Practical HPLC

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Practical HPLC
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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)
• Purity
• Viscosity
• Refractive index
• Boiling Point
• Toxicity
• UV Transparency/UV-Cutoff
• Solubility

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Solvent Miscibility
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Solvent UV-Cutoff/Transparency
Solvent
UV Cutoff (nm)
Acetonitrile
190
Water
190
UV cutoff is the wavelength at which absorbance
equals 1, measured in a 1 cm cell with air as a
reference.
Cyclohexane
195
Hexane
200
Methanol
210
Ethanol
210
Diethyl Ether
220
Dichloromethane
220
Chloroform
240
Carbon Tet
265
Tetrahydrofuran
280 (220)
Toluene
285
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Mobile Phase Preparation

• Major Steps
• 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
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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 non-aqueous 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.

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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.
• Dont change the flow direction, this will
  decrease column performance.

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Column Installation
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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
• Do not pressure shock the column.
• 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
• 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.
• Dont 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

You are Ready for Sample Analyses
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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.