Praktická MS a separace malých molekul

Transkript

Vzdělávací program Ekotech 2012
Praktická MS
a separace malých molekul
Petr ŠIMEK
1
Pracovní postup analýzy
Analytical problem
Sampling
Sample
l preparation
i
Instrumental analysis
Separation
Ch
Chromatography/Electrophoresis
h /El
h
i
Detection
S
Spectroscopy

Data acquisition/processing

INFORMATION

Management
2
SEPARACE MALÝCH MOLEKUL
Basic considerations






Real samples, particularly from complicated matrices (body
fluids, plant and animal tissues, soil etc.) require very often
another efficient step to separate components from each other.
other
So that each individual component
p
can be identified.
The separation properties of the components in a mixture are
constant under constant conditions.
Separation generally means selective transfer of each analyte
between two p
phases,, usuallyy called
STACIONARY (STATIC) phase
STACIONÁRNÍ FÁZE
MOBILE (DYNAMIC) phase
MOBILNÍ FÁZE
3
Princip separace
Sample Mobile
extract phase
•
••• •••
•••
••
.
Separation system
100
95
90
••••••••••••••••••••••••••••
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••
••
••
••
•
••
••
•• •
••
85
80
75
70
65
60
Detector
55
Sorbent or
electrical field
50
45
40
35
30
25
20
15
10
Forces p
participating
p
g in the separation
p
process:
p
5
6.3
30
6.3
35
6.4
40
6.4
45
6.5
50
6.5
55
6.6
60
6.6
65
TIM
E
Non-bond (dipole, induced dipol)
Ion-ion
[mobile phase- analyte – sorbent] 
Electrical field [buffer-analyte
[buffer analyte - el.
el field] 
CHROMATOGRAFIE
ELEKTROMIGRAČNÍ
METHODY
4
SEPARACE
Classification of separation methods

According to driving force
Chromatography
Electromigration methods (Elfo)
Chromatography








Distribution of analytes between phases
Liquid – solid
Planar chrom. (PC, TLC)
Liquid – solid
Gel permeation chrom. (GPC)
Liquid – solid
Ion exchange chrom. (GPC)
Fluid – solid
Supercritical fluid chrom.
chrom (SFE)
Liquid – solid
Liquid chrom. (NP-LC)
Liquid – liquid
Liquid chrom. (RP-LC)
Gas - liquid
Gas chromatography (GLC)
Gas – solid
Gas chromatography (GSC)
5
SEPARACE
Electromigration methods



El t i field
Electric
fi ld – liquid
li id
Capillary
C
ill
zone electrophoresis
l t h
i
(CZE)
Electric field – liquid/micelles
q /
Micellar electrokinetic chrom.
(MEKC)
Electric field – liquid/sorbent Electrochromatography
6
Chromatografie
Sample Mobile
extract phase
•
••• •••
•••
••
Separation system
.
100
95
90
••••••••••••••••••••••••••••
•••••••••••••••••••••••••••
••••••••••••••••••••••••••••
•••••••••••••••••••••••••••
••••••••••••••••••••••••••••
••
••
••
••
•
••
••
•• •
••
85
80
75
70
65
60
Sorbent
Detector
55
50
45
40
35
30
25
20
15
10
5
6.3
30
6.3
35
6.4
40
6.4
45
6.5
50
6.5
55
6.6
60
6.6
65
TIM
E
Chromatography is a separation method that relies on differences in
partitioning
p
g behavior between a flowing
g mobile phase
p
and a stationaryy phase
p
to separate the the components in a mixture.
As a result of these differences in intermolecular interactions arising from the
individiual structures the analytes show different mobilities
Sample components will become separated from each other as they travel
7
through the stationary phase.
Princip
p chromatografické
g
separace
p
Distribuce of analytu
y mezi dvě fáze

The distribution of analytes between phases can be described quite
simply. An analyte is in equilibrium between the two phases;
Amobile

Astationary
The equilibrium constant, K, is termed the partition coefficient; defined
as the molar concentration of analyte in the stationary phase (s)
divided by the molar concentration of the analyte in the mobile phase
(m).
KD= c [A]s /c [A]m
8
Intermolecular Forces
Interaction
Factors
Energy
gy of
interactions
Ion-ion
g ; size
Ion charge;
400-4000 kJ/mol
/
Ion-dipole
Ion charge; dipole 40-600 kJ/mol
moment
Hydrogen bond
Hydrogen bond
25-200 kJ/mol
Dipole dipole
Dipole-dipole
Dipole moment
5 25 kJ/mol
5-25
Dipole-induced
dipole
Dipole moment;
polarizability
2-10 kJ/mol
Induces dipole
polarizability
0.05-40 kJ/mol
9
Intermolecular Forces
1. Hydrophobic interactions (van der Waals)
2. Dipol-dipol (hydrogen bonding)
3. Electrostatic interactions (ion – dipole)
10
Chromatografie
Basic characteristics of the chromatographic process are deduced
from graphical result of the separation – chromatogram.
Peaks A,B
tR1
10
Retention time
(Migration time in CE)
.
A
95
90
85
80
tR2
75
Signal
70
65
B
60
55
50
45
40
35
30
25
20
15
10
0
1
2
3
TIME
TIME
4
5
6
7
11
Chromatografie
g
Úvod
Dead retention time – mrtvý retenční čas
Each analyte in a sample will have a different retention time. The
time taken for the mobile p
phase to pass
p
through
g the column is called
the column dead time tM.

The time is often replaced by analogous terms the retention
volume (vR) and the column dead volume (vM) that operate with
elution volumes rather than the time scale.

12
Chromatografie - Selectivita
A
.
10
95
B
90
85
80
75
70
65
60
55
50
Termodynamika procesu 45
40
35
30
25
20
15
10
Pozice píku na chromatogramu
0
1
2
3
TIM E
4
5
6
7
Peak position on the chromatogram is a result of interaction
between sorbent and analyte in the two phase system
tR thus reflects thermodynamic part of the separation process
 SELECTIVITY of the separation system, expressed in
Selektivita
 RESOLUTION
Rozlišení
2 (tR(A) - tR(B) )
R=
wh/2(A)+ Wh/2(B)
Optimal resolution  optimized sorbent, mobile phase composition
and temperature
13
Chromatografie - Selectivita
Capacity factor (relative retention, kapacitní faktor)
Vyjádření migrační rychlosti analytu kolonou
The retention factor, k', called the capacity factor is often used to
describe the migration rate of an analyte on a column.
column
The k', for analyte A is defined as;
k'A = (t R - tM )/ tM
t
R
and tM are easily obtained from a chromatogram.
When k' < 1, the analyte elution occurs in the column tM so that
the analyte is not retained on the column and is eluted together
with other components not retained
retained.
When k' < 10, elution and thus analysis takes a very long time.
Ideally,
Id
ll k' < 2,
2 5 > - the
h best
b
compromise
i in
i the
h terms off
separation effiency, running cost and analysis time.
14
Ch
Chromatografie
fi ‐ Selektivita
S l ki i
S
Separation
ti
f t α (separační
factor
č í faktor )
Míra dělení dvou analytů
The selectivity parameter is a measure of the spacing
b t
between
t
two
peaks
k expressed
d as:
α = kB/kA
kB capacity
it factor
f t off the
th analyte
l t B
kA capacity factor of the analyte A
15
Chromatografie
g
– Kinetika separačního děje
j
What else can we deduce from the peak shape ?
Elution curve of the peak shows detector
response in time and kinetic part of the
separation process
(a)
Initial separation of two components
(b)
Improved resolution of the two component
peaks - improved separation thermodynamics
((c))
= increased
i
d retention
t ti shifts
hift
.
10
95
90
85
(c)
Improved kinetics of separation
80
= improved peak shape
55
75
70
65
60
50
45
40
35
30
25
20
15
10
0
1
2
3
TIME
4
5
6
7
16
Chromatografie – Kinetika ‐Tvar píku
Kinetika Tvar píku
What can we deduce from peak shape ?
Elution curve of the peak shows
character of a normal Gauss distribution
In practice  we don
don’tt operate with statistics
and standard deviation, but we measure
Peak width of the peak at the half height wb/2
or
Peak width of the peak at the baseline wb.
((c))
17
Chromatografie – Kinetika Kinetika –Tvar
Tvar píku
píku
Assymetry factor
A 1 = B/A
As
As2 = N0.5/N0.1
USP tailing factor
T = w0.05/2C
W0.5
P k width
Peak
idth att 50% peak
k height
h i ht
W0.05
Peak width at 5 % peak height
N0.5
Plate number at 50% peak height
N0.1
Plate number at 10% peak height
18
Chromatografie
g
– Kinetika – Tvar píku
p
Assymetry factor
As1 = B/A
As2 = N0.5
/N0.1
0 5/
01
USP tailing factor
TF = w0.05/2C
B
Peak front distance at 10% peak height
A
Peak tail distance at 10 % peak height
C
Peak
k front
f
di
distance at 5 % peak
k height
h i h
D
Peak tail distance at 5 % peak height
19
Chromatografie – Kinetika Kinetika – Tvar píku
Tvar píku
Assymetry factor
As1 = B/A
As2 = N0.5
/N0.1
0 5/
01
USP tailing factor
T = w0.05/2C
Peak shape (kinetics) expressed as peak width (w) and peak position
(termodynamics) expressed as retention time tR characterize
Separační účinnost
20
Účinnost separace
Schopnost kolony separovat složky směsi. Mírou činnosti Počet teoretických pater kolony N
Teoretické patro = pomyslná část kolony, ve které dochází k ustavení rovnováhy mezi stacionární a mobilní fází 21
Účinnost separace
p
Výškový ekvivalent teoretického patra = délka pomyslné části kolony, ve které dochází k ustavení rovnováhy mezi stacionární a mobilní fází Height equivalent to the theoretical plate /
HETP = H= L/N
L
L = column
l
l ht
lenght
N= number of plates, plate count
Historický původ – destilace analytů na destilační koloně
HETP je funkcí vlastností jak kolony, tak analytu. Very good separation  H > 3000 TP/m
22
Účinnost separace
p
Počet teoretických pater kolony N
Theoretical Plate Concept
Plate number N is estimated from a chromatogram by analysis
of retention time for each analyte and its standard deviation as a measure for peak width, provided
a measure
width, provided that the elution curve
represents a Gaussian curve. 23
Separační
p
účinnost
A theoretical plate (počet teoretických pater) N in many separation processes is a hypothetical zone or stage in which
in which two phases of a substance (analyte), establish
a substance (analyte), establish
an equilibrium with each other.
The performance of many separation processes depends on having a series of
equilibrium
ilib i
stages
t
and
d is
i enhanced
h
d by providing
b
idi more such stages. In other
h t
I th words, d
having more theoretical plates increases the efficacy of the separation process be
it either a distillation, absorption, chromatographic, adsorption or similar process. 24
Chromatografie – separační účinnost
Separation efficiency is affected by non-ideal contributions expressed in van Deemter
equation
Eff t off mobile
Effect
bil phase
h
velocity
l it on the
th separation
ti
efficiency
ffi i
A = Eddy diffusion (vířivá difuze)
B = Longitudinal diffusion (podélná molekulární
difuze)
C = Mass transfer kinetics of the analyte between
mobile and stationary phase (odpor proti
přenosu hmoty ve stac. a mobilní fázi)
u = Linear velocity
Minimum – maximum separation
maximum separation efficiency at optimum mobile phase
optimum mobile phase velocity
25
Chromatografie ‐ hodnocení separace
.
10
95
Peak parameters used for quantitation properties:
Peak height, peak area (plocha, výška píku)
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
Optimization of the separation process to get maximum of:

15
10
0
1
2
3
TIME
4
5
1. Separation efficiency expressed by the N value
 the narrow “sharp” peak shape
2. Selectivity
Resolution R to separate peak top one from another


3. Time
Analysis in real time

4. Costs
Economical issue
26
6
7
Typy SEPARACÍ
Typy SEPARACÍ
Classification of separation methods

Accc. to driving force
Chromatography
Electromigration methods (Elfo)
Chromatography








Distribution of analytes between phases
Liquid – solid
Planar chrom. (PC, TLC)
Liquid – solid
Gel permeation chrom. (GPC)
Liquid – solid
Ion exchange chrom.
chrom
(GPC)
Fluid – solid
Supercritical fluid chrom. (SFE)
Liquid – solid
Liquid chrom. (NP-LC)
Liquid – liquid
Liquid chrom. (RP-LC)
Gas - liquid
Gas chromatography (GLC)
Gas – solid
Gas chromatography (GSC)
27
Intermolecular forces
1. Hydrophobic interactions (van der Waals)
2. Dipol-dipol (hydrogen bonding)
3. Electrostatic interactions (ion – dipole)
28
CHROMATOGRAFIE
29
PLANÁRNÍ CHROMATOGRAFIE

Paper
p ((PC)) and Thin-Layer
y Chromatography
g p y ((TLC))
tenkovrstvá
The oldiest chromatographic methods (Tswett, 1904).
Separation of plant pigments on a cellulose paper gave name the the methodology.
 A solid‐liquid technique ‐ solid (stationary phase) and a liquid
(mobile phase).  Stationary phase is planar ‐
Stationary phase is planar ‐ paper or thin‐layer of porous paper or thin‐layer of porous
sorbent [silica gel (SiO2 x H2O), alumina (Al2O3 x H2O)], cellulose.  Mobile phase –
M bil h
t i ll
typically solvent mixtures used (butanol, l t i t
d (b t
l
methanol, water, dichlormethane, diethylether etc. )
Like‐dissolves‐like principle is valid
p
p
 Detection – UV lamp, instrumentation ‐ densitometers
30
PLANAR CHROMATOGRAPHY

Operation principle
Spotting a TLC plate with
a sample
The plate
Th
l t is
i placed
l
d into
i t a
developing chamber
saturated with mobile phase.
The MP move-up by capillary
action. After the MP reaches the
edge,
g , the plate
p
is removed and
dried. Typically, under UV lamp
the spots are observed and the
chromatogram evaluated.
31
Thin-Layer CHROMATOGRAPHY (TLC)
Tenkovrstvá chromatografie, na tenké vrstvě
Ope ation
Operation


Stationary phase immobilized on a glass or plastic plate.
The sample, either liquid or dissolved in a volatile solvent, is deposited as a spot on the stationary phase. The constituents of a sample can be identified by simultaneously running standards with the unknown.

The bottom edge of the plate is placed in a solvent reservoir, and the solvent
moves up the plate by capillary action. 
When
h the
h solvent
l
f
front reaches
h the
h other
h edge
d off the
h stationary phase, the
h
h plate is
l
removed from the solvent reservoir. The separated spots are visualized with
ultraviolet light or by placing the plate in iodine vapor. The different components in the mixture move up the plate at
plate at different rates due to differences
to differences in their
in their
partioning behavior between the mobile liquid phase and the stationary phase.
32
Thin-Layer
Thin
Layer CHROMATOGRAPHY (TLC)

TLC Chromatogram
RF Retardation factor, analyte characteristics
RF (PE) = dA/dM
Front
dM
Start
dA
33
Thin Layer CHROMATOGRAPHY (TLC)
Thin‐Layer CHROMATOGRAPHY (TLC)
Operation
RF
Retardation factor,
factor analyte characteristics
RF= dA/dM
Advantages/Applications




Veryy simple,
p , cost-effective
For simple experimental design in organic synthetic labs,
pharma and anywhere for simple control of purity
E ll t productivity
Excellent
d ti it in
i qualitative
lit ti and
d semiquant.
i
t analysis.
l i
Tens of samples can be analysed simultaneuosly
The p
plate can be stored
Limitations

Separation efficiency is lower than at GC or HPLC
34
Gel permeation chromatography (GPC)
Gel permeation chromatography (GPC)
Size exclusion chrom. (gel filtration)


GPC (solid – liquid chrom.) uses porous particles to separate
molecules of different sizes in the column arrragment. It is generally
used to separate biological molecules,
molecules and to determine molecular
weights and molecular weight distributions of polymers. Molecules
that are smaller than the pore size can enter the particles and
therefore
h f
h
have
a longer
l
path
h and
d longer
l
transit
i time
i
than
h
larger molecules that cannot enter the particles.
Molecules larger
g than the p
pore size
cannot enter the pores and elute
together as the first peak
(dead retention volume )
in the chromatogram.
This condition is called total exclusion.
35
Gel permeation chromatography (GPC, SEC)

G l - inert
Gel
i t polymer
l
sorbent
b t (supressed
(
d ion
i and
d dipol
di l interactions)
i t
ti
)
Features

Primarily, desalting (rather than separation) of macromolecules (not
subject of this talk)

Separation of low molecular compounds such as pesticides from fat
matrices on special gels (e.g. Bio-Rad Biobeds)

Very mild method suitable for separation of labile analytes

Separation is not nearly dependent on the mobile phase compositon,
temperature

The method is approved by regulatory institutions and used in
agricultural and hygiene laboratories that analyse fatty matrices
36
p
g p y(
,
)
Application
Analysis of organosphosphorus pesticides in lanolin by GPC and gas
chromatography
Experimental data
GPC glass column 450x15 with a gel
Biobeads SX-3 stationary phase, MP
dichloromethane, 4 ml/min.
Pesticide in lanolin were injected via
a 2 ml loop.
Detection, UV, 254 nm.
Lanolin is a g
grease of sheep
p origin
g
used as a moisturizer in cosmetics
and pharma.
37
p
g p y(
,
)
Application
Analysis of organosphosphorus pesticides in lanolin by GPC and gas
chromatography
Recovery percentages of the nine
pesticides analysed.
GC chromatogram of the injection of the
fraction of the pesticides eluted from the
gel permeation chromatography column
of a lanolin spiked solution.
38
ION EXCHANGE CHROMATOGRAPHY
ION EXCHANGE CHROMATOGRAPHY (IEC, IC, iontoměničová, iontové výměny)



Ion chromatography is a solid-liquid technique,
technique that separates
molecules based on their ion-ion interactions (chemisorption
mechanism).
Adsorption of an analyte on the resin is reversible and depends
on the strenght of the electrostatic forces.
Anionic functional group of the resin binds cation group of an
analyte and vice versa; the cation resin the opposite negatively
charged analyte.
Anion exchanger
with counterions
Cation exchanger
with countrerions
39





When a charged analyte is applied to an exchanger of the
opposite
it charge,
h
it is
i adsorbed.
d b d Neutrals
N t l and
d ions
i
off the
th same
charge are not retained.
Binding
g of the charged
g analyte
y is reversible
Adsorbed analytes have different affinity to the ion exchanger
and can be desorbed with pH or a salt gradient (ion strenght
effect).
effect)
Stationary phase - ion exchange resins
Cation exchanger
g – negatively
g
y charged
g functional group
g p
(sulfonate) is covalently bound to the resin.
Anion exchanger – positively charged functional group is
covalently bound to the resin (usually a quaternary ammonium
group).
Experimental parameters of ion resins: particle size, ion form
(counterions add specific selectivity for each resin) and purity
40
When a charged analyte is applied to an exchanger of the opposite charge, it is adsorbed. Neutrals and ions of the same charge are not retained.
i d

Binding of the charged analyte is reversible 
Adsorbed analytes
y are eluted with a salt or pH gradient
p g
Experimental procedure

Choice of a proper ion exchanger by consultation of a reference catalogues e g Bio‐Rad Co
catalogues, e.g. Bio‐Rad Co. 
Preparation of the ion exchanger in proper ionic form (washing with counterion). Ionic form examples ‐ (CEX ‐ hydrogen, sodium, AEX chloride acetate)
chloride, acetate)

Sample introduction

Elution of the analyte by pH or ion strenght gradient 
Conductive detectors are required for the detection of small ionic molecules. Conductivity of the mobile phase must be minimized by ion supression (by another IEX column or membrane) to neutralize/remove MP ions. Novel ion supressors have been developed for sensitive detection of ionic compounds (see Dionex Co. website).

41
ION EXCHANGE CHROMATOGRAPHY (IC)
( )
Applications
Fast analysis of inorganic anions
and organic acid anions, including
polyphosphates, using hydroxide
gradient elution.
Anion exchange column Ion Pack IP11
(Dionex Co.). Gradient, 0.2-38 nM NaOH
in 15 min.
min
42
Applications



Its greatest utility is for analysis of anions (of organic as well
inorganic acids) for which there are no other rapid analytical
methods.
Traditionally used for amino acid analysis in amino acid
analysers and for analysis of cations
Ion-exchange
Ion
exchange effect is also utilised in mix
mix-bed
bed SPE sorbents and
in extraction of amino acids and amines from biological matrices
combined with chromatographic techniques (see later)
Limitations

Limited ion-exchanger
g capacity,
p
y, lower separation
p
efficiency,
y,
applications field for ionic substances, peptide mapping and
analysis of proteins
43
ELECTROMIGRATION METHODS (EMM)
Elektromigrační metody


Separation by electromigration methods is based on differences in
analyte velocity in an electric field.
Positively charged ions migrate towards a negative electrode and
negatively-charged ions migrate toward a positive electrode. Ions
have different migration rates depending on their total charge, size,
and shape,
shape and can therefore be separated.
separated
44
ELEKTROMIGRATION METHODS
There are 6 basic electromigration methods differing in separation
mechanism and a medium in a separation capillary:
Separated particles

Capillary Zone Electrophoresis (CZE)
ions

Capillary Gel Electrophoresis (CGE)
ions (macromolecules)

Micellar Electrokinetic Capillary
molecules ions
molecules,
Chromatography (MEKC)

Capillary Electrochromatography (CEC)
molecules, ions

Capillary Isoelectric focusing (CIECF)
ions (mostly macro- )
Capillary Isotachophoresis (CITP, ITP)
ions

Summary term “Capillary electrophoresis” (CE) is used for
CZE CGE,
CZE,
CGE MEKC,
MEKC CEC
45
CAPILLARY ELECTROPHORESIS (CE)
Basic instrumentation of capillary electrophoretic methods (CE)

Capillaries ‐ typically of 25‐100 µm inner diameter; 0.5 to 1 m in length. Fused silica coated with polyimide film  flexible. Cooled for heat dissipation
silica coated with polyimide film 
flexible Cooled for heat dissipation



The applied potential is 20 to 30 kV. For safety reasons one electrode is
usually at ground and the other is biased positively or negatively. Positively charged ions start to migrate towards a negative electrode and
negatively‐charged ions migrate toward a positive electrode (electrophoretic
migration). The mobility of an ion in a particular medium is constant and characteristic of that ion. Ions
h
f h
h
have
d ff
different
migration rates depending
d
d on their
h
total charge, size, and shape, and can therefore be separated. However during the electrophoretic process a second transport effect called electroosmotic flow occurs.
Schematic of capillary
electrophoresis
46
Electroosmotic Flow



The surface of the silicate glass capillary contains negatively‐charged
functional groups that attract positively‐charged counterions (electric, Helmholz bilayer). bilayer)
The positively‐charged ions migrate towards the negative electrode and
carry solvent molecules in the same direction. This overall solvent
movement is
i called
ll d electroosmotic
l
i flow
fl . During a separation, uncharged molecules move at the same velocity as the electroosmotic flow ((with veryy little separation). Positively‐charged
p
)
y
g
ions move faster and negatively‐charged ions move slower.
Si Si Si
O- O- O-
47
Capillary Electrophoresis (CE)
νEOF = E.ε ξ/η = E.μEOF
Rel. permitivita (ε), viskozita (η) roztoku
Potenciálový rozdíl mezi vnitřní a vnější hranicí difúzní vrstvy
se nazývá elektrokinetický potenciál – zeta (ξ)
48
Capillary
p
y Electrophoresis
p
((CE))

Due to the electroosmotic flow, all sample components migrate
towards the negative electrode.
(-)
++
++
Positive
NN
NN
----
Neutral
Negative
(+)
Electroosmotic flow (EOF) > electrophoretic mobility
EOF is the driving force the electrophoretic process.
process
Compare:
p
The driving force of the separation process in chromatography
is mechanical p
pressure
ess e of the mobile phase
49
Capillary Electrophoresis (CZE)
(-)
++
++
Positive
NN
NN
----
Neutral
Negative
(+)
• Vnitřní povrch kapiláry získává negativní náboj a uvolňované kationty vytvářejí pozitivně nabitou vrstvu asociovanou s molekulami vody , která migruje směrem ke katodě a unáší všechny částice zastoupené v elektrolytu
• Elektroosmotický tok převládá ve vhodném pufru o pH > 4 při vložení dostatečně vysokého napětí a průměru kapiláry < 100 um
p
p
p y
50
Capillary
p
y Electrophoresis
p
((CE))

Graphical output of CE is similar to a chromatogram and is called
electropherogram
T i l electropherogram
Typical
l t h
Separation of anions by CZE
51
Elution Curves in Chromatography vers. CE
Application of pressure as the driving force of separation results in
frictional forces building up where the mobile phase is in contact
with solid surfaces.
surfaces
These frictional forces result in drops of velocity in the mobile
phase at these interfaces and this creates a parabolic flow
profile
fil in
i the
th capillary.
ill
 The MP velocity is large in the centre but drops to zero next to
the walls. This p
profile results in the solute zones being
g broadened
as the move through the capillary, reducing efficiency of the
separation.
Hydrodynamic flow
in chromatography
Electroosmotic flow
in CE
52
p
y
p
( )
Plug profile
in CE
Parabolic profile in
chromatography
IIn CE the
th electroosmotic
l t
ti flow
fl
is
i distributed
di t ib t d across the
th llength
th off the
th
system and results in no pressure gradients, except very close to the
wall.
As a result, electrically driven systems offer
much higher separation efficiency
than comparative pressure driven equivalents
equivalents.
Important consequence:
Peaks in CE are narrower than in chromatography 
Resolution of components: CE> Chromatography
53
Main sources of zone broadening/distorsion

Longitudinal diffusion

Joule heating

Sample adsorption


Comment
Defines the fundamental limit of efficiency
Solutes with lower diffusion coefficients
form narrower zones.
zones
Leads to temperature gradients and
laminar flow.
Interaction of solute with capillary walls
usually causes severe peak tailing
Mismatched conductivities Analytes with higher conductivities than
the running buffer result in fronted peaks
p and buffer
of sample
Analytes with lower conductivities than
running buffer result in tailed peaks
Electrodispersion
Differences in sample zone and running
buffer conductivity
54




The most widelyy used type
yp of CE because of its simplicity
p y and
versatility. As long as a molecule is charged it can be separated by
CZE. This makes the applications for CZE very diverse being used for
peptide, ion and enantiomer analysis.
The easiest form of CE to perform because the capillary is only filled
with buffer.
buffer Separation occurs as solutes migrate at different
velocities through the capillary.
Another advantage of CZE is that it separates anions and cations in
the same run, something that some of the other modes of CE do not.
By its principle CZE cannot separate neutral molecules.
55
How to affect the separation process in CZE?
1.Buffer
Absolutely vital for CE separation
Critical parameters:
G d buffering
Good
b ff
capacity in the
h pH range off choice
h
Low absorbance at the wavelength of detection
Low
o mobility
ob ty (t
(that
at is,
s, large,
a ge, minimally
a y ccharged
a ged ions)
o s) to minimise
se cu
current
e t
generation (< 100 A )
Biological buffers such as Tris or borate are particularly useful
56
1.BUFFER - pufr
Data of commonly used buffers
Name
pKa
p
Phosphate 1
Citrate 1
Formate
Succinate 1
Citrate 2
Acetate
Citrate 2
Succinate 2
MES
2.12
3.06
3.75
4.19
4.74
4 75
4.75
5.40
5.57
6 15
6.15
Name
ADA
Imidazole
Phosphate 2
HEPES
Tricine
Tris
Morpholine
Borate
CHAPSO
Phosphate 3
pKa
6.60
6
60
7.00
7.21
7.55
8.15
8.30
8.49
9.24
9.60
12.32
57
2.The pH
strongly affects the EOF and the charge of the analytes.
Cation
Anion
Complete disociation in alkaline medium results in the
largest analyte electrophoretic mobility. Opposite, acid
conditions result in zero analyte
y mobility.
y
Complete disociation in acid medium results in the
largest analyte electrophoretic mobility. Opposite, acid
conditions result in zero analyte mobility.
mobility
Altering
gp
pH is useful when solutes have accessible p
pI's,, such as with p
peptides
p
and proteins. Working above or below the pI (isoelectric point) will change
the analyte charge and cause the analyte to migrate either before or after the
pI an analyte
y p
posseses a p
positive charge
g and migrates
g
to the
EOF. Below its p
cathode, ahead of the EOF. Above the pI the opposite occurs.
58
Capilla Zone Elect
Capillary
Electrophoresis
opho esis (CZE)
Applications


CZE electropherogram brings both qualitative and quantitative information on
the separated and detected components
Excellent resolution of components
components, speed of analysis
Application to all ionizable analytes
Limitations





Not amenable to neutral analytes
Not suitable pro preparative isolation of compounds
Problems with sample injections (low sample capacity, typically injection of nl
volumes))
Nonvolatile buffers used are often not compatible with spectroscopic
detection systems
Quantitative analyses
Q
y
is less reproducible
p
owing
g to EOF instabilities
59
Micellar electrokinetic chromatography(MEKC)



MEKC was developed for electrophoretic analysis of neutral
molecules
A pseudo-stationary phase is introduced using micelles of
surfactants which analytes partition into and out of.
Surfactants are molecules which contain both a hydrophobic
and hydrophilic part in their structure. In aqueous solution the
hydrofobic heads under certain concentration (called CMC =
critical micellar concentration) form aggregation structures micelles with protected from water by hydrophilic tails
arranged around the outside
The structure of a micelle
(SDS – anion tenside is most common]
60
Anionic surfactant
Sodium dodecyl sulfate 9SDS)
61
SDS micelle
i ll – schematic
h
ti structure
t
t
Hydrophobic core with negatively charged micelle surface
62
Mi ll electrokinetic
Micellar
l t ki ti chromatography(MEKC)
h
t
h (MEKC)
Mechanism
•
Neutral species which would normally move with EOF can be kept
inside the micelles.
•
The more the analyte
y interacts with the micelle the less time it spends
p
being swept along by the EOF. Thus neutral hydrophobic compounds
will spend a long time within the micelles and hence be 'retained' much
longer.
Some surfactants
Anionic
SDS
Cationic
DTAB
TDAB
Non-ionic
Brij 35
CMC (mM)
=polyoxyethylen-23-laurylether
Triton X-100
Aggregation number n
82
8.2
62
14.6
4.4
61
64
0.1
40
0.24
140
63
Capillary Micellar Electrokinetic Chromatography
64
Advantages/Applications


Introduction of micelles as another - pseudostacionary phase
brings extra separation dimension and capability to separate
neutral analytes
Simultaneous separation of mixtures of different polarity
Simultatenous separation of benzyl alcohols and phenols
65
Applications
pp
Determination of amino acids in serum by MEKC
Electropherogram of human
serum amino acids
Procedure from J.CH. 979 (2002), 227
1.Derivatization of AA with dinitrofluorbenzene in borate buffer (pH=(9.5)
(p ( )
2.CZE in 30 mM sodium tetraborate (pH=9.8)- isopropanol- 30% Brij 35
(825:150:25)
3.Capillary 75m I.D., 300 mm, 28 kV
4.Temperature 15°C, detection UV, 360 nm.
66
Electrochromatography (EC)
Současná chromatografické a elektromigrační separace






Another extension of CZE with mixed electrophoretic and
chromatographic separation mechanisms
Capillary is filled by stacionary phase as in liquid
chromatography
Driving force remains an electrical field which transports
analytes by EOF through a stationary phase to the detector.
Features
Relatively new approach employing separation efficiency of EOF
with selectivity of LC
Monolithic stationary phases (polybutylmetacrylate) are used
67
Electrochromatography
g p y
• EOF is also generated on the surface of a stationary phase
• EOF in EC is slower than CZE
• EOF enables efficient simultaneous separation both ionic, polar and nonpolar
analytes
68
Electrochromatography
Applications
Particularly chiral separations and other industrial separations
69
Separace
p
modelových
ý analytů
y
O
OH
NH2


Separation of leucine and naphtalene analytes in
chromatographic and electrophoretic systems.
Which method can be used for separation ?
Planar chromatography
Gel permeation
p
chromatography
g p y
Ion exchange chromatography
Capillary electrophoresis
70
Praktické náměty
y
O
OH
NH2
leucin

aromát
Uvažte možnosti separace modelových a vašich „oblíbených“ analytů
U
žt
ž ti
d l ý h
ši h blíb ý h“
l tů
některou z moderních separačních technik
Planar chromatography
rychlá paralelní analýza, kontrola čistoty
Gel permeation chromatography
separace polymerů, HW vs. LW, odsolení
Ion exchange chromatography
proteiny, AAs, anionty, kationty
Capillary electrophoresis
iontové sloučeniny, proteiny, nukleové kys.
71

Praktická MS a separace malých molekul

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