From X-rays to electron beams there are many analytical techniques to distinguish the elements

of the periodic table. For options to distinguish materials that have almost every element in

common, we need chromatography. Liquid and gas chromatography are widely applicable for

characterizing soft materials, pharmaceuticals, petrochemicals, adhesives and specialty

chemicals.

This article will focus on liquid chromatography since our previous article already described gas

chromatography. From a high-level perspective, chromatography is the science of separating

the components of a system such that positive identification can occur for the target

compounds. The separation takes many forms and is controlled by the columns attached to the

instrument. Equally diverse are the number of detectors that exist for positive identification.

Since chromatography column choice is a highly specialized skill set, this article will focus on

usage and applications.

Liquid chromatography is about selective filtration and creating gradients to separate soluble

compounds. If a compound is not soluble or is highly combustible, then gas chromatography

might be more appropriate. The compound of interest or target molecule will begin its journey in

some type of solvent which is known as the mobile phase. When the mobile phase is non-polar

(hexanes, octanes etc.) the columns (stationary phase) will need to be polar in order to cause

separation. This configuration with non-polar mobile phase is normal phase chromatography.

When the mobile phase is polar (acetonitrile, water etc.) and the stationary phase is nonpolar,

this is known as reverse phase chromatography.

The next steps are for highly experienced chromatographers to select the proper columns for

the instrument, set up solvent gradients to maximize the effectiveness of the separation and run

baseline method development to be able to accurately quantify the amount of target compound

in the sample. There is a true artistry in being able to develop methods to separate compounds

effectively. It takes decades of experience and storage closets full of columns and solvents.

Once the separation is completed and the target compound is isolated, the appropriate type of

detector needs to be used for positive identification. Detectors can utilize UV light, visible light,

photo diode arrays, refractive index, evaporative light scattering, multi angle light scattering,

mass spectrometers, conductivity, fluorescence, chemiluminescence, optical rotation and

electrochemistry. The selection of detector depends on the chemistry of the target molecule.

LC, HPLC or UPLC

When considering liquid chromatography there are a few categories of instruments available that

are distinguished by their operating pressures. Ultra-High Performance Liquid Chromatography

(UPLC) operates at 15,000 PSI while High Performance Liquid Chromatography (HPLC) HPLC

operates under 6,000 PSI. The oldest systems operate under 500 PSI but those are quite rare

nowadays. From a functionality point of view, any chromatography system can detect a

molecule once it is separated. The reason that higher pressures are used is to speed up the

separation times. A UPLC can separate a compound 2 to 3 times faster than an HPLC. If you are

in the market for method development, developing a UPLC method maximizes the cost savings

over the long term with faster analysis times.

Strengths and Limitations

Liquid chromatography is an excellent tool for separating closely related compounds. The

limitation is that for each target compound a method needs to be tailored so that quantification

is accurate, and separation occurs. This means that there is no such thing as a quick scan with

HPLC, there will always be some method development. As chromatographers know, equivalent

columns from different manufacturers are not identical. How the column is packed impacts its

effectiveness. This means that the chromatographer needs to stock columns from multiple

manufacturers to be able to support a wide variety of analytical work.

Liquid chromatography applications are centered on the detection of organic, biological and

pharmaceutical products. The analysis can be used to monitor the stability of drug products

over time and temperature. Alternatively, analysis can be done on sample to detect foreign

contaminates, such as measuring water for types of organic contamination. Chromatography is

often used with food and nutraceuticals as a means of monitoring product integrity. The final

usage is for isolation of materials where the chromatography is used to separate materials after

chemical reactions.

Discovery of

Liquid Chromatography

Mikhail Tsvet

(Italy-1872) was a botanist

at the University of

Warsaw. He developed a

technique to study the

separation of plant

pigments that he named

chromatography from the

Greek khrōma "color" and

graphia "description of".

First Commercial HPLC

Archer J.P. Martin (England-1910) and Richard

L.M. Synge (England-1914) developed partition

chromatography during their research at the Wool

Industries Research Association in Leeds. It was

introduced to characterize monoamino-acids in

proteins with two liquid phase chromatogram. They

received the Nobel Prize in 1952 for their work.

Introduction of

High Pressure LC

After two years of research at

Yale University, Csaba Horváth

(Hungary-1930) introduced, at

the Six International

Symposium on Gas

Chromatography in Rome, a

new liquid chromatography

system using high pressures

and reducing separation time

that he will later name HPLC.

Partition Chromatography

Waters Corporation, created by James Waters

in Framingham, MA in 1958, specialized itself

in the development of liquid chromatographs.

Following some prototyping work, the

company introduced the first successful

commercial HPLC in 1967, the ALC-100.

1941

1966

1967

1903

2004

HOW DID WE GET HERE?

By the beginning of the 21st

century, scientists were reaching

the limits of the HPLC systems.

To break the separation barrier,

Waters introduced Acquity UPLC

which, by using even higher

pressure and sub 2µm particles

in the column, was able to

increase both measurement

speed and peak capacity.

Enhanced Performance

with UPLC

High Performance Liquid Chromotography

HOW DOES IT WORK?

Technical Background

Figure 1. Ensemble of equipment that can make up a single liquid chromatography system

Figure 2. Modes of operation for liquid chromatography

Figure 3. UPLC System the leading edge of technology in chromatography

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