Spectroscopy and its type

Introduction
• Spectroscopy deals with the production, measurement, and
interpretation of spectra arising from the interaction of electromagnetic
radiation with matter.
• The methods differ with respect to the species to be analyzed (such as
molecular or atomic spectroscopy), the type of radiation–matter
interaction to be monitored (such as absorption, emission, or
diffraction), and the region of the electromagnetic spectrum used in the
analysis.
• Quantitative and qualitative analyses.
Quantum Nature of Matter
• Atoms and molecules, under normal conditions, exist predominantly in
the ground state, which is the state of lowest energy. Ground state
atoms and molecules can gain energy, in which case they will be
elevated to one of their higher energy states, referred to as excited
states.
• Electronic, Vibrational, and Rotational Energy Levels
• Eatom = Eelectronic (line spectrum)
• Emolecule = Eelectronic + Evibrational + Erotational (band spectrum)
 ENERGY LEVEL TRANSITIONS IN
SPECTROSCOPY

• The absorption of radiation by an atom or molecule is that process in

which energy from a photon of electromagnetic radiation is transferred
to the absorbing species.
• Emission is essentially the reverse of the absorption process, occurring
when energy from an atom or molecule is released in the form of a
photon of radiation. This emission process is referred to as either
fluorescence or phosphorescence
THE ELECTROMAGNETIC SPECTRUM
high Frequency (n) low
high Energy low

0.78-1000 mM

X-RAY ULTRAVIOLET INFRARED MICRO- RADIO FREQUENCY

WAVE

Nuclear
Vibrational
Ultraviolet Visible magnetic
Infrared (Mid-IR)
resonance
2.5 mm 15 mm
1m 5m
200 nm 400 nm 800 nm
BLUE RED

short Wavelength (l) long

UV-visible molecular absorption
spectroscopy

Instrumentation
Transmission and absorbance and losses

• The reduction in the intensity of light

transmitted through a sample can be
used to quantitate the amount of an
unknown material.

P P Psample
T T 
P0 P0 Pblank
P0 P0 Pblank
A   log T  log A   log T  log  log
P P Psample
 Measure

Amount of light absorbed by the dissolved substance

• Qualitative
• Quantitative

 Qualitative:
• The absorption of light indicates the presence of substance

 Quantitative:
• The amount of light absorbed measures the concentration of the dissolved substance.
Beer Lambert’s Law
• Quantitative relationship between
P0
absorbance and concentration of A  log   bc
analyte P
  molar absorptivity
• Absorption is additive for mixtures b  pathlength
• Units of ℇ (L/mole.cm) c  concentration
• b (cm)
Really: Al = lbc
• c (mole/L)
Beer’s Law is always wavelength-specific

Amixture  A1  A2  ...  An
Amixture  1bc1   2bc2  ... n bcn
Light sources for UV-vis
• Deuterium lamp and Tungsten lamp
• Most common UV and Visible light
source
• Continuum from 190-400 nm and
380-700
Wavelength Selectors/Monochromator

 Spectroscopic instruments need a wavelength selector to

provide a range of wavelengths to impinge on the sample.
The narrower the range of wavelengths chosen by the
wavelength selector, the more likely Beer’s law is to be
followed. This also leads to improved sensitivity and
selectivity.
 The closest type of radiation to a purely monochromatic
source is a laser.
 With normal optical instruments the wavelength selector
provides a range of wavelengths centered at a nominal
wavelength.
 Radiation Detectors and Transducers

 A detector is a device that indicates the existence of some physical

phenomenon. Examples are the human eye, photographic film, the
mercury level in a thermometer, etc.

 A transducer is a special type of detector that converts a physical

phenomenon, e.g. light intensity, pH, mass, etc., into electrical signals
that can be amplified, manipulated, and eventually turned into
numbers that are related to the magnitude of the original signal.
 1 𝑂𝐷 𝑜𝑓 𝑡𝑒𝑠𝑡 𝑣𝑜𝑙 𝑚𝑎𝑑𝑒
𝐶𝑎𝑙𝑐𝑢𝑙𝑎𝑡𝑖𝑜𝑛𝑠 = × × 𝑐𝑜𝑛𝑐. 𝑜𝑓 𝑠𝑡𝑑 ×
𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑂𝐷 𝑜𝑓 𝑠𝑡𝑑. 𝑣𝑜𝑙 𝑡𝑎𝑘𝑒𝑛
 Measurement of absorbance using
spectrophotometer

Turn on the spectrophotometer

Set the desired wavelength

Take an empty cuvette and filled it with distilled water (blank sample)

Wipe the surfaces of the cuvette with lint-free tissues

Fill another cuvette with test sample

 Again wipe the surfaces of the cuvette using lint-free tissues

Place the blank and test sample into the sample holders

Close the sample chamber door completely

Read the absorbance

Take out the sample and prepare the next sample

Repeat the steps to measure the absorbance of all test samples

 For estimation of
carotenoids and chlorophyll

Homogenized with 10
mL of an acetone–
1g of fresh weight hexane mixture (2:3) Centrifugation
(solvent) for 2 minutes
to uniform mass.

Wavelength: Chl a,
Chl b, β-carotene and
Supernatent Take the absorbance lycopene content at
636, 646, 470 and 663
nm
 Vitamin C
10 g of sample

Homogenised with
Absorbance at 521
metaphosphoric acid
nm
and acetic acid

Addition of H2SO4:
Filtered/centrifuge
colored solution

Addition of bromine
Kept at 37°C for 3 h
water

Addition of 2,4- Addition of thiourea

dinitrophenyl to remove excess
hydrazine bromine
Total Polyphenols
Standard
Extraction using
1-2g of sample procedure will
80% methanol
follow

Standard gallic
Absorbance at acid solution at
760-765 nm various
concentrations
 Total Antioxidant Activity

Test Principle Standard Color change Absorbance

DPPH (2,2- Antioxidant reaction Antioxidant capacity Purple to yellow 517 nm

Diphenyl-1- with an organic in % color
picrylhydrazyl) radical
Radical assay

FRAP, ferric Antioxidant reaction Trolox/ Fe (II) Colorless to blue 593 nm

reducing antioxidant with a Fe(III) color
power assay complex

ABTS radical Antioxidant reaction ABTS radical Colorless to blue- 734 nm

scavenging assay with an organic scavenging activity green color
radical (%)

ORAC, oxygen Antioxidant reaction Trolox Loss of fluorescence 420 nm

radical absorbance with peroxyl of fluorescein
capacity radicals, induced by
2,2-azobis-2-
amidino-propane
(AAPH)
 Spectrophotometric Tests for Carbohydrates
Test/method Color Wavelength
change
Anthrone test Blue green 620 nm
color

DNSA Reddish 540 nm

(Dinitrosalicylic brown color
acid) method:
reducing sugars
Phenol sulphuric Stable 490 nm for
acid method yellow-gold hexoses
color 480 nm for
pentoses
Nelson Blue color 520 nm
Somogyi’s
method: reducing
sugars
 Spectrophotometric Tests for Proteins
Method Wavelength Color change Reaction

Bradford Method 595 nm Brown to blue

The Lowry 750 nm Blue

Method

Bicinchoninic 562 nm Apple greenish

Acid Method to purple

Biuret Method 540 nm Violet purplish

• Detect the presence of synthetic food colors adulteration in sweets (Kaju Pista Leechi,
Burfi, Moti choor ka Laddoo, Boondi ka Laddoo) and jam.

• Extraction:
2gm of sample + 10 ml distilled water Centrifuge Supernatent

Results: It was found that 9 out of 10 sweet

samples are found to be adulterated, it was
shown that dark orange color sweet is not
adulterated by synthetic food color.
 Sample extraction:
• 5g dry weight
• Extracted 80% aq. Methanol for 45 min

 Total phenols:
• The total phenolic content decreased significantly from 7.08 mg
GAE/g to 5.42 mg GAE/g after direct sun drying.
• High temperature sun and low water content lead to polyphenol loss
• Fruit samples: Apples, oranges, lemons, tangerines and grapes.
• Titrimetric and spectrophotometric methods.
• The titrimetric method was carried out by an iodimetric back-titration.
• The absorbance is measured spectrophotometrically at 530 nm.
• The results obtained from this study revealed that there is no significant difference between the
two methods except in the case of tangerines and grapes in which they were showed differences
in the amount of vitamin C
• Spectrophotometric method has been preferred because it is simple and fast method.

Spectrophotometer Titration
Vitamin C (g/L)
 Wheat sample:
• Wheat kernels were milled to fine flour using a coffee grinder

 Extraction:
• One gram of wheat flour was mixed with 10 mL 80% ethanol for 20 min
• Addition of 5 mL 6 M NaOH.
• The mixing was continued for 3 h in the dark in a shaker.
• The sample was acidified with 6 M HCl to pH = 2.
• The final slurry was centrifuged at 4000 rpm for 30 min to collect the supernatant.

 Total phenols:
• At 765 nm using UV-Vis spectrophotometer

 Total phenol content: 1984 µg GAE/g

 Whole milk (250 ml) boiled

Addition of 2 M citric acid

Casein Serum (150 ml)

Addition of 5 ml saturated NaCl solution

Partition with n-hexane

Sample preparation Treated with anhydrous sod. sulphate

Separation of casein and serum from milk: using
2M citric acid
Filtered and transparent filtrate taken

• UV-Vis Spectrophotometer Concentration Standard

• Lactose : 489 nm 9.32% Lactose
• Vitamin B1: 490 nm 388 ppm Standard vitamin B1
• Vitamin B2: 445 nm 64 ppm Standard vitamin B2
• Vitamin B6: 292 nm 116 ppm Standard vitamin B6
 Sample
• Oyster mushroom, white button mushroom, chicken, black
Cut into small pieces
Bengal goat, brown shrimp, Mola carplet, Rohu fish, Reba
carp, slender rasbora, banded gourami, and egg albumin
Homogenization
• The highest amount of protein present in brown shrimp, i.e.,
85.982 mg/mL. Weigh 1g of protein

Mixed with 9 mL of sucrose solution.

Centrifugation at 4,000 rpm for 6 minutes.

The supernatant was collected and stored at
4°C.
Protein analysis: 1 ml of supernatent and 1 ml
of water
4ml of alkaline copper solution: vortex

Incubate for 10 min

Add 0.5 ml Folin Ciocalteau phenol: incubate
for 30 min
• Samples: Green tea and black tea (Lipton Tea, Himalayan Tea, Taaza Tea,
Nice Tea) and soft drinks (Thums Up, Pepsi)

• The caffeine was extracted using chloroform as an extractant

• Wavelength: 273 nm

• Findings:
• Maximum caffeine content was 45.6 mg/g in the Nice black tea sample and
the minimum was 0.161 mg/ml in Pepsi soft drink sample
Extraction: 1 g horse gram flour + 100 mL 95% methanol

Concentration in control Standard

Phytic acid: 410 nm 10.22 mg/g Phosphorous
Tannin: 760 nm 11.75 mg/g Tannic acid
Oxalate: titration method 3.13 mg/g
Total phenol content: 760 nm 4.6 mg GAE/g Gallic acid
Total flavonoid content: 510 nm 3.9 mg GAE/g Gallic acid
DPPH assay: 517 nm 52 %
• Yeast: Zygosaccharomyces spp. and Candida spp.
• The determination of optical density (OD) was carried out in spectrophotometer at 600 nm
(OD600), which automatically measured the OD at every 120 min interval for 1,440 min (24
h).
 Limnophila aromatica: spice and a medicinal herb
 Extraction: 50%, 75%, and 100% of methanol, ethanol, and acetone in water were used as
solvent in the extraction of L. aromatica.
 Results: The extract obtained by 100% ethanol showed the highest total antioxidant activity,
flavonoid content, phenolic content and DPPH (2,2- diphenyl-1-picrylhydrazyl) radical
scavenging activity.
• The highest phenolic content : 40.5 mg GAE/g
• The highest flavonoid content: 31.11 mg quercetin equivalent/g
• The highest DPPH radical scavenging activity: 90%
The wavelength range of LED was 380-840 nm
Instrument configurations
• Single-beam
• Double-beam
• Multichannel
Single-beam UV-vis spectrometers

Skoog, Fig. 13-13

Good light throughput, but

what if the source
power fluctuates?
Double-beam in time UV vis spectrometers
• Beam is split in two, but measured by same detector

“in time” because

the beam appears in 2
places over one cycle in
time
- Sample
- Reference
- Sample
- Reference

What if the source

power fluctuates?

Skoog, Fig. 13-13

Attributes of UV-visible absorption for quantitative
analysis
1) Applicable to organic and inorganic species
2) Good detection limits: 10-100 mM or better
• Possible need for larger slit widths to achieve best sensitivities
3) Moderate to high selectivity
4) Accuracy: 1-3% or better
5) Ease and convenience (Rs) of data acquisition
Thank You