Chromatography 3

Question 1

Each peak on a chromatogram represents what?

Answer 1

Each peak represents a separated component of the mixture

Question 2

The chromatograph gives the analysts a number of important details regarding……..

Answer 2

the separation and identification of the components of the mixture;

Question 3

What is Retention time?

Answer 3

The time taken for a solute to reach the detector after injection (to).

Question 4

List two terms used to Determine how good the chromatographic separation is.

Answer 4

1. Resolution

2. Efficiency

Question 5

What is Resolution?

Answer 5

This is a measure of how well the peaks have separated.

Resolution is needed to be able to use peak area quantitatively

Question 6

what is the formula for Resolution?

Answer 6

R = 2(tR2-tR1)/wb1 +wb2

By convention, resolution (R) = twice the distances between the two peak maxima 2(tR2-tR1) divided by the sum of base width of the two peaks (wb1+wb2).

Question 7

The greater the value of R, the better the resolution of the two compounds. true/false?

Answer 7

true

Question 8

R value of approximately 1.5 means?

Answer 8

good base line separation

Question 9

R value of approximately 1.0 means?

Answer 9

resolution not fully complete (only about 90%)

Question 10

R value less than 1.0 means?

Answer 10

the separation is really not good enough to allow the analysis to make any quantitative analysis of concentration.

Question 11

The BP assay of betamethasone 17-valerate states that it must be resolved from betamethasone 21-valerate so that the resolution factor is > 1.0. Which of the following ODS columns meet the specification? SEE TABLE ON CHROMATOGRAPHY 3 LECTURE SLIDE 8

Question 12

Why use an Alternative form of the resolution equation ?

Answer 12

Can be used if the base line is difficult to measure

Question 13

What is the alternative form of the resolution equation?

Answer 13

Rs = 1.18 x (tR2-tR1)/Wh1 + Wh2

Question 14

The retention times of the two peaks observed in the resulting chromatogram were 3.9 minutes and 4.1 minutes. The width of each peak at half height was 0.1 minutes and 0.05 minutes respectively. What is the resolution between the two peaks?

Answer 14

1.57

Question 15

In chromatography, what does a resolution value of 0.5 between peaks tell you?

Answer 15

Peaks are poorly resolved

Question 16

state the ways to improve resolution?

Answer 16

1. Increasing the column length leads to an increase in the number of theoretical plates (N), and gives better resolution. However this has a bad effect on efficiency since there is more time for diffusion to occur, broadening the bands.
2. By altering the mobile and stationary phases you can change the selectivity of the column for different solute molecules and can therefore improve upon the resolution of compounds in a mixture
3. Anything that increases efficiency and lead to narrowing of peaks (such as low sample concentration) will improve resolution.

Question 17

The broader the chromatographic peak is relative to…….

Answer 17

relative to its retention time, the less efficient the column is at eluting it.

Question 18

Define efficiency of a column?

Answer 18

Efficiency (n) is equal to the number of theoretical plates (N) per meter

Question 19

State Theoretical plates (N)

Answer 19

This is a region of a column in which a single equilibrium of solute molecules occurs between the stationary and mobile phases. The height of this segment is H, and the number of such equilibrium in a column is N.

Question 20

The more theoretical plates (N) there are in within a set length of a column, the better the efficiency (n) of the column. True/false?

Answer 20

True

Question 21

State the equation for Efficiency of a column?

Answer 21

n= 5.54 x (tR)2/L x (Wh)2

note- the 2 is squared not multiplied by 2. see slide 13

width at half height rather than width at base is used in the equation

Question 22

A standard operating system procedure states that a column must have an efficiency of >30,000 theoretical plates/m. Which of these 15cm columns meets the specifications?

(a) Retention time of analyte 6.4 min, Wh 0.2 min.
(b) Retention time of analyte 5.6 min, Wh 0.2 min
(c) Retention time of analyte 10.6 min, Wh 0.6 min.

Answer 22

(a) 37819.7 plates/m
(b) 28955.7 plates/m
(c)11527.3 plates/m
only column a meets the specification

Question 23

A standard operating system states that a chromatography column must have an efficiency of >20,000 theoretical plates. Which of the following 20 cm columns meets this criteria?

Question 24

State the Factors effecting efficiency of a column, and causing band broadening in HPLC.

Answer 24

1. Chromatographic peaks have widths and this means that molecules of a single compound, despite having the same properties, take different lengths of time to travel through a column.
2. The longer an analyte takes to travel through a column, the more individual molecules making up the sample spread out and the broader the band becomes
3. The more rapidly a peak broadens the less efficient the column is

Question 25

Why Factors effecting efficiency of a column, and causing band broadening in HPLC occurs?

Answer 25

1. Multiple path effect: Packaging of a column is not perfectly uniform, therefore a solute molecule could take a different path through a column and travel through at different speeds. This is known as the multiple path effect, and lead to band broadening
2. Non equilibrium mass transfer: Normally when a solute is partitioned between a mobile and stationary phase it is near the surface of both, but not always. By random chance, some molecules may travel further into the stationary phase, or be to far away from the surface of the stationary phase to partition at any one moment in time. This leads to variable times for partitioning in and out of the stationary phase.
3. Sample size: Ideally, the sample size should have no effect on the ratio of solute in the stationary phase compared to the mobile phase and as you increase solute concentration, CS and CM should increase proportionally. This will lead to a perfect Gausian peak
   However if you increase the solute concentration too much, the stationary phase may become saturated. This leads to a tailing peak. Occasionally fronting can be seen (rare)
4. Diffusion is the movement of solute molecules from high concentration to low concentration. Therefore it works to broaden peaks even when the mobile phase is flowing.
   Diffusion occurs mostly in the mobile phase and is grater in gas chromatography, compare to liquid chromatography.
5. Diffusion -The longer a compound is on the column the more opportunity the molecules have had to diffuse. The random effects of non-equilibrium mass transfer are also more apparent over time since there have been many more partitioning events occurring. Hence you will tend to see peaks broaden over time.

Question 26

State parameters that can increase column efficiency in liquid chromatography;

Answer 26

Very low flow rate
Large particle size of stationary phase
Thick stationary phase coating
Irregularly shaped particles of stationary phase
Low Temperature
Uneven Stationary phase coating
Low diffusion coefficient in the mobile phase
Low diffusion coefficient in the stationary phase