lecture 3&4 - Polarity, Extraction and Basic Chromatography Flashcards
what is a dipole moment
the sum of individual bond polarities and lone pair contributions within the molecule
explain the difference between hydrophilic and hydrophobic
hydrophilic = attracted to water and repels fats
hydrophobic = fat loving, repels water
what’s the rule about polar and non-polar extraction
like dissolves/extracts into like
list 3 properties of polar molecules
hydrophilic, dissolve into polar solvents and have higher boiling/melting points than non-polar molecules (due to dipole-dipole interactions)
the greater the strength of a dipole interaction…
the greater the dipole moment and therefore the higher the boiling/melting point
why is hydrogen bonding so strong
hydrogen is very small and doesn’t have electrons to protect its nucleus
name 3 properties of non-polar molecules
hydrophobic, dissolve into non-polar (organic) solvents, and have lower bps and mps than polar molecules
what type of forces occur between non-polar molecules
Van Der Waals - very weak, temporary and easy to break
why do we use extraction?
not all samples can be directly analysed by chromatography and therefore we need to extract the analyte and discard the matrix. We can also remove contaminants or concentrate a sample
what is the aim of extraction
sample sustainability
name 3 problems associated with extraction
- samples large number of analytes
- small amounts of analytes - extraction can lead to a large loss of evidence
- chemical sustainability
what is liquid - solid extraction
extracting a solid powder in a liquid e.g. a street drug laced with adulterants needs to be separated
what is liquid-liquid extraction
where a liquid is partitioned into another liquid
explain the process of liquid liquid extraction
using a separating funnel (O-ring and a stand) add the sample and the solvent and shake. Open the valve to release built up pressure and place into the stand to allow to settle into separate layers (an aqueous layer and a non-polar layer). Repeat a number of times and then remove the separate layers into two conical flasks, and evaporate to dryness under nitrogen.
what does immiscible mean
do not mix
what does a positive log P value suggest
the drug will be extracted into the organic layer, and therefore is non-polar
what does a negative log P value suggest
the drug will extract into the aqueous layer and therefore is polar
what type of extraction produces Log P values
liquid liquid extraction
give an example of an acidic drug and why its acidic
aspirin - it is a carboxylic acid (only has one ionisation centre)
what happens to an acidic drug when its placed into water?
it is deprotonated and forms a COO- ion
the proton then binds to water molecules forming H3O+ ions (an acidic solution)
for extraction of acidic drugs what pH do you use?
2 units below the pKa
name an example of a basic drug and why?
amphetamine - has an amine group
what happens when a basic drug is placed into water?
the NH2 group is protonated into NH3, and the water molecules will lose a hydrogen, turning them into HO- ions ( a basic solution)
what pH do you use to extract basic drugs in liquid liquid extraction
2 pH units above pKa
give an example of an amphoteric drug
morphine
how many ionisation centres does an amphoteric drug have?
2
what pH do you use to extract amphoteric drugs in liquid liquid extraction
the average pKa of both ionisation centres - the isoelectrical point
list 3 disadvantages of liquid liquid extraction
- time consuming
- large volumes of solvents used - wastage
- sample reconstitution - HPLC grade solvents need to disposed of specially
what is the rule regarding chromatography and polarity
Chromatography is governed by polarity
what is TLC used for?
to separate components of a mixture such as inks
what is the stationary phase in TLC
a solid microparticulate stationary phase (silica) bound to a supportive backing (aluminium)
how can we view non-coloured compounds on a TLC plate
impregnate the silica stationary phase and use a UV lamp
what is the range for the diameters of the particles in the stationary phase in TLC, and why?
10-60um - the smaller the particle = better the separation = better results
what is the acceptable range for Rf values?
0.2-0.8mm
what is the Rf equation
distance travelled by the component (from the baseline to the centre of the dot)/
Distance travelled by the mobile phase (solvent front)
whats the method to setting up a TLC plate
Pour a small amount of the mobile phase into the solvent tank and allow it to equilibrate draw a pencil line on the TLC plate and add a number of crosses equidistant from each other for the samples. Use a capillary tube to add to samples allowing time for them to dry in-between. Place in the solvent tank and wait 30 mins. Then measure Rfs.
how does the mobile phase travel up a TLC plate
Capillary action
what is the mobile phase of TLC
a mixture of analytical grade solvents
what do the locating reagents do in TLC, to allow the separated components be visible
bind to functional groups within your components
what does ninhydrin bind to in TLC
amino acids and produces purple spots - used in fingerprint detection
what does the locating reagent Anisaldehyde/ antimony trichloride bind to in TLC?
steroids - produces a variety of coloured spots
what does the locating reagent vanillin/ sulphuric acid bind to in TLC?
alcohol functional groups - producing pink, blue or green spots
what do we use TLC for in forensics
screening for drugs and forged documents ink analysis
what is gas chromatography used for
the separation of a complex mixture of volatile components (based on boiling points)
what are the carrier gases used in GC?
pressurised, purified, inert gases, such as; nitrogen (capillary), helium (capillary) or hydrogen (packed) - depend on the column used
what are the stationary phases of GC
high boiling point liquids, waxes, and oils - they have different polarities
what does an autosampler do in GC?
draws up the required amount of sample into the syringe and then it is injected into the injector
where does the injector release the sample in a GC?
the sample inlet which will then release it into the column inside the oven
where does separation occur during GC
inside the column
name the 3 injectors that can be used on capillary columns in GC
split
splitless
On-column
name the two GC injectors used on packed columns
flash vaporisation and On-column
what happens when a split injector is used in GC
only 2% of your sample reaches the column as the rest is vented to the atmosphere to prevent overloading. It also reduces sensitivity.
what happens when you use a splitless injector in GC
all of your sample is condensed on the top of your column - where the temperature is just above the boiling point of the carrier gas. Your sample is trapped until the temperature is increased at the top of the column. This allows for the experiment to be much more sensitive, however it also allows for overloading of the column.
what happens when you use an On-column injector on a capillary column in GC
all of the sample is condensed in a cooled zone on the top of the column. The sample is then volatilised by programmed heating, and then can be released into the carrier gas. This minimises degradation to thermally labile components
what happens when you use an On-column injector on a packed column in GC
the sample is injected onto a packed bed and it minimises degradation to thermally labile components
what happens when we use a flash vaporisation injector in GC and what can we not use it on?
the sample is injected into a hot zone above the column, where the temperature is around 20-50 degrees above the column temperature, causing the sample to immediately breakdown and volatize - DO NOT USE ON THERMALLY LABILE COMPONENTS
what are the properties of a capillary column
long (up to 100m), narrow (extremely small diameter), coiled tube made of quartz, with a stationary phase coating the inner wall
which column is more efficient; packed or capillary?
capillary
what are the properties of a packed column in GC?
short tubes made of glass with a large diameter, packed with a granular solid made of silica particles (on top of the silica particles is a thin coating of a liquid stationary phase)
what is the isothermal method regarding GC
the temperature remains the same throughout the analysis
what is temperature programming in regards to GC?
where the temperature of the fan oven is set to change during analysis
what do GC detectors produce
a chromatogram - a plot of retention time against relative abundance
what is retention time
the time taken for your separated component to elute from the column
name 2 universal GC detectors
Thermal conductivity & Flame ionisation
how do thermal conductivity detectors work?
they detect changes (decreases) in the thermal conductivity of the carrier gas, which can then be compared to a reference flow of carrier gas
how does a flame ionisation detector work?
the carrier gas elutes from the column and is mixed with hydrogen and air and then is burnt. This results in the production of ions which are collected at a negative electrode, which produces a current which is directly proportional to the concentration of the sample
name 3 specific GC detectors
- Nitrogen-Phosphorus
- Electron Capture
- Mass spectrometer
Explain what a nitrogen-phosphorus detector detects and how it does this?
Detects nitrogen and phosphorus containing groups. It is a modified flame ionisation detector that has a ceramic bead that is heated to 800 degrees. The modification allows for Nitrogen detection to be increased by a factor of 50 and Phosphorus by a factor of 500.
what does an electron capture detector detect
specific to electronegative elements - group 7 elements e.g. chlorine, iodine etc.
how does a mass spectrometer detector work?
samples are broken down into ions and fragments of a specific mass to charge ratio that can be identified
what does HPLC do
separate a complex mixture into its component parts at room temperature
what column is used in HPLC
a packed column
how is the sample transported to the column in HPLC
by a pressurised flow of liquid mobile phase
name the two types of separation used in HPLC and which is more commonly used
normal phase (not commonly used) and reverse phase (95% of the time)
what mobile and stationary phase is used in normal phase separation in HPLC
mobile = a non-polar liquid - a mixture of hydrocarbons with an alcohol or chlorinated solvent
stationary = a polar substance - an unmodified silica
what mobile and stationary phase is used in reverse phase separation in HPLC
mobile = polar solution e.g. methanol and water (must contain water)
stationary = non-polar - a modified silica (C18 or C8)
what type of bottle is the sample placed into for HPLC
Schott
what does a degasser do in HPLC
pumps helium into the mobile phase
what do the pumps do in HPLC
2 reciprocal pumps ensure the mobile phase is pumped around the HPLC instrument at a constant and reproducible flow rate, with a high pressure
what does the column compartment do in a HPLC machine
allows for temperature control
what detector is used with a HPLC machine
a UV visible detector set to 254nm
if the analyte is polar what should the mobile and stationary phase be?
mobile = non-polar stationary phase = polar
what is solvent programming in HPLC
changing the composition of the mobile phase during the analysis
name the two types of HPLC columns
conventional & microbore
name the 4 features both HPLC columns share
- stainless steel
- variety of lengths
- contain different stationary and mobile phases
- mean particle diameters 3.5 and 10um
what is the operating pressure and flow rate of conventional column in HPLC
OP = 500-3000 psi
FR = 1-3 ml/min
what is the operating pressure and flow rate of a microbore column in HPLC
OP = 1000-5000 - increases sensitivity
FR = 10-100µl/min - costs less
name the 4 HPLC detectors
- mass spectrometer
- UV visible absorbance
- Fluorescence
- Refractive index
what can a mass spectrometer detector do in HPLC that no other detector can
provide positive identification
what detector would you use to detect steroids in HPLC
Fluorescence
which is the only HPLC detector that is universal
Refractive index detector as it looks at the RI changes in your mobile phase as your sample elutes from the column
what are the forensic uses of GC and GCMS
Drug, accelerant, paint, plastic, polymer analysis
what are the forensic uses of HPLC and LC-MS
Drug and Ink analysis - without the need for chemical intervention
give 4 examples of polar solvents used in HPLC
water, acetic acid, methanol, ethanol
