In a previous post last week, I introduced the basics of spectroscopy and covered different spectroscopic techniques. This week, let’s dive deeper and understand advanced spectroscopic techniques.

ORD

Optical rotatory dispersion (ORD) spectroscopy is an analytical technique that helps us understand chiral molecules like proteins and metal complexes. It measures how much a chiral molecule rotates plane polarised light at different wavelengths.

This rotation is not always constant. It changes with wavelength. This wavelength dependent change in optical rotation is called optical rotatory dispersion spectroscopy.

The results are obtained in the form of a curve of wavelength against optical rotation.

Principle– Chiral molecules interact differently with polarized light because their 3D arrangement is asymmetric. Near an absorption band, the optical rotation changes sharply, producing a characteristic ORD signal called the: cotton effect

Applications

·  Determining stereochemistry

·  Studying protein conformation

·  Identifying chiral compounds

Circular Dichroism (CD)
Circular Dichroism (CD) is another type of spectroscopy. It measures the difference in absorption of left and right circularly polarized light by a chiral molecule.

Instead of measuring rotation like ORD, this measures differential absorption. Plane-polarized light vibrates in one plane.

Circularly polarized light rotates like a helix:

• Left circularly polarized light (L-CPL)
• Right circularly polarized light (R-CPL)

A chiral molecule absorbs these two differently. That absorption difference is: Circular Dichroism

Where:

• A_L​ = absorption of left circularly polarized light
• A_R​ = absorption of right circularly polarized light

CD is widely used in biology because proteins are chiral. Different protein secondary structures produce characteristic CD spectra:

• α-helix
• β-sheet
• random coil

Each gives a distinct signal. So CD helps determine: protein folding and conformation

Fluorimetry

Also known as fluorescence spectroscopy, it is an analytical technique that detects and measures fluorescent light emitted by a substance. It involves exposing a sample to a specific wavelength of light which excites the molecule. As a result, the sample emits light of a longer wavelength.

Principle– electrons absorb incoming light, jump to an excited state, and rapidly emit energy as photons as they return to the ground state. The difference between the peak excitation wavelength and the peak emission wavelength is known as the Stokes Shift.

Applications

·  Pharmaceutical Analysis: Quantifying active ingredients and trace impurities in drugs.

·  Medical & Forensics: DNA sequencing, protein analysis

·  Environmental & Food: Detecting pollutants, vitamins, and minerals in agricultural products

Mass spectrometry (MS)

It is an analytical technique. It can do the following:

• identify molecules
• determine molecular mass
• analyze protein structure
• study metabolites and biomolecules
  

It works by:

1. converting molecules into ions
2. separating them based on mass-to-charge ratio (m/z)
3. detecting the ions
   

Advanced MS techniques include:

• MALDI
• ESI
• MS/MS
• iTRAQ
  

General applications

• clinical diagnostics
• proteomics
• biomarker discovery
• drug research
  

Technique	Full Form	Principle	Ionization Method	Key Feature	Main Applications	Advantages	Limitations
MALDI	Matrix-Assisted Laser Desorption/Ionization	Sample mixed with matrix absorbs laser energy and gets ionized	Laser-based soft ionization	Produces mostly singly charged ions with minimal fragmentation	Protein identification, peptide mass fingerprinting, microbial analysis	Good for large biomolecules, simple spectra, gentle ionization	Less suitable for complex mixtures, limited coupling with LC
ESI	Electrospray Ionization	Charged liquid droplets release ions as solvent evaporates	Spray-based soft ionization	Produces multiple charged ions	Proteomics, metabolomics, LC-MS, drug analysis	Highly sensitive, works with liquid samples, ideal for LC coupling	Sensitive to salts/contaminants, complex spectra due to multiple charges
MS/MS	Tandem Mass Spectrometry	Selected parent ions are fragmented and analyzed again	Usually combined with MALDI or ESI	Structural analysis through fragmentation patterns	Protein sequencing, biomarker discovery, clinical diagnostics	High specificity and structural information	More complex instrumentation and data analysis
iTRAQ	Isobaric Tags for Relative and Absolute Quantification	Chemical tags label peptides for simultaneous quantification during MS/MS	Uses ESI or MALDI with MS/MS	Quantifies proteins across multiple samples simultaneously	Quantitative proteomics, cancer research, pathway analysis	Multiplexing capability, accurate protein quantification	Expensive reagents, ratio compression can affect accuracy

Atomic Absorption Spectroscopy (AAS)

It is an analytical technique used to:

• detect and quantify metals
• measure trace elements in samples
  

Applications:

• environmental analysis
• clinical laboratories
• food testing
• pharmaceutical industries
• toxicology
  

Commonly analyzed metals include:

• lead (Pb)
• mercury (Hg)
• cadmium (Cd)
• iron (Fe)
• copper (Cu)
• zinc (Zn)
  

Basic Principle

Atoms absorb light of a specific wavelength. When metal atoms in the ground state are exposed to light:

• they absorb energy
• electrons jump to a higher energy level

The amount of light absorbed is proportional to the concentration of the metal.