Chromatography: HPLC, HPSEC, MS, LC-MS/MS, GC Isariya Techatanawat, PhD Director of Bioequivalence Study Group, Research and Development Institute, The Government Pharmaceutical Organization

Chromatography Chromatography is a technique for separating and/or identifying the components in a mixture. Components in a mixture have different tendencies to adsorb onto a surface or dissolve in a solvent. All chromatographic methods require one static part (stationary phase) and one moving part (mobile phase).

Type of Chromatography Adsorption Chromatography Partition Chromatography Ion Exchange Chromatography Size Exclusion Chromatography

1. Adsorption Chromatography Solid stationary phase Liquid or gaseous mobile phase Each solute has its own equilibrium between adsorption onto the surface of the solid and solubility in the solvent, the least soluble or best adsorbed ones travel more slowly. The result is a separation into bands containing different solutes.

1. Adsorption Chromatography

2. Partition Chromatography Stationary phase is a non-volatile liquid. Mixture to be separated is carried by gas or liquid as mobile phase. Solutes distribute themselves between the moving and the stationary phases, with more soluble component in mobile phase reaching the end of chromatography column first.

2. Partition Chromatography

3. Ion-Exchange Chromatography (IEC) Ion-exchange chromatography based upon electrical charge. Likes may repel, while opposites are attracted to each other. Stationary phases are characterized by nature and strength of acidic or basic functions on their surfaces and the types of ions that they attract and retain.

3. Ion-Exchange Chromatography (IEC) Opposite charge are electrostatically bound to the surface. When the mobile phase is eluted through resin, electrostatically bound ions are released as other ions are bonded preferentially

Ion-Exchange Chromatography (IEC) Cation exchange is used to retain and separate positively charged ions on a negative surface. Anion exchange is used to retain and separate negatively charged ions on a positive surface.

3. Ion-Exchange Chromatography (IEC)

3. Ion-Exchange Chromatography (IEC) Adsorption based on binding of opposite charges. Different components (virus, proteins, DNA) have different charges, which means strength of binding varies from component to component.

4. Size Exclusion Chromatography Mixture passes as a gas or a liquid through a porous gel. Pore size is designed to allow the large solute particles to pass through uninhibited. Small particles permeate gel and are slowed down. The smaller the particles, the longer it takes for them to get through column. Separation is according to particle size.

4. Size Exclusion Chromatography

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

Principles of Liquid Chromatography

HPLC HPLC is improved form of column chromatography. Smaller particle size for column packing material Provide greater surface area for interactions between stationary phase and the molecules. Allow better separation of mixture component

HPLC Instead of solvent being allowed to drip through a column under gravity, it is forced through under high pressures. To separate, identify, quantitate compounds. Compounds in trace concentrations as low as parts per trillion [ppt] may also easily be identified.

HPLC Normal Phase HPLC Reversed Phase HPLC

Normal Phase HPLC Stationary phase is polar (eg silica particles). Mobile phase is non-polar solvent (eg. Hexane).

Normal Phase HPLC Stationary phase is polar and retains polar yellow dye most strongly. Non-polar blue dye is won in the retention competition by the mobile phase and elutes quickly.

Reversed Phase HPLC Stationary phase is non-polar. Silica is modified to make it non-polar by attaching long hydrocarbon chains to its surface (eg. C8 or C18 carbon atoms). Mobile phase is polar solvent (eg. mixture of water and methanol).

Reversed Phase HPLC The most strongly retained compound is non-polar blue dye, as its attraction to non-polar stationary phase. Polar yellow dye is won in competition by the polar, aqueous mobile phase, moves the fastest, and elutes earliest.

HPLC

HPLC Separation

What is Chromatogram?

HPLC: Qualitative Analysis

HPLC: Quantitative Analysis

HPLC Chromatogram

Isocratic LC System

Gradient LC System

Gradient LC System

Preparative Chromatography

High Performance Size Exclusion Chromatography (HPSEC)

Size Exclusion Chromatography (SEC) Smaller molecules penetrate more of the pores on their passage through the bed. Larger molecules may penetrate pores above a certain size so they spend less time in the bed. The larger molecules elute first, while the smaller molecules travel slower [because they move into and out of more of the pores] and elute later.

Size Exclusion Chromatography (SEC)

Size Exclusion Chromatography (SEC) Biomolecules could be separated based on their size, by passing, or filtering, them through a controlled-porosity packing. Stationary phases are synthesized with a pore-size distribution. Mobile phases are good solvents for analytes and may prevent any interactions [based on polarity or charge] between analytes and stationary phase surface.

Size Exclusion Chromatography (SEC) Large components such as virus typically elute just after void volume. Contaminating proteins and nucleic acids elute at volume greater than void volume.

SEC: Exclusion Limit SEC resin are often characterized by their exclusion limit. Exclusion limit is the molecular weight of the smallest molecule which cannot enter the pores of the matrix. All molecules bigger than the exclusion limit elute in the void volume.

Typical steps in SEC Equilibration – prepare column for binding target biomolecule/virus Load – apply biomolecule/virus to column Elution – collect purified biomolecule/virus from column Cleansing/Sanitization – ensure no viable microorganisms present Storage – fill column with solution that minimizes biological growth and maintains integrity of resin

Separation by SEC

HPSEC

HPSEC

HPSEC Chromatogram shows how much material exited the column at any one time, with the higher molecular weight, larger polymer coils eluting first, followed by successively lower molecular weight (and therefore smaller) chains emerging later. The primary separation is according to elution volume.

HPSEC Chromatogram is compared to calibration that shows the elution behavior of a series of polymers for which the molecular weight is known. Molecular weight distribution of the sample is calculated.

Calibration graph used to determine polymer MW from its retention time

Average molecular weights of polymer nearly symmetrical

Process flow for the preparation of clinical flu vaccine Expansion of Vero cells Infection Harvest and Clearance Benzonase TFF AIEX SEC Purified Vaccine Bulk

Mass Spectrometry (MS)

Mass Spectrometry Measures mass-to-charge ratio of ions to identify and quantify molecules.

Mass spectrometer 1. Ion source: Sample molecules are ionised. 2. Mass analyzer: Sample molecules are separated according to their mass/charge ratio (m/z). 3. Ion detector: Separated ions are detected and sent to data system. m/z ratios and their relative abundance is presented as m/z spectrum .

Ionisation methods Electrospray Ionisation (ESI) Matrix Assisted Laser Desorption Ionisation (MALDI) Atmospheric Pressure Chemical Ionisation (APCI) Chemical Ionisation (CI) Electron Impact (EI) Fast Atom Bombardment (FAB) Field Desorption / Field Ionisation (FD/FI) Thermospray Ionisation (TSP)

Mass Analyser To separate ions formed in ionisation source according to their mass-to-charge (m/z) ratios. Mass analysers: quadrupoles, time-of-flight (TOF) analysers, magnetic sectors , and both Fourier transform and quadrupole ion traps.

Ion Detector To monitor ion current and amplify it. Signal is transmitted to data system where it is recorded as mass spectra . m/z values of ions are plotted against their intensities to show number of components in sample, molecular mass of each component, and relative abundance of various components in sample.

Mass Spectrometer

Mass Spectrum

Mass Spectrum

Mass Spectrometry m/z spectrum shows dominant ions at m/z 556.1, which are consistent with the expected protonated molecular ions, (M+H+). Measured molecular weight is 555.1 Da.

Mass spectrum of sulfamethazine acquired without collision-induced dissociation exhibits little fragmentation

Mass spectrum of sulfamethazine acquired with collision-induced dissociation exhibits more fragmentation and more structural information

Mass Spectrum

Mass Spectrometry Samples (M) with molecular weights greater than 1200 Da give rise to multiply charged molecular-related ions such as (M+nH)n+ in positive ionisation mode and (M-nH)n- in negative ionisation mode.

Mass Spectrometry Proteins have many suitable sites for protonation as all of the backbone amide nitrogen atoms could be protonated theoretically, as well as certain amino acid side chains such as lysine and arginine which contain primary amine functionalities.

Applications for Mass Spectrometry Field of Study Applications Proteomics Determine protein structure, function, folding and interactions Identify a protein from the mass of its peptide fragments Detect specific post-translational modifications throughout complex biological mixtures Quantitate (relative or absolute) proteins in a given sample Monitor enzyme reactions, chemical modifications and protein digestion Drug  Discovery Determine structures of drugs and metabolites Screen for metabolites in biological systems Clinical  Testing Perform forensic analyses such as confirmation of drug abuse Detect disease biomarkers (newborns screened for metabolic diseases) Genomics Sequence oligonucleotides Environment Test water quality or food contamination Geology Measure petroleum composition Perform carbon dating

Tandem (MS/MS) mass spectrometers More than one analyser. quadrupole-quadrupole magnetic sector-quadrupole quadrupole-time-of-flight geometries.

LC-MS LC-MS/MS

LC-MS vs LC-MS/MS

LC-MS/MS

Full scan mass spectrum of ginsenoside Rb1 showing primarily sodium adduct ions

Full scan product ion (MS/MS) spectrum from the sodium adduct at m/z 1131.7

Subsequent full scan product ion spectrum (MS3) from the ion at m/z 789.7

LC-MS/MS

Identification of proteins

Identification of proteins

Biochemical Applications Rapid protein identification using capillary LC/MS/MS and database searching

Molecular Weight Determination

Determining molecular weight of green fluorescent protein 27,000 Dalton with 238 amino acids.

Rapid protein identification using capillary LC/MS/MS and database searching

Protein identification Full scan MS/MS spectra from doubly charged parent ion m/z 807.2 and matching theoretical sequence identified by database searching

Identification of structurally similar aflatoxins

Analysis of peptides using CE/MS/MS

Gas Chromatography (GC)

Gas Chromatography Mobile phase is gas. Stationary phase can either be solid or non-volatile liquid. GC involves a sample being vapourised and injected onto the chromatographic column. Sample is transported through the column by the flow of inert gaseous mobile phase.

Gas Chromatography

GC: Instrumental components Carrier gas Carrier gas must be chemically inert. Commonly used gases: nitrogen, helium, argon, carbon dioxide. Sample injection port Temperature of the sample port is usually about 50°C higher than the boiling point of the least volatile component of the sample.

GC: Instrumental components Columns Packed columns Capillary columns Column temperature Oven temperature is kept constant for a straightforward separation

GC: Instrumental components Detectors Concentration dependant detectors: Signal is related to concentration of solute Not destroy the sample Dilution of with make-up gas will lower response Mass flow dependant detectors Destroy sample Signal is related to rate at which solute molecules enter the detector. Response is unaffected by make-up gas

Detector Type Support gases Selectivity Detectability Dynamic range Flame ionization (FID) Mass flow Hydrogen and air Most organic cpds. 100 pg 107 Thermal conductivity (TCD) Concentration Reference Universal 1 ng Electron capture (ECD) Make-up Halides, nitrates, nitriles, peroxides, anhydrides, organometallics 50 fg 105 Nitrogen-phosphorus Nitrogen, phosphorus 10 pg 106 Flame photometric (FPD) Hydrogen and air possibly oxygen Sulphur, phosphorus, tin, boron, arsenic, germanium, selenium, chromium 103 Photo-ionization (PID) Aliphatics, aromatics, ketones, esters, aldehydes, amines, heterocyclics, organosulphurs, some organometallics 2 pg Hall electrolytic conductivity Hydrogen, oxygen Halide, nitrogen, nitrosamine, sulphur  

Gas Chromatography

References http://www.waters.com/ http://www.chemguide.co.uk https://https://www.thermofisher.com Chromatography. The Royal Society of Chemistry. An Introduction to Mass Spectrometry. The University of Leeds. Mass Spectrometry in Biotechnology Gary Siuzdak , Academic Press 1996 SiuzdakBiotechnology” An Introduction to Gel Permeation Chromatography and Size Exclusion Chromatography. Agilent Technologies. Size-exclusion Chromatography of Polymers. Encyclopedia of Analytical Chemistryilent Chromatography for influenza vaccine manufacture: Principles, Equipment and Design. NC State University Basics of LC/MS. Agilent Technologies. Gas chromatography. Sheffield Hallam University. http://teaching.shu.ac.uk/hwb/chemistry/tutorials/chrom/gaschrm.htm