Instrumentation

Question 1

describe preparative column chromatography

Answer 1

This involves a column packed with quite large stationary phase molecules (e.g. silica or alumina). The sample mixture is coated onto the top and the eluent is flushed down by the liquid mobile phase, taking the sample with it. Because of the large particle size efficiency is bad. Generally used for preparative work, to separate products, and not for analytical determinations.

Question 2

High performance liquid chromatography (HPLC) is?

Answer 2

High performance liquid chromatography (HPLC) is an extension of liquid column chromatography where particle size is smaller and the eluent is pumped around the column under pressure rather than relying on gravity.

Question 3

HPLC can be used for ________________

Answer 3

HPLC can be used for most forms of liquid chromatography i.e. Adsorption, partition, ion exchange, and gel permeation.

Question 4

The column packing material may be ____________, ____________,and ___________________

Answer 4

Microspheres –particulate packing with thin surface area 🡪 low mass transfer and high efficiency. Low capacity since only surface is used for partitioning. Used in ion exchange chromatography.

Porous microsphere- these particles are completely permeable, but very small (3,5 and 10μM) to give a large surface area. Also have good efficiency due to low mass transfer, but have a higher capacity.

Bonded phases –where silica has different groups bonded to the surface. This gives a wide range of possible stationary phases e.g.

Question 5

Silica gel and ODS silica gel are _________________

Answer 5

two of the most commonly used packing for normal and reverse phase chromatography respectively. The extent to which a compound is retained depends primarily on polarity (silica gel) and lipophilicity (reverse phase ODS silica gel).

Question 6

Name two of the most commonly used packing for normal and reverse phase chromatography respectively

Answer 6

SILICA GEL and ODS SILICA GEL

Question 7

Most drug molecules have both ______________ and ___________

Answer 7

lipophilic and polar groups.

Question 8

The other factor which influences the degree of retention of a particular compound is__________________

Answer 8

the nature of the mobile phase.

Question 9

Generally, the more polar the mobile phase the more ____________________

Answer 9

quickly it will elute a compound from silica gel column

Question 10

Generally, the more lipophillic a mobile phase the more ___________________

Answer 10

quickly it will elute a compound from a reverse phase OSD column

Question 11

In normal phase chromatography four commonly used solvents are:

Answer 11

hexane

DCM

isopropanol

methanol

In other of increasing Polarity/ strength
H>D>I>M

Question 12

In reverse phase chromatography, four commonly used solvents are:

Answer 12

water

methanol

acetonitrile

tetrahydrofuran

In other of increasing strength

Question 13

An ideal detector for HPLC should have;

Answer 13

Rapid and reproducible response to solutes
A linear response
Stability of operation

Question 14

There are two major classes of detectors for HPLC these are;

Answer 14

Optical detectors

Electrochemical detectors

Question 15

There are three important optical detectors;

explain each one

Answer 15

Refractivity index (RI) monitors :

UV/VIS Detector

• Fixed wavelength detectors
• Diode array spectrometers

Florescence detector
-

Question 16

explain Refractivity index (RI) monitors

and state its advantage and disadvantage

Answer 16

Refractivity index (RI) monitors :
These monitor the difference between the refractive index of a reference stream (the mobile phase solvent), compared to the eluent emerging from the column. Any solute in the elute, cases a change in RI.

Visible light is shone through a double cell containing both the reference and sample.
If the two are the same then the light is refracted to a certain extent into a photo-detector.
When the solute elutes from the column this alters the RI and this is picked up by the photo-detector.

If the two are the same then the light is refracted to a certain extent into a photo-detector.
Advantage
1. It is universal

Disadvantages – not very sensitive (need quite high concentrations of solute sensitive to about 1μg of compound); sensitive to temperature changes. Only really used if there is no other choice of detector.

Question 17

explain Fixed wavelength detectors

and state its advantage and disadvantage

Answer 17

fixed wavelength detectors:
fixed wavelength (at which you expect the solute to absorb) is selected and the absorption of that wavelength of light is monitors in the sample coming from the column. 

Advantages – Response is linear providing the concentration is not too high; it is not temperature sensitive; it is much more sensitive than RI (sensitivity up to 0.01μg)

Disadvantages – can only use for species that absorb at a set know wavelength in the UV/Vis region. You can’t really analyse unknown samples.

Question 18

explain Diode array spectrometers

Answer 18

These detectors enable you to measure absorbance of the sample coming from the column over all wavelengths simultaneously, thereby enabling you to analysis for more than one component (which absorb at different wavelengths).

Light of many wavelengths is passes through onto a holographic mirror which splits light up into all its component wavelengths and focuses them on many microdiodes, switching them all on. If some wavelengths are absorbed by the sample then not all of the wavelengths pass through and therefore some diodes switch off.

it measures absorbance at different wavelengths

Question 19

explain Florescence detector

and state its advantage and disadvantage

Answer 19

Similar to the UV/Vis detector except that it detects the light fluorescing for naturally florescent solutes (when light of the correct wavelength is shone on it).

Advantages - This is more sensitive than both RI and UV/Vis and you can detect very low concentrations (at the ng level for highly fluorescence compounds).

Disadvantages – you can only use it if the sample naturally fluoresces (very few do). However you can derivatise molecules to make them fluoresce. Phenols, amine, amino acids can all be made to fluoresce by reacting them with the following molecule;

Question 20

explain types of Electrochemical detectors

Answer 20

Conductivity detector
This is used in ion exchange chromatography where the sample components are ionic and the mobile phase has very low conductivity. It is useful for detecting anions and cations.

Redox electrochemical detector
These detectors are useful for solutes than can be oxidised or reduced.

Question 21

The extent to which a compound is retained depends primarily on ———————– and ——————-

Answer 21

Polarity(silica gel) and Lipophilicity(reverse phase ODS silica gel)