09 11 2006 Chromatography

WenHsin Huang
Principles of Chromatography
Principles of
Chromatography •Introduction to Analytical Separations
Introduction to –Solvent Extraction
Analytical Separations –What is Chromatography?
–A Plumber’ s View of Chromatography
–Efficiency of Separation
Wen-Hsin Huang
–Why Bands Spread
Ph. D.
NDMC
2006/9/7 WenHsin Huang, NDMC 1 2006/9/7 WenHsin Huang, NDMC 2
Simple Separations Solvent Extraction

Solute particles Solute particles Solute particles
•Extraction only have one now have two choose preferred
–transfer of solute from one phase to another choice of solvent choices of solvent solvent
–phase can be gas, solid, liquid
•Liquid/liquid extraction
–2 immiscible solvents used
add second
–typically aqueous solvent and organic solvent immiscible
solvent
•you know water and oil don’
t mix
–organic solvents less dense than water
Legend: shake
•diethyl ether, toluene, hexane aqueous solvent
–organic solvents more dense than water organic solvent
•chloroform, carbon tetrachloride, dichloromethane solute particles
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WenHsin Huang
Solvent Extraction Phase Partitioning

If we have a solute (S) that is partitioned between two
phases (1 & 2) then we can write an equilibrium expression
•Like dissolves like for this equilibrium.
–solute chooses solvent most like itself
–polar compounds (and ionic compounds) S1  S 2
choose water (which is very polar)
–nonpolar compounds choose nonpolar organic AS [S ]
solvents K 2  2
AS1 [ S1 ]
Partition Coefficient (K)
•
Convention: Organic phase = phase 2
Distribution Coefficient Example of Extraction with n-Hexane

The distribution coefficient for compound Z between
The distribution coefficient (D) is used in place of the n-hexane and water is 6.25. Calculate the percent Z
partition coefficient when dealing with a species that has remaining in 25.0 mL of water that was originally
more than one chemical form. 0.0600 M in Z after the extraction with the following
volumes of n-hexane.
A) One 25.0-mL portion 13.8%
total _ concentration _ in _ phase _ 2
D B) Two 12.5-mL portions 1.90%
total _ concentration _ in _ phase _ 1
C) Five 5.00-mL portions 4.99 x 10-3%
D) Ten 2.50-mL portions 2.49 x 10-7%
It is more efficient to do several small extractions

rather than one large extraction.
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WenHsin Huang
Solvent Extraction (pH effects) Solvent Extraction (pH effects)

•Charges of acidic and basic species change •Distribution of HA between two solvents
with pH –two equilibria to consider
–neutral species generally more soluble in •Ka equilibrium
organic solvent
•D equilibrium
–charged species generally more soluble in
aqueous solution
organic HA H+ + A-
•Distribution coefficient (D) describes
distribution of species between two phases
–Takes into account all forms of a compound Ka
(i.e. H2A, HA-, A2-) aqueous HA H+ + A-
–Different from partition coefficient (K)

•Distribution of HA between two phases
•Distribution of acids and bases between two
phases
[H][ A ] K [HA ]
total conc. in phase 2 (organic) Ka   [ A ]  a 
[HA ] [H ]
D=
total conc. in phase 1 (aqueous) •Also have K expression for [HA]
[HA]2 [B]2 [HA ]2

D= D= K
[HA]1 + [A-]1 [B]1 + [BH+]1 [HA ]1
•Substitute and rearrange to get
K[H]
•Ionic species only found in aqueous layer D 
[H ] K a Eq’
n
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WenHsin Huang

D for an acid (HA) D for a base (B) •Adjust pH to effectively extract an acid or
base (and its conjugates) into aqueous layer
K[H] KK a •Consider base B with a pKa of 9.0
D  D Eq’
ns
[H ] K a K a [H] –pH < 9.0, B hydrolyzes so majority of B (in the
form BH+) in water layer (low D value)
•Distribution of acids and bases between two 0
phases is pH-dependent [ B]
K  org -2
log D
[ B ]aq pKa
•To find D, need to know
[ H ][ B]aq -4
–pH of solution Ka  mainly mainly
[ BH ]aq -6 BH+ B
–Ka of acid or conjugate acid [ B ]org K Ka
D  KB
–K of acid or base [ B ]aq [ BH ]aq K a [ H ] 2 4 6 8 10 12
2006/9/7 WenHsin Huang, NDMC 13 2006/9/7 WenHsin Huang, NDMC
pH Fig.14
History of Chromatography Tswett’

s Experiment
Mikhail Tswett is credited with the
invention of chromatography. Ground up plant leaves and added
petroleum ether.
Developed a technique that separated
various plant pigments (e.g.,
chlorophylls and xanthophylls) by
passing solutions through glass Filled column with chalk.
columns filled with finely ground
M. S. Tswett
CaCO3.
Russian Scientist
(1872-1919)
The separated species appeared as Method forgotten for many years.
colored bands on the column.
Hence Greek chroma = color and graphein = writing

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WenHsin Huang
What is Chromatography?
Tswett’s
•Method to separate components in a
Experiment mixture based on different distribution
coefficients between the two phases
•Same principle as solvent extraction (like
dissolves like), but one phase is “
stationary”
and one phase is “ mobile”
–no longer working with organic and aqueous
layers in a separatory funnel
–working in a column (which is just a tube)
What is Chromatography?
Types of Chromatography
•Two phases: mobile and stationary
•Mobile phase is solvent moving through •Adsorption Chromatography
column
•Partition Chromatography
–liquid (Methanol, water, buffer)
•Ion-exchange Chromatography
–gas (He, H2, N2)
•Molecular Exclusion Chromatography
•Stationary phase fixed inside column
–viscous liquid coated on inside of column •Affinity Chromatography
–solid particles packed inside column
•Solutes have different affinities for mobile
phase and stationary phase
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WenHsin Huang
Adsorption Chromatography Partition Chromatography

Stationary phase Mobile phase Stationary phase Mobile phase
Solid Liquid or Gas Liquid on solid support Liquid or Gas
How it separates How it separates

–solute adsorbs on to –solute dissolves into
stationary phase surface liquid coating
Molecular Exclusion
Ion-Exchange Chromatography
Chromatography
Stationary phase Mobile phase
Anions or cations covalently Liquid Stationary phase Mobile phase
bound to solid stationary phase Porous gel Liquid
How it separates
–small molecules
How it separates
trapped in pores of
solute ions of opposite charge stationary phase
attracted to stationary phase Also called
gel filtration,
gel permeation,
or
size-exclusion
chromatography
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WenHsin Huang
Classification Specific Method Stationary Phase Type of equilibrium

Liquid Liquid-liquid or Liquid adsorbed onto Partition between immiscible
Affinity Chromatography chromatography
(LC) (Mobile
partition solid liquids
phase a liquid) Liquid-bonded Organic species bonded Partition between liquid and bonded
phase to solid surface surface
Stationary phase Mobile phase
Liquid-solid, or Solid Adsorption
Immobilized molecules on Liquid adsorption
liquid or solid stationary phase
Ion exchange Ion-exchange resin Ion exchange
Size exclusion or Interstices of polymeric Sieving

How it separates molecular- solid
exclusion
–Molecules with specific
Affinity Covalently bonded SelectiveAf f
ini
ty–( egs pe ci
fi c
shape dock with ligands Chromatography molecule (eg antibody) Protein/antibody bind together)
Gas Gas-liquid Liquid adsorbed onto Partition between gas and liquid
chromatography solid surface
(GC) (Mobile
phase a gas) Gas-bonded Organic species bonded Partition between gas and bonded
phase to solid surface liquid
Gas-solid Solid Adsorption

Basic Principles Simple Column

Two phases considered:
Fresh eluent
1) Mobile Phase: solvent moving through the column. Initial band

of A and B
2) Stationary Phase: stays in place inside of the column.

Column
Packing
“
Eluent” COLUMN “Eluate”
Porous Disk
Process is called “
elution”
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WenHsin Huang
Chromatogram Chromatographic Terms

A chromatogram is a
graph showing the tm = “dead time”which is
detector response the minimum time for
(proportional to mobile phase to pass
through the column
concentration) as
solutes emerge from a
tr = “retention time”
chromato-graphic which is the time required
column as a function of for the solute (analyte) to
time or volume. pass through the column.
Detector
t’r= “adjusted retention
time”describes time
•
All solutes spend equal time in mobile phase solutes spend in stationary
phase
t rt r t m
Chromatographic Terms A Plumber’

s View…
•kcapacity factor describes how long
= “relative retention” solute retained on column
which is the ratio of
t t
adjusted retention times.
time spent in stationary phase
t k'  r m =
 r 2 tm time spent in mobile phase
t r
1
>1 always
then separation
•Higher kindicates solute retained longer
between two components
•If k= 0, solute unretained
k’= “capacity factor” •If k= 1, solute spent same amount of time
time _ in _ stationary t r in stationary phase as in mobile phase
k
 
time _ in _ mobile tm
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WenHsin Huang
A Plumber’
s View… Partition Coefficient Relationship
•kdescribes time spent in two phases
time _ in _ stationary moles _ in _ stationary tr
•kalso describes number of moles of solute k
  
time _ in _ mobile moles _ in _ mobile tm
partitioned between two phases
CV [S ] V V t
k'  s s Eq’
n k
 s s K s  r
Cm Vm [ S ]m Vm Vm t m
•K Partition coefficient compares t k  K
concentration of solute in one phase to  r 2  2  2
t r
1 k1 K1
concentration of solute in the other
[ S] C V
K  2  s  k' K s
What this says is that analytes with different partition
Eq’
n coefficients will have different retention times.
[S]1 Cm Vm
A Plumber’
s View… Review
Partition coefficient describes C
•q fraction of solute in mobile phase K s
conc. of solute in SP and MP Cm
molesm Cm Vm
q 
moless molesm Cm Vm Cs Vs Capacity factor describes t t V
1 1 time solute spends (or moles of k'  r m K s
q  solute) in SP and MP
tm Vm
CV V
1 s s 1 K s
Cm Vm Vm
Relative retention compares
1 k' retention times (or partition t ' k' K
q (1 q)   r 2  2  2
1 k' 1 k ' coefficients or capacity factors) t r1' k '1 K 1
fraction in MP fraction in SP of solute 1 and 2
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WenHsin Huang
Chromatographic Peaks Resolution of peaks

•Solutes elute from a column in a Gaussian peak
shape Resolution is a measure t ( t t )
of how well two peaks are resolution  r  r1 r 2
–w width of peak at base separated. w av ( w 1 w 2 ) / 2
–w1/2 width of peak at half-height
R=0.50 R=0.75
As tr grows,
average
resolution
retention time
between two
 
h peaks 
w1/2=2.35
improves.
1/2h R=1.00 R=1.50
w=4
time
t0 tr
Peak width (w) is defined as the baseline width (4).
Plate Height (H)

Resolution Example
Bands broaden as they travel through the column.
Two solutes have retention times and widths of tr1 =

235 s, w1 = 8 sec; tr2 = 250 s, w2 = 10 s.
Resolution = 1.7
Plate height (H) relates the amount of broadening to

the linear distance traveled.
2
H
H = plate height
x = distance traveled
2 = variance of peak x
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WenHsin Huang
Evaluating Separation Efficiency Evaluating Separation Efficiency

•“N”in chromatography is analogous to “
n”
•Plate theory in liquid-liquid extractions
–Breaks separation up into many discrete stages
–each extraction represents a theoretical plate
–Stages represent individual equilibria
[S]2
•N (Theoretical plate) represents each
equilibrium between MP and SP
[S]1
•Each time a solute molecule
•Higher N corresponds to better separation
enters the SP from the mobile –indicates solute molecules enter SP a higher
phase is a theoretical plate number of times
Evaluating Separation Efficiency Chromatographic Peaks

•N is dependent on •Distribution of retention times for a solute
–column •Peak shape represents differences in
–analyte behavior of solute molecules during
–retention time of analyte separation
–width (at base or at half-height) of peak average
2 2 retention time
t 16t  
N  r 2  2r N is dimensionless so h
 w w1/2=2.35
measure tr and w (or w1/2)
2 1/2h
5.55t in same units (i.e. time)
N 2 r w=4
w 1/ 2 time
t0 tr
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WenHsin Huang
Evaluating Separation Efficiency Evaluating Separation Efficiency

•Do not want solute band to spread out as it
travels through column •H related to N and length of column (L)
•H (Plate height) represents the relationship L Report H in units
between the width of a solute band to the H of length (i.e. cm)
N
distance traveled through column •Resolution related to N
•Lower H corresponds to better separation
(higher efficiency)  k '2 
N 1
R  

 
•H also called HETP 4  1 k'av 
 Eq’
n
–Height Equivalent to a Theoretical Plate –R is dimensionless
–One solute equilibrium between SP and MP is –k
2 = capacity factor for solute retained longer
one theoretical plate (N) on column (higher tr)
–Equilibrium occurs in one HETP
Evaluating Separation Efficiency Number of Theoretical Plates (N)

N 1  k '2 
R  
 

The number of theoretical plates (N) increases as
4   1 k ' av 
Eq’
n the efficiency of the separation increases.
•Increase L to increase R
16t 2 t 2
L
H N
L N  2r  r 2 Derived
N H w 
•Increase to increase R or
t ' k' K
 r 2  2  2 5.55t 2 t 2
t r1' k'1 K 1 N  2 r  r2
–to change , must change nature of MP and SP w1 
2
to change relative affinity analytes have for SP
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WenHsin Huang
Example Factors Affecting Resolution

The greater the resolution, the better the separation.
The more theoretical plates, the better the separation.
A solute with a retention time of 302 s has a width of
11 s on a column that is 15 m long. Find both N and N   k 2 
1 
H for this separation. R  

 

4    
 ave 
1 k
R N R  L
N = 1.2 x 10-4
H = 1.2 mm
 1 R0
k 2 0 R0
Principles of Chromatography Why Do Bands Broaden?

Solute invariably spreads apart as it travels
•Introduction to Analytical Separations (diffusions) through a chromatographic
–Solvent Extraction column.
–What is Chromatography?
–A Plumber’ s View of Chromatography The observed variance is a sum of variances
from all the broadening mechanisms.
–Efficiency of Separation
Why Do Bands Spread
obs
2
i2
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WenHsin Huang
Where Does Broadening Occur? Where Does Broadening Occur?

Inside the column:
Outside the column: Multiple paths
Width of injection plug Longitudinal diffusion
Mixing in detector dead volume Equilibration time
Pump Injector Pump Injector

I I
Column Detector Column Detector
Multiple Paths (A) Multiple Paths (A)

•Solute molecules can choose many different Band of 3 solute
paths through packed columns molecules traveling Band spreads
through packed column
open tubular packed
column column
layer of SP SP particles
coated on packed inside
inside of column column
time
Occurs only in packed columns.
Compare to solute Compare to solute Smaller particles reduce the plate height.
traveling through traveling through HA
a straw a bean bag • Open tubular columns do not provide multiple paths for solute
during tm (no A term)
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WenHsin Huang
Longitudinal Diffusion (B /ux) Equilibration Time (Cux)

•S molecules travel through column in a band •Finite time required to allow solute molecules to
•[S] varies throughout band equilibrate between MP and SP
–some solute molecules get ahead of band •If flow rate too high, MP will “ leave behind”
–some solute molecules lag behind band molecules in SP (i.e., Solute in stationary remains
“stuck”while solute in mobile phase moves forward.)
Caused by diffusion of solute in the mobile phase. •Keep SP thin to decrease equilibration time
Faster flow rate means less time, thus less diffusion.
MP slow MP
Direction
start H B/ux so, of travel
SP eq. SP
increase ux (flow rate)
as band bandwidth bandwidth
to reduce problem
travels
2006/9/7 WenHsin Huang, NDMC 57 2006/9/7
H Cux so decrease ux to reduce problem
WenHsin Huang, NDMC 58
Van Deemter Equation Band Spreading

Putting the three factors together yields the Van
Deemter equation that helps predict how the •Three terms in van Deemter equation
column flow rate will affect the theoretical plate correspond to three sources of band
height. B
H A  Cu x spreading or broadening
ux
•A term Multiple Paths term
Multiple Equilibration (also called Eddy Diffusion)
Paths Longitudinal Time
Diffusion •B term Longitudinal Diffusion term
•
Break equation up into A, B, and C terms
•C term Equilibration Time term
–A H independent of flow rate
–B/ux H inversely proportional to flow rate
–Cux H proportional to flow rate
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WenHsin Huang
van Deemter plot Column Type Affects H

open tubular packed
•Plot dependence of H on individual terms
column column
•Sum and find optimum ux to use to minimize band
spreading layer of SP SP particles
coated on packed inside
inside of column column
H
B Cux •Advantages •Advantages
ux –useful for large-scale
–high resolution (low H)
Hmin analyses
–high sensitivity
A –low analysis time •Disadvantages
Flow •Disadvantages –A term in van Deemter
uopt rate equation increases H
–not useful for large-scale
analyses WenHsin Huang, NDMC 62
Asymmetric Band Shapes Asymmetric Band Shapes

•Overloaded column (high [S])
–so much S enters SP that the SP starts to look
•Expect Gaussian peak shape from solute S more like S than it looks like original SP
eluting from column, independent of [S]total –most S molecules retained longer on column
•Separation based on K Observed peak
C
K s Band shape in chromatogram
Cm
•In reality, K changes as [S]total changes
•If [S] is too high or too low, peak shape
Majority of S retained Faster-moving (less
deviates from Gaussian shape
on column longer retained) S reaches
detector first
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WenHsin Huang
Asymmetric Band Shapes Asymmetric Band Shapes

•Underloading (low [S])
•Load less sample to reduce overloading
–“hot sites”on SP more available when less S
problem
molecules trying to enter SP
–some (minority) S get “stuck”on hot sites •Protect groups on SP to reduce number of
–some S retained longer on column
hot sites to reduce tailing problem
Observed peak Constant K (slope) results
Band shape in chromatogram overloaded in ideal peak shape
Cs
tailed
Slower-moving (more
Minority of S retained Cm
retained) S reaches Fig. 23-19
on column longer
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Chromatographer’s Triangle Quantitative Analysis

Relationship •In general, detectors can tell us
–“
Yes, something is eluting from the column.”
Resolution •Use calibration methods to determine how much
of a compound is eluting from column
Finding Peak Area
Peak Area
•A = ½ wbaseh
•A w½ h
-b
Speed Capacity
x= m •cut-and-weigh
concentration (M) •computer integration
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WenHsin Huang
Quantitative Analysis Example

•Generally use an internal standard
–internal standard is a known amount of a When 1.06 mmol of 1-pentanol and 1.53 mmol
compound different from analyte of 1-hexanol were separated by GC, they gave
–compare analytical signal from analyte to relative peak areas of 922 and 1570 units,
analytical signal from internal standard respectively. When 0.57 mmol of pentanol was
–not the same as method of standard additions added to an unknown containing hexanol, the
–internal standards discussed in Ch. 4 relative chromatographic peak areas were
Ax area of analyte peak 843:816 (pentanol:hexanol). How much
Ax A As area of standard peak
F s [X] concentration of analyte
hexanol did the unknown contain?
[ X] [ S]
[S] concentration of standard
2006/9/7
F response factor
WenHsin Huang, NDMC 69 2006/9/7 WenHsin Huang, NDMC 70
Liquid Chromatography LC Columns

GUARD
•Analyte
–Soluble in MP (more likely than being volatile) •Constructed of steel or plastic
•Mobile phase •5 –30 cm length
A
–Liquid •1 –5 mm i.d. N
•Methanol, Water, Acetonitrile, Hexane
•Easily contaminated and degraded A
–Plays more active role in separation
•Guard column L
•Stationary phase Y
–Contains same SP
–Liquid (partition) or solid (adsorption) T
–Dust, other particles, strongly adsorbed
•High Performance Liquid Chromatography(HPLC) solutes retained on guard column
I
C
–High pressure used to force solvent through column (as –Expendable A
opposed to gravity) –Extends life of analytical column L
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WenHsin Huang
LC Columns LC Columns
•Almost exclusively use packed columns •Packing particle diameter (dp) crucial
–Typical particle diameters are 3 –10 m
•Solutes move much slower through liquids
–Decrease particle diameter to decrease H
than through gases
•Provide more uniform flow (low A)
–Time needed to diffuse to SP in open tubular •Less time needed for solute to get to particle (low C)
column is too long 60 10m
40
H (m)
Fig. 21-14 20 5m
S S
10
3m
0 2 4 6 8
Flow WenHsin
rate (mL/min)
Huang, NDMC 74
LC –Adsorption Chromatography
Microporous Silica Particles LC –Adsorption Chromatography
•Solid SP –Silica gel Aggregate of Particles Sponge-like Structure
•Solvent molecules compete with solute molecules

–Pure, spherical, microporous
particles for sites on SP
•Permeable to solvent MP
•Very high surface area Flow SP
•Use when pH < 8 OH OH Solvent
OH
Solute
O Si O Si
Silica gel with silanol O Si O
groups on surface Si O
HO O O
Si Si Fig. 21-10
Si
Deprotonated silanols O Si O •Solute is displaced by solvent molecule
(Si –O-) are “hot sites” OH
OH OH
that can lead to tailing OH
NDMC 19
WenHsin Huang
LC –Adsorption Chromatography Isocratic vs. Gradient Elution

•Forcible desolvation of solute by solvent is nearly •Isocratic elution
independent of solute identity –One solvent or one constant solvent mixture used as
–Dependent on solvent identity MP throughout entire separation
•Gradient elution
•Eluent strength (
o)
–Adjust solvent mixture through separation
–Ability of solvent to displace solute
–Adjusting eluent strength
•Elutropic series –May speed up separation process
–Relative eluent strengths of common solvents
–Must know about relative polarity of SP/MP •Table 21-2 includes 
o values of solvents for
adsorption chromatography on silica

•Eluent strength increases as MP becomes more
like SP –polarity, o
Isocratic vs. Gradient Elution Isocratic vs. Gradient Elution

Fig. 21-18
Hexane 
°=0.01 &
Fig. 21-19 •Previous example illustrated balance of good
isocratic resolution and reasonable retention times
elutions
Acetonitrile 
°=0.52
•Gradient elution may help
–Improve resolution
–Shorten retention times (and total analysis time)
Start w/ 100% Benzene –Improve peak shapes
then add Acetonitrile gradient
elution
NDMC 20
WenHsin Huang
LC –Partition Chromatography
LC Phases
•Liquid SP coated on solid support
–Often, liquid SP covalently attached to surface •Normal-phase Chromatography
of silica gel particle –SP polar
CH3 –MP weakly polar or non polar
Si –More polar solvent has higher 

o
O Si R
•More attracted to SP and able to displace solute
CH3 •Reversed-phase Chromatography
support liquid SP –SP nonpolar or weakly polar
–MP polar
Common polar R groups Common nonpolar R groups
(CH2)3NH2 (CH2)17CH3 (or C18) –Less polar solvent has higher 
o
•More attracted to SP and able to displace solute

(CH2)3CN (CH2)7CH3 (or C8)
(CH2)2OCH2CH(OH)CH2OH (CH2)3C6H5
LC Phases HPLC Instrumental Design

•Normal-phase developed first •Already covered columns
•Focus now on
•Reversed-phase is more common
–Injector
–Many choices of polar solvents in which solutes –Detectors
are commonly soluble
•Water, methanol Pump Injector
–Nonpolar SP less likely to have hot sites
•Less peak tailing I
Column Detector
NDMC 21
WenHsin Huang
HPLC 6-way Injection Valve Detectors

Fig. 21-17 •Questions to ask when evaluating an LC
detector:
waste waste
–Is the detection universal?
Sample loop Sample loop
–Is the detector response linear?
–What is the limit of detection (LOD)?
column
–Is the detector useful with gradient elution?
•In other words, is the detector insensitive to solvent
composition?
UV Detector (w/ flow cell) UV Detector

•Common HPLC detector
–Many solutes absorb UV light
•Will cover during spectrophotometry
Eluate out
–Any UV spectrophotometers discussed previously are
Flow cell
good LC detectors
•UV cutoff wavelengths
Light
source Detector –Below given , MP absorbs UV radiation
–MP absorbance overwhelms analyte absorbance
Eluate in
Fig. 21-20
pathlength
NDMC 22
WenHsin Huang
UV Detector Fluorescence Detector

•Applicable for UV chromophores •Similar idea to UV detector
Essentially linear •Few molecules fluoresce
–Will discuss deviations from linearity in CH 19 •Derivatize solutes with fluorescent tag
LOD ~ 0.1 ng –Derivatize mixture of solutes before separation
Good detection method with gradient –Derivatize solutes as they elute from column
elution before detection
•Post-column Derivatization
–Use solvents that do not absorb UV radiation
Refractive Index Detector

Fluorescence Detector
•Compare refractive indices of eluate & reference
Applicable to fluorophores
Essentially linear
–Will discuss deviations from linearity
(Reference) (MP solvent & analyte = eluate)
LOD ~ 0.001 ng
Fine with gradient elution •Cannot use with gradient elution
–Use solvents that do not fluoresce –reference composition cannot exactly match solvent
composition during experiment
NDMC 23
WenHsin Huang
Refractive Index Detector Comparison of LC Detectors

Type Approx LOD Approx Comments
Linear range
10 -10 g 10 4
Essentially universal UV-VIS For light absorbing
compounds
LOD ~ 100 ng Fluorescence 10-14 g 10 5 For Fluorescent
Essentially non-linear compounds
–Linear range very small MS 10-7-10-9 g 10 5 Universal detector
Disaster with gradient elution Electrochemical -8

10 g/mL 10 5
Specific detector; for
conductometric all ions
Finicky
Electrochemical 10- 10-10 –11 g 10 5 Specfic detector;
–Need constant T amperometric electroactve
compounds
Liquid Chromatography (LC)

Especially high-performance liquid chromatography (HPLC).
Thet erm“ high-pe rfor

ma nc e”refe r
stot
he
use of packed columns with very small
packing particles (diam. 5-10 m) -
giving greatly enhance resolution.
Note: several types of LC. In

addition to partition (as described
so far) - there are also ion-
exchange and size-exclusion
chromatography using liquid
mobile phases. We will concentrate
only on partition.
2006/9/7 WenHsin Huang, NDMC 95
NDMC 24

09 11 2006 Chromatography

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09 11 2006 Chromatography

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WenHsin Huang

2006/9/7 WenHsin Huang, NDMC 1 2006/9/7 WenHsin Huang, NDMC 2

Simple Separations Solvent Extraction

Solvent Extraction Phase Partitioning

Distribution Coefficient Example of Extraction with n-Hexane

It is more efficient to do several small extractions

Solvent Extraction (pH effects) Solvent Extraction (pH effects)

Solvent Extraction (pH effects) Solvent Extraction (pH effects)

[HA]2 [B]2 [HA ]2

2006/9/7 WenHsin Huang, NDMC 11 2006/9/7 WenHsin Huang, NDMC 12

Solvent Extraction (pH effects) Solvent Extraction (pH effects)

History of Chromatography Tswett’

Hence Greek chroma = color and graphein = writing

Adsorption Chromatography Partition Chromatography

How it separates How it separates

2006/9/7 WenHsin Huang, NDMC 21 2006/9/7 WenHsin Huang, NDMC 22

Classification Specific Method Stationary Phase Type of equilibrium

Size exclusion or Interstices of polymeric Sieving

Gas-solid Solid Adsorption

Basic Principles Simple Column

1) Mobile Phase: solvent moving through the column. Initial band

2) Stationary Phase: stays in place inside of the column.

2006/9/7 WenHsin Huang, NDMC 27 2006/9/7 WenHsin Huang, NDMC 28

Chromatogram Chromatographic Terms

Chromatographic Terms A Plumber’

Chromatographic Peaks Resolution of peaks

Plate Height (H)

Two solutes have retention times and widths of tr1 =

Plate height (H) relates the amount of broadening to

Evaluating Separation Efficiency Evaluating Separation Efficiency

Evaluating Separation Efficiency Chromatographic Peaks

Evaluating Separation Efficiency Evaluating Separation Efficiency

Evaluating Separation Efficiency Number of Theoretical Plates (N)

Example Factors Affecting Resolution

2006/9/7 WenHsin Huang, NDMC 49 2006/9/7 WenHsin Huang, NDMC 50

Principles of Chromatography Why Do Bands Broaden?

2006/9/7 WenHsin Huang, NDMC 51 2006/9/7 WenHsin Huang, NDMC 52

Where Does Broadening Occur? Where Does Broadening Occur?

Pump Injector Pump Injector

2006/9/7 WenHsin Huang, NDMC 53 2006/9/7 WenHsin Huang, NDMC 54

Multiple Paths (A) Multiple Paths (A)

Longitudinal Diffusion (B /ux) Equilibration Time (Cux)

Van Deemter Equation Band Spreading

van Deemter plot Column Type Affects H

Asymmetric Band Shapes Asymmetric Band Shapes

Asymmetric Band Shapes Asymmetric Band Shapes

Chromatographer’s Triangle Quantitative Analysis

Finding Peak Area

Quantitative Analysis Example

Liquid Chromatography LC Columns

2006/9/7 WenHsin Huang, NDMC 71 2006/9/7 WenHsin Huang, NDMC 72

•Solvent molecules compete with solute molecules

LC –Adsorption Chromatography Isocratic vs. Gradient Elution

adsorption chromatography on silica

2006/9/7 WenHsin Huang, NDMC 77 2006/9/7 WenHsin Huang, NDMC 78

Isocratic vs. Gradient Elution Isocratic vs. Gradient Elution

2006/9/7 WenHsin Huang, NDMC 79 2006/9/7 WenHsin Huang, NDMC 80

Si –More polar solvent has higher 

•More attracted to SP and able to displace solute

LC Phases HPLC Instrumental Design

2006/9/7 WenHsin Huang, NDMC 83 2006/9/7 WenHsin Huang, NDMC 84

HPLC 6-way Injection Valve Detectors

2006/9/7 WenHsin Huang, NDMC 85 2006/9/7 WenHsin Huang, NDMC 86

UV Detector (w/ flow cell) UV Detector