HPLC

1
HPLC

• Liquid Chromatography

Chapter 28
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HPLC
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
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Advantages of HPLC
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.

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HPLC Chromatograph of Muscadine Grape Juice
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HPLC
SOLVENTS
Includes both liquid phase and solid materials
(Buffers) dissolved in the liquid.
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HPLC

• Solvent properties affecting detection
• Solvent properties affecting separation
• Solvent properties affecting flow
• Viscosity
• Miscibility

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HPLC
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) Vo
latility needed for HPLC Mass Spectrometry (triflu
oroacetic acid is a typical volatile buffer)
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HPLC
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.
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HPLC
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
partially charged
Basic Compound
Relative Retention Time
pK
fully charged
a
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pH
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HPLC
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.
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HPLC
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.
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HPLC
Viscosity - resistance to flow
Difficult to force high viscosity solvents
through the column. Mixing solvents can
drastically change viscosity
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HPLC
Viscosity of Water-Organic Solvent Mixtures
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HPLC
Viscosity vs. Pressure
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HPLC
EXAMPLE
P 250 L ? F / Dp2 Dc2
column length 15 cm, column diameter .5 cm,
particle diameter 5 mm, 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
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HPLC
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HPLC
Peripheral Properties

• Toxicity
• Flammability
• Reactivity solvent should not react with sample
• Cost
• Disposal can be more than purchase cost

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HPLC
Geometry of HPLC Columns Diameter Length Particle
Size
What is the effect on pressure?
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HPLC
P 250 L ? F / Dp2 Dc2 Where P pressure drop
in psi. F flow rate (mL/min) L column length
(cm) Dp particle diameter (mm) ? solvent
viscosity (cP) Dc column diameter (cm)
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HPLC
Geometry of HPLC Columns Diameter Length Particle
Size
What is the effect on Theoretical Plates?
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HPLC
What is the effect of column geometry on
Theoretical Plates?
Remember that separation is best on columns with
high number of theoretical plates.
NL/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!
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HPLC
What is the effect of column geometry on
Theoretical Plates?
Decreasing column diameter by half
For comparison purposes, lets keep the mobile
phase velocity constant. Therefore, flow would
be reduced 4X and analysis wont 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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HPLC
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.
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HPLC
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!
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HPLC
Are your peaks coeluting again?