Chapter 13.2 Flashcards
The continual process where the components undergo adsorption to the stationary phase and desorption back in the mobile phase.
Occurs at different degrees which depends on the:
- Depending on the strength of their attraction to the stationary phase and mobile phase
What is HIGH - PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) or high pressure liquid chromatography
HPLC is a chromatography technique based on column chromatography.
Highly sensitive and therefore used for separation and
identification of complex mixtures of similar compounds.
Can separate compounds with relative molecular mass as high has 1000 or more.
What are the two main differences between column chromatography and HPLC?
- The particles in solid used in HPLC are 10-20x smaller than in column chromosomes
- this allows more frequent desorption and adsorption of components = better separation of similar compounds - The small particle size used in HPLC creates a considerable resistance to the flow of the mobile phase and so the SOLVENT is PUMPED through the column under HIGH PRESSURE
* sometimes the solids are bonded with chemicals on the surface to improve separation of particular classes of compounds.
How does HPLC work? Explain in steps
- Works similar to a column chromatography
- The components are usually detected by passing the Eluent stream through a beam of UV light.
- So when an organic compound passes in front of the beam of light a reduced signal is picked up by the detector.
- Amount of light received by the detector is recorded on a chart that moves slowly at a constant speed or sent to a computer.
- Resulting trace = chromatogram
- each component forms one peak in the chromatogram
Look at textbook diagram
Understand what the diagram is trying to say
What is HPLC used for?
Separation and identification of similar compounds Eg
- containments that are soluble in water
- drugs in blood
- hydrocarbons in oil samples
- Analyse presence and concentration of dioxins, insecticides, pesticides and oils spills in water.
- presence of pesticides in food
What is Gas Chromatography?
An extremely sensitive chromatography technique used to separate and identify VAPOURISED compounds.
Limited to components that are readily vapourised without decomposing and molecules with relative molecular mass less than 300.
Can detect as little as 10^-12grams of a component
What is the process of Gas chromatography?
- Mobile phase is unreactive and often is Nitrogen or an inert gas.
Called the carrier gas. - A small amount of sample is injected into the top of the column through an INJECTION PORT.
- Injection port is heated to VAPOURISE the sample which is then SWEPT by the carrier gas into the column.
- The column = series of loops of glass, or metal that has an internal diameter of 4mm and is 2-3Metres long in total
- SOLID ACTS AS A STATIONARY PHASE.
Column is HEATED and can be packed with POROUS SOLID COATED WITH AN ESTER OR LIQUID HYDROCARBON with high boiling point.
Or PACKED with an adsorbent solid such as SILICA GEL OR ALUMINA. - As sample passes through the the instrument, while the components of the sample repeatedly interact with the stationary phase and are swept forward by the carrier gas.
- Components that absorb least strongly to the stationary phase are swept out first by the gas. As the components emerge from the end of the column, they are sensed by a detector.
Look at diagram of a GAS chromatography
Draw the diagram
What is GC used for?
Since it is extremely sensitive it makes the GC ideal for the analysis and trace of contaminants in samples or detection of small amounts of portent components.
Eg urine samples taken from athletes competing in major events, to make sure they are not being benefited by illegal, performance enhancing drugs.
Explain Qualitative analysis. How is purity tested?
What chemical are present.. IDENTIFY components present in the sample and determine PURITY.
A solution Of A PURE COMPOUND that is thought to be one of the components is injected into HPLC OR GC under sane conditions.
- chromatogram of the standard is then compared with chromatogram of the sample.
What is the RETENTION TIME?
The time taken for a component to pass through the HPLC OR GC column p.
- Is The characteristic of the component for the conditions of the experiment
- used in the same way as the retardation factor value in paper and thin layer chromatography.
- the same compound will have same retention time if the conditions (temp, mobile phase, stationary phase, flow rate and pressure ect) remain the same.
Can be used to identify the components causing the peaks on a gas or liquid chromatogram.
What is the procedure called spiking ?
A way in which a compound can also be identified by adding a known compound to the sample.
If peaks = size and simultaneous time then same compound.
Explain Quantitative analysis .
How much of each chemical is present?.. determine concentration of each individual components.
It is calculated by It’s peak area is compared with the peak areas of samples of the same chemical known at known concentrations.
What is a standard solution?
A solution with an accurately Known concentrations is called a STANDARD SOLUTION
How to know concentration? Of GC and HPLC ANALYSED COMPONENTS.
By plotting the peak areas against concentrations of standard solutions.
A CALIBRATION CURVE can be drawn and used to determine unknown concentrations.
X value = concentrations
Y value = peak area
The information obtained from a GC and HPLC is
Retention time
And
PEAK AREA
Look at diagrams of calibration curves
Understand how to read them
The decision to use a particular technique depends on a number factors including;
- properties of the components
- amount of sample available for analysis
- concentration of the component in sample
- sensitivity of the technique
- time taken for analysis
- cost of equipments
Technique; Paper and thin layer chromatography Typical substance used? Typical samples? Advantages? Disadvantages? Comments?
Typical substance used; polar, water soluble substances.
Typical samples; drug detection, dyes in foodstuffs
Advantages; cheap, basic lab equipment needed, easy to perform
Disadvantages; poor precision and accuracy
Comments; samples need to be coloured or visible under UV light, otherwise samples can be visible using stains
Technique; HPLC Typical substance used? Typical samples? Advantages? Disadvantages? Comments?
Typical substance used; medium - high molecular mass organic compounds, Eg Enzymes and pesticides.
Typical samples; food,drugs, biological samples
Advantages; high sensitivity and precision, small sample size, readily automated
Disadvantages; moderately expensive instrument, trained technician needed to operate
Comments; samples must be soluble in a suitable environment
Technique; GC Typical substance used? Typical samples? Advantages? Disadvantages? Comments?
Typical substance used; low molecular mass organic compounds Eg aspirin or acetone
Typical samples; water, gases, drugs, biological samples
Advantages; high sensitivity and precision, small sample size, readily automated
Disadvantages; moderately expensive instrument, trained technician needed to operate
Comments; samples must be vaporised with decomposing
GC - sniffing for explosives
Sniffing for explosives
•Hand-held devices based on gas chromatography can be used to ‘sniff’ air from around bodies or bags, and can detect amounts of explosive material in quantities less than a nanogram.
This is much less than the amount that remains on the hands or suitcase of a person who has handled explosives, even after the person has cleaned their hands.
GC and drug testing
In horse racing, the use of illegal ‘go-fast’ or ‘stopper’ drugs is now relatively rare because of their ease of detection by GC-MS.
What Variables Have to be controlled for GC?
- Flow of gas
•Temperature of column
•Density of column
What is Gas chromatography used for long…
Crime scene testing
Gas chromatography is used to test evidence such as blood or hair from a crime scene. This allows investigators to understand the crime better and to develop theories on what exactly happened and where the victim has been earlier, based on the material found.
Forensic pathology
Gas chromatography (GC) has been widely used in forensic pathology to identify the type of compounds and fluids present in the human body, post death. This testing can help detect the presence of alcohol or drugs or poisonous substances in the body at the time of death, thus assisting in determining the possible motive and cause of death.
Arson investigation
GC is a low cost technique used to identify ignitable / flammable liquids from fire debris. On comparison with a list of flammable liquids publically available, the exact kind of liquid used can be concluded. Mass spectrometry (MS) characterization of the separated components yields better and more precise results
