Lecture11-12 Flashcards
Comparing problems with GC and HPLC:
1.both difficult separation (possible) 2. both speed 3. both automation (possible) 4. adaptation of system to separation problem (by change in stationary phase) (by change in stationary and mobile phase) 5. application restricted (lack of volatility, thermal decomposition) (insolubility)
HPLC restricted to analytes that are soluble to elute analyte: ____ ____ and suitable ____ ____ must be chosen.
stationary phase
mobile phase
Gas chromatography:
Mobile phase: ____
stationary phase: _____________
analyte: _________
gas
usually a nonvolatile liquid, but sometimes a solid
gas or volatile liquid
Three important differences between GC and HPLC:
- Diffusion coefficient of the sample in mobile phase much smaller in HPLC than in GC (reduces speed of analysis by HPLC). 2. viscosity of mobile phase higher in HPLC than GC (high flow resistance in HPLC vs GC). 3. compressibility of mobile phase under pressure negligibly small in HPLC (not in GC) -> not dangerous.
Gas Chromatography: can only cope with analytes that are _____/ can be _____ intact at higher temperature. only ______ of known organic compounds can use this method.
volatile
evaporated
~20%
Separation process in GC: ____ ____ transported through column by ____ ____ phase = ____ ____ (high purity,
gaseous analyte gaseous mobile carrier gas stationary nonvolatile liquid fine solid
Carrier gas could be: ___, ___, ___. Sample injected to injector oven through a ___ ___ ___. Column -> ____ enough to provide sufficient ____ ____ for elution of analytes.
He, N2 or H2
Silicone rubber septum
hot
vapor pressure
Open tubular columns (____): most frequently used ____ ____ in GC (___ ___) compared to packed columns they offer: ___, ___, ___ and ___.
A-term: ____; ____
capillary columns
stationary phase fused-silica
higher resolution, shorter analysis time, greater sensitivity, lower sample capacity.
flow-rate; amount of sample injected.
Outer wall of column: ____ ____ (____um thick); inner column: ____ ____ (____ um thick), up to ____oC
WCOT
SCOT
PLOT
stationary phase 0.1-5 polyimide coating 0.1-5 350oC Wall-coated open tubular column Support-coated open tubular column Porous-Layer open tubular column
Particles’ diameter ____ mm cannot use MS as detector (need ____).
____ column -> ____ resolution
____ pressure -> _____ sample capacity
Resolution = root(N) /4 * (r -1)
As column length _____, so does # of theoretical plates N.
>0.32 vacuum narrower higher higher less N = plate number r = unadjusted relative retention increases
As GC column ages -> ____ ____ can be lost, ____ group (____) exposed and tailing can increase “____”.
stationary phase
silanol
Si-OH
bleeding
Retention determined by ____.
Strong retention on ____ ____ ____(H-bonding)
volatility
polar stationary phase
To reduce tendency of stationary phase to “bleed” from column at high temperature, ____ bond (____ attach) it to ____ surface good idea: ____ ____ ____ (k of standard or N).
chemically covalently silica monitor column performance
High operating temperatures -> ____ phase will ____ -> ____ bleed -> ____ background in detector -> ____ signal-to-noise and detector ____.
____ liquids ____ volatility at elevated temperature; potential: offer ____ ____. Solid for ____ ___ columns: ____, ____(____) and ____ ____.
stationary decompose slow
elevated reduced contamination
Ionic low novel selectivities
porous OT polymers alumina(Al2O3) molecular sieves
Packed columns: contains ____ ____ ____ coated with ____ stationary phase or solid itself = ____ ____. ____ sample capacity, but give ____ peaks, ____tr and ____ resolution.
fine solid particles non-volatile stationary phase
Better broader longer less
Despite inferior resolution used for ____ ____. (ie. to isolate mg amounts)
Solid support: often ____ that has been ____ to reduce ____ ____ to ____ ____.
preparative separations
silica silanized hydrogen bonding polar solutes
Temperature and pressure programming: in GC temperature often ____ ____ separation, ____ analyte vapour pressure.
____ temperature at different rates, ____ retention time and ____ peaks.
increased during increases
Increase decreases sharpens
Most GC columns come with 2 temperature labels:
- ____ temperature limit: ____ kept at this for a long time without damaging it.
- ____ temperature limit: ____ ____
Isothermal column
Programmed few minutes
Carrier gas: ____ compatible with most detectors -> frequently used.
____ often not used as > 4% explosive. Sample injection through ____ ____ -> sample not immediately ____ + ___ washed needle and ____ plug expels solvent from needle (many ____)
He H2
rubber septum evaporated
solvent air autosamplers
Split injection: if analytes of interest __0.1% of sample -> complete injection contains ____ ____ material -> split injection (____ on column).
Proportion of sample that ____ ____ reach column: split ratio (_______). Quantitative analysis can be ____ because ____ ____ may not be ____ from run to run.
> too much 0.2-2%
does not (50:1 - 600:1)
inaccurate split ratio reproducible
Splitless injection: analytes of interest ____ of sample -> ____ ____. most appropriate (___ on column).
Initial cold temperature 40C ___ BP of solvent -> solvent ____ -> ____ peaks.
< trapping sharp
On-column injection: preferred for quantitative purposes and for samples that ____ above their ___. Detectors: qualitative analysis: use ____ ____ (MS) and compare results to spectral library. Co-chromatography (___): ____ compound added to unknown sample if added compound identical with component -> ____ peak area ____
decompose BP
mass spectrometry spiking
authentic relative increases
____ = tentative with 1 column, but firmer when carried out on several columns/ different ____ ____. Quantitative analysis based on ____ of ____ ____. It’s ____ response concentration range: ____ which peak area ~ to ____ of component.
Identification stationary phases
area chromatographic peak
linear over quantity
Thermal conductivity detector: since ____ sensitive than other detectors used for ___ columns -> useful for packed columns and operated with ___.
Electrical resistance ____ and voltage across filament changes -> ____.
Measures ability of a substance to conduct heat analyte -> ___. He high____.
less OT He
increases signal
hotter
thermal conductivity
Flame ionization detector: column eluate burned in a mixture of ___ and ___: ___ atoms -> ___ radicals. DL 100-fold lower than ____ ____ ____, linear response range: ___. Electron capture detector: particularly sensitive to ___ containing molecules, conjugated carbonyls, nitriles, nitro compounds and organometallic compounds, but relatively insensitive to ____, ____ and ____
H2 and air carbon CH
thermal conductivity detector 10^7
carbons, alcohols and ketones.
Carrier gas must either be ___ or ____. Gas ionized by ____ electrons (___). formed electrons attracted to ___ -> produce a small ____ when analyte with high ____ ____ enter -> they capture some ____ and decrease ____.
____ -> extremely sensitive (comparable to MS)
N2 or 5% CH4 in Ar. high-energy beta-rays anode current electron affinity electrons conductivity ECD
Other detectors: ____ ____ detector measures ____ ____ from phosphorus, sulfur, lead, tin or other selected elements.
flame photometric
optical emission
GC-MS: MS is a sensitive detector that provides both ____ and ____ information (identification of spills). Selected ion monitoring or selected reaction monitoring -> measure one component in a complex ____ of poorly ____ compounds. Lower detected limit by factor ____ compared to m/z scanning.
qualitative quantitative
chromatogram
separated 100-1000
m/z 293 (____ ion) selected by mass filter Q1, passed on to ____ cell Q2, ____ ____ ion m/z 264 Q3. Sample preparation: process of ____ sample into a form suitable for analysis. ____ ____ ____ extracts compounds from liquids, air or even sludge ___ using a solvent. Key component: ____ thich film of stationary phase as used in GC. Procedure of analysis expose ____ to sample -> analyte is ____.
precursor collision fragmentation prominent transforming Solid phase micro-extraction without 10-100um fiber extracted
The Chromatographic process: to increase ____ in chromatography: increase the rate at which ____ ____ ____ between stationary and mobile phase. In GC with ___ ___ (OT) column -> ____ established rapidly by ____ diameter of column -> ____ can diffuse quickly between ____ and ____ phase.
efficiency solute molecules equilibrate
open tubular equilibration reducing
molecules channel stationary
Continue for the Chromatographic process: since ____ in a liquid is several-fold ____ than in gases -> ____ of solvent channel too great for analyte molecules to traversed in short time -> not ____ to use __ columns in ____. HPLC conducted with ____ columns -> ____ ____ don’t have to diffuse far to encounter ____ phase.
diffusion slower diameter
feasible OT HPLC
packed solute molecules stationary
Small particles -> ____ efficiency, but require high ____. It needed to force solvent through ____ ____ (contains fine ____/____). Particle size: ____. The ____ the particles, the ____ the pressure.
high pressure packed column particles/silica
1.7-10um smaller higher
Smaller particle size leads to: ____ plate number, ____ pressure, ____ optimum run time and ____ detection limit. 2004, equipment became available to employ ____ um particles with pressures up to ____ MPa (____ bar, ____ psi) -> substantially ____ resolution and ____ run time -> less ____ phase consumption.
higher higher shorter lower
1.5-2 100 1000 15000
increased decreased mobile
Another penalty for small particle diameter is increased ____ ____: _____ of a column up to ___ oC warmer than ____ wall -> to avoid band ____ column diameter ____ mm for ___ um particles. The column: ____ columns (____cm): stainless steel can be easily clogged if small dust particles enter column(____), if you filter ____ phase through ___ um filter and use a ____ column (same ____ phase as main column).
frictional heating centre 2 outer broadening <=2.1mm 1.7um HPLC 3-30cm (sample/solvent) mobile 0.5um guard stationary
The stationary phase: most common support: ____ ____ particles. Bare silica -> ____ chromatography. ____ groups form weak bonds (____, ____, ____ and ____) which is referred to as ____ with any molecule in ____.
microporous silica
adsorption
Silanol (dipole-dipole, interactions, H-bonding and pi-complex bonding)
adsorption vicinity
Absorption strengths: saturated hydrocarbons < ____ < aromatics < ____ < nitro compounds < ____~ aldehydes ~ ____ < alcohols < ____ < amides < ____.
olefins ethers esters ketones sulfoxides carboxylic acids
If molecule has several functional groups -> most ____ one determines ____ properties. ____ gel surrounded on all sides by ____ phase -> sample molecule can only be ____ if it ____ more strongly than solvent. All sample molecules arranged on ____ surface so their ____/ ____ close to ____ group. Strength of ____ also depends on ____ factors -> ideal to separate ____.
polar retention Silica
mobile absorbed interacts
silica functional group / double bond silanol
interaction steric isomers
