Lecture 1 Flashcards
What are the 2 types of separations?
- Preparative
- Analytical
Give examples of preparative separations
- Acid-base workup (pKa)
- Recrystallisation (solubility)
- Distillation (volatility)
- Column chromatography (affinity)
or combination of these sequentially
Define preparative separation
It separates a mixture with multiple components e.g. reaction mixture on a larger scale.
The target molecule is separated from byproducts and waste
Define analytical separation
It separates a mixture with multiple components e.g. river using a sample.
The species of interest is separated from the rest of the sample.
Give examples of analytical separation
- Thin layer chromatography (affinity)
- High performance liquid chromatography (affinity)
- Gas chromatography
- Capillary electrophoresis (mobility in electric field)
Describe chromatography
- the molecules can move between the stationary phase and mobile phase
- the affinity of the molecules for each phase determines the position of this equilibrium
- the equilibrium position controls the rate at which the molecules progress through the system
- we can separate a mixture based on differing affinities of molecules for each phase
How is a chromatogram created?
- The two species (βanalytesβ) have different properties so they will have different affinities for the stationary and mobile phases
- They will be retained differently in the chromatographic system so they proceed through the system at different rates
- They will elute (pass the detector, and exit the chromatographic system)
at different times. - The chromatogram shows two well- separated peaks with clear baseline in between them: the two species have been effectively separated. We would say that the peaks/species are well-resolved
Define Retention time
- This is the amount of time it takes for a specific compound to elute.
- It is very useful
for identifying what the analyte is, since the retention time for a compound should always be the same (as long as the conditions are identical). - However, other compounds may have a similar or identical
retention time under those conditions.
Give some conditions that influence Rt
- Relative affinity of the analyte for the different phases
- The length of the column: π‘π β πΏ
- Mobile phase flow rate: π‘π β 1/ππππ€ πππ‘π
Define peak width
the width of the peak, measured at the baseline
Define column length
- the length of the chromatographic equipment.
- βColumnβ is often used as a shorthand for the setup of a support, a stationary phase and a mobile phase
Define resolution
A measure of how good the chromatographic method is, for separating two specific
components of a mixture.
Peaks are well-resolved if Rs > 1.5
Give some conditions which influence resolution
It depends on:
* Retention time and peak width of the two components:
π
π = 2(π‘ππ΄βπ‘ππ΅)/π€π΄+ππ΅
* Column length: π
π β βπΏ
Define area
- The area of the peak, which can be used to quantify the analytes, since the peak area should be proportional to the amount of analyte in the sample, although the constant of proportionality will likely be different for each analyte.
- In order to extract reliable quantitative data, chromatography experiments must be carefully standardised.
What is HPLC?
- HPLC is an analytical method for separating mixtures of molecules that are soluble. It uses a very narrow column,
packed with the stationary phase. - The mobile phase
in HPLC is pumped through
the column at very high pressure.
What is the mobile and stationary phase in HPLC?
- Stationary phase= typically tiny particles of a solid material, which can be chemically
modified in different ways in order to retain different molecules more, or less, as required. - Mobile phase= solvent, or a mixture of solvents, blended to achieve the required polarity
How are substances separated in HPLC?
- Separation of molecules in an HPLC column is based on partition of the analytes between the stationary phase and the liquid mobile phase.
- This partition depends on the strength of the molecular interactions
between the analyte molecules and the molecules of the stationary and mobile phases. - The best separation results are generally achieved by matching the polarity of the analytes to the stationary phase, and using a mobile phase with contrasting polarity.
What are the 2 types of HPLC?
- normal phase HPLC
- polar stationary phase e.g. silica
- non-polar mobile phase e.g. hexane - reverse phase HPLC
- non-polar stationary phase e.g. C18 silica
- polar mobile phase e.g. water
What is isocratic mode?
mobile phase composition
remains the same throughout
What is gradient mode?
mobile phase composition changes throughout
Name the 3 HPLC detection methods
- UVvis detection
- Fluorescence detection
- Mass Spectrometry (HPLC-MS)
What is UVvis detection?
It relies on analytes absorbing electromagnetic radiation (i.e. light) in the UVvis region, approximately 200-800nm.
Assuming that the analyte absorbs radiation in the UVvis region, the amount of light it absorbs is related to the concentration of analyte present, according to the Beer-Lambert law
What is the Beer-Lambert law?
A = Ξ΅ . c . l
A= absorbance (no units)
c= concentration of analyte in solution (moldm-3)
l= path length (m, cm, dm etc)
Ξ΅= molar absorption coefficient, a constant, for a specified compound at a specified wavelength of light (dm3 molβ1 cmβ1 or m2 molβ1)
How does UVvis detection work?
- The analyte is travelling through the column, and elutes past the UVvis detector
- the UVvis detector shines light across the column, and the analyte passes by as it elutes.
- the analyte absorbs radiation in the UVvis region, which the UVvis detector registers and the instrument records as a peak in the chromatogram.
- Since the UVvis absorbance is proportional to the concentration of the analyte, the analysis can be made quantitative β typically by comparison of the recorded peak area with an appropriate calibration curve.
Give a potential problem of UVvis detection
Analyte doesnβt absorb at chosen WL
- UVvis detectors use a fixed-wavelength light source at 254 nm, as this will not be absorbed by most common RP-HPLC solvents (water, acetonitrile, methanol), but it will detect most organic compounds with
conjugated structures (conjugated C=C bonds, aromatic rings, mesomeric groups).
- However, if the analyte of
interest does not have any of these structural features, it will not be detected.
How do you solve the problem of the analyte not absorbing at chosen WL?
- using a variable-wavelength UVvis light source, which allows the selection of a different wavelength for monitoring. (with care that this wavelength is not absorbed by the solvent)
- derivatising the analyte, e.g. pre-treating the mixture in order to synthetically attach a chemical group which absorbs UVvis light.
Why is it important that the right WL is chosen for UVvis detection?
- Different compounds absorb light at different wavelengths, according to their electronic structure. - - For the detector to work well, it needs to be able to measure absorbance of light at the appropriate wavelength for the
analytes of interest β which means being equipped with a light source of an appropriate wavelength. - solvents are also compounds, and will also absorb UVvis light β so the wavelength needs to be chosen so that it
is absorbed by the analytes of interest, but not by the HPLC mobile phase.
Give another potential problem of using UVvis detection in HPLC
Signal is too weak
- It is impractical to reliably measure values of A below 1x10β4.
- For a particular analyte, the value Ξ΅ may be low;
- For a particular mixture/sample, the concentration of the analyte of interest (the value of c) may
also be low.
How might the problem of the signal being too weak be solved in UVvis detection?
we can change the design of the detector to maximise the path length from a simple detector to a Z-cell detector
(see booklet)
What is fluorescence detection in HPLC?
some molecules
have the property that, when irradiated with a particular wavelength of light, they absorb it and then emit light at a higher wavelength. Detection of this emitted light allows for the identification and quantification of much
lower concentrations of analyte.
What is mass spec detection in HPLC?
- This detection method involves interfacing the HPLC instrument with a Mass Spectrometer, so that the column
effluent (i.e. the liquid mobile phase, containing the analytes) exits the column and enters a Mass Spec instrument. - This has the advantage of yielding molecular mass data and structural information about the
analyte molecules, as well as the analyte retention times (from the HPLC). - If the identities of the analyte molecules are not already known with absolute certainty, this extra information can be
very valuable.
Define sensitivity
β the ability to tell the difference between different amounts of analyte
Define limit of detection
the minimum amount of an analyte which can be detected
Define precision
reproducibility (multiple analyses of the same sample should give the same resultsβ¦)
Define accuracy
closeness of the determined value to the true value being quantified
Why is solid phase extraction used/helpful?
- SPE is often used to pre-concentrate analytes before analysis
- its very useful when the concentrations of the analytes of interest are likely to be low
How does SPE work?
SPE works by passing the sample through an SPE disk, which is essentially a few centimetres of material similar to the stationary phase in HPLC.
- Step 1; a large volume of
sample mixture is passed through the SPE
disk (βloadingβ)
- The SPE material is chosen to strongly retain the analytes of interest, which are present in a very low concentration.
- Passing a large volume of sample through results in a relatively large amount of analyte being loaded into the SPE material
Step 2; In the second stage of SPE, the SPE disk is eluted with a different solvent, which has a higher affinity for the analytes.
- This draws the analytes out of the SPE material, and elutes them for analysis (comparable to changing the mobile phase in HPLC).
Define βstandardisedβ
this means subjecting the results of the analytical technique to a careful comparison with
authentic data, to check that the results are reliable.
There are many different methods of standardisation, but
they can generally be categorised as internal or external.
Define external standardisation
relies on data collected from standards analysed under similar conditions, but as different samples
Give the general method for external standardisation
- Make up standard solutions, at different concentrations, using an authentic sample of the analyte.
- These standards are then analysed, and the detector response is measured, to generate a βcalibration curveβ, which plots the detector response (e.g. GC peak area) as a function of analyte concentration.
- The unknown sample is then analysed under the same conditions.
- The detector response is measured, and
compared with the calibration curve to determine the concentration of the analyte in the unknown sample.
Define internal standardisation
relies on the addition of a known amount of an extra compound (the βinternal
standardβ) to the sample.
How is the internal standard molecule chosen?
- behaves similarly to the analyte, but can be distinguished from it, either by tr or detection method.
- is not already present in the sample (and you need to run a βblankβ analysis to check this is true).
- Adding a known amount of the internal standard (IS) to the sample means that there is a fixed, known reference point in the analysis. When the IS is added to the sample, the ratio of IS to analyte is fixed; the detector response can then report the ratio, from which the amount of analyte can be calculated. The use of
internal standard can be very useful in minimising errors associated with sample handling.
