Liquid chromatography

Question 1

Planar chromatography

Answer 1

separation techniques based on the distribution of analytes between a liquid mobile phase and solid/liquid stationary phase. Uses either paper or coated plates

Question 2

Thin layer chromatography

Answer 2

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

Question 3

Apparatus of TLC

Answer 3

Thin layers of silica gel, alumina, cellulose, ion-exchange or gel permeation material supported on glass, aluminium foil or plastic plates

Question 4

Applications of TLC

Answer 4

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

Question 5

Disadvantages of TLC

Answer 5

Migration characteristics sensitive to conditions; plates easily damaged and low quantitative precision

Question 6

Sample application for TLC

Answer 6

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

Question 7

Plate development in TLC

Answer 7

Glass tank with 0.5 cm deep solvent. Solvent allowed to rise to within 1 cm of top of plate

Question 8

Detection of separated components

Answer 8

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

Question 9

Qualitative analysis of TLC

Answer 9

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

Question 10

Quantitive analysis of TLC

Answer 10

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

Question 11

Two-dimensional TLC

Answer 11

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

Question 12

High Performance Liquid Chromatography (HPLC), principles

Answer 12

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).

Question 13

HPLC, apparatus and instrumentation

Answer 13

Solvent delivery system
injection port;
stainless steel columns 
flow through detector
recorder

Question 14

HPLC applications

Answer 14

used largely for the separation of non-volatile substances including ionic and polymeric analytes; complementary to gas chromatography

Question 15

Disadvantages of HPLC

Answer 15

Columns very sensitive to settling of column packing and the accumulation of strongly adsorbed materials or particulate matter; no universal detector available

Question 16

HPLC instrumentation: mobile phase

Answer 16

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)

Question 17

HPLC instrumentation: pump

Answer 17

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)

Question 18

HPLC Injector

Answer 18

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

Question 19

HPLC column materials

Answer 19

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

Question 20

Normal phase HPLC columns (Stationary phase)

Answer 20

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

Question 21

Reversed phase HPLC columns (stationary phase)

Answer 21

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

Question 22

Mobile phase - polarity (non-polar to polar) (16)

Must learn

Answer 22

Aliphatic hydrocarbons
Alkenes
Aromatic hydrocarbons 
Halides 
Sulfides
Ethers
Nitro compounds
Esters = aldehydes = ketones
Alchols = amines
Sulfones 
Amides 
Carboxylic acids

Question 23

HPLC separations - polarity of mobile/stationary phase

Answer 23

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)

Question 24

Ion-exchange chromatography (use and theory)

Answer 24

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

Question 25

Size-exclusion chromatography

Answer 25

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

Question 26

HPLC detectors

Answer 26

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

Question 27

Photodiode array detector (PDA)

Answer 27

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

Question 28

capillary electrophoresis

Answer 28

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