Download HPLC - sedatture

KROMATOGRAFİ
Sedat Türe
HPLC
Liquid
Chromatography
ÖZET
The original development of HPLC used higher pressures
than previously used ----High Pressure Liquid Chromatography
However, over the years the preferred term has become:
High Performance Liquid Chromatography
Advantages of HPLC






High resolution
Speed
Re-usable columns
Great reproducibility
Control of physical parameters
 flow rate, polarity, packing efficiency, and particle size.
Easy automation of instrument and data analysis.
HPLC Chromatograph of
Muscadine Grape Juice
SOLVENTS
Includes both liquid phase
and solid materials (Buffers)
dissolved in the liquid.
•Solvent properties affecting detection
•Solvent properties affecting separation
•Solvent properties affecting flow
•Viscosity
•Miscibility
Solvent Properties Affecting Detection
UV Cutoff -Solvent may interfere with detection
For peptide analysis UV = 215 nm. Solvents that
absorb UV at this wavelength would not be good
candidates for the mobile phase.
Refractive Index of Solvent vs Sample for
Refractive Index detection (Carbohydrates)
Volatility needed for HPLC Mass Spectrometry
(trifluoroacetic acid is a typical volatile buffer)
BUFFERS
1)Buffers are needed to control the pH differences caused by
the sample matrix.
2)Buffers are used to control the ionization of compounds
and therefore their retention by the column.
Retention Time and pH in Reversed Phase
When an acid or a base is ionized it becomes much less
hydrophobic and will elute much earlier. Acids lose a
proton and become ionized (negative charge) as pH increases.
Bases on the other hand, gain a proton and acquire a positive
charge as pH decreases.
not charged
Basic
Compound
Relative Retention Time
partially charged
fully charged
3
4
pKa
5
6
pH
7
8
9
SOLVENT SELECTIVITY
The less time a compound spends in the stationary phase,
the faster it will move through the column (less retention time).
If two compounds are added to the column, the ratio of their
retention times is called the selectivity.
The higher the selectivity, the better the separation.
Selectivity can be increased by adjustment of the mobile and
stationary phases.
Solvent Selectivity Triangle
Representing 3 “Polarity” factors
1) Each dot in the triangle
represent a different solvent
2) Solvents can be grouped
based on their type of polarity
3) Solvents and solvent mixtures
are available for just about any
separation you may desire.
Viscosity - resistance to flow
Difficult to force high viscosity solvents through
the column.
Mixing solvents can drastically change viscosity
Viscosity of Water-Organic Solvent Mixtures
Viscosity vs. Pressure
The higher the solvent viscosity, the harder it is for the
solvent to move through a column, and the more pressure in
required to move the solvent at a specific velocity. The pressure
required to move a solvent through a column can be estimated by
the following formula:
P = 250 L  F / Dp Dc
2
Where P = pressure drop in psi.
L = column length (cm)
= solvent viscosity (cP)
2
F = flow rate (mL/min)
Dp = particle diameter (m)
Dc = column diameter (cm)
EXAMPLE
P = 250 L  F / Dp2 Dc2
column length = 15 cm, column diameter =.5 cm,
particle diameter = 5 m, flowrate = 2.0 mL/min
For water
n = 1.0 250 x 15 x 1.0 x 2 / 52 x .52 = 7125/6.25 = 1200 psi
For methanol n = 0.54 250 x 15 x .54 x 2 / 52 x .52 = 2025/6.25 = 648 psi
For 60% water n = 1.9
250 x 15 x 1.9 x 2 / 52 x .52 = 7125/6.25 = 2280 psi
40% methanol
SOLVENTS
Water
Methanol
Tetrahydrofuran
Propanol
Acetonitrile
Hexane
Ethyl Acetate
Chloroform
UV
Cutoff
190
205
212
210
190
195
256
245
Miscible
with
Viscosity Polarity Water?
1.00
0.55
0.55
2.3
0.38
0.31
0.45
0.57
10.2
5.1
4.0
4.0
5.8
0.1
4.4
4.1
--Y
Y
Y
Y
N
N
N
Peripheral Properties
– Toxicity
– Flammability
– Reactivity solvent should not react with sample
– Cost
– Disposal can be more than purchase cost
Geometry of HPLC Columns
Diameter
Length
Particle Size
What is the effect on pressure?
P = 250 L  F / Dp2 Dc2
Where P = pressure drop in psi.
L = column length (cm)
 = solvent viscosity (cP)
F = flow rate (mL/min)
Dp = particle diameter (m)
Dc = column diameter (cm)
Geometry of HPLC Columns
Diameter
Length
Particle Size
What is the effect on Theoretical Plates?
What is the effect of column geometry
on Theoretical Plates?
Remember that separation is best on columns with high
number of theoretical plates.
N=L/H
where N is number of plates, H is plate height
and L is Column Length
Therefore, doubling the column length will double N
but this will double analysis time and pressure!
What is the effect of column geometry
on Theoretical Plates?
Decreasing column diameter by half
For comparison purposes, let’s keep the mobile phase
velocity constant. Therefore, flow would be reduced 4X
and analysis won’t take any longer!
halving the column diameter can also increase N slightly
This reduces the amount of solvent used by 4X but also
reduces the amount of sample that can be injected by 4X.
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What is the effect of column geometry
on Theoretical Plates?
Decreasing particle size by half
Will increase pressure by 4X
However, halving the particle size can double N
Decreasing particle size and making the column half as long
will keep N the same but cut sample time in half and solvent
use in half.
In general small diameter columns with
small particles are best for rapid separation,
….but require higher pressures, smaller samples, and can plug easier.
The problem with plugging should not be underestimated
and care should be exercised in keeping the sample, mobile
phases, and columns CLEAN!