insmeth lecture 7 - chromatography
DESCRIPTION
Introduction to ChromatographyTRANSCRIPT
What is chromatography?
Chromatography (from Greek word chromos) - a family of laboratory techniques for the separation of mixtures. It involves passing a mixture which contains the analyte through a stationary phase, which separates it from other molecules in the mixture and allows it to be isolated. Which means ...
Chromatography is the physical separation of a mixture into its individual components.
We can use chromatography to separate the components of inks and dyes, such as those found in pens, markers, clothing, and even candy shells. Chromatography can also be used to separate the colored pigments in plants or used to determine the chemical composition of many substances.
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CHROMATOGRAPHY
Chromatography basically involves the separation of mixtures due to
differences in the distribution coefficient (equilibrium distribution) of sample components between 2 different phases.
One of these phases is a mobile phasemobile phase and the other is a stationary phase.
RECALL – SOLVENT EXTRACTION
Definition:
Different affinity of these 2 components to stationary phase causes the separation.
Concentration of component A in stationary phase
Concentration of component A in mobile phase
Distribution Coefficient (Equilibrium Distribution )
Gas ChromatographyUsed to determine the chemical composition of unknown substances, such as the different compounds in gasoline shown by each separate peak in the graph below.
Paper ChromatographyCan be used to separate the components of inks, dyes, plant compounds (chlorophyll), make-up, and many other substances
Liquid ChromatographyUsed to identify unknown plant pigments & other compounds.
Thin-Layer ChromatographyUses thin plastic or glass trays to identify the composition of pigments, chemicals, and other unknown substances.
Examples of Chromatography
CHROMATOGRAPHYCHROMATOGRAPHY
Chromatography is used to separate and analyse small amounts of mixtures
Methods involve a stationary phase and a mobile phase.
There are several forms of chromatography
TYPE STATIONARY PHASE MOBILE PHASE
paper solid (filter paper) liquid
thin layer (tlc) solid (silica) liquid
column solid (silica) liquid
high pressure liquid (hplc) solid (silica) liquid
gas liquid (glc) solid or liquid gas
PAPER CHROMATOGRAPHYPAPER CHROMATOGRAPHY
Stationary phase chromatography paper
Mobile phase suitable solvent (water, ethanol, organic solvent)
Separation As the solvent moves up the paper it dissolves thecomponents and moves them up the paper. The
more soluble a component is, the further it moves.
Place small a spot of the mixture to be analysed (and any possible component for comparison purposes) on the paper. Dip the paper in the solvent.
PAPER CHROMATOGRAPHYPAPER CHROMATOGRAPHY
Stationary phase chromatography paper
Mobile phase suitable solvent (water, ethanol, organic solvent)
Separation As the solvent moves up the paper it dissolves thecomponents and moves them up the paper. The
more soluble a component is, the further it moves.
Place small a spot of the mixture to be analysed (and any possible component for comparison purposes) on the paper. Dip the paper in the solvent.
Allow the solvent to rise up the paper. Each component dissolves in the solvent. Those which are more soluble travel further up the paper.
PAPER CHROMATOGRAPHYPAPER CHROMATOGRAPHY
Stationary phase chromatography paper
Mobile phase suitable solvent (water, ethanol, organic solvent)
Separation As the solvent moves up the paper it dissolves thecomponents and moves them up the paper. The
more soluble a component is, the further it moves.
Place small a spot of the mixture to be analysed (and any possible component for comparison purposes) on the paper. Dip the paper in the solvent.
Allow the solvent to rise up the paper. Each component dissolves in the solvent. Those which are more soluble travel further up the paper.
Finished chromatogram
PAPER CHROMATOGRAPHYPAPER CHROMATOGRAPHY
Rf value Under similar conditions, a component should always travel at the same speed.
Its identity can be found by comparing the distance it moves relative to the solvent.
Rf = distance travelled by the component = Y distance travelled by the solvent X
X Y
PAPER CHROMATOGRAPHYPAPER CHROMATOGRAPHY
Rf value Under similar conditions, a component should always travel at the same speed.
Its identity can be found by comparing the distance it moves relative to the solvent.
Rf = distance travelled by the component = Y distance travelled by the solvent X
Comparison can be a problem if…
a) components have similar Rf values
b) the unknown substance is new and there is no previous chemical to compare it with
X Y
MOBILE PHASE
• The solvent moving through the column
• Either a liquid or a gas
STATIONARY PHASE
• The one that stays in place inside the column
• Most commonly a viscous liquid chemically bonded to the inside of a capillary tube or onto the surface of a solid particles packed in the column
ELUENT AND ELUATE
• Eluent (IN) – fluid entering the column
• Eluate (OUT) – fluid emerging from the end of the column
• Elution – the process of passing liquid or gas through a column
GENERAL TYPES OF GENERAL TYPES OF CHROMATOGRAPHYCHROMATOGRAPHY
Thin Layer ChromatographyHere the mobile phase is a liquid
Flowing past a thin layer of powder on a solid support.
Substances that are less attracted to the solid or are more soluble in the liquid move faster.
And so move further up the plate by the time that the process has been stopped by taking the plate out of the liqiud. - larger Rf
Rf = distance moved by substance distance moved by solvent front
For substances that are very soluble in the liquid Rf will be close to ....
For substances that are rather insoluble in the liquid Rf will be close to ....
1
0
SP – Solid
MP – Liquid/Gas
Stronger adsorption, slower travel time
SP – Liquid bonded to solid surface
MP – Gas
Solute equilibrates bet SP and MP (GC)
SP – Solid (resin)
MP – Liquid
Electrostatic attraction exists
ION-EXCHANGE CHROMATOGRAPHY SO3
-Na
+
Separation in Ion-exchange Chromatography is based on the competition of different ionic compounds of the sample for the active sites on the ion-exchange resin (column-packing).
MECHANISM OF ION-EXCHANGE CHROMATOGRAPHY OF AMINO ACIDS
SO3-
SO3-
Na+
COO-
H3N+
Na+
COOHH3N
+
pH2
pH4.5
Ion-exchange Resin
H3N
+
SO3-
SO3-
SO3-
SO3-
SO3-
SO3-
H3N+
COOH
OH
COOH
COOH
H3N+
H3N+
OH
COO-
Na+
H3N+
COO-
Na+
Na+
H+ OH
- = H2O
H+ OH
- = H2O
Na+
Na+
pH3.5
Mobile PhaseStationary Phase
Exchange Resin
pH4.5
Chromatography of Amino AcidsChromatography of Amino Acids
Aka Gel Filtration/Permeation
SP – Solid
MP – Liquid/Gas
Separates molecules by size
GEL-PERMEATION CHROMATOGRAPHY
Gel-Permeation Chromatography is a mechanical sorting of molecules based on the size of the molecules in solution. Small molecules are able to permeate more pores and are, therefore, retained longer than large molecules.
SP – Solid
MP – Liquid
Very selective
Ex. Antibodies, proteins
LIQUID COLUMN CHROMATOGRAPHY
A sample mixture is passed through a column packed with solid particles which may or may not be coated with another liquid.
With the proper solvents, packing conditions, some components in the sample will travel the column more slowly than others resulting in the desired separation.
The 4 basic liquid chromatography modes are named according to the mechanism involved:
1. Liquid/Solid Chromatography (adsorption chromatography)
A. Normal Phase LSC (SP = P; MP = NP)
B. Reverse Phase LSC (SP = NP; MP = P)
2. Liquid/Liquid Chromatography (partition chromatography)
A. Normal Phase LLC
B. Reverse Phase LLC
3. Ion Exchange Chromatography
4. Gel Permeation Chromatography (exclusion chromatography)
FOUR BASIC LIQUID CHROMATOGRAPHY MODES
LIQUID SOLID CHROMATOGRAPHY
Si - O - H
Normal phase LS Reverse phase LS
Silica Gel
The separation mechanism in LSC is based on the competition of the components of the mixture sample for the active sites on an absorbent such as Silica Gel.
WATER-SOLUBLE VITAMINS
1. Niacinamide 2. Pyridoxine
N
CONH2
N
CH2OH
CH2OH
HO
H3C
3. Riboflavin N
NNH
N
CH2
HOCH
HOCH
HOCH
CH2OH
O
OH3C
H3C
ClN
S
N
NH3C
CH2
NH2
CH3
CH2CH2OH
4. Thiamin
WATER-SOLUBLE VITAMINS
0 5 10 15 20
Column: u Bondapak C18 Solvent: MeOH Sample: Water-Soluble Vitamins
Inject1
2
3
4
LIQUID-LIQUID CHROMATOGRAPHY
ODPN(oxydipropionylnitrile)
Normal Phase LLC Reverse Phase LLC
NCCH3CH2OCH2CH2CN(Normal)CH3(CH2)16CH3 (Reverse)
The stationary solid surface is coated with a 2nd liquid (the Stationary Phase) which is immiscible in the solvent (Mobile) phase. Partitioning of the sample between 2 phases delays or retains some components more than others to effect separation.
Gas Liquid Chromatography
Here the mobile phase is an unreactive gas ( eg Nitrogen) flowing through a tube.
And the stationary phase is a nonvolatile liquid held on particles of a solid support.
The red molecules are more soluble in the liquid (or less volatile) than are the green molecules. Which molecule will be eluted first?
In practice the Column is contained in a thermostatic oven. (Why ?)
About 1μL of liquid is injected into one end of the column.
As each component reaches the other end it is detected and registered on a chart recorder.
The Retention Time is characteristic of a particular substance. (for the same column, temperature, gas flow etc.)
The area under each peak indicates the relative quantities.
Oven
Detector
Injection port
Nitrogen cylinder
Column
Recorder
Chromatogram of petrol
Suggest identities of some of the unlabelled peaks.
SOLVENTS
Polar Solvents
Water > Methanol > Acetonitrile > Ethanol > Oxydipropionitrile
Non-polar Solvents
N-Decane > N-Hexane > N-Pentane > Cyclohexane
SELECTING AN OPERATING MODE
Sample Type LC Mode Positional isomers LSC or LLC
Moderate Polarity Molecules LSC or LLC
Compounds with Similar Functionality LSC or LLC
Ionizable Species IEC
Compounds with Differing Solubility LLC
Mixture of Varying Sized Molecules GCC
Schematic Diagram of Liquid Chromatography
1. Ultraviolet Detector
200-400nm 254 nm
2. Refractive Index Detector
Universal Detector
DetectorsDetectors
High Performance Liquid Chromatography
High Performance Liquid Chromatography
Chromatogram of Organic Compounds from Fermented Cabbage
Chromatogram of Orange Juice Compounds
THE CHROMATOGRAMTHE CHROMATOGRAM
• A graph showing the detector response as a function of elution time
Retention Time, tr
Time required for the sample to travel from the injection port through the column to the detector.
Response
Retention Time
5 10 15 20 25
A
B
C
D
Retention Volume, Vr
Volume of the MP required to elute a particular solute from the column
Retention Time of MP, tm
The minimum time the unretained mobile phase travels through the column
Adjusted Retention Time, tr’
The additional time required for solute to travel the length of the column beyond the required by the unretained solvent
tr’ = tr - tm
Relative Retention ()
The ratio of the adjusted retention time of two components (selectivity)
= tr2’/tr1’The greater the , the greater the separation between two components
Response
Retention Time
X
X
X
1 3 6
2
1
0
– Selectivity
SelectivitySelectivity
Capacity Factor, k’
For each peak in the chromatogram, k’ is mathematically equal to
k’ = tr – tm / tm
The longer the component is retained in the column, the greater the k’May be used to assess the performance of the column
Sample Problem
1. A mixture of benzene, toluene and methane (MP) was injected into a gas chromatograph. Methane gave a sharp spike in 42 s, whereas benzene required 251 s and toluene was eluted in 333 s. Find the adjusted retention time and capacity factor for each solute and the relative retention.
Answers - Sample Problem
tr’benzene = 209 s
tr’toluene = 291 s
k’benzene = 5.0
k’toluene = 6.9
= 1.39
EFFICIENCY OF EFFICIENCY OF SEPARATIONSEPARATION
RESOLUTION
• How close two bands in a chromatogram can be to each other and still be seen as two peaks
• The difference in the retention times of adjacent peaks divided by their width
Resolution = tr/wav
• wav = average width of the 2 peaks
HEIGHT EQUIVALENT TO A THEORETICAL PLATE
Length of a column necessary for the attainment of compound distribution equilibrium (measure the efficiency of the column).
Theoretical plates (N) = 16 ( )X
Y2
X
Y
RESOLUTION
EXAMPLES OF THEORETICAL PLATE, SELECTIVITY AND HEIGHT EQUIVALENT
TO A THEORETICAL PLATE
1
2
3
4V
V
V
V
W W W W
2
1
0
1
2
4
3 4
3
V
V0 = 1.02(Minutes) V1 = 4.92 V2 = 6.59 V3 = 8.17 V4 = 9.14
W1 = 1.0 (Minutes) W2 =1.0 W3 = 1.0 W4 =1.0
GENERAL FACTORS INCREASING RESOLUTION
1. Increase column length
2. Decrease column diameter
3. Decrease flow-rate
4. Pack column uniformly
5. Use uniform stationary phase (packing material)
6. Decrease sample size
7. Select proper stationary phase
8. Select proper mobile phase
9. Use proper pressure
10. Use gradient elution