Exam II Content

What are the primary components of gas chromatography?

• gas cylinder, column, injector, detector, and data system

What is the importance of the injector?

• the port is at a higher temperature so the sample vaporizes instantly when injected

• the whole system is kept at an elevated temp to keep everything in the gas phase

Advantages of the Open Tubular (Capillary) Columns over packed columns in GC

• higher efficiency (more theoretical plates) → produce narrower peaks

• better resolution → peaks are narrower so they can be separated more easily

• faster analysis

• greater sensitivity → narrow peaks give higher peak heights, making small amounts of analyte easier to detect

What are the 3 main types of carrier gases?

• Helium, Hydrogen, and Nitrogen

What is the main advantage and disadvantage of Helium as a carrier gas?

• A: inert, good efficiency

• D: expensive

What is the main advantage and disadvantage of Hydrogen as a carrier gas?

• A: fast separations

• D: flammable

What is the main advantage and disadvantage of Nitrogen as a carrier gas?

• A: cheap, good for electron capture

• D: slow separations

What is the general elution problem?

• when a mixture contains compounds with very different boiling points so one temp cannot give a good separation for both early and late eluents

What are ways to solve the General Elution problem?

1. Temperature programming → gradually increase temp

2. Use different columns/stationary phase

Gradient Program

• changing the composition of MP during the separation

• does not work for GC because since the carrier gas does not interact strongly with analytes, changing the composition would not significantly affect separation

What are the 2 main parameters for retention time?

• boiling point (volatility) and polarity (interaction with the stationary phase)

What is the relationship between boiling point and retention time?

• higher boiling point → higher retention time

Order these from least to most polar: Water, acetonitrile, methanol, THF, hexane

Hexane, THF, Methanol, Acetonitrile, Water

Normal phase: Order these from weakest to strongest eluent: Water, acetonitrile, methanol, THF, hexane

Hexane, THF, Acetonitrile, Methanol, Water

Common Stationary Phases and their characteristics

• Diphenyl: nonpolar, intermediate van der waals forces, high temperature

• Cyanopropylphenyl: intermediate, dipole-dipole + van der waals, lower temps

• Carbowax: strongly polar, dipole-dipole, hydrogen bonding, van der waals

• Biscyanopropyl: strongly polar, dipole-dipole + van der waals

What are the 3 most common GC detectors?

Thermal conductivity, flame ionization, and electron capture

Thermal Conductivity Detector

• GC

• Nondestructive

• Linear

• Nonspecific- least sensitive

Fluorescence Detector

• LC

• Nondestructive

• Linear

• Specific

UV-Vis Detector

• LC

• Nondestructive

• Linear

• Nonspecific

Electron Capture Detector

• GC

• Nondestructive

• Nonlinear

• Specific → extremely sensitive

Flame Ionization Detector

• GC

• Destructive

• Linear

• Specific → very sensitive

Split Injection vs. Splitless Injection

• in a split injection only a small fraction enters the column

• split injection has high concentration samples

• in a split injection there is low risk for column overload

• split injection → lower sensitivity

Kovats Retention Index

• standardized value based on the retention times of n-alkanes that allows retention data to be compared across different GC systems and helps identify all compounds

Advantages of Open Tubular (Capillary columns) over packed columns in LC

• higher efficiency → more theoretical plates, narrower peaks, and better separation

• less Eddy diffusion → no packing particles so MP flows more uniformly

• lower pressure requirements → less resistance to flow

• better resolution

Disadvantages of Open Tubular (Capillary columns) over packed columns in LC

• small sample capacity, low flow rates, and more difficult instrumentation

Isocratic Elution

mobile phase composition remains constant throughout entire separation

LSC vs LLC

• LSC is solid absorbent and LLC is liquid film on support

• LSC uses adsorption while LLC uses partitioning

• LSC has surface interactions and LLC has solubility differences

Normal phase vs Reversed phase LC

• in normal phase the stationary phase is polar but in reversed it is nonpolar

• in normal phase the mobile phase is nonpolar and in reversed it is polar

• in normal phase nonpolar compunds elute first and vice versa

• in normal phase polar compounds elute last and vice versa

What are the most common LC Detectors?

UV-Vis, Refractive Index, and Fluorescence

UV Vis Detector

• very good linear range

• good sensitivity

• moderately universal (only compounds that absorb UV)

Refractive Index Detector

• moderate linear range

• low sensitivity

• most universal

Fluorescence Detector

• moderate linear range’

• highest sensitivity

• least universal (only fluorescent compounds)

The Partition Coefficient (K)

• how a compound distributes between 2 immiscible phases

• large K: solute prefers the stationary phase → longer retention time

• small K: solute prefers the mobile phase → shorter retention time

The Distribution Coefficient (D)

• similar to K but includes all forms of the solute present

• K and D are only the same when only one form of the compound exists

Organic Acids D vs pH plots

• at low pH → mostly neutral HA; D is large

• at high pH → mostly ionized A- ; D decreases

• the partition coefficient K corresponds to the plateau region where compound is fully neutral

• the pKa occurs at the midpoint of the curve where pH=pKa

Organic Bases D vs pH plots

• at low pH → mostly protonated BH+; D is small

• at high pH → mostly neutral B; D increases

Solvent Extraction vs Partition Chromatography

• in solvent extraction there are one or few partition steps but in partition there are many repeated partition steps

• solvent uses a separatory funnel but partition uses a chromatography column

• solvent has coarse separation but partition uses high resolution separation

Theoretical Plates (N)

• are a measure of column efficiency in chromatography

• more theoretical plates → narrower peaks

Van Deemter Equation

H=A+B/u+Cu

• A: Multiple paths (Eddy diffusion)

• B: Longitudinal diffusion → faster flow rates don’t give solute band as much time to diffuse (broaden) → solute bandwidth is proportional to the square root of time → D is proportional to temp

• C: Equilibration (resistance to mass transfer) → inversely proportional to temp

What is fronting?

• occurs when the front (leading edge) of a peak becomes stressed out instead of being symmetric

• most common cause is column overload

What is Tailing?

• when the back of the peak becomes stretched out

• caused by strong adsorption to the stationary phase (interact strongly)

Retention Time (tr)

• total time it takes a compound to travel from injection point to the detector

Adjusted Retention Time (t’r)

• retention time corrected for dead time

Migration Time

the amount of time it takes a solute to reach the detector in Capillary Electrophoresis

Reverse phase: Order these from weakest to strongest eluent: Water, acetonitrile, methanol, THF, hexane

• Water, methanol, acetonitrile, THF, Hexane

Ion exchange Chromatography

• separates compounds based on charge

• common applications are protein purification, amino acid analysis, pharmaceuticals

Electroosmosis

• the movement of liquid through a capillary when an electrical field is applied

• only effective in small diameter capillaries because of large surface area to volume ratio, the charged layer affecting a larger portion of the solution and allows heat to dissipate quickly

Ways to increase the number of Theoretical Plates

• increase column length

• smaller stationary phase particles

• improve column packing quality

• optimize flow rate

LC Separation on C18 SP Column

• SP is nonpolar

• most polar compounds elute first

Electrophoretic Mobility

• describes how fast an ion moves in an electric field due only to its charge and size

Apparent mobility

• the actual mobility observed experimentally in the capillary

Capillary Electrophoresis has very high efficiency because…

• no stationary phase (separation occurs in electric field)

• all molecules move at same velocity

• very small diameter → allows efficient heat dissipation and use of high electric fields

• minimal eddy diffusion

What are the most common detectors in Capillary Electrophoresis

UV-Vis, Fluorescence, and Conductivity

Advantages of Capillaries for Electrophoresis

• much higher efficency

• faster separations

• better heat dissipations

• very small sample consumption

• quantitative analysis

Relationship between resolution and theoretical plates

Rs scales to √N

Internal Standard Method

• quantitative technique where a known amount of a compound (the internal standard) is added to all samples, calibration standards, and blanks

• a constant, known amount of internal standard is added to every solution

• chromatogram is run and peak areas are measured

• calibration curve is made using known concentrations

• ratio used to determine analyte concentration

To increase N in CE…

• increase applied voltage

• use a longer capillary

• reduce diffusion

• use smaller injection volumes

Problem with large relative retention

• very long analysis times

• peak broadening

• poor peak shape and sensitivity