What is chromatography? Chromatography is a powerful separation method that is usually composed of mobile phase and a stationary phase. This method.

INTRODUCTION TO CHROMATOGRAPHIC SEPARATIONS

What is chromatography?

Chromatography is a powerful separation method that is usually composed of mobile phase and a stationary phase.

This method is used to separate and identify the components of complex mixtures.

Works by allowing the molecules present in the mixture to distribute themselves between a stationary and a mobile phase to varying degrees.

Those components that are strongly retained by the stationary phase move slowly with the flow of mobile phase.

In contrast, components that are weakly held by the stationary phase travel rapidly.

As a consequence of these differences in mobility, sample components separate into discrete bands that can be analyzed qualitatively and/or quantitatively.

Classification of Chromatographic Methods

Can be categorized based on the followings:

1. Based on physical means The way stationary and mobile phase are

brought into contact

2. Based on the types of mobile phase

Either gas, liquid or supercritical fluid

3. Based on the kinds of equilibria involved in the solute transfer between the phases.

Interaction of analyte between stationary and mobile phases

Classification of Chromatographic Methods

Stationary phases is held in narrow tube;

mobile phase moves by pressure or gravity

E.g. – gas chromatography (GC)

– supercritical-fluid chromatography (SFC)

Stationary phase is supported on a flat plate or in the interstices of a paper; mobile phase moves through capillary action or gravity

E.g. – thin-layer chromatography (TLC)

– paper chromatography (PC)

Column chromatography

Planar chromatography

* Based upon physical means

Column chromatography can be further differentiated based on the types of mobile phases and the kinds of equilibria involved in solute transfer between the phases

Mobile Phase

i) Gas

Gas Chromatography

ii) Liquid

Liquid Chromatography

iii) Supercritical fluid

Supercritical-fluid Chromatography

Types of Chromatography on The Basis of interaction of The Analyte with Stationary Phase

Adsorption – for polar non-ionic compounds Ion Exchange – for ionic compounds

Anion – analyte is anion; bonded phase has positive charge

Cation – analyte is cation; bonded phase has negative charge

Partition – based on the relative solubility of analyte in mobile and stationary phases Normal – stationary phase polar, the mobile phase nonpolar Reverse – stationary phase nonpolar, the mobile phase polar

• Size Exclusion – stationary phase is a porous matrix sieving

Classification of Chromatographic Methods

Chromatography

Partition

Adsorption

Ion-exchange

Size-exclusion

Liquid-liquid

Gas-liquid

Liquid-solid

Gas-solid

Liquid-solid

Liquid-solid

PARTITION CHROMATOGRAPHY

Partition chromatography Accomplished by selective & continuous

transfer of the components of the mixture back & forth between a liquid stationary phase and a liquid mobile phase as the mobile phase liquid passes through the stationary phase liquid

Stationary phase: liquidMobile phase: liquid or gas

Partitioning distribution (by dissolving) of the

components between 2 immiscible phases: Relative solubilities of the components in the

mobile and stationary phase

e.g. stationary phase – polar Polar components will retain longer than the

non-polar components. Non-polar components will move quickly

through stationary phase & will elute first before the polar components, and vice-versa.

Partition chromatography

The stationary phase actually consists of a thin film adsorbed (stuck) on or chemically bonded to the surface of a finely divided solid particles.

Partition chromatography

If the mobile phase is gas, the volatility (vapor pressure) and solubility in stationary phase plays an important role.

ADSORPTION CHROMATOGRAPHY

Adsorption (Affinity) Chromatography Components of the mixture selectively

adsorb (stick) on the surface of a finely divided solid stationary phase.

As mobile phase (gas/liquid) carries the mixture through the stationary phase, the components of the mixture stick to the surface of it with varying degrees of strength & thus separate

Stationary phase: solid Mobile phase: gas or liquid

ION-EXCHANGE CHROMATOGRAPHY

Ion-exchange chromatography

Method for separating mixture of ions Sample: aqueous solution of inorganic

ions / organic ions Stationary phase – small polymer resin

“beads” usually packed in a glass tube These beads have ionic bonding sites on

their surfaces which selectively exchange ions with certain mobile phase compositions as the mobile phase penetrates through it.

Ion-exchange chromatography

Ions that bond to the charged site on the resin bead are separated from ions that do not repeated changing of the mobile phase composition.

The usual procedure is to initially use a mobile phase with all the ions in the mixture bond & then to change the mobile phase in a stepwise fashion so that one kind of ion at a time is removed

Done until complete separation achieved

SIZE-EXCLUSION CHROMATOGRAPHY

Size-exclusion chromatography

Also called gel permeation chromatography

Technique for separating dissolved species on the basis of their size

Stationary phase: porous polymer resin particles (molecular sieves)

The components to be separated enter the pores of these particles & are slowed from progressing through this stationary phase.

Size-exclusion chroamtography

Separation depends on the sizes of the pores relative to the sizes of the molecules to be separated

Small particles are retarded to a greater extent than large particles (some of which may not enter the pores at all) & separation occurs.

TERMINOLOGIES IN CHROMATOGRAPHY

Terminologies in chromatography

Elution: a process in which species are washed through a chromatographic column by addition of fresh solvent

Mobile phase: is one that moves over or through an immobilized phase that is fixed in place in a column or on the surface of flat plate

Stationary phase: a solid or liquid that is fixed in place. A mobile phase then passes over or through the stationary phase

Retention time: is the time interval btw its injection onto a column and the appearance of its peak at the other end of the column

Migration Rates of Solutes

Distribution constant, K

Retention time, tR

Capacity factor,k’

Selectivity factor,

Distribution constant, K

In chromatography, the distribution equilibrium of analytes between the mobile and stationary phases can often be described quite simple.

Let say, we have analyte A. The distribution equilibrium is written as:

A mobile A stationary Therefore, the equilibrium constant K is called

distribution constant and is defined as:

K =

c – Molar concentration

cstationary

cmobileK is also called partition coefficient or partition ratio

Retention Time, tR

Time required for the sample to travel from the injection part through the column to the detector.

A typical chromatogram for a two-component mixture. The small peak on the left represents a species that is not retained on the column & so reaches the detector almost immediately after elution is started.

tM - time taken for the unretained species to reach the detector.- sometimes called dead time- Rate of migration of the unretained species is SAME as

the average rate of motion of mobile phase molecules.- So, tM can be expressed as the time required for an average molecule of the mobile phase to

pass through the column.

Retention Factor (Capacity factor), k’

term used to measure the migration rates of analytes on columns.

k’A = KA (VS / VM) [unitless] for

analyte A

How is k’A related to tR and tM?

k’A =

tR – tM tM

When k’A is 1.0, separation is poorWhen k’A is > 30, separation is slowWhen k’A is 2-10, separation is optimum

Selectivity Factor,

is defined as: =

=

=

A measure of the relative migration rates of species A and B with a stationary phase material in chromatography

KB

KA

distribution constants

k’B

k’A

capacity factors

tR(B) – tM tR(A) – tM

retention times

tM

tR

tR

Response

Retention time , min

1 3 6

tR – tM tM

k’ =tR(B) – tM tR(A) – tM

=

Column Efficiency

Two related terms widely used as quantitative measures of chromatographic column efficiency:

i) Plate height, Hii) Number of theoretical plates, N

The relationship between H and N is:

N =

The efficiency of chromatographic columns increases as the number of plates becomes greater and plate height become smaller.

LH

Number oftheoretical plates

Column length

Plate height

Efficient column has small plate height

Experimentally, H and N can be approximated from the width of the base of the chromatographic peak.

The equation:

N = 16

tR

W

2

N can be calculated using tR and W To obtain H, the length of the column must be known

Another method for approximating N is to determine W½, the width of the peak at half its maximum height.

N = 5.54 tR

2

W½

Resolution, Rs

A measure of the separation of two chromatographic peaks.

Baseline resolution is achieved when Rs = 1.5

Rs = 2[tR(B) – tR(A)] WA + WB

Effect of Capacity Factor & Selectivity Factor on Resolution

Relationship btw the resolution of a column and the capacity factor k’, selectivity factor and the number of plates N is given by this equation:

Rs = √N - 1 k’ 4 1 + k’

Simplified: Rs = √N

Effect Resolution on Retention Time

Relationship btw the resolution of a column and retention time:

tR = 16Rs2H ( 1 + k’)3

u - 1 (k’)2

2

Simplified: tR = Rs

2

Example

Length of column: 30 cm Peak widths (at base) for A & B were

1.11 & 1.21 min respectively. Calculate:

i) column resolution, Rsii) the average number of plates,

Niii) the plate height, Hiv) length of column to achieve

Rs 1.5

tM

tR

tR

Response

Retention time , min

1 3 6

1.30 min

16.40 min

17.63 min

i)

Rs = 2(17.63 min – 16.40 min)

(1.11 min + 1.21 min) = 1.06

ii)

N = 16 16.40 min 1.11 min

= 3.49 x 103

Rs = 2[tR(B) – tR(A)] WA + WB

N = 16

tR

W

2

2

N = 16 17.63 min 1.21 min

= 3.40 x 103

iii) H = L / N

= 30 cm / 3.44 x 103 = 8.7 x 10-3 cm

iv) (Rs)1 √N1

(Rs)2 √N2

2Therefore, calculatethe N average

Nave = 3.44 x 103

=

1.06 = √ 3.44 x 103

1.5 √ N 2

N2 = 6.9 x 103

L = N x H = 6.9 x 103 x 8.7 x 10-3

= 60 cm

BAND BROADENING

Band broadening reflects a loss of column efficiency.

The slower the rate of mass-transfer processes occuring while a solute migrates through a column, the broader the band at the column exit.

Some of the variables that affect mass-transfer rates are controllable and can be exploited to improve separations.

Table 26.2 lists the variables that influence the column efficiency.

Their effect on column efficiency, as measured by the plate height will be described in the following slides

VARIABLES AFFECTING COLUMN EFFICIENCY

VARIABLES AFFECTING COLUMN EFFICIENCY

Mobile phase flow rate Particle size Diameter of column Film thickness

EFFECT OF MOBILE PHASE FLOW RATE ON PLATE HEIGHT

From both the plots for LC and GC, we can see that both show a minimum in H at low linear flow rates.

EFFECT OF PARTICLE SIZE ON PLATE HEIGHT

Refer to figure 26-11 , page 773 The numbers to the right is the

particle diameters The smaller the particle size, the

more uniform the column packing, then the more tolerant to the change in mobile-phase velocity.

For packed column, the most important variables that affect column efficiency is the diameter of the particles that making up the packing.

While for open tubular column, the diameter of the column itself is an important variables.

Refer to table 26-3, the mobile phase mass-transfer coefficient CM is known to be inversely proportional to the diffusion coefficient of the analyte in the mobile phase DM.

EFFECT OF DIAMETER OF THE COLUMN ON PLATE HEIGHT

CM is proportional to the square of the particle diameter of the packing material, d1p (packed column).

CM is proportional to the square of the column diameter, d2p (open tubular column).

As a conclusion, the bigger the column diameter, the smaller the diffusion coefficient DM. therefore, we can say that increase in column diameter will increase the plate height.

When stationary phase is an immobilized liquid, the mass-transfer coefficient Cs is directly proportional to the square of the thickness of the film on the support particles d1l and inversely proportional to the diffusion coefficient Ds of the solute in the film.

With thick films and smaller diffusion coefficient, analyte molecule travel slower. As a result, slower rate of mass-transfer and an increase in plate height.

EFFECT OF FILM THICKNESS ON PLATE HEIGHT

Application of Chromatography Qualitative analysis Quantitative analysis

Qualitative Analysis

Based on retention time

Provided the sample produce the peak at the same retention time as a standard under identical conditions

Quantitative analysis

Analysis based on Peak Height The height of chromatographic peak is obtained

by connecting the base lines on either side of the peak by a straight line and measuring the perpendicular distance from this line to the peak.

Analysis based on Peak Area Peak areas are usually the preferred method of

quantitation since peak areas are independent of broadening effects.

Most modern chromatographic instruments are equipped with computer or digital electronic integrator that permit precise estimation of peak areas.

Calibration Method(also known as external method)

- Involve preparation of series of standard solutions that approximate the

composition of the unknown.- The peak heights or areas are plotted as a function of concentration.- The concentration of the component(s) to be analysed is determined by comparing the response(s) peak(s) obtained with the

standard solutions.

Internal Standard Method

- Equal amounts of an internal standard substance is introduced into each standard and sample.- The internal standard should not react with the substance to be examined; it must be stable

and must not contain impurities with a retention time similar to that of the substance to be examined.- The concentration of the substance to be examined is determined by comparing the ratio

of the peak areas (or heights) due to the substance to be examined and the internal standard in the test solution with the ratio of the

peak areas (or heights) due to the substance to be examined and the internal standard in the

standard solution.

Area Normalization Method

In the normalization method, the areas of all eluted peaks

The percentage content of one or more components of the substance to be examined is calculated by determining the area of the peak(s) as a percentage of the total area of all the peaks, excluding those due to solvents or any added reagents and those below the disregard limit.

TAILING AND FRONTING OF CHROMATOGRAPHIC PEAKS

A common cause of tailing and fronting is a distribution constant that varies with concentration.

Fronting also arises when the amount of sample introduced onto a column is too large.

TYPES OF COLUMN

There are two types of column:

Packed Column Capillary Column

PACKED COLUMN

Packed column Modern packed columns are fabricated

from glass or metal tubing. These tubes are densely packed with

uniform, finely divided packing material or solid support, coated with a thin layer (0.05 to 1 µm) of the stationary liquid phase.

The tubes are usually formed as coils.

CAPILLARY COLUMN Capillary column

Also known as open tubular column

Early wall –coated open tubular (WCOT) column were constructed of stainless steel, aluminium, copper or plastic.later, they are made from glass.

The most widely used capillary columns are fused-silica wall-coated (FSWC) open tubular columns.• Column constructed of fused silica tubing.• Polyimide coating gives it strength (outer layer). This

resulting columns are quite flexible and can be bent into coils.

• These capillaries have much thinner walls compared to glass columns.

• Liquid stationary phases coated or chemically bonded.

Most applications utilize FSWC open tubular column (replacing WCOT glass column).

Recently, 530-µm capillaries, sometimes called megabore column have appeared on the market.• These columns maintain the features of

capillary column.• These columns tolerate sample sizes that are

similar to those for packed column.• However, the resolution with these columns is

lower compared to capillary column (resolution is higher with column of smaller inner diameter).

• Advantage of megabore column over packed column is their lack of bleeding (loss of stationary phase with time).