4103FSBMOL - Lecture 3/4 - Polarity, Extraction and Basic Chromatography. Flashcards
What thing is Chromatography goverened by?
Polarity of molecules. Tells you how much of an affinity the molecules have.
What 2 things is the Polarity of Molecules based off?
- Dipole Moment.
and/or
- Asymmetrical Structure.
What is the definition of a Dipole Moment?
The Dipole Moment (µ) - the sum of inidividual bond polarities and lone pair contributions within a molecule.
Which type of molecules (Polar or Non-Polar) require a dipole moment? Why?
Polar Molecules - they have one element which is more electronegative leading to a δ+ve and a δ-ve.
How does a Dipole Moment occur between 2 molecules of Chloromethane?
Chlorine is highly electronegative and loves electrons, so all the electrons in the bond between the methyl group and the chlorine group surround the chlorine atom, producing a permanent negative dipole (δ-). This leads to the methyl group being deficient in electrons giving it a permanent positive dipole (δ+).
What are some of the Properties of Polar Molecules?
- They are Hydrophilic - water loving, fat hating. They dissolve and extract into polar solvents.
- They have higher boiling and melting points than Non-Polar molecules - due to the dipole-dipole forces.
What Units are used for a Dipole Moment?
Debeyes (D) - higher dipole moment = higher boiling point.
Why are Hydrogen Bonds (such as that found in water) so strong?
They are found in Polar Molecules - attach to Oxygen, Nitrogen and Fluorine Groups. This leads to the hydrogen being deficient of electrons and in Water, the oxygen’s being in surplus due to having a higher electronegativity. This leads to a permanent negative dipole on oxygen, nitrogen or fluorine groups and a permanent positive dipole on hydrogen groups for example which have a lower electronegativity. Hydrogen is very small and has a lack of electrons attracting it (no electrons protecting the nucleus) - this leads to the hydrogen bonding occurring very close to the hydrogen’s nucleus. This is why Water, Ammonia and Hydrogen Fluoride have abnormally high boiling points!
What are some of the Properties of Non-Polar Molecules?
- They are Hydrophobic - fat loving, water hating. They dissolve and extract into non-polar or organic solvents.
- They have lower boiling and melting points than polar molecules due to the weak (temporary) Van der Waals Forces/ London Dispersion Forces. They can be easily broken and so less energy is required to break the bonds.
What is Extraction used for?
When samples can’t be directly analysed by Chromatography. A sample is made up of the analyte of interest and a matrix. Most biological matrices such as blood, plasma and semen and vaginal secretions can’t be injected into a HPLC or GC. The analyte of interest needs to be extracted out of the matrix into a solvent, to be able to analyse it by chromatography.
What type of samples can be extracted?
We need to make sure samples are pure so contaminants can be removed. Also, they need to be concentrated otherwise we can’t analyse them.
Why might we need to use extraction for samples from a Crime Scene?
At a Crime Scene, samples may be below the limit of detection for GC or HPLC, so we need to extract them to concentrate up the samples.
What is the main aim of Extraction?
To be able to make a Sample suitable to analyse with GC and HPLC.
What are some of the problems with Extraction?
- It requires samples with a large number of analytes - its difficult to extarct only 1 analyte.
- Small amount of substance (analyte) - extraction can remove some sample.
- Because of Chemical Similarity, its hard to extract just 1 analyte.
3
What are the 4 different Extraction Methods?
- Liquid-Solid Extraction.
- Liquid-Liquid Extraction.
- Solid Phase (SPE) - Level 5.
- Solid Phase Micro Extraction (SPME) - Level 6.
What is Liquid-Solid Extraction?
Extracting a solid powder into a liquid (e.g. extracting cocaine (non-polar) into a non-polar solvent).
What is Liquid-Liquid Extraction?
Where a liquid sample is partitioned into another liquid (use of a seperating funnel - to separate a mixed solution into 2 layers, which are split apart into 2 seperate conical flasks). The aqueous (polar) layer is found on the bottom usually, and a non-polar solvent layer on top (they are immiscible). You then evapourate to dryness using nitrogen to separate the solvents from the analytes. Dependent on the Polarity of the analyte and the pH.
What does Log(P) determine?
How Hydrophobic/ Hydrophilic or Non-Polar a drug is. Hydrophobic analytes extract into the oil (the upper non-polar layer), and hydrophilic analytes extract into the water (the lower polar layer).
What does a Positive Log(P) value mean?
It will be Non-Polar and extracted into the Organic (non-polar) layer.
What does a Negative Log(P) value mean?
It will be Polar and extracted into the Aqueous (polar) layer.
Give an example to help explain how Log(P) values work.
Log P values indicate how easily a drug will cross lipid membranes and reach the site of action. Because Methadone and THC are fat soluble, they will easily cross the blood brain barrier and therefore Methadone will bind to the opioid receptors in your brain and THC combines to cannabis receptors in the brain.
How can Log(P) values help us to explain how drugs can be traced in our fingerprints?
Drugs such as Methadone and THC are fat soluble and are stored in the fat stores. They will be released into the bloodstream periodically giving the effects of the drugs. They pass the blood sweat barrier which allows us to trace drugs in the sweat on your hands (fingerprints).
What 4 things can drugs be?
Acidic, Basic, Neutral or Amphoteric.
What does it mean if a drug is acidic?
It contains acidic functional groups such as the Carboxyllic Acid (COOH) group.
What does it mean if a drug is basic?
It contains basic functional groups such as the Amine (NH2) group.
What does it mean if a drug is neutral?
It is electrically neutral and doesn’t contain any acidic or basic functional groups/ or charges cancel each other out.
What does it mean if a drug is amphoteric?
It contains both acidic and basic functional groups. These functional groups are ionisation centres and can be protonated or deprotonated.
What is the difference between Ionised and Non-ionised drugs?
- Ionised drugs are soluble in Polar Solvents.
- Non-Ionised drugs are soluble in Non-Polar Solvents.
What type of drug is Aspirin?
(Acidic, Basic, Neutral, Amphoteric)
Explain how it is.
Acidic.
It has a COOH (Carboxyllic Acid Group). When Aspirin is put into water it is deprotonated (loses a Hydrogen atom from the COOH group) making a COO- ion. The proton binds to the water molecules forming H3O+ ions (acidic ions).
How many pH units should Acidic Drugs be extracted from the pKa value?
2 pH units below the pKa value.
What type of drug is Amphetamine?
(Acidic, Basic, Neutral, Amphoteric)
Explain how it is.
Basic.
When it is put into water the NH2 part is protonated to NH3+ and the water becomes OH- ions as its lost a proton leading to a basic solution.
How many pH units should Basic Drugs be extracted from the pKa value?
2 pH units above the pKa value.
What type of drug is Morphine?
(Acidic, Basic, Neutral, Amphoteric)
Explain how it is.
Amphoteric.
It has Acidic and Basic functional groups/ properties. It has 2 pKa values and so you balance the pKa values and you extract at that pH, where the isoelectric point is, so it becomes electrically neutral.
What are the Disadvantages of Liquid-Liquid Extraction?
- It’s Time Consuming.
- Large Volumes of Solvent are needed (this is analytical grade and very expensive).
- Sample Reconstitution (HPLC Grade Solvent).
- Analytical Grade Liquids need special waste disposal - expensive.
How does Gas Chromatography work?
The sample is transported by a mobile phase (carrier gas) in the GC column. The column is either lined with a stationary phase or is lined with the stationary phase on the inner walls. Separation occurs in the column based off the components affinity for the stationary phase. They are then detected by the detector.
Does the Polarity of the Sample have to be the same or different to the Polarity of the Mobile Phase?
The polarity of the sample must be the same as of the stationary phase.
Does the Polarity of the Sample have to be the same or different to the Polarity of the Stationary Phase?
The polarity of the sample must be different to the stationary phase.
What happens if the Mobile Phase and Stationary Phase on a GC Column are the same polarity?
A mixture will go on and off the column if the mobile phase is the same polarity as the stationary phase.
What does TLC stand for?
Thin Layer Chromatography.
What Stationary Phase is used in TLC?
A solid Microparticulate stationary phase bound to a backing.
What Mobile Phase is used in TLC?
A blend of Analytical Grade Solvents.
What is TLC used for in Forensic Science?
The separation of inks into its component dyes and pigments (for document analysis/ forgery), and screening for drugs.
How do you set up and run Thin Layer Chromatography?
- 0.5cl of your Mobile Phase is poured into your TLC Development Tank and it is let to equilibriate for 10 minutes.
- Prepare your TLC Plate by drawing a line 1cm from the bottom of the plate (don’t remove any stationary phase).
- Add crosses to the line at equal distances and add each sample seperately into double open-ended capillary tubes and dab each sample onto seperate crosses.
- Repeat the dabbing according to the SOP (Standard Operating Procedure) - spot, dry, re-spot, dry etc.
- Place the TLC Plate into the tank and leave to develop.
- Draw a pencil line where the solvent reaches. You may also need to draw around the spots if they are light (e.g. light yellow) which may be harder to see if they fade.
What is the name of the line at the bottom of the TLC Plate that you draw?
Base Line.
What is the name of the line at the top of the TLC Plate that you draw?
Solvent Front.
What is the name of the Movement of which the mobile phase travels up the TLC Plate?
Capillary Action.
What does a High Affinity for the Stationary Phase mean?
The components will travel a short distance up the TLC Plate.
What does a Low Affinity for the Stationary Phase mean?
The components will travel a long distance up the TLC Plate.
What is a Rf value?
It stands for ‘Retention Factor’ and can be used to TENTATIVELY identify the unknown spots from known Rf values.
How do you calculate the Rf Value on a TLC plate?
Rf value = distance travelled by the substance/ distance travelled by the solvent front.
Measure to halfway of the spot.
Drugs aren’t coloured apart from which one?
The Blue Pill.
What do you have to do to a TLC plate to calcaulate the Rf values of drugs?
Chemically Enhance the spots.
What Rf Value range is for optimum separation?
0.2 - 0.8.
What unit do you measure Rf values in?
Millimetres (mm) not centimeters (cm).
What is the backing of a TLC plate usually made of?
Glass or Aluminium.
Which is better - glass or aluminium - for the backing of the TLC Plate?
Aluminium is better as you can cut it to the required size needed.
What diameter size range must the Microparticulate stationary phase range between?
10-60µm.
Does a smaller or larger particle size lead to better seperation on a TLC Plate?
Smaller.
What do we use as our TLC Stationary Phase? Why?
We use Silica as our Stationary Phase. It can be used for the separation of Drugs, Inks, Fat-Soluble Vitamins and Amino Acids.
Why can’t we touch the TLC plate with our hands (no gloves)?
Because our sweat has Amino Acids in it and so it will contaminate the results if we touch the TLC Plate.
What can we impregnate into the Silica Stationary Phase to allow us to see the components?
What piece of equipment do we also have to use to see them?
Fluorescent Dyes (which emit at 1 wavelength - 254nm). It allows us to use a UV Lamp to view the compounds/ components if they aren’t coloured.
What are the different type of Stationary Phases you can use for TLC?
- Hydrocarbon Modified Silica - Non-Polar Compounds.
- Cellulose - Amino Acids and Carbohydrates.
- Alumina - Hydrocarbons, alkaloids (drugs), food dyes and lipids.
- Sephadex Gels - Polymers, Proteins and Metal Complexes.
What can we do to the TLC Mobile Phase?
To make things better.
Change the mixture of the high purity (analytical grade) solvents slightly to adjust the solvent strength to help optimise separation and achieve an accurate Rf value.
What binds to functional groups/ structures/ components within TLC spots to make them coloured?
Locating Reagents.
What can be used to enhance Locating Reagents?
Heating them.
What chemical can we use to bind to Amino Acids to allow us to see them?
Ninhydrin.
What colour does Ninhydrin go with Amino Acids?
Purple.
What is Ninhydrin used for in Forensics?
A fingerprint developer (binds to the amino acids in our sweat).
What chemical can we use to detect steroids in TLC?
Anisaldehyde/ antimony trichloride.
It produces various colour spots.
What can we use to detect Alcohols in TLC?
Vanillin/ Sulphuric Acid.
What colours of Spots are produced with Alcohols and Vanillin/ Sulphuric Acid?
Blue, Pink and Green Spots.
What do we need to add to enhance fats for TLC?
We need to Spray with Concentrated Sulphuric Acid in a Fume Cupboard.
What do we use to analyse Inks in Forged Documents?
TLC.
Can TLC be used for the Screening of Drugs?
Yes, but it isn’t used in the routine analysis of drugs.
What does GC stand for?
Gas Chromatography.
What type of components is Gas Chromatography used for?
The separation of a complex mixture of Volatile components.
What is the separation in GC based off?
Boiling points and the affinity of your sample for the stationary phase.
What is the mobile phase used in GC?
A Carrier Gas (has no part in separation) is just used to transport the sample to the column.
What is the stationary phase used in GC?
A liquid or packed solid stationary phase which lines the inner wall.
What does a Long Retention Time mean?
The sample likes the stationary phase, and moves slower in the GC Column.
What does a Long Retention Time mean?
The sample doesn’t like the stationary phase, and moves faster in the GC Column (elutes quicker).
What is the Carrier Gas usually made of in GC?
A pressurised and purified inert gas (non-toxic and non-flammable) - usually Nitrogen, Helium, Hydrogen or Acetylene.
What are the 2 different types of GC Column?
- Capillary.
- Packed.
What Carrier Gas(es) is/are used for a Capillary Column?
Nitrogen and Helium.
What Carrier Gas(es) is/are used for a Packed Column?
Hydrogen.
What is the Stationary Phase usually made of in GC?
High Boiling Point Liquids, Waxes and Oils which have specific polarities to allow us to analyse different components.
The ______ inert and ______ volatile a substance is, the __________ it travels through the GC Column.
1. more/less.
2. more/less.
3. faster/slower.
- More.
- More.
- Faster.
This means the Retention Time will be Lower - in the column less time.
What type of Detector does the GC have?
A Hydrogen Flame Burning Detector - measures the conductivity of the flame.
On a graph produced by a GC, what does the Area under the peak equal?
The amount of Sample.
________ drugs must be used with a ________ stationary phase and vice versa.
- Polar/Non-Polar.
1. Polar/Non-Polar.
Polar and Polar
or
Non-Polar and Non-Polar.
Important!
What are the different types of Non-Polar Stationary Phase which can be used for GC?
(not sure whether we need to know)
- Methyl polysiloxane (100%).
- Methyl (95%) and Phenyl (5%) polysiloxane.
What are the different types of Polar Stationary Phase which can be used for GC?
(not sure whether we need to know)
- Polyethylene Glycol.
- Cyanopropyl polysiloxane.
- Phenyldimethylsilicone (50%).
What does GC-MS stand for?
Gas Chromatography - Mass Spectrometry.
How does a GC-MS machine work?
- An auto-injector has a needle which penetrates the septum in the GC vial. It draws up a required amount of sample into the syringe and then injects this into the injector.
- The injector releases it into the sample inlet and the sample is released into the carrier gas stream which takes it to the column.
- The column sits in an oven and separation occurs within the column and the components get detected by the detector (e.g. the mass spectrometer) once they elute from the column.
- They reach the Ion Source in the MS and get sent to the mass analyser which the ions bounce around so we can detect them.
What is one of the difference between a GC and an MS which has to be resolved by an Ion Source in the MS?
Gas Chromatography runs at Atmospheric Pressure, whereas Mass Spectrometry runs under a vacuum. This means the sample needs to go through an ion source in which they are broken into ions which are passed through tiny holes until they reach vacuum.
What different Injector(s) can be used for GC Capillary Columns?
- Split Injector.
- Splitless Injector.
- On-Column (Capillary Column) Injector.
What different Injector(s) can be used for GC Packed Columns?
- On-Column (Packed Column) Injector
- Flash Vapourisation.
What is a GC Splitless Injector?
It involves all the sample being condensed on the top of the column. The temperature of the column is just above the boiling point of the carrier gas. The carrier gas flows through the instrument where the sample is trapped on top of the column. The Temperature is increased at the top of the column and the sample is injected into the stream of carrier gas.
What is an Advantage of a GC Splitless Injector?
All the sample reaches the column much more sensitively than a Split Injector.
What is a Disadvantage of a GC Splitless Injector?
It will overload your capillary column.
What is a GC Split Injector?
What percentage of the sample gets to the column?
It is an injector where only 2% of your sample reaches the column (rest goes into the atmosphere).
What is an Advantage of a GC Split Injector?
It prevents overloading of a capillary column.
What is a Disadvantage of a GC Split Injector?
It is not as sensitive as the Splitless Injector.
What is a GC On-Column (Capillary Column) Injector?
All the sample is condensed into a cool zone at the top of the column and is volatised using programmed heating and released into the carrier gas stream.
What is the name of the On-Column (Capillary Column) septum the needle is inejcted into?
Duckbill Septum.
What is an Advantage of a GC On-Column (Capillary Column) Injector?
This type of injection minimises degradation to thermally labile components - used for samples degraded by heat.
What is a GC On-Column (Packed Column) Injector?
Very similar to On-Column (Capillary Column) Injector, but the sample is injected into a packed bed which it reduces degradation to thermally labile components.
What is a GC Flash Vapourisation (Packed Column) Injector?
It is opposite to an on-column injector as the sample is injected into a heated zone above the column. It is 20-50°C above the column temperature.
What type of compounds shouldn’t you use with Flash Vapourisation?
One’s degradeable by heat - it rapidly volatises the sample - causes decomposition of thermally labile components.
What are Capillary Columns made out of and what lengths are they usually?
They can have lengths up to 100 metres and they are made out of long narrow tubing made of quartz. The stationary phase coats the inner wall.
Why do we use Split-injection with Capillary Columns?
To prevent overloading of the column.
Why is a Capillary Column the most effective column?
Because it has the smallest diameter of capillaries and the longest length possible with the thinnest layer of stationary phase thickness.
What are Packed Columns made out of and what lengths are they usually?
They are shorter tubes (max of 2 metres). They have a larger diameter and are made of glass because they have to be packed with a granular solid made of silica particles. On top of the granular solid is a thin layer of stationary phase.
How can you improve the efficiency of Packed Columns?
Using the smallest diameter of silica particles within the packed bed and the thinnest coating of stationary phase on top of the silica particles.
What are the GC Columns housed in?
An electric fan oven which the temperature can be changed.
What is it called if the temperature of the GC oven doesn’t change throughout analysis?
Isothermal Method.
What is it called if the temperature of the GC oven does change throughout analysis?
Temperature Programming Method.
In what form does the GC output its data?
A Chromatogram of retention time (in mins) on the x-axis and relative abundance on the y-axis.
What is the Definition of Retention Time?
The time taken for the separated component to elute from the column.
Practice Question:
Which GC Injector prevents overloading of the Column?
- Split.
- Splitless.
- Flash Vapourisation.
- On-Column.
- Split Injector.
Practice Question:
You have been given a mixture of thermally labile compounds to analyse, which GC Injector would you use for analysis?
- Split.
- Splitless.
- Flash Vapourisation.
- On-Column.
- On-Column.
Practice Question:
Which GC Injector causes decomposition of thermally labile components?
- Split.
- Splitless.
- Flash Vapourisation.
- On-Column.
- Flash Vapourisation.
What are the 2 classifications of GC Detector?
Universal and Specific Detectors.
Name the GC Universal Detectors.
- Thermal Conductivity Detector.
- GC-FID.
What does the Thermal Conductivity Detector do?
It detects changes in the thermal conductivity of the carrier gas when we compare it to a reference flow of carrier gas. When the compounds elute from the column, they reduce the thermal conductivity of the carrier gas. This is a detectable signal and so it enables us to detect the sample.
What does GC-FID stand for?
Gas Chromatography - Flame Ionisation Detector.
What does the GC-FID do?
Carrier gas elutes from the column and mixes with hydrogen and air, and is burnt. Produces ions collected at the negative electrode which produces a current that is directly proportionate to the concentration of the sample.
Which is the most important Universal GC Detector?
The GC-FID.
Name the GC Specific Detectors.
- Nitrogen-Phosphorus Detector.
- Electron Capture Detector.
- Mass Spectrometer.
What does the Nitrogen-Phosphorus Detector do?
It detects Nitrogen and Phosphorus Containing groups. It is a modified Flame Ionisation Detector in which its made of a ceramic bead, made from caesium and rubidium salts, its heated and placed between the burner jet and the collector electrode.
What temperature is the Nitrogen-Phosphorus Detector heated to?
800°C.
What factor do you get Nitrogen detection with in a Nitrogen-Phosphorus Detector?
An Increase Factor of 50.
What factor do you get Phosphorus detection with in a Nitrogen-Phosphorus Detector?
An Increase Factor of 500.
What does the Electron Capture Detector do?
It is specific for detecting electronegative elements (group 7 of periodic table) that attract electrons (halogens and sulphur).
What does the Mass Spectrometer do?
Samples are broken down in the GC and elute from the column where they are broken down into ions and fragments of specific mass to charge ratio which allows us to identify components.
Which is the most important Specific GC Detector?
The Mass Spectrometer as it is the most Specific for identifying components.
What are the different types of Mass Spectrometer which you can attach to a GC?
- Quadrupole.
- Time of Flight (TOF).
- Iron Trap.
They can also be attached to Liquid Chromatography and HPLC instruments.
In what form does the Mass Spectrometer output its data?
In a Mass Spectrum.
What are the most important parts of a Mass Spectrum?
- The last peak is the Molecular Ion Peak (M+) - tells you the m/z value for the molecular structure.
- The tallest peak is called the Base Peak (B+) - this tells you the m/z value of the most stable ion.
What do you have to compare values from the Mass Spectrum to?
Values from a Database in order to identify unknowns.
Practice Question:
An unknown powder has been found at a crime scene, which is suspected to be Amphetamine. GC has been chosen for analysis. Using the structure of Amphetamine (contains a secondary amine group and a benzene ring), which 3 specific detectors would you use and why?
Amphetamine Contains nitrogen: the electron capture detector can be used as nitrogen is highly electronegative. Also, the Nitrogen-Phosphorus detector can be used due to the nitrogen, that increases by a factor of 50, and the Mass Spectrometer can be used to positively identify the structure.
What does HPLC stand for?
High Performance Liquid ** **hromatography.
What Temperature does HPLC work at?
Room Temperature.
How is the sample in HPLC transported to the column?
By a pressurised flow of liquid mobile phase.
What is the HPLC column packed with?
A solid microparticulate stationary phase.
What are the components in a HPLC column sperated by?
Their affinity for the stationary phase.
What is HPLC used for?
To find the relative amounts of different components in a mixture.
If the mobile phase in HPLC is more polar than the stationary phase, the more polar components of a mixture will tend to move __________ than the less polar ones.
Quicker or Slower.
Quicker.
How does the liquid mobile phase move around in HPLC?
Using high pressure pumps which creates a pressurised flow.
What are the 2 phases in which HPLC can be used?
The Normal Phase and the Reverse Phase.
Is the Mobile Phase Polar or Non-Polar in Normal Phase HPLC?
Non-Polar (mixture of hydrocarbons/ chlorinated solvents and an alcohol).
Is the Stationary Phase Polar or Non-Polar in Normal Phase HPLC?
Polar (unmodified silica).
What happens in Normal Phase HPLC?
As the mobile phase is non-polar, the non-polar components will have a reduced affinity for the stationary phase and will elute faster. The polar components will have more affinity for the stationary phase and will elute slower.
Is the Mobile Phase Polar or Non-Polar in Reverse Phase HPLC?
Polar (simplest is Water).
Is the Stationary Phase Polar or Non-Polar in Reverse Phase HPLC?
Non-Polar (Can be modified silica and a C8 or C18 column will be used).
Simplest is methanol.
For Reverse Phase HPLC Separation, what MUST the Mobile Phase contain?
Water as it’s the Most Polar Solvent Available!
What happens in Reverse Phase HPLC?
The non-polar components will have a high affinity for the stationary phase - elute slower. Polar components - less affinity - elute faster.
What are the name of the Bottles which the mobile phase is contained in on the top of the HPLC column?
Schott Bottles (manufacturer of the glass bottles).
What is the De-gasser we use at LJMU for the HPLC machine?
Helium.
Why do we pump the de-gasser into the mobile phase for HPLC?
To remove air in the mobile phase to avoid the creation of an airlock.
How do you remove an airlock in a HPLC instrument and what is the downside of it?
Open the waste bottle and waste the HPLC mobile phase until the airlock is removed. It is VERY EXPENSIVE!
What lies under the Degasser in the HPLC machine?
Reciprocal Pump(s) - we have 2. These ensure the mobile phase is pumped around the system at a constant and reproduceable flow rate and pressure.
What constant and reproduceable flow rate and pressure do the reciprocal pumps in HPLC work at?
Around 15,000Psi and 1ml/ minute.
What lies under the Reciprocal Pumps in the HPLC machine?
The Auto-sampler.
What type of HPLC Column and Detector(s) do we use at LJMU?
A Conventional Column with a UV-Visible Detector.
What wavelength is the UV-Visible detector in the HPLC machine set at at LJMU?
254 nanometers (nm).
What other detectors can be used with the HPLC instrument at LJMU?
A HPLC-DAD Detector, a Refractive Index Detector, a Mass Spectrometer (LCMS with a quadrupole and a LCMS with TOF (Time of Flight)).
What is the Stationary Phase we use in HPLC dependent on?
The Polarity of the Analyte of interest (e.g. for the normal phase you would use Unmodified Silica as you are looking for polar components).
What Stationary Phases can you use for Normal-Phase HPLC?
Why are these used?
- Unmodified Silica.
- Aminopropyl (C3H6NH2).
Because they are Polar which is what the stationary phase needs to be in Normal Phase HPLC.
What Stationary Phases can you use for Reverse-Phase HPLC?
Why are these used?
- Octadecyl silica (C18) - C18 H37.
- Octyl Silica (C8) - C8 H17.
- Propyl silica (C3) - C3 H7.
Because they are Non-Polar which is what the stationary phase needs to be in Reverse Phase HPLC.
What is the Most Common Stationary Phase for Reverse Phase HPLC?
C18 Column (silica backbone with akyl group and 18 carbons attached).
C8 is the second most common.
Why is the choice of mobile phase very important in HPLC?
Because it can lead to separating errors with components and errors with retention times.
In HPLC, does the sample have to have the same polarity as the mobile or stationary phase?
Stationary Phase.
In HPLC, does the sample have to have a different polarity to the mobile or stationary phase?
Mobile Phase.
What happens if the Stationary Phase and Mobile Phase in HPLC have the same polarity?
There will be NO Separation!
What is the name of the process of changing the composition of the mobile phase during analysis?
Solvent Programming.
How do you blend a Non-Polar Mobile Phase for HPLC?
You need to use a number of hydrocarbon based samples (non-polar) with an alcohol such as ethanol (polar).
How do you blend a Polar Mobile Phase for HPLC?
Water (Polar) will be your main component and you will add an alcohol (polar).
Give examples of 3 different Non-Polar Mobile Phases for HPLC.
- Hexane.
- Cyclohexane.
- Chloroform.
(Hydrocarbons)
Give examples of 3 different Polar Mobile Phases for HPLC.
- Water.
- Methanol.
- Ethanol.
How much sample can sample loops hold?
Between 1 and 100 micro litres (μL) of a sample.
How do sample loops work for HPLC?
It is controlled by a computer - when you want to inject your sample, the value will open and the mobile phase will move into the sample loop which will then carry the sample to the column.
What are the 2 different types of column for HPLC?
Conventional and Microbore.
What are the HPLC Columns made of and what do they contain?
Stainless Steel Pipes containing different stationary phases and having different mean particle diameters.
What are the different mean particle diameters that can be used in HPLC Columns?
3, 5 or 10μm.
What is the operating pressure and flow rate for a Conventional HPLC column?
- Operating Pressure = 500-3000Psi.
- Flow Rate = 1000-3000μL/min-1.
What is the operating pressure and flow rate for a Microbore HPLC column?
- Operating Pressure = 1000-5000Psi.
- Flow Rate = 10-100μL/min-1.
Which is the HPLC Column of Choice?
Microbore.
Why is Microbore the HPLC Column of Choice?
- It has better separation.
- We also use less mobile phase, this costs us less and means we are being more environmentally friendly.
- It also has a higher operating pressure which means it has better sensitivity.
What is the HPLC Detector of Choice?
Mass Spectrometer.
Why is the Mass Spectrometer the HPLC Detector of Choice?
It always provides a positive identification.
What is the order of the Best to the Worst Detector for HPLC?
Best:
1. Mass Spectrometer.
2. UV-Visible Absorbance Detector.
3. Fluorescence Detector.
4. Refractive Index.
Worst:
Why would we use a UV-Visible Absorbance Detector for HPLC?
It can measure a single wavelength or Solid State Diodes.
Why would we use a Fluorescence Detector for HPLC?
It is good for analysing fluorescent compounds such as Steroids.
Why would/wouldn’t we use a Refractive Index Detector for HPLC?
It looks at the Refractive Index changes in the mobile phase as the sample elutes from the column.
It is the only Non-specific detector (we wouldn’t really use it)!
What is TLC mainly used for in Forensic Analysis using Chromatography?
Ink and Drug Analysis.
What is GC and GC-MS mainly used for in Forensic Analysis using Chromatography?
Drug, Accelerant, Paint, Explosive and Polymer Analysis.
You need to Chemically enhance drugs for GC.
What is HPLC and LC-MS mainly used for in Forensic Analysis using Chromatography?
Drug and Ink Analysis.
Practice Question:
What is meant by a “reverse phase” system in HPLC?
A reverse phase system uses a polar mobile phase (e.g. methanol and water) and a non-polar stationary phase (e.g. a C18 column). In reverse HPLC the most polar components will elute first and the more non-polar components will elute after.
Practice Question:
You wish to determine the concentration of a thermally unstable compound in a mixture by capillary GC, which injector would you use and why? (2 marks).
On-Column injection would be best (1 mark) because this reduces degradation to thermally labile compounds. (1 mark).
For normal compounds, would use Split-injection as it prevents overloading of the column. (2 marks).
