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Spectrophotometry (Handbook of spectrophotometry (Optical (Raman…
Spectrophotometry
Handbook of spectrophotometry
Sample preparation
Overall goal: to collect smaller portions of the original object that can be readily analysed
Collection of liquids and solids
often liquid and solids are inappropriate for analysis
the size is too large to analyse entirely (ocean, polycrystall)
solution:
random sampling
- mix the bulk, remove sub-unit
heterogeneous solid: powder mixed + random removal
awkward shape prevents from fitting in instrument
solution:
composition sampling
- collect small portions of different phases in the same proportions as in the bulk
if sample has distinct segregation of various sections
false measurements due to masking agents or interferences
heterogeneous sample always different from original object
so we need appropriate size and number of samples -
statistics
Sampling of liquids
If liquids
Inmixed solution
Random sampling
Flowing (changing durung experiment)
at
reqular time intervals
Storage to provide inertness between sample and container
Real time analysis
small bypasses integtated into system
ensure that the microfluidic system doesn't change the flow of the reaction
Sampling solids
metalls
coring to different depth
due to oxidation take sample from the center (the part that wasn't expoded to air)
heterogeneous
coring to different depth for several times to detect homogeneous layers
Preparation samples for analysis
Solids
as a big chunks Obtain a fine powder by milling
analysis suits best for liquids, so dissolve
Acid digestion for metalls
if sample doesn't dissolved heat the acid (nonoxidazing)
to dissolve metals that form oxid layer (Al) use oxidazing acids
use
digestion bomb
- Teflon container that can be sealed and heated in microwave
remove organics
dry ashing
combustion in 02 in oven
fusion with alkalai metals (
p. 29 and above
)
wet ashing (
p 29 and above
)
Liquids
remove any solid particles by filtration, centrifugation
Extraction
liquid/liquid extraction
two liquids with different affinity (immiscible) creating distinct boundary
solute has to has either very smal or very large partition coefficient (solibility) in one of liquids
liquid with soluted analyte/ supporting solute (charcoal)
analyte is adsorbed onto solid
already long used in pollution
Complexation
if there are components in solution that can cause false results on chemical reaction
masking - forming chelating agent
reaction forms stable complex that cannon undergo certain chem reaction
precipitation
reaction forms precipitation either with analyte or interference for further discardtion or analysis
depends on temperature, pH, chem composition
can gain exellent results
(p30)
Cromatographic separation
liquid flow across the solid and either elutes or deposites analyte from/ on solid
as it was in extraction analyte has different solubility in liquid and solid phases
Types of interactions between analyte and solid matrix
adsorbtion
based on polarity of solid matrix and solute
1 more item...
Partition
solid matrix impregnated with liquid. Then solute with different liquid immiscible with the impregnated flows across the solid. So what? No idea
ion exchange
ion exchange resin and aqueos solution separate ions of same charge
1 more item...
thin-layer
glass coated with adsorbent (silica gel), dried
1 more item...
size exclution
solid matrix with well defined pores
1 more item...
Electrophoresis
based on the ability of particles travel in the electric field
also depends on friction between particles and solvent
Intro
Problem: sample preparation relies upon what equipment is availavle, rather than on what questions to be answered
the Right question: can my sampling method provideme with answers I want
storage and transportation should account for analysis purposes
Analytical methods made great leap over past 20-30 years
But sampling methods did not kept the pace
poorly collected sample will still yield a poor result, no matter how sophisticated the techniqs are
An
Artifact
is something not present in sample but is introduced during the sampling or work-up procedure
Optical
IR
Ranges
Far-IR
- 25 mkm (400 cm-1) - 1 mm (10 cm-1)
Mid-IR
- 2.5 mkm (4000 cm-1) - 25 mkm (400 cm-1)
Near-IR
- 800 nm (12500 cm-1) - 2.5 mkm (4000 cm-1)
Advantages
Rapid, easy to handle, sensitive, a lot techs for gases, liquids and solids
convinient quantitive and qualitive analysis
complications in analysis broad by weak (intensity) overlaped vibrations -
individual vibrations
directly bonden with molecular structure -
molecular fingerprint
vibrarions are changes in molecular dipole moment
direction of vibration allows distinguish
stretching - changes of bond length
deformation - changes of bond angles
Types of vibrations
individual
- substructures within molecule
characteristic
- presence of functional groups, identity of molecule
Mixture analysis - concentrations of components correspond to intensity of bands according to Beer-Lamberl law (
p 72
)
Raman
As only very small percentage of molecules relax to virtual state the intensity of Raman scatering is very low and requires high quality equipment to detect
can be exited in UV, Vis or NIR ranges
It is a change in polarization of electron clouds on molecule
Raman=linear Raman if exciting light is up to 1 kW
If abowe 1 MW - hyper-, stimulated coherent anti-stokes (non-linear)
Number, shape and intensity of bands is directly related to the molecular structure
Limitation
- fluorescence (even when trace of impurity may fluoresce so it is
impossible to obtain spectrum
10^7 times stronger than Raman scattering
To avoid it NIR range often prefered (not much of electronic transitions within it) but
intensity of NIR Raman scattering is lambla*10^-4
Instrumentation
Basic for optical spec - monochromator, interferometer, polychromators
impossible to build device covering all the wavelength ranges
Fluorescence Spectroscopy
Why use?
biophysics and biochemistry
chemical and physical properties ot the surroundings
photophysical properties
in vivo
no gaseous samples
Beckman history
great interest in vitamin A lead to a rush
scientists had to develop their own for special needs
1976 year of spectrophot production in Bechman
after release du had lowest stray light and the best resolution in UV than any other device
thousands of papers on application of du (name of spect) had been published in subsequent year
model remained unchanged for 23 years!