Appendix- Pipes and connectors used in HPLC - Sample preparation for HPLC analysis - Preventive maintenance of HPLC

Selection of pipeMaterialStainless StealTeflonPEEK (polyether ether ketone)Size0.1 mmI.D. x 1.6 mmO.D. 0.3 mmI.D. x 1.6 mmO.D.0.5 mmI.D. x 1.6 mmO.D.0.8 mmI.D. x 1.6 mmO.D.

Pipe MaterialStainless Steel (whole connection can be applied)Withstand up to thousand kg/cm2 pressureCorrosion in the acidic condition ( particularly less than pH 2) or high concentration of salt conditionsPEEK (whole connection can be applied)Withstand up to 250 kg/cm2 pressure and whole pH range (pH 1-14)Week against high soluble organic solvent such as chloroformTeflon (back pressure coil, reaction coil, drain tube)Strong chemical resistanceWithstand up to only 5 kgf/cm2 pressure

Pipe Size0.8 mmI.D. connection for from pump to injectordrain pipe0.5 mmI.D. connection for from pump to injectorreaction coil0.3 mmI.D. connection for from injector to columnconnection for from column to detectorback pressure coil0.1 mmI.D. resistor coil

ConnectorMale Nut SUS / Ferrule Mainly for pump, injector connectionwithstand up to 400 kgf/cm2Once ferrule is fixed, can not adjust again

Male Nut PEEK ( handy connector) Mainly for column , detector connectioneasy to handlewithstand up to 250 kgf/cm2easy to make a dead volumeMale Nut SUSFerruleMale Nut PEEK

Dead VolumeDead volume will cause a broad peak.Sample Injector ConnectionColumn ConnectionDetector ConnectionDead VolumeGood

Sample Preparation for HPLCRemoval of insoluble materialFiltrationCentrifuge (Precipitation)Control of concentrationConcentrationDilutionExtractionLiquid phase extractionSolid phase extractionSolvoysis (Hydrolysis)Derivatization for detection

FiltrationMembrane filter of 0.45 mm mesh will be used before injection in order to remove the insoluble material.

UltrafiltrationSeparation of high molecular weight compounds such as protein and low molecular weight compound

Centrifuge

Liquid Phase Extraction

Liquid-Liquid ExtractionExtracting solventn-HexaneCarbon tetrachlorideCyclohexaneChloroformDichloromethane1,1-DichloroethaneDiethyl etherEthyl acetate

Liquid-Liquid Extraction pH controlAnalyte un-ionized for successful extractioncarboxyl group (-COOH) amine group (-NH2)Mixing and emulsion formationManual or mechanical mixingMinimize emulsions by less vigorous mixingCentrifuge

Removal of ProteinsSerum 100 uLAcetonitrile for denaturation of proteinsIS (20 ug/mL) 100 uL Shake (10 sec)Centrifugation (12,000 rpm 3 min)Supernatant (10 uL Injection of HPLC)

Extraction of steroids from serumSerum 1mLExtraction (CH2Cl2 10 mL)1.25N NaOH 1 mLHydrocortisone acetate (IS) 0.1 ugOrganic phase0.1N HCl 1 mLOrganic phaseOrganic phaseH2O 1 mLDry with Na2SO4FiltrationEvaporationMobile phase 100 uLHPLC Aqueous phase

Solid Phase Extraction

Benefit of SPESolid phase extraction (SPE)ConvenientInexpensiveTime - savingReduction of organic solvent volume alternative to liquid - liquid extraction, the method traditionally used for cleaning and concentrating analytical samples

Extraction TubesNormal Phase (silica gel, almina, florisil, CN, NH2, Diol)Reversed Phase (C18, C8, C4, Phenyl)Ion Exchange (SAX, WAX, SCX, WCX)

Derivatization for detectionPost-column Derivatization - automatizationPre-column Derivatization - good separationSample itselfDerivatized SampleReagentColumn

Detector

Column

Detector

o - phthaldialdehyde (OPA)Primary amine groupDetection : Fluorescence (Ex=350nm, Em=460nm)Reaction Temperature : AmbientReaction speed : FastReagent stability : Less stable Derivatized product stability : Less stable

Preventive MaintenanceMobile PhaseSolvent gradeHow to change mobile phaseDegassingInjectorColumnDetector

Mobile phase - solvent grade -Water gradePure waterDistilled waterDeionized waterAbsorptionWavelength (nm)pure waterdeionizedwaterDue to existence of impurity,deionized water shows higherabsorption.

What's happening in use of deionized water ?Ghost peak will be appeared in gradient elution!Gradient curveGhost PeakH2O / MeOHgradient

ODS Column

Difference of analytical and HPLC gradeMethanol Acetonitrile Hexane

Cut off point If the solvent has back ground absorption,Linear range will be smaller.Noise will be bigger.Baseline stability will be poor.

Cut off point for HPLC grade solvent

TetrahydrofurnButylated hydroxytolune (BHT) as an oxidation inhibitor is containing in the analytical grade of THF. BHT has strong UV absorption. UV-detector is not suitable for analytical grade of THF.BHT is not available in HPLC grade. To prevent the explosion , THF should be kept in the dark and cool place, after seal is opened.

Chloroform3% ethanol is containing analytical grade of Chloroform to prevent a generation of phosgene which is strong toxic gas. Ethanol will influence the separation of normal phase mode.HPLC grade of chloroform is not containing ethanol. Once seal is opened, ethanol should be kept in the dark and cool place.

How to change the mobile phaseBuffer solutionWaterAqueous Organic Solvent2-propanolNon-aqueous Organic Solvent( methanol, acetonitrile etc. )( hexane, chloroform )

How to change the mobile phaserinse of suction filterSuction FilterPipe20 cm Do not touch directly

DegassingOff line degassingOn line degassingHe purgingVacuum ChamberHePumpTeflon tubeVacuum ChamberPump

Reasons for Column FailurePlugged Fit or Column PackingAdsorbed Sample & Solvent ImpuritiesMechanical Shock, forming VoidsChemical Attack of Packing Material

Plugged Fit or Column PackingBuffer solution must be filtered by 0.45 um membrane filter.Sample must be filtered by 0.45 um membrane filter.Connect the column reversibly and flow the washing solvent slowly.Open the column end and wash the filter (or change the filter).

Adsorbed Sample & Solvent ImpuritiesImpurities will cause a tailing peak.Wash a column using high soluble solvent.only 1 cm will be pollutedLC columnRemove this parts and repack the packing material.

Causes of Tailing PeaksBuilt-up of garbage on the column inletExtra column effects (dead volume) Sample OverloadIncorrect solvents for the sampleSecondary retention effectsSilanol groupResidual heavy metal

Incorrect solvent for sampleBetter do not select a high soluble solvent as a sample solvent.Better inject small amount of sample.

Solubility effectLow soluble solventHigh soluble solvent

Secondary retention effectsSilanol GroupEven modified silica gel (e.g. ODS, C8), residual silanol group are still remained on the surface area.Silanol group will strongly absorb amine compounds, therefore tailing will be happened.C18C18C18C18silica coreOHO-Silanol groupnegative charge

End cappingTo suppress the silanol group effect, an end capping type of column will be selected.C18C18C18silica coreO-TMSO-TMSTMS treatmentTMS : trimethylsilyl group[End capping type][Non-End capping type]C18C18

Secondary retention effectsResidual heavy metal Normal silica gel has heavy metal as an impurity. This heavy metal will have an interaction with chelate compounds, therefore tailing will be happened.High pure type of silica gel are available.

Mechanical Shock, forming VoidsDo not give the shock to column such as drop of column or sudden release of pressure. These mechanical shock will form voids.Do not open drain valveduring operation

VoidsVoids will cause split peaks.voidHard to repair!!!

Chemical Attack of Packing MaterialCaution : Must check the vale in terms of every items of polymer based column

Detector- bubbles problems -To solve bubbles problems, resistor coil will be very effective.0.3 mmID x 2 mL pipe as a resistor coil is used.Before attaching this coil, must check whether detector's cell can withstand pressure.

Points to be considered for maintenance Pumpcheck a leak of plunger sealwash a behind plunger seal for bufferInjectorwash an injection port after every injectionselect a suitable washing solventColumndo not keep a buffer solutionDetectorcheck a life time of lamp or electrode

2021