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Transferring LC-UV to LC-MS.

Transferring LC-UV to LC-MS. Things to think about. All methods have 3 parts. Some questions to ask before transferring a method. So that we know when were’ done!. What is the structure? Will MS see it ( S cience). What is the point -Quantification or Qualitative?

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Transferring LC-UV to LC-MS.

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  1. Transferring LC-UV to LC-MS. Things to think about.

  2. . All methods have 3 parts

  3. Some questions to ask before transferring a method.So that we know when were’ done! What is the structure? Will MS see it (Science). What is the point -Quantification or Qualitative? What is the dynamic range needed? What are the method requirements to be considered good? What type of changes can be made (Politics)? What type of data do you expect to get (History)?

  4. Modes of chromatography Where are we?

  5. How Can We Affect Chromatographic Resolution intentionally or by accident? System Hardware differences-What all are we changing? Efficiency Selectivity Retentivity Initial Increase N Increase k Increase 

  6. Chemical Factors That Affect Selectivity Changes here can be the biggest issue • Elution solvent • Stationary phases • Particles • Surface chemistry • Mobile phase pH Column Chemistries Organic Solvent CreatingSelectivity α Mobile Phase pH Gradient Slope Temperature

  7. So about MP ANon volatile vs volatile buffersThis is typically where the fun starts.Mobile Phase modifiersit is really about the column and compound structure

  8. UV & PDA Detector • Detection mechanism • Analyte must have UV chromophore, with double bonds–C=C-, -C=CH-CH=C- • A chromophore absorbs the light at a specific wavelength,lmax. • Solvent may change throughout separation - gradient • Not very temperature sensitive • Not flow sensitive

  9. Mass Spectrometers (MS) • Detection Mechanism • Measures the mass-to-charge ratio (m/z) • Compounds must have a charge (+ or -) associated with them to be detected. • The compound can acquire a charge, e.g. acid contributing H+ (protonation). • MS detection occurs in a high vacuum environment . • In LC/MS analytes must go from a liquid to a gaseous environment. • Mobile phase must be volatile so it can be removed.

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