Analytical Session 1 Flashcards
Chromatography
Method of separating a mixture between two phases. Used for quantitative and qualitative analysis
To achieve separation
the analytes need to spend different lengths of time being retained by the stationary phase
Mode
How the analytes interact with stationary phase
What are the modes of separation?
the four modes: adsorption,partition, ion exchange, and size exclusion.
Chromatogram
a graph showing separating the components of a mixture by chromatography.
A good chromatogram will have:
All analytes separated
Well separated peaks
Symmetrical, sharp peaks
Short run times
normal phase chromatography
has a polar stationary phase (eg silica gel) and a non-polar mobile phase (eg hexane)
reverse phase chromatography
has a non-polar stationary phase and a polar mobile phase.
In thin-layer chromatography, the retention factor (Rf) is used
to compare and identify compounds.
The solvent system that gives the best resolution during TLC
can also be used to purify the compound using column chromatography
Phase
a distinct and homogeneous form of matter (i.e. a particular solid, liquid, or gas)
Analytes
a substance whose chemical properties are being identified and measured.
Sample binds to stationary phase bearing strong negative charges, and dissolves in mobile phase with high electrolyte concentration
Ion exchange chromatography
Sample equilibrates between a liquid trapped inside porous particles and mobile liquid outside the particles
size exclusion chromatography
Sample equilibrates between liquid bound tightly to stationary phase particles and a mobile phase
: partition chromatography eg gas chromatography (based on polarities)
The mode used in TLC
adsorption chromatography (based on polarities)
An ion exchange chromatography, what are the forces involved?
Electrostatic forces
absorption chromatography, what are the forces involved?
VanderWaal forces are involved. Polar interactions from dipole dipole forces and hydrogen bonding.
In adsorption chromatography a very nonpolar mobile phase as used
FALSE The mobile phase can be polar or nonpolar in absorption chromatography
In reversed-phase chromatography, the mobile phase is more polar than the stationary phase.
TRUE In normal phase chromatography the mobile phase is less popular than the stationary phase
In partition chromatography the stationary phase is a solid
FALSE In gas chromatography The stationary phase is a liquid
A cation exchange resin binda and exchanges positive ions
TRUE Once the positive ions have bound to the cation exchange resin, the mobile phase can be changed and the bound ions will be released
Chromatography is a technique for the SEPARATION of a mixture, in which the sample is introduced into a system of two different PHASES.One of the latter is continually being added to the system and is called the MOBILE PHASE. Differences in ADSORPTION/PARTITIONS/ INTERACTIONS shown by the sample components due to their structures and properties cause them to travel at different speeds.
analysis, separation, determination, substances, chemicals, phases, adsorption, distribution, partition, liquid, mobile phase, system, narrowness of peaks or bands, degree of peak or band separation]
Adsorption chromatography:
Stationary phase
Mobile phase
Interaction
Diagram
Uses
Solid eg silica gel
Liquid eg petroleum ether and acetone
Polarity VdV
TLC/ HPLC
Normal and reverse phase applies to both
Adsorption (TLC) and Partition (GLC)
Diagram of adsorption
See “analytical”
Adsorption chromatography forces
Hydrogen bonding (VdV interactions)
Polarity of silica
Has an OH group, makes the overall molecule polar
Which is the non polar phase in reverse phase
The sp is non polar eg C18 and the mp is polar eg methanol and water
In reverse phase chromatography of a non-polar sample, will increasing the polarity of the mobile phase cause the retention time to rise or fall?
In reverse phase the sp in non polar eg c18, making the mobile phase less like the sample will increase retention times
Describe the important functional differences between both the stationary phase and the interactions leading to sample binding and separation, in the four cases of adsorption, partition, ion exchange and size exclusion chromatography.
Table
Name the four principal classes of chromatography based on the interaction of components with the stationary phase. In each case, describe the type of interaction that results in retention of components, and also the basis for separation of different components.
Table
What modes can use normal and reverse phase chromatography
Partition and adsorption
Principles of size exclusion chromatography
mp continuously added.
Smaller molecule elute quicker (stick in a forest)
Liquid liquid extraction
Pre analytical testing to prepare for analysis to isolate components in sample
Based on differential solvent
eg spinach pigments in a solvents add another immiscible solvent when they are put together they created two layers when slightly agitated. Can decant sample and solvent of interest
What happens when 100% Hexane:0% Ethyl Acetate is used as the solvent?
The components remain on the baseline.
The solvent in TLC is also known as the
Mobile phase
Good separation occurs when rf
<1
Retardation factor is the ratio of
Distance moved by substance from base line to distance moved by the solvent from base line
In TLC, initially the sample is
Not in contact with mobile phase
True or False: Hexane is more polar than Ethvl Acetate.
False
In TLC, you allow the solvent to travel
To nearly the top of the plate.
Atomic absorption spectrometry v AES
AAS
Energy and wavelengths absorbed by atoms
AES
Energy and wavelengths emitted by excited electrons
What happens during when a sample absorbs radiation
When a sample is exposed to light energy that matches the energy difference between two levels, the electrons will be excited and the electrons will be promoted to the higher orbital. Energy is absorbed by the sample and a spectrometer records the degree of absorption at different wavelengths and the results are plotted, absorbance (A) versus wavelength
this is known as a spectrum.
Lambda max
the wavelength at which there is a maximum adsorption
Example - Isoprene
The absorbance at 222 nm is due to excitation of electrons from pi to pi star
Absorption coefficient
It is simply the absorption of light at a particular wavelength by a 1 mol/litre solution with a 1 cm light path. It is the factor a or ε seen in the Beer Lambert equation.
Hplc experiment
In GLC, the two phases are Gas and Liquid. In HPLC, the phases are both liquid. Both can be changed to improve resolution and define the optimal conditions for separation. In this experiment Reverse Phase HPLC will be used. The mobile liquid (phase) is a polar mixture of 70 % methanol and 30 % water. The stationary phase is a non-polar long-chain alkane, octadecylsilane C18H38, which is bonded to the silicon beads which fill the column. Partition of the sample occurs between the mobile and stationary phases, depending on the polarity of its constituents.
Internal standard practical
For your lab, a series of standards is prepared, having varying concentrations of Ethanol (Alcohol), but each having the same concentration (10%) of Propanol. Each of these is used to obtain a chromatogram, from which the ratio
Peak area Ethanol
Peak area Propanol
is obtained. A standard curve is drawn of this peak area ratio versus Ethanol
concentration.
Samples are also spiked with 10% Propanol, and the peak area ratio is obtained for each. This ratio can be used to obtain the % Ethanol from the standard curve. The Ethanol concentration of the actual samples can then be found by adjusting for the Propanol dilution and any other dilutions carried out.
Beer lambert experiment
This experiment (and absorption spectroscopy in general) derives from the fact that most molecules will absorb electromagnetic radiation (visible and invisible ‘light’). Different compounds vary in the extent to which they absorb electromagnetic radiation and the wavelengths which they absorb. This experiment demonstrates these two points.
Theory
A=εcl
A = Absorbance
ε = Molar absorption coefficient (which reflects the extent of absorption by individual molecules in the solution)
l = Light path length through the solution
c = Concentration of test solution
The Beer-Lambert Law states that when light is shone through a solution, the amount of light absorbed depends on the nature of the molecules in the solution, the length of the light path through the solution and the solution concentration where:
Absorbance is directly proportional to
Concentration (A C), since in any experiment ε and l are constant.
Beer lambert experiment 2
This experiment concerns the ability of different molecules to absorb radiation (in this case visible light) to different extents at different wavelengths. A visual (graphical) display of this absorption pattern is called an absorption spectrum.
Tabulate your results. Plot a graph with all your results using Excel, absorbance on the vertical axis and wavelength on the horizontal axis.
This video shows how to create a graph (spectrum) of absorbance versus wavelength.
These graphs are called spectra, and the differences in complexity are due to differences in molecular structure of the two compounds. Find a published spectrum for carotene in a book or on the net for your report, and discuss yours and the published version
While the compounds used above absorb visible light (which is why they are coloured), in practice more compounds absorb ultraviolet light, or uv as well as visible light.
the very significant difference in double bond arrangement that gives rise to the differences in spectra. Include the spectra/observations in your report.
This experiment illustrates the fact that absorption intensity and spectrum are critically dependent on molecular structure.
Session 8. Determination of Fe in Cu-Ni alloy by Atomic Absorption Spectrophotometry
A standard source of radiation, consisting of the atomic spectrum of Iron, is passed through a solution of Iron, which is atomised in an oxy-acetylene flame. Some of this radiation is absorbed by some of the Iron in the flame. This Absorbance obeys the Beer-Lambert Law.
We prepare standard solutions of known concentrations, obtain a standard curve from their Absorbances, and use this to find the Concentration corresponding to the Absorbance of a sample solution.
Session 9. Standard Addition Method in AAS to determine the Fe content of a Cu-Ni Alloy
Prepare the sample so it has a concentration of approximately 4 ppm Fe. Measure its Absorbance, and then add increasing amounts of standard Fe solution to the sample, measuring Absorbance after each addition. Plot the results on a Gran’s Plot. The concentration of Fe in the sample will be indicated by the point where the line crosses the x-axis as in the example below:
Session 3. Use of Gas Liquid Chromatography to:
1) Identify the number of components in a mixture of homologous alcohols
2) Identify each individual component
3) Determine the percentage composition of the mixture
Chromatography is based on the principle that different molecules interact to a different extent with different kinds of substances. This fact can be used to separate mixtures of various substances.
In GLC, the two phases are Gas (mobile phase) and Liquid (stationary pahse). In GLC chromatography, the stationary phase is generally a non- volatile liquid, which coats an inactive, pulverized, solid material packed within a very long (30 meters) thin (1/4 of a millionth of a meter) column. The mobile phase is generally an inert gas such as Nitrogen or Helium and is referred to as a carrier gas. Our gaseous phase is Helium.
The components of the mixture to be separated must be volatile. A very small amount of solution (perhaps 6/10 of one millionth of a liter) is injected into the injection port of the chromatography system using a syringe. The mixture is immediately vaporized and carried by the carrier gas into the column. The column, like the injection port and the detector, is kept at a controlled temperature inside an oven so that the mixture remains in vapour form. From the time the materials are injected into the instrument until they reach the detector, they are being retained by the liquid in the column (the stationary phase).
The time each substance is retained is called its retention time and is usually represented in minutes. Experimental conditions (for example, temperature and choice of column) are chosen so that each component of the mixture has a different retention time and, therefore, reaches the detector separately and appears as a peak on a chromatogram. There are several types of detectors, but the one used in this experiment is a flame ionization detector. This detector essentially counts carbon atoms, and therefore, the area under each peak represents the amount of component in the mixture. Thus, both qualitative and quantitative information can be gathered.
Liquid liquid
WAPE
The lower phase is largely water and acetone, and the.
upper the petroleum ether since it has low density. Run off the lower phase into the original conical flask for discarding and collect the upper phase in a clean container
