Chapter 23
Analytical Separations
1
What is Chromatography
• We have looked briefly at distillation and more
fully at extraction. How does this apply to
chromatography?
• Both separations were based on multiple
equilibria.
• For Distillation this was a evaporation /
condensation step. (in a vertical column)
• For extraction this was a solvent extraction step
(in a piece of glassware).
2
Chromatography
• Each step has enhanced purity of one of the
compounds.
• To improve this equilibrium step in distillation we
force interaction between the vapor and liquid.
This is done a variety of ways but one common
way is to place plates in the column to collect the
liquid.
• This has become a key term in separations.
• It now means a separation step.
3
Chromatography
• Gas Chromatography based on volatility.
• Liquid Chromatography based on partitioning.
4
History
•
•
•
•
•
•
•
Pliny the Elder
Purification of water in antiquity.
Tswett Plant Physiologist - Russian 1906
Martin & Synge Nobel Prize
Craig
Van Deemter
Giddings
5
History
•
1848 Way and Thompson
: Recognized the phenomenon of ion exchange in solids.
•
1850-1900 Runge, Schoenbein, and Goeppelsroeder
: Studied capillary analysis on paper.
•
1876 Lemberg
: Illustrated the reversibility and stoichiometry of ion exchange in aluminum
silicate minerals.
•
1892 Reed
: First recorded column separation: tubes of kaolin used for separation of
FeCI3 from CuSO4.
•
1903-1906 Tswett
: Invented chromatography with use of pure solvent to develop the
chromatogram; devised nomenclature; used mild adsorbents to resolve
chloroplast pigments.
6
History
• 1930-1932 Karrer, Kuhn, and Strain
: Used activated lime, alumina and magnesia
absorbents.
• 1935 Holmes and Adams
: Synthesized synthetic organic ion exchange resins.
• 1938 Reichstein
: Introduced the liquid or flowing chromatogram, thus
extending application of chromatography to colorless
substances.
• 1938 Izmailov and Schraiber
: Discussed the use of a thin layer of unbound alumina
spread on a glass plate.
• 1939 Brown
: First use of circular paper chromatography.
7
History
• 1940-1943 Tiselius
: Devised frontal analysis and method of displacement
development.
• 1941 Martin and Synge
: Introduced column partition chromatography.
• 1944 Consden, Gordon, and Martin
: First described paper partition chromatography.
• 1947-1950 Boyd, Tompkins, et al
: Ion-exchange chromatography applied to various
analytical problems.
• 1948 M. Lederer and Linstead
: Applied paper chromatography to inorganic
compounds.
8
History
• 1951 Kirchner
: Introduced thin-layer chromatography as it is practiced today.
• 1952 James and Martin
: Developed gas chromatography.
• 1956 Sober and Peterson
: Prepared first ion-exchange celluloses.
• 1956 Lathe and Ruthvan
: Used natural and modified starch molecular sieves for molecular
weight estimation.
• 1959 Porath and Flodin
: Introduced cross-linked dextran for molecular sieving.
• 1964 J. C. Moore
: Gel permeation chromatography developed as a practical method.
9
Resources
• Journals
–
–
–
–
Journal of Chromatography
Journal of Chromatographic Science
Analytical Chemistry
Trade Journals
•
•
•
•
LC-GC
American Laboratory
Today’s Chemist at Work
Other Free-bees
10
What happens
11
Terms
• Stationary Phase - The part of the system that
does not move.
• Mobile phase – The part of system that moves
• Elution – Eluent (in), eluate (out)
• Packed column
• Open tube column.
12
Mechanisms
13
14
15
The Chromatogram
16
Terms of Chromatography
• Chromatogram - The instrumental output. A
signal as a function of time (or volume)
• Retention Time - How long a compound stays
in the column. (tr) or could be expressed in terms of
volume (Vr)
• Dead volume Vm
or could be expressed as a time (tm)
– Volume to get through the system even without any
interaction. A constant for a given column.
• Adjusted retention time tr’
–
tr ’ = t r - tm
17
Terms

a
alpha Relative Retention or Relative
volatility, I will also refer to this as a separations
factor.
•
a = (tr2’ / tr1’)
• Capacity factor – measure of the amount of
extra time a compound stays in the system
beyond the tm. Will correlate with the
equilibrium constant.
–
k’ = (tr – tm)/tm
18
Retention time and partition coefficient
• Capacity factor can be restated as the ratio of
the time a compound is in the stationary phase
over the time the compound is in the mobile
phase.
• This can be converted to moles. Thus the
capacity factor is molesstat / molesmobile
• This allows us to write k’ the following way
•
k’ = CsVs / CmVm
19
Relationships
• Recall that K = Cs/Cm
• So k’ = K (Vs/Vm) = (tr – tm) / tm = tm’ / tm
• Relative Retention can also be expressed as
 a = (tr2’/tr1’) = k2’/k1’ = K2/K1
• To convert between volume and time one just
needs the flow rate as a conversion factor.
20
Terms
• Flow rate uv (ml/min)
• Vr = tr * uv
• Some types of chromatography will use volume
and others time. However time is preferred.
21
Scale Up
• Chromatography is known mostly as an
analytical procedure. Separation of micrograms
of material. The object of the game is to
separate and quantify.
• The system can be scaled up to separate at the
gram scale.
• Develop an analytical scale separation and then
scale it up.
22
Scaling Rules (1)
• Keep column length the same.
• Cross-sectional area of column proportional to mass on
column.
mass2  radius 2 

 
mass1  radius1 
2
23
Scaling Rules (2)
• Maintain constant linear flow rate. (This will mean that the volume
flow rate will change.)
volume flow2 mass2

volume flow1 mass1
24
25
The Peak
• Ideal chromatographic peaks are Gaussian in
peak shape.
• This comes directly from the Craig Model.
• We know certain facts about Gaussian peaks.
26
Efficiency of Separation
27
Resolution
• The more peaks we can resolve the better the
separation.
• How do we quantify Resolution.
28
Good Resolution
29
Poor Resolution
30
Factors for Resolution
• Two
– The separation of the
peaks
– The widths of the peaks
• Both separations are the
same but the widths are
wider for the bottom
example.
31
Resolution
• Resolution = Dtr / wave = 0.589Dtr/w1/2 ave
32
Diffusion
33
34
Diffusion
• A fundamental process. Leads to broadening of peaks
in separation methods.
• Flux (mol/m2s) = J = -D(dc/dx)
35
Diffusion
• Broadening of band by diffusion.
• c concentration (mol/m3)
• t is time
• x distance along column
• Standard deviation of the band will be
m
 x 2 /( 4 Dt )
c
e
4Dt
  2 Dt
36
Plate Height
• Terms
– Linear flow rate ux
– Distance peak has traveled along the column
– Time on column then would be t = x / ux
x
• 2 = 2Dt = 2D(x/ ux) = (2D/ ux)*x = Hx
• 2D/ux is the plate height giving us
• H = 2 / x
37
Plate Height is a Measure of Column
Efficiency
• The longer a compound is in the column the wider the
peak.
• Narrow peaks will allow us to resolve peaks coming out
at nearly the same time.
• Different compounds passing through a column at
different times might have different plate heights since
they will generally have different diffusion coefficients.
• Plate theory calls for constant plate height since diffusion
is ignored in this model.
38
Typical Plate Heights
• GC ~0.1 to 1 mm
• HPLC ~ 0.01 mm
• CZE ~ 0.001 mm
39
Plates
• N = L/H = Lx/2
=
L2/2 = 16L2/w2
t 
16t
N
  
w

 
2
r
2
2
r
2
40
What if is difficult to measure the width of a
baseline?
• We could potentially measure the width at half
height and knowing it is a Gaussian peak derive
the following.
2
r
2
1/ 2
t
N  5.55
w
41
Asymmetric Peaks
41.7(t r / w0.1 )
N
A / B  1.25
2
42
Factors Affecting Resolution
• Resolution can also be expressed with the
following equation.
N
R
4
 a  1  k 


'

a
1

k


ave 
'
2
R N  L
43
44
45
Band Spreading
• We have gone to a great deal of effort to
separate our peaks. We can see that diffusion
is working against us.
• We measure this spreading as the standard
deviation squared (Variance). 2
• Variance comes from many sources but we can
express it as a sum.

2
obs
             
2
1
2
2
2
3
2
4
2
i
46
Outside the Column
• Injector, detector, tubing and tubing junctions.

2
injection

2
det ector

Dt 

2
12
47
Van Deemter Equation
• Tells us the contribution to H of three sources.
• Recall that we want a minimum number for H!
H  A
• A Multiple paths
B
x
 C x
B Longitudinal diffusion C
Equilibration time
• ux is the linear flow rate
48
49
Optimum Flow Rate
• You can see from the previous plot that that best
flow rate for your system.
• Where the H value is minimum
• How do we find this point.
• Run about 20 or more experiments at different
flow rates, find H and then plot the resulting
curve. Pick Hopt from this plot.
50
Optimum Flow Rate
• Or ………
• Make three injections, find the values of A, B and
C.
• Find the minimum point.
• How?
51
Optimum Flow Rate
• Take the derivative
of the van
Deempter
equation.
• At the minimum point
the derivative will be
zero so:
dH
B

C
dux
ux
uopt
B

C
52
A Term – multiple paths
(eddy diffusion)
53
Longitudinal Diffusion
2 Dm
  2 Dmt 
ux
2

2
2 Dm B
HD 


L
ux
ux
54
55
Equilibrium Time
(Mass Transport)
H mass transport  Cu x  Cs  CM u x
2
d
Cs 
2
'
3 k  1 Ds

Cm 
2k
'

1  6k  11k
'


24 k  1
'
2
'2
2
r
DM
56
Mass Transport Band Spreading
57
Heat as a separations tool.
58
Comparison of open tubular and packed
columns.
• Open tube columns
–
–
–
–
Higher resolution
Shorter Analysis time
Increased sensitivity
Lower sample capacity
59
Open Tubular Columns
• At a constant pressure
• Flow rate is proportional to cross sectional area
• Flow rate is inversely proportional to the column length
area
flow a
length
For open tubular column this means that we can get
Increased linear flow rate and/or a longer column
Decreased Plate height, which means improved better resolution
60
Comparison
61
Asymmetric Bands
62