360 - Chromatography

Question 1

What is the mobile phase

Answer 1

a liquid or gas that carriers the sample through the stationary phase

Question 2

What is the stationary phase

Answer 2

a solid or liquid which interacts with the sample and the mobile phase

Question 3

Chromatography can be separated into 2 groups -

Answer 3

planar and columnar based on the geometric shape of the support material.

Question 4

Planar chromatography

Answer 4

the stationary phase is supported by a flat surface such as a glass plate

Many POCT dry slide tests use the principle of planar chromatography

Thin layer chromatography is used in the biochemical genetics labs to detect inherited disorders of amino acid metabolism

Question 5

Column chromatography

Answer 5

the stationary phase is supported by a column

can be made of materials such as glass and stainless steel

two groups - based on the type of mobile phase used
- liquid chromatography (LC) the mobile phase is a liquid while the stationary phase may be a solid or another liquid
- gas chromatography, the mobile phase is a gas while the stationary phase is usually a liquid

Question 6

Chromatogram

Answer 6

a plot of the detector response as a function of the time or volume of the mobile phase that is needed to elute a compound from a column

Question 7

Retention time (tR)

Answer 7

the interval between specimen injection and the solute reaching the detector; retention time helps identify an analyte

Question 8

Retention volume (VR)

Answer 8

the volume of eluent carrier gas admitted to the column between the injection of the sample and the emergence of the peak maximum of the specified component

Question 9

How can analyzes be identified using a chromatogram?

Answer 9

Analytes can be identified by comparing their retention time or retention volume to a known analyte separated under thee same conditions

NOTE: In order to identify an analyte the peaks of the analyte peak must be separated or solved from adjacent peaks. The measurement of this separation is the resolution.

Question 10

Resolution (Rs)

Answer 10

a measurement of the separation of two neighbouring peaks; it is calculated by subtracting the earlier peak retention time or volume from the later peak and dividing the by the average baseline peak widths (Wb) of the two peaks

Question 11

T or F. The larger the resolution the greater the separation between the two peaks

Answer 11

T! Ideally, a Rsvalue of 1.5 or greater indicates complete separation of the two peaks

Question 12

T or F. The goal is to have the detector signal return to baseline in between peaks

Answer 12

T, however as the resolution increases the amount of time or volume of mobile phase used to separate the two analytes increases, this will affect turnaround time

Question 13

Peak resolution is influenced by the …

Answer 13

efficiency of the column, an analyte’s retention factor (k), and the separation factor (α) for two analytes

Question 14

Efficiency

Answer 14

a measure of chromatographic performance that usually is related to the sharpness of peaks of a chromatogram

Question 15

Theoretical plate #

Answer 15

• efficiency of a column is defined by this (N)
• the number of times an analyte transitions from the mobile phase to the stationary phase and back to the mobile phase
  
  • the greater the number of these steps the easier it is to separate two analytes with similar retention times or volumes

Question 16

T or F. Decreasing the length of a column will increase efficiency

Answer 16

F! Increasing the length

Question 17

Efficiency can be manipulated by …

Answer 17

Increasing length of column

altering the flow rate of the mobile phase

using a smaller diameter support material or smaller diameter column e.g. capillary column

Question 18

Why do we NOT want analytes to spend a long time in a column

Answer 18

The longer it spends there, the more it will diffuse = wider bands

Question 19

Retention factor (k)

Answer 19

a measure of retention in the chromatograph; it is the ratio of the volumes of the mobile and stationary phase in the column

Question 20

Small k vs large k value

Answer 20

a small k value indicates the analyte is poorly retained by the column

a large k value indicates the analyte is retained by the column and longer analysis time is required

Question 21

How is the retention factor calculated?

Answer 21

retention factor is calculated by subtracting the void time or volume from the analyte’s retention time or volume divided by the void time or volume

Question 22

a measure of the degree of separation

Answer 22

Separation factor (alpha)

equal to the ratio of the retention factors for two adjacent peaks, where the larger retention factor (k) is the numerator

Question 23

Separation factor relationship with retention factor

Answer 23

If two analytes have the same (equal) retention factor, the separation factor is one. As the value of the separation factor increases (>1) the degree of separation between the two analytes increases

Question 24

T or F. peak area calculations are more accurate than peak heights

Answer 24

T!

Question 25

External calibration/ external std technique

Answer 25

a set of standards of known concentration are processed and run with the sample

standard curve (calibration curve) is generated and the sample peak is compared to the curve to calculate the concentration

Question 26

Internal calibration/ internal std technique

Answer 26

a compound, the internal standard, is added in a known concentration to both the external standards and sample

standards and sample are processed and analyzed in the same run

calibration curve is generated by plotting the ratio of external standard to internal standard [this curve is used to determine the concentration of the analyte]

This technique helps normalizes any variation caused by pre-treatment steps

internal standards must be pure and of a known concentrations; must not normally be found in the sample and should elute close to the analyte

Question 27

T or F. The SA of the particle in packed columns is inversely proportional to pore diameter

Answer 27

T!

Question 28

types of SEC

Answer 28

classified by mobile hase
- aqueous = gel filtration chromatography
- organic =gel permeation chromatography

Question 29

most common type of high pressure pump

Answer 29

dual piston reciprocating pump
- product pulsation in moile phase

Question 30

problem caused by pulsation

Answer 30

can increase baseline signal
- solve by pulse dampers

Question 31

guard column

Answer 31

between injector and analytical column; protects it

trap particulate matter and strongly retained sample components

bind components that would irreversibly bind to analytical column

Question 32

column

Answer 32

before analytical column

• saturates mobile phase w silicates
  = conditions mobile phase before main HPLC column

Question 33

different types of HPLC detectors

Answer 33

• absorbance = compound w chromophores absorb UV or visible light
• fluorescence = compounds w fluorophores
• electrochemical = redox rxns
• conductivity = ionic solutes
• refractive index = universal detector
• mass spec = universal in full scan mode or selective ion monitoring mode

Question 34

these detectors are commonly used in HPLC

Answer 34

absorbance dectectors

Question 35

advantages of gradient elution method

Answer 35

retained sample components eluted quicker

reduced separation time

decreases band broadening

Question 36

inlet filters

Answer 36

on solvent reservoir lines

can filter out particulates

Question 37

As the size of particle in liquid chromatogaphy decreases, the resolution _______ but the pressure _____________.

Answer 37

resolution increases

pressure also increases

Question 38

pre-column

Answer 38

placed before columns w silica support particles to prevent silica particle from dissolving in the mobile phase

packed w silica-based particles; saturates mobile phase w silicates