Part 2: Instrumentation Notes

What is chromatography?

• separates a mixture into its components

  • small/large organic molecules (DNA, proteins, polysaccarides)

What does the mobile phase do for chromatography?

• carries sample through a stationary phase

  • different molecules move through at different rates

What is the retention time in chromatography?

• how long a compound takes to travel through the column

• under fixed conditions it is characteristic of the compound - so it identifies it

What is the peak area in chromatography?

• proportional to how much the compound is present- so it quantifies it

• each peak is roughly triangular → area is easy to compute

What is the equation for peak area?

• = ½ x base x height

What are the different phases in chromatography?

• mobile phase

  • carriers sample through stationary phase

• stationary phase (column)

What does Gas chromatography do?

• separates compounds that are volatile in the gas phase

• offers low limits of detection and VERY high resolving power

• separates on basis of chemical/physical properties

What is the mobile phase in Gas chromatography?

• carrier gas

  • helium

  • hydrogen

  • nitrogen

  • argon

What is the main step in gas chromatography?

• liquid sample is injected into a heated injection port whose temperature is above the compounds boiling point

  • sample vaporizes instantly

What is the column like in gas chromatography?

• very long (several meters) coiled capillary

  • its length is what gives GC its high resolution

  • letting it separate steroids that differ only slightly

What are the different detectors in gas chromatography?

• thermal conductivity (TCD)

• flame ionization (FID)

• electron capture (ECD)

• mass spectrometer can also serve as detector

What is the main limitation of gas chromatography?

• older technology, replaced with liquid chromatography and mass spectrometry

• it can not analyze non-volatile molecules (proteins)

What is High performance liquid chromatography (HPLC)?

• separates molecules in the liquid phase under high pressure

  • pressure allows very small stationary-phase particles which greatly increases surface area and resolution

What are the four basic components of HPLC?

• solvent delivery system

• sample induction system

• column

• detector

What is the role of the solvent delivery system in HPLC?

• pump

• pushes mobile phase through the column

• multiple pump heads run out of phase so solvent is always flowing

What is the role of sample-introduction system in HPLC?

• injector

• a fixed loop injector loads the sample and flushes it onto the column

What is the role of column in HPLC?

• heart of the system

• guard column sits in front of it to trap debris and protect the expensive analytical column

What detectors are used for HPLC?

• spectrophotometer (absorbance)

• photodiode array

• fluorescence

• electrochemical

• mass spectrometer

What does the reverse phase of HPLC do?

• separates by hydrophobic interaction

• used for: non-polar organic compounds

What does the normal phase of HPLC do?

• separates by: polarity

• best for: polar compounds

What does the ion-exchange (cationic/anionic) phase do for HPLC?

• separates by: magnitude / charge of the ion

• best for: polar and charged compounds, proteins, DNA

What does the gel filtration zone do for HPLC?

• separates by: size

• best for: macromolecules

What does the affinity mode do for HPLC?

• separates by: specific (often immunogenic) binding to a ligand

• best for: isolating one target (antibody-antigen pair)

What is affinity chromatography?

• most selective mode for HPLC

• column carries an immobilized ligand that captures only its target while everything else washes through

• application of immunological principles to separation

What occurs in reverse-phase columns?

• a carbon chain is bonded to silica

  • C4 is short and less hydrophobic

  • C18 is long and VERY hydrophobic

What is C18 used for in Reverse-phase columns?

• longer chain

• resolves closely related compounds better

• is the most broadly useful single column

What is isocratic elution in HCLP?

• mobile phase solvent composition that stays constant throughout the run

What is gradient elution in HCLP?

• the solvent strength changes during the run which sharpens separation of complex mixtures

• ex: 100 nM → 1 M NaCl for proteins

What is Mass Spectrometry?

• most powerful identification technique

• a detector most often coupled to a gas or liquid chromatography

What is the defining feature of Mass Spectrometry?

• stored Library

  • it not only measures a compound but matches its spectrum against a database of essentially every known drug, steroid, protein, and toxin to identify it outright

What is the principle of Mass spectrometry?

1. sample is converted to gas-phase ions which are then moved through magnetic/electric field

2. field separates ions by mass-to-charge ratio (m/z)

3. pattern of ions produces gives identification, number of ions = concentration → quantification

How do ions move in Mass Spectrometry?

• field separates ions by their mass-to-charge ratio (m/z)

  • smaller ions move faster

  • larger ions move slower

what is base peak in mass spectrometry?

• most abundant ion in the spectrum

• assigned a relative abundance of 100%

What is the molecular ion in mass spectrometry?

• unfragmented ion of the original molecule

What ions are analyzed by Mass spectrometry?

• cations

  • formed by electron loss or proton attachment

  • very reactive so the analyzer is kept under high vacuum

What are the different ion sources for mass spectrometry?

• electron ionization and chemical ionization

• electrospray ionization

• MALDI

How does electron ionization (EI) and chemical ionization (CI) work for Mass spectrometry?

• used when the sample arrives as a gas (from a GC)

How does electrospray ionization (ESI) work for Mass Spectrometry?

• common when an HPLC is coupled to th MS

How does the MALDI (matrix-assisted laser desorption/ionization) work for mass spectrometry?

• a soft ionization technique

• analyte is placed on a UV-absorbing matrix and struck by a UV laser (337 or 355 nm) which vaporizes it into a plume of ions

• ideal for proteins / paired with time-of-flight analyzer

what are examples of mass analyzers?

• Quadrupole

• Time-of-flight (TOF)

• Magnetic sector

• ion trap

• ion cyclotron

What does the Quadrupole mass analyzer do?

• How it separates ions: four rods with tuned voltages steer one m/z at a time to the detector

• use: proteins

What does the magnetic sector mass analyzer do?

• how it separates ions: magnetic field defects ions by m/z

• use: organic molecules

What does the ion trap mass analyzer do?

• how it separates ions: traps / releases ions selectively

• use: organic molecules

What does the ion cyclotron resonance mass analyzer do?

• how it separates ions: ion orbit frequency in a magnetic field

• use: high-resolution research

How does tandem mass spectrometry (MS/MS) work?

• two analyzers can be linked in series

• ions are first separated by m/z

• a selected ion is then fragmented and re-analyzed in the second stage

what occurs in tandem MS?

• fragmentation takes place AFTER the first m/z separation

• lets you identify/ quantify each drug in a mixture individually

What is a detector?

• some form of electron multiplier

• software converts the signal to a digital form

What does the data system do for a detector?

• most powerful part

• matches the measured spectrum against a computerized reference library to identify the compound in seconds

What is GC-MS used for?

• drugs of abuse

• anabolic steroids

• pesticides

• inborn errors of metabolism

• standard way to confirm drugs of abuse in toxicology lab

What is the LC-MS used for?

• steroids

• immunosuppressant drugs

• inborn errors of metabolism

• proteins

What is Proteomics used for?

• identifying / quantifying proteins in a complex mixture

• single bacterial protein passing through MS ca identify organism

  • making MS invaluable for rapid identification of sepsis, where speed is needed

What is mass-to-charge ratio (m/z)

• quantity the analyzer separates ions by

What is a molecular ion?

• unfag,ened ion of the original moleucule

What is a Base Peak?

• most abundant ion (relative abundance = 100%)

What is Tandem MS (MS/MS)

• ions fragmented and re-analyzed after a first m/z separation

How is Monoisotopic vs. average mass?

• mass from the lightest isotopes vs. the weighted average isotopic mass

What is fluorescence polarization (FPIA)

• measures change in polarization with molecular size

• use: drugs, drugs of abuse, steroid hormones

What is Turbidimetry?

• measures decrease in transmitted light (180*)

• use: bacterial growth, higher level proteins

What is Nephelometry?

• measured scattered light (90*)

• use: specific proteins, immunoassays

What is gas chromatography used for?

• measures: retention time of volatile compounds

• use: drugs / toxin screens

What is HPLC?

• measures retention time in liquid phase

• use: steroids, drugs, proteins

what is mass spectrometry

• measures mass-to-charge ratio (m/z)

• use: drug confirmation, proteomics