
 o There is a particular molecule attracted/ attached with stationary phase
and the rest are washed/excluded.
GAS CHROMATOGRAPHY
- Used to pass mixture of volatile solutes through a column containing the
stationary phase.
- Mobile phase here is an inert gas either a nitrogen, helium, or an argon.
- Separation of solute is based on the relative differences in the solutes vapor
pressures and interactions with a stationary phase.
- The more volatile the solute is, it will elute faster from the column.
- A solute that selectively interacts with the stationary phase elutes from the
column after with lesser degree of interaction.
- Carrier gas- mobile phase, supplies gasses.
- Injector port- where we introduce sample to be separated.
- Oven- causes to evaporate volatile compounds.
 Analysis of peppermint oil on two GC phases
o They usually use two columns: OV-5 TYPE/BPX-5 or POLAR
CARBOWAX
 They give KOVATS INDEXES/I-VALUES
 I VALUES- constant number for characterizing in unknown
compounds.
 OV-5 TYPE- is used based on molecular weight and shape of our
sample.
 A higher value of a molecular weight of the sample will also
give a high I-VALUES.
 POLAR CARBOWAX- used for highly selective polar compounds
such as menthol, menthone.
o Compounds with same polarity with column will elute slowly which then
gives higher I-VALUES with longer retention time.
 Governing capillary GC performance
o Carrier gas type/flow
 Hydrogen and helium usually gives higher efficiencies at high flow
rates as compared with nitrogen.
 Separation of samples is efficient when we use hydrogen or helium.
o Column Temperature- as column temperature increases, degree of
resolution of component decreases.
 Lower temperature will produce a better resolution.
o Film thickness phase loading- the greater the volume of the stationary
phase, the more a solute will partition in it.
 A thicker films are usually used for very volatile materials to
increase the retention time and to increase the resolution between
analytes without increasing column length.
o Internal diameter- the smaller the internal diameter of the capillary column
the more efficient the column is for a given stationary phase film thickness
on the capillary wall.