Fichas
Chromatography: applications
Fichas del conjunto (45)
- 1Planer Chromatography• Planer Chromatography
– Thin Layer Chromatography (TLC)
– Paper chromatography
• Column Chromatography
– Open column
– GC
– HPLC - 2TLC• Speed & cost
• Proceed column experiments
• Drug industry – product purity
• Clinical, biological and chemical
studies - 3TLC• Solvent evaporation must be
prevented –why?
• Sample spots must be above
solvent mixture
• Paper wick is often used -
why? - 4Preparation of TLC plate• Aqueous slurry of finely ground solid
onto glass or plastic plate or
microscope slide
• Binder is used to facilitate adhesion
–Calcium sulphate hemi-hydrate
(CaSO4
(0.5H2O))
• Dry or heated
• Pre-coated plates commercially
available - 5Plate development• Most plates have 5 x 20 or 20 x 20 cm
dimension
• Dissolve sample in organic solvent
• Apply sample (a drop) at one edge
– Capillary tube or micropipette
• Allow to dry
• Place in a closed container saturated with
vapours of developing solvent
• After development remove plate and dry
• Multiple development is possible - 6Locating analyte on plate• Non-specific spry agents
– Sulphuric acid and iodine – organic
compounds- dark
• Specific reagents
– Interact with functional groups
• Ultraviolet light
– Incorporate fluorescent material in the
stationary phase
• Usually phosphor which emits green
fluorescence when irradiated with UV light
(254 nm, Hg lamp)
– Components absorbing at 254 nm quench the
fluorescent material - 7Densitometeres• Absorption measurement UV
• Fluorescence
• Radiochemical detection
• Documentation
– Rf values
– Shape and colour of spots - 8TLC- advantage• Simultaneous analysis of multiple
standard and samples under similar
condition
• Bands can be removed and purified
• Preparative scale can be achieved
– 0.5-2 mm thickness as oppose to 250 µM
of analytical - 9Silica gel normal TLC platesa is 5, b is 1, c is 2 , d is 3, e is 4
- 10Salicylic acid analysis by silica gel TLCSalicylic acid is polar need to displace it with a polar solvent to lift it up
c = 2, methanol is b, d is 1
- 11Limit tests• Concentrated solution of analyte
Vs. dilute solution of impurity
• E.g. 5% solution of analyte (A) with
the following specification:
– No additional spot
– Analyte plate should not be more
intense than that of 0.05% solution (the bigger the spot means the more concentrated it is)
we accept the drug if it is lower than the acceptable level.
– 1% limit is being conducted!
- 12Open column chromatography• Open column Vs. high
performance/pressure liquid
chromatography (HPLC)
(gravity is how it works)
• Adsorption, ion-exchange, size
exclusion, affinity etc modes are
possible
- 13Packing column and sample application• Work with gravity!
• What packing material can be used?
• Air bubbles – packing material slurred with
solvent and poured gently into column
• Column should not be run dry!
• Elution
– Isocratic from Greek, ischros – ‘equal
strength’
– Stepwise Vs. gradient elution - 14Adsorption of compounds• -CH=CH-
• -OCH3
• -CO2R
• R2C=O
• -CHO
• -SH
• -NH2
• -OH
• -COOH
as a general rule - carboxylic acid is most polar, followed by an OH group, then aldeyde, and ketone then ester. Ionised groups are the most polar and soluble like salts - 15Gas chromatography (Gasliquid)First chromatographic method
developed commercially
• Relatively easy to produce stable flow
and pressure for mobile phase –
carrier gas
• All we need is a compressed air,
pressure regulator and a valve - 16Gas chromatography (Gasliquid)
- 17GC carrier gas• Gas selection is critical:
– Column separation process
– Detector performance
– Must be inert to column material and
sample
– Smallest diffusion coefficient - best
column performance
–Hydrogen and helium are ideal
career gases - 18Columns• Data is available for vast number of
compounds
• Classified by tubing diameter and
packing type
– Conventional - stainless steel or glass
tube of 6-20 feet in length and 0.125-
0.25” OD
– Preparative – greater than 10 feet in
length and over 0.25” OD
– Capillary – 10-100 metres in length and
0.1-0.5 mm ID - 19Types of columns expected to know the comparison
- 20Packed vs. capillary columns
- 21Liquid phases: Dimethyl silicone the stationary phase is liquidSeparation is based on:
- polarity
- molecular weight
Retention index available - for n-alkanes:
- OH group is equivalent to two methylene unit
- Branching reduced retention time
- 22Predict elution order: dimethyl silicone column count number of carbons which is heavier, branching helps it to come further
- 23Polyethylene glycol a stationary phase• Highly polar liquid phase
• Similar molecular weight but varying
degree of polarity or double bonds - 24Temperature programming for gas chromatography• Column is placed in an
oven (up to
320oC)
– Constant temperature –
isothermal
• tR (rentention time) increases with
number of carbon!
– Variable – temperature
programming - 25Detectors• Destructive Vs. non-destructive
• General Vs. some/very discriminating
• Requirements
– Sensitivity
– Response to concentration
– Linear range
– Low sensitivity to variations in flow,
pressure and temperature - 26Flame ionisation detector (FID)• Most commonly used detector
• Burning carbon containing compounds
with oxygen/hydrogen flame
• Destructive method - sample must be
combustible
• Mode of detection
– Production of ions in a flame results in a current that can be measured
– Ions change the resistance of flame producing
signal - 27Other detectors• Thermal conductivity detector
• Electron capture detector
• Nitrogen phosphorus detector
• Mass spectrometric detector
• FTIR detector - 28Derivatisation of sample• Very polar samples are not volatile!
• Strongly adhered to stationary phase - tailing
• Silylation
– Replace acidic hydrogen by alkylsilyl group
such as SiMe3
– Derivatives are less polar, more volatile and
more thermally stable
• Using trimethylchlorosilane
– R-OH + Cl-SiMe3 R-O-SiMe3 + HCl
• Acylation and alkylation are also used for derviatisation - 29Analysis of hyoscine
- 30HPLC (not working with gravity, uses a pump system under high pressure so you cant use a glass column)
- 31Mode selection• Adsorption, ion-exchange, size-exclusion, affinity, etc.
• Adsorption:
– Water insoluble or non polar – normal phase
– Polar of water soluble or insoluble –reverse phase
– Solvent selection – mixing solvents
Reverse phase solvents
Methanol -- acid
Acetonitrile -- base
Tetrahydrofuran --large dipole
Water -- polarity adjustment
• All are low viscosity, available in high purity, uv transparent,
miscible in each othe - 32Gradient elution• Alter solvent mix during analysis
– Reduce analysis time
– Improve resolution
– Improve peak shape
– Improve sensitivity
• Stepwise vs continuous
• Appropriate for adsoption and ion exchange
chromatography but not size exclusion
chromatography - 33Gradient elution• Polarity of a starting solvent resolves the
first few components of a mixture
• Final polarity should resolve the last few
species in timely manner
• Construct various blending rates to
separate components - 34Solvents• Must be HPLC grade
• Filtered through 0.2 µm filter – filtration
increases column and pump life
• Degassed – reduce the chance of bubble
formation
– Displace with less soluble gas
– Apply vacuum
– Heat the solvent - 35Guard column• Small column between injection system
and analytical column
• Prevent entry of materials that might stay
on analytical column from sample or
solvent – used to extend column life
• Same packing material as analytical
column - 36HPLC column• The originally irregular silica and alumina
columns are now replaced by a range of
synthetic regular shape packing:
– Porous – channels through packing
– Superficially porous rough surface
– Smooth bead like
• Packing size – efficiency and pressure
requirement increases as packing size
decreases - 37Detector system• Absorption of sample in UV/Vis range
– Solvent must not absorb significantly at the
measured wavelength
– Filter photometres:
• Single λ
• Variable wavelength
• Multiwavelength
• Refractive index detector
• Electrochemical detector - 38Refractive index detector• Bulk property
detector – general
purpose
• Based on refraction of
light as it passes from
one media to another
• Presence of solute
changes the refractive
index of the solvent
- 39HPLC based assays• Calibration based assay using
external standard
– Varying concentration of analyte
injected
– Calibration curve constructed from peak area
– Unknown sample injected
– Concentration of unknown determined
from calibration curve - 40Calibration based assays using internal standard• Most common assays used by BP
• Solution 1
– Known con. of pure standard (to be analysed) plus related
compound (internal standard)
• Solution 2
– Sample in the absence of internal standard extracted – check for
the absence of internal standard
• Solution 3
– Sample extracted with same concentration of internal standard as
solution 1
• Peak ratio solution 1 = area of sample peak/ area of
internal standard
• Peak ratio solution 3 = sample peak/internal standard
• Con. In sample = peak ratio sol. 3 x con. Standard/peak
ration sol 1 - 41Validation of quality control test• Suitable test methods validated for a given
equipment
• Validity of results checked before
materials are released or rejected - 42Hyphenated methods• Combination of chromatographic and
spectral method
– Chromatography – purified samples
– Spectroscopy – qualitative information
– Gas chromatography
• Mass spectrometry- GC-MS
• Infrared spectrometry – GC-FTIR
• Atomic emission – GC-AES
– Liquid chromatography
• Mass spectrometry – LC-MS
• NMR- LC-NMR - 43GC-MS and LC-MS
- 44in gas chromatographyits about molecular weight, the smaller the further it will go
- 45a reverse phaseis non polar
so the most polar one comes out first
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- Fichas sobre Chromatography: applications, creado por megan trent el 02/02/2021.
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- megan trent
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