ANALYTICAL SEPARATIONS

Precipitation

Gravimetry

•Precipitation•Filtration•Washing•Drying or ignition•Measuring•Calculation

Separating species by distillation

Determination of ammonia

Determination of carbon dioxide

Extraction

Distribution between organic and water phase

Separation of metal ions as chelates

Ions are: soluble in water insoluble in non polar-organic phase

O(C6H5 ) NH4

+

NO

O(C6H5 )3 Fe

NO

Cupferron: ionic ferric cupferrate: neutral

Separation of Fe3+ ion

Separating ions by ion exchangeCation exchange:xRSO3

-H+ + Mx+ (RSO3-)xMx+ + xH+

solid soln solid soln

Anion exchange:xRN(CH3)3

+OH- + Ax- [RN(CH3)3]xAx- + xOH-

solid soln solid soln

where: Mx represents a cation and R a part of resin containing sulfonic acid group

where: Ax- represents an anion and R a part of resin containing trimethyl ammonium group

After ion exchange cations or anions are on the resin, it should remove them

Chromatography

Mobile phase

Stationary phasesolid liqiud

Gas chromatographyGC

gas GSC GLC

Supercritical chromatography

SFC

Supercritical fluid

SFC SFC

Liquid chromatographyLC liquid

TLCIC

GPC,SEC

PC

Normal phase

(HPLC-NP)

Reversed phase

(HPLC-RP)

Liquid chromatographyLC

liquidCE

GEL ELFO

Classification of chromatographic methods

Frontal chromatography

Modes of chromatographic separation

Displacement chromatography

Elution chromatography

1. Physical interactions-sorption: adsorption

absorption (solvation, distribution)chemisorption

-hydrofil-interactions

-hydrofob-interactions

-interactions based on size exclusion

2. Chemical interactions-acid-base interactions

-complex formation

-H-bond interactions

3. Biochemical interactions-biochemical affinity

Interactions in chromatography

Consequences:•Analytes are moving with different rates (differential migration)•In the course of chromatographic process band are wider and wider (band broadening)

The chromatographic process

Retention data

Retention time: tR

Dead time: tM (t0)

Reduced retention time: tR’ = tR - tM

Retention volume:

Reduced retention volume:

FtV RR

MRMRRR VVFttFtV

where: F, volumetric flow rate (cm3/min)

The average linear rate of solute migration, (usually cm/s)

RtL

where L is the length of the column, tR retention time

RtLu

The average linear velocity of the mobile phase molecules, u

The relationship between migration rate and distribution constant

The rate as a fraction of the velocity of mobile phase:

phasemobileinspendsoluteoftimeoffractionu

This fraction equals the average numbers of moles of solute in the mobile phase at any instant divided by the total number of moles of solute in the column:

solutesofmolestotalphasemobileinsoluteofmolesu

The total number of moles of solute in the mobile phase is :

nM = CM x VM

in stationary phase: nS = CS x VS

MMSSSSMM

MM

VCVCu

VCVCVC

u/11

M

S

MA

SAC C

Caa

K

Therefore:

Since distribution constant:

therefore:

MSC VVKu

/11

•Time spended of analyte in the stationary phase relating to the mobile phase

The retention factor: k’

k’: relative number of moles of analytes in the stationary and mobile phase

k’ = nS/nM

Other definition of retention factor for analyte A:

M

sAA VV

Kk

AKu

11

AMR ktL

tL

11

where: KA is the distribution constant for analyte A

Substitution to equation earliers:

Rearranging:

M

MRA t

ttk

Selectivity factor:

1

2

R

R

A

B

tt

kk

Always greater than 1.0

Column efficiency and band broadeningThe plate theory of chromatographyOne theoretical plate (N): the part of the column, where quasi-equilibrium takes place between stationary and mobile phase

2L

22

2t

R

σL

σtN

54,5162

wtN R

2

2/1

wtR

Where: standard deviation and 2

Variancew = 4

Gauss equation:

HETP: Height equivivalent to the theoretical plate (H)

NLH

The rate theory of chromatography (van Deemter)

ApedC

Porous silica particleparticle size (diameter): dP

Theory of band broadening1. Eddy diffusion term (A) multiple path effects

uB

uDC Md

2. The longitudal diffusion term (B/u)

uCMM

2pM

DudC

uCSM

2pMS

DudC

3. Mobile phase mass transfer term (CM/u)

4. Stationary phase mass transfer term (CS/u)

uCuu

A S MCBH

H

u

The van Deemter equation of chromatography

The equation has an optimum (Hopt) where the column efficiency is highest.This optimum has been found at a linear velocity: for gas chromatography at. 0.1 – 0.5 cm/sfor liquid chromatography at: 1.0 – 5.0 cm/s

At high linear velocities equation can be estimated as:

uCu

A SBH

)(21

21

12

ww

ttR RRs

Resolution

'k1'k

α1αN

41R

2

22S

Resolution expressed with the terms of plate number, selectivity and retention factors

'k1'k

α1αN

41R

2

22S

Methods to increase resolution

'k1'k

α1αN

41R

2

22S

Effect of increase of retention factor on resolution

How to increase retention factor:•By decreasing eluent strength

Effect of increase of separation factor on resolution

'k1'k

α1αN

41R

2

22S

How to increase separation factor:•By change chemical quality of the mobile phase•By change quality of column

'k1'k

α1αN

41R

2

22S

Effect of increase of plate number on resolution

How to increase theoretical plate number:•Decrease of the flow rate (u)•Increase of the column length (L)