Chromatography CacPPPTHD 10 2014

Chromatography

Chromatography

Chromatography

Chromatography

The history of modern chromatography What is a chromatographic method Classifying Analytical Separations General Theory of Column Chromatography Applications

Chromatography

The history of modern chromatography

1872 - 1919 The Russian botanist Mikhail Tswettused a column packed with a stationary phase of calcium carbonate and a mobile phase of petroleum ether to separate colored pigments from plant extracts.1941 Martin and Singe established the importance theory for chromatographic separations (Nobel Prize in 1952)

Since then, chromatography in its many forms has become the most important and widely used separation technique

Chromatography

What is a chromatographic method

Chromatography is a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary (stationary phase), while the other (the mobile phase) moves in a definite directions

Chromatography

Classifying Analytical Separations Analytical separations may be classified in

three ways: (1) by the physical state of the mobile phase

and stationary phase; (2) by the method of contact between the

mobile phase and stationary phase; (3) or by the chemical or physical mechanism

responsible for separating the samples constituents.

Chromatography

Classifying Analytical Separations (1) Analytical separations by the physical

state of the mobile phase and stationary phase: The mobile phase is usually a liquid or a gas, and

the stationary phase is a solid or a liquid film coated on a solid surface.

Chromatographic techniques are often named by listing the type of mobile phase, followed by the type of stationary phase. (For example, gasliquid chromatography: the mobile phase is a gas and the stationary phase is a liquid.)

If only one phase is indicated, as in gas chromatography, it is assumed to be the mobile phase.

Chromatography

GC instrument

Chromatography

Schematic diagram for a typical gas chromatograph

Chromatography

GC

Chromatography

Chromatography

Packed columns Capillary columns the most widely used

Wall-coated open-tubular (WCOT) wall-coated open tubular consist of a capillary tube whose

walls are coated with liquid stationary phase. Support coated open-tubular (SCOT)

support coated open tubular consist of a capillary lined with a thin layer of support material such as diatomaceous earth, onto which the stationary phase has been adsorbed.

Porous layer open-tubular (PLOT) porous layer open tubular columns in which a thin layer of

adsorbent is affixed to the inner walls of the capillary.

Stationary Phase

Chromatography

Stationary Phase

Chromatography

Stationary Phase

An example of a trimethylsilyl deactivating group.

Glass surface

Chromatography

Stationary Phase

Chromatography

Classification of Chromatographic Techniques

GC

Typical gas chromatogram of comples mixture using a capilary column

Chromatography

Gas Chromatography

Mobile Phase Chromatographic Columns Stationary Phases Sample Introduction Temperature Control Detectors for Gas Chromatography Quantitative Applications

Chromatography

What compounds Can Be Determined by GC

- All gases

- Most nonionized organic molecules, solid or liquid, containing up to about 25 carbons

- Many organometallic compounds (volatile derivatives of metal ions may be prepared)

Chromatography

Chromatography

Schematic diagram of a high-performanceliquid chromatograph

Chromatography

Chromatography

High-Performance Liquid Chromatography

Mobile Phases HPLC Columns Stationary Phases Sample Introduction Detectors for HPLC Quantitative Applications

Chromatography

Classifying Analytical Separations (2) Analytical separations by the method

of contact between the mobile phase and stationary phase Column chromatography:

The stationary phase is placed in a narrow column through which the mobile phase moves under the influence of gravity or pressure

The stationary phase is either a solid or a thin, liquid film coating on a solid particulate packing material or the columns walls.

Planar chromatography: The stationary phase coats a flat glass, metal, or

plastic plate and is placed in a developing chamber. A reservoir containing the mobile phase is placed in

contact with the stationary phase, and the mobile phase moves by capillary action.

Chromatography

Chromatography

Classifying Analytical Separations (3) Analytical separations by the chemical

or physical mechanism responsible for separating the samples constituents Adsorption chromatography:

solutes separate based on their ability to adsorb to a solid stationary phase.

Partition chromatography: A thin liquid film coating a solid support serves as the

stationary phase. Separation is based on a difference in the equilibrium

partitioning of solutes between the liquid stationary phase and the mobile phase.

Chromatography


or physical mechanism responsible for separating the samples constituents. Adsorption chromatography: solutes separate based on

their ability to adsorb to a solid stationary phase. Partition chromatography: a thin liquid film coating a

solid support serves as the stationary phase. Separation is based on a difference in the equilibrium partitioning of solutes between the liquid stationary phase and the mobile phase.

Chromatography


or physical mechanism responsible for separating the samples constituents. Ion exchange chromatography: Stationary phases

consisting of a solid support with covalently attached anionic (e.g., SO3) or cationic (e.g., N(CH3)3+) functional groups. Ionic solutes are attracted to the stationary phase by electrostatic forces.

Size-exclusion chromatography: Porous gels are used as stationary phases. Separation is due to differences in the size of the solutes. Large solutes are unable to penetrate into the porous stationary phase and so quickly pass through the column. Smaller solutes enter into the porous stationary phase, increasing the time spent on the column.

Chromatography

Classifying Analytical Separations Not all separation methods require a stationary

phase. Electrophoretic separation: charged solutes migrate

under the influence of an applied potential field. Differences in the mobility of the ions account for their separation.

Chromatography

Classifying Analytical Separations (3)a) Adsorption chromatography

b) Partition chromatography

c) Ion-exchange chromatogIonraphy

d) Size-exclusion chromatography

Chromatography

General Theory of Column Chromatography

Progress of a column chromatographicseparation showing the separation oftwo solute bands.

Chromatography


chromatogram A plot of the detectors signal as function of elution

time or volume. retention time

The time a solute takes to move from the point of injection to the detector (tr).

retention volume The volume of mobile phase needed to move a solute

from its point of injection to the detector (Vr). Dividing the retention volume by the mobile phases flow rate, u, gives the retention time.

baseline width The width of a solutes chromatographic band

measured at the baseline (w).

Chromatography

void time The time required for unretained solutes to move

from the point of injection to the detector (tm). void volume

The volume of mobile phase needed to move an unretained solute from the point of injection to the detector.


Chromatography


tr : retention time

tm : void time

W : baseline width

Chromatography

Chromatographic ResolutionresolutionThe separation between twochromatographic bands (R).

Three examples of chromatographicresolution

Chromatography

Chromatographic Resolution- Example

In a chromatographic analysis of lemon oil a peak for limonene has a retention time of 8.36 min with a baseline width of 0.96 min. g-Terpinene elutes at 9.54 min, with a baseline width of 0.64 min. What is the resolution between the two peaks?

Chromatography

capacity factor

capacity factor A measure of how strongly a solute is retained by the

stationary phase (k ).

adjusted retention time The difference between a solutes retention time and

columns void time (tr).

Chromatography

capacity factor

In a chromatographic analysis of low-molecular-weight acids, butyric acid elutes with a retention time of 7.63 min. The columns void time is 0.31 min. Calculate the capacity factor for butyric acid.

Chromatography

Column Selectivity

selectivity factor The ratio of capacity factors for two solutes showing

the columns selectivity for one of the solutes ().

Chromatography

Column Selectivity - Example In a chromatographic analysis of low-molecular-

weight acids, butyric acid elutes with a retention time of 7.63 min. The columns void time is 0.31 min. The retention time for isobutyric acid is 5.98 min. What is the selectivity factor for isobutyric acid and butyric acid?

butyric

isobutyric

Chromatography

Column Efficiency Column efficiency provides a quantitative

measure of the extent of band broadening band broadening

the increase in a solutes baseline width as it moves from the point of injection to the detector.

theoretical plate Martin and Synge treated the chromatographic

column as though it consists of discrete sections (theoretical plate) at which partitioning of the solute between the stationary and mobile phases occurs.

With N: theoretical plates

H: the height of a theoretical plate

L: the column length

Chromatography

Column Efficiency The number of theoretical plates

The number of theoretical plates depends on both the properties of the column and the solute.

The number of theoretical plates for a column is not fixed and may vary from solute to solute.

Columns with more theoretical plates are more likely to separate a complex mixture.

tr: the retention time

W1/2: the width of the chromatographic peak at half its height

W: the width of the chromatographic peak

Chromatography

Theoretical plates - Example A chromatographic analysis for the chlorinated

pesticide Dieldrin gives a peak with a retention time of 8.68 min and a baseline width of 0.29 min. How many theoretical plates are involved in this separation? Given that the column used in this analysis is 2.0 meters long, what is the height of a theoretical plate?

Chromatography

Nonideal Behavior

fronting A tail at the beginning of a chromatographic peak,

usually due to injecting too much sample (a). Tailing

A tail at the end of a chromatographic peak, usually due to the presence of highly active sites in the stationary phase (b).

Chromatography

Optimizing Chromatographic Separations

kB, (the effect of solute Bs capacity factor)

NB: the number of theoretical plates (the effect of column efficiency)

: the influence of column selectivity

(1) (2) (3)

Chromatography

Using the Capacity Factor to Optimize Resolution (3)

Increasing kB (when kB small) when the original value of kB is 1, increasing its

value to 10 gives an 82% improvement in resolution; a further increase to 15 provides a net improvement in resolution of only 87.5%.

However, improvement in resolution obtained by increasing kB generally comes at the expense of a longer analysis time.

Chromatography


Increasing kB by decreasing the columns temperature in gas

chromatography At a lower temperature a solutes vapor pressure

decreases (it spends more time in the stationary phase). Therefore, its capacity factor increases.

Temperature programming in gas chromatographyThe process of changing the columns temperature to enhance the separation of both early and late eluting solute accomplished.

Chromatography

A typical temperature program

(a)

(b)

(c)

(a) = initial temperature and time(b) = ramp (C/min)(c) = final hold time and temperature

Some GCs will allow for far more complex temperature programming

Chromatography


Increasing kB By decreasing the solvent strength in liquid

chromatography When the mobile phase has a low solvent strength,

solutes spend proportionally more time in the stationary phase, thereby increasing their capacity factors.

Gradient elution in liquid chromatographyThe process of changing the mobile phases solvent strength to enhance the separation of both early and late eluting solutes.

Chromatography


Chromatography

Using Column Selectivity to Optimize Resolution (2)

A second approach to improving resolution is to adjust alpha, a.

when a is nearly 1, it usually is not possible to improve resolution by adjusting kB or N.

changing a from 1.1 to 1.5 improves resolution by 267%.

In gas chromatography, adjustments in a are usually accomplished by changing the stationary phase,

In liquid chromatography, changing the composition of the mobile phase is used.

Chromatography


The variation in retention time with mobile phase pH

Chromatography


the change in alpha with mobile phase pH

Chromatography

Using Column Efficiency to Optimize Resolution (1)

Increase the length of the column (increase retention time)

Decrease the height of a theoretical plate To determine how the height of a theoretical plate

can be decreased, it is necessary to understand the experimental factors contributing to the broadening of a solutes chromatographic band.

The height of a theoretical plate is determined by four contributions: multiple paths, longitudinal diffusion, mass transfer in the stationary phase, and mass transfer in the mobile phase.

Chromatography

Chromatography Effiency The van Deemter Equation

uCu

BAH

The net height of a theoretical plate is a summation of three terms: Multiple Paths Longitudinal Diffusion Mass Transfer

u: the average linear velocity of the carrier gas in cm/s (or the liquid mobile-phase velocity for liquid chromatography)

Multiple path term

Longitudinal dispersion term

Mass transferterm

A, B and C are constants for a given system

Chromatography

Multiple Paths

Molecules passing through a stationary phase that differ in path and length

pdHp 2Where Hp= contribution to theoretical plate

= constant associated with consistency of packingdp= average diameter of packing material

Open tubular column Hp=0

A term

Chromatography

Longitudinal Diffusion

u

DH md 2

Where Dm = the solutes diffusion coefficient in the mobile phase= constant relating to column packingm = mobile phase velocity

B term

decreases by increasing the flow rateu

B

Chromatography

Mass TransferDiffusion of the solute between the mobile and stationary phase interface

uDk

dqks

s

fH 22

)'1('

Where df =thickness of stationary phase

dc= the columns diameterdp= average diameter of packing materialDs= solutes diffusion coefficient in stationary phaseDm= solutes diffusion coefficient in mobile phaseq = constant related to packing materialk = capacity factor

uddfn

mDm

cpH ),(22

the exact form of Hm is unknown

C term

Chromatography

Mass transfer

M

p

Dd

C2

61

Where DM = the solutes diffusion coefficient in the mobile phasedp= average diameter of packing material

The type and amount of liquid phase (of the stationary phase), temperature

For example, C decreases : thin stationary liquid phase film to minimize diffusion within this phase

Cu decreases by decreasing the flow rate

Chromatography

Gas Chromatography Effiency The van Deemter Equation

uCu

BAH

The net height of a theoretical plate is a summation of three terms: Multiple Paths (Eddy diffusion) Longitudinal Diffusion Mass Transfer

H: The height of a theoretical plate

u: the average linear velocity of the carrier gas in cm/s (or the liquid mobile-phase velocity for liquid chromatography)

Multiple path term

Longitudinal dispersion term

Mass transferterm

A, B and C are constants for a given system

Chromatography

Multiple Paths

Hp: The contribution of multiple paths to the height of a theoretical plate,

dp is the average diameter of the particulate packing material

is a constant accounting for the consistency of the packing

Chromatography

Longitudinal Diffusion

longitudinal diffusion One contribution to band broadening in which solutes

diffuse from areas of high concentration to areas of low concentration.

Because a solutes diffusion coefficient is larger in a gaseous mobile phase than in a liquid mobile phase, longitudinal diffusion is a more serious problem in gas chromatography

Dm: the solutes diffusion coefficient in the mobile phase

u: the mobile phase velocity: a constant related to the column packing

Chromatography

Mass transfer

Diffusion of the solute between the mobile and stationary phase interface

M

p

Dd

C2

61

Where DM = the solutes diffusion coefficient in the mobile phasedp= average diameter of packing material

The constant C term is the interphase mass transfer term and is due to the finite time required for equilibrium of the solute to be established between the two phases as it moves between the mobile and stationary phases.

The type and amount of liquid phase (of the stationary phase), temperature

The term Cu decreases by decreasing the flow rate

For example, C decreases : thin stationary liquid phase film to minimize diffusion within this phase

for LC, smalle particles, thin stationary phase films, low-viscosity mobile phases and high temperatures.

Chromatography

Van Deemter Curve

Chromatography

van Deemter Curves

Chromatography

HPLC - Example A 2.013-g sample of dried soil is extracted with

20.00 mL of methylene chloride. After filtering to remove the soil, a 1-mL portion of the extract is removed and diluted to 10 mL with acetonitrile. Injecting 5 mL of the diluted extract into an HPLC gives a signal of 0.217 for the PAH fluoranthene. When 5 mL of a 20.0-ppm fluoranthene standard is analyzed using the same conditions, a signal of 0.258 is measured. Report the parts per million of fluoranthene in the soil.

Chromatography

Quantitative Measurements

Peak area ratio(EtOH/PrOH)

0.24945

0.619154

1.1918

1.86534

2.39374

1.17868

The blood alcohol concentration is 0.142% (wt/vol)

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Chromatography CacPPPTHD 10 2014