HPLC

Question 1

Hvad står HPLC for?

Answer 1

High Performance Liquid Chromatography

Question 2

Hvilke to metoder findes til injection?

Answer 2

Manuel/automatisk

Question 3

Hvad er fordelen ved højere temp?

Answer 3

Hurtigere runs, større detectability og nogle stoffer bliver bedre opløst

Question 4

Hvad sker der i den kolonnen/ stationære fase?

Answer 4

Seperationen

Question 5

Hvad er pumpens funktion?

Answer 5

skaber højt tryk i den mobile fase og samplen (højt tryk - konstant flow)

Question 6

Detektoren måler..?

Answer 6

Retentionstiden

Question 7

I reversed phase HPLC er den mobile fase…?

Answer 7

Polært solvent

Question 8

Hvad består den stationære fase af i reverse fase?

Answer 8

nonpolære eller lipofile perler

Question 9

Hvordan filtreres prøven inden HPLC’en?

Answer 9

gennem et 0,5 um filter

Question 10

Hvad skal laves på forhånd for at bestemme koncentrationer?

Answer 10

Standard kurve

Question 11

Arealet under kurven er propertionalt med…?

Answer 11

Koncentrationen af stoffet

Question 12

Hvilken matematisk analysemetode anvendes til at bestemme koncentrationerne af stoffet?

Answer 12

Lineær regression

Question 13

Lipofile molekyler tiltrækkes af hinanden. Hvad hedder kraften der tiltrækker nonpolære stoffer (lipofile) til hinanden?

Answer 13

London dispersion krafter

Question 14

Hvad kaldes tiden som samplen residuerer i den stationære fase relativt til dens tid i den mobile fase?

Answer 14

Retentionsfaktoren

Question 15

Hvordan kan man sikre sig en god sample seperation? (efter samplen er forberedt korrekt)

Answer 15

Der anvendes en kolonne og en mobil fase med forskellig polaritet

Question 16

Hvordan ser et godt peak ud?

Answer 16

Det er symmetrisk og perfekt separeret

Question 17

Hvis der ikke sker separation, hvordan ser peaket ud så?

Answer 17

kun 1 stort peak

Question 18

Hvis en prøve har været opbevaret under ekstreme forhold, og der derefter kommer mindre peaks, hvad betyder det så?

Answer 18

lavere koncentrationer

Question 19

Perlerne i kolonnen kaldes for…?

Answer 19

Den stationære fase

Question 20

Hvad er nogle af de mest brugte mobile faser i reverse phase?

Answer 20

Methanol og acetonitril

Question 21

Hvordan laves en standard/kalibreringskurve?

Answer 21

Kendte koncentrationer køres gennem systemet og der laves lineær regression på peaks

Question 22

hvordan er stationær og mobil fase i reversed-fase?

Answer 22

hydrofobisk stationær; polær mobil

Question 23

forholdet mellem hvor lang tid det tager en prøve at passere gennem den stationære fase sammenlignet med hvor lang tid det tager at passere gennem søjlen med den mobile fases hastighed kaldes ?

Answer 23

Retentionsfaktoren (k)

Question 24

i reverse-fase HPLC injiceres en prøve indeholdende 2 forbindelser. Forbindelse A er mere lipofil end forbindelse B. hvilken forbindelse vil eluere først fra kolonnen?

Answer 24

B

Question 25

Hvad kaldes ratioen af retentionsfaktorer af peaks?

Answer 25

selektivitet

Question 26

Kolonne effektiviteten kan måles ved at beregne teoretisk 'plate' nummer ved at sammenholde .... og ...

Answer 26

retentionstid ; bredde ved baseline

Question 27

det teoretiske 'plate' nummer kan øges ved at..?

Answer 27

Gøre størrelsen af perlerne i den stationære fase mindre

Question 28

Når der anvendes mindre perler i den stationære fase, medfører det... ?

Answer 28

højere tryk

Question 29

Fortæl om normal fase hplc

Answer 29

Uses non polar MP like he ans uses polar SP - silica bonded with polar functional group like Cyano useful for separation of polar compounds as Sp is polar Polar compounds retained longer on SP and higher retention times eluent strength (how quickly it elites) is increased by adding more polar solvent. More polar MP = Higher éludent strength of MP = faster solute elute from column as polar solute will attract to it most non polar elute first as polar react with SP

Question 30

Fortæl om reversed phase hplc

Answer 30

Use polar MP (methanol/water) and non-polar SP(C18) use to seperate non-polar compounds ans more non polar will elute last a éludent strength is increased by adding less polar solvent popular as peaks tend to be narrow and symmetrical mist polar solutes elute first as non polar react with SP

Question 31

Basale komponenter i instrumentet

Answer 31

-Solvent delivery system (a pump) -sample injection valve -pre column or guard column -high pressure column detector -computer

Question 32

Fordele v. HPLC

Answer 32

- Wider range of possible mobile phase properties - wider choice of SP and detectors than GC - suitable for non - volatile components - useful for analytes that decompose at high temps

Question 33

Typer af detektorer i HPLC

Answer 33

- spectrophotonetric - UVVIS - electrichemical - mesures current due to oxidation of solute - others - refractive index detector or LS-Ms (more difficult than GC-MS)

Question 34

Faktorer der påvirker separationen

Answer 34

- Adjust the retention factor (interaction between the solute and SP/MP) by changing solvent strength or no. plates - adjust the selectivity by changing temp and MP/SP composition

Question 35

Solvent krav for HPLC

Answer 35

Rene; uden partikler; ikke for viskøs

Question 36

Beskriv den analytiske kolonne

Answer 36

Packed with SP normally made if stainless steel 5-30cm length 1-5mm diameter 25,000- 100,000 N heating column - reduces retention time, improves resolution, degraded SP narrow columns are preferred as require less sample & generate less waste

Question 37

Isokratisk vs gradient elution?

Answer 37

Iso occultes when the elution is performed with a single solvent or solvent mixture at a time. Run at a solvent composition of 100% methanol and then 90% gradient involves changing the solvent composition during the elution process. Start with 20% methanol ans than gradually change to 80% in 15 minutes. Useful to seperate solutes with widely differing polarities as it ensures they can all be examined in one run and sharpens peak if différent polarities ans retention times, use gradient

Question 38

Sample introduktion/indførelse

Answer 38

Samples are dissolved in MP and passed through a filter to remove impurities before injection Injection is via a port that has an injection valve and sample loop to waste or column in modern HPLC, sample injection is automated - precise and accurate sampling

Question 39

Pre-kolonne eller 'guarding column'?

Answer 39

Protects analytical column | has same packing material as analytical column

Question 40

Optimering af resolution?

Answer 40

For ions, adjust ph of MP for others, adjust MP polarity. Changing from a more polar to a less polar decreases retention time in Reversed phase ans changing from less polar to more polar decreases retention time in normal

Question 41

Ion-Exchange Chromatografi (IEC eller IC)

Answer 41

Baseret på udveksling af ioniske analytter med mod-ioner af de ioniske grupper der er attached til SP

Question 42

Size exclusion chromatography (SEC)

Answer 42

separationsmetode kun baseret på analyttens molekylære størrelse

Question 43

Forklar tailing

Answer 43

Stofferne kommer ikke ud samtidigt, så peaket bliver bredere