CHROMATOGRAPHY

fundamentals, methods, instrumentation, trends SEPARATION METHOD CATEGORISATION

1. Separations based on distribution of sample components between two

Zdeněk Šimek phases

Materials for individual study 2. Separations based on migration rates differences of sample components
a) through semi-permeable membrane
b) in force field

force field:
- electrophoresis
membrane separations :
- thermal diffusion
Masaryk University in Brno - ultrafiltration (hydrostatic pressure)
- mass spectrometry
Faculty of Science - reverse osmosis (hydrostatic pressure)
- ultracentrifugation
Research Centre for Toxic Compounds in the Environment - dialysis (concentration differences on membrane sites)
- gravitation
- electrodialysis (electric potential differences)
RECETOX

1 2

Crucial factor of sample component separation is

SEPARATIONS BASED ON
DISTRIBUTION OF SAMPLE COMPONENTS BETWEEN TWO PHASES DISTRIBUTION CONSTANT
measure of component affinity
(C A )1 to contacted phases in
Types of interfaces separation methods abbreviation K D (A)  equilibrium
(C A ) 2
gas - liquid distillation
gas chromatography GLC
Equilibrium
gas - solid sublimation static and/or dynamic
gas chromatography GSC
molecular sieve

liquid – liquid extraction Phase1 organic adsorbent ion stationary

liquid chromatography LLC, exchanger
SEC (GPC) solution
liquid - solid fractional crystallization water gas solution mobile
precipitation
Phase 2
liquid chromatography LSC, IEC
molecular sieve, inclusion
Extraction Adsorption Ion exchange Chromatography
3 4
SEPARATION REQUIREMENT K D ( A)  K D ( B )  K D ( C ) CHROMATOGRAPHY

separation factor A,B Definitions

separation ratio KD( A)
separation coefficient  A,B 
elution ratio
retention ratio
K D( B ) Method of separation, identification and subsequent quantification of
relative volatility - distillation components more or less complex mixtures.
M.S. Cvet (1903-1906) Stationary phase: CaCO3
High easy separation : higher concentration of component A in phase 1 Sample: leaf pigments
and component B in phase 2

Two extremes:
Physical method of separation in which the components to be separated are
 KD(A)= 6 KD(B) = 2 =3 components A and B easy penetrate into distributed between two phases, one of which is stationary (stationary phase,
phase 1 SP) while the other moves (mobile phase, MP or ‘eluant’) in a definite
direction IUPAC (1993)
 KD(A)= 0,6 KD(B) = 0,2  = 3 components A and B remain mainly in
phase 2 5 6

Common chromatographic arrangement CHROMATOGRAPHY

Mobile phase Two basic processes occur in the column

(eluent)
1. The components are moved apart as
Sample
a result of their relative affinities for
(component A and B)
the stationary phase 2. The spread (dispersion) of the
A
zones (peaks) is constrained so
Chromatographic bed that solutes can be eluted
(column filling) discretely

B
Sample
mixture
Frit injection Peaks
(porous disk) separated
Outgoing liquid
separation system
effluent
B column,
7
thin layer,.. 8
 SEPARATION OF COMPONENTS Chromatographic process
IN CHROMATOGRAPHIC SYSTEM

Components are moved through system in the mobile phase direction.

stains, strips Chromatography takes for separation of components:
Result: chromatographic zones of separated components
multiple repeated set up of components equilibrium between two phases
Detection: - detector at the end of chromatographic system
stationary (SP) and mobile (MP)
- physical property of effluent and sample component

Record: chromatogram: chromatographic zones

mobile phase flow

response
component transferring
peaks waves profile of analyte
from the SP to the MP at
concentration in the MP
the back of the peak
profile

profile of analyte component transferring

concentration in the SP from the MP to the SP at
stationary phase the front of the peak
9
profile 10
time, volume

INTERACTIONS IN CHROMATOGRAPHIC SEPARATION SYSTEMS

INTERACTION with MOBILE PHASE
Equilibrium is created by various physical - chemical interaction
INTERACTION with STATIONARY PHASE
(qualitatively and quantitatively) between
component and mobile phase
component and stationary phase 1. Adsorption - adsorption chromatography - stationary phase = adsorbent
 mobile and stationary phase - components are caught on the surface of stationary phase

a) physical adsorption – reversible, energy 0,3 -3 kJ/mol

Types of interaction microscopic view Acting forces:

(intermolecular forces)
macroscopic view polar (specific) non-polar (non-specific, dispersion)
• van der Waals:
•adsorption ind. dipole – ind. dipole between permanent dipoles (columbic) between electroneutral molecules
•dilution dipole-dipole, dipole - induced dipole permanent dipole vs. induced dipole induced dipoles
•chemisorption • electrostatic
•precipitation ion-dipole, ion-ion
alcohols, carboxylic acids aliphatic hydrocarbons
•sieve effect • hydrogen bridge
sulfonic acids aromatic hydrocarbons
b) chemisorption – irreversible retention of component part
energy comparable with energy of covalent or ionic bond 40 - 400 kJ/mol
11 12
 characterisation of physical adsorption
ADSORPTION ISOTERM INTERACTION with MOBILE PHASE
INTERACTION with STATIONARY PHASE continue
effect of component concentration in environment on amount of adsorbed the
component in equilibrium
Attention ! chromatography is dynamic process !!!!!!!!! 2. Partition
dissolution in two phases  partition equilibrium creation
CS = D . Cm D - distribution coefficient  KD liquid bonded on the inert surface - film of bonded stationary phase
invalid for real systems Cs, Cm – concentration in SP, MP
3. Precipitation
Freundlich: CS = k . Cm1/n k,n - constants precipitant bonded on stationary phase
separation of component according to solubility product Ksp
Langmuir: CS = w . z . Cm / (1+wCm) w - adsorption coefficient
z – number of free interaction sites 4. Sieve effect
on the surface Inclusion:
valid for chromatography ions of similar dimensions and same charge are caught in crystal lattice
K+ in NH4MgPO4

wcm<< 1 isotherm shape

Occlusion
Cs wcm>> 1 component, which is not a part of crystal lattice is caught in cavities
water in AgNO3
peak shape
Cm 13 14

STATIONARY AND MOBILE PHASE – general information

CHROMATOGRAPHIC METHOD CLASSIFICATION

STACIONARY PHASE general: sorbent
1.According to mobile phase
-solid particles diameter 1 - 100 ( 200, 300 ) m
-thin liquid layer on solid particles
Gas - gas chromatography - GC
-thin liquid layer on inner wall of capillary
Arrangement: thin layer liquid - liquid chromatography - LC
tube - column
capillary - capillary column Supercritical fluid – supercritical fluid chromatography - SFC
supercritical
SFC: fluid
- supercritical temperature and pressure p liquid
diffusion solid
MOBILE PHASE density viscosity coefficient CO2, SF6, Xe, NH3
[g.cm-3] [g.cm-1. s-1] [cm2 . s] CO2 phase diagram
Higher values of diffusion coefficients
• gas 0,001 0,0001 0,1 in comparison with similar liquids, gas
• supercritical fluid 0,1 – 1 0,0001 – 0,001 0,001-0,0001 Lower viscosity T
• liquid 1 0,01 <0,00001
15 16
 CHROMATOGRAPHIC METHOD CLASSIFICATION

CRITICAL VALUES 2. According to stationary phase:

Solid: Adsorption chromatography

Critical temperature - Tc:
only one phase exists in a system of Separation is based mainly on differences between the adsorption affinities of the
liquid-gas over Tc sample components for the surface of an active solid.
substance is in the fluid stage
GSC - gas adsorption chromatography
Critical pressure-pc: LSC - liquid adsorption chromatography
Tlak needed to condensation of fluid
substance at critical temperature Liquid: Partition chromatography

How to prepare supercritical fluid Separation is based mainly on differences between the solubility of the sample
1. Substance in liquid form is exposed to temperature and pressure to form components in the stationary phase (gas chromatography), or on differences
equilibrium with its vapour : two phase exist between the solubility of the components in the mobile and stationary phases (liquid
chromatography).
2. Created system is closed in tube and temperature is increased over Tc to
form only one phase disregarding the pressure GLC - gas partition chromatography liquid on support
LLC - liquid partition chromatography liquid on support –
17 18
immiscible with MP

CHROMATOGRAPHIC METHOD CLASSIFICATION

2a . Special types
3. According to main separation mechanism
Exclusion chromatography
Separation is based mainly on exclusion effects, such as differences in molecular
size and/or shape or in charge.
SEC - size exclusion chromatography may also be used when separation is based on  adsorption
molecular size.  partition
GPC - gel permeation chromatography were used earlier to describe this process  ion exchange
when the stationary phase is a swollen gel.  ion paired
IEC - ion-exclusion chromatography is specifically used for the separation of  affinity
ions in an aqueous phase.  gel permeation adenin
………. aminopurin
Ion exchange chromatography, Ion chromatography - IC
Separation is based mainly on differences in the ion exchange affinities of the
sample components.
competition of analyte and mobile phase ions for ionic groups bonded on the
stationary phase surface

Affinity chromatography - AFC

Particular variant of chromatography in which the unique biological specificity of
the analyte and ligand interaction is utilized for the separation.
„affinant“ bonded on chromatographic support specific biological/biochemical
interaction of antidote-antigen, enzyme-substrate, hormone-receptor 19 20
 5. According to method of component transport through separation system
 method of injection
4. According to experimental set-up Elution
chromatography
Column Chromatography A procedure in which the mobile phase is continuously passed through or along the
A separation technique in which the stationary bed is within a tube. chromatographic bed.
The sample is fed into the system as a finite slug.
Packed Column: The particles of the solid stationary phase or the support coated with a liquid Sample components are more retained as mobile phase components
stationary phase may fill the whole inside volume of the tube or be concentrated on or along
the inside tube wall leaving
A procedure in which the mobile phase contains a compound (the Displacer) more
Open-Tubular Column:
strongly retained than the components of the sample under examination.
An open, unrestricted path for the mobile phase in the middle part of the tube
The sample is fed into the system as a finite slug.
Mobile phase is more retained as sample components
Planar Chromatography Open-Bed Chromatography.
Displacement
A separation technique in which the stationary phase is present as or on a plane.
Frontal chromatography
Paper Chromatography, PC: The plane can be a paper, serving as such or impregnated by a chromatography
substance as the stationary bed
Thin Layer Chromatography, TLC: Layer of solid particles spread on a support, e.g., a glass A procedure in which the sample (liquid or gas) is fed continuously into the
plate chromatographic bed. In frontal chromatography no additional mobile phase is used.
Sample solution act as „mobile phase“
21
Solvent is less retained as sample components 22

ELUTION CHROMATOGRAPHY ELUTION:

isocratic - composition of the mobile phase remains constant during the
Position of zone: elution process.
Step 1: Step 2: Step 3: magnitude of interaction
Sample injection Component zone
stepwise - composition of the mobile phase is changed in steps during a
Component identification of component
into the flow of development in zone elution
single chromatographic run.
mobile phase mobile phase flow by mobile phase gradient - composition of the mobile phase is changed continuously or
stepwise during the elution process.

1,2 3 4 1 2 3 4
small amount
of component
mixture in a Vr
solvent
Vr’
Vm
mobile phase

elution of components in separated zones

in the order of interaction magnitude with Area/height:
stationary phase separation of amide C1- C4 lower content of
quantitative parameter
components are segregated by clear MP: ethylacetate/heptane ethylacetate in heptane
mobile phase zone
Eluted components
The best separation of sample components 23 24
dissolved in mobile phase
 DISPLACEMENT CHROMATOGRAPHY DISPLACEMENT CHROMATOGRAPHY

Step 1: Step 2: Step 3: c [mol/l] Result 1:

Sample injection Component zone Component zone A, A+B, B, B+C, C, C+D, D
development in displacement by
mobile phase flow mobile phase

D
sample Inserted displacers
C X,Y,Z
D
mobile phase B affinity between A, B, C
„displacer“ A volatile
A B C displacer

detector response
C
Sample components are displaced one another Result 2:
according to affinity to stationary phase. Mobile B
A, A+X, X, X+B, B, B+Y, Y,
phase has greatest affinity. A
Y+C, C, C+Z, Z, Z+D, D
Zone interfaces are diffuse (unequal flow, diffusion,
non-homogenous bed, non-homogeneity of displaced
interaction zones X,Y,Z evaporate, distil
Preparation, sampling of gases and a vapours 25 26

FRONTAL CHROMATOGRAPHY FRONTAL CHROMATOGRAPHY

Step 1: Step 2: Step 3: Step 4: Step 5:

Continuous feed of A+B+C
Component Saturation of bed Feed of Elution of all
component mixture zone A (all components) clear MP components sorption step
in MP perforation A+B

A+B+C
MP A
A+B

A A+B+C
desorption step
B+C
mobile
phase MP
C

A+B+C in MP C in MP clear only: first component (A with lowest affinity to SP)

A+B in MP B+C in MP last component (C with highest affinity to SP)
A in MP A+B+C in MP other components in mixtures
27 28
quantity – area defined by integration curve
 Example of a result of chromatographic analysis
SELECTED TERMS RELATED
CHROMATOGRAM
to THE CHROMATOGRAPHIC PROCESS
and THE THEORY OF CHROMATOGRAPHY continued
Response of detector

tR - retention time of compound retained in the system

compound moves slowly than mobile phase
dR - retention distance
tM - retention time of compound not retained in the system
dead retention time , hold-up time
compound moves as quickly as mobile phase

Fm - flow rate of mobile phase - [cm3/ s, ml/min, l/ min]

the volume of mobile phase passing through the column in unit time.
VM - hold-up volume, dead volume = Fm . tM
often equal to Vm – volume of mobile phase in column, the sample injector,
the detector, and connectors..
VR - retention volume of retained compound = Fm . tR

L - length of chromatographic bed , column length

u - mobile phase velocity, linear velocity u = L / tM [cm / s]

Time of analysis29 uA - retained compound A velocity = L / tR,A
30

KD doesn't reflect how components are shared in two phases !!!!

SELECTED TERMS RELATED
to THE CHROMATOGRAPHIC PROCESS
Better:
and THE THEORY OF CHROMATOGRAPHY Partition Ratio
Capacity Ratio
retention factor k (kA for component A) Capacity Factor
Mass distribution Ratio
„Equilibrium“ (component distribution between two phases) found in literature
Amobile phase Astationary phase (n A )1 (c A )1.V1 V V
kA    K D , A 1  K D , A SP
(n A ) 2 (c A ) 2 .V2 V2 VMP
Distribution constant - crucial factor of separation
- ratio of total concentrations of component A in two phases
Meaning of k

(c A )1 (n A )1.V2 (n A ) SP .VMP chromatography • measure of the time the sample component resides in the stationary phase relative
Generally: K D, A    to the time it resides in the mobile phase
(c A ) 2 (n A ) 2 .V1 (n A ) MP .VSP • how much longer a sample component is retarded by stationary phase than it would take
to travel through column with velocity of mobile phase
n (n A ) O .VW • equilibrium ratio of component amount in SP and MP
c extraction
V (n A )W .VO
• migration speed of analyte in chromatographic bed

31 • comparison of components interactions in MP and SP ( KD requires VMP a32VSP)

ATTENTION!!!!!
RF - retardation factor
Chromatographic bed, Chromatographic column is dynamic system The fraction of the sample component in the mobile phase at equilibrium; it is
related to the retention factor and other fundamental chromatography terms
it is impossible to reach equilibrium in particular step of component transportation
uA (n A ) m
RF   n = m / Mr
u (n A ) m  (n A ) s
TIME can be observed only, (nA)s  0 analyte molecule all the time in MP = no retention  RF  1,0
(nA)m  0 analyte molecule remains in SP  RF  0
time witch molecule spends in both phases during transport through
chromatographic system. RF – measure of presence probability of compound A in MP

TIME depends on interaction with mobile and stationary phase

Relation between RF a k ?????
different interaction
 different time spent in column kA = (nA)s / (nA)m
 different time spent between inlet and outlet of chromatographic system
(n A ) s / k A 1
R F ( A)  
(n A ) s / k A  (n A ) s 1  k A
RETENTION TIME tR
33 34

ADJUSTMENT OF SEPARATION PROPERTIES

HOW TO OBTAIN k ???
Quality of separation of sample components
u tt
M  t R , A  1  V R , A  1  V R , A  V M  V R , A  t R'
'
u  u .R F  k  R, A
1 k
A
A t tM VM VM VM tM Suitable combination of retention times difference
L L M
 and zone width of separated components
tR ,A tM (1  k )
VR - VM = V´R - adjusted retention volume
1 1
 tR - tM = t´R - adjusted retention time
tR ,A t M (1  k )
Influence of zone width
t M  t M .k  t R , A
(transport of analyte through column)
kinetic aspect of separation
kA can be obtained from chromatogram !!!!!!!
Insufficient zone resolution
- velocity ratio of analyte and mobile phase
- ratio of times spent in chromatographic system for retained and unretained solute

If VM = Vm VR , A  VM VS
 KD
VM VM Influence of magnitude of interaction of
components with MP and SP
VR  VM  K DVS
(migration velocity)
VR'  K DVS thermodynamic aspect of separation
Retention volume depends on volume of stationary35phase 36
 KINETIC ASPECT OF SEPARATION EFFICIENCY OF CHROMATOGRAPHIC SEPARATION
Efficiency of chromatographic column
c0 t0
Zone broadening
during movement of component through the column Ability of system to separate sample components to independent zones of
individual component during elution.
Injection of analyte into the middle of column
Measure of efficiency – peak Expression – Number of theoretical plate N
c1 t1
width 
Plate - part of chromatographic bed or column
Zone broadening caused by diffusion makes possible to establish dynamic equilibrium between
c2 t2 component fractions in mobile and stationary phase.
The faster equilibrium, the higher plate number
 standard deviation
2 = 2Dt Einstein
From theory of chromatographic plate:
2 – square of distance covered by component molecule V, t, L
2 2 2
2 in time t ~ standard deviation t  V   L  standard deviations
N   R    R     in consistent units
peak width in the inflection points= 2  t  V  L 
the segment of the peak base intercepted by tangents drawn 2 = 2 D. t Higher  lower number of separated components in time
4
to the inflection points on either side of the peak = 4  37 lower separation efficiency
38

HOW TO CALCULATE EFFICIENCY OF CHROMATOGRAPHIC SEPARATION?

Effective plate number - Neff
dR, tR, VR A number indicative of column efficiency calculated by using the adjusted
Peak width measurement: retention volume (time) instead of total retention volume (time).

a) peak-width at base
2 2 2
 t'   d  dM   
  n t R  t M
2
 16 R   16 R
2
t  d  
N  16 R    R 
  Y  4 N eff
 Yt 
 Y



 t
 R 
 Yt   Y  Y0,607h 2
b) peak-width at half height Yh/2 : sample N is usually calculated for one meter of column
Yh/2
injection 10 000 plate / meter
h

2
 d 
N  5,545 R  Y  2,355
 Yh / 2  Comparison of efficiency of different columns:
0,607h

c) peak-width at inflection points (at 0,607 height) Height Equivalent to One Plate H plate height
h/2

Y  2
2
 d 
N  4 R 
 Y0,607  L LYt 2  2 [m]
H  
dR – retention distance
N 16t R2 L

N - depends on retention time Yt, Y
39 40
peculiar to individual component
 HF : the eddy diffusion
Why is zone (peak) broadened ????

xxxxxxxxxxxxxxxxx

1. Theory of chromatographic plate – unable to describe separation correctly

2. Dynamic theory - van Deemter

Four factors causing zone broadening:

Different speed of sample particles v in broad and narrow „canals“
1. eddy diffusion in mobile phase - HF High number of streamlines, different local speed

2. molecular (longitudinal) diffusion in mobile phase - HL Eddy diffusion is proportional to

dimension and shape of stationary phase particles
3. resistance to mass transfer in the stationary phase - HS
Lower particle: higher bed homogeneity
4. resistance to mass transfer in the mobile phase - HM 
lower differences in „canals“
H = HF + HL + HS + HM
41
Flow-round: different for spherical particles and irregular 42

The eddy diffusion is not proportional to linear velocity of mobile phase

Zone dispersion 2 during chromatographic process involving high number of 
Chromatografické metody I
random steps , HF ~ u  parallel line with x-axis

generally 2 = l2.n l = average length of random steps

n = number of random steps

speed of moving zone u H

molecule remains time te in streamline with speed u*
deviation from midpoint of zone te(u*-u) - length of random step
difference u*-u is proportional to average speed
l = (u*-u) te
number of steps n = L/teu
u*-u = u
L/teu is proportional to particle diameter dp
n = L/ute = L/dp
HF = 2dp
HF = 2dp
 = constants of
proportionality
 - geometrical factor
dp - diameter of sorbent particle L 2 = 2dpL = 2dpL
u
43 44
 HL : molecular diffusion

Chromatografické metody
(capillary column)I
u = L / tM
2 = 2Dm L / u
diffusion in „free“ mobile phase
•at both sides of zones
H = 2 / L
•transport of molecules from
HL = 2 Dm / u
higher concentration area to
HL = 2Dm / u packed column
lower concentration area
 - factor expressed impossibility of „free diffusion in
packed columns
proportional to quality of bed (canal shape,...)
diffusion proceeds:
•in the direction of mobile phase flow and in opposite direction
•only in longitudinal axis of column H HL ~ u  hyperbola
total time of molecule diffusion in
Fick‘s law: dn dc mobile phase:
 D Dm : GC ~ 0,1 (1,0 ) cm2 . s -1
dt dx • independent on retention,
• identical with hold-up time LC ~ 10-5 cm2 . s -1  can be vanished

2 - square of distance covered by

Einstein's law   2 Dmt M
2
component molecule in time 45 46
t ~ standard deviation
u

HM : mass transfer in mobile phase

HS : mass transfer in stationary phase

Molecules penetrate into layer of SP and back.

Molecule entering deeper falls behind
molecule entering only under surface.

Sample zone in MP runs faster

than zone in SP

Different speed near surface or capillary wall

d p2 qd 2f u k
HM  u HS 
Dm Different speed of sample DS k  12
molecules in MP towards to
 - factor dependent on packing type  1,3 SP surface q - configuration factor – packing geometry
HM  u  line df - stationary phase width
DS - diffusion coefficient of molecule in SP
HS ~ u  line
47 48
TOTAL EFFICIENCY OF CHROMATOGRAPHIC SYSTEM B/u

H = H F + HL + H S + HM
H
A
2 D m qd 2f u k  d p2 u C.u
H  2d   
D S ( k  1) 2
p
u Dm
B
H  A  Cu
B/u u
A Cu u
van Deemter
B for GC van Deemter
H  A   Cu H
u High Dm in gas phase  low HM
1 1
H  HL  HS  for LC Giddings H  HL  HS  Giddings
1 1 1 1
 
HF HM
HF HM
50
max efficiency u

2
2 D m qd f u k  d p2 u
H  2d p    PEAK RESOLUTION
u D S ( k  1) 2 Dm Measure of relative separation of two adjacent zones - peaks
B GC
H  A  Cu A: open tubular column  eliminated
u t R j  t Ri
B: significant owing to high diffusion coefficients 2 Z
R ij  Rij 
0 ,5 Yi  Y j 
(high diffusion in gas phase)
C: thin films  lower Yi  Y j
H: ~ 0.1 mm tRj
dimensionless number
LC (HPLC)
tRi Z tR – retention times
A: packed columns, high-homogenous particles, high pressure  lower
Y – peaks width on base.
B: significant owing to relatively high diffusion coefficients in liquid phase
(lower than in GC)
the higher value Rij , the better separation
C: thin films  lower
Rij = 1,0 - zones overlap by 2%
H: ~ 0.1 mm

CE
A: open tubular column  eliminated
B: significant owing to relatively high diffusion coefficients in liquid phase Yi Yj
C: only one „phase“ , no equation between phases  eliminated
H: ~ 0.001 mm 52
 Effect of chromatographic parameters on resolution
PEAK RESOLUTION
Measure of relative separation of two adjacent zones - peaks - difference of elution times ~ thermodynamics of separation
- zones (peaks) width ~ kinetics of separation

full separation t R j  t Ri ki - retention factor (capacity ratio) of

R ij 
0 , 5 Y i  Y j 
component i
 - ki / kj - separation factor
t R  t M (1  k ) n (k j  ki )
R1,2 = 1.0 R1,2 = 1.5 R ij 
4 (1  k i )
2
 t 
n  16  R 
 Y t 
overlap 2% overlap 0,1%
Yt 
4 Rij 
n
  1 k i
n
tR 4 1  ki

t M (k j  ki ) n   1  1  k j
Rij  n Rij   
If Y2=Y1 4t Ri 4    kj

53 54

G A S CH R O M A T O G R A P H Y
THREE INDEPENDENT TERMS AFFECTING RESOLUTION
Mobile phase: gas – inert – transport of components – carrier gas
hydrogen, nitrogen, helium, argon, CO2
according to type of detection, influence on separation efficiency

R 
n
  1  k i Carrier gas DG (30 oC)  (50 oC)  (150 oC)

1  k
ij
4 i
H2 0.277 94 112
He 0.248 208 249

N2 0.073 188 227

KINETIC TERM THERMODYNAMIC
CAPACITIVE TERM
TERM Ar 0.059 242 296
can be changed by: CO2 0.059 162 206
can be changed by:
can be changed by:
Diffusion coefficients DG (cm2s-1)
of n-octane
•amount SP in the column and viscosity  (P) of most used carrier gases
•mobile phase velocity •change of MP and/or SP
•temperature
•column length
•change of MP and/or SP H2
•sorbent particle diameter
disadvantage: explosive in mixture of air - Attention
•diffusion coefficients
advantage: fast separation (low analysis time),
55 separation of strong retained components 56
Sample components to be separated, main application:
• gas mixtures
• volatile organic compounds b.p. < 400°C
(requirement for sample conversion into gas phase)
Interaction with stationary phase:
• Adsorption gas chromatography GSC
• Partition gas chromatography GLC
Elution:
• isocratic constant temperature
• gradient variable temperature
INSTRUMENTATION
in Gas Chromatography
Sources of carrier gas:
gas cylinders
generators (molecular sieves)
couplings, connections :
metal capillary, ideal gas tightness
gas purifying :
moisture, low molecular hydrocarbons and oxygen removing
flow regulation :
a) mechanical regulators
b) electronic regulators
gas flow : 1 - 100 ml/min.,
gas-solid phase
gases, liquids (low Mr)
film of non-volatile liquid
on the surface of solid carrier
57
 1 - 2 % accuracy
 0.2 % repeatability of set value
59
INSTRUMENTATION
in Gas Chromatography
Gas Chromatograph
Components
• carrier gas source
• sample introduction (injector)
• chromatographic column
• thermostated compartment
• detector
• data station
58
Isocratic GC - Isotherm TEMPERATURE MODES
Two basic characteristics:
Chromatografické metody I
retention data  number of methylated groups (-CH2-)
(vapour pressure, boiling point)
T – column temperature
 1/Tc (log tR = f (1/Tc) c
linear dependence of homologous series
(n-alkanes, n-alkylbenzenes, …)
disadvantages :
 it is not possible to find temperature suitable for separation of components with b.p.
difference than 100 °C
 poor separation of early eluted peaks
 poor delectability of later eluted peaks (broadened peaks)
Gradient GC – Temperature programming
• analytes retention times decreasing in samples with broad range of boiling points
• improving of delectability
• large volumes of injected samples
• optimum: temperature slightly over average b.p. of sample components
temperature stability: ± 0,1°C, change: about 1°C, temperature range: up to 450°C
60
 INJECTORS for GC

Function: Methods of sample injection

 Sample injection on the column beginning as a narrow zone
 Conversion of sample into the gas phase
 Mixing of sample and carrier gas ahead of column entry  over the column inlet – packed column
 on-column – capillary columns
Requirements (ideal) :
 injection without decreasing of separation efficiency
 injection without temperature degradation and sample adsorption
 injection without discrimination according to b. p, polarity or Mr
 injection with total recovery of all sample components

Injection devices
depend on sample state
gases - gastight syringes, injection valves (volume up to 1 ml)
liquids - syringes, autosamplers (volume 0.5 - 5 µl)

61 62

Syringe injection methods Syringe injection methods

solvent flush air flush

cold needle hot needle

Fast injection with
minimum delay in injector space
following fast extraction from injector.
Liquid remains in needle.
Small amount of solvent is full field into the Syringe is full field more than required
syringe, following by air, sample and again volume and tip into required volume.
Needle remains in hot injector space
air.
approx. 5 sec before sample injection.
Liquid sample is retracted in to the syringe.
Polar compounds absorbable on glass
Samples with high boiling point of
and/or needle Sample is fast injected in to the GC and
components.
piston is released
Hot needle increases evaporation speed.
Syringe is fast taken out the injector.
Injection of sample in needle and syringe
Complete sample injection, low loss of
cylinder.
volatile compounds, excellent repeatability
63 64

Methods of sample injection into capillary columns
SPLIT – flow splitter
 high number of components
 < 10% of sample
SPLIT LESS – without flow splitter
 diluted samples
 approx. 80% of sample
Splitter closed 1 - 2 minutes, sample enters column any longer
Use :
• qualitative analysis at high
injection ways :
chromatographic resolution
• into hot column
• less for quantitative analysis
• into cold column
(temperature 20 - 30 C lower, than
Disadvantages : b.p. of solvent used)
• discrimination of compounds with
higher b.p.
• loss of injected sample according to advantages :
split opening • analysis of diluted samples without
preconcentration
(the whole sample is injected into a
column)
• analysis of impure samples
(change of injection liner and
retention gap)
65 66

ON COLUMN INJECTOR
- direct into capillary column Retention gap
under b.p. of solvent, thermolabile components
reduction of the length of submerged zone in capillary column
injection ways (deactivated fused silica capillary, 1 - 10 m)

• direct on column (micro syringe with thin needle,

• injection into short column with i.d. 0.53 mm)

temperature 20 - 30 C lower, than b.p. of solvent used

difficulties:

• Sample could be broken into separated parts.

• Bubbles generated et the inlet of column separate sample into different pars
of column depending on way of solvent evaporation
• Zone with different concentration are formed, each behaves as individual
injection.
• Chromatogram with more peaks of the same component.
67 68
 GAS CHROMATOGRAPH

PTV INJECTOR (programmed temperature vaporization) injection port detector port capillary column fan
(FID) wound around holder
Control of temperature of injector body with liner packed with
chromatography bed.
advantage combination of split, split less and on-column injectors. control panel

advantages
• minimum discrimination caused by injection from needle of microinjector
• minimum discrimination according to b.p. of analytes
• not necessary to use special needle as for on column injection
• large volume injection oven
• elimination of solvent and low molecular compounds before analysis
• retention of non-volatile compounds in injection liner
• high repeatability of retention times and peaks areas.

69 70

COLUMN FOR GC / STATIONARY PHASES

GAS CHROMATOGRAPH PACKED COLUMNS:

N2 inlet tubes: Al, Cu, Ni, stainless steel, glass,

Air inlet 2-6 mm, 1-5 m
(detector)
H2 inlet
particles: 0,13 až 0,40 mm
(detector)
adsorbents (GSC): silica gel, alumina, active carbon,
molecular sieve,
porapak (styren-divinylbenzen copolymer)

bonded phases (GLC): non-volatile, chemically inert liquids

siloxane, polyethylenglykole, esters, hydrocarbons
heated fan (squalan, C30H62),silicone
He inlet
(carrier gas) inert carriers: siliceous earth, modified siliceous earth

Properties of packed columns:

worse resolution
higher volume of stationary phase, preparative purposes
71 72
low-boiling components, less retained gases
 Chromatographic carriers for liquid stationary phases
Column packing device
Sorbents
Graphitized active carbon (heated to 3000 K)
non-porous, non-specific, high inert material
adsorbent and solid carrier
Carbopack , Carbotrap, Spherosil, Spherocarb

Chromatographic carriers Silica gels

for stationary phases particles based on silicon dioxide
activity depends on amount of adsorbed water
Porasil, Chromosil, Chromosorb T, Chromosorb G, Supelcoport
Porous/nonporous particles of
suitable chromatographic properties Activated alumina
particles based on aluminium oxide
Particles: - 0.5 - 0.1 mm Siliceous earth
- narrow distribution of particle diameter Siliceous earth micro amorphous silicate
- low specific surface (1 - 7 m2.g-1) rock (formation) treated by calcinations at
Molecular sieves
suppression of adsorption 1000 C, sintered particles separated
aluminosilicate, zeolite
according to size
- sufficient porosity (0.1 - 1 ml.g-1)
good wetting by liquid SP Graphitized molecular sieves
separation of permanent gases and low molecular organic compound
Carbosieve, Carboxen, Supelcarb
Properties: - inert Polymers
chemical reactivity – peak deformation STY-DVB, Acryl ester, Polystyrene, EVB-DVB, ACN-DVB
or disappearance Chromosorb (polar copolymer), Durapak, Porapak, Tenax
specific adsorption – peak tailing 73 Teflon PTFE 74

Liquid stationary phases for GC

Liquid stationary phases for GC Product Tempe Use Properties

Type name rature
°C
requirements:
High molecular Non-polar Squalan  300 Hydrocarbon easy oxidizable
hydrocarbons Apiezon s Non-polar
- low volatility (vapor pressure 1 - 10 Pa) compounds

- suitable chemical composition (from non-polar to polar phase)

Perfluorinated Polar Fomblin  250 Halogens High reactivity
- good solubility of separated components in the liquid phase alkanes Fluorinated Fluorad Amines Low temperature
alkylesters Phenols stability
- different solubility of separated components Carboxylic
acids
- no chemical reaction with analytes • High molecular hydrocarbons
• Perfluorinated alkanes Polysiloxanes - R2SiO – OV  350 Non polar- High temperature
Variable SE polar stability
• Polysiloxanes polarity easy oxidizable
• Polyethylenglykoles
• Polyfenylethers, Phtalates, Polyethylenglykoles Polar Carbowax 220 oxygen Low temperature
• Liquid crystals
Superox  300 compounds stability
HO(CH2CH2O)n CH2CH2OH Alcohols easy degradable
75 76
 Selected commercially available polysiloxane phases Liquid crystals

Mark Type Structure  polar liquids

 strong columbic interaction of ions with analyte
OV-1 Dimethylsiloxane CH3  positional isomers
OV101 Dimethylsiloxane CH3 Cation Anion
OV-7 Phenylmethyldimethylsiloxane C6H5 (20%) Tetrabutylamonium Perfluorooktansulfonate
4-toluensulfonate
OV-17 Phenylmethylsiloxane C6H5 (50%)
Tetrafluoroborate
OV-25 Phenylmethyldiphenylsiloxane C6H5 (75%) Picrate
OV-210 Trifluoropropylmethylsiloxane CH2CH2CF3 (50%)
Tetrapentylamonium 4-toluensulfonate
OV-225 Cyanopropylmethylsiloxane C6H5(25%)
Tributylbenzylphosphonium Chloride
OV-275 Dicyanoalkylsiloxane C3H6CN(20%)
Ethypyridinium Bromide

77 78

Bonded liquid phases CAPILLARY COLUMNS

carrier stacionary phase polarity temperature use

(w/w) limit (C)  capillaries with low inner diameter wetted by thin layer of liquid
Porasil C 3-hydroxypropionitrile middle 135 hydrocarbons,  efficiency 100 times higher than packed columns.
(3%) aromates Marcel Golay 1957
Porasil C Carbowax 400 non-polar 175 alcohols
(7.86%)
• glass, fused silica, organic polymers (PAD, PES, PTFE, FEP)
Porasil C n-Octanol polar 175 alcohols, • metals (stainless steel, Ni, Al, Cu)
hydrocarbons
• i. d. 100 – 530 m (700)
Porasil S Carbowax400 non-polar 230 hydrocarbons • length 15 – 100 m
(16.75%)
• layer 0.1 - 10 m
Porasil S Carbowax 4000 polar 230 aromates,
chloraromtes
Porasil F Carbowax 400 non-polar 230 waxes,steroids, PAHs
(1.41%)
According to stationary phase immobilization
• Porasil C (100 m2/g, pores 30 nm) • classical capillary columns
• Porasil S (300 m2/g) disperse forces-bonded stationary phase,
• Porasil F (10 m2/g, pores 300 nm) permanent dipoles, induced dipoles, hydrogen bonds, ….

• capillary column with bonded phase

79 SP chemically bonded to inner wall of capillary column. 80

100% dimethylpolysiloxane
trifluoropropyl-methylpolysiloxane (trifluoropropyl)
phenyl –dimethylpolysiloxane
diphenyl –dimethylpolysiloxane
phenylmethylpolysiloxane
cyanopropyl-phenyl –dimethylpolysiloxane
polyethylene glycol
CAPILLARY COLUMNS FILLED in WHOLE VOLUME
Advantage:
column capacity improving
packed column with floating and irregular packing
capillary tube drawn from tube field by bulk
chromatography carrier followed by wetting of stationary
phase
packed column with regularly loaded bad
capillary packed in ultrasonic bath using inert gas
CAPILLARY COLUMNS with NON- FILLED FREE SPACE
PLOT (Porous Layer Open Tubular column)
thin layer of solid sorbent (10 µm) on inner wall
WCOT (Wall Coated Open Tubular column)
thin film (0.01 - 1 µm) of SP created directly on inner surface of
column,
small inner diameter (narrow bore) - pod 0,1 mm I.D.
conventional diameter - 0.32 - 0.15 mm I.D.
big inner diameter (wide bore) - 0.53 mm I.D.
FSOT (Fused Silica Open Tubular column)
- type of WCOT, thinner film, smaller diameter
- rigidity increased using polyimide laser, flexible
- low reactivity
SCOT (Support Coated Open Tubular column)
thin layer of carrier (1 - 5 µm) wetted by SP on
inner wall of capillary
81 82
DEACTIVATION
elimination of negative effect of silanol groups from inner
surface of capillary
silylation
aliphatic or cyclic silylation dyes
polycondensation
reaction of silanol groups with deposited thin layer film of
Carbowax or silicone stationary phase at high temperature
esterification
reaction of silanol groups with aliphatic alcohols C4 - C10 and
tetraethylglycole at high temperature
polyimide layer
layer of polyimide (1 - 5 µm) on the inner surface of capillary
83 84
Retention time in gas chromatography

 B  p 2  p o2
u  p    o  i 

u  po po
 u  po  j
 Gas compressibility - change of mobile phase volume
 Decry's law for liquid flow trough nonporous bed   o L  2p p

 
p - middle pressure in column
B
dp Bo - specific permeability constant 2 pi3  po3
u   p u( p) - velocity at middle pressure
 
o
o - antiparticle porosity
 o dz  - liquid viscosity 3 pi2  po2
dp/dz – fall in pressure in the flow direction j - James-Martin‘s compressibility factor
 pi 
2

 B  pi a po - absolute pressure at inlet and outlet 3     1 
u   o   pi  p o  L - column length  po  
  o L  j  
 pi 
3

2     1 
  po  
pro GC
 Bo
u  p o   

 p i2  p o2


  o L  2 po L (1  k ) L (1  k )
tR  
85
u u ( p0 ) j 86
u(po) – velocity at pressure po,

Hold-up volume
Detectors - classification
Dead volume
according to the basis of response:
retention volume - corrected to 0°C concentration-sensitive - response depends on change of sample concentration in
- correlated to mass unit (ms) of stationary phase Vg detector (g/ml) TCD, ECD PID
surface unit (S) of adsorbent VS mass-flow-sensitive - response depends on mass component in detector (g/s)
FID, TID, FPD,
Vg 
j ( t R  t M ) F m . 273
m s .T c
ml . g 
1
according to detector selectivity
universal - response to every component in the effluent except the mobile phase

 
j ( t R  t M ) F m . 273 selective - response to a related group of sample component in the effluent
VS  ml . g .m  2 specific - response to a single component or to a limited number of components having
S .T c similar chemical characteristics

according to detection principle

Values of specific retention volumes (times) affected by experimental errors:
ionization of molecules (FID, TID, PID, ECD, HID)
Vg – dependent on accurate mass determination of used SP bulk physical properties of molecules (TCD)
loss of liquid SP as o result of volatility optic properties of molecules (FPD)
electrochemical properties of molecules (HECD)

87 88
 Signal noise - changes of output signal (base line) not caused by eluted
sample component
Detectors - requirements a) short-time - frequency higher than eluted peak frequency
b) long-time - frequency similar to eluted peak frequency
• high sensitivity
• low limit of detection
• good stability and repeatability of signal

detector signal
• low noise and drift of signal
• fast response independent on flow of MP
A – peak area
• linear response over several orders
F – flow through detector
w – sample amount
sensitivity – slope of calibration curve
S = A . F/ w ( detector with concentration response)
S = A / w ( detector with mass response)

Limit of detection - the lowest concentration or amount of detected time

compound which caused signal magnitude three times
higher than magnitude of detector noise
Signal drift – frequency lower than frequency of eluted peaks
89 90

Flame ionization detector - FID

Linear dynamic response range of detector working principle

 hydrogen-air flame between two electrodes

Range of concentration or mass flow of analyte with linear response  difference in potentials  500 V
 burning of organic compounds – ion generation
 ions increase flame conductivity
signal

 generated current equal to concentration of organic compound in carrier gas

•organic compounds detection

Upper limit • limit of detection (up to 10-11 mol)
• linear dynamic range (up to 107).
five percent deviation of signal • low sensitivity for :
from linearity inert gases,
hydrogen, nitrogen, oxygen,
Lower limit chlorine, ammonia,
S/N=2

peak height equal to double noise sulphane, water, CO2

height of signal/noise ratio • small hold-up volume
concentration
91 92
 Termionic ionization detector - TID
Alkali-flame ionisation detector - AFID (FID with alkali metal) Photoionization detector - PID
Nitrogen phosphorus detector – NPD
• selective for N, P, S, B
and halogenated compounds
• pesticide, drugs
• selected metals (Sb, As, Sn, Pb)
• limit of detection (10-13 - 10-14 mol)
• linear dynamic range (105 )

High noise
Low signal stability

working principle • compounds absorbed UV radiation

 similar to FID detector • limit of detection (10-14 mol)
 carrier gases N2, O2, air, air + H2 • linear dynamic range (107 ).
 alkali metal salt (Rb2SO4) working principle
in bead on platinum wire or ceramic ring  absorption of radiation emitted from UV lamp
 difference i potentials  180 V  ion current detection in ionization chamber
 plasma production in bead  800C  high amount of ions, high current  non-destructive detector
93 94

Electron-capture detector - ECD

Thermal conductivity detector -TCD (catarometer)
• detection of compounds generated stabile ions:
alkylhalogenide, carbonyle compounds, nitrile, • organic and inorganic compounds
organometalic compounds, water vapour • non-hydrocarbons gases
compounds with P and S, NO2,, oxygen, ozone • low-molecular hydrocarbons
• halogenated compounds • limit of detection (10-8 mol)
• limit of detection (10-15 mol) • linear dynamic range (104)
• linear dynamic range (105)

Electrodes (anode, cathode)

working principle -emitter

 carrier gases N2, H2, He
 proportional counter for measurement of intensity
of radiation
 beta-source (63Ni, 3H on Pt or Ti layer). working principle
 ionization of carrier gas caused by electron from  emitter  thermal conductivity changes of carrier gas outgoing from column
 strong production of „thermal electrons“.  heated Pt, W, Au fibre
 absorption of thermal electrons during collision with positive charged particles  measurement of temperature changes of sensor (thermistor, metal fibre)
 decreasing of number of negative charged particles  current decrease
95 96
 Atomic emission detector - AED
Flame photometric detector - FPD

• selective for compounds with S or P

working principle
 similar construction as FID,
 separated optical system and photomultiplier
 excitation of molecules in flame
 emission of specific irradiation
• particles S2 or POH
• chemiluminiscence blue (394nm) Selective detection of
green (526nm). elements in organic molecules
compounds N, halogens, B, Se, Ge (analysis of elements in
• limit of detection (10-13 g P/s, 10-12 g S/s) solute).
• linear dynamic range (104 ).
working principle
plasma source – excitation of atoms (C, H, D, O, N, S, P and halogens)
detection of emitted irradiation
97 98

Comparison of most used GC detectors

Chemiluminiscence detector – CLD Limit of detection
Detector Signal generation Signal production Advantages Disadvantages (g analyte /ml
type carrier gas)

TCD thermal
conductivity of
change of
electrical
universal, simple, wide
dynamic range, non-
low
sensitivity
10-8
(10-100 ppm)
gaseous analyte resistance of destructive number of
filament in stream compounds
of analyte

FID ionization of
analyte in H2/air
current of ions high sensitivity, wide
dynamic range, broad
destructive 10-13

flame applicability

TID ionization of
analyte in H2/air
current of ions selective for org. P or N 10-13
Not for N a P
flame

ECD decreasing of
ionisation of carrier
current of ions
decreased in the
selective for org.
compound with
narrow
dynamic
10-15

gas caused by presence of electronegative function range

radioactive source organic molecule groups, non-destructive,
working principle high sensitivity

chemically produced vibration- or electron- excited particles MS ionization of ions of analyte, universal. complex price 10-12
analyte separation mixture of organic
excited particles emit photons according to compound, speed, high
mass/charge ratio sensitivity, identification of
compounds
99 100
Detectors for GC
range of applicability LIQUID CHROMATOGRAPHY

Differences in comparison with GC:

• compressibility of mobile phase is not considered

• lower influence of temperature on retention characteristics
• active role of mobile phase
• 85 % compounds are non-volatile , bad volatile, unstable

„Classical“ LC:
Open system, large-size particles > 100 m, MP flow controlled by gravitation
time consumed separation – a number of hours
fractions analyzed separately Cl-, Br-,J-
low efficiency
injection amount

101 102

GC theory  LC theory  technique for faster LC separation  HPLC

HIGH PERFORMANCE LC
CHOMATOGRAPHIC SYSTEMS IN LIQUID CHROMATOGRAPHY
Result: low particles 3-10m 30-90 thousand plates per meter
• homogenous filling – narrow particle distribution
• homogenous film of stationary phase
according to dominant mechanisms of interaction of sample
components with SP and MP
H = H F + HL + H S + HM
LSC - liquid adsorption chromatography
2 Dm qd u
2
k d u 2
B
H  2 d p    H  A  Cu
f p
LLC - liquid partition chromatography
u DS ( k  1) 2
Dm u
SEC - size exclusion chromatography
dp : lower eddy diffusion:  A GPC - gel permeation chromatography
Dm: lower molecular diffusion in the liquid:  B
 u, df, Ds : faster mass transport between phases :  C IC - ion chromatography
1
H  HL  HS 
1 1

better for HPLC - Giddings HF HM 104
 LSC - Liquid adsorption chromatography
Alumina (aluminium oxide):
Stationary phases for LSC - solid particles
◘ surface - hydroxyl groups and electron-acceptor centres
particle character irregular spherical spherical - strong electrostatic field, creation of induced dipoles
◘ pH = 8 - 11 - separation of weak acidic compounds from neutral
particle property fully porous fully porous surface porous
- strong acids – chemisorption (no requested phenomenon)
specific surface 100-500 m2/g 100-500 m2/g 5-15 m2/g ◘ activation - 400 °C 6 - 16 hod, activity control with water addition
capacity high high low ◘ capacity - lower than silica gel
permeability lower high high
efficiency lower high high Silica gel:
◘ surface - hydroxyl (silanol) groups
area of applicability preparative analytical analytical
- creation of H-bridges
price low high high ◘ pH  8 - chemically labile
◘ pH 3 – 5 - strong retention of basic compounds
Basic material for SP: - polar adsorbents - silica gel (protonated basic compounds + dissociated silanols)
- aluminium oxide ◘ activation - 180°C 3 hour
Two forms: wide pores > 10 nm surface area: 100 - 500 m2/g
narrow pores < 10 nm > 500 m2/g
Adsorption of analytes increases with increasing surface activity and area
Polymeric packing decreases with increasing mobile phase polarity
- stabile in broad range of pH: 2 - 12 depends on geometrical distribution of functional groups
105 106
- HEMA

MAGNITUDE of ANALYTES POLAR INTERACTIONS and ADSORBENT

depends on analytes polarity and MP polarity

Retention order of compounds Mobile phase for LSC

LLC - Liquid partition chromatography
on polar adsorbents Organic solvent and/or water
 different polarity
Aliphatic hydrocarbons MP polarity must be lower than SP polarity - separation of analytes between two immiscible phases (liquids)
Aromatic hydrocarbons analytes penetration through phase interface into whole space of SP – absorption
Halogenated compounds NORMAL PHASE CHROMATOGRAPHY - similar as extraction
depends on type of substituent

Ethers Advantage over LSC:

SYSTEMS
Tertiary amines - retention depends on stationary phase amount
NPLC, NP-HPLC
Nitryl k = KD VS/Vm
Nitro compounds Liquid stationary phases: - immobilization of different amount of SP
Polarity of MP for LSC
Esters of carboxylic acids
• mechanically immobilized on suitable carrier (silica gel) – physically bonded
Heptane
Ketones low solubility in mobile phase  saturation column
Pentane
Aldehydes
Cyclohexane
Primary amines
Benzene
• chemically bonded on reactive carrier
Amides of carboxylic acids - insoluble in mobile phase
Ethyl ether
Alcohols - possible use of gradient elution
Dichloromethane
Phenols - possible temperature changes
Acetone
Carboxylic acids
2-propanole
Sulfonic acids
water 107 108
 Chemically bonded stationary phases

Bonds with surface silanols 1. No polar, hydrophobic 2. Polar

hydro carbonic

C18

C18 ec
 Si - O - C
 Si - O - Si - C C8ec
 Si - C C8
 Si - N - C

C4 SA

SB
109 C2 110

REVERSED PHASE CHROMATOGRAPHY SYSTEMS

„ENDCAPPING“
SP – non-polar MP viscosity changes
Surface of silica gel covered with SP - long hydrocarbon chain as a function of organic/water ratio
with free silanol groups usually C18, C8, C4 backpressure

MP- polar - acetonitrile

Polar protective group - tetrahydrofuran
in carbon chain - dioxane
strongly interact with - diethylether
silanol groups - methanol
- propanol

Inserted polar group mixture with water: decreasing of high

elution strength of organic solvents
Surface coverage
with high density
Modification of mobile phase properties:
of alkyl groups
change of pH: increasing/decreasing of solute ionisation and a polar
groups at the surface of stationary phase
increasing pH: - higher retention of basic sample components
- lower retention of acid sample components
cross-linked carbon skeleton 111 112
Reversed phase chromatography systems Effect of higher water content > 95% in MP

Effect of SP surface modification on the • plainly visible deterioration of

retention of aromatic hydrocarbons column efficiency
Analytes separated in
reversed phase systems • non-polar alkyl chains lose
their

increase of retention
n - alkanes “brush-type-structure“
aromatics
halogenated hydrocarbons • drastically decrease of
ethers retention times and
nitro compounds resolution
esters
amines
amides
acids
sulfonic acids

113 114

SEC - Size exclusion chromatography

- separation according to size of molecules

- mechanical separation according to particle hydrodynamic diameter

SP: particles with high number of pores

- defined pore size
- pores filed by MP
- no interaction with MP and analyte
MP: good dissolution of analyte

Small molecules - diffusion into the SP

- highest retention
Middle and large - only widest pores Exclusion limit: - pores accessible for molecules from certain size KD = 0
Larger than pore diameter - no retention Total exclusion: - size area over exclusion limit
- Mr, min - exclusion limit
VR´ = A - B log Mr,A Total penetration: - pores accessible for all molecules KD = 1

115 116
Materials for GPC (SEC) Materials for GPC (SEC)

Gels:
hard - aero gels, inorganic materials, non-swelled porous glass Sephadex

semi-hard, soft - xero gels, organic materials, swelled Sephadex

Sepharose
hybrid - MP change causes only low volume changes Spheron
C O N H2
Hydrophilic: Hydrophobic: Sephacryl
cross-linked dextran PS/DVB Nucleogel, Supelcogel CO N H2 C O N H2

(epichlorhydrin) Sephadex acrylate, polyvinylacetate CONH C O N H2

• stabile polymers with high pressure

(N,N -methylbisacrylamid) Sephacryl stability CONH

Sepharose • minimum changes in volume caused C O N H2

glycolmethacrylate Spheron • by MP polarity change
polyamides • suitable for different temperatures CO N H2

117 118

IC – Ion chromatography
GPC application

molecule mass determination separation of ions and charged particles

biochemical analysis of macromolecules SP: Ion exchangers:
chemically bonded ion groups on the surface of
oligomeric compounds Chromatogram of styrene oligomers n =1-14
inorganic carrier (silica gel) or organic carrier (PS/DVB)
MP:
Excluded molecules aqueous solution of salt of different pH and ionic strength

elution volume  volume

Styrene monomer IC modes:
of MP in interparticle
space Vz
size of small molecule 1. common ion exchange:
equal to molecule MP Sample and MP ions competition for ionic
elution volume  hold-up sites on the SP surface
volume VM
SP pore volume Na+ + ~~ SO3H  ~~ SO3Na + H+
VM - Vz = Vp Vz VM
MP: acid (diluted HCl) tR = f(pH, I, cH+ )
exclusion volume hold-up volume
119 120
 IC modes (continue)
Ion chromatography interaction scheme

2. Acid-base reaction
separation of weak acids
~~ CH2N+(CH3)3OH- + CH3COOH  ~~ CH2N+(CH3)3CH3COO- + H2O

3. Ligand exchange
Competition of sample and MP components for metal on SP surface
retention
SP ion exchanger with bonded metal element
MP ligand created complex compound with bonded metal
Sample creates similar complex as MP component

Solutes retentions
ratio of complexes constants of bonded metal with MP and sample ligand
elution Separation of amino acids - bonded metals: Cu, Ni
- mobile phase ligand: NH3
- sample ligand: NH2 – groups of amino acids

121 122

Effect of pH mobile phase change on

basic, neutral and acidic compounds retention in reversed phase system The choice of suitable bonded phase

C18
Rules CN
Retention times are changed with the
chains length of bonded phase.

Retention order of main compounds

on „aliphatic“ SP (C18, C8, C4) is the Phenyl
C8 same.

The change of elution order or

resolution of zones can be better
realised using phenyl or CN phase
C4 Column:
than mobile phase change. ACE 250 x 4.6 mm i.d., 5 m

Higher resolution can be achieved on Mobile phase:

Resolution changes 20% 25 mM KH2PO4, pH6
R = 3,0  1,4 SP with low particle diameter. 80% MeOH
Flow rate: 1.0 mL/min
caused by 0,1 pH change 1. Norephedrine, 2. Norteiptiline, 3. Toluene, 4. Imipramine, 5. Amitriptyline Temperature: 22°C

123 124
INSTRUMENTATION in COLUMN LIQUID CHROMATOGRAPHY Instrumentation in column liquid chromatography (continue)

Mobile phases reservoir with degasser

Scheme of
liquid chromatograph

Detectors

Injector

Pump

Thermostated oven
with column holder

Control and data station

125 126

Instrumentation in column liquid chromatography (continue) Instrumentation in column liquid chromatography (continue)

1. Mobile phase pumps

common analytical flow range: 0,1 - 10 ml/min flow precision < 2% Piston pumps
types:
pulsed - piston
- membrane
non-pulsed - linear „syringe“

pump accessories
pulse dampener - capillary resistor
- two (or more) pump coupling
- programmed change of piston speed

gradient - two linear syringes

programmer - continual mixing of two or more component of MP

127 128
Instrumentation in column liquid chromatography (continue) Instrumentation in column liquid chromatography (continue)

Syringe pump
Membrane pump
Flow/pressure profile

Pulse dampener

129 130

Instrumentation in column liquid chromatography (continue) Instrumentation in column liquid chromatography (continue)

2. Sample injection
High pressure
gradient direct injection - septum, onto the MP flow
- stop flow
Low pressure - poor reproducibility
gradient
injection valve
automatic injection

131 132
Instrumentation in column liquid chromatography (continue) Instrumentation in column liquid chromatography (continue)

Construction details of selected column for analytical LC

3. Columns Type, proportions ( according to application)
metals
stainless steel diameter [mm] length [mm]
glass Preparative > 50 500 - 2000 Stainless steel
PEEK 10 - 50 250 - 1000
Semi preparative 8 - 10 150 - 500
„Classical“ (SEC, GPC, IC) 6 - 10 150 - 1000
Analytical 2 - 6(4,6) 50 - 300
with guard column
Micro columns 0,5 - 2 50 - 1000 PEEK

Capillary columns  0,3 100 - 1000

packed
open tubular
with SP chemically bonded on the capillary wall

133 134

Instrumentation in column liquid chromatography (continue) DETECTORS for HPLC

Photometric detector
4. Detectors 1. Optical - photometric
- universal - fluorescence
Types UV - VIS detector:
- selective - refractometric
2. Electrochemical - voltammetric - fixed wave length: 254 nm
- conductimetric
- filter detector : 254, 280, 313, 340, 365, 405, 436, 546 nm
3. Mass
- continuously changed wave length : 190-400 (600) nm
Requirements - fast, linear concentration response
- high sensitivity - fast record of optical spectrum: DAD - Diode Array Detector
- low noise photodiodes
- minimal effect of change pressure, MP flow and temperature simultaneous detection and quantification at different 
- minimal contribution to peak broadening simultaneously obtained chromatogram and optical spectrum
- gradient elution

135 136
 DETECTORS for HPLC (continue)

DETECTORS for HPLC (continue) Absorbance maximum wave length for compounds with selected groups

chromophor wave length [nm] absorption coefficient

Scheme of UV-VIS detector acetylide -C=C 175-180 6,000

aldehyde -CHO 210 1,500
Scheme of diode array detector - DAD
aminee -NH2 195 2,800
azo group -N=N- 285-400 3-25
bromidee -Br 208 300
carboxyle -COOH 200-210 50 - 70
disulphide -S-S- 194 5,500
ester -COOR 205 50
ether -O- 185 1,000
ketone >C=O 195 1,000
nitrate -ONO2 270 12
nitrile -C=N 160 -
nitrite -ONO 220 - 230 1000-2000
nitro group -NO2 210 high
137 138

DETECTORS for HPLC (continue) DETECTORS for HPLC (continue)

Scheme of fluorescence detector Voltammetric (amperometric) detector:

- oxidizable / reducible compounds
- current is recorded according to impressed voltage
between working polarizable and auxiliary electrode
- property: good MP conductivity
Phenoles, thioles, peroxides, aromatic amines, ketones
aldehydes, nitrocompounds, nitriles, esters

Conductivity detector
- ionic compounds
- dominant in IC
- two electrodes connected to alternating current

139 140
DETECTORS for HPLC (continue) Comparison of selected detectors for HPLC

Electrode lay-out in
electrochemical Detector limit of detection dynamic range gradient temperature
detectors g/ml application effect
photometric 10-9 104 yes low

fluorescence 10-10 103 yes low

refractometric 10-6 104 no high

voltammetric 10-10 104 no low

conductivity 10-8 104 no low

141 142

DETECTORS for HPLC (continue) DETECTORS for HPLC (continue)

Evaporative Light Scattering Detector (ELSD)

Transport detector
working principle

effluent from column is dispersed by

spray into small droplet
droplets are evaporated and leave an
analyte into the form of fine particles
suspended in a gas
suspended particles absorb radiation
of primary source
absorbed radiation is proportional to
component amount

143 144
DETECTORS for HPLC (continue) DETECTORS for HPLC (continue)

Low Angle Laser Light Scattering (LALLS) Detector Multiple Angle Laser Light Scattering (MALLS) Detector

working principle working principle

• Laser beam goes through set of optical •dispersed irradiation is collected
elements and is focused on inlet cell
under more angels than LALLS (till
window of detector
•Ring form mask situated between outlet
16, not in the direction of primary
cell window of detector and transferring beam)
lens selects beams dispersed on eluate
•significant decrease of effect of
particles under small angle and prevents
primary laser beams to enter
beam dispersed by effluent
photomultiplier impurities
• Dispersed beams
• recording of relation of mean
are focused to
photomultiplier
quadratic value of molecule diameter
and molecular
ass

145 146

DETECTORS for HPLC (continue) DETECTORS for HPLC (continue)

Multi–Electrode Array Detector Multi–Electrode Array Detector

working principle
Porous large surface, permeable for mobile phase
Record of coulometric response of electrochemical reaction on electrode

 Limits of detection – femtomoles

 3D resolution of co-eluted peaks
 gradient elution

147 148
 efficiency
separation velocity
Trends in preparation of packing of HPLC column Trends in preparation of packing of HPLC column - continue
chemical/pressure
stability
miniaturization „Perfusion“ packing
Separation of peptides and proteins
Lower particles - dp on surface porous particles
C 18 75 x 2.1 mm i.d.
 10 m  5 m  3 m  2 m  < 2 m diffusion pores
 decreased time of transport of analyte into and from pores through-hole pores
 efficiency increasing
 increasing of permeability of chromatographic bed  flow of MP through particles
 increasing of pressure  special pump construction and  mass transport speed increasing
injection requirements (max 450 bar  5000 bar)  narrower zones, higher efficiency
 decreasing of analysis time
 UPLC ultra-high pressure liquid chromatography
 UHPLC ultra-high performance liquid chromatography

5m

Non-porous particles Surface porous particles

column length 25 cm non-porous silica gel (NPS)
viscosity MF 1,0 cP – thin layer
3m non-porous resin (NPR) dp 5 m
dp 1,5 – 2,5 m • separation of bio molecules
m • high mass transport speed and separation • high mass transport speed
1000psi = 70 bar • low capacity of column • acceptable column capacity
149 150
• good recovery

Monolithic packing
Polymeric monolithic columns
Column packing created as „uninterrupted“ homogenous porous phase
•„uninterrupted“ cross-linked porous polymer
• polymethylacrylte, metylacrylat copolymer, PS-DVB
Types:
• agglomeration of polyacrylamide particles
• polyacrylamide block
• agglomeration of micro particulate silica gel bed
• PS-DVB block
• silica gel rod Separation of oligonucleotide on
• membrane of different types polymeric monolithic column
CIM DEAE
a) disk monolithic column
b) cylindrical monolithic columns in glass and ion exchanger
stainless steel shell poly(glycidylmethacrylate-ethylenglycol)
dimethacrylate
Silica gel rods:
disk: ø16 mm, thickness 3mm
• through-hole pores, diffusion pores, to be modified (C18,...)
• efficiency equal to columns with particles 3-5 m monolithic cylinders Fm = 6 ml/min
• pressure drop 30-40% in comparison with 5 m particle packing 
151 152
• column coupling for higher efficiency
Non-silica gel stationary phases
•PS-DVB cross-linked copolymers
lower efficiency than silica gel,
high resistance against pH
Silica gel packing with very low content of trace metals
Future trends in LC
•decreasing of interaction of metal with separated compounds
•effect on acidity of residual silanols

Hydroxyapatite • Lower particles of silica gel packing (max 3 mm) in short columns
interaction with sensitive bio molecules (7,5-15 cm) (LC/MS).

•Zirkonia (ZrO2) • Columns with low dimensions with inner diameter lower than 100 m
high stability against high pressure and temperature, with low particles (proteomics, LC/MS).
resistant against high pH (to pH 14)
surface without silanols, contain centres Lewis acidic centres
strong affinity to Lewis base (hydroxyl, phosphate, fluoride,..)
• Monoliths in small diameter columns.

•Graphitised carbon • Monoliths in 100 mm column diameter created in situ

Hybrid inorganic-organic stationary phase hydroxyapatite particle • TiO2 particles

• polymerization of tetrachloro- or tetraethoxysilane • Chip column „lab-on-a-chip“,

creation of silica gel polymer with silanols on the surface column „packing“ created directly on inner capillary column wall
electroosmotically generated liquid flow - see CEC capillary electrochromatography
•triethoxysilane connected with ethylene
(RO)3SiCH2CH2Si(OR)3
153 154
high pH stability

Column format trends in HPLC

Efficiency of capillary columns
Scheme of LC column on chip and part HETP corresponds to particle
of packed capillary size
150 x 0,5 mm 3,7 m

Analysis of BSA digest on „ChipLC column“

Column dimensions:
40 x 0,075 x 0,050 mm 5m particles

Separation on capillary column

packed with micron particles
370 x 0,030 mm i.d. 1m

detection
inner part of injection valve

155 156
Other separation examples
Separation of peptides on nanoflow
HPLC column
C 18 150 x 0,075 x 0,050 3,5 m
injection 100 fmol 1 l
Selected examples of chromatographic separations
Selected examples of chromatographic separations
Separation of mixture of BSA digest,
myoglobine a alfa-lactalbumine
on monolithic capillary column
PS/DVB monolith 50 x 0,2 mm
Fm = 2,5 l/min
detector cell volume 3nL
157 Optima 17 - Phenylmethylpolysiloxane 158
Optima 5 – 5% diphenyl-95% dimethylpolysiloxane
Selected examples of chromatographic separations
159 160
 Selected examples of chromatographic separations Selected examples of chromatographic separations

161 162
Permabond SE-54-HKW –polysiloxane type

Selected examples of chromatographic separations Selected examples of chromatographic separations

Optima 240 – 33%cyanopropyl-methyl-67%dimethylpolysiloxane 163 Optima 1 - dimethylpolysiloxane 164

 Selected examples of chromatographic separations Selected examples of chromatographic separations

Optima delta-6: síťovaný mrthyl-phenyl-polysiloxan 165 166

Selected examples of chromatographic separations Selected examples of chromatographic separations

167 168
Optima 5 – 5%diphenyl-95% dimethylpolysiloxane
Selected examples of chromatographic separations Selected examples of chromatographic separations

169 170

Selected examples of chromatographic separations Selected examples of chromatographic separations

171 172
Selected examples of chromatographic separations Selected examples of chromatographic separations

173 174

Selected examples of chromatographic separations Selected examples of chromatographic separations

Polyethylene imine on silica

strongly basic polymer anion
exchanger

175 176