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Chromatography Testing

Liquid chromatography testing

Chromatography is the collective term for a family of analytical techniques where a mixture is separated into its individual components. The mixture, dissolved in a ‘mobile phase’, is passed through a stationary phase, where the individual components of the mixture are separated. Once separated, the components of interest may be further identified and quantitated.

Chromatography analytical techniques and expertise provided by Galbraith include:

Gas Chromatography (GC)

A Gas Chromatograph is a chemical analysis instrument used for separating volatile and semi-volatile compounds in a complex sample. The stationary phase in GC is contained in a narrow -bored column and the mobile phase is a gas. The volatile and semi-volatile components of a sample mixture are separated based on their varying partition behavior between the mobile phase and the stationary phase.

Samples are introduced into the GC by first dissolving the sample in a suitable solvent and then injecting the subsequent sample mixture via a syringe - or - by heating the sample in a closed container and transferring the resulting headspace to the GC. The headspace technique is often preferred because 1) it is less prone to interferences from the sample matrix, 2) it is a much “cleaner” technique in that the sample matrix itself is not injected into the GC, thereby reducing maintenance on the GC, and 3) larger sample masses can be used often resulting in lower detection limits.

GC’s are equipped with flame ionization detectors (FID), Mass Spectrometry (MS) detectors or thermal conductivity detectors (TCD). FID’s provide excellent sensitivity and linearity for most volatile and semi volatile compounds. Detection limits for most compounds using the FID are typically in the range of 1-5 micrograms per gram of sample (1-5 ppm).

GC-MS also provides excellent linearity for most volatile and semi-volatile compounds with the advantage of identifying unknown compounds that are present in the sample mixture. The identification of the unknown compound(s) is made by comparison of an unknown MS scan to the MS scans within the NIST Mass Spectral Library. This library contains the MS scans of over 200,000 different compounds.

Galbraith offers two general types of testing services with GC-MS: Tentatively Identified Compound (TIC) testing and quantitative analyses. TIC analysis provides the user information concerning the volatile and semi-volatile component of a sample mixture. Quantitative analyses involve a multi-point calibration of the instrument for a particular component of the sample. These types of analyses give the concentration of a particular analyte(s) of interest. In many cases, the GC-MS can provide a lower limit of quantitation than that obtained by GC-FID.

The TCD is also a versatile detector that can be used to quantify many volatile and semi-volatile compounds. However, Galbraith generally relies on this detector for measuring moisture content.

High Performance Liquid Chromatography (HPLC)

High-performance liquid chromatography is a form of column chromatography used frequently in analytical chemistry. HPLC is used to separate components of a mixture by using a variety of chemical interactions between the compounds within the substance being analyzed and the chromatography column.

In isocratic HPLC the analyte is forced through a column of the stationary phase by pumping a liquid (mobile phase) at high pressure through the column. The sample to be analyzed is introduced in a small volume to the stream of mobile phase and is retarded by specific chemical or physical interactions with the stationary phase as it traverses the length of the column. The amount of retardation depends on the nature of the analyte, stationary phase and mobile phase composition. The time at which a specific analyte elutes (comes out of the end of the column) is called the retention time and is considered a reasonably unique identifying characteristic of a given analyte. The use of pressure increases the linear velocity (speed) giving the components less time to diffuse within the column, leading to improved resolution in the resulting chromatogram.

A further refinement to HPLC has been to vary the mobile phase composition during the analysis; this is known as gradient elution. The gradient separates the analyte mixtures as a function of the affinity of the analyte for the current mobile phase composition relative to the stationary phase. The choice of solvents, additives and gradient depend on the nature of the stationary phase and the analyte. Often a series of tests are performed on the analyte and a number of generic runs may be processed in order to find the optimum operating conditions for a specific analyte.

[Courtesy of http://en.wikipedia.org/wiki/High_performance_liquid_chromatography]

UV/VIS & Refractive Index Detector
Ultraviolet-visible spectroscopy (UV/VIS) uses light in the visible and adjacent near ultraviolet (UV) and near infrared (NIR) ranges. In this region of energy, molecules undergo electronic transitions. The Beer-Lambert law states that the absorbance of a solution is directly proportional to the solution's concentration. Thus detectors that use UV/VIS spectroscopy can be used to determine the concentration of a solution.

Another common HPLC detector is the Refractive Index Detector (RI). RI detectors are commonly used when a substance will not adsorb in the UV range. Many carbohydrates are examples of these substances. RI detectors take advantage of the difference between the refractive index of the compounds of interest and that of the mobile phase.

[Courtesy of http://en.wikipedia.org/wiki/UV/VIS_spectroscopy]

Ion Chromatography (IC)

Ion-exchange chromatography is a process that allows the separation of ions and polar molecules based on the charge properties of the molecules.

Ion exchange chromatography retains analyte molecules based on ionic interactions. The stationary phase surface displays ionic functional groups that interact with analyte ions of opposite charge. This type of chromatography is further subdivided into cation exchange chromatography and anion exchange chromatography:

A sample is introduced into a sample loop of known volume. A buffered aqueous solution (mobile phase) carries the sample from the loop onto a column that contains some form of stationary phase material. This is typically a resin or gel matrix consisting of agarose or cellulose beads with covalently bonded charged functional groups. The target analytes (anions or cations) are retained on the stationary phase but can be eluted by increasing the concentration of a similarly charged species that will displace the analyte ions from the stationary phase. The analytes of interest must then be detected by some means, typically by conductivity or UV/Visible light absorbance.

[Courtesy of http://en.wikipedia.org/wiki/Ion_exchange_chromatography]

Thin Layer Chromatography (TLC)

Thin layer chromatography (TLC) is a widely-used chromatography technique used to separate chemical compounds. It involves a stationary phase consisting of a thin layer of adsorbent material, usually silica gel, aluminum oxide, or cellulose immobilized onto a flat, inert carrier sheet. A liquid phase consisting of the solution to be separated dissolved in an appropriate solvent is drawn through the plate via capillary action, separating the experimental solution, whereas TLC can be configured for quantitative work, at Galbraith it is primarily used in identification tests and limit tests.