Chromatography Techniques

What is the mobile phase in a GC

The carrier gas, usually helium, hydrogen, nitrogen, or argon

What compounds can be used in a GC?

Volatile compounds

What are the volumes of an injection in a GC?

1-10uL

What are the three kinds of columns that can be used in a GC?

1. Packed columns with solid stationary phase (charcoal, molecular sieves)
2. Packed columns with inert solid support coated with liquid stationary phase
3. Open tubular columns with stationary phase bonded directly onto surface of fused silica capillary column

Name the detectors used with a GC

Thermal Conductivity Detector (TCD)
Flame Ionization Detector (FID)
Electron Capture Detector
Flame Photometric Detector
Mass Spectrometric Detector (Mass Spec)

Give the characteristics of TCD

Measures change in resistivity of heated wire in gas stream, non-destructive, universal, poor sensitivity, requires reference flow, no structural info

Give the characteristics of FID

Most widely used detector, analyte ionized in H2/air flame and ion current is detected. More sensitive than TCD, almost universal, destructive, no structural info

Give the characteristics of electron capture detector

Radioactive source ionizes sample. Good for NO2, P-containing analytes, or halogens. Destructive, selective, no structural info

Give the characteristics of flame photometric detector

Analytes burned in flame, selective for Sulfur and Phosphorous compounds, destructive, no structural info

Give the characteristics of mass spec

mass-->charge of whatever is injected, relatively universal, destructive, expensive, gives structural info

What are the two types of experiments (relating to oven temp in GC)?

Isothermal- oven remains at a single temperature
T gradient- temperature raises during the run (more volatile things elute first, less volatile elute after)

Fundamental difference between GC and LC?

Samples interact with both m.p. and s.p.

What is the mobile phase and stationary phase in liquid chromatography?

mobile phase: liquid- analytes dissolve
stationary phase: solid (LSC)- column-analytes absorb
liquid (LLC)- HPLC-analytes dissolve

What are the two types of LLC?

Normal phase (m.p. is less polar than s.p. and less polar analytes elute first)
Reverse phase (m.p. is more polar than s.p. and more polar analytes elute first)

What are the two compositions of the mobile phase for LC?

Isocratic- similar to isothermal in GC where solvent composition (whether mixture or not) remains the same
Gradient- Like T programming in GC, can be either linear ramp or gradient

What are the two types of stationary phase for LC?

Normal phase, m.p. non-polar so non-polar analytes elute first
Reverse phase, m.p. polar so polar analytes elute first

What are the requirements of HPLC?

Flow rate needs to be accurately controlled, columns are made of stainless steel packed with stationary phase to withhold pressure from the column

Name the detectors used with LC

uv-vis absorption, fluorescence, mass spec

Give the characteristics of uv-vis

analytes can absorb light, with absorption proportional to concentration. Sensitive and relatively universal, limited molecular information, non-destructive

Give the characteristics of fluorescence

Very selective!!! Fluorescence proportional to concentration, while most things don't fluoresce they can be derivatized to form fluorescent compounds. Tags can be added pre or post column depending on tag.

More mass spec characteristics (for LC)

universal- better for polar molecules, basically same as GC mass spec.

Name the other types of LC

Ion exchange and size exclusion chromatography

Give the characteristics of ion exchange

s.p. is a polymer with either positively or negatively charged side-groups, separation depends on charge density of analytes where small compact ions bond more tightly than large ones
Dications bond more strongly than monocations
Useful for amino acids and peptides

Give the characteristics of size exclusion chromatography

s.p. is a cross linked polymer with large, uniform pores. Smaller molecules become trapped in pores while larger ones are excluded, retention is inversely proportional to molecular mass. BIG size differences needed

What is qualitative analysis?

Performed by comparing retention times of analyte to those of known standards under the same conditions. Requires pure reference compounds. More than one compound can have the same retention time so not the best, but mass spec helps.

What is quantitative analysis?

Performed by comparing peak heights to calibration curve. Needs standards of known concentration that are run on the instrument under known conditions. Peak height or area is used to make curve, and then unknown is run under same conditions.

How are internal standards useful for quantitative analysis?

Ratio of the peak areas or peak heights of standard and analyte eliminates sample to sample variation in injection volume

What are the two major types of electrophoresis?

gel and capillary electrophoresis

Give the characteristics of gel electrophoresis

gel is an x-linked polymer (agarose or polyacrylamide), voltage is applied across the gel and anions migrate towards anode, useful for DNA and protein separation

Give the characteristics of capillary electrophoresis

gel is replaced with capillary fused with silica filled with buffer solution. Can withstand higher higher voltages than gel so gives better separation. Doesn't work with neutral compounds, can be used with uv-vis, fluorescence, and ms detectors

What are the two different flows for capillary electrophoresis?

Electrophoretic flow and electro-osmotic flow

Give the characteristics of electrophoretic flow

cations attracted to cathode, anions to anode. Capillary gives ability to apply larger voltages without without having to worry about melting capillary (gel would melt)

Giver characteristics of electro-osmotic flow

Silica capillary has anionic side groups above pH 1-2, cation buffer attracted to wall of capillary forming electrical double layer. Next layer is rich in cations and attracted to cathode

How does the combination of electrophoresis and electro-osmotic flow lead to the separation of anions and cations

electro-osmotic (eo) is generally stronger than electrophoresis (ep), so eo flow brings everything to the detector quicker while ep gives separation between different ions