Lecture 1-4

Question 1

What does Plate Theory describe?

Answer 1

Provides a theoretical treatment of zone broadening and migration of solutes in a column

Question 2

Why are they called “Theoretical Plates?”

Answer 2

Because equilibrium between the SP and the MP occurs in each unit

Question 3

What is Smax?

Answer 3

the maximum rate of solute escape as the peak leaves the chromatographic column

Question 4

What is the relationship between moles and peak area?

Answer 4

moles is proportional to peak area, increasing m, increases peak area

Question 5

What is the relationship between S(max) and peak height?

Answer 5

the maximum rate of solute escape is proportional to peak height

Question 6

What is column efficiency?

Answer 6

the efficiency of a column is best judged by HETP (height equivalent theoretical plate, at a fixed column length, more plates result in smaller plate height and more efficiency

Question 7

What is the partition coefficient, K?

Answer 7

K describes the solutes affinity for the SP, the large the K value, the more affinity for the SP

Question 8

What causes peak tailing?

Answer 8

1) Strongly polar groups present on SP which retain solute more on polar sites than on other sites
2) Solute/SP interactions > solute/solute interactions

Question 9

What causes fronting?

Answer 9

1) injection of excessive amount of solute to the column

2) Solute/SP interactions

Question 10

What are the shortcomings of plate theory?

Answer 10

1) Diffusion is never instantaneous
2) Spreading of the chromatographic zone by longitudinal diffusion from one Theoretical plate to another does not occur at all (False)
3) Column is assumed to consist of a discrete number of volume units (False)
4) MP flows through the column in a continuous fashion

Question 11

What are migration rates dependent on?

Answer 11

they are dependent on the magnitude of the equilibrium constants for the reactions by which the solutes distribute themselves between the MP and SP

Question 12

What is retention time?

Answer 12

the time it takes after sample injection for an analyte peak to reach the detector

Question 13

What is dead time?

Answer 13

the time it takes an unretained solute to pass through the column

Question 14

What is the capacity factor?

Answer 14

tells how much solute is retained compared to unretained compound

Question 15

What is the selectivity factor?

Answer 15

a fraction consisting of the partition ratios of two retained species on a chromatographic column, by convention the more strongly held species is in the numerator, the greater the difference in retention times as well as capacity factors, the higher the selectivity

Question 16

What is resolution?

Answer 16

provides a quantitative measure of its ability to separate two analytes
Rs = 1.5
Rs > 1.8 too much separation

Question 17

What is resolution proportional to?

Answer 17

Rs is proportional to square root of N and the square root of L

Question 18

What is the cross-sectional area of a column proportional to?

Answer 18

it is proportional to the mass of an analyte, this affects the speed of analysis because volumetric flow rate is proportional to cross sectional area

Question 19

What is peak asymmetry?

Answer 19

measured at 10% the peak height,
As > 1, BC>CA, peak tailing
As BC

Question 20

What is peak capacity (PC)?

Answer 20

it is defined as the number of peaks that can fitted into a chromatogram between the dead point and the last peak, each peak being separated by 4 st.dev.

Question 21

Impact of PC

Answer 21

1) Larger the tr/tm greater PC

2) If k’

Question 22

Describe the effect of efficiency, selectivity factor, retention factor on column resolution

Answer 22

At fixed alpha, N values —–> increase k’ , increase in Rs
At fixed k’, N values —–> increase in alpha, increase in Rs
At fixed alpha, k’ ——> increase in N, increase in Rs
–Improvement in Rs, for increasing N is not as dramatic as for increasing alpha

Question 23

What does measuring at the baseline represent?

Answer 23

When peak width is measured at 2.4% of the peak height, this peak represents 99.7% of the molecule in a band

Question 24

Describe the variation in H

Answer 24

depends on the average linear velocity of the mobile phase, H is a measure of the ratio of bandwidth and retention time, easiest way to minimize H is to optimize MP velocity

Question 25

What is the relationship between H and B?

Answer 25

H is inversely proportional to MP velocity when it is associated with B

Question 26

What is the relationship between H and C?

Answer 26

H is directly proportional to MP velocity when it is associated with C

Question 27

More on H with B and C

Answer 27

at u = 0, B/u is largest –> B/u decreases with increase in u
at u = 0, Cu is zero –> C increases with increase in u

Question 28

What is optimum velocity?

Answer 28

the minimum value of H at some value u, depends on B and C

Question 29

Why does the plate height increase in the Van Deemter plot? (ie efficiency decreases at low flow rate)

Answer 29

solute spends a long time in the column and is broadened by longitudinal diffusion (B term increases)

Question 30

Why does the plate height increases at high flow rate?

Answer 30

not enough time for the solute to equilibriate between MP and SP, therefore C, the mass transfer is poor (increases) due to high mobile phase velocity

Question 31

Why can longer columns not be used in HPLC?

Answer 31

high pressure drop

Question 32

What are the two main reasons for Eddy diffusion?

Answer 32

1) multiple paths adopted by the same molecules

2) stagnant pools of MP retained in SP

Question 33

What causes multiple paths?

Answer 33

non-homogenous packing of SP is the main reason for multiple paths

Question 34

What causes stagnant pools?

Answer 34

a poorly packed column

Question 35

What does the A term depend on?

Answer 35

the A term is independent of mobile phase velocity, but depends on the stationary phase particles, which may inhomogenously packed

Question 36

What causes inhomogenous packing?

Answer 36

1) particle size or diameter
2)packing consistency
good separation and minimum band broadening will be achieved using small particles with narrow size range that are uniformly packed

Question 37

What is diffusion?

Answer 37

random motion that tends to spread molecules uniformly

Question 38

Where does longitudinal diffusion take place?

Answer 38

along the column axis and parallel to the movement of the mobile phase
–this forward and backward diffusion will let some molecules move ahead while others lag behind the band center

Question 39

Why is the B term inversely proportional to the mobile phase?

Answer 39

when the MP flow rate is faster, the time between injection and the detection is shorter
therefore, shorter time, less time the molecules spread out by longitudinal diffusion

Question 40

What is D(m)?

Answer 40

the mobile phase diffusion coefficient and is directly proportional to B/u

Question 41

What is Cm? Cs? (RMT)

Answer 41

Cm is resistance to mass transfer in the mobile phase

Cs is resistance to mass transfer in the stationary phase

Question 42

What is mass transfer?

Answer 42

when there is a slow equilibriation of solute between MP and SP which results in band broadening

Question 43

C term and particle diameter

Answer 43

increasing the particle diameter increases C, and therefore decreases N. Increase in Dm decreases the C term

Question 44

Mass transfer at slow MP velocity

Answer 44

the adsorption/desorption process to and from SP is usually resulting in poor mass transfer

Question 45

Mass transfer at high MP velocity

Answer 45

molecules will not be able to desorb the solute from SP
therefore C,su is proportional to u
as k’ decreases, Hs increases

Question 46

Why is longitudinal diffusion inversely related while mass transfer is directly proportional to the flow rate of the MP?

Answer 46

LD – molecules move in direction parallel to MP flow

MT – molecules move perpendicular to MP flow, faster MP flow rate, less time to equilibriate with MP