Transcript Practical HPLC

Practical HPLC
In This Section, We Will Discuss:
How to set up an HPLC System for a sample injection including:
 Solvent Handling
 Mobile Phase preparation
 Priming the HPLC
 Column Handling - Equilibration
 System Performance Checks
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Solvent Handling
Solvent Characteristics (Specifications):
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Purity
Viscosity
Refractive index
Boiling Point
Toxicity
UV Transparency/UV-Cutoff
Solubility
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Solvent Miscibility
Name
Acetic Acid
Acetone
Acetonitrile
Benzene
Butyl Alcohol
Carbon Tetrachloride
Chloroform
Cyclohexane
Cyclopentane
Dichloroethane
Dichloromethane
Dimethylformamide
Dimethyl Sulfoxide
Dioxan
Ethylacetate
Ethyl Alcohol
Di-Ethylether
Heptane
Hexane
Methyl Alcohol
Methylethyl Ketone
I-Octane
Pentane
I-Propyl Alcohol
Di-Propylether
Tetrachloroethane
Tetrahydrofuran
Toluene
Trichloroethane
Water
Xylene
Immiscible
Miscible
2-Propanol is an excellent
intermediate solvent
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Solvent UV-Cutoff/Transparency
Solvent
UV Cutoff (nm)
Acetonitrile
Water
Cyclohexane
190
190
195
Hexane
Methanol
Ethanol
Diethyl Ether
200
210
210
220
Dichloromethane
220
Chloroform
Carbon Tet
240
265
Tetrahydrofuran
280 (220)
Toluene
285
UV cutoff is the wavelength at
which absorbance equals 1,
measured in a 1 cm cell with air
as a reference.
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Mobile Phase Preparation
Major Steps:
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Measure appropriate volume of each solvent
Mix solvents
Add buffers and additives*
Filter mobile phase
Degas mobile phase
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Priming HPLC System
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Priming HPLC System
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Priming HPLC System
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Priming HPLC System
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Priming HPLC System
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Priming HPLC System
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Priming HPLC System
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Priming HPLC System
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Priming HPLC System
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Priming HPLC System
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Priming the HPLC
Flow
Purge Valve
Waste Capillary
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Sample Preparation
At a minimum - filter samples:
 Nylon - hydrophilic nature works with aqueous and solvent based
samples, autoclavable to 121ºC, pH range 3-12, no concentrated
acids.
 PTFE- a hydrophobic membrane which is highly resistant to
solvents, acids, and alkalis. This filter is generally used for nonaqueous samples. pH range 1-14.
 Cellulose Acetate- good filter for aqueous biological samples
with very low protein retention. pH range 4-8.
 PVDF- highly resistant to most solvents, exhibits low protein
binding. pH 2-12.
 Ultrafilter Membranes- molecular weight cut-off filters for
biological samples.
 Nitrocellulose- exhibits high protein retention.
 Solid Phase Extraction.
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Sample Preparation
 Dissolve the sample in the mobile phase or in a solvent weaker than the
mobile phase.
 The sample volume should be kept as small as possible.
Sample in Mobile Phase
Sample in Stronger Solvent
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Column Storage
 Avoid any physical stress to the column.
 Close on both ends to avoid dryness.
 Store the column well flushed
with the appropriate solvent.
 Record the history of the
column .
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Column Installation
 Each column has a defined flow direction!
 The flow direction is shown by the arrow or direction of writing.
 Don’t change the flow direction, this will decrease column performance.
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Column Installation
What’s needed:
The right connectors to avoid
any future leaks
or dead volume.
Guard column to protect
the main column
The right tools
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Column Installation cont.
Practical hints:
• Finger tighten
• 1/4 turn with wrench
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Column Equilibration
Equilibrate with mobile phase
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Do not pressure shock the column.
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5-10 column volumes for reversed-phase equilibration.
• Assures reproducible results.
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Column Check
 New columns should be delivered with a performance
certificate.
 Each additional use should be documented including:
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Back pressure
Mobile Phase
Temperature
Sample type
Storage condition (Solvent)
Based on that history the column can be checked with
defined compound mixture.
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Column Care and Handling
 Wash the column after use with selected solvents; flush highly retained
sample components from the column, eliminate buffers.
 Do not store a column in 100% water. Microbes may grow and clog the
column.
 Do not store the column in 100% Acetonitrile.
 Don’t open the column and repack the material if you want to maintain
performance.
 Use the column at its optimal flow rate - avoid high flow rates.
 Do not operate silica or bonded phases for extended periods at high
temperature.
 Keep the pH of the mobile phase in an appropriate range for the column.
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System Check - Routinely
Principle:
The HPLC system (including the column) can be checked out using a defined
test sample and method. Use at least three replicates.
Preparations for a system check:
• HPLC system is primed with mobile phase.
• Column is equilibrated.
• Detector shows a stable response.
• There are no leaks.
• System is ready for injection
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System Check - routinely cont.
Test sample requirements:
• Sample is well characterized.
• Detector response is known.
• Sample contains multiple components.
Test design:
The test sample is analyzed using a defined test method. The results are
compared with the expected results. If the results are in the defined range,
than the system is ready for use.
This is not comparable to an OQ test or PV test!!
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Summary
•Prepare mobile phase
•Prime the HPLC system
•Install the column
•Turn on the detector (warm-up at least 20 minutes for UV)
•Equilibrate the column
•Prepare the samples
•Record the detector response - stable response
•Perform a system check using a test sample and test method
•Compare the results with the expectations (limits)
•Document the results (Control Chart)
•Record any failures/errors if appropriate
•If system check is OK, then
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Review
1. You are running a routine analysis when you notice a periodic
perturbation in the baseline. The pressure reading is fluctuating
up and down. What is the problem? How would you correct it?
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Review
2. You decide to run a reversed-phase analysis on an instrument in
lab. The previous operator does not indicate the solvents last used
on the instrument. You place water in channel A and turn on the
pump. You cannot get a stable baseline. Suggest a possible reason
for this dilemma.
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