Agilent Effect of Resolution Mass Accuracy on Empirical Formula Confirmation Identification of Unknowns Manual
Contents
1. Author Doug McIntyre is an applications chemist at Agilent Technologies in Santa Clara California U S A www agilent com chem Agilent Technologies Inc 2004 Information descriptions and specifications in this publication are subject to change without notice Agilent Technologies shall not be liable for errors contained herein or for incidental or consequential damages in connection with the furnishing performance or use of this material Printed in the U S A May 14 2004 5989 1052EN Agilent Technologies2. Thus resolution and resolving power have an inverse relationship In practice the terms resolu tion and resolving power are used somewhat inter changeably and it is common to see references to a resolution of 10 000 although that would actually be a very bad mass resolution but a good resolving power For simplicity this overview discussed masses in terms of unified atomic mass units u This implies singly charged ions For multiply charged ions the same principles and formulas apply but the correct units would actually be the mass to charge ratio m z Agilent Technologies Low resolving power S Target mass z Z 53 2 lt eee I oa High resolving power I oa I Target mass o i i 1 1 1 2 1 i 2 lt Interference Figure 1 The high resolving power of a TOF mass analyzer helps to reduce the chances of having the mass peak of inter est merged with an interfering ion from the sample or the background Resolving power is mass dependent For a given degree of separation AM that can be distin guished resolving power is lower at lower masses and higher at higher masses For example 100 At 100 u RP 9 gg 2 000 1000 At 1000 u RP gt ox 20 000 Nominal mass Nominal mass is the mass of a given empirical for mula using the integer mass numbers of the most abundant isotope of each element C 12 H 1 O 16 N 14 Cl 35 Purine C5 H4N and ace
3. 33 3 25 0 20 0 600 u 1000 u 1 7 1 0 3 3 2 0 5 0 3 0 6 7 4 0 8 3 5 0 10 0 6 0 11 7 7 0 13 3 8 0 15 0 9 0 16 7 10 0 Effect of Resolution and Mass Accuracy on Empirical Formula Confirmation and Identification of Unknowns Accurate mass measurements Accurate mass generally refers to a mass mea surement that is accurate enough to unambigu ously distinguish two compounds with identical nominal masses For 200 u a mass error of 5 ppm or less is required to make an accurate mass mea surement 0 001 u For 1000 u a mass error of 1 ppm would be required to achieve the same accurate mass measurement The example compounds purine and aceto phenone differ in mass by 0 0139 u At a nomi nal mass of 120 u this difference translates to 116 ppm At 120 u the Agilent LC MSD TOF has amass accuracy of approximately 4 ppm so it can easily distinguish between the two compounds Other contributions to confirmation and identification For a given mass accuracy as molecular mass increases the number of possible elemental com positions also increases For a totally unknown compound above about 600 u the number of pos sible elemental compositions which fit the accu rate mass data is too large to definitively assign an elemental composition However other infor mation such as starting materials the isotope dis tribution the number of possible nitrogens and the number of unsaturated bonds in a compound may help to limit some
4. of the elemental composi tion possibilities to those that make chemical sense In the case of a target compound the expected empirical formula is known and can be compared against the measured accurate mass data to confirm identity Figure 4 A second nebulizer and automated calibrant delivery system continuously introduce reference mass compound at a very low level into the LC MSD TOF for continuous mass correction Agilent Technologies Design for mass accuracy One of the ways to improve the mass accuracy of TOF mass spectrometers is to periodically intro duce a known reference mass compound into the MS This allows mass correction based on any dif ference between the known exact mass of the ref erence mass compound and the mass measured by the instrument One difficulty is that traditional TOF mass spectrometers tend to have very narrow dynamic ranges as little as one order of magni tude Thus a reference mass compound must be introduced at a very specific high concentration It can persist in the system and act as a contami nant interfering with later measurements The Agilent LC MSD TOF features an analog to digital ADC acquisition system The ADC acquisi tion system provides several orders of magnitude of dynamic range Combined with an automated cali brant delivery system and a second nebulizer this allows the continuous introduction of a reference mass compound at a concentration low enough that it i
5. Effect of Resolution and Mass Accuracy on Empirical Formula Confirmation and Identification of Unknowns Technical Overview Doug Mcintyre Agilent Technologies Introduction When synthetic chemists generate new com pounds they need to quickly confirm that the compounds they created are the compounds they intended to create Multiple compounds with different empirical formulas can share a common nominal molecular weight Both very good mass resolution and mass accuracy are required in order to positively distinguish between compounds with the same nominal mass and confirm com pound identity Traditionally sufficient mass resolution and mass accuracy were only available in expensive difficult to use instruments such as double focusing magnetic sector mass spectrometers Recent improvements in time of flight TOF technology have significantly increased TOF mass resolution and mass accuracy The Agilent LC MSD TOF mass spectrometer benefits from these improve ments Its outstanding mass resolution and mass accuracy combined with the powerful and easy to use atmospheric pressure ionization sources make it a good solution for empirical formula con firmation of small molecule organic compounds This overview discusses some of the theory and concepts behind mass resolution and mass accu racy and how they affect the ability to unam biguously confirm empirical formulas of small molecule organic compounds or identif
6. acetophenone have identical nominal mass 120 they have different empirical formulas purine C5 H4N and acetophenone CgHgO Their exact mass difference is 0 0139 u In order to resolve purine and acetophenone a mass spectrometer would need a resolving power of 120 RP 120 0575 120 0436 8633 If a mass spectrometer does not have sufficient power to resolve this difference it cannot posi tively confirm without additional information which of the two molecules is present Design for resolving power The Agilent LC MSD TOF includes a number of design elements designed to enhance its resolving power These include e A beam shaper and related ion optics that reduce variations in ion position and energy before they enter the mass analyzer e One dimensional harp grids oriented in the direction of ion travel in both the pulser and ion mirror reflectron e A mechanical design that automatically creates proper alignment parallelism Effect of Resolution and Mass Accuracy on Empirical Formula Confirmation and Identification of Unknowns Mass Accuracy Accurate Mass and Limita tions on Empirical Formula Confirmation and Identification of Unknowns As demonstrated previously high resolution is necessary to positively confirm identities and to identify unknowns High resolution alone how ever is not sufficient A mass spectrometer must also have a high mass accuracy Mass accuracy Mass accu
7. racy is the difference between the theo retical calculated mass of a compound and the mass measured by the mass spectrometer see Fig ure 3 This is also called mass error Mass accuracy error can be expressed in either parts per million ppm or milli mass units mmu Parts per million is not an absolute measurement it varies according to the mass to charge ratio For example at 200 u a 1 milli mass unit 0 001 u mass error is 5 ppm At 600 u the same 1 milli mass unit error is 1 7 ppm The errors are mass Agilent Technologies Calculated Measured Mass Mass Mass I I Error Low resolving power Solving power Abundance Mass Figure 3 Mass accuracy is independent of resolving power but the LC MSD TOF exhibits outstanding resolving power and mass accuracy dependent and get larger at the low masses or smaller at the high masses The relationship of milli mass unit mmu mass error to parts per million ppm mass error at a given mass is shown in Table 2 Table 2 Relationship of milli mass units mmu to parts per million ppm at a given mass to charge ratio ppm 100 u 200 u 300 u 400 u 500 u 1 mmu 10 0 5 0 3 3 2 5 2 0 2 mmu 20 0 10 0 6 7 5 0 4 0 3 mmu 30 0 15 0 10 0 75 6 0 4 mmu 40 0 20 0 13 3 10 0 8 0 5 mmu 50 0 25 0 16 7 12 5 10 0 6 mmu 60 0 30 0 20 0 15 0 12 0 7 mmu 70 0 35 0 23 3 17 5 14 0 8 mmu 80 0 40 0 26 7 20 0 16 0 9 mmu 90 0 45 0 30 0 22 5 18 0 10 mmu 100 0 50 0
8. s unlikely to interfere with analyses The LC MSD TOF gets the benefit of continuous mass correction without the drawbacks n Sample nebulizer Reference nebulizer b Effect of Resolution and Mass Accuracy on Empirical Formula Confirmation and Identification of Unknowns Mechanical and electrical changes caused by tem perature changes can also have a negative effect on mass accuracy The LC MSD TOF features a flight tube made from a special ultralow thermal expansion alloy that minimized flight path changes due to temperature changes It also fea tures mechanical and electronic temperature com pensation in the flight tube and electronics Summary Mass resolution and mass accuracy are both criti cal aspects of MS performance With sufficient mass resolution and mass accuracy a mass spec trometer can positively confirm elemental compo sition or identify unknows Even if mass resolution and mass accuracy are not sufficient for positive identification accurate mass measurements can Agilent Technologies reduce the number of likely candidates enough that positive identification can be made based on a combination of accurate mass measurements and other information The Agilent LC MSD TOF design includes unique design features that enhance both its mass resolu tion and mass accuracy It offers the power of accurate mass measurements with the ease of use of much simpler less powerful instruments
9. to phenone CgHgO have the same nominal mass of 120 u On a typical quadrupole mass analyzer under unit mass resolution conditions the purine and acetophenone ions are indistinguishable Effect of Resolution and Mass Accuracy on Empirical Formula Confirmation and Identification of Unknowns a N b Figure 2 Structures of a Purine and b Acetophenone Average mass Average mass is the mass of a given empirical formula calculated using the atomic weights of each element in the compound e g C 12 011 H 1 0080 O 15 9994 N 14 0067 Cl 35 453 The atomic weight is a weighted average of the natu rally occurring stable isotopes of an element Natural variations in the isotopic ratios of some elements limit the precision of some atomic weights The average mass for purine is 120 11 u The average mass for acetophenone is 120 15 u Typically the average mass is reported as the molecular weight in reference books such as the Merck Index Monoisotopic mass Monoisotopic mass is the mass of a given empirical formula calculated using the exact mass of the most abundant isotope of each ele ment C 12 000000 H 1 007825 O 15 994915 Agilent Technologies N 14 003074 Cl 34 968851 The monoisotopic mass of purine is 120 0436 u The monoisotopic mass for acetophenone is 120 0575 u Monoiso topic mass is sometimes referred to as exact mass Resolving compounds with identical nominal masses Although purine and
10. y unknowns j Agilent Technologies Effect of Resolution and Mass Accuracy on Empirical Formula Confirmation and Identification of Unknowns The Effect of Mass Resolution on Empirical Formula Confirmation and Identification of Unknowns Mass resolution has a large influence on whether a mass spectrometer can unambiguously determine elemental composition for confirmation of empiri cal formulas or identification of unknowns The better the mass resolution the less likely it is that a mass peak of interest will be merged with an interfering ion from the sample or background This section explains some of the basic concepts and terminology related to mass resolution and demonstrates how the mass of the analyte affects the mass resolution required Mass resolution and resolving power Mass resolution R is the degree of separation AM between two adjacent ions in the mass spec trum that can be distinguished at a given mass M The ability to resolve singly charged ions one mass unit 1 u apart is called unit mass resolu tion A typical quadrupole mass spectrometer has a resolution from a few tenths of a mass unit to one mass unit e g 0 25 1 u A typical TOF mass analyzer can resolve mass differences of a few hundredths of a mass unit to a few tenths of a mass unit e g 0 025 0 25 u Resolving power RP is the nominal mass M to be measured divided by the difference in the masses to be identified AM RP M AM
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