Chromatography by Y1CSu7R

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									Teachers’ Chemistry Conference
17-19th April 2011
                                        Dr. Ellie Hurst
                                            Separation Science

                                      Department of Chemistry

                                            Durham University

                                       Tel: +44(0)191 334 2187

                                                             Chromatography- Teachers’ conference
                                                                               17th-19th April 2011

                          Introduction to Chromatography
What is chromatography? What is it used for?
An analytical separation technique; separation based on the affinity of compounds for the stationary
(column) and mobile phase (eluent). Changing the stationary and/or mobile phase will alter the
degree or order of separation of a specific mixture.


Analytical scale (femtograms (10-15 g)-milligrams (10-3 g)) - purity checks, monitoring reactions,
identification, quantification etc.

Preparative scale-(milligrams to kilograms), used for larger scale isolation of pure compounds.

Applications: Lots!!

Biological/Biochemical                                   Petroleum industry (e.g. Motor racing)

Pharmaceutical                                           Food industry

Medical                                                  Forensic Science

Drug testing                                             Home land security

Environmental                                            Organic Chemistry

   Geological                                               Confirmation of synthesis product(s)

   Climate change                                           Mixture analysis

   Pesticide residues                                       Identification of unknowns

   Land/water contamination                                 Quantitation

Who discovered it?
In 1906 a Russian botanist named Mikhail Tsvet
passed a solution of coloured leaf pigments through a
column packed with chalk particles. The individual
pigments separated from each other and were
identified as coloured bands, hence the name chroma
(colour) and graphy (writing)                                        Figure 1. Mikhail Tsvet


                                            Chromatography- Teachers’ conference
                                                              17th-19th April 2011

                      Theory of Chromatography
How does it work?

Using solvent as the mobile phase, a mixture of compounds (solute) can be
pushed through the stationary phase (e.g. silica); the separation of the
individual components in the mixture will depend on their affinity for each


                                                           Chromatography- Teachers’ conference
                                                                             17th-19th April 2011

                         Methods Of Chromatography
How can we use it?
          Paper chromatography
                                                                      methanol   acetone

                                          Separation of pen inks using different solvents

               ratio of fronts R f  
                                          distance from start tocentreof spot
                                          distance from start tosolvent front
The Rf value allows us to compare the separation of different components, under the same

     Thin layer chromatography (TLC)

    e.g. Thin layer chromatographic separation of spinach pigments

                                                                          solvent front


                                                                           chlorophyll A
                                                                           chlorophyll B




                                                                    Chromatography- Teachers’ conference
                                                                                      17th-19th April 2011

           Column chromatography

         Separation of ink using a silica
         packed column

                                   bellows                                                   sand
                                                  silica packing material

         Collect fractions one at a time                                             cotton wool plug
         and analyse further…

Paper, TLC and column chromatography are all techniques that are easily replicated in the
laboratory. This is especially useful for coloured compounds (unless you have a UV lamp). However,
it is useful to also understand how these methods can be scaled up and be used in a more
sophisticated and diverse manner for industrial or research purposes...


                                                           Chromatography- Teachers’ conference
                                                                             17th-19th April 2011

                                   Industrial techniques
  In industry... chromatography    needs to be quicker and more reproducible than the single-use
  practical methods described above. Gas Chromatography (GC) and High pressure (sometimes called
  High performance) Liquid Chromatography (HPLC) enable samples to be run in quick succession with
  accurate measurements. HPLC systems use a solvent pump to mix the solvent and increase the
  pressure through the stationary phase (column). GC systems use a carrier gas to elute compounds
  from the column.

   Main components of HPLC instrument; what are their purposes?

                                                                              Eluent retains / separates
                                                                              components (mobile

     Column- stationary
     phase- retains and
     separates components                                                        Autosampler
                                                                                 (detects and injects

Detector(s) produce a
signal when a
component passes
through it
                                                                                    Pump (solvent
                                                                                    manager, mixes
                                                                                    ratio of solvents)

                                               Figure 2. A HPLC system


                                                                Chromatography- Teachers’ conference
                                                                                  17th-19th April 2011

                                    Figure 3. Diagram of a HPLC system

Figure 3 shows that two different solvents can be run through the system at the same time; by
changing the ratio of solvent (and therefore the polarity) through an LC run, the separation of
components can be improved further; this is called a GRADIENT run. If the ratio of the solvents was
kept constant, this would be called an ISOCRATIC run.

 Figure 4. Various LC columns available; different types for different experiments e.g. external coatings may
depend on inert or high pressure experiments, different internal packing for separation of specific compounds
                             and different lengths of column to aid separation.


                                                          Chromatography- Teachers’ conference
                                                                            17th-19th April 2011

Once the compounds have been eluted from the column, the compounds can be detected and
analysed by a computer program. There are various detectors utilised in analytical chemistry, which
can be used on their own but often a combination of two or more detectors gives more accurate
results and better identification. Some examples of detectors are shown below:


                       Mass Spectrometer (MS)


                       Light scattering

                       Refractive index



Types of LC conditions:
There are two types of LC conditions; Normal or reverse phase- these depict the polarities of
stationary and mobile phases as shown in Figure 5 and 6.

                              Figure 5. Normal-Phase Chromatography


                                                            Chromatography- Teachers’ conference
                                                                              17th-19th April 2011

                              Figure 6. Reversed-Phase Chromatography

 Note: The separation of the yellow, blue and red components have
                       reversed! Why is this?
    Example chromatogram:

                           Figure 7. Gradient reverse phase LC chromatogram

Figure 7 shows a typical chromatogram from a HPLC column. The eluent (mobile phase) is made up
of water and acetonitrile. The different compounds are eluted from the column as the polarity of the
mobile phase changes on a gradient run (i.e. from 0% to 100% acetonitrile) and will have different
retention times (x axis). Different compounds will have a different response (y axis) even if they are
the same concentration (see compounds 1 and 5 for example, in Figure 7). This is because some


                                                             Chromatography- Teachers’ conference
                                                                               17th-19th April 2011

compounds might stick to the column for longer (broad peaks) or be eluted more quickly from the
column (sharp peaks), therefore the area under the peaks should be calculated and the compounds
should always be calibrated before measuring.

Gas Chromatography-          similar theory to LC but sample is vaporised (by high temperature),
injected onto a column (stationary phase) and the compounds are eluted by a gas mobile phase.

Samples are separated by their chemical properties and boiling point.

                                                                                    Carrier gas pushes
                                                                                    compounds through the


                                                                                          Oven with column

                                        Figure 8. A GC system

  Figure 9. Diagram to show a GC system. Note that a GC column is much longer and narrower than an LC
               column (Figure 4) as there is no need to withstand high pressure (unlike HPLC).


                                                               Chromatography- Teachers’ conference
                                                                                 17th-19th April 2011

Types of separation
There are various ways to separate the compounds in a mixture, by polarity, electrical charge or size.
The methods chosen will depend on the type of molecule. Some examples are shown in Figure 10.


                         e.g. Non polar molecules will be retained on apolar columns

                                                Electrical Charge

        Needs positive/neutral particles to displace anions: Needs negative buffer to displace cations

                                                 Molecular Size

                                                           Larger molecules elute faster

                 Figure 10. Selectivity achieved by various modes of chemical separation


                                                                        Chromatography- Teachers’ conference
                                                                                          17th-19th April 2011

                          Applications of Chromatography
Drug testing: Sports.
      Most sports tested for drugs that enhance performance e.g. horse racing, dog racing etc

                                             • Edgar Davids (Dutch footballer)
                                               Tested positive for nandrolone in 2001,
                                               because of contaminated meat?

                                             • Alain Baxter (GB alpine skier)
                                               Tested positive for methamphetamine in
                                               2002 at Winter Olympics, because of a
                                               Vicks inhaler?

                                             • Floyd Landis (American cyclist)
                                               Tested positive for testosterone after
                                               winning 2006 Tour de France – because
                                               of ???

           100                                        9 min - Testosterone                              SRM ESI+
                 Chiral HPLC                                                                            289 > 97
                                                               11 minutes - Epitestosterone



                                                          OH                                OH


                                  O                                 O

             0                         Testosterone                            Epitestosterone                        Time
                   2.00    4.00       6.00     8.00    10.00    12.00      14.00    16.00    18.00   20.00   22.00

          Typically the ratio of testosterone:epitestosterone is 4:1.
          The cut-off in sporting events is 6:1. Floyd Landis had


                                                          Chromatography- Teachers’ conference
                                                                            17th-19th April 2011

          Drug identification and quantification by HPLC

          Blood/urine analyses e.g. drink driving tests.

          Accelerants identified by GC e.g. pattern recognition of an unknown liquid such
           as diesel or petrol. (See figures Figure 11 andFigure 12)

          Unknown flammable liquids or gases are further identified by GC-Mass

                               Figure 11. GC Chromatogram of Diesel

Diesel is made up of hydrocarbons, straight chain alkanes from decane (nC10) to hexaicosane

Note: Extra peaks around nC17-18; indicates the presence of FAMES (Fatty acid methyl esters) which
are often found in modern diesels; increases lifetime of diesel hence being more environmentally
friendly i.e. bio-diesel.


                                                  Chromatography- Teachers’ conference
                                                                    17th-19th April 2011

     A GC method

                       Figure 12. GC Chromatogram of Petrol

Petrol is made up of hydrocarbons with some low level additives which are specific to a
manufacturer. The GC method is set up to ramp up the temperature over time which aids
separation. Link to FRACTIONAL DISTILLATION- e.g. lower fractions such as petrol, lighter
fluid; higher fractions include diesel and oils/fats. Work out which component is which by
boiling points / retention times and/or comparisons with reference materials- using ramped
GC method- opportunity to mention optimisation of methods in industry?


                                                              Chromatography- Teachers’ conference
                                                                                17th-19th April 2011

Food science
Analysis of pesticides used to promote growth or to protect natural foodstuffs; LC used to show the
presence of foreign components as well.

   Water, carrots, lettuce, potatoes...

…everything must be tested on a year by year basis with targeted inspection for particular problems
(e.g. Trace levels of potentially harmful dioxins (Figure 13) were discovered in liquid eggs from
Germany early in 2011).

                                          Figure 13. Dioxin


                                                              Chromatography- Teachers’ conference
                                                                                17th-19th April 2011

                                         Experiments to try
    1. Separation of ingredients in drinks/ Sweets e.g. Coca-Cola, Skittles, can be achieved on
       either filter papers, flash column or on TLC plates.

                          Figure 14. Separation of food dyes using filter paper

    2. How much vitamin C in OJ? How much Vitamin C in an orange?

                                  Figure 15. Vitamin C (ascorbic acid)

This experiment to shows how to quantify the amount of a specific compound using LC methods, i.e.
by using calibration curves of known concentrations of vitamin C and extrapolating the data. Table 1
outlines an example:


                                                               Chromatography- Teachers’ conference
                                                                                 17th-19th April 2011

Table 1. Example calibration data for experiment 2.

             Concentration of Vitamin C / ppm                               Area under peak

                              0                                                    0

                              5                                                  879

                              10                                                 2069

                              25                                                 4079

                              50                                                 5101

 Unknown- from the extract of one segment of an orange                           3025

Use a graph to find out the concentration of the whole orange...

    3. CSI-whodunit? Which pen wrote the ransom note?- ID by coffee filter papers or TLC

Types of black ink in ballpoint pens will differ from different manufacturer’s e.g. different ratios or
type of components. Use TLC to separate inks and identify the ink in a ransom note.

                                     Figure 16. Example of a TLC tank

Method: On a piece of paper, use each pen (including suspect’s pen) to colour in a black square. Cut
out a small piece and place in a test tube. Add a few drops of methanol or ethanol and pipette out
the coloured liquid into a vial. Spot the liquid onto a TLC plate. Repeat for each pen. Cut a piece out
of the ransom note (make sure you include the bit with the most ink on!) and extract in the same
manner. Run the TLC in 50% methanol (or ethanol) and 50% water. Take the plate out when it is 1
cm from the top. Calculate the Rf values and identify the suspect!!


                                                            Chromatography- Teachers’ conference
                                                                              17th-19th April 2011

    4. Why do leaves turn brown in autumn?

Try a TLC of a brown and a green leaf (of the same species), they will have different ‘spots’ or
components. This is because when leaves appear green, they contain an abundance of chlorophyll.
Chlorophyll masks other pigment colours. Anthocyanins, in turn, mask carotenoids. As summer
turns to autumn, decreasing light levels cause chlorophyll production to slow. However, the
decomposition rate of chlorophyll remains constant, so the green colour will fade from the leaves.
At the same time, anthocyanin production in leaves increases, in response to surging sugar
concentrations. Leaves containing primarily anthocyanins will appear red. Leaves with good
amounts of both anthocyanins and carotenoids will appear orange. Leaves with carotenoids but
little or no anthocyanins will appear yellow. In the absence of these pigments, other plant chemicals
also can affect leaf colour (see Figure 17). An example includes tannins, which are responsible for
the brownish colour of some oak leaves.

             Pigment Class           Compound Type                         Colours

               Porphyrin                chlorophyll                        green

               Carotenoid              carotene and               yellow, orange, red

               Flavonoid                  flavone                          yellow

                                          flavonol                         yellow

                                       anthocyanin            red, blue, purple, magenta

                             Figure 17. Leaf compounds and their colours


                                                             Chromatography- Teachers’ conference
                                                                               17th-19th April 2011

   5. ABC of chromatography

A fun way of highlighting chemicals different properties is by making the alphabet using TLC. As all
the chemicals/ food dyes behave differently on the stationary phase, the different Rf values can be
sued to make up letters and ultimately spell out words. An example of how to make the letter ‘A’ is
shown in Figure 18.

                Figure 18. How to make the letter 'A' using food dye separations on TLC.

                                    Figure 19. The alphabet by TLC

       Details of the solutions required for this experiment can be found in the useful links section
       at the end of this handout.


                                                            Chromatography- Teachers’ conference
                                                                              17th-19th April 2011

   6. TLC of spinach

   By extracting the liquid from spinach (experimental handouts available on request) you can
   discuss the properties of the compounds that separate. By highlighting the functional groups,
   the order of separation can be explained by the difference in the compounds’ polarities (see

                                   Figure 20. TLC plate of spinach

     How can we predict the identification of these compounds??
Figure 21 shows that the polarities of the compounds depicts how the spinach mixture will separate.


                                                                         Chromatography- Teachers’ conference
                                                                                           17th-19th April 2011

Increasing polarity



                                    Figure 21. Structures of the compounds found in spinach

                      Experiments 1-6 are just some ideas to use across all age ranges but there are many more


                                                            Chromatography- Teachers’ conference
                                                                              17th-19th April 2011

Useful links:
1. search tool for information about compounds, e.g. chemical
   properties, structures.

2. some ideas for practical


4. more information about the egg/dioxin scare in Jan

5. using TLCs of food dyes to spell words

6. and


i accessed 11/4/11
iii accessed 11/4/11
 Determination of Methyl Esters in Diesel Oils by Gas Chromatography – Validation of the Method
R. Wawrzyniak, W. Wasiak, M. Frąckowiak, Chem. Pap., 2005, 59 (6b) 449—452

      The ABC's of Chromatography: A Colourful Demonstration. Robert C. Reynolds and Robert N.
Comber J. Chem. Educ., 1994, 71 (12), p 1075




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