b Describe two advantages of thin-layer chromatography over paper chromatography.
9 Thin-layer chromatography was used to investigate the colourings in two food dyes. Two chromatograms were obtained under identical conditions.
These are shown below.
origin solvent front
A B C
D E F
food dye 1 food dye 2
CHAPTER 1 MODERN ANALYTICAL CHEMISTRY a Explain why the colourings in the food dyes separated during the
chromatography procedure.
b Which components (A to F) are found in both food dyes?
10 a Explain why column chromatography rather than thin-layer chromatography would be used if samples of the components are required for further analysis.
b Explain why paper or thin-layer chromatography, rather than column chromatography, might be used for the initial investigation of a mixture.
A UV–visible spectrophotometer is used to analyse samples that absorb in the ultraviolet (200–400 nm) and visible (400–800 nm) regions of the electromagnetic spectrum. This analysis is applicable to coloured substances, which absorb visible light. In addition, many colourless substances, especially organic compounds, absorb radiation of higher energy than visible light (ultraviolet light), as their electrons are excited between electronic energy levels and so may be analysed by UV–visible spectroscopy.
Figure 1.7.1 Schematic diagram of the UV–visible spectrophotometer.
radiation source
slit
detector
chart recorder monochromator
motor
sample cell
reference cell
mirror mirror
semitransparent mirror rotating mirror
(beam chopper)
The schematic diagram in fi gure 1.7.1 allows us to identify the key stages in UV–visible spectroscopy. As in infrared spectroscopy, a double beam spectrophotometer is used for greater accuracy. The sample in this case is in solution. Light to be passed through the sample is generated by a light source that provides radiation in both the visible and ultraviolet ranges. The selection of wavelength for analysis occurs via a monochromator, which selects and focuses the chosen wavelength. The light beam is split into two beams and pulsed. One beam passes through the sample while the other beam passes through a reference cell.
The detector converts the light signal to an electrical signal, comparing the sample beam and the reference beam to remove any absorbance due to the solvent. The recorder uses the electrical signal to produce a recording of the absorbance of the sample.
The sample and reference cells are usually square-shaped and made from a special type of clear glass that is able to transmit UV radiation. Care must be taken in handling these cells that no fi ngerprints distort the optical clarity of the cell.
1.7 VISIBLE AND ULTRAVIOLET SPECTROSCOPY
HLUV–visible spectroscopy plays a signifi cant role in analysis, particularly in biochemical and environmental applications. Examples include the analysis of hemoglobin in blood and the analysis of nitrates and phosphates in water samples. UV–visible spectrometry has two main uses: qualitatively it is used to identify unknown compounds in solution by comparing known absorption spectra with the spectrum of the unknown, and quantitatively it may be used to determine the concentration of a known compound in solution. This
technique is quite sensitive; it is capable of measuring concentrations as low as 10–5 mol dm–3 with around 98% accuracy.
When used qualitatively, recordings are made of the absorbance of the sample over a range of wavelengths, producing an absorbance spectrum. Comparison of this spectrum with the spectra of known substances allows identifi cation of the unknown.
For quantitative use, the instrument is set to one wavelength. This wavelength is chosen by examining the absorption spectrum. Usually, the wavelength corresponding to the maximum absorbance will be used, provided that other substances present in the sample do not absorb this wavelength. Any interference by other substances absorbing can be a source of error in UV–
visible spectrophotometry. Using the selected wavelength, the absorbance of a set of solutions of known concentration is recorded and a calibration curve constructed. The unknown concentration is found by interpolating this curve once the absorbance of the unknown is determined.
absorbance
wavelength (nm) absorption of
purple light
absorption of red light
400
300 500 600 700 800
200
Figure 1.7.2 An absorbance spectrum shows the absorbance of light by the sample over a range of wavelengths. This is the spectrum of a green Ni2+ solution.
Figure 1.7.3 Which wavelength would be chosen for use in the quantitative analysis of a compound with this absorption spectrum?
absorbance
wavelength (nm) 400
300 500 600 700 800
200
As in AA spectroscopy, the greater the concentration of the solution being analysed, the greater the absorbance of the solution. Similarly the path length, the distance through the solution that the light must travel, also infl uences the absorbance, as does the molar absorptivity of the substance. A substance with a high molar absorptivity is very effective at absorbing light.
An example of a substance with a very high molar absorptivity is β-carotene, the strongly coloured organic substance that is responsible for the orange colour of carrots.
The Beer–Lambert law relates absorbance, A, to the molar absorptivity, ε (in dm3 mol–1 cm–1), the path length, l (in cm), and the concentration of the solution, c (in mol dm–3):
A = εlc A.8.6
Determine the concentration of a solution from a calibration curve using the Beer–Lambert law. © IBO 2007
CHAPTER 1 MODERN ANALYTICAL CHEMISTRY A calibration curve can be plotted for absorbance against path length, or molar
absorptivity, but it would most commonly be plotted for absorbance against concentration in order to determine the concentration of an unknown solution.
Worked example 1
The concentration of iron in a sample of lake water was determined by UV–
visible spectroscopy. Iron, present as Fe2+ ions, was reacted to form an orange-yellow complex. The absorbance of a series of standard solutions and a sample of the lake water were measured at 480 nm.
Determine the concentration of iron in the lake water in ppm.
Concentration of Fe2+ (ppm) Absorbance
0 0
5.0 0.17
10.0 0.33
15.0 0.49
20.0 0.65
Sample 0.35
Solution
Figure 1.7.4 The calibration curve for the absorbance of iron.
0 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7
5 10
concentration of Fe2+ (ppm)
absorbance
15 20
From the calibration curve, the concentration of iron in the lake water can be seen to be 10.9 ppm.
CHEM COMPLEMENT
Colorimetry
Colorimetry is a much simpler, less expensive, but less accurate form of absorption spectrometry. Its design is very similar to UV–visible spectrometry, but the wavelength used is selected by a filter rather than a monochromator. In addition, the light source used provides only visible light, not ultraviolet radiation. Comparison of the schematic diagrams in figures 1.7.1 and 1.7.5 highlights the similarities and the differences.
sample solution light source
detector recorder coloured filter
I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I
Figure 1.7.5 Schematic diagram of a colorimeter.
Selection of the wavelength to be used for analysis in colorimetry involves the use of coloured filters. Recall the concept of complementary colours from Chemistry: For use with the IB Diploma Programme Higher Level, chapter 3. When we view an object, our eyes receive light reflected by the object. This reflected light is composed of the wavelengths not absorbed by the object. We see the complementary colour of the colour absorbed. For example, copper(II) solutions absorb wavelengths in the orange region, and reflect those in the blue-green region. The more concentrated a solution, the more of a certain colour that has been absorbed, making the colour appear darker. In colorimetry, the filter used is the complementary colour to that of the solution being analysed.
While it is unlikely that a school laboratory would have a UV–visible spectrophotometer, a colorimeter is a much more affordable and compact instrument and can be used for a range of practical investigations.
400 nm 700 nm
435 nm 605 nm
480 nm 595 nm
490 nm 560 nm 500 nm 580 nm
Figure 1.7.6 Complementary colours are opposite
one another on a colour wheel. Figure 1.7.7 Solutions of known concentrations are used to calibrate a colorimeter.
PRAC 1.4
Determination of phosphate in washing powder
CHAPTER 1 MODERN ANALYTICAL CHEMISTRY In UV–visible spectrometry, the substance being analysed is often coloured.
Many non-coloured substances, however, can be reacted with reagents to produce a coloured complex that can be analysed spectroscopically. In this way, a wider range of elements and compounds can be analysed using UV–visible spectrometry.
The chemistry of transition metal complexes is studied in detail in Chemistry:
For use with the IB Diploma Programme Higher Level, chapter 3; however, it is useful to consider here the factors that affect the colour of transition metal complexes.
Transition metal ions have one or more unpaired electrons in their
d subshells. The ions are also highly charged and quite small. A ligand is a small molecule with an electron pair that can be donated to a transition metal ion in a coordinate (dative) bond. Typically two to six ligands may surround a transition metal ion in a complex. Common ligands include water, H2O;
ammonia, NH3; the chloride ion, Cl–, and the cyanide ion, CN–.
The d orbitals of a transition metal ion that has formed coordinate bonds with ligands in a complex will be affected by the ligands surrounding the ion.
Repulsion occurs between the non-bonding electrons of the ligand and the electrons in the d orbitals of the metal ion. Instead of being all the same energy, as they would be when the ion is isolated, the orbitals are split into two groups. The closer a ligand can get to the metal ion the further apart the d orbitals will split. The amount of splitting depends on four factors:
1 Nature of the transition metal ion. Different transition metals have different ionic radii, so even if the charges on the ions are the same, the attraction of the ion for the electrons of the ligand will vary from one transition metal to another.
2 Charge on the transition metal ion. An ion with a greater charge will attract