A318Lecture12IntroChromatographicSephandout_000

8/13/2019 A318Lecture12IntroChromatographicSephandout_000

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Introduction to Chromatographic

SeparationsSeparation of complex mixtures

Sample transported in a mobile phase gas, liquid, supercritical fluid

Mobile phase / sample passes over/through an immiscible stationary

phase that is fixed in place in column or on solid surface

Mobile and stationary phases chosen so that sample components will

distribute themselves between the two phases to varying degrees

Components strongly retained by the stationary phase will travel slower &

elute at longer times than those weakly retained

Each component separates into a discrete band or zone that can be

qualitatively or quantitatively analyzed

Chromatography Classifications

Introduction

26A

By means of contact:

Column Chromatography stationary phase held in narrow tube

through which mobile phase is forced under pressure (our focus)

Planar Chromatography stationary phase supported on a flat plate or

in a paper; mobile phase travels by capillary action or gravity

By mobile/stationary phase (Table 26-1):

Liquid Chromatography (LC) mobile phase is a liquid; stationary

phase can be adsorbed liquid, bound organic species, solid, ion-

exchange resin, liquid in a polymeric support

Gas Chromatography (GC) mobile phase is a gas; stationary phasecan be adsorbed liquid, bound organic species, or solid

Supercritical Fluid Chromatography (SFC) mobile phase is a

supercritical fluid; stationary phase is bound organic species


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Column Chromatography - Elution

Introduction

26A

detector

t0

t1

t2

t3

t4

packed

column

sample

A + B

mobile phase

AB

B

B

A

A

B

detector

signal

time

AB

addition of mobile

phase washes

sample through

column

B spends more

time in stationary

phase thanA

chromatogram

Chromatography Characteristics

Introduction

26A

Analyte dilution band broadening during separation leads to dilution of

analyte; requires greater detector sensitivity

concentration

A

B

t1

BA

t2

distance migrated

Chromatogram plot of detector response (concentration) vs. migration

time or mobile phase volume is used for qualitative (peak position) or

quantitative (peak area) analysis


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Migration Rates & Zone BroadeningEffect on Resolution

Introduction

26A

Improved separation changing elution time (b) or decreasing broadening

(c) can each be used

Variables that influence relative migration rates (26B) and zone broadening

(26C) can be described mathematically

detectorsignal

Time

(a) unresolved (overlapping) peaks

(b) Increase band separation

(c) decrease band width

Migration Rates of Solutes

Introduction

26B

For a single soluteA involved in transfer between mobile and stationary

phases, an equilibrium is reached whose constant K is the distribution

constant, partition ratio, or partition coefficient.

Amobile Astationary K =cScM

Linear chromatography K is constant over a wide concentration range

Retention time (tR) time for analyte to reach detector

Dead time (tM) time for unretained species to reach detector

Average linear rates for analyte (v) and mobile phase (u):

v = L/tR u = L/tM where L is length of column packing

Also,v = u X

moles of solute in mobile phase

total moles of solute

and since cV = moles


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Retention Time & Partition Coefficient

Introduction

26B

v = u Xc

M

VM

cMVM + cSVS= u X 1

1 + cSVS /cMVM

1

1 + KVS /VMv = u X

combine with definition of K to get:

which is the rate of solute migration as a

function of partition coefficient & volumes

of stationary & mobile phases

Retention factor (kA) describes migration rates of a solute on a column

kA =KAVS

VM

tR- tM

tMkA =

tR & tM obtained from expt.;

ideally 2 1

this is correct

Zone Broadening & Column Efficiency

Introduction

26C

Kinetic (rate) theory of chromatography explains peak shapes quantitatively

based on a random-walk mechanism well discuss qualitatively.

Peaks are Gaussian in shape due to thousands of transfers between mobile

and stationary phases; elution occurs only in mobile phase

- extended residence in stationary phase longer retention times

- extended residence in mobile phase shorter retention times

- longer time in column broaderpeaks; faster flow narrowerpeaks

Plate theory of chromatography provides terminology: plate height H &

number of theoretical plates N, related byN = L/H (L is column length)

plate theory based on analogy of separation to a series of distillation plates

at which solute reaches equilibrium between phases abandoned b/c it

does not account for peak broadening


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Column Efficiency / Plate Height

Introduction

26C

Plate Height is directly related to variance (2) or breadth of Gaussian peak

H =L

2

L

(L + 1)(L - 1) Analyte profile at

end of packing

Plate height contains ~34% of analyte (1 )

Based on time, variance is defined as 2, related to by = /(L/tR)

At the base, a peaks width W = 4, and thus = LW/4tR

Plate height can be defined as H =LW2

16tR2

Number of theoretical plates N = 16(tR/W)2

Using width at half height (W1/2), N = 5.54(tR/W1/2)2

H & N used by manufacturers / researchers to measure column

performance comparison can be done using same compound.

Zone Broadening / Kinetic Variables

Introduction

26C

van Deemter plot of H vs. u (linear flow rate) shows efficiency dependence

on flow rate, other variables (diffusion coefficients, particle diameter,

stationary phase thickness, retention factor, etc.) listed in Table 26-2

H,

cm

u, cm/s

AB/u

Cu

Multipath Term (A) each molecule

takes a different path through column

Longitudinal Diffusion Term (B/u)

solute molecules diffuse to regions of

lower solute concentration in front of

& behind zone; inversely proportional

to mobile phase flow rate

Mass-Transfer Terms (Cu) for Stationary Phase (CS) & Mobile Phase (CM) solute molecules require time to reach the surface of either stationary or

mobile phase, depending on thickness of each phase & diffusion

Zone Broadening reduced by using smaller particles, narrower columns,

lower temp. in GC, thinner liquid stationary phases

A318Lecture12IntroChromatographicSephandout_000

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