Liquid chromatography Flashcards
Planar chromatography
separation techniques based on the distribution of analytes between a liquid mobile phase and solid/liquid stationary phase. Uses either paper or coated plates
Thin layer chromatography
Very common example of planar chromatography. Used to separate mixtures of microgram quantities of analyses due to differences in solubility, adsorption, size or charge. Small particle sizes of adsorbent (10 to 60 mm) gives resolution at better than functional group level
Apparatus of TLC
Thin layers of silica gel, alumina, cellulose, ion-exchange or gel permeation material supported on glass, aluminium foil or plastic plates
Applications of TLC
a very widespread use largely for qualitative analysis of both organic and inorganic materials. Especially useful for checking purity, monitoring production processes or early step in characterising complex mixtures. Can be used preparatively
Disadvantages of TLC
Migration characteristics sensitive to conditions; plates easily damaged and low quantitative precision
Sample application for TLC
Spotting: analyses dissolved in a small volume of volatile solvent to prevent spreading of the spot during application. Accurately measured volumes can be applied using micropipettes or syringes. For qualitative analyses drawn out melting point tubes used. larger samples applied as a streak when TLC is used preparatively to isolate compounds for further analysis
Plate development in TLC
Glass tank with 0.5 cm deep solvent. Solvent allowed to rise to within 1 cm of top of plate
Detection of separated components
Coloured analytes observed visually after developing the plate. Colourless compounds visualised by physical or chemical means. Specific classes of analyte revealed by reactions of sprayed reagents with functional groups or metal ions. Spraying with conc sulphuric acid then heating plates to 200C for few minutes causes charring to reveal dark spots; very sensitive
Qualitative analysis of TLC
components identified by comparison of their retardation factors (Rf) with values of standards run under identical conditions
Rf = d(analyte)/d(solvent)
Spots can be removed from plates and components solvent extracted for analysis by other methods: GC, HPLC, IR, UV/vis, MS
Quantitive analysis of TLC
Estimates of concentration achieved by comparison of spot intensity with standards of known conc. Most accurate determinations achieved by removing spots from plate for analysis by other means
Two-dimensional TLC
Resolution can be improved by using 2-D TLC. Sample spotted at corner of plate which is developed in one solvent. Plate then removed, dried and developed in a second solvent system
High Performance Liquid Chromatography (HPLC), principles
Separation of mixtures of analytes (microgram to gram quantities) by passage of the sample through a column containing a solid stationary phase by means of a pressurised flow of liquid mobile phase.
Components migrate through the column at different rates due to different relative affinities for the stationary and mobile phase (based on adsorption, size or charge).
HPLC, apparatus and instrumentation
Solvent delivery system injection port; stainless steel columns flow through detector recorder
HPLC applications
used largely for the separation of non-volatile substances including ionic and polymeric analytes; complementary to gas chromatography
Disadvantages of HPLC
Columns very sensitive to settling of column packing and the accumulation of strongly adsorbed materials or particulate matter; no universal detector available
HPLC instrumentation: mobile phase
HPLC instruments comprise one or more solvent reservoirs (binary, ternary and quaternary) each containing around 0.5 to 1 L of different solvent (or mixtures of solvents). Solvents must be degassed (sparring with He) to avoid pumping problems. Elution can be under constant solvent compositions (isocratic) or varied under instrument control (gradient elution)
HPLC instrumentation: pump
high performance corrosion resistant pumps deliver solvents at flow rates ranging 0.1 to 10 ml min-1 with high reproducibilities (0.5%). Typical back pressures range from 3000 to 6000 psi (200 to 400 bar)
HPLC Injector
Sampling loops most widely used. Interchangeable loops allow injection volumes to be varied (5 to 500 micro litres)
loop filled while isolated from flow between pump and column
value moved to introduce sample into main flow to column
HPLC column materials
straight lengths of precision made stainless steel tubing. typically 10 to 25 cm long and 4-5 mm i.d. column densely filled with spherical porous micro-particulate packing materials, typically 3, 5, or 10 micro m diameters
Normal phase HPLC columns (Stationary phase)
Silica gels
Packing has polar surface due to the high abundance of silanol (si-OH) groups. Adsorption is the major mechanism for separation. Separatons are achieved by eluting with solvents capable of competing with analytes for adsorption sites.
Analytes elute in order of increasing polarity
Gradient elution involves elution with solvent compositions of gradually increasing polarity
Reversed phase HPLC columns (stationary phase)
employs bonded phases produced by chemical modifications of silica gel.
>75% of HPLC reversed phase. Mobile phase used generally aqueous based with varying cons of organic solvents such as MeOH, CH3CN and tetrahydrofuran
Mechanism of separation involves portioning at analytes into the organic layer introduced by chemical modification
With hydrocarbon phases (e.g. ODS) the order of elution of analytes is the reverse of their polarity
Gradient elations involve solvent compositions of decreasing polarity
Mobile phase - polarity (non-polar to polar) (16)
Must learn
Aliphatic hydrocarbons Alkenes Aromatic hydrocarbons Halides Sulfides Ethers Nitro compounds Esters = aldehydes = ketones Alchols = amines Sulfones Amides Carboxylic acids
HPLC separations - polarity of mobile/stationary phase
Matching polarity of the analyte to the appropriate stationary phase
Normal phase = elations achieved by increasing polarity of solvents (mobile phase)
Reversed phase = elations achieved by decreasing polarity of solvents (mobile phase)
Ion-exchange chromatography (use and theory)
Synthetic ion exchange resins are high molecular weight polymers containing a high proportion of ionic functional groups, e.g. SO3-H+, -NH3+OH-, -CO2-H+
Used to separate inorganic and organic ions, and organic acids and metal ions
Ion exchange preparations based on interactions of ionic groups on resins with analyses of opposite charge in mobile phase. Separations highly dependent on the pH and presence of competing ions in the fluent. Eluted analytes detected using conductivity detectors
Size-exclusion chromatography
Size exclusion or gel chromatography is the newest liquid chromatography procedure to be introduced whereby analytes are separated on the basis of molecular size
Packings for size exclusion chromatography consist of small ( 10 micro m) silica or polymer containing particles and network of pores into which analyse and solvent molecules can diffuse
no chemical or physical interactions between analytes and stationary phase
Most commonly used to estimate molecular weights of natural polymers e.g. proteins
HPLC detectors
Same requirements as GC, however, no universal detectors like for GC (FID)
most based on absorption of UV or visible radiation
UV.vis photometric detectors respond to the presence of an absorbing species in the eluent from the column. Obey Beer-Lambert so sensitivity depends upon the nature of chromophores present in analytes. analytes with high extinction coefficients display highest sensitivities. the linear range is reasonably high
Solvents for eluents must be carefully chosen to be compatible with UV absorbances of analytes
the zig zag path for the fluent through the cell ensures minimal volume ( 10 micro l or less) and max sensitivity
Photodiode array detector (PDA)
provide more spectral info but more expensive
light beam passing through sample is spread out into a spectrum by a diffraction grating. the light beam is detected by an array of several hundred photodiodes mounted on a silicon chip
full adsorption spectra (200-700 nm) recorded every 0.1 s
advantage: full uv/vis spectra recorded as analytes emerge from the column
data displayed in 3D chromatogram of time/absorbance/wavelength
capillary electrophoresis
high voltage (10 to 30 kV) causes the surface of quartz capillary to develop -ve charge due to ionisation of si–OH groups. this attracts +vely charged counter ions
highly solvated +ve ionic analytes migrate to -ve electrode
oerall solvent movement is called electro-osmotic flow
during a separation, uncharged molecules move at the same velocity of the electro-osmotic flow (v. little separation). Positively charged ions move faster and negatively charged ions move slower than the solvent flows.
applicable to low and high molecular weight charged species e.g. proteins
