This article highlights the differences between spectroscopy and chromatography. Both spectroscopy and chromatography are extremely useful techniques that aid in chemical analysis. Chromatographic isolation and purification of chemical substances from a complex sample mixture is a major pre-requisite for their spectroscopic examination. Therefore, spectroscopy and chromatography complement each other and together make a scientist’s life easier.
But, having said that, particular differences do exist when we compare the two techniques one on one. What are these differences and why do they matter? Let’s find out through this article.
What is spectroscopy
Spectroscopy is an analytical characterization and structure determination technique. It is based on the interaction of a chemical substance with different regions of the electromagnetic spectrum. Electromagnetic radiations are passed as a stimulus through the sample mixture. The targeted chemical molecules respond to the stimulus by absorbing, emitting, or scattering the incident radiations. This response is recorded and consequently used to determine the atomic structure and/or chemical composition of the target compound.
What is chromatography
Chromatography is an analytical separation, purification, and identification technique. It is based on two distinct phases i.e., a stationary phase and a mobile phase. The stationary phase is held on a plane or packed into a column. The mobile phase is the solvent or gas that sweeps past the stationary phase, carrying the analytical components with it. Different components separate on the basis of their interaction with the stationary phase versus their dissolution in the mobile phase.
What is the difference between spectroscopy and chromatography
The key differences between spectroscopy and chromatography lie in the working principle of the two techniques and their end goal of the analysis.
‘Spectroscopy is a structural analysis technique. It depends on the interaction of analyte molecules with electromagnetic radiations. Contrarily, chromatography is an analytical separation and purification technique that depends on the relative interaction of analyte molecules with the stationary phase versus the mobile phase ‘.
All the significant differences between spectroscopy and chromatography are summarized for you in the table given below.
| Difference | Spectroscopy | Chromatography |
| Origin | Spectroscopy originated back in 1672 when Issac Newton did his optics experiment. He passed sunlight through a prism and the sunlight split into its constituent colors of different wavelengths | Mikhail Tsvet introduced chromatography in 1903. He separated different plant pigments on a piece of paper. Distinctly colored spots appeared at different positions on the paper |
| Purpose | Structure determination | Separation, purification, and identification of chemical components |
| Working principle | It is based on the interaction of chemical molecules with the electromagnetic spectrum except for Mass spectrometry (MS) which is based on the interaction of molecules with simultaneously applied electric and magnetic fields |
It is based on the interaction of chemical components with a stationary phase versus a mobile phase |
| Distinct phases | No distinct phases are needed in spectroscopy | Two distinct phases are an essential requirement of chromatography i.e., a stationary phase and a mobile phase. The stationary phase could be a solid or a liquid. Conversely, either liquid or gaseous mobile phases are used in chromatography |
| Instrument | A spectrometer or a spectrophotometer is used to perform spectroscopy. The spectrophotometer mainly consists of : 1. A radiation source 2. Diffraction grating 3. Monochromator 4. Sample cell 5. Detector 6. Recorder |
A chromatographic system such as HPLC is made of the following main components: 1. Solvent reservoir 2. Pressure pumps 3. Sample injector 4. Analytical column 5. Detector 6. Recorder |
| Analyte interaction | The electrons present in the molecule absorb photons of radiant light and interact with it by undergoing electronic, vibrational, and/or rotational transitions from a low to a high energy level. No chemical bonding or polar, non-polar concept exists in spectroscopy | No energy transitions are involved in chromatography. The analyte components interact with the stationary and the mobile phase by chemical bonding or intermolecular forces of attraction. The intermolecular forces of attraction include hydrogen bonding, dipole-dipole interactions, ionic interactions, etc. Similarly, polar molecules dissolve in polar solvents (ethanol, methanol, water) while non-polar analyte molecules dissolve in non-polar mobile phase solvents (n-hexane, benzene, toluene, etc.) |
| Level of analytical interactions |
In spectroscopy, the analyte molecules interact with radiant light based on their electronic energies. The electronic transitions depend on the functional groups present within the structure of the molecule. So, it is a more in-depth interaction | Surface-level interactions are involved in chromatography. The analyte molecules interact with the stationary or mobile phase using intermolecular energies and/or based on the functional groups present externally. Other than that, the molecules may interact based on their size or shape as well, as in size exclusion chromatography (SEC) |
| Sample preparation | A highly pure sample is introduced at the spectrophotometric port, either in solid form or dissolved in an inert solvent. The sample can be purified through chromatography prior to spectroscopy | The sample mixture is dissolved in the mobile phase and either applied as a spot on the TLC plate or introduced into the column through a syringe injector |
| Sensitivity | Spectroscopy is conducive to structural analysis, so it is a more sensitive protocol than chromatography | Chromatography is used for the isolation and purification of molecules from a sample mixture. Although there are highly sensitive chromatographic techniques like HPLC, UHPLC and SFC but generally chromatography demand less sensitivity as compared to spectroscopic analysis. Chromatography is often used as a first step when a new product is synthesized. An unknown sample mixture can be separated into its constituent components through chromatography followed by their identification prior to the structural confirmation of every single molecule using spectroscopy |
| Applications | i) Spectroscopy is used for atomic structure determination in research and development ii) It is used for structural identification and confirmation of drugs in the pharmaceutical sector iii) It finds a diverse range of applications for studying the structure of biomolecules such as proteins and nucleic acids. | i) Chromatography is used for the separation of analyte molecules from a complex sample mixture in the chemical laboratory and in industry · ii) It is helpful for identifying impurities, separating enantiomers in medicinal development, and for quality control in the pharmaceutical sector iii) Chromatographic techniques such as gel electrophoresis are used in biomedical and forensic applications for protein separation and DNA fingerprinting. |
Spectroscopy coupled with chromatography
Both spectroscopy and chromatography allow qualitative as well as quantitative analysis. Qualitative separation of analyte molecules can be performed in a chromatographic column but as it approaches the stage of quantitative identification and measurement, chromatography needs the support of a detector. Spectrophotometric detectors can be coupled with a chromatographic column to facilitate the detection process. The most commonly used spectrophotometric detectors with chromatography are the UV-Vis spectrophotometer and the mass spectrometer.
UV-Vis spectrophotometer is often coupled with a liquid chromatographic column. Analyte molecules eluting out of the column reach the UV-Vis spectrophotometer which helps the identification of all components individually by exploiting their light absorbing properties. The detector response is plotted as a graph of absorbance versus wavelength, called the UV-Vis spectrum. If the spectrum is recorded for multiple components eluting out of the column one by one and plotted as a function of absorbance versus retention time then it is known as a chromatogram.
Similarly, a mass spectrometer is often coupled with a GC column and this setup together is called GC-MS. A GCMS interface is required to couple the two systems together. Gaseous analyte molecules reach the mass spectrometer where they are ionized, accelerated, and identified.
In conclusion, spectroscopy and chromatography are complementary techniques that make any scientific exploration successful. These two techniques are not substitutes for each other. They serve different purposes, function differently, and have distinct end goals. Both are equally important for the research and development of new and existing chemical substances.
Commonly used spectroscopic and chromatographic techniques
Some commonly known spectroscopic techniques are:
- Ultraviolet-visible (UV-Vis) spectroscopy: It is used for structural identification of organic molecules that absorb radiations in the ultraviolet (200-400) nm or visible (400-800) nm region of the electromagnetic spectrum. Conjugated organic molecules such as those containing C=C or C=O absorb UV-Visible radiations and undergo electronic transitions. A specific wavelength is absorbed while the remaining are transmitted. The absorbed wavelength helps in determining the structure of tested molecules.
- IR spectroscopy: It is used for functional group identification of molecules. Molecules absorb infrared radiations (4000-625 cm-1) and undergo vibrational transitions in IR spectroscopy. The absorbed frequencies are used to determine the functional groups present in the molecule.
- NMR spectroscopy: It is used for structure determination depending upon the magnetic properties of certain atomic nuclei such as 1H and 13C nuclei. The nuclei change their nuclear spin state on absorbing radiowaves ( > 103 nm) under the influence of an externally applied magnetic field. The absorbed frequencies in NMR spectroscopy help determine the chemical environment of the molecule.
The most widely useful chromatographic analysis techniques include:
- Thin-layer chromatography (TLC)
- High-performance liquid chromatography (HPLC)
- Gas chromatography (GC)
To learn more about spectroscopy, you may like our article: What is spectroscopy-Everything you need to know about spectroscopy and for chromatography, you can check out What is chromatography.
References
1. (1990). “Chromatography and Spectroscopy.” Analytical Chemistry 62(11): 663A-669A.
2. Braithwaite, A. and F. J. Smith (1999). Chromatography and spectroscopic techniques. Chromatographic Methods. Dordrecht, Springer Netherlands: 366-398.
3. M.Younas (2017). Organic Spectroscopy and Chromatography.
4. Smith, B. C. (2019) “Spectroscopy Versus Chromatography for Potency Analysis.” Cannabis- Science and technology 2.
