Chromatography Techniques

Sketch a gas chromatograph

What are the major components of a GC system?

carrier gas, injector, column, oven, detector

Describe the characteristics of GC carrier gases

- mobile phase
- high in purity (99.99% or better)
- usually He (most common), H2, N2, or Ar (inert gases)

What is the most common carrier gas for GC? What makes a carrier gas a good mobile phase?

He gas, inert

What can impurities cause in GC?

degradation of the column and cause false signals

How does an injector work?

sample is vaporized in the injector and injected into the gas stream, 1-10 microliters per injection, 30-50 degrees hotter than the highest oven temp., samples often split before reaching the column

What are the ONLY type of compounds GC can be used for?

volatile (b.p. less than 200, if can smell it can put it in GC)

What are the three types of columns used in GC?

packed with solid stationary phase, packed with inert solid support coated in liquid stationary phase, and open tubular

What are GC columns packed with solid stationary phases used for?

purifying gas, rare

Describe the properties of a GC column packed with an inert solid support coated in a liquid stationary phase

- 1-8mm in diameter
- made of glass or stainless steel
- 2-10m in length
- packing material 5-10 micrometers in diameter
- liquid stationary phase ADsorbed
- varying polarity (more polar = higher boiling point)
- MUST be thermally stable and non-volatile and not react with the analyte

What percent of compounds can be analyzed using GC?

5-10%

Describe the characteristics of an open tubular column in GC

- no solid support
- stationary phase bonded directly to the interior surface of the capillary column
- columns made of fused silica
- 100-500 micrometer diameter
- 10-100 micrometer thickness
- 10-100m long
- different functional groups for different polarities
- more efficient separation

What material is a GC column with a solid stationary phase packed with?

charcoal, molecular sieves

What material is a GC column packed with an inert solid support coated in liquid stationary phase made of?

glass, stainless steel

What material is a GC open tubular column made of?

fused silica

List characteristics of an ideal detector

- low limit of detection
- wide linear response range (can detect a wide range of concentrations)
- uniform response to a variety of compounds
- simple calibration
- rapid response and recovery (

What does a thermal conductivity detector measure?

the change in resistivity of a heated wire in the gas stream due to the introduction of a compound

What are the pros and cons of a thermal conductivity detector (TCD)?

- Pros: universal, non-destructive
- Cons: low sensitivity (anything can make a signal), no structural info
- ?: requires a reference flow (mobile phase only)

What is the most widely used GC detector?

flame ionization detector (FID)

How does GC-FID work?

analytes are burned in H2/air flame, analytes ionized and ion current detected

What are the pros and cons of GC-FID?

- Pros: more sensitive than TCD, almost universal
- Cons: destructive, no structural info

Which is more sensitive: FID or TCD?

FID

Which method is preferred: TCD or FID?

FID

How does an electron capture detector (ECD) work?

a radioactive source (63Ni) ionizes the sample, ions and e-'s generated, analytes containing electronegative atoms can "capture" the e-'s which reduces the current; can get a "dip" in the signal because the e-'s are sucked back up

What type of compounds is GC-ECD good for?

halogens, NO2, or P-containing analytes

What are the pros and cons of GC-ECD?

- pros: selective
- cons: destructive, no structural info

How does a flame photometric detector work?

analytes are burned in flame, increased temp creates an excited state, photons are emitted from samples (detected by photo multiplier tube)

What are the pros and cons of a flame photometric detector?

- pros: selective
- cons: destructive, no structural info

What compounds is a flame photometric detector selective for?

S and P

How does a mass spectrometer work?

the sample is fed into a "jet separator" to remove the majority of the carrier gas, analyte is ionized by e- impact, ions detected by m/z ratio

What are the pros and cons of a mass spectrometer detector?

- pros: gives structural info (qualitative data) on analytes, gives molecular weight (mass spectrum shows the fragments of a molecule with weights), mostly universal
- cons: EXPENSIVE ($30,000+), destructive

Why is GC done at an elevated temperature?

to keep gases from condensing in the column

What are the two conditions for temperature in GC runs?

isothermal and gradient

What is an isothermal GC run?

the oven temperature remains constant throughout the run

Describe a temperature gradient in a GC run

the temperature ramps during a run (not constant), more volatile substances elute first (at low temp), less volatile elute after

What is the difference between liquid and gas chromatography (besides the liquid/gas)?

In LLC, samples interact with BOTH the mobile and stationary phases

How does an analyte interact with the mobile phase of LC?

the analyte dissolves in the liquid mobile phase

How does an analyte interact with the stationary phase of LC?

In LSC (liquid-solid chromatography), the analytes are absorbed by the solid. In LLC (liquid-liquid chromatography), the analytes dissolve in the liquid.

What are the two types of LC?

normal phase and reverse phase

What is normal phase LC?

- the mobile phase is LESS polar than the stationary phase
- less polar substances elute first
- can increase the polarity of the mobile phase to elute more polar analytes
- solvents MUST be miscible

What is reverse phase LC?

- the mobile phase is MORE polar than the stationary phase
- more polar substances elute first
- gradient decreases the polarity of the mobile phase

Describe the mobile phases of LC

- one or more solvents mixed together
- must be degassed (remove bubbles)
- composition of the solvent is an adjustable parameter (isocratic = solvent composition remains the same throughout the run, similar to isothermal function in GC)
- can have temperature gradients: linear ramp or gradient

What is the stationary phase of LC normally made of?

silica-based beads with liquid outside

What are the stationary and mobile phases of a normal phase LC made of?

- stationary: SiO2, Al2O3
- mobile: non-polar substance like hexane, pentane, benzene, etc.

What are the stationary and mobile phases of reverse phase LC made of?

- stationary: silica-based polymer, long chain alkanes
- mobile: polar substances like H2O, CH3OH, etc.

Describe the requirements of sample introduction in HPLC

- mobile phase supply system
- need a way to mix several solvents in varying ratios
- need a way to accurately control flow rate
- need a way to inject sample into flowing stream w/o losing pressure
- column must be able to withstand high pressure (up to 1000 psi for normal flow and up to 5000 psi for Ultra PLC)

What are HPLC columns made of? Why?

many made of stainless steel to withstand the pressure

What are the two types of pumps used in HPLC?

syringe pump and reciprocating pump

What is a syringe pump used for in HPLC?

lower pressure

What is a reciprocating pump used for in HPLC?

higher flow/pressure

Which pump is more commonly used in HPLC?

reciprocating

Which HPLC pump has better control?

reciprocating

How do injectors introduce samples into the stream in HPLC without losing pressure?

use a sample loop - inject a sample into the loop which may now seamlessly incorporate the sample into the mobile phase flow without interruption

What are the two types of columns used in HPLC?

- stainless steel columns: (1-4mm diameter), 5-150cm long, packed with 3-10 micrometer solid support
- nano-bore columns: can be much smaller (0.36mm diameter), can be made of fused silica

How does a refractive index detector work?

compares the refractive index of the solvent versus the solvent + the analyte

What are the pros and cons of a refractive index detector?

- pros: most universal HPLC detector, non-destructive
- cons: can't be used with solid gradient, no structural info

How does uv-visible absorption work?

analyzes the light absorption of analytes (analyte must absorb light while mobile phase doesn't, can't analyze more than one substance at a time)

What are the pros and cons of uv-vis?

- pros: sensitive, relatively universal, non-destructive
- cons: limited molecular info

How does fluorescence work?

some molecules can absorb light at one wavelength and re-emit at another, fluorescence is proportional to concentration

What are the pros and cons of fluorescence?

- pros: very sensitive (zero background), non-destructive
- cons: very selective (most things don't fluoresce)

How does fluorescence tagging work?

most compounds won't fluoresce but can be derivatized (tagged) to form fluorescent compounds

What are the two options for fluorescence tagging? How do you determine which to use?

- pre-column and post-column
- depends on the tag you want to use

Give an example of a fluorescence tag and tell when you would tag the compound (pre- or post-column)

- ninhydrin: reacts with primary amines so can tag amino acids, amine R-group does not end up in the product so ninhydrin must be used POST-column
- dansyl chloride: since R-group retained can do pre- OR post-column
- anthranilic acid: tag for carbs, can be used pre- OR post-column

Give pros and cons of using HPLC-MS

- pros: universal (better for polar molecules), compatible with electrospray ionization, lots of structural info
- cons: destructive

What is ion exchange LC?

separation depends on the charge density of the analytes: small compact ions bind more tightly then larger more diffuse ions, dications bind more strongly than mono-cations

Describe the stationary phase of ion exchange LC?

polymer with either positively or negatively charged side groups, substituted polystyrenes often used

What is size exclusion chromatography?

the stationary phase is a cross-linked polymer with very large uniform pores, smaller molecules get trapped in the pores whereas large ones pass through, retention inversely proportional to molar mass

What kinds of molecules is size exclusion chromatography used for?

proteins, polymers

How is qualitative analysis performed?

compare the retention time of the analyte to those of known standards under the same conditions
- requires pure reference compounds
- more than one compound can have the same retention time
- NOT best for true qual., but MS has improved it dramatically

How is quantitative analysis performed?

compare peak heights or areas

What are the two types of analysis used?

calibration curves and standard addition

What is a calibration curve?

- creates standards of known concentration
- run on instrument under known conditions
- use peak height or area to create curve
- run unknown under same conditions and interpolate

Name some concerns in analysis

- linearity of Beer's Law
- variability of injection volume

What approaches can be taken to address concerns in analysis?

- dilutions: can always dilute, can't always concentrate
- internal standards: add compound with similar structure to analyte to each standard and unknown in known concentrations, ratio of peak areas or heights eliminates sample to sample variation in injection volume

Sketch a liquid chromatography setup

Sketch capillary electrophoresis

What are the two types of electrophoresis?

gel and capillary

How does gel electrophoresis work?

The sample is put in an x-linked polymer, a voltage is applied, and anions traveled down toward the anode.
- only works for charged species
- separation based on charge density (size)
- separation proportional to applied voltage, but resistive heating limits voltage

What is gel electrophoresis good for separating?

DNA and proteins

What voltage can be applied in gel electrophoresis?

less than 500 volts

How does capillary electrophoresis work?

buffer solution instead of gel, small sample volumes, higher voltages (leads to VERY efficient separations, millions of theoretical plates), doesn't work for neutrals, many detectors available

What voltages can capillary electrophoresis be used for?

High voltages, 10s of kilovolts

What detectors are available for use with capillary electrophoresis?

uv/vis, fluorescence, MS

What are the two types of flow in capillary electrophoresis?

electrophoretic flow (ep) and electroosmotic flow (eo)

What is electrophoretic flow?

cations attracted to cathode, anions attracted to anode

What is electroosmotic flow?

silica capillary has anionic side groups above pH 1-2: cations in buffer attracted to wall of capillary forming an electric double layer, next layer rich in cations and attracted to cathode, results in net buffer flow towards cathode

What is separation based on in capillary electrophoresis?

combination of ep and eo flows results in separation of anions from cations (and neutrals), separation within charge classes is by charge density

What are the mobile and stationary phases of capillary electrophoresis?

stationary: NONE!
mobile: buffer