LIQUID CHRMATOGRAPHY P URDUE UNIVERSITY 液相色谱 P普渡大学.docVIP

LIQUID CHROMATOGRAPHY PURDUE UNIVERSITY INSTRUMENT VAN PROJECT DETERMINING CAFFEINE CONCENTRATIONS IN SOFT DRINKS (Revised: 5-18-93) INTRODUCTION Chromatography is a separation technique that was first used by the Russian botanist Mikhal Tsvet. Around the turn of the century he was attempting to separate a mixture of liquids by pouring them into a column of powdered chalk. After a while he noticed bands of colors corresponding to the different components of the mixture. Tsvet named the technique chromatography from the Greek for color writing. Despite the name, chroma-tography can be applied to uncolored substances as well as those that are colored. There are several separation methods that fall under a general category known as chromatography. All of the methods have one common principle that distinguishes them from other physical and chemical methods of separation. This common feature is both a stationary and mobile phase. These two phases are incapable of being mixed, and come in contact with the sample that is to be separated, numerous times. The sample is carried through the system by the mobile phase and the interactions that occur are due to the different physical and chemical properties of the sample components. Separation is then a result of these different properties which affect the rate at which the components pass over the stationary phase. This rate, or retention time (Rt), is determined as a result of the UV detector seeing each component as it elutes from the column. It will then record these different substances as different peaks relative to the rate which they elute out of the column. The size of the peak indicates the amount of the component in the sample. The High Performance Liquid Chromatograph (HPLC) instrument that you will use for this laboratory exercise follows these same principles. Your HPLC has a column that has been carefully packed with C -18 particles that are less then 10 micrometers in diameter. This is the stationary phase