In paper chromatography, coloured compounds can be separated into many colours, but in some cases, colourless spots are involved. When this happen, chemicals like NINHYDRIN will be sprayed to those substances, and those substances will form coloured complexes with these coloured compounds. The solutes can be identified in 2 ways:
1. Comparison to the reference compounds 2. Calculation of Retardation factor = D moved by centre of solute spots D moved by front of mobile phase Sometimes a 2-way chromatography is carried out to ensure that all the solutions are fully separated. C.
Thin layer Chromatography TLC- Simple, Cheap and Reliable Stationary Phase: Thin layer of silica Mobile Phase: Liquid (Silica Gel) TLC is a very similar technique as paper chromatography, but it is 3 times faster than paper chromatography, works with small amount of sample, a wider range of mixtures separated. In the stationary phase, the silica is heated to remove water, these acts as a polar solid and the solutes are transported by adsorption in mobile phase. (When dry, both phases will be likely to occur) However, silica will attract water and the presence of water becomes the stationary phase and the solutes are separated by partition.
Actual uses: Clinical diagnosis / Forensic testing / Quality control. D. Gas/Liquid Chromatography Use for very small sample Stationary Phase: THE SAMPLE itself Mobile Phase: Inert Gases or carrier gases * GLC is worked by partition, Flows thru the column of stationary phase Separated due to different solubility, Or volatility, If the stationary phase is non polar > Volatility. Stationary is polar > polar molecules retain> Take longer to appear. The sample will be detected by the detector and appear on chromatogram.
Chromatogram tells us how much of a component is present, the area under the graph being related to the amount. So how to Determine the % composition of a mixture by GLC: The area of a peak = 1/2 (Base i?? Height) Therefore, a chromatogram must show all the components and the detector are respond equally to all components. Green Tea Separation in GLC ? Retention time: Time between injection and appearance of a component.?
The retention time is specific to particular components for the same conditions: Same carrier gas Same flow rate Same stationary phase Same temperatureE. Mass Spectroscopy A high resolution one (Double focusing) In the detection of mass spectra, the following condition used in the experiment must be constant: The same Temperature The same ionizing voltage.
The same type of instrument Analysis of an ion determine theAr The mass spectrum for zirconium Number of isotope:This will be given in the exam So we will be able to distinguish 2 compounds have similar Mr by using mass spectra analysis. E. g. Mr= 44 Propene, C3H8, & ethanal, CH3CHO In a high resolution mass spectrometer, the molecular ion peaks for the two compounds give the following m/z values up to 4 d. p. C3H8 44. 0624 CH3CHO 44. 0261 M peak / M+1 peak / M+2 peak / M+4 peak Peak Original ions Mr of isotope.
Cancer Treatment Urine analysis F. Electrophoresis Biological analysis Electrophoresis is separation of charged particles by their movement in an electric field. The visual presentation is by using Electropherogram. Although the amino acid solution is colourless, its position after a time can be found by spraying it with a solution of ninhydrin. If the paper is allowed to dry and then heated gently, a coloured spot will be observed.
In zone Electrophoresis, the mixture is a solution or gel, The supporting medium is a filter paper made by cellulose, Separated by the charge and the size of compounds. A highly charged ion will move faster in an electric field. A larger ion moves slowly across the supporting medium. The electrophoresis need to be worked in a stable environment. The temperature and pH need to be controlled by adding buffer solution. Normal amino acids will be look like the one on the left. If there is an internal transfer of a hydrogen ion, a zwitterion will be released. If the pH has increased, the zwitterions will then become,
If the pH has decreased, the zwitterions will then become, One of the uses for electrophoresis will be used for genetic fingerprint: 1. Take a DNA strand 2. Cut Restriction Enzyme 3. Separated by Electrophoresis 4. Transferred to Nylon membrane 5. P-32 DNA probes bind with DNA band 6. X Ray exposed, Results. There are also other uses for electrophoresis: Forensic Science / GM Food / Medical Research / Establishing Relationships G. Atomic Emission Spectroscopy Interaction of EMR This type of spectroscopy concerns the electromagnetic radiation with matter. The EMR have different frequency and different energy.
E. g. the radio waves change the orientation of spinning nuclei in a magnetic field and infrared causes changes in vibrational energy. C The molecular H (g) at a low pressure is bombarded with electrons in a discharging tube. The bombardment dissociates the hydrogen molecules to give atom as well as excited atoms so that they are no longer in its ground state. The excited atoms relax and the EMR seen as a pink glow. This radiation is then passing thru a spectrometer which splits the EMR according to frequency.
Hydrogen Absorption Spectrum Black lines on continuous spectrum EMR absorbed. Hydrogen Emission Spectrum Discrete lines EMR missing from the absorption spectra. When the energy of the photons exactly matches the energy difference between the ground state and an excited state, the radiation is absorbed by the atoms. An atom in an excited state will rapidly relax this means it will revert back to its ground energy level.
So the EMR used this characteristic to let the atom to lose a photon. Calculate the ionization energy For hydrogen the convergence limit is 3. 30 x 1015 Hz, so we can calculate the IE by E=h x f:E = 6. 63 x 10-34 x 3. 30 x1015 x 6. 02 x1023 E = 1318 kJ mol-1 Quantitative Analysis of flame emission spectroscopy This is a method that we used for finding how much metal ion is present in clinical diagnosis. E. g. determination of Na+ in blood:
1. Moisten a nichrome or platinum wire with conc. HCl, 2. Dip into the sample and place the wire to a Bunsen flame, 3. If Na+ is present, a yellow / orange colour will be observed. H. UV/Visible absorption spectroscopy changes in electronic structure An Electromagnetic spectrum see section G on p. 9.
Here are 2 definitions that you need to know: Chromophore are unsaturated groups of atom in organic molecules that absorb radiation mainly in the UV/Vis regions.Conjugation the system of alternate double bond and single bonds in a molecule. Organic molecules with extended conjugation absorb radiation in the visible part of the spectrum and are therefore colored. The wavelength at which organic molecules absorb radiation depends on how tightly their electrons are bound. Bonds the shared electrons in single bonds do not easily give absorption spectra. But the electrons in double and triple bonds are more loosely held and easier to get excited.
Lone pair Lone pair that is localized around atoms (O2 / N) is loosely bound and are absorb in the same region of the spectrum. Ultraviolet and visible radiation are absorbed by the outmost electrons in organic molecules, which then move from lower to higher energy levels. The outmost electrons can be: Electrons directly involved in bond formation, Unshared outer electrons. ?It is the movement of electrons between molecular orbital give rises to UV/Vis spectra.? Some of the organic molecules have colours, because, they absorb certain frequencies of radiation within the visible region.
(Wavelength 400 750 nm) The frequencies of radiation absorbed by various chromophores can be used for the identification. The wavelengths and the intensities of the absorption bands due to chromophores are altered by the conjugation in molecules and delocalized electrons. In benzene, the electrons are evenly distributed around the rings. The conjugation of chromophores in a molecule causing ? orbital interact with each other then become delocalized > Formation of new orbital > Energy gap reduced > absorption bands shift to longer wavelength > intensity increase.
Effects of additional Chromophores will shift to an organic molecule more from the UV region to the visible region. See table below for more details. Number of CH=CH Region of Electronic transition Colour 1 / 2 UV Colourless 3 Visible Yellow 15 Visible Dark Green Dyes and colour change in acid base indicators 1. Phenolphthalein Acidic = Colourless // Alkaline = Red 2. Methyl Orange Acidic = Red // Alkaline = Yellow 3. Azo Dyes coloured due to presence of chromophores Benzene Diazonium + Napthalene-2-ol > Red azo dyes + HCl I. Combined spectral Techniques Analysis of mixture and compound.
Now, we will be able to use evidence from N. M. R. / I. R. / Mass Spectra to suggest a probable structure for an unknown compound. The following questions are from the OCR text book methods of analysis and detection. See page 50 -53 in OCR Chemistry Methods of Analysis and Detection. J. Infrared Spectroscopy (IR) using wave numbers Here, IR used the characteristics that different bonds in compounds absorb different frequencies in the infrared region of the spectrum. In infrared analysis the wave numbers are used instead of frequencies or wavelengths.
In IR, note the following: Compound Things to note Alcohol Peak at 3400 (OH) Carboxylic acid Peak at 2500-3500 (Broad for OH) Peak at 1700 (C=O) Peak at 1000 -1250 (C-O) Carbonyl Compounds Peak at 1700 (C=O) Ester Peak at 1700 (C=O) Example of an IR spectrum Ethanoic Acid K. Nuclear Magnetic Resonance Spectroscopy (NMR) -Splitting Pattern The peak in the NMR gives many information of compound; this is a summary in a table of what it tells you: What it tells you How this is shown in the spectrum How many types of proton present
The number of distant peak = number of proton How many of each type of proton The relative peak area under the peak = relative number of proton What type of proton The value of ? and ten use the table provided in the exam The number of chemically different protons adjacent to a particular type of proton By the splitting of the distant peak. If there is n chemically different type of protons next to a particular type of proton then the peak for that proton is split to n+1 peaks Use D2O with -OH group Deuterium is an isotope of hydrogen that does not produce an NMR peak.
If an alcohol or carboxylic acid is shaken with D2O, the H of the OH group is replaced by D and the peak disappears from the spectrum. Example of a NMR spectrum C4H6O2 1. 4 peaks and 4 Cs, 2 of them are in the same environment. 2. The peak just over 50 must be C-O 3. 2 peaks around 130 must be 2 carbons at either end of a C=C. 4. The peak at just less then 170 is the C in a C=O. 5. By using IR and Mass Spectroscopy, we can work out the structural formula. L. Additional Notes and Materials Colour wheel can be used to determine a compounds colour: Colours nm Red 625-740.
Yellow 565-590 Green 520-565 Cyan 500-520 Blue 435-500 Magneta 380-435 Colours directly opposite each other on the colour wheel are said to be complementary colours. Blue and yellow are complementary colours; red and cyan are complementary; and so are green and magenta. E. g. Beta-carotene absorbs throughout the ultra-violet region into the violet but particularly strongly in the visible region between about 400 and 500 nm with a peak about 470 nm and if we use the table above, we know it will absorb blue colour so the colour we will see will between red and yellow.
Bibliography and Reference 1. http://www. chemguide . co. uk 2. A-Z Chemistry Hand book 3. Revise A2 Chemistry by Helen Eccles and Mike Wooster 4. OCR Text Book Methods of analysis and detection 5. OCR Past Examination Paper 2002 ~ 2007 6. Sherborne School resources 7. Mr. N. C. Scorer Teachers in chemistry of Sherborne School 8. Yahoo! Image Search Engine ?? ?? ?? ?? OCR Chemistry A 2815/04 Methods of Analysis and Detection 1.