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HPLC
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Chapter 1HPLC Theory
Content
HistoryThe Chromatographic principleThe HPLC systemHPLC parameters & terms
V0, k, α, N & RThe Resolution equationStationary phase, physical propertiesUnderstanding the van Deemter curveColumn dimensions
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Definition of Chromatography
Analytical Science of Chemical SeparationsSeparate one compound from another at the molecular level in a liquid or gas stateCHROMATO = Color / GRAPHY = writing
First documented by the Russian Scientist Tswett, 1903-1906
Sample - Ground-up plant extract.Poured into a glass tube packed with calcium carbonate (column) - colored bands develop as the extract percolated down through the column.Compounds had separated.
Tswett´s Chromatographic System
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Principle of Chromatography
Mobile phase
Stationary phase
V
VV
VV
V
VV
V V
V
V
Elution
V
VV
V
V
V
XX
XX
XX
A
A
A
A
A
A
X
XX
X
X
X A
AA
A
A A
X
X
XX
XX
A
A
AA
A
A
Injection
Interaction
Type of Chromatography as a Function of Molecular Weight
Size Exclusion ChromatographyGel Permeation/Gel Filtration
Liquid ChromatographyIon Exchange
Liquid ChromatographyDistribution/Adsorption
Gas Chromatography
Molecular weight (MW)~50 ~500 ~10.000 ~2.000.000
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The Chromatographic System
Solvent
Pump
AutoSampler
HPLC Column
Detector
Waste
Data System
What is a Chromatogram?
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Terminology – Mobile Phase
The mobile phase is a liquid for HPLC.Total representative sample needs to be soluble in the liquid. Carrier of the sample, moving it through the stationary chromatographic packing material.
Terminology – Stationary Phase
The chromatographic packing material which is held in a fixed position within column hardware.Performs the chemical separation.
Also called the packing material, the chromatographic material, or the adsorbent.
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Stationary vs. Mobile Phases
The stationary phase and the mobile phase will have OPPOSITE properties to set up a competition for sample analytes.
Chromatographic Parameters
V = Retention volumeV0 = Void volumetr = Retention timek = Retention factor or capacity factorα = Selectivity or separation factorN = Plate countW = Peak widthR = Resolution
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Elution Volume and Peak Width
V0 = Elution volume of unretained compoundequals roughly one column volume
V1 = Elution volume of compound 1V2 = Elution volume of compound 2
W2= Peak width of compound 2 in terms of volume, measured where the tangent crosses the baseline
k = Retention FactorA Measure of Retention
TIME .5 1 2 5
V0
V1V2 V3
Describes how far the peak is from V0No dimension, independent of flow & column size
0.5k1 = 1-0.5 = 1
0.5k2 = 2-0.5 = 3
0.5k3 = 5-0.5 = 9
V0k1 =
V1-V0
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The Retention Factor, kk depends on the equilibrium of the analyte
between the stationary phase and the mobile phasek is an equilibrium constant
A(S)
A(M)
B(S)
B(M)
Mobile phase (M)
Stationary phase (S)
The Capacity Factor, k
The capacity factor depends on the polarity of themobile phase and column stationary phase.
The polarity of the mobile phase can be altered bychanging the percentage of organic in a mobile phase consisting of water and organic solvent.
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Changing k by Changing Mobile Phase Polarity
80 % MeOH
40 % MeOH
50 % MeOH
60 % MeOH 30 % MeOH
Column : Symmetry C18 4.6x50mmMobile phase : Methanol/phosphate bufferSample : Caffeine
AcetaminophenAspirinSalicylamide
α = SelectivityA Measure of Peak Separation
TIME .5 1 1.5 5
V0
V1V2 V3
Ratio between k-values for two adjacent peaksDefines the position of two peaks relative to each otherDepends on the “chemistry” of the system
1-0.5α1,2 = 1.5-0.5 = 2
1.5-0.5α2,3 = 5-0.5 = 4.5
α1,2 = V2-V0
k1α1,2 =
k2 or V1-V0
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α = SelectivityMobile Phase
40% Methanol60% Water
33% Acetonitrile67% Water
25% THF75% Water
Column : μBondapak C18
α depends on thechemistry of themobile phase,i.e.which organic solventis selected
R = ResolutionDegree of Magnitude of Separation
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R = ResolutionDegree of Magnitude of Separation
TIME .5 1 2 5
V0
V1V2 V3
R =V2 - V1
½(W1 + W2)
R1,2 =2 - 1
½(0.5 + 0.5)
R2,3 =5 - 2
½(0.5 + 1.0)
= 2
= 4
N = Theoretical PlatesA Measure of Efficiency
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Calculation of Column Efficiency5σ Method
7018.=RV
plates 8240
1.0318.7025N
2
5
=
⎥⎦⎤
⎢⎣⎡=
1.03W =
height4.4%
height4.4%
plates 3334
N5
=
⎥⎦⎤
⎢⎣⎡=σ
2
441631625
..
6316.=RV
1.44W =
Good Column Bad Column
Inje
ct
Inj e
ct
Efficiency Expressed as HETP
H = HETP (Height Equivalent to Theoretical Plate){Need H to be as Small as Possible so you can ‘fit” more “plates” into a column}
LNHETP =
N= # Plates
Plate
(From Distillation Theory) 5 Plates 10 Plates(Larger H) (Smaller H)
L columnlength
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Resolution Equation
Selectivity
Efficiency
Capacity
R = 1/4 α - 1α
k1+kN
What happens to R if N is increased 2x or 3x ?
Resolution EquationThe Efficiency Term
R = 1/4 α - 1α
k1+kN
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R = 1/4 α - 1α
k1+kN
What happens to R if k = 1, 2, 10 or 20 ?
Resolution EquationThe capacity term
Resolution Depends on k
k Value k Term k Resolution?0 0 01 1/2 0.502 2/3 0.673 3/4 0.75 10 10/11 0.9120 20/21 0.95
k
k Ideal Range ?
Res
olut
ion
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What happens to R if α = 1.1 or 1.4 ?
Resolution EquationThe selectivity term
R = 1/4 α - 1α
k1+kN
0.29
0.09
=−
=
=−
=
⎟⎠⎞
⎜⎝⎛ −
4.114.14.1
1.111.11.1
1
α
α
αα
Resolution Depends on α
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α, k, N Control Resolution
Initial
Increase N
Increase k
Increase α
Stationary Phase -Physical Properties
Column construction materialsParticle shape Particle size
The van Deemter plotParticle pore sizeColumn dimensions
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Column Construction - Materials
Stainless SteelGlassPlastic
HDPE (High Density PolyethylenePEEK (Polyetheretherketone)
Filters / Frits
Stationary Phase
EndFittings
Column Construction
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Frit
Spherical Particle Irregular Particle
Packed Bed (Stationary Phase)
Packing of the Column
Direction of flow to pack column
Types of Packing Materials
Silica (inorganic)Silicone Dioxide
Similar to beach sand
Alumina (inorganic)Aluminum Oxide
Zirconia (inorganic)Polymeric (organic)
PlasticHybrid (inorganic/organic)Carbon
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Particle Size of Packing Material
Particle size is measured in:
Microns = 10 –6 metersFor analytical columns: 2.5 – 5 μm (10 μm)
Highest Efficiency Columns are Packed with Smallest Particle Sizes!
Particle Size DistributionThe range of particle sizes presentExample: 5 μm (3 μm - 7 μm)
Narrower distribution gives higher performance, but are more costly to produce
3 5 7 4 5 6Microns
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Common Particle for HPLCChromatographic Silica is by far the most commonly used particle for HPLCMany different types of silica based packing materials are manufactured by many different Chromatographic performance is greatly determined by how the silica particle is made.
Surface Attribute of ParticlesHigh surface area particles
Key to high performance for all porous chromatographic sorbents
Particles are highly porous [similar to the pores in a sponge]
Pore size is measured in atomic size units, called Angstroms Å (10-10 meters)
Typical analytical packing material particles have pores ranging from 60-125 Å
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Comparison of Porosity and Surface Area
Many small pores60-125 Å
High surface area300-500 m2/g
Good for small molecules and pharmaceuticals
Few large pores250-4,000 Å
Lower surface area50-150 m2/g
Good for larger molecules and proteins
Plates vs. Flow Rate10000900080007000600050004000300020001000
1.0 2.0 3.0Flow Rate{mL/min}
N OptimumOnly one flow rate gives maximum efficiency
Every column will have its’ own specific curve – depending on dimensions and particle size
Typical Column Behavior For N (Plate Count)
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Why Does the Flow Rate Change the Plate Count?
van Deemter Equation
A= Interparticle channels - velocity independentB=Molecular diffusion axially -
inversely proportional to velocityC=Mass transfer kinetics - directly proportional to velocity
H = HETP (Height Equivalent to Theoretical Plate){Need H to be as Small as Possible so you can “fit”more “plates” into a column}
µ = Linear Velocity (speed) of Mobile Phase (mm/sec)
H = A + B + µCµ
Linear Velocity of the Mobile Phase - u
Flow RateArea
cm3/min.cm2 cm3 = mL
T e r m in o lo g yL in e a r V e lo c ity (c m /s e c ) - - S p e e d a t w h ic h th e m o b ile p h a s e is f lo w in g th r o u g h th e c o lu m n - - fu n c t io n o f th e F lo w R a te a n d C o lu m n In te r n a l D ia m e te r
L in e a r V e lo c ity (c m /s e c )
0 .1 0 .3 0 .5 0 .8 1 .0
4 .6 m m ID
m L /m in0 .8 2 .4 4 .0 6 .2 7 .8
4 .0 m m ID
m L /m in0 .6 1 .8 3 .0 4 .8 6 .0
2 .0 m m ID
m L /m in0 .1 5 0 .4 5 0 .7 5 1 .2 1 .5
Linear Velocity u = =
= cmmin.
or cmsec.
or mmsec.
Cross-sectionalArea of ID
{ }
Column
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Smaller particles, spherical, well packed => less interstitial volume => tight band, sharp peak ==> less A
A
A
Larger particles, +/or irregular shape => larger interstitial channels/more interstitial volume (between particles) => broader band, broader peak, less efficiency ==> more A
No impact of linear velocity
Particle Size A
H EfficiencyH = A + B + uCu
Sample/Analyte “BANDS” and van Deemter A Term (Interstitial Volume or Channels)
B
B
Slower linear velocity u, (or, flow rate), more axialdiffusion of band => broader peak ==> more “B” Term
Higher linear velocity u, less time for axial diffusion of band => sharper peak ==> less “B” Term
u B H Efficiency u
H = A + B + uCu
Sample/Analyte “BANDS” and Van Deemter B Term (Diffusion of Band)
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Sample/Analyte “BANDS” and Van Deemter C Term (mass transfer)
X
XX
X
Stationary phasemass transfer
Mobile phasemass transfer
Bandspreading
X X
X
XBandspreadingBandspreading
X X X X
Influence of particle size
X
XXX
Sample molecules Sample
molecules
Solvent
Packingmaterial
Packingmaterial
Solvent
C
High linear velocity, u, causes “spreading” of analyte molecules (band) due to effect on mass transfer (into and out of the stationary phase– not enough time to adsorb/partition)=> broad bands => broad peaks == > more “C” Term
C
Lower linear velocity, u, causes reduced “spreading” of analyte molecules => narrower bands => sharper peaks == > less “C” Term
u Cu H EfficiencyH = A + B + uCu
Sample/Analyte “BANDS” and Van Deemter C Term (Mass Transfer)
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Hei
ght E
quiv
alen
t to
Theo
retic
al P
late
Linear Velocity
HET
P
u
Lowest HETP => Optimum Plate Count
Note: Once past optimum,as flow rate increases so doeslinear velocity - HETP Increases - plate count DECREASES
Note: Minimum HETP point is related to viscosity. As viscosity increases,the minimum HETP point occursat slower linear velocity
{cm/sec}
HETP PLATESH
H = A + B + uCu
The Van Deemter Curve
Comparison of the Van Deemter Plots for 5 µm and 2.5 µm Particles
2
4
68
10
12
14
16
18
20
22
24
26
28
30
0 0.5 1 1.5 2 2.5 3 3.5 4
Linear Velocity (mm/sec)
H (µ
m)
5 µm XTerra® Particle
2.5 µm XTerra® Particle
(50/50, acetonitrile / water mobile phase) Alden
Speed with maximum efficiency (resolution) should be achievable with smaller particles
Benefit of Smaller Particle* Reduction in HETP* Higher Linear Velocity Range
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Effect of Temperature on Column Efficiency -- Van Deemter
0 1 2 3 4 5 6 7 8 910
15
20
25
30
35
HET
P, H
[µm
]
5°C 25°C
60°C
As temperature is increased, lower HETP, higher N, higher linear velocity, reduced viscosity, improved masstransfer
u Linear velocity, u [mm/s]
Column Dimensions
Length: Millimeters or Centimeters
Internal Diameter: Millimeters
Internal diameter Analytical : 1mm - 4.6mmPreparative : 7.8mm - 50mm
Length2cm - 30cm20mm – 300mm
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Summary of Column Geometry
DiameterSmaller diameter INCREASES sensitivity.Larger diameter INCREASES column Capacity.
LengthLonger column has a HIGHER resolving power and capacity.Shorter Column has FASTER speed of separation and LOWER capacity.
Summary of Theory
DefinedV0, k, α, N & R
Learned the effect of k, α, and N on resolutionLearned the importance of particle size and flow on column efficiency (van Deemter plot)Looked at the effect of column dimensions