QUALITY CONTROL Quality-Control Analytical Methods: Gas Chromatography Tom Kupiec, PhD Since GC is a gas-based separation technique, it is limited to Analytical Research Laboratories components that have sufficient volatility and thermal stability. Oklahoma City, Oklahoma Practical Aspects of Gas Chromatography Theory Introduction To understand GC and effectively use its practical applica- Chromatography is an analytical technique based on the tions, a grasp of some basic concepts of general chromato- separation of molecules due to differences in their structure graphic theory is necessary. Chromatographic principles, and/or composition. Chromatography involves moving a sam- including retention, resolution, sensitivity and other factors, ple through the system to be separated into its various compo- are important for all types of chromatographic separation, and nents over a stationary phase. The molecules in the sample will were discussed in volume 8, issue 3 (May/June 2004) of the have different interactions with the stationary support, leading International Journal of Pharmaceutical Compounding.1 to separation of similar molecules. Chromatographic separa- The GC section of United States Pharmacopeia (USP) 27 tions can be divided into several categories based on the Chapter <621> outlines the basic theory and separation tech- mobile and stationary phases used, including thin-layer chro- nique of GC. A compound is vaporized, introduced into the matography, gas chromatography (GC), paper chromatography carrier gas and then carried onto the column. The sample is and high-performance liquid chromatography (HPLC). then partitioned between the gas and the stationary phase. The GC is a physical separation technique in which components compounds in a sample are slowed down to varying degrees of a mixture are separated using a mobile phase of inert carrier due to the sorption and desorption on the stationary phase. gas and a solid or liquid stationary phase contained in a col- The elution of the compound is characterized by the partition umn. The separation is based on the interactions of the vapor- ratio kD′, which is a dimensionless quantity also called the ized components in a mixture with the stationary phase as they capacity factor. The partition ratio can also be thought of as the are moved along by the mobile phase. ratio of the time required for the compound to flow through the column (the retention time) to the elution time of an unre- tained compound. The value of the capacity factor is depen- Continuing Education dent on several elements of the chromatographic system, GOALS AND OBJECTIVES including the chemical nature of the compound; the nature, “Quality-Control Analytical Methods: Gas Chromatography” amount and surface area of the stationary phase; the column Goal: The goal of this presentation is to provide compounding temperature; and the gas flow rate. Capacity factor is essential pharmacists with auxiliary information concerning the use of gas for separation by GC because separation is only possible if the chromatography for quality control of compounded preparations. compounds in the sample have different capacity factors. Objectives: After reading and studying the article, the reader will be able to: Samples and Sample Preparation 1. Review the basic theory behind chromatographic separation. A variety of sample types can be successfully analyzed by GC. 2. Discuss the basic instrumentation and components of gas Unlike HPLC, which is used to separate larger molecules, GC chromatography. is best suited for analysis of samples with smaller molecules. 3. Discuss the different analysis methods by gas Another important characteristic of samples for GC analysis is chromatography. that they must be volatile. If a compound is not volatile a tech- 4. Discuss the applications, strengths and limitations of gas nique called derivatization can be used to increase its solubility chromatography. To complete this continuing education program, go to www.ijpc.com. The and add to its volatility. Derivatization of samples involves a program is presented by the IJPC in partnership with P*ceutics Institute @ chemical reaction that alters the molecular structure of the an- PCCA, which is accredited by the American Council on Pharmaceutical alyte of interest to improve detection. Different types of com- Education as a provider of continuing pharmaceutical education. Upon pounds called derivatizing agents can be used to increase the successful completion of 70 percent of the questions and completion volatility of sample components. However, if possible it is best of the evaluation, an ACPE statement of credit for one (0.1 CEU) to avoid derivatization to keep the separation simpler. credit will be immediately available for printing. The cost is $15. International Journal of Pharmaceutical Compounding 305 Vol. 8 No. 4 July/August 2004 QUALITY CONTROL auto sampler system. A major source of precision errors in GC Figure 1: Schematic of a Gas Chromatograph. is poor injection technique. Auto samplers are very effective and help ensure that precisely the same sample volume is in- Heater Oven Heater jected every time, thereby eliminating injection errors. The injector temperature is also important for separation. The temperature of the injector is used to rapidly vaporize the liq- uid sample into a gaseous phase that can be carried to the column for separation. The temperature of the injector site Compressed Injector Column or Detector Recorder can be varied to help optimize separation. Different sample Gas (Carrier Stationary or Computer components will dictate what temperature is necessary Gas) Phase for vaporization. Hardware Carrier Gas In the early days of GC experiments, the carrier gas was seen The hardware components used in typical GC systems in- merely as the mass transport system. However, it is becoming clude an injector, a carrier gas, a column (stationary phase), an clearer that carrier gas is integral to the chromatographic oven, a detector and a recorder or information processor process. Several inert gases can be used as the carrier gas or (Figure 1). Several components have variable settings that can mobile phase of GC. Hydrogen, helium and nitrogen are all be used to optimize the analysis of different sample types. common carrier gases. Each carrier gas has its benefits and Injectors systems for which it is best suited. For example, helium is the Introduction of the sample into a GC system is a critical step most common gas used with GC/mass spectrometry systems. in separation. The reproducibility of the amount of sample in- Before the carrier gas can be used, it is important to ensure jected is important to ensure the reproducibility of results. A that it contains no oxygen because oxygen can have detrimen- sample can be injected manually into the system or by using an tal effects on the stationary phase of GC. Also, the chemical nature of the carrier gas has an effect on the efficiency of the GC column. The pressure at which the carrier gas is moving influences the retention time of samples on the column. In- creasing the pressure decreases the retention time. Varying both the carrier gas and the pressure at which the gas is exert- ed on the column can ensure that the sample has ample time to interact with the stationary phase and improve the separation. Column/Stationary Phase The column and stationary phase are responsible for the ma- jority of the separation of sample components. Interaction between the mobile phase, stationary phase and sample com- ponents determines how components are separated, so selec- tion of columns and stationary packing material is critical. There is a great deal of variation in commercially available columns and packing material. Depending on the components to be separated, the mobile phase being used and the desired degree of separation, different combinations of column type, column length and packing material can be used to achieve optimal results. Columns can be classified by column diameter and packing material. The three main types of GC columns are (1) conven- tional, (2) preparative and (3) capillary. Columns can be either packed or open. Packed columns can contain either a porous or nonporous stationary phase. A multitude of different mate- rials are used to pack columns. Each material has its own prop- erties, limitations and effective separation parameters. The capillary column is the most frequently used column for GC separations. Both conventional and capillary columns have ad- vantages and disadvantages. For example, more packing mate- rials are available for conventional columns, but capillary columns give improved sensitivity. 306 International Journal of Pharmaceutical Compounding Vol. 8 No. 4 July/August 2004 QUALITY CONTROL When selecting a column, a major choice is among nonpolar, that are well resolved, both peak height and area are propor- moderately polar or polar columns. Special-phase columns tional to the concentration. Three different calibration meth- such as chiral columns can also be utilized to separate isomers. ods, each with its own benefits and limitations, can be used in Other important factors to consider are packing material, col- quantitative analysis: (1) the external standard (std), (2) the in- umn length and column diameter. ternal standard (IS) and (3) the standard addition method. For Columns can be the most expensive component of a chro- GC, the most commonly used quantitative methods are the IS matographic system, so proper maintenance and use can help and standard addition methods. For our present purpose, only control cost. For proper column care it is best to consult the the IS method is discussed. manufacturer’s guidelines. The IS is an effective method because it tends to yield the Oven most accurate and precise results of all the quantitative meth- ods. With this method, an equal amount of an IS, a component The column resides in an oven, and temperature, which that is not present in the sample, is added to both the sample greatly affects the effectiveness of the chromatographic separa- and calibrator solutions. The IS selected should be chemically tion, is an extremely important factor used in controlling GC.