UMD CHEM 425 - Labreport (15 pages)

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Labreport



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Labreport

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Pages:
15
School:
University of Maryland, College Park
Course:
Chem 425 - Instrumental Methods of Analysis
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1 Introduction For this experiment we used High Performance Liquid Chromatography HPLC in order to separate and identify an unknown mixture of different aromatic compounds We were given four solutions for this experiment 1 methylnapthalene 1 ethylnapthalene 1 napthol and an unknown consisting of one or more of the 3 aromatics dissolved in acetonitrile ACN Like other types of chromatography HPLC is able to determine the identity of a mixture by separating its components based on the analyte mixture s relative affinities for the mobile and stationary phase of the column There are two types of partitioning chromatography normal phase and reverse phase Normal phase corresponds to a non polar stationary phase and a polar mobile phase while reverse phase follows the opposite motif Since we were interested in separating aromatics this experiment employed a reverse phase separation which is more commonly used This is due to recent technological improvements such as gradient elution that allow reverse phase to cover a very large range of analytes1 There are four commonly used types of HPLC adsorption ion exchange size gel exclusion chromatography and partition Adsorption chromatography is used for solid analytes and is able to separate compounds based on competitive surface interactions between the sample and the mobile phase molecules1 Adsorption chromatography usually employs normal phase parameters and uses a polar stationary phase and a non polar mobile phase For the stationary phase adsorption chromatography uses acidic silica in order to retain basic analyte components such as amines When trying to retain acids or phenols a basic alumina or a very basic magnesia stationary phase is used depending on the analytes relative acidity1 One disadvantage associated with adsorption chromatography is it s inability to separate homologous series of analytes well such as hydrocarbons and carboxylic acids Size exclusion chromatography is used in order to 2 separate large MW compounds such as proteins and polymers via a sieving mechanism rather than a partitioning one1 Size exclusion contains a stationary phase consisting of porous silica or polymer particles with pore sizes ranging from 40 2 500 When running the analyte through a size exclusion column molecules that are larger in size are able to pass through more quickly than smaller molecules that get caught via the sieving mechanism This allows researchers to determine the relative weight of the analyte by comparing retention time to known standards For our experiment we used partition chromatography which allows for the separation of compounds in a liquid solution such as our unknown mixture via a liquid mobile phase Partition chromatography is the most commonly used type of HPLC and is used to separate analytes with a molecular weight of less than 3 0001 Since we are attempting to separate naphthalene derivatives we used a reverse phase partition HPLC Our non polar stationary phase consisted of silica particles that are coated with C 18 that allow them to be bonded to the surface of support1 In order to fully understand the mechanism behind partition HPLC one must first consider its instrumentation A diagram is illustrated below in Figure 11 3 1 Figure 1 Block Diagram of the Low Pressure Gradient HPLC System used in our experiment Prior to analysis it is imperative that the solvents used for the mobile phase must be free of any gases degassed This is to ensure that any previously dissolved gases that might evolve from mixing the analyte with the mobile phase does not interfere with column analysis Another requirement for HPLC is high purity of the solvent reservoirs used for the mobile phase in addition to maintaining dust free filters1 For our experiment we utilized a reciprocating pump for our solvent delivery system which allowed for us to pump the mobile phase at any desired flow rate ranging from 0 1 10 mL min with high reproducibility The solvent is usually pumped through the column at pressures ranging from 1 000 5 000 psi1 A diagram of a reciprocating pump is illustrated below in Figure 21 More advantages of an HPLC pump include a pulse free output its ability to tolerate pressures up to 6 000 psi and high resistance to corrosion Other types of HPLC pumps include displacement pumps with no changing solvent and pneumatic pumps which only perform isocratic single solvent analysis1 The reciprocating pump is one 4 advantage of using HPLC over other types of chromatography because it allows for a larger analysis range high performance due to the pumps ability to perform a gradient elution in addition to an isocratic delivery 1 Figure 2 Block Diagram of reciprocating pump with labeled components Common mobile phases for HPLC include any miscible combination of water with various organic solvents such as ACN2 When introducing the analyte in question into the flow stream there are a few things one must consider For starters there must be reproducibility in the way the sample is introduced Second there is the possibility of band broadening occurring on the chromatogram if the sample is injected for a lengthy amount of time Third the sample injection system has to be able to withstand the high pressures that accompany the HPLC1 In order to account for these issues HPLC injects the analyte into a sample loop that allows the instrument to control the amount introduced in the system by disposing all excess volume into a waste vent This loop is illustrated below in Figure 31 By using a sample loop the analyte can successfully be introduced into the system without depressurizing it In addition by using a set small volume band broadening is reduced and high reproducibility is achieved1 5 1 Figure 3 Illustration of Sample Injection Loop in the load position In regards to the stationary phase i e the column HPLC has a very high theoretical plate count N of 40 000 60 000 plates meter1 Each theoretical plate corresponds to an equilibrium or separation stage so HPLC s high theoretical plate count attests to its high performance3 Analytical HPLC columns such as the one we used are usually between 15 250 mm in length and have a diameter of approximately 1 0 4 6 mm1 Stainless steel columns are the most commonly used type of HPLC column while glass is used for the separation of biological molecules and PEEK polymer columns are used for cost effective separations4 Normal phase HPLC uses a column filled with tiny polar silica particles allowing for non


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