HPLC 2009

High Performance Liquid
Chromatography (HPLC)
Learning outcomes
At the end of the class we should be able:
 To differentiate between different types of LC.

 To differentiate HPLC from GC and LC.
 To learn the different components of HPLC.
 To differentiate between Normal phase and
reverse and also between isocratic elution and
gradient elution.
Liquid Chromatography (LC)
 In HPLC we use the mechanism of LC to separate
mixtures in solution.
 Two different phases are used to separate
components of the mixture: stationary and mobile
phase.
 Quick solution elution comparison: Basic column
vs. HPLC (force of gravity vs. forced pressure
medium).
 The solute separates on the column via interactions
based on different physical mechanisms.
Types of Liquid Chromatography
Liquid Chromatography
Adsorption Partition Size Exclusion Affinity Ion Exchange
Competition Competition Molecular “Lock and Key” Competition

between liquid between liquid sieving mechanism between liquid
mobile phase mobile phase and ionic
and solid and “liquid” stationary phase
adsorbent stationary phase
The classification of chromatographic modes is based

according to the retention mechanism
The concepts of HPLC...
 Capacity or Retention factor
k’R = (tR - tm) / tm = (VR - Vm) / Vm
 Selectivity =t2 - t0) / (t1 - t0)
= (V2 - Vm) / (V1 - Vm) = k’2 / k’1
 Resolution R = 0.25*((-1)/)(N^0.5)(k’/(1+k’))
 Efficiency (plate height) H= L / N
N = number of theoretical plates
What we have...”Waters”
ISOCRATIC Pump
Detector
Injector
Mobile
phase
Column
Instrument Components
 In terms of online LC, the choice of phase separation
processes is more limited than in traditional HPLC
 Ion exchange – high strength salt buffers
 Size exclusion – not very useful for small molecules,
not very selective
 LC/MS is dominated by reversed phase (95%)
chromatography, particularly C18
 Normal phase chromatography may still be useful for
some compounds (e.g. small highly organic
molecules), including those that have poor solubility
in water and partially aqueous mobile phases
Normal Phase and Reversed Phase
 Normal phase: polar stationary phase and a less polar

solvent. Separates on the basis of polarity – gradient
elutions start with a non-polar solvent and move
gradually to a more polar solvent. The polar analytes
are the most retained.
 Reversed Phase (C8, C18): the stationary phase is
nonpolar or weakly polar and the solvent is polar.
Gradient elutions usually start with a predominantly
aqueous mobile phase and move gradually to a more
organic mobile phase.
 RP – the smaller molecules with lowest carbon
number elute first
Reversed Phase Separation
Based on hydrophobicity (approximates carbon number) Separation
based on the differential solubility in aqueous and organic media
CH3
H3C CH3
7 Carbons
8 Carbons
6 Carbons
Particle Diameter
 Has a greater effect on resolution than length
 Small particles – short analysis times
 Short columns with small particles ideal
 5m is standard size
 3m better, but restricted range of packings
available
 Downside is high back pressure and issues
with retention of small particles inside the
column, blockages
Sensitivity Considerations and Column ID
SAME INJECTION VOLUME
1mm ID column
2mm ID column
4.6mm ID column
Normal Phase HPLC
Various solvents can be

used based on the best
polarity ratio of the solvent
mixture in the separation -
the solvents are the
“mobile phase” ..Gradient syt.
This system is “isochratic” –

only one solvent is used!
Electrochromatograms showing the comparison of isocratic and
gradient elution for the separation of the 16 PAHs.
Injector Port
Normal Phase HPLC
Load and Inject

positioning
Overview of an HPLC run
 Choose solvents
 System Initialization
 Injection and Data Acquisition
 Data Analysis
 Shutdown
 Important Hints,Tips, and Reminders for
HPLC
 Troubleshooting
Choosing Solvents
 Look in literature for similar separation; find solvent ratios for the
mobile phase based on polarity
 Run a quick TLC plate to determine best proportion for best
separation. When you find the right solvent do the following:
 With a 0.22 micron nylon filter; filter the solution into a filtration
flask using a water aspirator or vacuum line.
 First pour a small amount of solution into the HPLC filter
apparatus. Swirl and rinse out the filtration flask; put the
filtrate into appropriate waste container.
 Pour rest of the solution through the filter.
 Before adding the filtrate into the solvent reservoir be sure to

rinse out the solvent reservoir as well with small amounts of
the filtered solvent!
System Initialization
 Turn on the pump. Turn on UV detector (15 min. warmup time).
Make sure the injection port is in the “LOAD” position.
 Organize benchtop and be sure that all lines are secure in
intput/output ports on all components. Insert solvent line into
desired solvent bottle.
 First check for air bubbles in the lines and if you are not using the
same solvent as the run before you must prime the pumps
 Turn on On-Line solvent degasser and pump. Slowly increase
the flow rate over 10 minutes by adjusting the flow rate knobs on
the pump starting from 0.1 ml/min up to 1.0 ml/min over a 10
minute time span (i.e. Increase rate 0.1ml/min per minute).
 Set the UV detector sensitivity and the desired wavelength.
Injection and Data Acquisition
 With a 25l syringe extract the full 25l of your solvent. Inject this
rinse injection onto a Kimwipe or any tissue paper. Repeat this several
times.
 Now extract a full 25l, invert the syringe, check for air bubbles and
depress the plunger over a Kimwipe to the 20l mark as the load
volume of the LOAD loop is 20l (see tag).
 Insert the needle into the injection port and inject the sample into the
LOAD loop. Leave the syringe in port for next step; hold it gently.
 When ready rotate the sample port to INJECT mode and
immediately! click anywhere on the integrator to start the data
acquisition.
 Remove syringe from port after the completion of the analysis.
 Watch for peaks; time zero will glide across the screen from right to
left.
Data Analysis
 The chromatogram provide the following
information of each peak
 Retention time
 Peak area
 Peak height
 % of peak area
Shutdown
 Slowly bring the flow rate on the pump back to
zero.
 Shut off the detector, pump and degasser
sequentially. Leave solvent line in the solvent
reservoir.
Important Hints,Tips, and Reminders...
 Be sure to filter all HLPC solvents with the HPLC filters
and glass ware.
 Prime the pump before using the instrument upon a
solvent change!
Practical Hints and Troubleshooting
High Back Pressure

Causes Solutions
Plugged frits before guard Back flush column
column or main column. cartridge, replace frits
Irreversibly retained Follow regeneration

contaminants on column head. procedure in the column
cleaning figure on next silde
And most importantly to prevent back pressure…

PRE-FILTER ALL SOLVENTS BEFORE USING
General Regeneration Chart
The relevant
operation for
this HPLC...
(from www.
http://www.alltechweb.com/Technical/ColumnCare/Manual_Insert1.htm)
Regeneration Procedure
Regeneration procedures entail flushing the column with a series of

solvents which are increasingly more similar in polarity to the stationary
phase. As the contaminants lose attraction for the packing, they enter the
solvent stream and are carried out of the column. Since most adsorption
takes place on the head of the column, reversing the column direction
provides a shorter " exiting " path. Particulates that may have collected
on the inlet frit,contributing to higher pressure, can often be dislodged,
thus avoiding frit replacement. The figure provides wash procedures for
silica-based packings. If the solvents suggested are not readily available
in your lab, solvents of similar elutropic strength can be substituted. As
long as every solvent introduced into the column is miscible with the
solvent currently inside the column. Buffers and ion pairing reagents can
precipitate upon addition of organic solvents, and therefore should be
completely removed by flushing with 10 column volumes of mobile phase
with these salts removed. The number following each wash step
indicates the number of column volumes that should be used for that
solvent. To determine one column volume for your column dimensions
refer to the chart at bottom left corner below.
(from www. http://www.alltechweb.com/Technical/faq/hplc/hplcfaqs9a.htm)
Tailing or Double Peaks

Causes Solutions
Mismatch between sample and Modify mobile phase.
mobile phase or packing Change column if
possible
Void at head of column or cartridge Reverse flow through

Particulates on inlet frit column or cartridge. Run
effluent directly to waste.
www.alltechweb.com Do this only with Instructor
supervison.
After column lifetime has expired dispose of the catridge only ;

the end fittings are perfectly fine!
Symptom Cause Remedy
Noise Spikes Bubbles passing through cell Degas mobile phase and/or apply
backpressure to the sample cell
High pressure fitting not making Tighten Fittings
an air tight seal
Excessive Noise Sample cell windows are contaminated Flush cell with solvents to remove
contamination
Bubble trapped in sample cell Increase backpressure on flow cell
UV lamp is sputtering Replace UV lamp
Pump flow pulsing or inconsistent Shut off pump. If baseline quiet:
service pump
Excessive Drift Insufficient warm-up Allow detector to warm-up for 30 min.
Sample cell is contaminated Flush cell with solvents to remove
contamination
Column is not equilibrated Allow time for column to equilibrate
UV absorbing components in mobile phase Replace solvents with HPLC/UV grade
(Excerpts taken from the Beckman 110B Solvent Delivery Module Manual)

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High Performance Liquid

 To differentiate between different types of LC.

Adsorption Partition Size Exclusion Affinity Ion Exchange

Competition Competition Molecular “Lock and Key” Competition

The classification of chromatographic modes is based

 Normal phase: polar stationary phase and a less polar

SAME INJECTION VOLUME

Various solvents can be

This system is “isochratic” –

Load and Inject

 Before adding the filtrate into the solvent reservoir be sure to

High Back Pressure

Irreversibly retained Follow regeneration

And most importantly to prevent back pressure…

Regeneration procedures entail flushing the column with a series of

Tailing or Double Peaks

Void at head of column or cartridge Reverse flow through

After column lifetime has expired dispose of the catridge only ;

Symptom Cause Remedy

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