Chromatographic Separations Basics

8/9/2019 Chromatographic Separations Basics

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Chromatographic Separations

Introduction & Basics

Read all of Skoog – Chapter 26.

Common analytical problem identify and !uantify "# component

in a mi$ture.

Ideally

• Completely selecti%e method to analye each component

indi%idually in the mi$ture

•

In absence of such a method' separate the analyte(s) prior toanalysis to a%oid selecti%ity issues

Separations *ethods

• +istillation

• ,$traction

• Chromatography

• ,lectrophoresis

Introduction to separations li!uid-li!uid e$traction

he solute / S is partitioned bet0een 2 li!uid phases 1# and 12

,!uilibrium constant or

artition coefficient or

+istribution constant 3 / 4S524S5#

#


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So 0hat gi%es a better separation of solute bet0een the 2 phases –

# large e$traction or se%eral small ones7

Solute 8 has 3 / 9 bet0een toluene and 0ater (485 in toluene / 9$485 in 0ater). Start 0ith #:: m; of :.:# * a!ueous solution of 8

and e$tract 0ith toluene.


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The more equilibria a mixture attains between 2 different phases

the greater the separation.

Instrumental separations methods (i.e. chromatography) designed

to gi%e the ma$imum number of e!uilibria (theoretical plates).

Chromatography operates on the same principle as e$traction' but

one phase is held in place (stationary phase) 0hile the other mo%es

past it (mobile phase).

he interaction of the solute 0ith the stationary phase to a large

e$tent dictates the distribution constant 3 . he nature of this

interaction is one 0ay to generally categorie chromatographicmethods. >or a solute 8 3 / 485stat 1485mobile 1

9


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he basics remain the same regardless of the type of interaction

dictating the distribution constant.

?ote that your te$t in able 26-# also categories chromatographic

methods by the type of mobile phase• @C / gas chromatography' gaseous mobile phase

• ;C / li!uid chromatography' li!uid mobile phase

• S>C / supercritical fluid chromatography' supercritical fluid

mobile phase

Belo0' 2 substances 8 and B are sho0n eluting do0n a column

packed 0ith stationary phase. *obile phase is continuously added

such that elution continues until the substances are eluted from theend of the column.

If 3 / 485stat 1485mobile 1

• hen 3 for solute 8 A 3 for solute B

• 8s solute partitions bet0een the stationary phase and fresh

mobile phase' bands from the 2 solutes begin to separatefrom one another as a result of successi%e e!uilibria bet0een

mobile and stationary phase

• ,ach e!uilibrium achie%ed bet0een mobile and stationary

phase is a theoretical plate (holdo%er terminology from

distillation theory)


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• Since solute mo%ement can occur only in the mobile phase'

the a%erage rate at 0hich a solute migrates do0n the column

depends on the fraction of time it spends in the mobile phase'

dictated by 3 for that solute

8 chromatogram is a graph of detector response as a function of

elution time

2 factors affect column performance (separations)

#. 8s solutes elute do0n the column' band separation occurs

due to successi%e e!uilibria bet0een phases (differences in

migration rates – good to ma$imie)

2. 8s solutes elute do0n the column' each solute bandine%itably broadens – good to minimie

Band (one) separation – 8n e!uilibrium treatment (Ch. 26

Section B)

he partition or distribution constant (3) is not readily measured'

but the retention time is' and it is directly related to 3.

=


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+efinitions

• t*

• tR

• tR D

• retention or capacity factor k or k D /

• E (selecti%ity factor)

If Solute is in mobile phase all the time

Solute is in mobile phase =:F of the time and in stationary

phase =:F of the time

Solute is in mobile phase 2=F of the time and in stationary

phase G=F of the time

6


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If the solute spends 9$ as much time in the stationary phase

as the mobile phase' then 9$ as many moles of solute are in

the stationary phase compared to the mobile phase.

If kD A # then the solute elutes too !uickly' near t*

If kD " 2: then tR is too long causing %arious problems

Ideally kD bet0een # and #:' separation conditions are

adHusted to make that happen (discussed in Ch 26' Section +)

?o0 the last definition selecti%ity factor. he point of

chromatography is to effect a separation' 0hich is

fundamentally based on differences in partition coefficients

bet0een solutes.

E / 3 83 B / selecti%ity factor

(E " # by definition)

G


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0o factors contribute to ho0 0ell compounds are separated

#. +ifference in elution times bet0een peaks (already)e$plained by e!uilibrium theory. ;arger difference in 3' the

better the separation.

2. he 0ider the peaks' the poorer the separation. ?o0 to be

treated by rate theory.

Band broadening and column efficiency Rate theory Section 26C

Chromatography peaks are @aussian.

%erall uncertainty / J many random uncertainties

*ost common result / mean


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,arly on chromatography and band spreading 0as treated as an

e!uilibrium process using distillation theory. erminology' 0hich

can cause confusion' unfortunately remains.

Theoretical Plate – 0here a solute undergoes e!uilibrium bet0eenmobile and stationary phase.

Number of theoretical plates / ?

Plate Height = O

If ? / ;O and O / s2;

P


10/18

02 / #6s2Q s2 / 02#6

8t a gi%en mobile phase flo0 rate ; is proportional to tR for a

gi%en solute so

8 solute 0ith a retention time of :Gs has a 0idth at the base of

#9s' on a #2.2m long column. >ind ? and O.

Column separation efficiency increases as ? increases' and

increases as O decreases.

Compare ? and O only for the same compound.

Chromatography ? / #:: – #:':::

O / :.# – :.::# cm

Capillary electrophoresis ? #:6

O #:-9 cm

So far' column efficiency discussed by plate or e!uilibrium theory'

0hich cannot e$plain the follo0ing e$perimental data

#:


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he abo%e %an +eemter plot sho0s that there is an optimum flo0rate' and that plate height is %ery much a function of mobile phase

flo0 %elocity.


12/18

he longitudinal diffusion (Bu) term

*obile and stationary phase mass transfer (Cu) term

Breaking the %an +eemter plot into indi%idual contributors

#2


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Comparison of %an +eemter plots for gas chromatography (@C)

and li!uid chromatography (;C)

#9


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• 8t lo0 flo0 rates plate height decreases 0ith increasing flo0

rates from longitudinal diffusion term. ;arger effect in @C

• >or same reason plate heights smaller in ;C than @C.

he multipath 8 term

8 / : for no packing (common in @C' not ;C)

Summary

• 8ddressed migration rates and distribution constants (26B)

• 8ddressed one broadening (26C)

?o0 – optimiation of column performance (26+) by either

• 8ltering relati%e migration rates' or

• reducing one broadening

#


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he goal is to resolve 2 or more solutes in a mi$ture – dependent

on differences in retention time and one 0idth.

Resolution R s / TtR


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?ote that the abo%e e!uation can be rearranged to find ? for a

desired resolution

In practical terms' resolution is only important 0hen 3 8U3 B

,$ample 26-# on p. GGG re%ie0s many concepts.

he fundamental parameters of selecti%ity (E)' retention factor (k)

and theoretical plates (?'O) can all be %aried to achie%e a

separation.

Selecti%ity

heoretical lateslate Oeight

Retention factor easiest 0ay to impro%e resolution

#6


17/18

8 more general discussionK

radient !lution in li!uid chromatography – a systematic %ariation

of mobile phase composition to optimie k for a 0ide range of

solutes.

Temperature programming in gas chromatography – a systematic

%ariation of temperature to optimie k for a 0ide range of solutes.

#G


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@eneral applications of chromatography (Section 26>)

Vualitati%e analysis

• tR only !ualitati%e information. ?o structural information.

Strong indicator of presence of analyte' une!ui%ocal proof ofanalyte absence.

• Wseful for separation prior to ac!uiring structural information

using another techni!ue 0hich 0ould not be useful for a

mi$ture.

Vuantitati%e analysis

• eak areas

• Reproducible inHection %olumes (calibrations)

,nd of Chapter 26 !uestionsproblems

#-9' 6-#=' #G-#P' 2#

#N

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Chromatographic Separations Basics

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