Chromatography

Chromatographic separation is based on the principle that a sample can be separated in time by passing it through a column which contains material specifically designed to interact with the individual compounds of the sample in a unique way. The effect of this interaction with each compound varies, causing some compounds to be retained in the column longer than others.  With the proper design of the separation column, and use of a suitable detector, samples can be analyzed both qualitatively and quantitatively using chromatography.

Chromatographic techniques can be classified according to the mobile phase used, i.e. the substance used to transport the sample through the column.  The two general classes of chromatographic separation most often employed are gas chromatography (GC) and liquid chromatography (LC).  GC uses gas pressure (usually pure hydrogen, nitrogen or helium) to propel a volatile sample through the column to the detector.  LC uses a pure liquid solvent or a liquid solution to force a dissolved sample through the column to the detector.  The mobile phase solvent can be aqueous (so-called reverse phase LC) or organic (so-called normal phase LC).

The analytical sample, which is being propelled through the column by the mobile phase (gas or liquid), comes in contact with the stationary phase, i.e. the substance that interacts with the sample.  The extent to which compounds of the sample interact with the stationary phase is called retention.  Those compounds which are highly retained are detected last, while those that are not well retained are detected first.  The result of the compounds of the sample being differentially retained is to cause separation within the column, with the weakly retained compounds reaching the detector first, and the highly retained compounds reaching the detector last. 

A more detailed description of GC is presented here to help the customer decide which separation method is best suited for his or her sample. LC and GPC will be discussed in other documents.

GC is a type of chromatography used to separate compounds that can be vaporized without decomposition (i.e. stable volatile substances). Some uses of GC include purity determination of a particular substance, or separating the compounds in a mixture (the relative quantitative amounts of compounds can be determined).

As stated earlier, the mobile phase is a gas such as helium, hydrogen, or nitrogen. The stationary phase is an ultra-thin layer of liquid or polymer on a non-reactive solid support, inside a piece of cylindrical metal or glass called a column.  The most common types or columns are capillary columns, which are hollow in the inside.  Packed columns, which have no hollow center, are sometimes used as well but are much less common in modern analysis.  The gaseous compounds being analyzed interact with the walls of the column, which depending on the type of column, is coated with different stationary phases. This causes each compound of the sample to elute (i.e. travel through the column) at a different rate. The time required for a compound to elute is referred to as the retention time tR.

Instrumentation

A gas chromatograph is an instrument designed to separate volatile chemicals in a complex sample. A gas chromatograph uses gas flow through the column to transport the sample to the detector.  As the chemicals exit the end of the column, they are detected and identified electronically using a variety of methods (see below). The function of the stationary phase in the column is to interact (i.e. weakly bond) to different components of the sample, causing each component to reach the detector at the end of the column at a different time (retention time). Other parameters that can be used to influence the order or time of retention are the gas flow rate, column length and dimensions, and the temperature of the column.

In a GC analysis, a known volume of gaseous or liquid sample is injected into the injector port of the column through a self-sealing septum, employing a glass micro syringe. As the carrier gas propels the sample through the column, the individual components of the sample begin to interact with the stationary phase. The rate at which the molecules progress along the column depends on the extent of chemical adsorption, which in turn depends on the type of molecular component of the sample and on the type of stationary phase. Since each component of the sample has a different rate of transport through the column, the various components of the analyte mixture are separated as they move along the column and reach the detector at different times. The detector is specifically designed to monitor the outlet stream from the column; thus, the time at which each component reaches the detector and the quantity of that component can be determined experimentally. Components are qualitatively identified by the order in which they exit (elution order) from the column and by the retention time of the component.

The injector (or column inlet) introduces an analytical sample into the stream of carrier gas (mobile phase). The inlet is attached to the column head.

The most common inlet types are:

§  S/SL (Split/Splitless) injector; a sample is injected through a septum via a micro syringe into a small heated chamber, increasing the volatility of the sample. The carrier gas then either sweeps the whole sample (splitless mode) or a fraction (split mode) of the sample into the column. In split mode, a part of the sample/carrier gas mixture in the injection chamber is eliminated through the split vent. Split injection is used when working with samples with high analyte concentrations (>0.1% by weight) whereas splitless injection is best for headspace (gas above the sample in a closed vessel) and trace analysis with low amounts of analyte (<0.01% by weight).

§  On-column inlet; the sample is added directly into the column in its entirety without heat.

§  Gas source inlet or gas switching valve

§  P/T (Purge-and-Trap) system

GC Detectors

The most common detectors for GC are the flame ionization detector (FID), the thermal conductivity detector (TCD), and the mass selective detector (MS).  All are sensitive to a wide range of components, and work over a wide range of concentrations. While TCDs are essentially universal and can be used to detect any component other than the carrier gas (as long as their thermal conductivities are different from that of the carrier gas, at detector temperature), FIDs are sensitive primarily to hydrocarbons, and are more sensitive to them than TCD. However, an FID cannot detect water. Mass selective detectors are very sensitive, and have the added advantage of giving information regarding molecular weight as well as the characteristic fragmentation patterns of components which can assist the analyst in identifying the individual components of a sample.  Other detectors which can be employed are:

§  Catalytic combustion detector (CCD), which measures combustible hydrocarbons and hydrogen.

§  Discharge ionization detector (DID), which uses a high-voltage electric discharge to produce ions.

§  Dry electrolytic conductivity detector (DELCD), which uses an air phase and high temperature to measure chlorinated compounds.

§  Electron capture detector (ECD), which uses a radioactive Beta particle (electron) source to measure the degree of electron capture.

§  Flame photometric detector (FPD), which uses a photomultiplier tube to detect spectral lines of the compounds as they are burned in a flame.

§  Hall electrolytic conductivity detector (ElCD)

§  Helium ionization detector (HID)

§  Nitrogen phosphorus detector (NPD), a form a thermionic detector where nitrogen and phosphorus alter the work function on a specially coated bead and a resulting current is measured.

§  Infrared detector (IRD)

§  Photo-ionization detector (PID)

§  Pulsed discharge ionization detector (PDD)

§  Thermionic ionization detector (TID)

Method and method development

The method is the set of conditions in which the GC operates for a given analysis. Method development is the means of determining what conditions are suitable and/or optimized for the analysis required.

Conditions which can be modified to optimize an analysis include inlet temperature, detector temperature, column temperature and temperature program, carrier gas and carrier gas flow rates, the column's stationary phase, diameter and length, inlet type and flow rates, sample size and injection technique.

Choice of stationary phase

The polarity of the solute is an important consideration for the choice of stationary phase, which in an ideal case would have polarity similar to the components in the sample. Common stationary phase materials in capillary columns are cyanopropylphenyl dimethyl polysiloxane, carbowax polyethylene glycol, biscyanopropyl cyanopropylphenyl polysiloxane and diphenyl dimethyl polysiloxane. For packed columns other options are available.

Sample size and injection technique

Sample injection

The actual chromatographic analysis starts with the introduction of the sample onto the column. The development of capillary gas chromatography resulted in many practical problems with the injection technique. The technique of on-column injection, often used with packed columns, is usually not possible with capillary columns. The injection system in the capillary gas chromatograph should fulfill the following basic criteria:

1.     The amount injected should not overload the column.

2.     The width of the injected plug should be small relative to the diffusion (which causes spreading) due to the chromatographic process. Failure to adhere to this requirement will reduce the capability of the column to separate components. As a rule (the rule of ten), the volume injected, Vinj, and the volume of the detector cell, Vdet, should be about 1/10 of the volume occupied by the portion of sample containing the molecules of interest (analytes) when they enter the detector.

Injection technique

Some general requirements which a good injection technique should fulfill are:

§  It must be able to obtain the column’s optimized separation efficiency.

§  It must allow accurate and reproducible injections of small amounts of representative samples.

§  No change in composition should occur during the analysis

§  It should be applicable for trace analysis as well as for neat (undiluted) samples.

Column temperature and temperature program

The column(s) in a GC are mounted in an oven, the temperature of which is carefully controlled.  The rate at which a sample component passes through the column is directly proportional to column temperature. The higher the temperature, the faster the sample travels through the column. However, the faster a sample travels through the column, the less interaction with the stationary phase, which could impact the separation of similar components.

In general, the column temperature is a trade-off between the length of the analysis and the quality of separation.

A method which keeps the column at a fixed temperature for the entire run is called "isothermal." More often, however, the column temperature is increased over the course of the analysis. The starting temperature, rate of temperature increase (the "ramp") and the final temperature is called a "temperature program."

A temperature program allows components that elute early to separate satisfactorily, while shortening the time it takes for late-eluting components to reach the detector.

Data reduction and analysis

Qualitative analysis

Chromatographic data is basically presented as a graph of detector signal (y-axis) versus retention time (x-axis), which is a plot called a chromatogram. This provides a variety of peaks for a sample representing the components eluting from the column at different times. Retention time can be used to identify analytes if the method conditions are constant, and a reference sample is run consecutively.  Also, the pattern of peaks will be constant for a sample under constant conditions and can be used to identify complex mixtures of analytes. In most modern applications however, the GC is connected to a mass spectrometer or similar detector that is capable of identifying the analytes represented by the peaks by determining molecular weights.

Quantitative analysis

The area under a peak is related to the amount of analyte present in the chromatogram. By calculating the area under the peak using integration, the concentration of a component in the sample can be calculated. Concentration of the component of a sample can be calculated using a calibration curve which is a plot of component peak area vs. concentration of a known concentration of component. 

In the case of MS detection, software can be used to display and integrate peaks, and match MS spectra to a digital library of known spectra.

Application

In general, samples that vaporize below 300 °C (and therefore are stable up 300 °C) can be measured quantitatively. The samples are should not contain ionic species. Very small amounts of a substance can be measured.  It is often useful that the sample be compared to a reference standard, i.e. the pure, suspected substance.

Temperature programs can be modified to improve the chromatogram; for example to resolve substances that behave similarly during the GC process and may have overlapping peaks.

GC is very accurate if used properly and can measure picomoles of a substance in a 1 ml liquid sample, or parts-per-billion concentrations in gaseous samples.

The types of samples Associated Polymer Labs are capable of analyzing by GC include:

Plastics

Additives

Bioplastics

Polymers

Monomers and Oligomers

Elastomers

Rubbers

Composites

Epoxy

Cosmetics

Essential Oils

Waxes

Household Products (Consumer Products)

Bisphenol

Phthalates

Hydrocarbon gases

Volatile Organic Compounds ( VOC )

It is often determined that some components of the above sample types are of sufficient volatility and stability to be analyzed by GC.  These components might include:

1)      Monomers/Oligomers (Low Boiling)

2)      VOCs (Degassing from Samples)

3)      Additives

4)      Residual Solvents (Physisorbed)

5)      Trace Impurities

In some cases a headspace GC analysis (analysis of the volatile gases above the sample in a close, heated vessel) is useful in detecting the above components.  In other cases, dissolution of the sample in a suitable volatile solvent may be appropriate to analyze the sample.

SPME – Solid Phase Micro Extraction

The attraction of SPME is that the extraction is fast, simple, and done without solvents, and detection limits can reach parts per billion (ppb) levels for certain compounds.

Conclusion

GC is a versatile analytical technique for analyzing a variety of sample types.  Analysis is quick, quantitative, and reproducible.  The analysis is limited, however, to volatile components.  Typical GC uses FID or TCD detection, and retention time can be used to identify peaks if a suitable standard is run consecutively.  Using a mass-selective detector coupled to GC (GC-MS), compound identification can be achieved by determining molecular weights of the various components. Molecular weights up to 1000 amu can routinely be determined with GC-MS analysis.

Services Offered

Associated Polymer Labs (APL) offers a full spectrum of GC analytical services, and has the capability of developing custom methods for your particular needs.  GC analysis is often the first step in analyzing the volatile components of an unknown sample. Follow-up analysis by GC-MS is then used to identify the unknown components by molecular weight.