chromatography 1 (1)

8/20/2019 Chromatography 1 (1)

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Introduction to Chromatography

Chromatography is a separation technique based on the different interactions of

compounds with two phases, a mobile phase and a stationary phase, as thecompounds travel through a supporting medium.

mobile phase: a solvent that flows through the supporting medium

stationary

phase: a layer or coating on the supporting medium that

interacts with the analytes

supporting medium: a solid surface on which the stationary phase is

bound or coated

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What is Chromatography

1) Solvent Extraction :transfer of a solute from phase 1 phase 2

S (in phase1) S (in phase 2)

Partition coefficient

1 2

s

s K


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What is Chromatography

A solute equilibrates between a mobile and a stationary phase.

The more it interacts with the stationary phase, the slower it is moved along acolumn.

Xm Xs

Ks =

Solutes with a large Ks value will be retained more strongly by the stationaryphase.

s

m

X

X


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Interaction Between Solutes, Stationary

Phase, and Mobile Phase

• Differences in the interactions between the solutes and stationary and

mobile phases enable separation.

Solute

Stationary

phaseMobile phase

Degree of adsorption,

solubility, ionicity, etc.


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The analytes interacting moststrongly with the stationary

phase will take longer to pass

through the system than those

with weaker interactions.

These interactions are usually

chemical in nature, but in some

cases physical interactions can

also be used.

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How do we describe a chromatogram

A graph showing the detectors response as a function of

elution time :

band’s shapes, position, resolution.

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Theory of Chromatography

1.) Typical response obtained by chromatography (i.e., a chromatogram):

chromatogram - concentration versus elution time

Wh

Wb

Where:

t R = retention time

t M = void time

Wb = baseline width of the peak in time unitsWh = half-height width of the peak in time units

Inject

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Note: The separation of solutes in chromatography depends on two factors:

(a) a difference in the retention of solutes (i.e., a difference in their time

or volume of elution

(b) a sufficiently narrow width of the solute peaks (i.e, good efficiency for

the separation system)

A similar plot can be made in terms of elution volume instead of elution

time. If volumes are used, the volume of the mobile phase that it takesto elute a peak off of the column is referred to as the retention volume

(VR) and the amount of mobile phase that it takes to elute a non-

retained component is referred to as the void volume (VM).

Peak width & peak position

determine separation of peaks

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2.) Solute Retention:

A solute’s retention time or retention volume in chromatography is

directly related to the strength of the solute’s interaction with the mobile

and stationary phases.

Retention on a given column pertain to the particulars of that system:

- size of the column

- flow rate of the mobile phase

Capacity factor (k’): more universal measure of retention, determined

from t R or VR.

k’ = (t R –t M )/t M

or

k’ = (VR –VM)/VMcapacity factor is useful for comparing results obtained on different

systems since it is independent on column length and flow-rate. 11


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The value of the capacity factor is useful in understanding the retention mechanisms for a

solute, since the fundamental definition of k’ is:

k’ is directly related to the strength of the interaction between a solute with the stationary

and mobile phases.

Moles Astationary phase and moles Amobile phase represents the amount of solute present in each

phase at equilibrium.

Equilibrium is achieved or approached at the center of a chromatographic peak.

k’ =moles Astationary phase

moles Amobile phase

When k' is 1.0, separation is poor When k' is > 30, separation is slow

When k' is = 2-10, separation is optimum

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A simple example relating k’ to the interactions of a solute in a column is illustrated for

partition chromatography:

A (mobile phase) A (stationary phase)

KD

where: KD = equilibrium constant for the distribution of A between the mobile

phase and stationary phase

Assuming local equilibrium at the center of the chromatographic peak:

k’ =[A]stationary phase Volumestationary phase

[A]mobile phase Volumemobile phase

k’ = KD

Volumestationary phase

Volumemobile phase

As K D increases, interaction of the solute with the stationary phase becomes more

favorable and the solute’s retention (k’) increases 13


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k’ = KD

Volumestationary phase

Volumemobile phase

Separation between two solutes requires different KD’s for their

interactions with the mobile and stationary phases

since

peak separation also represents different changes in free energy

DG = -RT ln KD

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3.) Efficiency:

Efficiency is related experimentally to a solute’s peak width.

- an efficient system will produce narrow peaks

- narrow peaks smaller difference in interactions in order toseparate two solutes

Efficiency is related theoretically to the various kinetic processes that

are involved in solute retention and transport in the column

- determine the width or standard deviation (s) of peaks

Wh Estimate s from peak widths,

assuming Gaussian shaped peak:

Wb = 4s

Wh = 2.354s

Dependent on the amount of time that a solute spends in the column (k’ or t R )

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Number of theoretical plates (N): compare efficiencies of a system for solutes that have

different retention times

N = (t R /s )2

or for a Gaussian shaped peak

N = 16 (t R /Wb)2

N = 5.54 (t R /Wh)2

The larger the value of N is for a column, the better the column will be able to separate

two compounds.

- the better the ability to resolve solutes that have small differences in retention

- N is independent of solute retention- N is dependent on the length of the column

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Plate height or height equivalent of a theoretical plate (H or HETP): compare efficiencies of

columns with different lengths:

H = L/N

where: L = column lengthN = number of theoretical plates for the column

Note: H simply gives the length of the column that corresponds to one theoretical plate

H can be also used to relate various chromatographic parameters (e.g., flow rate, particle size,

etc.) to the kinetic processes that give rise to peak broadening:

Why Do Bands Spread?

a. Eddy diffusion

b. Mobile phase mass transfer

c. Stagnant mobile phase mass transfer

d. Stationary phase mass transfer

e. Longitudinal diffusion

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a.) Eddy diffusion – a process that leads to peak (band) broadening due to the presence

of multiple flow paths through a packed column.

As solute molecules travel through the column,

some arrive at the end sooner then others simply

due to the different path traveled around the

support particles in the column that result in

different travel distances.

Longer path arrives at end of column after (1).

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c.) Stagnant mobile phase mass transfer – band-broadening due to differences in the

rate of diffusion of the solute molecules between the

mobile phase outside the pores of the support

(flowing mobile phase) to the mobile phase within

the pores of the support (stagnant mobile phase).

Since a solute does not travel down

the column when it is in the stagnant

mobile phase, it spends a longer time

in the column than solute that

remains in the flowing mobile phase.

The degree of band-broadening due to stagnant mobile phase mass

transfer depends on:

1) the size, shape and pore structure of the packing material

2) the diffusion and retention of the solute

3) the flow-rate of the solute through the column 22


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d.) Stationary phase mass transfer – band-broadening due to the movement of solute

between the stagnant phase and the stationary phase.

Since different solute molecules

spend different lengths of time in the

stationary phase, they also spend

different amounts of time on the

column, giving rise to band-

broadening.

The degree of band-broadening due to stationary phase mass transfer

depends on:

1) the retention and diffusion of the solute

2) the flow-rate of the solute through the column

3) the kinetics of interaction between the solute and the

stationary phase 23


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e.) Longitudinal diffusion – band-broadening due to the diffusion of the solute along the

length of the column in the flowing mobile phase.

The degree of band-broadening due

to longitudinal diffusion depends on:

1) the diffusion of the solute

2) the flow-rate of the solute through

the column

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m optimum

Plot of van Deemter equation shows how H changes with the linear velocity (flow-rate) of

the mobile phase

Optimum linear velocity (mopt) - where H has a minimum value and the point of maximumcolumn efficiency:

mopt = rB/C

mopt is easy to achieve for gas chromatography, but is usually too small for liquid

chromatography requiring flow-rates higher than optimal to separate compounds 26


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4.) Measures of Solute Separation:

separation factor (a) – parameter used to describe how well two solutes are separated by

a chromatographic system:

a = k’2/k’1 k’ = (t R –t M )/t M where:

k’1 = the capacity factor of the first solute

k’2 = the capacity factor of the second solute,

with k’2$ k’1

A value of a$

1.1 is usually indicative of a good separation

Does not consider the effect of column efficiency or peak widths, only retention. 27


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resolution (R S) – resolution between two peaks is a second measure of how well two

peaks are separated:

RS =

where:tr1, Wb1 = retention time and baseline width for the

first eluting peak

tr2, Wb2 = retention time and baseline width for the

second eluting peak

tr2 – tr1

(Wb2 + Wb1)/2

Rs is preferred over a since both

retention (tr ) and column efficiency

(Wb) are considered in defining

peak separation.

Rs$ 1.5 represents baseline

resolution, or complete separation

of two neighboring solutes ideal

case.

Rs$ 1.0 considered adequate for

most separations. 28


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Types of Chromatography

1.) The primary division of chromatographic techniques is based on the type of mobile phase

used in the system:

Type of Chromatography Type of Mobile Phase

Gas chromatography (GC) gas

Liquid chromatograph (LC) liquid

2.) Further divisions can be made based on the type of stationary phase used in the system:

Gas ChromatographyName of GC Method Type of Stationary Phase

Gas-solid chromatography solid, underivatized support

Gas-liquid chromatography liquid-coated support

Bonded-phase gas chromatography chemically-derivatized support

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Types of Chromatography

Liquid Chromatography

Name of LC Method Type of Stationary Phase

Adsorption chromatography solid, underivatized supportPartition chromatography liquid-coated or derivatized support

Ion-exchange chromatography support containing fixed charges

Size exclusion chromatography porous support

Affinity chromatography support with immobilized ligand

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3.) Chromatographic techniques may also be classified based on the type of support material

used in the system:

Packed bed (column) chromatography

Open tubular (capillary) chromatography

Open bed (planar) chromatography

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• Mobile Phase: Liquid

• Stationary Phase Separation Mechanism

- Solid Adsorption

- Liquid Layer Partition

- Ion exchange resin Ion exchange- Microporous beads Size Exclusion

- Chemically modified resin Affinity

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Column Chromatography and Planar

Chromatography

Separation column

Packing material

Column Chromatography

Paper or a

substrate coated

with particles

Paper Chromatography

Thin Layer Chromatography (TLC)33


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PARTITION CHROMATOGRAPHY

Partition chromatography can be subdivided into

(i) liquid-liquid chromatography and

(ii) bonded-phase chromatography.

• With liquid-liquid, a liquid stationary phase is retained onthe surface of the packing by physical adsorption.

• With bonded-phase, the stationary phase is bonded

chemically to the support surfaces.

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PARTITION CHROMATOGRAPHY

Early partition chromatography was the liquid-liquid type;

now the bonded-phase method has become predominatebecause of certain disadvantages of liquid-liquid systems.

• One of these disadvantages is the loss of stationary

phase by dissolution in the mobile phase, which requires

periodic recoating of the support particles.

• Furthermore, stationary-phase solubility problems prohibit

the use of liquid-phase packings for gradient elution.

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Columns for Bonded-Phase Chromatography

The supports for the majority of bonded-phase

packings for partition chromatography areprepared from rigid silica, or silica-based,

compositions.

These solids are formed as uniform, porous,

mechanically sturdy particles commonly having

diameters of 3, 5, or 10mm. The surface of fully

hydrolyzed silica is made up of chemically reactive

silanol groups.

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The most useful bonded-phase coatings are

siloxanes formed by reaction of the hydrolyzed

surface with an organochlorosilane.

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Reversed-Phase and Normal-Phase Packings


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Reversed-Phase and Normal-Phase Packings

Two types of partition chromatography are

distinguishable based upon the relative polarities of

the mobile and stationary phases.

Liquid chromatography based upon highly polar

stationary phases such as water or triethyleneglycol

supported on silica or alumina particles; a relatively

nonpolar solvent such as hexane or I-propylether

then served as the mobile phase. This type of

chromatography is referred to as normal-phase

chromatography.

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Reversed-phase chromatography

The stationary phase is nonpolar, often a

hydrocarbon, and the mobile phase is relativelypolar (such as water, methanol, or acetonitrile).

In normal-phase chromatography, the least polar component is eluted first because it is the mostsoluble in the mobile phase; increasing the polarityof the mobile phase has the effect of decreasingthe elution time.

In contrast, in the reversed-phase method, the mostpolar component appears first, and increasing the

mobile phase polarity increases the elution time. 40


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Ion Exclusion Chromatography

• Separation of ionic solutes from weakly

ionized of neutral solutes using strong

cation and anion exchange resins

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Factors Affecting Retention in IC

• Degree of ionization of the solute

- solute pKa

- eluent pH

- organic modifier content• hydrophobic interactions between solute

and sp

• molecular size of the solute

• degree of X-linkage of the sp

• system temperature44


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Complexation Chromatography

• Separation based on the rapid

reversible formation of a complex

between metal ions and ligands• Suitable ligands are immobilized on sp

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Chiral Chromatography

• Separation based on the use of chiral

stationary phases which interact to

varing degrees with optical isomers of

the solute

• Systems differ little from conventional

HPLC systems

• Limited applications for a given sp

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GAS CHROMATOGRAPHY

Mobile phase is a gas!

Stationary phase could be anythingbut a gas

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Gas Chromatography (GC)

GC is currently one of the most popular methods for separating and analyzing

compounds.

This is due to its high resolution, low limits of detection, speed, accuracy and

reproducibility.

GC can be applied to the separation of any compound that is either naturally volatile (i.e.,readily goes into the gas phase) or can be converted to a volatile derivative.

This makes GC useful in the separation of a number of small organic and inorganic

compounds (They can be big compounds if you can make them small before separation!)

A simple GC system consists of:

1. Gas source (with pressure and flow regulators)

2. Injector or sample application system (sample inlet)

3. Chromatographic column (with oven for temperature control)

4. Detector & computer or recorder

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Carrier gas: He (common), N2, H2Pinlet 10-50 psig

Flow = 25-150 mL/min packed column

Flow = 1-25 mL/min open tubular column

Column: 2-100 m coiled stainless steel/glass/Teflon/fused silica, packed vs. unpacked

Oven: 0-400 °C ~ average boiling point of sample

Accurate to


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(a) Filling sampling loop

(b) Introduction of sample into column

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GC separates solutes based on their different interactions between mobile and stationary

phases.

solute’s retention is determined mostly by its vapor pressure and volatility

solute’s retention is controlled by its interaction with the stationary phase

Carrier gas – main purpose of the gas in GC is to move the solutes along the column, mobile

phase is often referred to as carrier gas (MUST BE INERT!).

Common carrier gas: include He, Ar, H2, N2

Carrier Gas or Mobile phase does not affect solute retention, but does affect :

1.) Desired efficiency for the GC System (Van Deemter!)

- low molecular weight gases (He, H2) larger diffusion coefficients

- low molecular weight gases faster, more efficient separations

2.) Stability of column and solutes

- H2 or O2 can react with functional groups on solutes and stationary

phase or with surfaces of the injector, connections and detector

3.) Response of the detector

- thermal conductivity detector requires H2 or He- other detectors require specific carrier gas compatibility

Mobile Phase

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Stationary phase in GC is the main factor determining the selectivity and retention

of solutes.

There are three types of stationary phases used in GC:

• Solid adsorbents

• Liquids coated on solid supports

• Bonded-phase supports

1.) Gas-solid chromatography (GSC)

- same material is used as both the stationary phase and support material

- common adsorbents include:

alumina

molecular sieves (crystalline aluminosilicates [zeolites] and clay)

silica

active carbon

Magni f ied Pores in act ivated carbon

Mobile Phases

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G

S lid Ch h


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Advantages:

- long column lifetimes

- ability to retain and separate some compounds not easily resolved by other

GC methods

geometrical isomers

permanent gases

Disadvantages:

- very strong retention of low volatility or polar solutes

- catalytic changes that can occur on GSC supports

- GSC supports have a range of chemical and physical environments

different strength retention sites

non-symmetrical peaks

variable retention times

Gas-Solid Chromatography

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G

Li id Ch h


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2.) Gas-liquid chromatography (GLC) LIKE DISSOLVES LIKE!

- stationary phase is a liquid coated on a solid support- over 400 liquid stationary phases available for GLC

- material range from polymers (polysiloxanes, polyesters, polyethylene glycols) to

fluorocarbons, and liquid crystals etc.

Based on polarity, of the 400 phases available only 6-12 are needed for most separations.

The routinely recommended phases are listed below:

Gas-Liquid Chromatography

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chromatography 1 (1)

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