Title: Practical HPLC
1Practical HPLC
2In 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
3Solvent Handling
- Solvent Characteristics (Specifications)
- Purity
- Viscosity
- Refractive index
- Boiling Point
- Toxicity
- UV Transparency/UV-Cutoff
- Solubility
4Solvent Miscibility
5Solvent 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
6Mobile Phase Preparation
- Major Steps
- Measure appropriate volume of each solvent
- Mix solvents
- Add buffers and additives
- Filter mobile phase
- Degas mobile phase
7Priming HPLC System
8Priming HPLC System
9Priming HPLC System
10Priming HPLC System
11Priming HPLC System
12Priming HPLC System
13Priming HPLC System
14Priming HPLC System
15Priming HPLC System
16Priming HPLC System
17Priming the HPLC
18Sample Preparation
- At a minimum - filter samples
- Nylon - hydrophilic nature works with aqueous and
solvent based samples, autoclavable to 121ÂșC, 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.
19Sample 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.
20Column 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 .
21Column 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.
22Column Installation
23Column Installation cont.
Practical hints
- Finger tighten
- 1/4 turn with wrench
24Column Equilibration
- Equilibrate with mobile phase
- Do not pressure shock the column.
- 5-10 column volumes for reversed-phase
equilibration. - Assures reproducible results.
25Column 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.
26Column 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.
27System 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
28System 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!!
29Summary
- 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
30Review
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?
31Review
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.