Chromatography: purifying your understanding

academics chemistry chromatography organic chemistry

Maybe you were studying for the MCAT or just in your college chemistry lab when it dawned on you: why are there so many different types of chromatography? What do they have in common and what are their differences? 

In all its forms, chromatography is an extremely useful tool for separation and purification of mixtures of different chemical substances. Separation allows us to further analyze components, and some everyday applications of this include drug testing from blood samples, food and beverage testing for ingredients that could make consumers sick, and even vaccine preparation! Therefore, chromatography is nicely paired with analytical chemistry, where methods such as mass spectrometry and NMR are used to determine the identity of an unknown substance. 

So, what do they all have in common? There is always a mobile phase (liquid or gas) and a stationary phase over which a sample is run. The key to separation lies in the difference in polarity of the mobile and stationary phases. Usually, it is the stationary phase that is more polar and the mobile is less or nonpolar. A sample consisting of a more polar substance (we’ll call substance A) and a less polar substance (substance B) will be separated out because A will prefer to be dissolved in the polar stationary phase while B will prefer to be dissolved in the less polar mobile phase. Remember: like dissolves like

The main forms of chromatography and their principles:

Paper Chromatography


  • Stationary phase: paper with water molecules**
  • Mobile phase: liquid solvent
  • What is measured: Retention factor
  • Takeaway: Greater the Rf, the less polar the compound

Paper is made of cellulose, a sugar and, therefore, a polar molecule. Furthermore, cellulose molecules will attract water vapor molecules, making the stationary phase relatively polar. Therefore, most of the time we think of an effective mobile phase as being a nonpolar solvent, such as hexane. Retention factor, or Rf, is calculated as the distance traveled by a compound over the distance traveled by the solvent. In our example here, nonpolar compounds will travel further with the nonpolar liquid solvent, giving them greater Rf values. Polar compounds will be attracted to the paper and will be less likely to move far with the mobile phase; they will have lower Rf values.  

Thin Layer Chromatography


  • Stationary phase: glass with silica gel, aluminum, or cellulose
  • Mobile phase: liquid solvent
  • What is measured: Retention factor
  • Takeaway: Greater the Rf, the less polar the compound

The experimental setup of TLC can be thought of as similar to paper, but with a different stationary phase.  Here, a surface such as glass or plastic is coated with a polar substance, the most common being silica gel, aluminum oxide, and cellulose. Rf is measured the same way here, with the same trend as above.

Gas/Liquid Chromatography


  • Stationary phase: Polar liquid coating the column
  • Mobile phase: Inert gas
  • What is measured: Retention time
  • Takeaway: Greater the retention time, the MORE polar

Here, the stationary phase is actually a liquid that lines the column through which a sample is run. The mobile phase is an inert gas, such as hydrogen, nitrogen, or helium, which carries the sample without reacting with it. A detector at the end of the column will pick up when each compound passes, generating a series of retention times. A greater retention time means the compound spent more time being attracted to the polar liquid coating the column, rather than traveling straight through with the inert gas. Therefore, greater retention time corresponds with more polar compounds. 

Column Chromatography


  • Stationary phase: Solid silica gel or alumina
  • Mobile phase: liquid solvent
  • What is measured: Order of elution
  • Takeaway: First eluents are the least polar

In column chromatography, a polar solid (silica and aluminum are used here as in TLC) is packed into a column and the sample is loaded on top. Then the liquid phase is poured on top and allowed to run downward. At the bottom, a stopcock is opened to allow the liquid to run through. The solvent will carry the least polar components of the sample the fastest, as they will have the least attraction to the silica. We collect the sample in fractions, with the first fractions being solvent alone, the next being solvent carrying the nonpolar components, and finally the fractions containing the more polar compounds. 

A Final Note

This has been a brief rundown of the most important types of chromatography that separate compounds based on polarity. Size-exclusion and ion exchange chromatography are two different forms that should be explored individually, as they separate based on size and charge, respectively. The secret to answering chromatography questions lies in a keen understanding of relative polarities of compounds, so, as always, be sure to brush up on your organic chemistry! 

Cassandra attended UVA on a four-year, full cost of attendance scholarship with a double major in Human Biology and Spanish. After graduating in May 2020, she went straight into the NYU Grossman School of Medicine, with dreams of becoming an adult or pediatric oncologist.


academics study skills MCAT medical school admissions SAT college admissions expository writing English strategy MD/PhD admissions writing LSAT GMAT physics GRE chemistry biology math graduate admissions academic advice law school admissions ACT interview prep language learning test anxiety career advice premed MBA admissions personal statements homework help AP exams creative writing MD test prep study schedules computer science Common Application mathematics summer activities history philosophy secondary applications organic chemistry economics supplements research grammar 1L PSAT admissions coaching law psychology statistics & probability dental admissions legal studies ESL CARS PhD admissions SSAT covid-19 logic games reading comprehension calculus engineering USMLE mentorship Spanish parents Latin biochemistry case coaching verbal reasoning AMCAS DAT English literature STEM admissions advice excel medical school political science skills French Linguistics MBA coursework Tutoring Approaches academic integrity astrophysics chinese gap year genetics letters of recommendation mechanical engineering Anki DO Social Advocacy algebra art history artificial intelligence business careers cell biology classics data science dental school diversity statement geometry kinematics linear algebra mental health presentations quantitative reasoning study abroad tech industry technical interviews time management work and activities 2L DMD IB exams ISEE MD/PhD programs Sentence Correction adjusting to college algorithms amino acids analysis essay athletics business skills cold emails finance first generation student functions graphing information sessions international students internships logic networking poetry proofs resume revising science social sciences software engineering trigonometry units writer's block 3L AAMC Academic Interest EMT FlexMed Fourier Series Greek Health Professional Shortage Area Italian JD/MBA admissions Lagrange multipliers London MD vs PhD MMI Montessori National Health Service Corps Pythagorean Theorem Python Shakespeare Step 2 TMDSAS Taylor Series Truss Analysis Zoom acids and bases active learning architecture argumentative writing art art and design schools art portfolios bacteriology bibliographies biomedicine brain teaser campus visits cantonese capacitors capital markets central limit theorem centrifugal force chemical engineering chess chromatography class participation climate change clinical experience community service constitutional law consulting cover letters curriculum dementia demonstrated interest dimensional analysis distance learning econometrics electric engineering electricity and magnetism escape velocity evolution executive function fellowships freewriting genomics harmonics health policy history of medicine history of science hybrid vehicles hydrophobic effect ideal gas law immunology induction infinite institutional actions integrated reasoning intermolecular forces intern investing investment banking lab reports letter of continued interest linear maps mandarin chinese matrices mba medical physics meiosis microeconomics mitosis mnemonics music music theory nervous system