4103FSBMOL - Lecture 3/4 - Polarity, Extraction and Basic Chromatography. Flashcards
1
Q
What thing is Chromatography goverened by?
A
Polarity of molecules. Tells you how much of an affinity the molecules have.
2
Q
What 2 things is the Polarity of Molecules based off?
A
- Dipole Moment.
and/or
- Asymmetrical Structure.
3
Q
What is the definition of a Dipole Moment?
A
The Dipole Moment (µ) - the sum of inidividual bond polarities and lone pair contributions within a molecule.
4
Q
Which type of molecules (Polar or Non-Polar) require a dipole moment? Why?
A
Polar Molecules - they have one element which is more electronegative leading to a δ+ve and a δ-ve.
5
Q
How does a Dipole Moment occur between 2 molecules of Chloromethane?
A
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+).
6
Q
What are some of the Properties of Polar Molecules?
A
- 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.
7
Q
What Units are used for a Dipole Moment?
A
Debeyes (D) - higher dipole moment = higher boiling point.
8
Q
Why are Hydrogen Bonds (such as that found in water) so strong?
A
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!
9
Q
What are some of the Properties of Non-Polar Molecules?
A
- 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.
10
Q
What is Extraction used for?
A
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.
11
Q
What type of samples can be extracted?
A
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.
12
Q
Why might we need to use extraction for samples from a Crime Scene?
A
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.
13
Q
What is the main aim of Extraction?
A
To be able to make a Sample suitable to analyse with GC and HPLC.
14
Q
What are some of the problems with Extraction?
A
- 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.
15
Q
3
What are the 4 different Extraction Methods?
A
- Liquid-Solid Extraction.
- Liquid-Liquid Extraction.
- Solid Phase (SPE) - Level 5.
- Solid Phase Micro Extraction (SPME) - Level 6.
16
Q
What is Liquid-Solid Extraction?
A
Extracting a solid powder into a liquid (e.g. extracting cocaine (non-polar) into a non-polar solvent).
17
Q
What is Liquid-Liquid Extraction?
A
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.
18
Q
What does Log(P) determine?
A
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).
19
Q
What does a Positive Log(P) value mean?
A
It will be Non-Polar and extracted into the Organic (non-polar) layer.
20
Q
What does a Negative Log(P) value mean?
A
It will be Polar and extracted into the Aqueous (polar) layer.
21
Q
Give an example to help explain how Log(P) values work.
A
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.
22
Q
How can Log(P) values help us to explain how drugs can be traced in our fingerprints?
A
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).
23
Q
What 4 things can drugs be?
A
Acidic, Basic, Neutral or Amphoteric.
24
Q
What does it mean if a drug is acidic?
A
It contains acidic functional groups such as the Carboxyllic Acid (COOH) group.
25
What does it mean if a *drug is **basic**?*
It **contains basic functional groups** such as the *Amine (NH2)* group.
26
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.
27
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.
28
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.*
29
What *type of drug* is **Aspirin**?
| (Acidic, Basic, Neutral, Amphoteric)
## Footnote
Explain how it is.
Acidic.
## Footnote
**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).**
30
How many pH units should **Acidic** Drugs be *extracted from the pKa value?*
**2** pH units ***below*** the pKa value.
31
What *type of drug* is **Amphetamine**?
| (Acidic, Basic, Neutral, Amphoteric)
## Footnote
Explain how it is.
Basic.
## Footnote
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.
32
How many pH units should **Basic** Drugs be *extracted from the pKa value?*
**2** pH units ***above*** the pKa value.
33
What *type of drug* is **Morphine**?
| (Acidic, Basic, Neutral, Amphoteric)
## Footnote
Explain how it is.
Amphoteric.
## Footnote
**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.***
34
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.***
35
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.
36
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**.
37
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**.
38
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.
39
What does **TLC** stand for?
**T**hin **L**ayer **C**hromatography.
40
What **Stationary Phase** is used in *TLC?*
A **solid Microparticulate** stationary phase bound to a backing.
41
What **Mobile Phase** is used in *TLC?*
A blend of *Analytical Grade* Solvents.
42
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.**
43
How do you set up and run Thin Layer Chromatography?
1. 0.5cl of your Mobile Phase is poured into your TLC Development Tank and it is let to equilibriate for 10 minutes.
2. Prepare your TLC Plate by drawing a line 1cm from the bottom of the plate (don't remove any stationary phase).
3. 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.
4. Repeat the dabbing according to the SOP (Standard Operating Procedure) - spot, dry, re-spot, dry etc.
5. Place the TLC Plate into the tank and leave to develop.
6. 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.
44
What is the *name of the line* at the ***bottom* of the TLC Plate** that you draw?
Base Line.
45
What is the *name of the line* at the ***top* of the TLC Plate** that you draw?
Solvent Front.
46
What is the **name of the Movement** of which the *mobile phase travels up the TLC Plate?*
Capillary Action.
47
What does a ***High Affinity* for the Stationary Phase** mean?
The components will **travel a *short* distance** up the TLC Plate.
48
What does a ***Low Affinity* for the Stationary Phase** mean?
The components will **travel a *long* distance** up the TLC Plate.
49
What is a **Rf value?**
It stands for **'Retention Factor'** and can be used to ***TENTATIVELY* identify** the unknown spots from known Rf values.
50
How do you **calculate the Rf Value** on a TLC plate?
Rf value = distance travelled by the substance/ distance travelled by the solvent front.
## Footnote
Measure to halfway of the spot.
51
Drugs aren't coloured apart from which one?
The Blue Pill.
52
What do you have to do to a TLC plate to calcaulate the Rf values of drugs?
**Chemically Enhance** the spots.
53
What Rf Value range is for optimum separation?
0.2 - 0.8.
54
What unit do you measure Rf values in?
**Millimetres (mm)** *not* centimeters (cm).
55
What is the **backing of a TLC plate** usually made of?
Glass or Aluminium.
56
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.
57
What **diameter size** range must the *Microparticulate stationary phase* range between?
10-60µm.
58
Does a *smaller or larger particle size* lead to *better seperation* on a TLC Plate?
Smaller.
59
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.
60
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.
61
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.
62
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.
63
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.*
64
What binds to functional groups/ structures/ components within TLC spots to *make them coloured?*
Locating Reagents.
65
What can be used to *enhance Locating Reagents?*
Heating them.
66
What **chemical** can we use to *bind to Amino Acids* to allow us to see them?
Ninhydrin.
67
What **colour** does *Ninhydrin go with Amino Acids?*
Purple.
68
What is ***Ninhydrin** used for in Forensics?*
A **fingerprint developer** (binds to the amino acids in our sweat).
69
What *chemical* can we use to **detect steroids** in TLC?
Anisaldehyde/ antimony trichloride.
| It produces **various colour spots.**
70
What can we use to *detect **Alcohols** in TLC?*
Vanillin/ Sulphuric Acid.
71
What **colours of Spots** are produced with *Alcohols and Vanillin/ Sulphuric Acid?*
**Blue, Pink and Green** Spots.
72
What do we need to add to *enhance **fats** for TLC?*
We need to ***Spray*** with **Concentrated Sulphuric Acid** in a *Fume Cupboard.*
73
What do we use to analyse Inks in Forged Documents?
TLC.
74
Can TLC be used for the Screening of Drugs?
**Yes**, but it *isn't used in the routine analysis* of drugs.
75
What does GC stand for?
**G**as **C**hromatography.
76
What *type of components* is Gas Chromatography used for?
The separation of a complex mixture of **Volatile** components.
77
What is the separation in GC based off?
Boiling points and the affinity of your sample for the stationary phase.
78
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.
79
What is the *stationary phase* used in GC?
A liquid or packed solid stationary phase which lines the inner wall.
80
What does a ***Long* Retention Time** mean?
The sample likes the stationary phase, and **moves slower** in the GC Column.
81
What does a ***Long* Retention Time** mean?
The sample doesn't like the stationary phase, and **moves faster** in the GC Column (elutes quicker).
82
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.***
83
What are the *2 different types of GC Column?*
* Capillary.
* Packed.
84
What *Carrier Gas(es)* is/are used for a **Capillary Column**?
Nitrogen and Helium.
85
What *Carrier Gas(es)* is/are used for a **Packed Column**?
Hydrogen.
86
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.
87
The ______ inert and ______ volatile a substance is, the __________ it travels through the GC Column.
| 1. more/less.
2. more/less.
3. faster/slower.
1. More.
2. More.
3. Faster.
| This means the *Retention Time will be Lower* - in the column less time.
88
What type of **Detector** does the **GC** have?
A **Hydrogen Flame Burning Detector** - measures the conductivity of the flame.
89
On a graph produced by a GC, what does the *Area under the peak equal?*
The amount of Sample.
90
________ drugs must be used with a ________ stationary phase and vice versa.
2. Polar/Non-Polar.
| 1. Polar/Non-Polar.
Polar and Polar
or
Non-Polar and Non-Polar.
## Footnote
Important!
91
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.
92
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%).
93
What does **GC-MS** stand for?
**G**as **C**hromatography **-** **M**ass **S**pectrometry.
94
How does a GC-MS machine work?
1. 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.
2. The injector releases it into the sample inlet and the sample is released into the carrier gas stream which takes it to the column.
3. 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.
4. 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.
95
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.
96
What **different Injector(s)** can be used for *GC Capillary Columns?*
* Split Injector.
* Splitless Injector.
* On-Column (Capillary Column) Injector.
97
What **different Injector(s)** can be used for *GC Packed Columns?*
* On-Column (Packed Column) Injector
* Flash Vapourisation.
98
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.
99
What is an **Advantage** of a *GC Splitless Injector?*
All the sample reaches the column **much more sensitive**ly than a Split Injector.
100
What is a **Disadvantage** of a *GC Splitless Injector?*
It will **overload** your capillary column.
101
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).
102
What is an **Advantage** of a *GC Split Injector?*
It **prevents overloading** of a capillary column.
103
What is a **Disadvantage** of a *GC Split Injector?*
It is **not as sensitive** as the Splitless Injector.
104
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.
105
What is the name of the On-Column (Capillary Column) septum the needle is inejcted into?
Duckbill Septum.
106
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.
107
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.***
108
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.
109
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.
110
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.
111
Why do we use Split-injection with Capillary Columns?
To prevent overloading of the column.
112
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.*
113
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.
114
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.
115
What are the GC Columns housed in?
An **electric fan oven** which the temperature can be changed.
116
What is it called if the **temperature of the GC oven *doesn't change*** throughout analysis?
Isothermal Method.
117
What is it called if the **temperature of the GC oven *does change*** throughout analysis?
Temperature Programming Method.
118
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.
119
What is the *Definition* of **Retention Time**?
The time taken for the separated component to elute from the column.
120
# **_Practice Question:_**
Which GC Injector *prevents overloading* of the Column?
## Footnote
1. Split.
2. Splitless.
3. Flash Vapourisation.
4. On-Column.
1. Split Injector.
121
# **_Practice Question:_**
You have been given a mixture of *thermally labile compounds* to analyse, which GC Injector would you use for analysis?
## Footnote
1. Split.
2. Splitless.
3. Flash Vapourisation.
4. On-Column.
4. On-Column.
122
# **_Practice Question:_**
Which GC Injector *causes decomposition of thermally labile components?*
## Footnote
1. Split.
2. Splitless.
3. Flash Vapourisation.
4. On-Column.
3. Flash Vapourisation.
123
What are the *2 classifications* of *GC Detector?*
Universal and Specific Detectors.
124
Name the *GC **Universal Detectors**.*
* Thermal Conductivity Detector.
* GC-FID.
125
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.
126
What does **GC-FID** stand for?
**G**as **C**hromatography **-** **F**lame **I**onisation **D**etector.
127
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.
128
Which is the **most important *Universal* GC Detector**?
The **GC-FID**.
129
Name the *GC **Specific Detectors**.*
* Nitrogen-Phosphorus Detector.
* Electron Capture Detector.
* Mass Spectrometer.
130
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.
131
What *temperature* is the *Nitrogen-Phosphorus Detector* heated to?
800°C.
132
What **factor** do you get **Nitrogen detection** with in a *Nitrogen-Phosphorus Detector?*
An **Increase Factor of *50.***
133
What **factor** do you get **Phosphorus detection** with in a *Nitrogen-Phosphorus Detector?*
An **Increase Factor of *500.***
134
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).
135
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.
136
Which is the **most important *Specific* GC Detector**?
The **Mass Spectrometer** as it is the most Specific for identifying components.
137
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.
138
In what form does the Mass Spectrometer output its data?
In a **Mass Spectrum**.
139
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.*
140
What do you have to compare values from the Mass Spectrum to?
**Values from a Database** in order to identify unknowns.
141
# **_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.*
142
What does **HPLC** stand for?
**H**igh **P**erformance **L**iquid ** **hromatography.
143
What *Temperature* does *HPLC* work at?
Room Temperature.
144
How is the sample in HPLC transported to the column?
By a pressurised flow of liquid mobile phase.
145
What is the HPLC column packed with?
A solid microparticulate stationary phase.
146
What are the components in a HPLC column sperated by?
Their affinity for the stationary phase.
147
What is HPLC used for?
To find the *relative amounts* of different components in a mixture.
148
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.
149
How does the liquid mobile phase move around in HPLC?
Using **high pressure pumps** which creates a pressurised flow.
150
What are the **2 phases** in which **HPLC** can be used?
The **Normal Phase** and the **Reverse Phase.**
151
Is the *Mobile Phase Polar or Non-Polar* in **Normal Phase HPLC**?
**Non-Polar** (mixture of hydrocarbons/ chlorinated solvents and an alcohol).
152
Is the *Stationary Phase Polar or Non-Polar* in **Normal Phase HPLC**?
**Polar** (unmodified silica).
153
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.***
154
Is the *Mobile Phase Polar or Non-Polar* in **Reverse Phase HPLC**?
**Polar** (simplest is Water).
155
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.
156
For *Reverse Phase* HPLC Separation, **what MUST the Mobile Phase contain**?
**Water** as it's the *Most Polar Solvent Available!*
157
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.**
158
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).
159
What is the **De-gasser we use** at LJMU for the HPLC machine?
Helium.
160
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.**
161
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!
162
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.
163
What constant and reproduceable **flow rate** and **pressure** do the *reciprocal pumps in HPLC* work at?
Around **15,000Psi** and **1ml/ minute.**
164
What lies under the *Reciprocal Pumps* in the HPLC machine?
The **Auto-sampler**.
165
What type of HPLC Column and Detector(s) do we use at LJMU?
A Conventional Column with a UV-Visible Detector.
166
What *wavelength is the UV-Visible detector* in the HPLC machine set at at LJMU?
254 nanometers (nm).
167
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)).
168
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).
169
What **Stationary Phases** can you use for *Normal-Phase HPLC?*
## Footnote
Why are these used?
* Unmodified Silica.
* Aminopropyl (C3H6NH2).
## Footnote
Because **they are Polar** which is what the stationary phase needs to be in **Normal Phase HPLC.**
170
What **Stationary Phases** can you use for *Reverse-Phase HPLC?*
## Footnote
Why are these used?
* *Octadecyl* silica **(C18)** - C18 H37.
* *Octyl* Silica **(C8)** - C8 H17.
* *Propyl* silica **(C3)** - C3 H7.
## Footnote
Because **they are Non-Polar** which is what the stationary phase needs to be in **Reverse Phase HPLC.**
171
What is the ***Most Common** Stationary Phase for **Reverse Phase** HPLC?*
**C18 Column** (silica backbone with akyl group and 18 carbons attached).
## Footnote
C8 is the second most common.
172
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.**
173
In HPLC, does the sample have to have the **same polarity** as the *mobile or stationary phase?*
Stationary Phase.
174
In HPLC, does the sample have to have a **different polarity** to the *mobile or stationary phase?*
Mobile Phase.
175
What happens if the *Stationary Phase and Mobile Phase in HPLC have the same polarity?*
There will be **NO Separation!**
176
What is the **name of the process** of *changing the composition of the mobile phase during analysis?*
Solvent Programming.
177
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).*
178
How do you *blend a **Polar** Mobile Phase* for HPLC?
**Water (Polar) will be your main component** and you will *add an alcohol (polar).*
179
Give examples of **3 different *Non-Polar* Mobile Phases** for HPLC.
* Hexane.
* Cyclohexane.
* Chloroform.
| (Hydrocarbons)
180
Give examples of **3 different *Polar* Mobile Phases** for HPLC.
* **Water**.
* Methanol.
* Ethanol.
181
How much sample can sample loops hold?
Between **1 and 100 micro litres (μL)** of a sample.
182
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.
183
What are the *2 different types of column* for HPLC?
Conventional and Microbore.
184
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.
185
What are the different mean particle diameters that can be used in HPLC Columns?
3, 5 or 10μm.
186
What is the **operating pressure and flow rate** for a *Conventional HPLC column?*
* Operating Pressure = **500-3000Psi.**
* Flow Rate = **1000-3000μL/min-1.**
187
What is the **operating pressure and flow rate** for a *Microbore HPLC column?*
* Operating Pressure = **1000-5000Psi.**
* Flow Rate = **10-100μL/min-1.**
188
Which is the **HPLC *Column* of Choice**?
Microbore.
189
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.**
190
What is the **HPLC *Detector* of Choice?**
Mass Spectrometer.
191
Why is the Mass Spectrometer the HPLC Detector of Choice?
It always *provides a **positive identification.***
192
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:_**
193
Why would we use a *UV-Visible Absorbance Detector* for HPLC?
It can measure a single wavelength or Solid State Diodes.
194
Why would we use a *Fluorescence Detector* for HPLC?
It is good for analysing fluorescent compounds such as Steroids.
195
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.
## Footnote
It is the only *Non-specific detector* (we wouldn't really use it)!
196
What is **TLC** mainly used for in *Forensic Analysis using Chromatography?*
Ink and Drug Analysis.
197
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.
198
What is **HPLC and LC-MS** mainly used for in *Forensic Analysis using Chromatography?*
Drug and Ink Analysis.
199
# **_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.
200
# **_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).
## Footnote
For *normal compounds*, would use Split-injection as it prevents overloading of the column. (2 marks).
