Hi High Per gh Performanc formance e Li Liqu quid id Chr - - PowerPoint PPT Presentation

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Hi High Per gh Performanc formance e Li Liqu quid id Chr - - PowerPoint PPT Presentation

Nov 13, 2023 309 likes •777 views

Hi High Per gh Performanc formance e Li Liqu quid id Chr Chroma omatogra tography phy Lis Smith Alder Hey Children's Hospital Nana Ghansah Great Ormond Street Children's Hospital HPLC HPLC HPLC is a separation technique based on a

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SLIDE 1

Hi High Per gh Performanc formance e Li Liqu quid id Chr Chroma

  • matogra

tography phy

Lis Smith Alder Hey Children's Hospital Nana Ghansah Great Ormond Street Children's Hospital

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SLIDE 2

HPLC is a separation technique based on a solid stationary phase and a liquid mobile

  • phase. Separations are achieved by partition,

adsorption, or ion-exchange processes, depending upon the type of stationary phase used

HPLC HPLC

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SLIDE 3

Ove Overvi rview of HP ew of HPLC LC

Solven ent(Mo t(Mobi bile le Phase) ) Pump Auto to sampler ler Column mn (Station tionary ary Phase) e) Detecto tector r –UV, , Fl Flores escen cence, ce, Elect ctrochem chemical ical ECD

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SLIDE 4

Wa Water ters s Al Allian liance ce

Mobile Phase Detector

Auto Sampler

Column Pump

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SLIDE 5

Th Ther ermo mo Ul Ulti timate mate 300 3000

Mobile Phase Pump Auto sampler Column UV Detector Florescence Detector Electrochemical Detector

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SLIDE 6

Why UHPLC ?

Small particle size (<2 µm) results in higher theoretical plate numbers for more resolution and/or faster separations. smaller particles cause less dispersion (band broadening) flow rate can be increased with less loss of efficiency compared to larger particles the chromatographic efficiency, N, is directly proportional to the ratio of column length and particle diameter, L/d This means that column length can be shorter without losing resolution Faster analyses using higher flow rates and shorter column Narrower columns are often used which also reduces eluent consumption smaller volumetric flow for same linear flow down the column 1ml/min on 4.6mm i.d. = 0.21ml/min on 2.1mm i.d.

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SLIDE 7

HPL HPLC vs U C vs UHPLC PLC

Traditional HPLC UHPLC Pressures up to 300bar 4000psi Pressures up to 1400bar 20,000psi Longer column larger particle size Shorter Column Smaller particle size Longer run times Shorter run times = higher throughput Uses more mobile phase Uses less Mobile Phase Cheap Expensive Often bail out with High Pressure Prone to Blockages

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SLIDE 8

Co Colu lumn mn Sele Selecti ction

  • n
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SLIDE 9

sa

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SLIDE 10

Re Reve vers rse e Ph Phase ase Ch Chro romat matograp

  • graphy

hy

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SLIDE 11
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SLIDE 12

Ed Eddy dy Dif Diffusi fusion

  • n
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SLIDE 13

Pl Plasma asma Ph Phenylalanine nylalanine + Ty Tyro rosine sine

Partisil column 250mm x4.6 10um Temperature 25C Mobile phase 60mL Acetonitrile &2mL 70%perchloric Acid in 1L Deionised water 100uL plasma & 200uL 10%perchloric Acid -mix & spin 200uL supernatant & 200uL internal std methyl phenylalanine in mobile phase Flow 1.0ml/min injection volume 10uL

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SLIDE 14

Pl Plasma ma Ph Phen enyl ylalan alanine ine + Ty Tyrosi

  • sine

ne

Phenomenex Kinetex C18 2.6um 100mm x4.6 Temperature 50C use a guard column

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SLIDE 15

Blood Spot Phenylalanine

Punch 2 blood spots Add 150uL 70%Ethanol /internal Std Mix for 1 min Inject 10uL elutent onto Partisil 250x4.6 10u column

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SLIDE 16

Blood Spot Phenylalanine

Punch 2x blood spots Add 150uL 80% ACN mix for 1min & spin Take 100uL of supernatant & dry under nitrogen at 37C for 2mins Reconstitute with 100uL Internal Std Inject 10uL onto Kinetex 100x4.6 2.6um temp 50C flow 1.4mL

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SLIDE 17

Column: Waters Picotag 4µm, 3.9 x 300 mm Temperature: 46ºC Flow rate: 1ml/min Injection volume: 20µl Mobile Phase A: Sodium Acetate buffer adjusted to pH6.55 with acetic acid Mobile Phase B: Acetonitrile/Methanol / Water (50:15:35)

Current LC method – Blood Spot Standard

Rs=1.15, 1.25

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SLIDE 18

A = 79 % B= 21 % COLUMN = ECLIPSE PLUS 2.1, 150MM X 1.8UM

V

min 1 2 3 4 5 6 7 8 9 mAU

  • 5

5 10 15 20 25 DAD1 A, Sig=254,8 Ref=360,100 (BRANCHCHAIN\BCA000117.D) 2.848 5.244 5.725 6.518 6.718 6.937 8.198

Rs=1.181

Flow rate = 0.95 ml/min (pressure = 1100 bar)

Valine Isoleucine Leucine Norleucine

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SLIDE 19

Each of the following items need to be optimised in order to generate a satisfactory chromatogram

  • Mobile phase composition
  • Bonded phase chemistry
  • Column and packing dimensions
  • Injection volume
  • Sample pre-treatment and concentration
  • Mobile phase flow rate
  • Column temperature
  • Detector parameters

Whether you are using HPLC or UHPLC the troubleshooting remains the same

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SLIDE 20

Go Good Hou

  • d Housek

sekeepin eeping

Use HPLC grade solvents for mobile phase Equilibrate column well before use Use a guard column to prolong the life of the column Never leave the lamp or ECD on without mobile phase going through the system Flush the system with 50% methanol after use Record daily maintenance & running pressures for each assay Record any instrument or assay problems and actions taken to resolve the issues.

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SLIDE 21
  • Replace solvents regularly- composition may change
  • Filter solvents-remove- particulate matter could damage

components

  • Degas solvents-removal of dissolved gases from the mobile

phase helps to prevent bubble formation, lead to loss of prime

Goo Good d Hou Housek sekeeping eeping

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SLIDE 22

Tr Trou

  • uble

ble sh shoo

  • oti

ting ng

Hi High Pres h Pressu sure re Lo Low Pres w Pressur sure Po Poor Chro r Chromato matograp graphy hy

slide-23
SLIDE 23

Hi High gh Pr Press essure ure

High Organic content of Mobile Phase-try slowly increasing flow when column first installed ?Blocked guard column-change filter ? Blocked column – try reversing column Does the pressure drop when the column is removed? Yes - try replacing column No - ?blockage in tubing – replace tubing…make sure you replace like for like otherwise chromatography will be effected!

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SLIDE 24

Lo Low Pr w Pres essu sure re

Loo

  • ok fo

k for r leaks aks Is the s the co column umn ins nstalled talled pro roperly? erly? Is Is th the pri rime me va valve ve op

  • pen?

n? Los

  • st

t Pr Prime me –so sonic nicate ate ch check eck va valve ves s (m (mak ake su sure re re re-installed installed co correctly) rrectly)

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SLIDE 25

Ch Chec eck k Val Valves ves

Pump Head

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SLIDE 26

Po Poor

  • r Chr

Chrom

  • matog

atograp raphy hy

Be Be Sy Systematic tematic Ch Check ck Mo Mobil bile e Phas hase e +Sol Solvent vent line ine Ch Check ck Co Column umn + Co Column lumn Te Temperature mperature Ch Check ck sample mple vi vial al +sample sample preparation eparation Ch Check ck Tub ubing ing and d co column umn fittings ttings –vo void/ id/dead dead vo volumes lumes

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SLIDE 27

Dea Dead Vol d Volum umes es

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SLIDE 28 m in 0.5 1 1.5 2 2.5 3 m AU 10 20 30 40 50 60 DAD1 A, Sig=254,4 Ref=360,100 (2010-02-05\EXPT-000005.D)

Ferrule depth incorrectly set

m in 0.5 1 1.5 2 2.5 3 m AU 20 40 60 80 100 DAD1 A, Sig=254,4 Ref=360,100 (2010-02-05\EXPT-000006.D)

Ferrule depth correctly set

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SLIDE 29

Pl Plasma asma Ph Phenylalanine nylalanine + Ty Tyro rosine sine pro robl blem em

Wrong Column!

Nova Pack Column Kinetix Column

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SLIDE 30

Pl Plasma asma Ph Phenylalanine nylalanine + Ty + Tyro rosine sine p pro roblem blem

Clue: No Internal Standard!

Vitamin A+E sample 75%methanol 25% Ether

Problem: Wrong sample vial injected

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SLIDE 31

Plas asma ma Phe henylal nylalanine anine + + Tyr Tyros

  • sine

ine pro roble lem

Wash line (50% Methanol) and MP lines switched

Solvent line B in Mobile Phase Solvent line C in 50% Methanol Wash Solvent line C in Mobile Phase

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SLIDE 32

Disap sappearing pearing Pe Peaks! aks! Vi Vitami tamin n A +E +E

Sample Preparation -Expired Hexane!

Expired Hexane No Vitamin E peak

Fresh Hexane

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SLIDE 33

Mo More D re Dis isap appea pearing ring Pe Peak aks! s! VMA VMA +H +HVA VA

ECD needs cleaning

Pre ECD clean Post ECD clean

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SLIDE 34

Cle Cleani aning ECD ng ECD

Cell 2 +1000mv for 3min

  • 500mv for 3mins

+1000mv for 3min Turn cell off for 10minutes with mobile phase running through Turn cell back on

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SLIDE 35

Is this ECD Dead? (VMA +HVA)

ECD cells turned off for 1.5hrs with MP still flowing

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SLIDE 36 Q2414 (2,1) HPLC,Instrument5.150213C5L,2,1,1 Acquired 13 February 2015 17:40:27
  • 20
  • 10
10 20 30 40 50 60 70 Response 10 20 30 40 50 60 Retention time ASP GLT HCYEIC ASA HYPRO SER ASN GLY GLN TAU HIS cit CIT + NH3 THR ALA ARG PRO AAB TYR VAL MET ILEU AILEU LEU NLEU PHE TRY ORN LYS AEC

Normal profile

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SLIDE 37 Q2414 (2,1) HPLC,Instrument5.150219C5LCAA,2,1,1 Acquired 19 February 2015 19:22:05
  • 30
  • 20
  • 10
10 20 30 40 50 Response 10 20 30 40 50 60 Retention time CSFASP CSFGLT HCYEIC ASA HYPRO CSFSER CSFASN CSFGLY CSFGLN CSFTAU CSFHIS cit CIT + NH3 CSFTHR CSFALA CSFARG CSFPRO AAB CSFTYR CSFVAL CSFMET CSFILEU CSFALIEU CSFLEU NLEU CSFPHE CSFTRY CSFORN CSFLYS AEC

Alanine and Threonine peak higher than usual Sloping baseline ? Contamination, Check blank

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SLIDE 38

Blank on working system , flat straight baseline

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SLIDE 39 BLANK (1,1) HPLC,Instrument5.150219C5LCAA,1,1,1 Acquired 19 February 2015 17:49:17
  • 30
  • 20
  • 10
10 20 30 40 50 Response 10 20 30 40 50 60 Retention time

Problem contamination in system ? Buffer A ? Buffer B, Needle wash, poor house keeping ,Millipore water system Negative peak Extra lump Extra lump Falling baseline

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SLIDE 40

Blank sample run with HPLC gradient – not straight as slide ?

On this occasion check blank Check samples

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SLIDE 41

Amino acid QC sample / where are peaks?

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SLIDE 42

Troubleshooting Has anything change ?

  • Initial conditions 100% buffer A psi usually 1800 psi, on this run 1600 psi
  • Buffers changed on day of run
  • Other machine chromatography fine with same buffer
  • Therefore Problem with that particular HPLC Machine ?
  • Possible cause ?
  • Answer
  • Buffer A and buffer B line switched

Phase organic buffer, wash sample off column

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SLIDE 43

A = 79 % B = 21 % COLUMN = POROSHELL EC-C18 2.7µm, 2.1 x 150mm BLOOD SPOT STD

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SLIDE 44

UHPLC MSUD Method

A = 79 % B = 21 % COLUMN = POROSHELL EC-C18 2.7µm, 2.1 x 150mm BLOOD SPOT STD

Chromatography usable, not great

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SLIDE 45

Poor chromatography ? split peaks why ? High pressure Answer Several problems, poor fitting connections= void volume, dirty frit ,column aged

Change column !

Split peak

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SLIDE 46

Use HPLC grade solvents and water Degas solvents Filter buffers Purge lines daily Change lamp as required Run seal wash , mainly aqueous Check wires and connections Guard column or inline filter Needle wash, mainly organic, check for bubbles leaks Filter samples

Prevention is better than cure!