HPLC Flashcards
Sequence of HPLC?
Mobile phase Pump Sample injector Column Detector Data system
What does the pump do?
Separation of compounds based on flow of mobile phase, if too variable problems with reproducing retention times, flow less than 1ml per minute so slow
What does the sample injector do?
Needs to deposit analytes in a narrow band in the top of the column, will not get symmetrical peaks otherwise, small quantity used 10 micro litres or less
What does the column do?
Maintain stable temperature although it plays little role in overall separation
Why does there need to be adequate fitting?
Narrow bore tubing needed to connect the parts, has to be used to avoid extra (dead) volumes affecting separation
Three most important HPLC stationary phases?
Silia
Chemically modified silica
Styrene divinylbenzene
Adsorptive centres on the surface of silica?
Free silanols Geminal silanols Associated (vicinal) silanols Silanols near metal cations Siloxanes
Characterisation of free silanols?
Slightly acidic, sorption sites for basic compounds (possibility of chemical tailing)
Characterisation of geminal silanols?
Not acidic
Characterisation of vicinal silanols?
Not acidic sorption sites for compounds with OH groups
Characterisation of silanols near metal cations?
Strongly acidic they increase heterogeneity of the surface and can badly affect the separation of basic compounds
Characterisation of siloxanes?
Products of the condensation of associated silanols
What is chemically modified silica?
Silanol groups on the surface of silica are chemically modified to give stationary phases with specific properties - do this for reversed phase
What is end capping?
Subsequent treatment with trimethylchlorosilane may reduce the number of silanol groups that remain unreacted for steric reasons, steric reasons OH not capped, provides secondary interactions, end capping block the extra functionality which is unwanted
Why does the silica need to be spherical?
Need spherical shape, want uniform interactions shape and size of particles in column will determine how molecules behave if different molecules would behave in different ways, experience band broadening
Chemical stability of silica bonded phases in high pH?
High pH leads to dissolution of the silica backbone
Chemical stability of silica bonded phases in low pH?
Low pH leads to the hydrolysis of the siloxane bond. The end capped small groups are the preferred site of attack. Therefore end capped phases can alter their properties when used at pH<3
Column lifetime in extreme conditions?
Column lifetime is decreased at high temperature and at high buffer concentration
Chemically modified silica pH range?
Chemically modified silica useful and stable in very narrow pH range, cannot use at high or low pH so between 3 and 8, limits applications
What is styrene divinylbenzene?
Cross linked polystyrene is formed as a result of copolymerisation of styrene and divinylbenzene, can be modified in any way we want
Amount of cross linking in styrene divinylbenzene?
The amount of divinylbenzene added for the reaction determine the degree of cross linking and hence the pore structure, can control particle size and porocity
pH range of styrene divinylbenzene?
Stable at pH 1-13
Structure of styrene divinylbenzene?
Can be microporous or contain a mixture of micro and macro pores
Size exclusion?
Chromatography can be used to separate on size called size exclusion chromatography, separation of polymers, proteins, macromolecules biological materials, needs to control porocity
Octadecyl reversed phase?
Obtained by incorporation C18H37 groups (stable alternative to reversed phase silica without unreacted OH groups)
Size exclusion chromatography and styrene divinylbenzene?
Styrene divinylbenzene giving well defined pores is the most important stationary phase for size exclusion chromatography
How are ion exchangers obtained from styrene divinylbenzene?
By incorporating suitable groups into the matriculates, widely applied in ion exchange chromatography used for ionic molecules
HPLC solvents?
Solvent A (aqueous) and B (organic) need to be fully miscible, don’t want to have two liquid phases want one mobile liquid phase. Two solvents needed for elution and to control separation process
HPLC mobile phase - isocratic?
The mobile phase remains constant over the course of the separation (band broadening)
HPLC mobile phase - gradient?
Alteration of the composition of the mobile phase during the course of the chromatographic run, (increase strength of solvent obtain nice peak shaped reduce retention time and band broadening)
HPLC mobile phases properties - buffers?
Used for ionic ionisable compounds, buffers are required in ion exchange chromatography and sometimes in reverse phase chromatography
How can buffers be used if ionic or ionisable compounds need to be separated?
Often necessary to keep a well defined pH of mobile phase, the analyses can be forced either into the non dissociated or ionised form depending on the mode of separation
pH of the buffer used?
The chosen pH of the buffer must be two units apart from the pKa of the compounds of interest in order to get the vast majority of the molecules in one single form
What are volatile buffers needed for?
Light scattering detection
Coupling with mass spectrometry
Preparative separations
Why is pH of mobile phase so important in ion exchange chromatography?
Ion exchange mechanism is controlled by pH
Why is pH of mobile phase so important in reversed phase HPLC?
pH influences retention time and peak shape, the pH level affects the degree of dissociation of acidic/basic compounds and free (residual) silanol groups on the surface of stationary phase
Mobile phase additives?
Small percentage of additives or modifiers added to mobile phase to improve peak shapes mention times and to modify selectivity, block functionalities if too polar
If analytes are weak acids what additive is added?
Acetic acid/acetate
If analytes are weak bases what additive is added?
Alkylamines
Advantages of increasing temperature in HPLC?
The performance of the column increases
Analysis time decreases
Need to if the mobile phase is viscous
Why does the performance of the column increase at higher temperatures?
The performance of the column often increases because of the decreases of mobile phase viscosity which improves mass transfer
Why does analysis time decreases at higher temperatures?
Analysis time deceases due to the possibility go using higher flow rates of the mobile phase due to the increase in diffusion coefficients
Why is it necessary to work at higher temperatures if the mobile phase is viscous?
Less pressure is needed to pump the mobile phase
What are the disadvantages of increasing temperature in HPLC?
Solvent or sample are more likely to decompose (silica is unstable doesn’t like very high temerature)
The vapour pressure of the solvent rises thus increasing the risk of bubbles in the detector (introduce a third phase into the analysis which will mess up the separation) - uneven baseline, ghost peaks
What is degasification?
Probability of bubbles made low as possible
What is the maximum temperature for silica and chemically bonded stationary phases?
Should not exceed 120 and 80 degrees C respectively
HPLC detectors?
Ultraviolet detector: Variable uv-vis detector, photodiode array detector (PDA) Fluorescence detector Refractive index (RI) detector Electrochemical (Amperometri) detector Conductivity detector Light scattering detectors Mass spectrometry
Variable UV Vis detector?
Allows for the choice of one wavelength in order to accommodate the absorption characteristics of a particular solute or group of solutes, UV vis only cannot differentiate compounds hence why use LC before to separate in time, couple chromatography with other techniques, LC helps with measurement process - grating (monochromator) allows a particular wavelength from the whole spectrum to be chosen
What does the deuterium lamp do in variable UV Vis detector?
Deuterium lamp emits UV spectrum tungsten halogen lamp emits in the near UV and visible range (340 - 850nm)
What does the photo diode do in variable UV Vis detector?
The first photodiode measures original signal and transfers light to electric current, if analytes are in it then observe a decrease in signal, higher decrease in signal higher concentration
Photo diode array detector?
Allows for an access to all of the wavelength simultaneously - an entire spectrum of light is passed through the detector cell. The light is then bounced off a grating and detected using a multiple array of photodiodes
HPLC fluorescence detector?
Measures emission of radiation from a molecule which has attained an excited electronic state after the absorption radiation
How does HPLC fluorescence detector first?
Analyte gets into excited state different wavelengths for different compounds emitted light is measured, not many compounds fluoresce so this is slightly more selective
Excitation sources in HPLC fluorescence detector?
Mercury vapour, xenon, deuterium, lasers
Applications of HPLC - fluorescence/UV detector?
Compounds that fluorescence of which fluorescing derivatives can be obtained
Due to small number of molecules which show fluorescence this type of detection ca be used for the detection of trace quantities of analyte in complex matrices
(how much light gets absorbed compared to what gets emitted)
