Transcript Spectrophotometry

Spectrophotometry
scattering
reflectance
Emmision
Absorbtion
transmitance
EMR
refractance
raman
Characteristics of Light
1. Wavelength = distance between two peaks in a wave
a. l (lambda) is the symbol
b. Meters = m is the unit
2. Frequency = number of complete waves passing a given point
per second
a. n (nu) is the symbol
b. Hertz = Hz = s-1 is the unit
3. Amplitude = A = measure of the intensity of the wave,
“brightness”
l and n are inversely proportional
If one increases, the other decreases
Complementary Colors
l of Max.
Absorption
Color
Absorbed
Color
Observed
380-420
420-440
440-470
470-500
500-520
520-550
550-580
580-620
620-680
680-780
violet
violet-blue
blue
blue-green
green
yellow-green
yellow
orange
red
purple
green-yellow
yellow
orange
red
purple
violet
violet-blue
blue
blue-green
green
UV : 10-400 nm
UV-C : 100- 280
UV-B : 280- 315
UV-A : 315- 400
CHROMOPHORIC STRUCTURE
Group
Structure
nm
Carbonyl
>C=O
280
Azo
-N = N-
262
Nitro
-N=O
270
Thioketone
-C =S
330
Nitrite
-NO2
230
Conjugated Diene
-C=C-C=C-
233
Conjugated Triene
-C=C-C=C-C=C-
268
Conjugated Tetraene
-C=C-C=C-C=C-C=C-
315
Benzene
261
Mo la r Ab s or pt iv ity
Ele c tr on i c Sp e c tr a
(l /m o l- cm )
5 54 8 7
4 43 9 0
3 32 9 2
2 21 9 5
1 10 9 7
N a c et y lin do l
0
2 00
W a v el en g th
2 10
2 20
2 30
2 40
2 50
2 60
2 70
2 80
2 90
3 00
(n m )
This spectrum of a sunscreen lotion is in the ultraviolet region.
UV-B is the shorter l or higher energy and thus more harmful to skin.
Comparison of atomic and molecular spectra
Atomic spectra
Molecular spectra
Adsorption spectra
Yes
Yes
Emission spectra
Yes
Yes
Energy required for
excitation
high
low
Spectral region
UV
mainly visible
Relative complexity of
spectra
simple
complex
All spectrometric methods use a range of wavelengths Instead of monochromatic
Range called bandwidth . A narrower bandwidth represents better performance
A   logT    b  c
T 
%T
;
100
Light
I0
A  2  log %T
I
Glass cell filled with
concent rat ion of solut ion (C)
BLANK TYPES : RGT/SAMPLE/DW / AIR
The Beer-Lambert Law
Photodiode Array Spectrophotometer
Double Beam Instrument
Source
E
Wavelength
selector
P0(l)
P(l)
sample
Detector
& readout
Cells
Quartz: 180 – 3500 nm
Si glass: 380 – 2000 nm
W – 350 -2200 nm
D2 – 160 – 380 nm
SPECTROPHOTOMETER
•
SOURCE
MONOCHROMATOR
Tungsten Lamp
SAMPLE HOLDER
DETECTOR
Tungsten evaporate ,black surface lamp
Tungsten (W) Filament
l: 350-2200 nm (VIS/near-IR)
Deuterium Arc Lamp &
Mercury Lamp
Intensity nonvariable in constant voltage
continues wave
D2 + electrical energy  D2*  D2 + hn
D2 lamp to cover entire UV region (200-400nm)
SILICA CARBIDE (Carborundum )
– IR SORCE LIGHT
Lasers
Light Amplification by Stimulated Emission of Radiation
highly monochromatic (bandwidths of 0.01 nm or less)
Lasers 100 - 10,000 nm , monochromatic, high power ( LINEAR WAVE)
Light Output for UV/Vis Sources
Intensity
Deuterium
Arc
200
Tungsten
Filament
400
600
Wavelength/nm
800
MONOCHROMATOR
SOURCE
SAMPLE HOLDER
DETECTOR
MC’s
Filter (used to reject/select broad bands of radiation,excitation
and emmision wavelength. filter is colored glass)
Prism (used in older instruments . Tilt prism to allow various l’s to exit
from acontinuous source
Grating (Czerny-Turner grating monochromator)
grooved, reflective surface(300/2000-cm) each groove is a “light source”
different l’s of light are bent at different angles.
Position slit to allow only one wavelength to pass after grating has spread
the spectrum via diffraction. Each made with specific line spacing(d) - silica
(SiO2) coated Aluminum
Infrared
Polychromatic
Ray
PRISM
Red
Orange
Yellow
Green
Blue
Violet
Ultraviolet
Polychromatic
Ray
Monochromatic Ray
monochromatic
Ray
SLIT
MONOCHROMATOR
SOURCE
Grating
SAMPLE HOLDER
DETECTOR
nl = d(sin I - sin Filters
r) I = constant; therefore l  r
r
I
d
SAMPLE HOLDER
SOURCE
MONOCHROMATOR
DETECTOR
Cuvettes
Flat surface best – better reproducibility
Cleaning: H2SO4 or Hcl+Ethilicalchohol+Water in 1 :3 : 4 ratio respectively.
(glass adsorb uv spectrum)
Take 1gm of Potassium dichromate, add a little distilled water and very slowly add
approximately 100ml of Conc.H2SO4. Keep the cells in this acidic solution for a
maximum period of 12hrs. Wash the cells thoroughly with distilled water before use.
Quartz
160-2500
Methacrylate 280-800
Glass
350-1000
Plastic( polystyrene) Visible region
NaCl or KBr
IR
Polyethylene Far IR
Standard : L=1 cm , V= 3 ml
Microplates are used when volumes are below 200 L
DETECTOR
SOURCE
MONOCHROMATOR
SAMPLE HOLDER
Detector converts incident light to an electrical signal that we can measure and process
l: UV/VIS
Phototube
hn
e-
amplifier
-V
Photosensitive, negatively charged electrode
( Photoemissive cathode ).
Emits electrons when struck by UV / VIS light.
Electrons move through vacuum to anode (+)
and produce current proportional to intensity.
Detector response is a function of l
therefore must blank at after each l change.
hn
l: UV/VIS
Photo Multiplier Tube - PMT
Similar to Phototube YET more sensitive
Electrons amplified via collection of
positively charged dynodes
Cascade of e-’s where >106 electrons are
collected for each photon striking the
cathode
-HV+ V
hn
e - e- eee-
e
e e- e e e e- - e
e
hn
-HV
-HV+ 2V
DETECTOR
Photodiodes
SOURCE
Si
Si
MONOCHROMATOR
Photodiodes I
Si
Si
P
Si
Si
Si
n-type Si
SAMPLE HOLDER
Si
Si
Si
Si
Al
Si
Si
S
Si
Si i
p-type Si
hn
l: UV/VIS
Photodiode Array
SOURCE
HOLDER
PhotodiodeSAMPLE
Array
MONOCHROMATOR
DETECTOR
Collect entire spectrum simultaneously . Collection of diodes for each
wavelength. Grating reflects dispersed radiation to all diodes. Faster yet less
sensitive than phototube
white light
Sample
Source
Grating Polychromator
Infrared methods uses thermal transducers e.g. Thermocouples
consist of a pair of junctions formed when two pieces of a metal
that are different are fused at the ends
Quality control of spectrophotometer
1-Wavelenght accuracy
2- linearity
3- photometric accuracy
1- Observation
2- Intensity of light source
2-1 mecury :313-365-405-436-546nm
2-2 deuterium : 486-656 nm
Holmium oxide: narrow SBW (less than8nm)blank:perch.A
λmax:279,287,333,360,418,536
3- Filter
1-Wavelenght accuracy
Didymium oxide: broad SBW (more than 8nm)blank:air
lmax 530 & 585 nm
potassium dichromat(257,350nm)
paranitrophenol(401nm)
4- Chemical solvents
ammonium cobalt sulphate(512nm)
cyanomethemoglobin(540nm)
2- linearity
(paranitrophenol-450),(ammonium cobalt sulphate- 512)
1
(cooper sulphate- 650),(cyanomethemoglobin-540)
(green food color-630),(nickel sulphate-550)
didymium oxide in 550nm against air
2
0(air) ---- 0.096 , 0.25(air) ---- 0.346
0.5(air) ---- 0.596 , 0.75(air) ---- 0.846
3- photometric accuracy
3-1 ammonium cobalt sulphate
400nm----0.012,450mn-----0.077
500nm----0.163,550nm-----0.077
3-2 potassium dichromat
350nm----0.536±0.005(blank : 0.01N H2SO4)
CHECKING THE PERFORMANCE OF THE SPECTROPHOTOMETER
1. Preparation of Blank:
Measure accurately 10 ml of “Anal R” quality HCl of 36-46% concentration. Add it
slowly to 400 ml distilled water filled in one litre volumetric flask. Make it up to mark with
distilled water and mix it up well.
2. Preparation of sample
Weigh exactly 22.2 gm of “Anal R” quality Cobalt Chloride(COCl2 ).Transfer into one
litre volumetric flask. Dissolve it in 1% Hydrochloric Acid (HCl) and make it up to the
mark.
3- measure the Transmittance/absorbance at wavelength 480 to 540 nm at 5nm interval.
4- Graph should be in increasing order up to 510-515nm wavelength and then in
decreasing order