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
NameAcetic AcidAcetoneAcetonitrileBenzeneButyl AlcoholCarbon TetrachlorideChloroformCyclohexaneCyclopentaneDichloroethaneDichloromethaneDimethylformamideDimethyl SulfoxideDioxanEthylacetate
Ethyl AlcoholDi-EthyletherHeptaneHexaneMethyl AlcoholMethylethyl KetoneI-OctanePentaneI-Propyl Alcohol
TetrachloroethaneTetrahydrofuranToluene
Buty
l Alc
ohol
I-Pro
pyl A
lcoh
ol
Di-P
ropy
leth
er
Tric
hlor
oeth
ane
Acet
ic A
cid
Acet
one
Acet
onitr
ile
Benz
ene
Car
bon
Tet
Chl
orof
orm
Cyc
lope
ntan
e
Cyc
lohe
xan
e Dic
hlor
oeth
ane
CH
Cl
2
2D
MF
DM
SOD
ioxa
nEt
hyla
ceta
te
Ethy
l Alc
ohol
Di-E
thyl
ethe
rH
epta
neH
exan
eM
ethy
l Al
coho
l MEK
I-Oct
ane
Pent
ane
2
2 C
H C
l
4
THF
Tolu
ene
Wat
erXy
lene
TrichloroethaneWaterXylene
Di-Propylether
ImmiscibleMiscible
2-Propanol is an excellent intermediate solvent
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Solvent UV-Cutoff/Transparency
Solvent UV Cutoff (nm)
Acetonitrile 190
Water 190
Cyclohexane 195
Hexane 200
Methanol 210
Ethanol 210
Diethyl Ether 220
Dichloromethane 220
Chloroform 240
Carbon Tet 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:
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 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.
Sample in Mobile PhaseSample 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
• 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. 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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