Principles and applications of TLC and HPLC

Question 1

Liquid chromatography

Answer 1

*Can be performed using planar or column techniques.
*Sample is dissolved in a liquid –then transported onto or into the chromatographic device.

Question 2

TLC

Answer 2

Thin layer chromatography (TLC) is similar to paper chromatography but instead of paper, the stationary phase is a thin layer of an inert substance (eg silica) supported on a flat, unreactive surface (eg a glass plate).

Due to capillary action the solvent will begin to move up the plate and take the sample with it. As the solvent moves up the plate the different components of the solvent move up the plate at different rates and the mixture is separated.

Put a line where the sample is on the mobile phase and where the solvent stops once it has finished travelling. These lines are then used to calculate the Rf value.

Question 3

HPLC

Answer 3

High performance liquid chromatography

Uses a liquid mobile phase to transport
the analytes (sample) through the column which is packed with a stationary phase material

HPLC is now one of the most powerful tools in analytical chemistry

Question 4

Advantages of TLC

Answer 4

TLC has some advantages over paper chromatography. For example:

*the mobile phase moves more quickly through the stationary phase
*the mobile phase moves more evenly through the stationary phase
*there is a range of absorbencies for the stationary phase

Question 5

Gas chromatography

Answer 5

The stationary phase is a very thin layer of an inert liquid on an inert solid support - such as beads of silica packed into a long thin tube (this flexible tube is coiled many times inside a thermostatically-controlled oven to keep it at a constant temperature).

GC is used to separate complex mixtures. It is much better at this than thin-layer or paper chromatography.

This is because it is more sensitive - allowing the determination not only of what chemicals are in the mixture, but also how much of each chemical there is.

The mixture to be analysed is injected into the stream of carrier gas. As it passes along the column (long thin tube) it separates into the different substances.

Substances with a greater affinity (attraction) for the mobile phase reach the detector at the end of the column more quickly. Substances with a greater affinity for the stationary phase move more slowly through the column.

Gas chromatography can be used to detect banned substances in urine samples from athletes, or by forensic investigators to detect the presence of fuels that may have been used to deliberately start fires.

A gas chromatogram might show the time along the x-axis and the strength of response along the y-axis.

The amount of time that a substance takes to pass through the column is called it retention time. The retention time of an unknown substance can be compared with standard reference data to help to identify it.

Three main pieces of information can be gathered from a gas chromatogram:
*the number of compounds in the mixture - represented by the number of peaks
*how much of each compound is present - represented by the height of the peak (higher = more)
*the retention time - indicated by the position of the peak

Question 6

GC VS HPLC (GC)

Answer 6

*Samples analyzed by GC must be volatile (have a significant vapor pressure below 250oC)
*Derivatization to increase analyte volatility is possible but cumbersome and introduces possible quantitative errors
*Most GC analytes are under 500 Da molecular weight for volatility purposes GC is amenable to smaller, more volatile analytes

Question 7

GC VS HPLC (HPLC)

Answer 7

*HPLC analysis has no volatility issues, however, the analyte must be soluble in the mobile phase
*HPLC can analyze samples over a wide polarity range and is able to analyze ionic samples. Mobile phase components are selected to ensure sample solubility
*HPLC has no real upper molecular weight limit and large proteins of many thousands of
Daltons may be analyzed. Solubility in the mobile phase may preclude the analysis of very large molecules
*Large proteins and peptides can be analyzed by HPLC

Question 8

Chromatogram

Answer 8

a visible record (such as a graph) showing the result of separating the components of a mixture by chromatography.

Question 9

How do chromatographs work

Answer 9

1. They separate mixtures of substances
2. They all have a stationary phase and a mobile phase
3. The mobile phase flows through the stationary phase and carries the sample with it
4. Different components in the sample will travel at different rates due to how they interact with the stationary phase, thereby separating them

Question 10

What makes the components of a solvent separate in TLC

Answer 10

1. How soluble the compound is in the solvent
2. How much of the compound sticks to the stationary phase

Question 11

Rf equation

Answer 11

Rf value= distanced travelled by component/ distance travelled by solvent