Chapter 4 - Fundamentals of LC in LC-MS - part 1

Question 1

GC separation involves a

Answer 1

mobile phase and a stationary phase.

Question 2

In GC, the stationary phase is usually

Answer 2

an immobilized liquid of varying hydrophobicity coated onto the inner walls of fused silica capillary tubing.

Question 3

GC has ________ options for improving selectivity when compared to LC.

Answer 3

less

Question 4

In LC, the mobile phase is usually

Answer 4

a liquid mixture of water and an organic solvent plus other additives.

Question 5

In LC, the stationary phase is usually

Answer 5

materials of varying hydrophobicity chemically bonded to particles of silica as a solid support.

Question 6

With reverse phase LC, the amount of water in the mobile phase will

Answer 6

determine how strongly a hydrophobic analyte is repelled into the stationary phase.

Question 7

An LC system consists of the following components:

Answer 7

1. mobile phase reservoir
2. vacuum degasser
3. pump
4. injector
5. column
6. detector
7. data system

Question 8

LC grade solvents are required in order to

Answer 8

prevent column degradation and minimize background signals.

Question 9

A filter in LC excludes _____ micro meter matter and in HPLC it excludes ______ micro meter matter.

Answer 9

> 0.5 (LC)

>0.2 (HPLC)

Question 10

Outlet degassing occurs when

Answer 10

the mobile phase moves from the small volume of the capillary tubing to the large volume of the flow cell.

Question 11

Most effective degassing technique

Answer 11

Offline refluxing (arduous and has risks, so rarely used)

Question 12

Least favored degassing technique

Answer 12

Offline ultrasonic degassing (least efficient, and risks loss of volatile mobile phase components).

Question 13

Degassing technique that removes 80% of dissolved gases.

Answer 13

Helium sparging

Question 14

Most popular degassing technique

Answer 14

Vacuum degassing (low pressure of vacuum chamber draws dissolved gases out through a gas permeable tubing)

Question 15

Isocratic elution

Answer 15

uses a constant mobile phase composition.

Question 16

RPLC uses a _________ stationary phase and a ________ mobile phase.

Answer 16

nonpolar, polar

Question 17

As the organic solvent content increases,

Answer 17

mobile phase polarity decreases, resulting in increase of affinity of the analytes for the mobile phase and a decreased retention time.

Question 18

General problems of isocratic elution

Answer 18

Early eluting analytes may be poorly resolved.
Late eluting analytes may have broad peak shape and low sensitivity.
Not always suitable to separate complex mixtures with a wide range of chemistry and polarity.

Question 19

3 fundamentals of an organic solvent that affect chromatographic selectivity.

Answer 19

Polarity (dipole moment)
Acidity (proton donor)
Basicity (proton acceptor)

Question 20

3 organic solvents commonly used in RPLC whose properties differ significantly.

Answer 20

Methanol
Acetonitrile
Tetrahydrofuran (THF)

Question 21

Nomograms

Answer 21

can be used to alter the separation such that the overall retention time remains similar, but the selectivity is altered.

Question 22

THF is the ________ while methanol is the __________ of the three organic solvents commonly used in LC.

Answer 22

strongest, weakest

Question 23

Gradient elution

Answer 23

gradually increases the content of organic solvent to increase eluting strength.

Question 24

Advantages of gradient elution:

Answer 24

Improved resolution
Increased detection
Ability to separate complex samples
Shorter analysis times
Decrease in column deterioration

Question 25

Disadvantages of gradient elution:

Answer 25

More expensive instrumentation | Re-equilibration of column after each analysis adds time to analysis time

Question 26

Most common length of LC column

Answer 26

15 cm

Question 27

Inner diameter of LC column is between

Answer 27

1-5 mm

Question 28

Most common LC column inner diameter

Answer 28

2.1 mm

Question 29

Most common support of LC stationary phase is

Answer 29

highly pure, spherical, microporous particles of silica that have a surface area of several hundred square meters per gram.

Question 30

Irregular particles provide poorer column efficiency than spherical particles due to

Answer 30

much poorer packing homogeneity.

Question 31

The smaller the packing material particle in LC column,

Answer 31

the lower the plate height, the better column efficiency, the higher the optimum linear velocity of the mobile phase.

Question 32

The presence of metal ions within silica can cause

Answer 32

peak tailing.

Question 33

The composition of silica used for LC is

Answer 33

SiO2 xH2O

Question 34

Most silica cannot be used above pH

Answer 34

8 because it dissolves in base.

Question 35

Silanol groups are protonated at pH

Answer 35

~ 2-3

Question 36

All silanol groups interact with

Answer 36

polar and ionic analytes to different extents.

Question 37

Acidic silanol groups give the strongest reaction and the

Answer 37

worst peak tailing.

Question 38

The siloxane bond (Si-O-SiR) hydrolyzes below pH

Answer 38

2 (generally limited to use between pH 2-8)

Question 39

Monomeric stationary phases

Answer 39

are highly efficient, but not as chemically robust as those produced from di- or trichlorosilanes (which are known as polymeric bonded phases).

Question 40

Manufacturers may choose to end-cap the column surface, which involves

Answer 40

reacting the silanol groups with a sterically small but highly reactive reagent that 'caps' the polar surface silanol with a non-polar trimethylsilyl group.

Question 41

Longer chain silica provide more steric hinderance and achieve

Answer 41

a lower surface coverage.

Question 42

RPLC is characterized by having a stationary phase that is

Answer 42

less polar than the mobile phase.

Question 43

In RPLC, ____ is the most popular stationary phase.

Answer 43

C18 (highly robust hydrophobic phase which produces good retention with non-polar analytes).

Question 44

Shortening the alkyl chain of RPLC stationary phase will

Answer 44

shorten the retention time.

Question 45

The use of more polar RPLC stationary phases show

Answer 45

altered selectivity compared to alkyl phases, and can interact with polar analyte functional groups via dipole-dipole interactions.

Question 46

Traditional reverse phase columns are not suitable for use with aqueous mobile phases above _____% water due to phase collapse (or self-association)

Answer 46

95

Question 47

Stability in highly aqueous mobile phases is achieved by

Answer 47

the adsorption of a layer of water at the silica surface.

Question 48

The method of introducing stability in 100% aqueous mobile phases is to use

Answer 48

polar embedded ligands.

Question 49

Polar embedded ligands contain a modification to the alkyl chain which is usually

Answer 49

an amide, carbamate, or other suitable polar functional group.

Question 50

Water wettable phase advantages

Answer 50

polar group that gives an alternative selectivity ability to separate polar, ionizable and highly basic compounds enhanced robustness over shorter chain and polar bonded phases