Unit 5 - Intro to Separation Science and Liquid Chromatography

Question 1

Separation science

Answer 1

separate and isolate components of the mixture, then determine amount of each analyte

Question 2

Liquid-liquid extraction

Answer 2

Separatory funnel separates two immiscible liquids and the analytes are separated between two phases. The partition coefficient depends on solvent, pH, ionic strength, and temp

Question 3

Solid-liquid extraction

Answer 3

Coated with hydrophobic/hydrophilic extraction phase. If coating is nonpolar, it’ll help extract nonpolar compounds from the solution. Same applies to polar

Question 4

Elution chromatography

Answer 4

Column has a stationary phase and mobile phase and the analyte partitions depending on its affinity towards the stationary and mobile phase. Depends on polarity

Question 5

Multistep phase separation

Answer 5

use counter-current extraction. Put sample in and certain molecules will prefer the mobile or immobile phase and move. When eq’m is achieved, repeat process until the molecules are separated and is pure

Question 6

Adsorption chromatography

Answer 6

Some molecules are attracted to the surface of the chromatography and are held back. Too much of these molecules will cause surface saturation and it won’t adsorb on the surface. Partition coefficient goes down because of overload

NOTE: the stationary phase is a solid and the mobile phase is usually liquid or gas

Question 7

Solid adsorption media examples

Answer 7

silica (SiO2) - polar, slightly acidic

alumina (Al2O3) - polar, neutral or slightly basic

Question 8

Thin layer chromatography (TLC)

Answer 8

Quick and simple; low cost
A mixture is spotted on silica or alumina (stationary phase) and the bottom plate is immersed in solvent and travels up the plate by capillary action (mobile phase) and allows the mixture to separate into spots or bands

Question 9

Column chromatography (purifies reaction products)

Answer 9

Column contains silica gel or alumina and load mixture on top of the column. Elute with organic solvent as mobile phase and the bands for each component separate out, which is then collected

(think of NH4+ experiment in lab)

Question 10

High Performance Liquid Chromatography (HPLC) and Ultra Performance Liquid Chromatography (UPLC)

Answer 10

Stationary phases are made up of small solid particles. As particle size decreases, separation efficiency increases because the column is more densely packed. Drawback is the very high back-pressure, where gravity is insufficient to move the mobile phase through the column and pumps are required

Question 11

Analytical and Preparative columns

Answer 11

Analytical columns are used for trace analysis and detection. For particles smaller than 10 micrometers

Preparative columns are used for purification of synthesized compounds. Usually larger than analytical columns

Question 12

Bonded stationary phases - silica particles

Answer 12

The silica particles are packed inside columns. These particles are coated with functional groups that modify their polarity

Question 13

Partition Chromatography (absorption)

Answer 13

There are two liquids that separate the solutes. One liquid is the stationary phase, and the other is the mobile phase, and the solutes prefer one or the other due to their partition coefficients. The solute is dissolved in the liquid phase that is coated on the surface of solid support

Question 14

Normal and Reverse phase HPLC

Answer 14

Normal phase HPLC separates polar molecules and is generally more stable than reverse phase. Its stationary phase is polar: silica, amino, cyano. Reverse phase separates non-polar molecules and is more commonly used. Its stationary phase it nonpolar: C18, C8, phenyl. The elution of analytes are inverted between normal and reverse phase HPLC

Question 15

What does sparging mean in HPLC instrumentation?

Answer 15

It is when helium is bubbled through the solvent reservoirs to sweep out dissolved air since helium is insoluble in most solvents

Question 16

Significance of the flow cell in HPLC UV-VIS detectors

Answer 16

They maximize sensitivity by increasing the path length term, b, in the Beer-Lambert Law, A=Ebc, because of its Z-shape. Sensitivity is determined by Eb

Question 17

Significance of retention time in HPLC chromatogram

Answer 17

analytes can be differentiated and identified on the bases of their retention time. The size of the peaks indicates something about the quantity of the corresponding analyte

Question 18

Dead time (t_m)

Answer 18

Time required for the mobile phase to travel the length of the column

Question 19

Retention time (t_r)

Answer 19

Time required for the analyte to elute from the column

Question 20

Adjusted retention time (t’_r)

Answer 20

Retention time corrected for dead time

Question 21

Retention factor (K)

Answer 21

A measure of the relative amount of time an analyte spends in the stationary phase

Question 22

Resolution

Answer 22

The degree to which two peaks are separated in a chromatogram

Question 23

Theoretical plates

Answer 23

They are a conceptual representation of a separation step. The longer the analyte is on a column, the larger the number of plates. The wider the peak, the lower the number of plates. More theoretical plates, better separation

Question 24

What causes tailing, a type of asymmetrical peak shape?

Answer 24

When there is more than one retention mechanism for the analyte
Other causes include when a sample dilution solvent is a stronger eluent than the mobile phase, contamination of the stationary phase, and sample overload

Question 25

What causes fronting, a type of asymmetrical peak shape?

Answer 25

Cause by bad column packing. Fixed by replacing column

Question 26

Band broadening

Answer 26

The longer the analyte stays in the column (retention time), the broader the peak shape becomes, but total area remains constant. Therefore, peak area is better for quantitative analysis than peak height

Question 27

how does plate height contribute to the peaks

Answer 27

More plates result in narrower peaks

Question 28

Eddy Diffusion

Answer 28

Different substances take different paths in the column length. Leads to the diffusion of mixtures through the column

Question 29

Longitudinal Diffusion

Answer 29

Diffusion of analyte from the concentrated middle region of the band and goes from high concentration to low concentration (center to the edges). molecules travel in the mobile phase quickly, analytes spend less time in the column

Question 30

Mass transfer effect

Answer 30

Temperature dependent. The higher the T, the faster it is to reach eq’m and results in small mass transfer effect. The trade-off is the increase in temperature affects longitudinal diffusion, since it depends on thermodynamics (faster to diffuse)

Question 31

Peak width vs. separation

Answer 31

Smaller peak widths are better, but the draw back is it might not be separated properly

Question 32

Effect of particle size on separation efficiency

Answer 32

As particle size decreases, monodispesrity increases, then the various paths through the column become more uniform in distance

As particle size decreases, diffusion lengths (the distance an analyte must travel to reach the stationary phase) decrease

Question 33

Totally porous particle vs superficially porous particle

Answer 33

Superficially porous particle provides shorter diffusion lengths and have better efficiency. Improve C term in van Deemter eq. (rate of mass transfer)

Question 34

Isocratic elution

Answer 34

the same mobile phase composition is used throughout the separation. Not efficient in eluting things with different hydrophobicity/polarity

Question 35

Gradient elution

Answer 35

the mobile phase composition is gradually changed during the separation. In reverse phase HPLC, the mobile phase is gradually made less polar

Temperature programming is the GC equivalent of the gradient elution in LC. T has a much greater effect in GC than LC

Question 36

Gas chromatography

Answer 36

Still partitioning, but temperature drives analytes into the gas phase. Analyte can remain in the vapor phase, condense on the stationary phase, and dissolve in the stationary phase

Question 37

GC capillary columns

Answer 37

long and narrow. Bonded phase on inner wall of capillary column

Question 38

Split/splitless injection

Answer 38

insert sample into GC and often uses a syringe in narrow columns. Rapid vapourization of sample. Some bled off to waste, some enters the column (valve controls these amounts)

Question 39

Flame ionization detector

Answer 39

Insensitive to most inorganic compounds and does not respond to fully oxidized carbons. Has very low detection limits. Good for trace analysis and is low cost. Has fast response.

Question 40

Significance of Flow rate in GC

Answer 40

optimizing it is very important for the efficiency of GC separations than LC separations. A flow rate that’s too high or low causes broadening, which we do not want

Question 41

Significance of Temperature in GC

Answer 41

Separates compounds into distinct identities. At an optimal temperature, the band is narrow and separate from other bands

Question 42

Examples of non-polar mobile phase in liq chrom

Answer 42

Hexanes
Diethyl ether
ethyl acetate
dichlorochromethane

Question 43

Examples of polar mobile phase in liq chrom

Answer 43

water
methanol
acetonitrile
propanol