Chromatography worksheet A Vocabulary stationary phase column retention time microsyringe device eluate analyte mobile phase theoretical plate chromatogram eluent sample injector flow volume 1__________ characteristic time it takes for a particular analyte to pass through the system 2__________ in many separation processes is a hypothetical zone or stage in which two phases, such as the liquid and vapor phases of a substance, establish an equilibrium with each other. 3__________ substance fixed in place for the chromatography procedure 4__________ phase that moves in a definite direction 5__________ substance to be separated during chromatography. It is also normally what is needed from the mixture 6__________ the visual output of the chromatograph 7__________ the mobile phase leaving the column 8__________ the solvent that carries the analyte 9__________ the matter analyzed in chromatography 10__________ a device used in conjunction with injecting samples 11__________ a glass tube with a diameter from 5 mm to 50 mm and a height of 5 cm to 1 m with a tap and some kind of a filter 12__________ an object or a piece of equipment that has been designed to do a particular job 13__________ a small pump with a plunger that fits tightly in a tube 14__________ amount of eluate passing through the column B Reading The aim of chromatographic techniques is to separate the sample into its components to quantify or produce a pure fraction. To obtain such separation, it is important to understand a few basic principles, which would help to improve and speed up the separation. Speed of analysis The retention time of a peak (i.e., how long it is held on the column before it is eluted) is a reflection of the speed of analysis. Efficiency The efficiency (i.e., how good a column is for separating different compounds) is described mathematically by the “theoretical plate number”. Retention Compounds have been retained on the column to separate by chromatography. Most of the sample components have interacted with the stationary phase (i.e., need to be retained). In chromatographic techniques a solute (analyte) is preferentially distributed between two phases: a stationary (fixed) and a mobile (moving) phase. High-performance Liquid Chromatography (HPLC) This is the most popular chromatographic technique used in clinical laboratories. It offers rapid and sensitive separation with accurate quantification. It can be applied for the analysis of a wide range of compounds. The main components of modern HPLC are pump, injector, column, detector, and data recording device. The sample prepared in a liquid is usually introduced on the column through the injector using a glass microsyringe. The column is the most important part of HPLC. It is packed under controlled conditions with very small but uniform particles. Silica-based particles, most commonly used, give good separation. To push the mobile solvent (mobile phase) through the small particles of the column, a pump capable of high pressure is used. Some of these offer very low flow volumes suitable for using very narrow columns. Ultraviolet absorption is the most commonly used detection technique in HPLC (UV/VIS detector). Other detectors used are fluorescence and electrochemical detectors, which produce better sensitivity for certain types of compounds. Read the text and answer the questions: 1 What is the aim of chromatographic techniques? 2 What is determines how quickly the analysis is done? 3 How do you measure efficiency? 4 What are the two main phases in chromatographic techniques? 5 What is the most crucial part of HPLC? 6 How do you put samples into the column? 7 In which phase do you use silica-based particles? Why is silica used? 8 What is the standard HPLC detection technique? C Listening Listen and complete each gap with one word. 1 The letters HP in HPLC stand for ‘___________’ or ‘high-performance’. 2 HPLC is used to analyse a mixture or to _________ a required product from others in a reaction mixture. 3 HPLC works on the same principle as _________ chromatography. 4 A liquid, called the ___________, moves past the solid, the ____________ phase. 5 In paper chromatography, the ___________ consists of water molecules bound to the cellulose in the paper. 6 The _______ carries different components of a mixture, called the ________, along with it at different rates. 7 The speed of __________ depends on their relative affinity for the ______________________. 8 If the _____ is more polar than _______, the more polar components of a mixture move more quickly than the less polar ones. 9 In HPLC, the _____________ is a solid packed into a _____________. 10 Unlike in paper chromatography, the (solvent) liquid is forced through the column by _____________ pumps. 11 Two __________ can be mixed in any proportions to give a mixture, the liquid phase, of suitable polarity for the separation. 12 Water is more ___________ than ethane nitrile (CH[3]CN). 13 The pumps produce a _____________ of 15,000 kPa 14 A single ____________ is injected into the solvent stream in the injection port via a hypodermic ___________. 15 Several ____________ can be run in succession, by loading them into this auto-sampler which will run them in order without any human intervention. 16 The pumps force the mixed solvent through the ____________. The solvent emerging from the column and carrying the separated components of the mixture passes into the detector. 17 In the detector, UV light of specific wavelength passes through the _____________ and is absorbed by all the components to be separated. D Grammar