Chapter 25
HPLC
High-Performance Liquid
Chromatography
1
Typical HPLC
2
Application
Microdialysis
3
Monitor Aspirin in Blood
4
Increased Efficiency
• In any method the object is to increase
mobile phase / stationary phase interaction.
• Decrease particle size.
– Better packing – slower flow.
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How Does the Smaller Size Help
7
Smaller is Better
• N ~ 3500 L (cm) / dp (mm)
• Smaller particle size leads to
– Higher plate number
– Higher pressure
– Shorter optimum run time
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Size Considerations
10
What is a bar?
• A measure of pressure.
• One bar = 1.01325 Atm
• One bar is 100 000 Newtons/m2
• Under water you gain about one atm for
each ten meters in depth.
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HPLC Columns
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Stationary Phase / Support
• Support is the scaffolding that the stationary
phase sits on.
• Support - Microporous Silica
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–
–
–
Solvent can get inside
Surface area of 100’s m2 / gram
Silica degrades above pH 8 so keep pH below.
Special supports have been developed for
higher pH
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Active Sites
Generally Bad
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Active Sites Can Lead to Tailing
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We Can Modify the Surface
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Bonded Phases
• SiOH + ClSi(CH3)2 - R = Si-O-(CH3)2 - R
• Change surface polarity
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–
–
–
–
–
Amino (CH2)3NH2
Cyano (CH2)3C#N
Diol
(CH2)2OCH2CH(OH)CH2OH
Octadecyl (CH2)17CH3
Octyl
(CH2) 7CH3
Phenyl
(CH2)3C6H5
17
New Technology
Monolithic Silica Columns
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Micrographs
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Elution
• Competition for the surface.
• Which substance has the greater surface
affinity – a solvent with a higher affinity
will push the analytes along.
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The Eluotropic Series
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Isocratic and Gradient Elution
• Isocratic - same composition
• Gradient - composition changes over time
• Method Development.
• A mixture of compounds. Separated with
acetonitrile (B) and aqueous buffer (A)
• 1) benzyl alcohol 2) phenol 3) 3’,4’-dimethyloxyacetophenone 4)
benzoin 5) ethyl benzoate 6) toluene 7) 2,6-dimethoxytoluene 8) omethyoxybiphenyl
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Too Long - Over Two Hours!
• We can do a gradient. Examine when we
get the best separations and then change
composition over time.
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Separation Design
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Separation Design
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Practical Issues
• Solvents
– Must be very pure, lack UV absorbing species
– Should be filtered
– Guard columns should be used to protect column from strong
absorbing compounds
– Solvents should be degassed – bubbles and oxygen (sparging)
– Normal phase solvents should be 50% saturated with water
– Gradients in reversed phase can require 10 – 20 column volumes to
return to starting conditions
• Add 3% 1-propanol to each solvent and you will cut this to 1.5 empty
volumes
31
Reducing Waste Solvent
(Save some money – be a hero)
• Shorter columns with smaller particles
• Switch from 4.6 mm to 3.0/2.0 mm id
columns
• In isocratic systems – use an electronic
recycler.
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Quality Assurance
• Inject a QC sample each day to insure that
you have consistent peak shapes and
retention times.
– Keep a log of your column performance
33
Symmetric Band-shape
• Asymmetry Factor A/B should rarely be worse than 0.9 to
1.5.
• Tailing a bigger issue and fronting generally.
–
–
–
–
–
–
Amines interacting with support active sites.
Add 30 mM TEA
Acidic compounds
Add 30 mM Ammonium Acetate
Or for mixes or unknowns – 30 mM triethylammonium acetate.
Persistent problem – add dimethyloctylamine or
dimethyloctylammonium acetate
These take a long time to wash on on changing mobile phases.
34
Other issues
• Voids can develop at the column inlet.
– Repack with fresh stationary phase to get rid of this
problem. (Might want to get new column)
• Columns should be washed to get rid of salts and
strongly adsorbed compounds
• Frits should be cleaned. Back wash or replace
• Samples should be dissolved in mobile phase or a
weaker solvent.
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Overloading
• Care should be taken not to overload the
injection on the column.
– Inject 10x less and see if peaks look any better.
• Reversed Phase can deal with 1 to 10 mg
sample per gram of silica. (About 10 cm on
a 0.46 mm column)
• Injection volume
– < 15% of the peak volume at baseline
37
Minimize Dead Volume
• Minimize connection tubing
• Ensure that fittings are proper matches.
38
Injection and Detection
• Pumps
– Piston Type under program control. Up to 400
bar (40 MPa or 6000 psi). Gradients made by
proportioning valves.
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Injection
• Sample loop filling either done manually or by and
autoinjector
41
Detectors
Mass or Concentration Types
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UV- Vis (Photometric)
Variable Wavelength Detector
43
Photodiode Array
All wavelengths at once
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Refractive Index
Universal Detection with major issues.
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Evaporative Light Scattering
•
•
•
•
•
Solutes less volatile than mobile phase
Light scattered from mass of analyte.
Poor linearity – polynomial calibration
Good with gradients. No solvent front.
Same buffers as used with mass spec.
Acetic acid, formic acid and TFA,
ammonium acetate, diammonium
phosphate, ammonia or TEA.
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Electrochemical
• Analytes that can be oxidized or reduced
– Phenols, aromatic amines, peroxides,
mercaptans, ketones, aldehydes etc.
• Can be very sensitive but are difficult to
work with.
50
Method Development
51
Eluent Strength Nomogram
52
Gradient Separations
• Same purpose as temperature or pressure
programming in GC. Speed up analyses.
• Increase the eluent strength as the run
progresses. That is increase the amount of
organic phase as the run progresses in
reversed phase methods.
53
Dwell Time
• Time it takes for the composition change at
the pump to reach the head of the column.
• Important on method transfer
• Can be determined by running a gradient of
0.1% acetone (detect at 260 nm) without a
column. Start gradient at time 0 and see
when the acetone starts to be absorbed.
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Since gradients require a post equilibrium
time then perhaps isocratic will work better.
• What is the span of all the peaks. That time span is Dt
– If Dt/tg > 0.25 then use a gradient. If less you can make
an isocratic system. (See next slide)
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Optimize Gradient
• Run a broad gradient. (5% to 100% over 40
to 60 min)
• Eliminate gradient before first peak and
after last peak. Run over same time.
• Cut time if above works well to save time.
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