Chromatography

About

Often in the course of research initiatives, there often comes a time that precipitates the requirement for complete qualitative (who, molecular structure) and quantitative (how much) analyses of the components contained within a complex sample matrix. Such situations are common to research programs in chemistry, biochemistry, biology, medicine, toxicology, oceanography, food and nutrition, environmental chemistry, biology, pharmaceutical development, etc. These qualitative and quantitative results on individual sample components are most often completely necessary and essential before information can be assimilated, conclusions can be drawn, hypotheses developed, and appropriate next steps developed. There are a notable number of well-documented approaches by which complex mixtures can be separated into their component parts, however, the most popular and proven technologies are most often linked to chromatographic separations.

Chromatography is an analytical technique in which two or more compounds in a mixture are physically separated by differentially distributing themselves between two phases: a stationary phase which can be a solid or liquid supported on a solid and a mobile phase, either a gas or a liquid which flows continuously around and through the stationary phase.  The separation of the individual components results from the relative difference in component affinity for the stationary phase.  In liquid chromatography (LC), the flowing or mobile phase is a liquid, in gas chromatography (GC) is a gas, whereas in supercritical fluid chromatography (SFC) is a supercritical or subcritical fluid.

It is well that these chromatographic technologies have the capability to separate the components of complex mixtures. However, once separated, the component structures must then be determined (qualitative) as well as their amounts quantitatively described. These processes are effectively executed through the judicial application of an array of detectors as well as information rich detectors that possess the capability of both detecting sample components as well as determining their structure and providing for quantitative determinations.  Detection of the separated components in GC, LC and SFC can be made by various means, one of the most sensitive being a mass spectrometer. The mass spectrometer can detect and record the relative masses and abundances of ions that are produced from compounds that have been separated using chromatography giving structural information, resulting in both qualitative and quantitative capabilities. Other common detectors are ultraviolet (UV) that can detect and quantitate components having a chromophore, and is commonly used with LC and SFC, while flame ionization detector (FID) which is commonly used with GC analysis can provide detection and quantitative capabilities.  Also, there are other detectors that are more selective (fluorescence for molecules having this physical property, nitrogen-phosphors detector(NPD), for molecules containing nitrogen and phosphorous, electron capture detector (ECD), for molecules having functional groups like nitrates, halogens, NH2,  etc.) and are employed with selected chromatographic techniques for the detection and quantitation of compounds having somewhat unique functionalities.

Speaking in generalities, most often GC is employed when sample analytes possess thermal stability and can be volatilized without decomposition at elevated temperatures, such as ~250°C. Most often LC is applied to samples with documented water solubility and thermal instability. SFC finds applications in both categories.

Instrumentation for analysis of small and relatively large molecules

Gas Chromatography

1. Agilent 7890 GC equipped with 5975 mass selective detector (MSD). The MSD uses Electron Impact (70 ev) for all ionization and can be operated from 10-800 amu mass range in both scan (most popular) and selected ion monitoring SIM (a bit more complex) modes.   
2. Agilent 6850 GC equipped with FID, NPD, and electron capture (ECD) detectors for analysis of compounds having selected functional groups.   FID is more a universal carbon-based detector while both NPD and ECD are selective detectors for the analysis of nitrogen, phosphorus, and halogenated or nitrated compounds, respectively.  

High Performance Liquid Chromatopgrahy (HPLC)

1. Agilent 1200 HPLC equipped with diode array (DAD) and refractive index detectors in series – HPLC/DAD/RI
2. Agilent 1200 HPLC equipped with Agilent Fluorescence detector (FLD)
3. Agilent 1100 HPLC equipped with evaporating light scattering detector (ELSD)
4. Agilent 1260 Preparative HPLC with DAD and fraction collector
5. Waters Acquity UPLC H-Class equipped with Waters TUV detector
6. Waters Acquity UPLC H-Class equipped with Waters Synapt Q-TOF (Time of Flight) mass spectrometer (Both electro-spray ionization, ESI, and atmospheric pressure chemical ionization, APCI, capabilities).

Supercritical Fluid Chromatography

1. Waters SFC equipped with Waters diode array detector (DAD) – SFC/DAD
2. Waters SFC equipped with Waters Synapt Q-TOF mass spectrometer
3. Waters SFC equipped with evaporating light scattering detector (ELSD)

Recent Chromatography Center Analytical Applications

1. Analysis of canabinols from supercritical fluid extraction (SFE) of natural products
2. Support for the production scale generation of flavor components
3. Quantitative analysis of perfluoromethylcyclohexane (PMCH) in gas samples using GC/MS
4. Characterization of the glucosides contained within selected flowering species using LC/UV and GC/MS
5. Characterization of the triglycerides present in seed oils
6. Quantitative analysis of pentane, hexane, methyl hexane and heptane in mine samples using SPME/GC/FID
7. Quantitative analysis of pioglitazone in their metabolites using LC/MS/MS
8. Determination of the active flavor ingredients in natural product extracts
9. Isolation of Free Amino Acids via Enzymatic Hydrolysis of Tobacco-Derived F1 Protein
10. Supercritical Fluid Chromatography with Evaporative Light Scattering Detection (SFC-ELSD) for Determination of Oligomer Molecular Weight Distributions
11. Separation of stereoisomers of 7-oxa-bicyclo [2.2. 1] heptene sulfonate (OBHS), a selective estrogen receptor modulator (SERM), via chiral stationary phases using SFC/UV and SFC/MS

Services

The Virginia Tech Department of Chemistry Chromatography Center provides both custom and routine chromatographic testing for a wide range of analytes and applications.  Whether it is for routine analytical testing or the development of challenging or specialized analytical methods for your large or small molecule, the Chromatography Center’s highly experienced analytical chemists use up-to-date, laboratory instrumentation to meet your research requirements and or quality standards. By integrating analytical method development and automation, turnaround times for both method development and analysis are very attractive. The basic qualitative and quantitative methods based upon GC, HPLC and/or SFC coupled with optimized detector selections allow wide flexibility to optimize your specific routine or research-based analysis needs. Specific analyses according to the latest compendial testing requirements or specific client-provided methods are routinely employed. In selected cases, development of specific target compound sample preparation procedures can be developed. For highly custom analyses involving possible detailed sample preparatory procedures or unique methods of analysis, please request a consultation with a technical expert.

Ordering Analysis

Sample submission is by appointment only. Users should send email to Mashraf@vt.edu to make appointments. After the appointment is confirmed users should deposit samples in the Sample Box located behind the lab 1001. For further information, please refer to the COVID-19 Standard Operating Procedure document here.