COURSE 146C Laboratory Experiment 5 Measuring Gas phase Basicities of Amino Acids using an Ion Trap Mass Spectrometer Please read the lab procedure before your experiment References 1 Sunderlin L S Ryzhov V Keller L M M And Gaillard E R Measuring GasPhase Basicities of Amino Acids Using an Ion Trap Mass Spectrometer J Chem Educ 82 1071 1073 2005 2 Cooks R G Patrick J S Kotiaho T and McLuckey S A Thermochemical determinations by the kinetic method Mass Spectrom Rev 18 287 339 1994 1 Experiment 5 Measuring Gas phase Basicities of Amino Acids using an Ion Trap Mass Spectrometer I Introduction Mass spectrometry MS is a commonly used technique in chemical research In particular applications of MS in biochemistry have increased vastly since the development of new ionization techniques suitable for noncovalently bound complexes especially electrospray ionization ESI The development of quadrupole ion trap mass spectrometry QIT MS in recent years has also expanded the range of MS applications For example MS has been widely used in peptide sequencing As interest in biochemistry and health sciences continues to increase it is also valuable to introduce experiments in biophysical chemistry into the physical chemistry laboratory In this experiment we will use ion trap MS to measure the gas phase basicities of amino acids You will learn the basic principle of ion trap mass spectrometer and the MS MS techniques II Fundamentals of Ion Trap MS The basic mechanism of MS involves ionization fragmentation separation and detection of individual molecules and fragments Molecules of a sample can be ionized by different techniques including electron impact EI fast atom bombardment FAB chemical ionization CI and electrospray ESI Among these techniques ESI is probably the softest ionization process available and permits the molecular weight determination of large molecules such as proteins or peptides As shown in Figure 1 the solution containing the targeted molecules will form fine mist of ionized droplet by passing through the exit of a fine needle held at an electrical potential around 4 kV The droplets will quickly lose their solvent in the chamber leaving behind an aerosol of protonated sample for spectrometric analysis These positive charged ions will accelerate along an electrical potential and pass through a magnetic field which causes them to be deflected to an extent which is dependent upon the mass to charge ratio m z Figure 2 Measurement of m z allows determining the mass of each charged species if the charge is known Figure 1 left Schematic of an electrospray source 2 Figure 2 Schematic showing the accelerating ions are deflected by magnetic field In this experiment we will explore the MS MS technique to study the basicities of amino acids and it is only possible if our instrument is a quardrupole MS This instrument has four voltage carrying rods running the length of the flight path of charged species Figure 2 Without external voltage the ions will travel parallel to the rods By applying of direct current and radiofrequency voltages to the rods the ions will undergo oscillation in their path Only ions with a particular m z ratio can have stable oscillation path and travel through the rods without hitting the rods and lost and therefore these ions with specific m z ratio can be separated from other ions These ions progress into a collision cell can be activated using collisions with inert gas atom such as argon and the fragments resulting from collision induced dissociation can again be MS analyzed Figure 2 Schematic of the arrangement of the quadrupole mass spectrometer III Discussion Under appropriate conditions the kinetics of the dissociation of molecular cluster ions can yield relative but quantitative thermochemical information on the constituent species Cluster ions bound via protons electrons or other atomic or polyatomic anions or cations can be isolated and their dissociations followed in a tandem mass spectrometry experiment The isolated internally excited proton bound dimers of organic molecules dissociate competitively to yield the individual protonated monomers to a relative extent which is quantitatively related to the difference in proton affinities of the two monomers This concept is extended to the studies of basicities of amino acids in this experiment 3 An acidic solution of two amino acids A and B will be electrosprayed into an ion trap protonated monomers AH and BH as well as the dimers AHA BHB and AHB will be formed A moderate quantity of the proton bound mixed dimer AHB can then be isolated After isolation collision induced dissociation CID of this ion leads to a competition for the proton between the two amino acids Measuring peak heights for the two products AH and BH using the mass spectrometer allows determination of the branching ratio for these competing reactions The branching ratio can be used to derive the difference in the free energies of protonation of the two amino acids G where R is the ideal gas constant Teff the effective temperature of ions AH and BH are concentration of protonated A and B ions 4 Procedure 1 One pair of amino acids will be provided by instructors 2 An acidic solution of two amino acids A and B is electrosprayed into an ion trap protonated monomers AH and BH as well as the dimers AHA BHB and AHB are formed 3 Identify the peaks in the mass spectrum 4 Isolate the proton bound mixed dimer AHB 5 Collision induced dissociation CID of this ion leads to a competition for the proton between the two amino acids 6 Measuring peak heights for the two products AH and BH using the mass spectrometer allows determination of the branching ratio for these competing reactions 7 To derive the difference in the free energies of protonation of the two amino acids based on the branching ratio 8 Repeat the measurements on other pair of amino acids 9 Compare the gas phase basicities of different amino acids Questions for Lab Report To be completed and handed in within one week 1 Search literatures to find out the structure molecular weight proton affinity and gas phase basicities of the amino acids provided 2 List and label all the peaks observed in the MS and MS MS spectra 3 We usually observe some minor peaks around MS peaks can you tell where s the minor peak originating from 4 Calculate the gas phase basicities of the amino acids and comparing to the literature reported values 5 What s the key approximation in the calculation of difference in the free