INTRODUCTION TO CHROMATOGRAPY

HISTORY

The Russian botanist Mikhail Tswett coined the term chromatography in 1906 to describe his experiments in separating different colored constituents of leaves by passing an extract of the leaves through a column

Analytic technique to discover chemical components:

a method of finding out which components a gaseous

or liquid mixture contains that involves passing it

through or over something that absorbs the different

components at different rates

ChromatographyWeb Dictionary:

Chromatography

Carrier Gas

Flow Control

Injector Port

Column

Column Oven

DetectorRecorder

CHROMATOGRAPHY COLUMNS

Packed Column: Typical HPLC columns but some gas chromatography columns also (especially older columns). The columns are packed with tiny particles.

Capillary Column: Typical gas chromatography column which consists of a small diameter tube coated on the inside with stationary phase.

CHROMATOGRAPHY THEORY

PARTITION COEFFICIENT

K = Co/Cw

Co is concentration in the organic phase (solvent)Cw is the concentration in the aqueous phase (water)

Remember from the solvent lecture……….

K = Co/Cw

Co is concentration in the organic phase (solvent)Cw is the concentration in the aqueous phase (water)

PARTITION COEFFICIENT

molar concentration in stationary phase molar concentration in mobile phase K =

PARTITION COEFFICIENT ETC.

mass in the stationary phase mass in the mobile phase

volume of mobile phasevolume of stationary phase

concentration in stationary phase concentration in mobile phase K =

k =

b =

PARTITION COEFFICIENT ETC.

k = K/b

If mass = volume x concentration then:

EXAMPLE:

K = 4b =k =grams inmobile phase =

Compound A: mass = 1 mgVol. Mobile Phase: 1 mLVol. Stationary Phase: 1 mL

Compound A: mass = 1 mgVol. Mobile Phase: 1 mLVol. Stationary Phase: 2 mL

K = 4b =k =grams inmobile phase =

Mobile Phase

StationaryPhase

14 0.2

0.58 0.11

If the mobile phase is moving, in which situation will compound A move faster through the column?

PARTITIONING IN A MOBILE PHASE

1.0 mg

0.16 mg

0.83 mg 0.83 mg

0.14 mg

0.69 mg 0.69 mg

0.12 mg

0.58 mg

0.10 mg 0.08 mg 0.07 mg 0.06 mg

0.28 mg

Theoretical Plates

0.16 mg0.03 mg

0.13 mg

0.14 mg 0.12 mg 0.08 mg 0.07 mg 0.06 mg

0.28 mg0.13 mg0.23 mg

0.05 mg 0.10 mg

0.23 mg

0.12 mg

0.29 mg

0.06 mg

0.29 mg

0.10 mg

0.32 mg

0.06 mg

Partitioning in a Mobile Phase

PARTITIONING IN A MOBILE PHASE

1.0 mg

0.00 mg

0.83 mg 0.83 mg

0.00 mg

0.69 mg 0.69 mg

0.00 mg

0.58 mg

0.03 mg 0.04 mg 0.07 mg 0.06 mg

0.28 mg0.17 mg0.05 mg 0.34 mg 0.28 mg0.01 mg0.00 mg0.00 mg

Note: These equilibrium steps to do not actually take place in the column, it is a continuous process.

ANALYTE PEAKS IN THE MOBILE PHASE

1.0 mg

0.00 mg

0.83 mg 0.83 mg

0.00 mg

0.69 mg 0.69 mg

0.00 mg

0.58 mg

0.03 mg 0.04 mg 0.07 mg 0.06 mg

0.28 mg0.17 mg0.05 mg 0.34 mg 0.28 mg0.01 mg0.00 mg0.00 mg

How would you make this broad peak more narrow?

ANALYTE PEAKS IN THE MOBILE PHASE

SEPARATION OF PEAKS

RETENTION

k = (tr – to)/ to

Where tr = the retention time of the compound, and to = the dead time

Higher values of k mean the analyte will stay in the column longer. The longer it stays, the more time there is for the peak will widen.

SELECTIVITY

a = kB/kA

the selectivity factor α and is an indication of how well the compounds will separate. Higher α means larger difference in retention time and more separation

EFFICIENCY

Efficiency is a factor that is typically used todescribe peak width.

High Efficiency - narrow peaks

EFFICIENCY

The term that is generally used to describe column efficiency is “number of theoretical plates” or N

N = L/H

Where: L =column lengthH = plate height (both in the same units)

N IN PRACTICAL TERMS...

Units for tr and to….?Units for W1/2 …..?

N can be measured from the peaks on a chromatogram..

N = 5.54 tr

w1/2( )

2

RESOLUTION

The purpose of chromatography is to separate or resolve compounds. The separation or distance

between two peaks is known as their resolution and is a function of the 3 factors discussed previously:

retention (the time it takes for the analytes to elute, related to k), selectivity (how

different the analytes are from each other and related to α), and efficiency (how good the column is,

related to N)

RESOLUTION

Rs = ¼ (-1/) (k/k+1) N½

The effect on Rs of:increasing a…?increasing k…?increasing N…?

Efficiency

Selectivity Retention

RESOLUTION

Rs = 2 (tR-B – tR-A)/(wb-A + wb-B)

Where: A and B are the two peakstR = retention time andwb = the peak width at the

base of each peak

Rs can also be calculated from actual measurements of peak retention times and measured peak widths

RESOLUTION

With a resolution value of 1.0, two peaks that overlap by about 4%. Values less than 1.0 indicate peaks that overlap, while at a resolution of 1.5, the

peaks are considered fully separated.

GOING BACK TO N….

N = L/HThe value of N is greatly dependent on the value of H.

The value of H depends primarily on four factors:1) the velocity of the mobile phase,2) eddy diffusion or multipath diffusion, 3) the diffusion of the compound in the mobile phase4) the transfer of the compound between the stationary phase and the mobile phase.

H - Theoretical Plate Height

H = A + B/u + (Cs + Cm) u

u = the average linear mobile phase velocity A is a term expressing multipath diffusionB/u is the term for longitudinal diffusionCs is the mass transfer term in the stationary

phase Cm is the mass transfer term in the mobile

phase

A Multipath

1 2

FlowDirection

Pathways of two molecules during elution. Distance traveled by molecule 1 is longer than that traveled by molecule 2, thus molecule 1 will take longer toelute.

The amount of spreading is affected by the nature of the column material and how well the column is packed. This factor is generally proportional to the particle size of the packing material. This factor must be taken into account for packed columns, but for capillary columns, this term is not needed since there are no particles.

B Longitudinal Diffusion

Flow

Flow

Molecules diffuse from areas of highconcentration to areas of low concentration.

Over time….

At low velocities longitudinal diffusion has a negative effect on resolution, but this effect is negligible at higher velocities. This term is very important in gas chromatography as diffusion coefficients in gasses are orders of magnitude higher than in liquids. In liquid chromatography, this term is typically close to zero relative to the other terms.

Equilibrium between the mobile and stationary phases is never realized

Mass Transfer Terms Cs & Cm

It takes time for analytes to move from the mobile phase into the stationary phase. Because no equilibrium is reached, some of the analytes are swept ahead of the of the main band.

It also takes time for molecules to move back out of the stationary phase, and some of the analyte molecules will be left behind by the rapidly moving mobile phase.

Mass Transfer Terms Cs & Cm

The faster the mobile phase moves, the less time there is for equilibrium between the phases and the mass transfer effect on peak broadening is directly

related to mobile phase velocity.

VAN DEEMTER PLOT

Linear Velocity, u

Pla

te H

eigh

t, H

Multipath Term, A

Mass Transfer (both), Cu

Longitudinal diffusion, B/u

A + B/u + Cu