Why Chromatography is an Important Technique in the Lab

Chromatography analysis is used to determine the presence and concentration of analytes in a sample.

Chromatography refers to a set of laboratory methods and techniques for the separation of mixtures. It involves passing a mixture that dissolved in a mobile phase through a medium known as the stationary phase. This separates the analyte to be measured from other components of the mixture and allows it to be isolated.

This technique may be preparatory or analytical in nature. Preparatory chromatography is performed to separate the components of a mixture for further analysis as well as for cleansing and purification applications. Analytical chromatography is usually done with smaller amounts of material and is used to measure the relative proportions of analytes in a mixture.

In chromatography analysis, chemical substances are introduced into a vertical glass tube containing an adsorbent. The various components of the substance move through the adsorbent material at different rates of speed according to their degree of attraction to it. This produces bands of color at different levels of the adsorption column.

Analysis techniques by physical state of the mobile phase fall into several categories. Gas chromatography (sometimes called gas-liquid chromatography) is a separation technique in which the mobile phase is a gas. Gas chromatography is always performed in a column, typically packed or capillary. Liquid chromatography is a separation methodology in which the mobile phase is a liquid and can be performed either in a column or a plane. Present day liquid chromatography analysis generally utilizes very small packing particles and a relatively high pressure; a method referred to as high performance liquid chromatography or HPLC.

Affinity chromatography is based on selective non-covalent interaction between an analyte and particular molecules. It is frequently used in biochemistry in the purification of proteins bound to tags.

Other techniques use a variety of separation mechanisms. Ion exchange chromatography employs the ion exchange mechanism to separate analytes. It is usually performed in columns but can also be useful in planar mode. Ion exchange chromatography uses a charged stationary phase to separate charged compounds including amino acids, peptides, and proteins.

Size exclusion chromatography analysis (also known as gel permeation chromatography or gel filtration chromatography) separates molecules according to their size (or more accurately according to hydrodynamic diameter or volume). Smaller molecules are able to enter the pores of the media and take longer to elute, while larger molecules are excluded from the pores and elute more rapidly.

Special methodologies are sometimes needed. Reversed-phase chromatography is an elution procedure used in liquid chromatography analysis, using a mobile phase which is significantly more polar than the stationary phase.

If the chemistry within a given column is insufficient to separate some analytes, two-dimensional chromatography can be used, making it possible to direct a series of unresolved peaks onto a second column with different properties. This method allows for the separation of compounds which are indistinguishable from one another when using one-dimensional chromatography methods.

Additional specialized analysis techniques include simulated moving-bed chromatography, pyrolysis gas chromatography, fast protein liquid, countercurrent and chiral.

How Advances in High Performance Countercurrent Chromatography Benefit the Chemists

Ever since chemists began using separating funnels to isolate compounds by partitioning, they have understood the potential benefits of liquid/liquid chromatography, known today as countercurrent chromatography (CCC). Yet despite this knowledge, solid/liquid chromatography techniques, such as HPLC or flash, have become the workhorses of purification. Until recently, CCC was primarily a technique for natural products or academic research and was hardly used in mainstream purification. Unfortunately, early CCC instrumentation was poorly engineered and suffered from slow speed of separation, a combination that led to negligible adoption as a complementary and orthogonal chromatography technique.

However a new generation of high-performance countercurrent chromatography (HPCCC) instrumentation has led to the rebirth of liquid/liquid chromatography in the 21st century and therefore offering a greater benefit to the chemists.

CCC can significantly improve a chemists productivity and separate compounds that were previously very difficult to isolate or uneconomical to produce. Due to the large difference in accessible stationary phase between liquid/liquid to solid/liquid chromatography – typically 70-80% compared to 5-10% – the loadings are dramatically higher, shortening the number of sample injections needed to process a batch. Furthermore, because both mobile and stationary phases are liquids, we gain two further important productivity benefits.

First, sample solubility issues are reduced because one’s options for injecting sample onto the column have been tripled. Using CCC, one can inject a sample into either of the individual mobile or stationary phases or a mixture of the two, whichever combination provides the highest loading per injection. The use of two liquids is also beneficial once the sample is on the column, because even if the sample crashes out of solution, it does not cause the column to block, stopping the chromatography.

Another productivity benefit is that with CCC there is no possibility of irreversible adsorption occurring either onto or into the stationary phase. Recoveries are always very high, and it is certain that the entire sample will elute from the column.

With all of the advantages that CCC can bring to the productivity of chemists, why has it been so poorly adopted?

The first generation of CCC instruments introduced in the early 1980s were known as high-speed countercurrent chromatography (HSCCC) machines and were poorly adopted for three reasons. The first was speed of separation – HSCCC instruments perform separations over a period of hours, rather than the tens of minutes typical HPLC. Secondly, the instrumentation was unreliable and therefore scientists quickly became hesitant to risk their valuable compounds. Finally, the range of equipment available was poor and typically only available at the preparative scale, requiring gram-size sample injections. This is a problem for chemists working in small-molecule synthetic chemistry, who initially may have had samples available only in hundreds of milligram amounts that took months to produce. Therefore, the entire quantity of a valuable sample would have to be injected – a risk a chemist is reluctant to take.

The combination of these factors ensured that CCC was only used as a technique of last choice, rather than adopted as a complementary and orthogonal liquid chromatography technique that could dramatically impact the productivity of chemists – a purification process that takes hours to perform is unacceptable.
Work commenced in the mid 1990s to improve the operating performance. The development of high-performance counter-current chromatography (HPCCC) overcame problems of the heat generated in the instrument’s bearing, no longer the need to bolt machines to the bench, rewind columns after a few runs, repair flying leads during a separation and no longer the need for working in the back room due to the noise, which was greatly reduced. They were also able to break through the 240g barrier which made it possible to develop robust analytical scale instruments, using small-bore columns, so that milligram quantities of compound could be processed.

The benefits of HPCCC to chemists not only improves the productivity at all scales it is also a technique that can be applied across the whole range of polarity and to both small and large synthetic molecules, peptides, and natural products. Because HPCCC is a high-capacity technique, it is becoming the first choice for scientists when they need to produce large quantities of target compounds.

This is especially attractive when a compound and its analogues are identified as a lead candidate required in ever-increasing quantities as they progress through the pharmaceutical development process. Using HPCCC instruments chemists are able to concentrate on their product development process, not purification/chromatography redevelopment, as scale increases.

Performing scale up of purification between different sizes of HPCCC instruments is quick and simple. One simply uses the volumetric ratio between the two column volumes one wishes to use to determine the new sample volume and mobile phase follow rate.

A Further significant benefit concerns sample solubility. Rather than the solubility of samples becoming a limiting factor, they tend toward irrelevance because the sample can be injected onto the column in either the mobile, stationary, or a mixture of both phases, without affecting the performance of the chromatography.

Chemists and scientists are now able to us HPCCC in their laboratories and use high capacity separation instruments on the benchtop. It is possible with pumps that work at 50mL/min to easily process up to 200g of crude material per day and potentially up to 400g. This is a significant advance in reducing the chromatography bottleneck caused by the throughput constraints of liquid/solid chromatography techniques or the solubility of samples. HPCCC can help solve these problems.

Different Kinds of Chromatography

Chromatography is a technique used to isolate the various components of a mixture and this makes its application in analysis of biomolecules very important. It is used to separate and analyse the complex DNA sequences and other compounds, and also the concentration of the samples. There are many types of chromatography used in the study of biomolecules which range from DNA/RNA to recombinant proteins and antibodies. Here are some types of chromatography that you should know about.

High Performance Liquid Chromatography

Small particles and High pressure is required to carry out this type of liquid chromatography. HPLC has many forms and its application revolves around drug analysis and other forensic applications. There are forms of HPLC which specifically deal with enzymology and purification of other biomolecules.

The reversed phase chromatography has a larger application in industry. In this the stationary phase is non-polar, while the solvent or mobile phase used is polar which is opposite to normal chromatography where stationary phase is polar and the mobile phase is non-polar. The advantage of reverse phase liquid chromatography is that it allows the separation of a large variety of samples, with a wide range of molecular weights and polarities involved. It is easy to use and results are attained rapidly.

Fast Protein Liquid Chromatography

FPLC is also a form of liquid chromatography and it specializes in separating proteins from complexes, as the name suggests. FPLC is popularly used in enzymology, with a complete setup designed especially for separation of proteins and other biomolecules. Cross linked agarose beads are used.

Aqueous- Normal Phase Chromatography

This type of chromatography has a special feature, it has a mobile phase which is somewhere between polar and non-polar. The mobile phase is based on an organic solvent and a small amount of water which results in it being semi polar.

Affinity Chromatography

This type is again used in the purification of proteins which are bound to tags. The proteins being analysed are marked or labelled with compounds like antigens or biotins. To get pure proteins in the end, the labels are removed; the labels are just there to provide accurate separation of proteins. The mechanism uses a property of biomolecules i.e. affinity for metals, hence various metals are used in the chromatography columns. Immobilized Metal Affinity Chromatography is an advanced and much refined version of affinity chromatography used in identification of biomolecules these days.

How is Ion Chromatography Used in the Laboratory

Ion chromatography (or ion-exchange chromatography) is a process that permits the separation of ions and polar molecules on the basis of the charge properties of the molecules.

It can be used for many types of charged molecules including large proteins, small nucleotides and amino acids. The solution to be injected is normally called a sample and the individually separated components are identified as analytes. It is frequently used in protein purification, water analysis and for quality control purposes.

Ion exchange chromatography retains analyte molecules using coulombic (ionic) interactions. The stationary phase surface displays ionic functional groups that interact with analyte ions of opposite charge. This category of chromatography can be further subdivided into cation exchange chromatography and anion exchange chromatography. The ionic compound consisting of the cationic species and the anionic species may be retained by the stationary phase.

Cation exchange ion chromatography retains positively charged cations since the stationary phase exhibits a negatively charged functional category. Anion exchange chromatography retains anions displaying a positively charged functional category. Note that the ion strength of either cations or anions in the mobile phase may be adjusted to shift the equilibrium position and, therefore, the retention time. An ion chromatogram can be used to show the chromatogram obtained with an ion exchange column.

A typical ion chromatography technique involves the introduction of a sample either manually or using an autosampler, into a sample loop of known volume. A buffered aqueous solution known as the mobile phase carries the sample from the loop into a column which contains some type of stationary phase material. This is normally a resin or gel matrix that consists of agarose or cellulose beads with covalently bonded charged functional groups. The target analytes (anions or cations) are retained on the stationary phase but may be eluted by increasing the concentration of a similarly charged species. This will displace the analyte ions from the stationary phase.

For example, in cation exchange chromatography, the positively charged analyte could be displaced by the introduction of positively charged sodium ions. The analytes of interest must then be detected by some method, which is normally by either conductivity or UV/Visible light absorbance.

In order to control an ion chromatography system, a chromatography data system is usually needed. Some of these chromatography data systems can also be used to control gas chromatography and HPLC systems.

Proteins have many functional groups that can have both positive and negative charges.

Ion chromatography separates proteins according to their net charge. This is dependent on the composition of the mobile phase. By adjusting the pH or the ionic concentration of the mobile phase, various protein molecules can be separated. For example, if a protein has a net positive charge at pH 7, then it will bind to a column of negatively-charged beads, but a negatively charged protein will not. Changing the pH so that the net charge on the protein is negative will cause it to also be eluted.

Accomplishing elution by changing the ionic strength of the mobile phase is a more subtle effect. It works because ions from the mobile phase will interact with the immobilized ions in preference to those on the stationary phase. This shields the stationary phase from the protein and vice versa. This allows the protein to elute. A preparative-scale ion exchange column is used for protein purification.

Ion chromatography is a powerful technique for ascertaining low concentrations of ions and is particularly useful in environmental and water quality studies.

What is Chromatography?

Loosely put, the term “chromatography” deals with the separation of substances within a mixture. Chromatography was invented by a Russian botanist who was studying materials in plant life by separating leaf pigments. By using chromatography, scientists are able to analyze a compound and figure out what elements substances constitute the makeup of that compound.

There are 4 major types of chromatography in use today:

1. Gas Chromatography is used in places like airports to detect bombs and also in crime scene investigation. Helium is used to separate elements from a compound by moving a gaseous mixture through absorbent material.

2. Liquid Chromatography is for testing water samples across the world. It analyzes metals and organic compounds in solutions to determine makeup.

3. Paper Chromatography is used for RNA fingerprinting, separating and testing histamines and antibiotics. This is the most common form of chromatography and uses a strip of paper to pull up the substances into the paper and separate them out from each other.

4. Thin Layer Chromatography is used in forensics and looks at the dye composition in fibers. It is also used to detect pesticides or insecticides in food.

Chromatography allows for separation of an element into the substances that makeup that element. In some cases, the compound looks to be made up of one substance to the naked eye. Utilizing the aspects of chromatography allows a scientist to decipher exactly what substances makeup any specific compound. Chromatography eliminates masking agents and even allows for the analysis of colorless and odorless substances.

The time saved by using chromatography to assess and analyze compounds in an environment benefits the user by allowing them time to delve deeper into other sections of an investigation. In the case of forensics, chromatography allows the investigator to better manage a scene by better managing the evidence gathered. Each of the above chromatography types has its own merits. The most widely used is paper chromatography. There are multiple uses for each of the chromatography types listed.

The Fundamentals of Liquid Chromatography

Chromatography is the collective term for a set of techniques used to separate mixtures. These techniques include gas chromatography (GC), thin layer chromatography (TLC), Size exclusion Chromatography (SEC), and high performance liquid chromatography (HPLC).

The Two Phases

Chromatography involves passing a mixture dissolved in a “mobile phase” through a “stationary phase”. The mobile phase is usually a liquid or a gas which transports the mixture to be separated through a column or flat sheet which has a solid stationary phase.

Liquid Chromatography

Liquid chromatography (LC) is a separation technique in which the mobile phase is a liquid. It can be carried out in either a column or a plane. LC is particularly useful for the separation of ions or molecules that are dissolved in a solvent.

Simple liquid chromatography consists of a column with a fritted bottom that holds a stationary phase in equilibrium with a solvent. Commonly used stationary phases include solids, ionic groups on a resin, liquids on an inert solid support and porous inert particles. The mixture to be separated is loaded onto the top of the column followed by more solvent. The different components in the mixture pass through the column at different rates because of the variations in the partitioning behaviour between the mobile liquid and stationary phases.

Liquid chromatography is more widely used than other methods such as gas chromatography because the samples analysed do not need to be vaporised. Also, the variations in temperature have a negligible effect in liquid chromatography, unlike in other types of chromatography.

High Performance Liquid Chromatography (HPLC)

Present day liquid chromatography that generally utilises tiny packing particles and a fairly high pressure is known as HPLC. It is basically a highly improved form of column chromatography often used by biochemists to separate amino acids and proteins due to their different behaviour in solvents related to the amount of electronic charge of each one.

Instead being allowed to drip through a column under gravity, the solvent is forced through under high pressures of up to 400 atmospheres, making the process much faster. Because smaller particles are used, with their sizes being determined by a particle size analyser, there is greater surface area for interactions between the stationary phase and the molecules flowing past it. This in turn allows for much better separation of the components in the mixture.

There are many advantages of HPLC. For one, it is an automated process that only takes a few minutes to produce results. This is a vast step up from liquid chromatography, which uses gravity instead of a high-speed pump to force components through the densely packed tubing. HPLC produces results that are of a high resolution and are easy to read. Moreover, the tests are easily reproduced via the automated process.

Unfortunately, there are also disadvantages of this technique. It is difficult to detect coelution with HPLC and this may result in inaccurate compound categorisation. The equipment needed to conduct HPLC is also costlier and its operation can be complex.

Thanks to rapid advances in technology, analytical instrumentation such as HPLC are increasing in popularity. For the most part, the efficiency of these techniques outweighs their disadvantages making them a popular choice particularly in the pharmaceutical and medicinal industries.