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.