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Quality-Control Analytical Methods: High-Performance Liquid Chromatography

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Quality-Control Analytical Methods: High-Performance Liquid Chromatography QUALITY CONTROL Quality-Control Analytical Methods: High-Performance Liquid Chromatography Tom Kupiec, PhD of liquid chromatography used to separate and quantify com- Analytical Research Laboratories pounds that have been dissolved in solution. HPLC is used to Oklahoma City, Oklahoma determine the amount of a specific compound in a solution. For example, HPLC can be used to determine the amount of morphine in a compounded solution. In HPLC and liquid Introduction chromatography, where the sample solution is in contact with a Chromatography is an analytical technique based on the sep- second solid or liquid phase, the different solutes in the sample aration of molecules due to differences in their structure solution will interact with the stationary phase as described. and/or composition. In general, chromatography involves The differences in interaction with the column can help sepa- moving a sample through the system over a stationary phase. rate different sample components from each other. The molecules in the sample will have different affinities and interactions with the stationary support, leading to separation Practical Aspects of HPLC Theory of molecules. Sample components that display stronger inter- In order to understand HPLC and to utilize its practical ap- actions with the stationary phase will move more slowly plications effectively, some basic concepts of chromatographic through the column than components with weaker interac- theory are presented here. tions. Different compounds can be separated from each other Chromatographic Principles as they move through the column. Chromatographic separa- Retention. The retention of a drug with a given packing mate- tions can be carried out using a variety of stationary phases, in- rial and eluent can be expressed as a retention time or reten- cluding immobilized silica on glass plates (thin-layer chro- tion volume. Retention or elution volume is the quantity of the matography), volatile gases (gas chromatography), paper (pa- mobile phase required to pull the sample through the column. per chromatography) and liquids (liquid chromatography). Retention time is defined as how long a component is retained High-performance liquid chromatography (HPLC) is a type in the column by the stationary phase relative to the time it re- sides in the mobile phase. The retention is best described as a Continuing Education column capacity ratio (k´), which can be used to evaluate the GOALS AND OBJECTIVES efficiency of columns. The longer a component is retained by the column, the greater is the capacity factor. The column ca- “Quality-Control Analytical Methods: High-Performance pacity ratio of a compound (A) is defined by the following Liquid Chromatography” equation: Goal: The goal of this presentation is to provide compounding T −T V −V pharmacists with auxiliary information concerning the use of high- k´ = A O = A O performance liquid chromatography for quality control of com- TO VO pounded preparations. where VA is the elution volume of component A and V0 is the Objectives: After reading and studying the article, the reader will elution volume of a nonretained compound. At constant flow be able to: rate, retention times (TA and T0) can be used instead of reten- 1. Discuss basic theory behind chromatographic separation. tion or elution volumes. 2. Discuss basic instrumentation and components of high- Resolution. Resolution is the ability of the column to separate performance liquid chromatography. peaks on the chromatograph. Resolution (R) is expressed as the 3. Discuss different analysis methods for high-performance liquid ratio of the distance between two peak maxima to the mean chromatography. value of the peak width at the base line 4. Discuss validation and maintenance of high-performance liquid (T −T )2 R= B A chromatography. wA +wB To complete this continuing education program, go to www.ijpc.com. The where TB is the retention time of component B, TA is the re- program is presented by the IJPC in partnership with P*ceutics Institute @ tention time of component A, wA is the peak width of compo- PCCA, which is accredited by the American Council on Pharmaceutical nent A and wB is the peak width of component B. If R is equal Education as a provider of continuing pharmaceutical education. Upon to or more than 1, then components are completely separated, successful completion of 70 percent of the questions and completion of the but if R is less than 1, then components overlap. evaluation, an ACPE statement of credit for one (0.1 CEU) credit Sensitivity. Sensitivity is a measure of the smallest detectable will be immediately available for printing. The cost is $15. level of a component in a chromatographic separation and is dependent on the signal-to-noise ratio in a given detector. International Journal of Pharmaceutical Compounding 223 Vol. 8 No. 3 May/June 2004 QUALITY CONTROL the eluent and is coated on an inert support. Ion exchange Figure 1. Schematic of a High-Performance Liquid chromatography has a stationary phase with an ionically Chromatograph. charged surface that is different from the charge of the sample. The technique is based on the ionization of the sample. The stronger the charge of the sample, the stronger the attraction to the stationary phase; therefore, it will take longer to elute off the column. Size exclusion is as simple as screening sam- Mobile Pump Injector Column Detector Data ples by molecular size. The stationary phase consists of mate- Phase for Display rial with precisely controlled pore size. Smaller particles get Reservoir Sample caught up in the column material and will elute later than larg- er particles. Several other types of chromatographic separation have been described, including ion-pair chromatography, Sensitivity can be increased by derivatization of the compound which is used as an alternative to ion-exchange chromatogra- of interest, optimization of chromatographic system or minia- phy and chiral chromatography (to separate enantiomers). turization of the system. Instrumentation Chromatographic Mechanisms As shown in the schematic diagram in Figure 1, HPLC in- Systems used in chromatography are often categorized into strumentation includes a pump, injector, column, detector and one of four types based on the mechanism of action, adsorp- integrator or acquisition and display system. The heart of the tion, partition, ion-exchange and size exclusion. Adsorption system is the column where separation occurs. Since the chromatography arises from interactions between solutes and stationary phase may be composed of micron-sized porous par- the surface of the solid stationary phase. Partition chromatog- ticles, a high-pressure pump is required to move the mobile raphy involves a liquid stationary phase that is immiscible with phase through the column. The chromatographic process be- gins by injecting the solute into the injector at the end of the column. Separation of components occurs as the analytes and mobile phase are pumped through the column. Eventually, each component elutes from the column as a peak on the data For Sterile Compounding display. Detection of the eluting components is important, and Why Build a cleanroom? the method used for detection is dependent upon the detector used. The response of the detector to each component is dis- played on a chart recorder or computer screen and is known as a chromatogram. To collect, store and analyze the chromato- graphic data, integrators and other data-processing equipment are frequently used. Mobile Phase and Reservoir The type and composition of the mobile phase affects the separation of the components. Different solvents are used for different types of HPLC. For normal-phase HPLC, the sol- MicroSphereTM 4x2 vent is usually nonpolar, and, in reverse-phase HPLC, the sol- MicroSphereTM 8x3 vent is normally a mixture of water and a polar organic sol- TM vent. The purity of solvents and inorganic salts used to make Now, MicroSphere the mobile phase is paramount. A general rule of thumb is to The cleaner, safer, more cost-effective AMERICA’S # 1 use the highest purity of solvent that is available and practical microenvironment for sterile compounding BARRIER ISOLATOR depending on the particular application. The most common and manipulating cytotoxic agents. solvent reservoirs are as simple as glass bottles with tubing connecting them to the pump inlet. CONSIDER THE ADVANTAGES Pumps NO CLEANROOM TO BUILD, NO GOWNING -Very cost-efficient High-pressure pumps are needed to push the mobile phase ASEPTIC - Class 10 - meets all regulations SAFE - Physical barrier between products and operators through the packed stationary phase. A steady pump pressure ERGONOMIC - Easy-to-operate, install and maintain (usually about 1000–2000 psi) is needed to ensure repro- MODULAR - Can be custom-designed- Many sizes/ options available ducibility and accuracy. Pumps are typically known to be ro- bust, but adequate maintenance must be performed to main- tain that characteristic. Inability to build pressure, high TEL: 1-800-ISO-2010 FAX: (514) 956-1032 pressures or leakage could indicate that the pump is not func- E-MAIL: [email protected] tioning correctly. Proper maintenance of the pump system will WEB SITE: isotechdesign.com minimize down time. INNOVATORS IN CLEAN AIR TECHNOLOGY 224 International Journal of Pharmaceutical Compounding Vol. 8 No. 3 May/June 2004 QUALITY CONTROL Injectors materials with C1, C2, C4, C6, C8 and C22 coatings are also The injector can be a single injection or an automated injec- available. Miscellaneous chemical moieties bound to silica, as tion system. An injector for an HPLC system should provide well as polymeric packing, are designed for purification of spe- injection of the liquid sample within the range of 0.1-100 mL cific compounds. Other types of column packing materials in- of volume with high reproducibility and under high pressure clude zirconia, polymer-based and monolithic columns. (up to 4000 psi). For liquid chromatography, liquid samples Theoretical plates relate chromatographic separation to the can be directly injected and solid samples need only to be di- theory of distillation and are a measure of column efficiency.
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