Instrumentation Quiz Notes:
A. UV/Vis

Region of EMR spectrum
o UV: 190-350 nm
o Vis: 350-700 nm
o HOMO  LUMO
 Shifts in  max – indicate changes in chemical environment that can given
hints about the structure
o
Want matched cuvette to minimize lightloss
o Quantitative Conc. Measurement
 A = -log T = εbc
o b – path length (cm)
o c – absorptivity conc. (g/L)
o ε – molar absorptivity (mol/L)
o T = P/Po
 Po – radiant power of the incident beam
 Practical usage - Po – radian power transmitted by blank
(everything in sample w/o chromophere, what
measuring)
 P – “ of the transmitted beam
1. Diagram of System
2. Source –Provides EMR
1. Visible: Tungston or Tungston-Halogen Lamp
i. black body radiator – it glows
ii. Metal can be heated to very high temps
iii. can run its hotter to get slightly lower 
iv.  close to visible and near IR (350-250 nm)
v. Some Tungston (W) is lost by sublimation
1. Adds small amounts of I2 captures sublimed W as WI2
2. Longer life and a little UV (slightly bluish tint)
vi. UV: Deuterium (isotope of hydrogen 2H)
a. Adv- won’t flood
b. Lamp gets hot and has to cool
c. D2 is high energy and high output when subjected to electric
arc discharge
d. D2 (g) + Earc  D*2  D.’ + D.” + h
Range
Range of UV
of KE’s
energies (not quantized
system)
e. D.’ + D.” – Fly off at varying amts of KE
vii. Xenon Arc lamp – in both the UV & Vis regions
1. Disadv: expensive and relatively short lifespan, less
dependable
2. Only used when need high intensity
a. Not needed in absorbance, helpful in fluorescence
2. Sample Holder – light bouncing through other end
i. Cuvette - must be transparent to  region you are using
1. Vis range – used plastic (cheap), pyrex, polystyrene
2. UV range – use fused silica, quartz
a. Also works in Vis range but more expensive
ii. Fiberoptic: Ncore > Ncladding
1. Dependent on critical angle
a. Light directed into core so it meets cladding at  >
critical , total internal reflection
3. Wavelength selection – transmit certain  band
i. Wavelength selector – gives S/N ratio
ii. Need a narrow band of ’s
1. Don’t want to keep the ’s that don’t absorb
iii. Get higher sensitivity  higher slope of Beer’s law plot
1. Beer’s law requires narrow  band, choose  max (largest
Energy)
2. Best sensitivity
3. Beer’s law would be obeyed best when  band is flat
4. Only obeys for monochromatic light
iv. Selectivity – ability to measure one compound in the presence of other
compounds
v. Filter removes large regions of ’s by absorbing them
1. Removes unwanted ’s
2. Types of filters
a. Wide band pass – 50 to 100 nm
b. Interference filter – (narrow ) 0.1 to 5 nm
i. Only certain  act constructively
c. Cut-off Filter (blocking)
i. Block entire region from getting in system
ii. Has sharp change form where it lets light
through ot by where it doesn’t
d. Notch Filter (opp. interference)
i. Blocks a very narrow range
ii. Ex: Laser light  want to hit sample but not get
into detector
vi. Dispersing Element – separates  spatially
1. prism
2. Grating – spreads out  in space by diffraction
a. Sheet w/marrow grooves w/ sharp, well defined shapes
b. Does the same thing as a prism
c. Can have an array of detectors that detect entire
spectrum
d. Used in 2 ways, depending upon detector set-up
i. Single detector (PMT)
1. Detects entire useful range
2. Need to isolate the desire  band using a
monochormoter
3. Exit slit- only one narrow band
4. Turn grating to change  band
ii. Multiple detector
1. actually from the focal plane
2. Detector selects by position not b/c of 
3. Get all the ’s but only sees one  per
detector, thus get entire spectrum in a
few seconds
4. Detector Design
i. Photoelectric effect (older way) – light strikes a photoemissive
surface and e- are ejected, allowing the signal to be converted from
light energy to electrical energy
1. Has single state phototube
a. Only one emissive surface
2. Photomultiplier tube ( in fluorimeter and LC)
a. To detect smaller light signals
b. Have multiple stages
c. Potentials guide path of ed. Collect at anode
ii. Semiconductor Detector
1. Electrical signal – transfer energy of EMR to
semiconductor crystals which creates e- hole
2. Band structures of solids – atoms are close to each other
and electrical orbitals overlap to form band of energy
orbitals
a. Diagram
3. Insulators - non-metals
a. Most covalent cmpds and pure ionic crystals (e.g.
Diamond, sulfur)
b. Very large band gap between valence and
conduction band
4. Conductors – metals
a. Higher Energy bands overlap to form broad valence
band
i. partially filled with eii. bands close together
b. E- move to positive end when Elec.field applied
c. As Temp. increases
i. lattice vibration increases
ii. resistance increases
iii. conductivity decreases
5. Semiconductors – Silicon and Germanium (nuclear
detectors)
a. Small band gap between valence Band (completely
filled @ 0 K) and conduction bands ( completely
empty @ 0K)
i. e- jump by absorption of UV light or nuclear
emission
ii. When e- jumps to upper band, holes left in
valence band can move
b. As Temp. increases (major diff. from conductor)
i. thermal promotion can exist to bridge Band
gap
ii. resistance decreases
iii. conductivity increases
iv. # e-/p+ pairs increases
Element Conductivity (-1 M-1) Band Gap (eV)
C
< 104
6
Si
increased
114
Ge
2
0.67
Sn
> 100
0.08
c. Intrinsic – pure material e - promotion form
valance to cond. Band
i. [e-]mobile = [p+]mobile
ii. same # of holes as ed. Extrinsic (Doped) – small conc. of impurities to
increase conductivity
i. impurity has similar size as host atom but
diff # valence e- which creates impurity
levels in the Band gap
e. n-Type: Dope Ge or Si (Grp IV) or As pr P (Grp
V), negative charge carriers move
i. extra valence e – cant fit into filled valence
bnad
ii. weakly bounded and easily excited to add eto conduction band
iii. Major charge carriers are e –
a. The hole stays fixed
b. [e-]mobile > [p+]mobile
f. p-type: Dope w/ Ge or Si (Grp IV) or B orIn (Grp
III)
i. deficiency of e-‘s compared to surrounding
atoms
ii. e- from valence band are easily transferred
to impurity atom
1. leave p+ in valence band
iii. Major carriers are p+’s , the holes are
moving (+ charge carrier)
g. Semiconductor junction – p-n type: p-type in
contact with n-type
i. At junction e’s from n-type fill p+’s in ptype
ii. thin depletion zone
1. Region in middle (intrinsic)  few
m’s when sticking 2 slabs together
2 ways to wire system:
iii. Forward Bias – current flows from (+) to ptype and (-) to n-type
1. Get pulsating DC
2. Used for rectification of AC, not in
EMR detection
iv. Reverse bias – external applied to ∆V, (+)
to n-type and (-) to p-type
1. Depletion zone thick
a. Enlarged zone compared to
just putting them together
2. Momentary movt. of charge, like
capacitor
a. Burst of current then nothing
3. Entering radiation creates e-/p+ pair,
which creates current
4. Charge carriers move to electrode
v. Photodiode (HP and Fiberoptics) – uses p-n
junction
How it works:
1. Prior to measurement – capacitor is
charged up
2. As exposed to radiation, charge is
lost
3. After it’s charge back up, the amt
need to charge it is recorded.
a. Difference from the blank
after holes removed
4. Make all (+) to reblank for next
measurement, thus holes go away
6. Single Beam Spectrophotometer (Beackman DU-2)
a. UV source: Tungsten lamp or deuterium w/ mirror
that can be rotated to allow different sources
b. Prism monochromator – for dispersing element
i. Turn prism to let different  through
ii. No ray detector
iii. Displaced vertically slightly so goes through
upper slit
c. Used in Vis and UV ranges
i. Single beam and single detector can only do
one  at a time
ii. Dispersed beam travels same path
d. Only one optical pathway
e. 3 step-process measurement at each  - Time
consuming to get entire spectrum
i. Dark current (0% T)
ii. Blank (100% T)
iii. Sample (sample % T)
7. Double Beam Spectrophotometer – w/o ray detector
a. Chopper device – turns at constant rate, when open
lets light through so breaks up the light
i. Alternates light, but only one beam at any
instant
ii. Alternative sending beam
b. Entrance and exit slit horizontal to each other
c. Constant comparison between reference and sample
i. Automatically incorporates the blank
8. B4054-reverse optics – all  passes through sample
a. Has upper beam director mirror and lower beam
director
b. Oscillates, some light to sample, some to reference
i. Curved mirror collects light from many
sources
B. Molecular Fluorescence
Molecular Fluorescence Events (aromatic or extended conjugated systems):
1. molecules absorbs an e- (EMR in UV) and get excited to upper electronic state
(vertical transition)  π to π* transition
2. The molecules lose energy via vibrational states within the excited electronic state
3. The electron makes a vertical transition down to the ground state (in visible
region), thus emitting light at a lower energy than the corresponding excitation
energy.
a. Instrument measure the intensity of light emitted (fluorescent intensity =
FI)
With an increase in energy state, the bond length is increased
Choosing λexc and λem:
1. First, you choose the λem to observe the fluoresced light – this has to be a
good guess
2. Then, vary the λexc (λ of light sending into the sample) to find out where
to get the most fluorescence
3. Max FI is usually at the λexc in the UV
Franck-Condon Principle:
1. nuclei cannot move during a transition (ONLY e-)
2. excitation occurs to an upper vibrational level within the excited electronic state
3. thus, there is ample time for the molecule to lose excess vibrational energy
4. emission occurs from the lowest vibrational level (v0) within the excited state to
an upper vibrational level (vn) within the ground state
5. Eem << Eexc and λem (visible) >> λexc (UV)
Quantitative Analysis:
Measure the FI as a function of [ ]
*at low [ ], the FI is proportional to the true concentration
*FI = K x P0 x c
where K is a constant that is dependent on instrumentation (detector
efficiency and monochromator throughput – amt of light able to pass) &
P0 is the radiant power of the beam coming into the sample from the incident
beam
Therefore, for a given [ ], the increase in FI is proportional to the radiant power of the
incident beam…..you can detect very small [ ]s by pumping up the incident beam
power.
UV/Vis Absorption
Uses Beer’s Law
A = εbc = -logT = -log (P/P0) = log (P0/P)
Overall Conclusion:
1. For fluorescence – a higher P0 gives a greater FI for a given [ ] – must use a very
intense source such as the Xe arc lamp
***can do a very low [ ] and still get a signal because the radiant power (P0) is
proportional to FI
2. For absorption work – a higher P0 does not change the absorbance – must use a
source with a reasonable intensity such as D2 in UV or W in the visible region
Graphing the log FI vs log [ ] gives a linear plot with a slope of 1.000 (ideally)
Log FI = log (K x P0 x c) = log (K x P0) + log c
Hook’s Effect: at high [ ] the linearity is lost and curves back downward
Possible Reasons:
1. FI = K x P0 x c is a limiting equation that depends on the assumption
that [ ] and absorbance is very low
2. Self-absorption – if emission band overlaps the excitation band, then
the fluoresced light will get absorbed on it way out of cuvette  can
happen due to impurities
Quenching – Anything that causes the FI to be less than what it should be (anything
that decreases the quantum yield,Ф)
Ф = # molecules fluoresced / # molecules absorbed light to begin with
The theoretical Max is 1, but this NEVER happens!!!
*** Ф is usually < 1 because there are many processed that compete with
fluorescence.
They compete kinetically and return the molecule to ground state.
1. Forms of competition w/ Flourescence
a. Internal conversion – return to the ground state w/o transition via
vibrational relaxation
i. If the excited state (upper well)overlaps the potential well (lower
well) and dips into ground state then its goes all the way back to
the ground state and the molecule doesn’t fluoresce
ii. Happens primarily in aliphatic cmpds
iii. Can minimiz:
1. By adding rigidity, mount sample on support
2. If more flexible, easier to change vibrational levels
b. External Conversion - collisions with solvent and impurities which take
away energy from the system (collisional deactivation)
i. Excited molecules transfer energy via collisions, if lose enough
then lose fluorescence
ii. Temp affects the # of collisions, raise temp will reduce
fluorescence
iii. Can minimize by
1. Lowering Temp
2. Controlling T (transmittance)
c. Intersystem Crossing – spin of e- in excited state gets flipped  triplet, 2
unpaired so now have the same spin
i. Low likelihood of fluorescence, possibility of phosphoresce
ii. Want to avoid:
1. Halogenated solids (any halogens)
a. Esp. bigger atom b/c of heavy atom effect due to
large e- clouds (Br,I)
2. Paramagnetic materials – ones or more unpaired ea. High spin transition metals will cause problems
b. Remove O2 (2 unpaired spins)
2. Instrumentation
a. Source – fluorescence wants high intensity source (UV)
i. Most common:
1. Hg lamps (254nm, 313 nm)
a. Very high intensity offsets not having optimal  exc
2. Xe arc source – use for fluorescence but not absorption b/c
don’t get anything for high intensity
3. Monochromator – need to select  exc and  em
a. not need with array detector
b. Sample holder – rectangular, 4 optical UV surfaces
i. Made out of quartz or fused silica
ii. Look at beam perpendicular
c. Wavelength selector – need 2 b/c have light going in and light going out
i. Emitted at 90°
ii. Often times excitation one is the filter
iii. Also possible emission   array detector
d. Detector - PMT or array detector
i. PMT (photomultiplier tube)
1.
2.
3.
4.
Detects intensity of light
Source intensity is monitored all the time
Can detect very low conc.
Beam attenuator - cuts it down so you can compare
a. Compare to cancel out any fluctuations
b. Beam splitter - 50% reflected and 50% transmitted
ii. Molecular Beacon – quencher is your friend (Dr. Tan)
1. Complementary base pairing, so Q (quencher) and F
(fluorescence) are next to each other
a. with a very small distance fluorescence is quencher
and the beacon is turned off
2. When target DNA added and pairs with DNA with
fluorescence tag, i.e. the complementary strand is present,
the loop opens and binds making Q and F far apart
a. Thus no quenching occurs and beacon is on
e. Qualitative analysis – very useful b/c very low LOD
i. Conditions must remain constant
C. Liquid Scintillation
Refers to when energy from a nuclear emission (α, β, or γ) causes a fluorescent
molecule (scintillator) to be excited  then the fluorescent intensity is detected by a
photomultiplier tube (PMT)
Mechanism:
1. energy transferred to scintillator
2. solute excited electronically (fluoresces)
3. light detected by PMT
4. larger input from radioactivity (β)  get many photons back per β
Solvent  other solvent molecules  1º solute (aromatic cmpd’s that can fluoresce)
…..instead: 1º solute  2º solute that emits at wavelengths that can be detected by
PMT easier
Nuclear Emission gives a large amount of energy such that one nuclear event can
excite many molecules!!!
Fluorescent Intensity (Pulse Height) = is directly proportional to the energy of nuclear
radiation deposited by nuclear emission.
Quenching = any process that decreases the number of number of photons detected by
the PMT (decrease quantum yield)
2 Broad Classes of Quenching:
A. Chemical Quench – usually not detected by energy transfer (PMT) &
problems with the energy transfer b/c of sample contamination
B. Color Quench – an absorption band that overlaps the fluorescence
a. Fluoresced light gets absorbed by impurity before leaving the
scintillation vial (not recorded)
Instrumentation Diagram:
PMT
vial
PMT
Analog-to-Digital
Convertor
Coincidence
circuit
ADC
PH
Analysis
Computer
passes only if both
PMT's give a signal
*need 2 PMT to discriminate between random events (both PMT must get a signal
from the vial at the same time)
*b/c can get Random Noise Events (chemiluminescence) which are single
photon events
A true nuclear event produces many UV photons (from the vial in all directions which
is why both PMT must get a signal)
……the # of UV photons is proportional to the energy deposited by the nuclear event
If the obtained signal (2 PMT) doesn’t meet the criteria, then the coincidence circuit
does not allow it to pass!!!
 but if allowed to pass, then coincidence circuit passes voltage to the ADC
which changes to voltage to a binary number
PH Analyzer (pulse height analyzer)
1. where pulse height is the magnitude of the binary number
2. PH analyzer allows us to make a PH spectra
3. Remember:
PH = Binary # = Voltage = Light intensity struck PMT = Energy of Nuclear event
4. There is a “counting window” in the pulses/time vs pulse height (of nuclear
energy)
5. outside the “counting window” is background
6. the plot looks like a semi circle indicating that the nuclear event has
continuous wavelength distribution
7. you can do a dual label dectection (3H and 32P) which would overlap their
semi-circle plots and you count the overlap (like a Venn Diagram)
Benefits of LSC:
1. Very high detection efficiency, which is very important for low energy β-emitters
(11C and 3H)
2. Counting Geometry – 100% efficient b/c sample is in the vial, so all radioactivity
is counted
3. Highly automated
4. Count filter paper, fibers, etc right into the vial
Draw Backs of LSC:
1. Expensive – cocktail, vials, disposal
2. Quenching – but it can be monitored
3. Sample Preparation – use benzene, thus aqueous soln aren’t as soluble
a. Reverse micelles, where add surfactant and turn themselves inside out
i. Ionic/polar = inside
ii. Hydrophobic = outside
D. Mass Spectroscopy
The purpose of Mass Spec is to:
1. Convert an unknown sample to is gaseous ion form  Difficult Part!!!!!
2. Separate the ions by mass/charge ratio (m/z) & measure # of each m/z ratio
3. Prepare a plot of Abundance vs (m/z)’s
Uses of Mass Spec:
1. Can determine the structure of the sample by using with other tools (such as
NMR, IR, or UV)
2. Qualitative Analysis from molecular weight and fragmentation patterns
3. Quantitative Analysis because used very low Limits of Detection
Different Connections for Mass Spec:
1. GC to MS
2. LC to MS (very common)
3. CE to MS
4. ICP to MS
Interpreting the Abundance vs (m/z)’s plot:
1. Parent ion peak-this is the peak that relates to the whole molecule and contains the
most abundant isotopes (1H, 12C, 14N, etc.)
2. P+1 Isotope peaka. this refers to peaks with a higher m/z ratio than the parent ion peak
b. contains ONE atom in the entire molecule that is an uncommon isotope
c. most commonly 13C or 2H
3. P+2 Isotope peaka. contains TWO atoms in the entire molecule that are uncommon isotopes
b. if chlorine or bromine present – have sizeable contributions from its
isotopes (66-33) and (50-50), respectively
4. Fragment low peak-see this when molecular ion peak breaks apart
5. Base peaka. Highest abundance peak
b. Simple one bond breakage of M+
6. Addition peaksa. Occurs in biological samples
b. When m/z ratio > M+
c. eg. Addition of H+ to M  (M+H)+
Addition of Na+ to M  (M+Na)+
Instrumentation:
[Inlet System]  [Ion Formation]  [Mass Analyzer]  [Detector]  [Readout]
-------------------------------------use Fast Atom Bombardment (FAB)
-------------------------------Always need to be under vacuum
-------------------------------------------------------------------------Sometimes all 4 parts need to be under vacuum
I.
II.
Inlet System-put in very small amount of sample (μg) [2 methods]
i. Open Tubular GC-MS
1. compounds must be volatile liquid and thermally stable
ii. Direct Insertion Probe (DIP)
1. improvement
2. uses less volatile and less thermally stable cmpd’s
iii. NOTE: both methods GC and DIP use neutral molecules
Ion Formation [4 methods]
i. Electron Impact (EI)
(MOST COMMON!!!!!!)
1. an electron (w/ hi KE ~ 70 eV) is accelerated and hits the
sample
2. Mg (s) + e-  Mg•+ + 2e3. creates a radical
ii. Fast Atom Bombardment (FAB)
1. used when inlet system and ion formation combined
2. used for MWs of 200-2000 Daltons
a. goop sample in glycerol
b. put on DIP
c. bombard sample with high kinetic energy atoms or
ions (Liquid Secondary Ion Mass Spec – LSIMS)
i. if ions, the usually Cs+ because low
ionization energy so formed easily
d. get sample back with M (g) and M+ (g)
3. Not all of sample ionized
4. needed improving because wanted to do MS for large
polypeptides and not have to break them into fragments.
iii. Matrix-Assited Laser Desorption & Ionization (MALDI)
1. Need matrix that absorbs strongly in UV
2. Mix macromolecule sample with solution of acidic
aromatic compounds
3. Spot on plate and put in Inlet System (vacuum)
4. Fire a N2 LASER b/c put out very high energy (337 nm)
that the aromatic cmpd’s will absorb
III.
5. Energy transferred to macromolcule
6. Some macromolecules become energized enough to come
off as (M+H)+ ----get the H+ from the matrix
7. Go to Time of Flight Mass Spec Analyzer
iv. Electrospray Ionization (ESI) ----- common method
1. Allows us to make many ions & serves as interphase
between the chromatograph and the MS
a. LCESIMS
2. LC solution put through needle with high voltage (~3kV)
and solution becomes spray
3. Solution aimed at the capillary tube----then rest of solvent
evaporates and rather than getting a couple of added H+s
like MALDI, you get 10 to 20 added H+s
4. The m/z ratio has been altered but the m/z is still low
enough for any mass analyzer
5. NOW, you may detect a small change in one AA residue
based on the weight obtained
a. Important for drug biotechnology
Mass Analyzer – separates ions by m/z ratio
i. Time of Flight Detection (TOF)
1. produce ions in pulses
2. they are accelerated to the same KE (thus the bigger ions
will move more slowly)
3. ions released into a 1 meter tube---determine the time to get
from one end to the other with a detector at the end
4. NOTE: used with MALDI
a. Very fast
b. Unlimited Mass Range (if the ions are a bigger, it
just tales a little longer)