Instrumental Quiz 4

Mobile Phase

dissolves mixture, carries components to stationary phase

stationary phase

column material, different components absorb/partition more or less

liquid-liquid separation

• most basic form of extraction

• extract compound from one phase to another

• removes analyte from matrix that has interferences in it to one that doesn’t or less

from aqueous to organic

analyte goes from aqueous phase to organic

column extraction

• solid is stationary phase liquid is mobile phase

• compounds elute at different rates because of differences in retention

tm

“dead time” or time to elute unretained solutes - takes time for the mobile phase to elute off the column

tr

retention time of components

thermodynamics of column chromatography

solute equilibrium between stationary phase and mobile phase impacts retention

factors that affect retention time

• peak width

• thermodynamics

  • all peaks would be the same height and width if this was the only cause

• multiple paths

• longitudinal diffusion

• mass-transfer

multiple paths

multiple paths pushes the solute through the column so molecules interact with the stationary phase differently

• different interactions = broader peaks because of more path options

longitudinal diffusion

Spreading of solute band peak width represents the efficiency of a column (H plate height and number of theoretical plates N)

• want the plate heiht to be small as a large number of theoretical plates is large

number of theoretical plates (N)

treating a chromatographic column as if it is made up of discrete, touching layers or the number of times an analyte goes between the mobile phase and stationary phase

mass transfer

solute is moving constantly

• the movement between phases may not maintain equilibrium

• i.e. some of the solute stays in mobile phase the whole time while other parts of the solute interacts with stationary phase from longer

van deemter vs. flow rate in column plot

• plotting each term of the equation vs. the flow rate indicates the optimal flow rate to maximize column efficiency

• use chromatogram with some sample to compute H from N

Things that increase N and increase resolution

• lengthen the column

• reduce stationary phase particle size (less paths and mass transfer decreasing plate height)

• reduce stationary phase thickness (decreases mass transfer, decreasing plate height)

• change the flow rate to the minimum H in the theoretical curve plate vs mobile phase velocity

• increase temperature for gas chromatography (lowers H

things that increase alpha

• for GC use a column with a stronger retention for solute B (increases k_B which increases alpha)

• for reverse phase LC, increase polarity of mobile phase or stationary phase (increases k_B)

• for normal phase LC, decrease polarity of mobile phase or stationary phase (increases k_B)

qualitative analysis with chromatography

• need to use standards of what you think is in sample

• can get information by putting good detector at the end (MS)

• can get okay information with okay detector (UV-Vis)

quantitative analysis with chromatography

• peak area

• can use external standards alone

• internal standards will account for instrument variability, but only get 1 pt calibration

• best is to use both external and internal standards

GC-FAME analysis

converts lipids in bacterial cell walls to esters and are then separated/detected by GC

• lipid profile used to identify bacteria

• create homogenous populations

• grow on streaked agar plate and harvest with loop and clean culture tube

• convert lipids to fatty acid methyl esters

• separate with phenyl methyl silicone columns, H2 mobile phase

• detect with flame ionization detector

esterification

reacting the carboxylic acid of the lipid with an alcohol and form an ester/water

GC gas delivery system

• carrier gas must be inert and sweeps sample from injector port onto column

• sample loops provide standard sample size

• injector port is a direct metal tube lined with silicon ionized glass (making it intert)

advantages of open tubular column compared to packed column

• better resolution (longer column)

• higher flow rates (shorter analysis and retention times)

• better sensitivity

packed columns in LC

give higher sample capacity (amount of analyte that can be separated w/o overloading)

pros of flame ionization detector for LC

• good detection limits

• sensitive

• operates over wide T range

• large dynamic range

cons of flame ionization detector GC

• destructive

• non-universal i.e. no alcohols, aldehydes or alkyl halides

How does flame ionization detector for GC work

• constant H2-air flame

• when organic analyte reaches flame, loses an electron

• ions are produced by combustion

• e- create an electric current that is part of the circuit between the jet and the collector electrode

pros of mass spec as detector for GC

• low detection limit

• universal

cons of mass spec as detector for GC

• destructive

• can be expensive

3 tactics for optimizing GC separations

• temperature programming (temperature ramping changes elution rate because elution depends on boiling point - lower bp elutes first)

• change stationary phase

• avoid overloading capacity (signified by peak tailing/falling - need to increase capacity or decrease amount of sample loaded)

qualitative analysis with GC

usually can’t use retention time to identify compound, but can pair it with a good detector like MS/IR that has a library

quantitative analysis with GC

can use calibrated peak area with internal standards (different tr) and spiking (standard addition)

advantages of HPLC

• separation

• no vaporization needed

• easier to do preparative work

• larger variety of modes to separate compounds (polarity, charge, size, etc)

preparative separation

separate compounds from a mixture w/ the idea you will use these going forward (need large capacity mLs to Ls)

analytical separation

goal is to characterize a mixture, not trying to collect purified compounds, need small capacity (microliters)

differences between LC and GC

• LC plate heights are smaller than GC

• LC velocities are smaller than GC

• LC columns are shorter than GC (N_GC»N_LC)

why are liquid plate heights smaller than GC?

gasses diffuse much more quickly than liquids meaning they have a higher diffusion coefficient meaning the plate height is orders of magnitude smaller than GC. Furthermore, at low linear velocities, plate height is determined largely from the longitudinal diffusion. Furthermore, due to this difference, the increase in diffusion coefficient, at larger linear velocities, there is more of a discrepancy between the two as B/u will be higher for GC than LC and make the plate height (H) larger in effect.

isocratic solution

elution with constant mobile phase composition

gradient elution

elution with varying mobile phase composition

increasing solvent strength LC

making the mobile phase more effective at moving the solute through the stationary phase (RP make the mobile phase more polar)

when is separation good for LC

0.5 < k < 20 and R > 1.5 for all peaks

Steps in HPLC Solvent Delivery System

1. sweeps sample onto and through column

2. mobile phase resevoirs are connected to degassers for solvent purging

3. solvent gradient mixes the solvents in the desired proportions at the time

4. mixing chamber ensures thorough solvent blending

5. pump ensures steady reproducible flow

Can add pulse damper to reduce flow fluctuations

HPLC injector valve

• fixed volume sample loop: based on length and can deliver 2-1000 microliters of sample

• load position where syringe fills size of the loop and excess goes to waste

• solvent from reservoir bypasses loop, and runs through column instead

• inject position: solvent from resevoir is diverted into loop, flushes loop contents onto column

Injection loop

• columns must be kept under high pressure which drives the mobile phase at a particular linear velocity through the small particles in the column

• can often fail and get leaks because the high pressure column exists in low pressure environment so the interface between environments can leak

guard columns

typically 10-50 mm long, <5 mm inner diameter, packed same stationary phase as analytical

analytical columns

30-150 mm long, 1-10 mm i.d. stainless steel packed with particles to perform analytical separation

preparatoty (prep) columns

longer, larger i.d. stainless steel packed with particles to perform preparatory separation

size exclusion

• packing material has canyones/pores

• small molecules hang out in canyons while large molecules come out first

• typically no gradient mobile phase needed

• gel permeation chromatography (term used by chemists who work with synthetic polymers/plastics often organic solvents)

• gel filtration chromatography (term usually used by biochemists separating biomolecules, usually aqueous solvents)

ion exchange columns HPLC

separation based on charge and related to ionic strength

• resin is negatively charged and interacts with the positively charged ions (cation exchange)

• can use positively charged ions (anion exchange)

• can be used for protein collection

• change in the salt concentration of the mobile phase to change the amount of ions available for exchanging

• lowest net charge at the selected pH elute first, highest charge are most strongly retained