Joshua Martinez

English

363, Bruce

Chromatography

Chromatography is a laboratory technique in which a mixture is separated into smaller

components when passed through a chemically inactive material using an apparatus. This laboratory

technique measures the retention times for analytes, a chemical substance that is being identified, to

pass through a column of absorbents. There are several types of chromatography techniques that use

different phases (solid, liquid or gas). The three major methods of chromatography are Column

Chromatography, Gas Chromatography and Thin-Layer Chromatography. This laboratory separation

method consists of mainly liquid substances, but gases and solids can be used depending on the

technique, which can be referred to as the mobile phases when mixed with a solvent. A solvent is a

liquid which has various components dissolved in it, forming a solution (Britannica, 2011). Essentially all

of the separation techniques utilize a mobile phase, the moving liquid or gas substance of the column,

that passes through a stationary phase, the nonmoving liquid or gas substance of the column, that sits in

the column of the chromatograph. The stationary phase selectively absorbs material in the mobile phase

as it passes through.

History

In the mid 1800’s, a professor and his student by the names of Christian Friedrich Schonbein and

Friedrich Goppelsroeder were the first to publish attempts to study the rate that different substances

travel through a filter paper ("History of chromatography", 2019). Instead of today’s methods which
measure different absorption levels, or the amount of light that is absorbed by the substance, as being

responsible for the movement, they thought capillary action was the reason for the movement of

substances. Capillary action is the movement of liquids through narrow spaces without any external

force assisting it (Saig, 2018).

The technique of column chromatography separation was first used in Russia by Mikhail Tsvet in

the early 20th century. He primarily used this technique to separate different plant pigments, which gave

the technique its name. The pigments that Tsvet was separating were chlorophyll (green pigments),

carotenes (orange and yellow pigments), and xanthophylls (yellow pigments) ("Chromatography", 2019).

From his research with separation of colors, the term chromatography was coined, Chromato- meaning

“color” and -graphy meaning the production of images (Online Etymology Dictionary, "chromatography

(n.)").

Later on, in the 20th century new types of this technique were developed. Two scientists by the

name Archer John Port Martin and Richard Laurence Millington Synge further developed

chromatography by establishing principles of partition chromatography. Partition Chromatography is a

type of liquid Chromatography which separates the components based on solubilities, ability to be

dissolved (International Union, 2006). These two scientists went on to win the Nobel prize in Chemistry

in 1952. From these new principles several other chromatographic techniques were developed

("Chromatography", 2019).

Column Chromatography

Column Chromatography, one of the most common techniques, separates specific compounds

depending on the type of stationary phase is used to pack the column. The column can separate the

eluent, or the mobile phase consisting of a solvent that dissolves the sample, based on the molecule’s
hydrophobicity (affinity for water substances), polarity (the “pull” atoms have on their electrons), or

electrostatic charge (either a positive or negative charge of the atom) (Torres, 2016). Different sized

columns can be used when packing the stationary phase depending on the amount of substance you are

attempting to separate. After packing the column, the eluent is placed on top of both the sample and

stationary phase, or the substance in the column that is not moving. The bottom end nozzle is then

opened to allow the sample to flow through the column. Depending on molecular properties, the

analyte which has a weaker interaction with the stationary phase will pass through it more rapidly while

the analytes with a stronger interaction will take a longer time to pass (Torres, 2016). The time that it

took the desired analyte to pass will then aid the researcher in determining what kind of molecules were

compromised of the analyte.

The above diagram is a depiction of column chromatography. The first column on the left is the
beginning of this technique with only the stationary and sample loaded. The mobile phase is then
placed on top on the sample forming the eluent and then as time passes the different analytes pass
through separately depending on their interactions. The first analyte is then collected as it passes
through, then the second is collected as it passes through (Torres, 2016).
Gas Chromatography (GC)

To the left is a diagram of a basic gas

chromatograph. Starting from the top,
we have the sample gas which is carried
by an inert gas, or unreactive gas, which
forms the mobile phase. The mobile
phase is then carried passed the
stationary phase in the column. The
detector then determines the retention
times of the contents that make it
through which will help the researcher
determine what the substance is ("Gas
chromatography", 2019).

Gas Chromatography, GC, is a method of separation used in order to separate volatile

compounds. Volatile compounds are organic compounds that, at standard room temperature of about

23C, have an elevated vapor pressure ("Volatile organic compound", 2019). In this technique, the

sample is mixed with a solvent and then vaporized and passed through the stationary phase in order to

separate. The mobile phase of this technique is compromised of an inert gas, or noble gas. Unlike the

other techniques of separation, the inert gas in the mobile phase does not interact with the analyte but instead

helps carry the molecules of the sample (Libretexts, 2019).

Inlets, also known as injectors, are the entrance attached to the column head for the sample to

be placed into the chromatograph. These inlets introduce the sample into a small heated chamber from

a syringe, the heat breaks down the sample and turns the liquid into a gas which is then carried to the

column by either the entirety, “split-less” injection, or in portions, “split” injection (Kumar, 2015).

Another type of inlet is the “on-column” inlet where the sample is introduced in its entirety without
heat. Programmed Temperature Vaporizing Injector introduces the sample just below its boiling point

through the split line. (Kumar, 2015)

Columns of GC are a long tubing where the stationary phase is held at can come in two different

types: packed columns or capillary columns, which is also known as being open tubular. Capillary

columns come in two different forms usually made of glass (Libretexts, 2019). One form of capillary

columns contains finely divided, inert, solid support material that is coated on the wall of the column

with the stationary phase, also known as wall-coated open tubular (Libretexts, 2019). The second form is

also known as a support-coated open tubular column. In these the columns are first coated with

extremely small layer of adsorbent solid that is treated with the stationary phase. Of these two capillary

columns the, the support-coated can hold a greater volume of stationary phase while the wall-

coated is more efficient in separation (Libretexts, 2019). Packed columns are usually made of

glass or metal with a larger diameter but do have a shorter length than capillary columns. Due

to the greater diameter and shorter length, the packed columns are way less efficient than wall-

coated open tubular columns (Libretexts, 2019).

Detectors are used at the end of the chromatogram to give a quantitative (being able to

measure) measurement of the analytes as they pass through with the inert gas (Libretexts,

2019). Regardless of the type of detector, there are two key parts that work together to

transduce (converts a physical amount into an electrical signal) (Libretexts, 2019). The first part is

a sensor at the end of the column and the second is an “electrical equipment used to digitize

the analog signal” for a computer to analyze the collected chromatogram (Libretexts, 2019).

Some types of detectors are Mass Spectrometer, Flame Ionization, Thermal Conductivity,

Electron-Capture, Atomic Emission, and Chemiluminescence detectors (Libretexts, 2019).

References

1. Britannica, T. E. (2011, February 07). Solvent. Retrieved from

https://www.britannica.com/science/solvent-chemistry

2. Capillary action. (n.d.). Retrieved from https://www.dictionary.com/browse/capillary--

action

3. Chromatography. (2019, February 15). Retrieved from

https://en.wikipedia.org/wiki/Chromatography

4. Chromatography (n.). (n.d.). Retrieved May 8, 2019, from

https://www.etymonline.com/word/chromatography

5. History of chromatography. (2019, February 23). Retrieved from

https://en.wikipedia.org/wiki/History_of_chromatography

6. International Union. (2006). Partition chromatography. Retrieved from

https://goldbook.iupac.org/html/P/P04436.html

7. Kumar, P. (2015, October 16). Top 12 Types of Chromatographic Techniques |

Biochemistry. Retrieved from

http://www.biologydiscussion.com/biochemistry/chromatography-techniques/top-12-

types-of-chromatographic-techniques-biochemistry/12730

8. Libretexts. (2019, February 23). Gas Chromatography. Retrieved from

https://chem.libretexts.org/Bookshelves/Analytical_Chemistry/Supplemental_Modules

_(Analytical_Chemistry)/Instrumental_Analysis/Chromatography/Gas_Chromatography

9. Saig, A. (2018, December 10). Retrieved from

https://davidson.weizmann.ac.il/en/online/askexpert/chemistry/what-capillary-action-

and-how-it-affected-gravity-ariel-michal
10. Thin-layer chromatography. (2019, February 08). Retrieved from

https://en.wikipedia.org/wiki/Thin-layer_chromatography

11. Thin Layer Chromatography. (n.d.). Retrieved May 8, 2019, from

http://faculty.sdmiramar.edu/fgarces/labmatters/chemtech/modules/tlc/tlcinterp.htm

12. Torres, J. (2016, August 07). The Basics of Running a Chromatography Column. Retrieved

from https://bitesizebio.com/29947/basics-chromatography-column/

13. Gas chromatography. (2019, April 27). Retrieved from

https://en.wikipedia.org/wiki/Gas_chromatography

14. Volatile organic compound. (2019, February 05). Retrieved from

https://en.wikipedia.org/wiki/Volatile_organic_compound