insmeth lecture 7 - chromatography

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.

http://members.shaw.ca/vict/chemistry_test3.htm

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