Introduction & the Beer- Lambert Lawweb.nchu.edu.tw/pweb/users/splin/lesson/9431.pdf · Calculate...

Modern Optical Spectroscopy

http://www.scientific-web.com/en/Books/ModernOpticalSpectroscopyWithExercises.html

Shu-Ping Lin, Ph.D.

Institute of Biomedical Engineering E-mail: [email protected]

Website: http://web.nchu.edu.tw/pweb/users/splin/

Introduction & the Beer-Lambert Law

mailto:[email protected]

http://web.nchu.edu.tw/pweb/users/splin/

Energy Absorption

The mechanism of absorption energy is different in the Ultraviolet, Infrared, and Nuclear magnetic resonance regions. However, the fundamental process is the absorption of certain amount of energy. The energy required for the transition from a state of lower energy to a state of higher energy is directly related to the frequency of electromagnetic radiation that causes the transition.

Wave Number (cycles/cm)

X-Ray UV Visible IR Microwave

200nm 400nm 800nm

Wavelength (nm)

Spectral Distribution of Radiant Energy

Region of the Electromagnetic Spectrum

V = Wave Number (cm-1)

l = Wave Length

C = Velocity of Radiation (constant) = 3 x 1010 cm/sec.

u = Frequency of Radiation (cycles/sec)

The energy of photon:

h (Planck's constant) = 6.62 x 10-27 (Ergsec)

V =u

C l=

E = h = hC

lu

C=

lu C = ul

Electromagnetic Radiation

Visible

Ultra violet

Radio

Gamma ray

Hz

cm

cm-1

Kcal/mol

eV

Type

Quantum Transition

Type

spectroscopy

Type

Radiation

Frequency

υ

Wavelength

λ

Wave

Number V

Energy

9.4 x 107 4.9 x 106

3.3 x 1010 3 x 10-11 1021

9.4 x 103 4.9 x 102

3.3 x 106 3 x 10-7 1017

9.4 x 101 4.9 x 100

3.3 x 104

3 x 10-5 1015

9.4 x 10-1 4.9 x 10-2

3.3 x 102

3 x 10-3 1013

9.4 x 10-3 4.9 x 10-4

3.3 x 100

3 x 10-1 1011

9.4 x 10-7 4.9 x 10-8 3.3 x 10-4

3 x 103 107

X-ray

Infrared

Micro-wave

Gamma ray emission

X-ray absorption, emission

UV absorption

IR absorption

Microwave absorption

Nuclear magnetic resonance

Nuclear

Electronic (inner shell)

Molecular vibration

Electronic (outer shell)

Molecular rotation

Magnetically induced spin states

Spectral Properties, Application and Interactions of Electromagnetic Radiation

Dispersion of Polymagnetic Light with a Prism

Prism - Spray out the spectrum and choose the certain wavelength (l) that you want by slit.

Polychromatic

Ray

Infrared

Red

Orange

Yellow

Green

Blue

Violet

Ultraviolet

monochromatic

Ray

SLIT

PRISM

Polychromatic Ray Monochromatic Ray

Electronic Spectroscopy Ultraviolet and visible

Where in the spectrum are these transitions?

Why should we learn this stuff? After all, nobody solves structures with UV any longer!

Many organic molecules have chromophores that absorb UV

UV absorbance is about 1000 x easier to detect per mole than NMR

Still used in following reactions where the chromophore changes (useful) because timescale is so fast, and sensitivity is very high.

Kinetics, esp. in biochemistry, enzymology.

Most quantitative Analytical chemistry in organic chemistry is conducted using HPLC with UV detectors

One wavelength may not be the best for all compound in a mixture.

Affects quantitative interpretation of HPLC peak heights

Ultra Violet Spectrometry

The absorption of ultraviolet radiation by molecules is dependent upon the electronic structure of the molecule.

So the ultraviolet spectrum is called electronic spectrum.

The absorption of light energy by organic compounds in the visible and ultraviolet region involves the promotion of electrons in , , and n-orbitals from the ground state to higher energy states. This is also called energy transition. These higher energy states are molecular orbitals called antibonding.

Electronic Molecular Energy Levels

The higher energy transitions ( *) occur a shorter wavelength and the low energy transitions (*, n *) occur at longer wavelength.

Ener

gy

*

*

n

*

*

n

*

n

*

Antibonding

Antibonding

Nonbonding

Bonding

Bonding

Spectrophotometer

An instrument which can measure the absorbance of a sample at any wavelength.

Light Lens Slit Monochromator

Sample Detector Quantitative Analysis

Slits

Instrument to measures the intensity of fluorescent light emitted by a sample exposed to UV light under specific conditions.

Emit fluorescent light as energy decreases

Ground state

Sample

90 ° C

Detector UV Light Source

Monochromator Monochromator

Antibonding

Antibonding

Nonbonding

Bonding

Bonding Energy

->

->

'

'

'

' '

n-> n

n-> '

Electron's molecular energy levels

Fluorometer

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Spectrophotometry

Key Concepts

• Lambert’s Law of Absorption

• Beer’s Law

• Beer-Lambert Law

• Absorption Cross-Sections

• Photometric quantities

• Spectrophotometer

• The Cary 50 Spectrophotometer


Lambert‟s Law of Absorption

xeI

I

Ix

I

-=

-=

0

d

d

The intensity I0 if a beam of light decreases exponentially as it passes though a uniform absorbing medium with the linear decay constant α.

Restatement: In a uniform absorbing medium, the intensity of a beam of light decreases by the same proportion for equal path lengths traveled.

The linear decay constant α is a characteristic of the medium. It has units of reciprocal length. α is the path length over which the intensity is attenuated to 1/e.

Photo: http://www-history.mcs.st-andrews.ac.uk/history/PictDisplay/Lambert.html

α

I0

I(x)

x

Lambert described how intensity changes with distance in an absorbing medium.

xeIxI -= 0)(

leII -= 0

I

l

xeII

xII

-=

-=

0

dd

Johann Heinrich Lambert 1728-1777

The distance traveled through the medium is called the path length.


Lambert’s Law of Absorption (base 10)

xkxeI

I -- == 100

Typically base 10 is used in photometry.

xkx IeII -- == 1000

10ln=k

k is the path length over which the intensity is attenuated to 1/10.

xk

I

I -=100


Lambert’s Law Example If one slab of absorbing material of thickness l reduces the intensity of a beam of light

to half.

α I0

2

110

0

== - lk

I

II

l

And three slabs will reduce the intensity of a beam of light to one eight.

Then two slabs of the same absorbing material will then reduce the intensity of a beam of light to one quarter.

4

1

2

110

2

2

0

=

== - lk

I

Iα I0 I

l

α

l

8

1

2

110

3

3

0

=

== - lk

I

Iα I

l

α

l

α I0

l

Beer found that Lambert’s linear decay constant k for a solution of an absorbing substance is linearly related to its concentration c by a constant, the absorptivity ε, a characteristic of the absorbing substance.

Restatement: The linear decay constant k is linear in concentration c with a constant of proportionality ε.

(August Beer, 1825-1863)

A colored absorber has an absorptivity that is dependent on wavelength of the light ε(λ). The absorptivity is the fundamental property of a substance. This is the property that contains the observable spectroscopic information that can be linked to quantum mechanics (also see absorption cross section.)

ck =

Typical units are: k cm−1; c M (moles/liter); ε M−1cm−1

Beer’s Law


Photometric Quantities

Transmittance (T)

Absorbance (A) (AKA optical density, O.D.)

0I

IT = usually given in percent

TI

IA loglog

0

-=

-= by convention, base 10 logs are used

In photometry we measure the intensity of light and characterize its change by and object or substance. This change is typically expresses as percent transmittance or absorbance.

Frequently when your primary interest is the light beam

Used almost exclusively when your interest concerns the properties of the material

Beer – Lambert Law

Glass cell filled with

concentration of solution (C)

IILight

0

As the cell thickness increases, the transmitted intensity of light of I decreases.

T- Transmittance

T = I0 - Original light intensity

I- Transmitted light intensity

% Transmittance = 100 x

Absorbance (A) = Log

= Log = 2 - Log%T

Log is proportional to C (concentration of solution) and is also proportional to L (length of light path through the solution).

I

I0

I

I0

I0

I

1

T

I

I0

Beer-Lambert Law

Lambert‟s and Beer‟s Laws are combined to describe the attenuation of light by a solution. It is easy to see how the two standard photometric quantities can be written in terms of this law.

lcII -= 100

lcA

TI

IA

=

-=

-= loglog

0

xcT

I

IT

-=

=

10

0

Transmittance Absorbance

http://en.wikipedia.org/wiki/Beer%E2%80%93Lambert_law

＃Beers Law states that absorbance is proportional

to concentration over a certain concentration range

A = absorbance = molar extinction coefficient (M-1 cm-1 or mol-1 L cm-1) c = concentration (M or mol L-1) l = path length (cm) (width of cuvette)

Beer-Lambert Law

Beer‟s law is valid at low concentrations, but breaks down at higher concentrations

For linearity, A < 1

1

Beer-Lambert Law

If your unknown has a higher concentration than your highest standard, you have to ASSUME that linearity still holds (NOT GOOD for quantitative analysis)

Unknowns should ideally fall within the standard range

25

Quantitative Analysis

A < 1 If A > 1:

Dilute the sample Use a narrower cuvette

(cuvettes are usually 1 mm, 1 cm or 10 cm)

Plot the data (A v C) to produce a calibration „curve‟

Obtain equation of straight line (y=mx) from line of „best fit‟

Use equation to calculate the concentration of the unknown(s)

26

Quantitative Analysis

Calibration curve showing absorbance as

a function of metal concentration

y = 0.9982x

R2 = 0.9996

0

0.2

0.4

0.6

0.8

1

1.2

0 0.2 0.4 0.6 0.8 1 1.2

Concentration (mg L-1)

Ab

so

rban

ce (

no

un

its)

Steps in Developing a Spectrometric Analytical Method

1. Run the sample for spectrum

2. Obtain a monochromatic wavelength for the maximum absorption wavelength.

3. Calculate the concentration of your sample using Beer Lambert Equation: A = ECL

Wavelength (nm)

Absorbance

0.0

2.0

200 250 300 350 400 450

Spectrometer Reading

Slope of Standard Curve = A

C

1 2 3 4 5

1.0

0.5

Concentration (mg/ml)

There is some A vs. C where graph is linear.

NEVER extrapolate beyond point known where becomes non-linear.

x

x

x

Spectrometric Analysis Using Standard Curve

1 2 3 4

0.4

0.8

1.2

Concentration (g/l) glucose

Avoid very high or low absorbencies when drawing a standard curve. The best results are obtained with 0.1 < A < 1. Plot the Absorbance vs. Concentration to get a straight line

Homeworks

1. Calculate the Molar Extinction Coefficient ε at 351 nm for aquocobalamin in 0.1 M phosphate buffer. pH = 7.0 from the following data which were obtained in 1 cm cell.

Solution C x 105 M Io I

A 2.23 100 27

B 1.90 100 32

2. The molar extinction coefficient (ε) of compound riboflavin is 3 x 103 Liter/Cm x Mole. If the absorbance

reading (A) at 350 nm is 0.9 using a cell of 1 Cm, what is the concentration of compound riboflavin in sample?

3. The concentration of compound Y was 2 x 10-4 moles/liter and the absorption of the solution at 300 nm using 1 cm quartz cell was 0.4. What is the molar extinction coefficient of compound Y?

4. Calculate the molar extinction coefficient ε at 351 nm for aquocobalamin in 0.1 M phosphate buffer. pH =7.0 from the following data which were obtained in 1 cm cell.

Solution C x 105 M I0 I

A 2.0 100 30


Cross-Sections and Absorptivity the connection to single particles and

molecules

The absorption of light by particles (and single molecules) is characterized by an absorption cross section C. In this model the particle is replaced by a perfectly absorbing sphere with a cross sectional area C. This cross section is a property of the particle and is not related to its geometric cross sectional area. The concentration of particles per unit volume is N.

=

=

=

-

3

3

cm

liter 1010ln

10ln

CN

NCk

NC

A

typical units are: C cm2; N cm−3

The cross section can be directly related to the molar absorptivity. NA is Avagadro‟s number. units are: C cm2; N cm−3; NA mole−1; ε M−1cm−1


Efficiency

The absorption efficiency Q of a particle is the ratio of its absorption cross section C to its geometric cross section Cgeo.

Absorption efficiency is dimensionless.

geoC

CQ =


Extension to Scattering and Extinction

Attenuation of light by absorption and scattering both obey Lambert‟s Law. Thus we can extend our treatment of absorption to scattering and extinction. (Recall that extinction is the effect of absorption + scattering.)

)cxcxA

QQQ

CCC

scaabsext

scaabsext

scaabsext

scaabsext

==

=

=

= The scattering efficiency can be much larger than unity.

Extinction paradox: Qext = 2 (Qabs = 1; Qsca = 1) for an perfectly absorbing particle very large compared to the wavelength of light.

Note:

•All of these quantities are in general wavelength dependent.

•Our discussion has not included the mechanism (cause) of absorption and scattering.

•There are many different mechanisms that cause of absorption and scattering.


Instrumentation

Spectrometer: measures I vs λ. Simply measures the spectrum of the light (e.g. emission spectroscopy).

Spectrophotometer: measures I/I0 vs λ. Measures how the sample changes the spectrum of the light (e.g. transmission, reflection, scattering, fluorescence).

All spectrophotometers contain a spectrometer.

-meter: the detector is electronic

-graph: light intensity recorded on film

photometer: measures I/I0 without λ selection.


The Spectrophotometer

Measures absorbance as a function of wavelength

Components: light source, monochromator, sample cell, detector, optical system.

monochromator sample cell detector

light source

slit

diffr

act

ion g

rating

balance the forces:

Computer controlled acquisition of absorption spectra

Cary 50 UV-Vis Spectrophotometer

sample

detector

light source

monochromator

Can you find the diffraction grating and the slit?

www.varianinc.com OU NanoLab/NSF NUE/Bumm & Johnson


Making a Measurement with the Cary 50

First, measure the baseline using a blank sample. This is raw I0. The blank sample is the cuvette with deionized water (everything but your nanoparticles). This corrects for any absorption due to the cuvette, water, and variations of the light intensity of the light source, monochromator, etc.

Second, measure the zero by inserting the beam block. This corrects the instrument for the detector background.

Third, measure your sample. This is the raw I. The Cary 50 automatically calculates the corrected intensities (I and I0) by subtracting the zero from each of the raw intensities.

Subsequent measurements do not require re-measuring the blank and zero, simply repeat step 3.

TA

zeroIraw

zeroIraw

I

IT

log

00

-=

-

-==


Applications of Spectrophotometry

Spectroscopy

Chemical Analysis: trace analysis, pH, forensic, in situ

monitoring, remote monitoring, geology, astronomy, ....

Particle size

Thin film characterization

Color matching

Optics

Sample Cells (Cuvettes) UV Spectrophotometer

Quartz (crystalline silica): works in the UV region below 350 nm, can be employed across the whole UV-Visible wavelength range, 190-1,000 nm

Visible Spectrophotometer

Optical glass: employed in the region above 300nm

Moulded plastic cells: used in the visible region above 350 nm, low cost

Common size of cells is the 1cm rectangular cell, which has an optical path length of 1cm and path width of 1 cm. Typically, these cells hold 2-3 ml of sample. When sample volume is an important issue Consider

micro cells

Light Sources

UV Spectrophotometer

1. Hydrogen Gas Lamp

2. Mercury Lamp

Visible Spectrophotometer

1. Tungsten Lamp

Uses for UV, continued

Knowing UV can help you know when to be skeptical of quant results. Need to calibrate response factors

Assessing purity of a major peak in HPLC is improved by “diode array” data, taking UV spectra at time points across a peak. Any differences could suggest a unresolved component. “Peak Homogeneity” is key for purity analysis.

Sensitivity makes HPLC sensitive

e.g. validation of cleaning procedure for a production vessel

But you would need to know what compounds could and could not be detected by UV detector! (Structure!!!)

One of the best ways for identifying the presence of acidic or basic groups, due to big shifts in l for a chromophore containing a phenol, carboxylic acid, etc.

l

“bathochromic” shift “hypsochromic” shift

The UV Absorption process

• * and * transitions: high-energy, accessible in vacuum UV (lmax <150 nm). Not usually observed in molecular UV-Vis.

•n * and * transitions: non-bonding electrons (lone pairs), wavelength (lmax) in the 150-250 nm region.

•n * and * transitions: most common transitions observed in organic molecular UV-Vis, observed in compounds with lone pairs and multiple bonds with lmax = 200-600 nm.

•Any of these require that incoming photons match in energy the gap corrresponding to a transition from ground to excited state.

•Energies correspond to a 1-photon of 300 nm light are ca. 95 kcal/mol

What are the nature of these absorptions?

Example: * transitions responsible for ethylene UV absorption at ~170 nm calculated with ZINDO semi-empirical excited-states methods (Gaussian 03W):

HOMO u bonding molecular orbital LUMO g antibonding molecular orbital

h 170nm photon

Example for a simple enone

π

π

n

π

π

n

π*

π

π

n

π* π*

π*

π*

π*

π* π*

π*

-*; lmax=218 =11,000

n-*; lmax=320

=100

How Do UV spectrometers work?

Two photomultiplier inputs, differential voltage drives amplifier.

Matched quartz cuvettes

Sample in solution at ca. 10-5 M.

System protects PM tube from stray light

D2 lamp-UV

Tungsten lamp-Vis

Double Beam makes it a difference technique

Rotates, to achieve scan

Diode Array Detectors

Diode array alternative puts grating, array of photosens. Semiconductors after the light goes through the sample. Advantage, speed, sensitivity,

The Multiplex advantage

Disadvantage, resolution is 1 nm, vs 0.1 nm for normal UV

Model from Agilent literature. Imagine replacing “cell” with a microflow cell for HPLC!

Experimental details

What compounds show UV spectra?

Generally think of any unsaturated compounds as good candidates. Conjugated double bonds are strong absorbers

Just heteroatoms are not enough but C=O are reliable

Most compounds have “end absorbance” at lower frequency. Unfortunately solvent cutoffs preclude observation.

You will find molar absorptivities in L•cm/mol, tabulated.

Transition metal complexes, inorganics

Solvent must be UV grade (great sensitivity to impurities with double bonds)

The NIST databases have UV spectra for many compounds

An Electronic Spectrum

Abso

rbance

Wavelength, l, generally in nanometers (nm)

0.0

400 800

1.0

200

UV Visible

lmaxwith certain extinction

Make solution of concentration low enough that A≤ 1

(Ensures Linear Beer‟s law behavior)

Even though a dual beam goes through a solvent blank, choose solvents that are UV transparent.

Can extract the value if conc. (M) and b (cm) are known

UV bands are much broader than the photonic transition event. This is because vibration levels are superimposed on UV.

Solvents for UV (showing high energy cutoffs)

Water 205

CH3CN 210

C6H12 210

Ether 210

EtOH 210

Hexane 210

MeOH 210

Dioxane 220

THF 220

CH2Cl2 235

CHCl3 245

CCl4 265

benzene 280

Acetone 300

Various buffers for HPLC, check before using.

Chemical Structure & UV Absorption

Chromophoric Group ---- The groupings of the molecules which contain the electronic system which is giving rise to absorption in the ultra-violet region.

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

Organic compounds (many of them) have UV spectra

From Skoog and West et al. Ch 14

One thing is clear

UVs can be very non-specific

It is hard to interpret except at a cursory level, and hard to say that the spectrum is consistent with the structure

Each band can be a superposition of many transitions

Generally we don‟t assign the particular transitions.

An Example--Pulegone

Frequently plotted as log of molar

extinction So at 240 nm, pulegone has a molar extinction of 7.24 x 103

Antilog of 3.86

O

Can we calculate UVs? Electronic Spectra

Wavelength (nm)

Molar Absorptivity (l/mol-cm)

220 230 240 250 260 270 280 290 300

0

10049

20097

30146

40194

50243

nacindolA

Electronic Spectra

Wavelength (nm)


220 230 240 250 260 270 280 290 300

0

10394

20789

31183

41578

51972

Nacetylindol

Semi-empirical (MOPAC) at AM1, then ZINDO for config. interaction level 14

Bandwidth set to 3200 cm-1

The orbitals involved

Electronic Spectra

Wavelength (nm)


200 210 220 230 240 250 260 270 280 290 300

0

11097

22195

33292

44390

55487

Nacetylindol

Showing atoms whose MO‟s contribute most to the bands

The Quantitative Picture

• Transmittance:

T = P/P0

B(path through sample)

P0

(power in)

P (power out)

• Absorbance:

A = -log10 T = log10 P0/P

• The Beer-Lambert Law (a.k.a. Beer‟s Law):

A = bc Where the absorbance A has no units, since A = log10 P0 / P

is the molar absorbtivity with units of L mol-1 cm-1

b is the path length of the sample in cm

c is the concentration of the compound in solution, expressed in mol L-1 (or M, molarity)

Beer-Lambert Law

Linear absorbance with increased concentration--directly proportional

Makes UV useful for quantitative analysis and in HPLC detectors

Above a certain concentration the linearity curves down, loses direct proportionality--Due to molecular associations at higher concentrations. Must demonstrate linearity in validating response in an analytical procedure.

Polyenes, and Unsaturated Carbonyl groups; an Empirical

triumph R.B. Woodward, L.F. Fieser and others

Predict lmax for π* in extended conjugation systems to within ca. 2-3 nm.

Homoannular, base 253 nm

heteroannular, base 214 nm

Acyclic, base 217 nm

Attached group increment, nm

Extend conjugation +30

Addn exocyclic DB +5

Alkyl +5

O-Acyl 0

S-alkyl +30

O-alkyl +6

NR2 +60

Cl, Br +5

Similar for Enones

O

x

b b O O

X=H 207

X=R 215

X=OH 193

X=OR 193

215 202 227 239

Base Values, add these increments…

Extnd C=C +30

Add exocyclic C=C +5

Homoannular diene +39

alkyl +10 +12 +18 +18

OH +35 +30 +50

OAcyl +6 +6 +6 +6

O-alkyl +35 +30 +17 +31

NR2

S-alkyl

Cl/Br +15/+25 +12/+30

b g d,

With solvent correction of…..

Water +8

EtOH 0

CHCl3 -1

Dioxane -5

Et2O -7

Hydrcrbn -11

Some Worked Examples

O

Base value 217 2 x alkyl subst. 10 exo DB 5 total 232 Obs. 237


Base value 215 2 ß alkyl subst. 24 total 239 Obs. 237

Distinguish Isomers!

HO2C

HO2C


Base value 253 4 x alkyl subst. 20 total 273 Obs. 273

Generally, extending conjugation leads to red shift

“particle in a box” QM theory; bigger box

Substituents attached to a chromophore that cause a red shift are called “auxochromes”

Strain has an effect…

lmax 253 239 256 248

Interpretation of UV-Visible Spectra

Transition metal complexes; d, f electrons.

Lanthanide complexes – sharp lines caused by “screening” of the f electrons by other orbitals

One advantage of this is the use of holmium oxide filters (sharp lines) for wavelength calibration of UV spectrometers.

See Shriver et al. Inorganic Chemistry, 2nd Ed. Ch. 14

Benzenoid aromatics

From Crewes, Rodriguez, Jaspars, Organic Structure Analysis

UV of Benzene in heptane

Group K band () B band() R band

Alkyl 208(7800) 260(220) --

-OH 211(6200) 270(1450)

-O- 236(9400) 287(2600)

-OCH3 217(6400) 269(1500)

NH2 230(8600) 280(1400)

-F 204(6200) 254(900)

-Cl 210(7500) 257(170)

-Br 210(7500) 257(170)

-I 207(7000) 258/285(610/180)

-NH3+ 203(7500) 254(160)

-C=CH2 248(15000) 282(740)

-CCH 248(17000) 278(6500

-C6H6 250(14000)

-C(=O)H 242(14000) 280(1400) 328(55)

-C(=O)R 238(13000) 276(800) 320(40)

-CO2H 226(9800) 272(850)

-CO2- 224(8700) 268(800)

-CN 224(13000) 271(1000)

-NO2 252(10000) 280(1000) 330(140)

Great for non-aqueous titrations

Example here gives detn of endpoint for bromcresol green

Binding studies

Form I to form II Isosbestic points

Single clear point, can exclude intermediate state, exclude light scattering and Beer‟s law applies

Binding of a lanthanide complex to an oligonucleotide

Quantitative analysis

More Complex Electronic Processes

• Fluorescence: absorption of radiation to an excited state, followed by emission of radiation to a lower state of the same multiplicity

• Phosphorescence: absorption of radiation to an excited state, followed by emission of radiation to a lower state of different multiplicity

• Singlet state: spins are paired, no net angular momentum (and no net magnetic field)

• Triplet state: spins are unpaired, net angular momentum (and net magnetic field)

UV Spectrometer Application

Protein

Amino Acids (aromatic)

Pantothenic Acid

Glucose Determination

Enzyme Activity (Hexokinase)

Flurometric Application

Thiamin (365 nm, 435 nm)

Riboflavin

Vitamin A

Vitamin C

Visible Spectrometer Application

Niacin

Pyridoxine

Vitamin B12

Metal Determination (Fe)

Fat-quality Determination (TBA)

Enzyme Activity (glucose oxidase)

Introduction & the Beer- Lambert Lawweb.nchu.edu.tw/pweb/users/splin/lesson/9431.pdf · Calculate...

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