Theory, Application of TLC, HPLC, CE Separation Methods: Part 2

Theory, Application of TLC, HPLC, CE Separation Methods: Part 2
September 2017 Pharmaceutical Analysis Course (#8002)

Why Use Chromatography?
Direct spectroscopic measurements Complicated via sample matrix hv hv Complex samples cause interference problems with direct spectroscopic measurements Chromatography can help improve quantitative and qualitative measurements by separating these components prior to making the spectroscopic measurements Increased time for analysis and subsequent costs

Try to Avoid Direct spectroscopic measurements Complicated via sample matrix hv hv Complex samples cause interference problems with direct spectroscopic measurements Chromatography can help improve quantitative and qualitative measurements by separating these components prior to making the spectroscopic measurements Increased time for analysis and subsequent costs

HPLC High Performance Liquid Chromatography (HPLC) is one of the most widely used techniques for identification, quantification and purification of mixtures of organic compounds. In HPLC, as in all chromatographic methods, components of a mixture are partitioned between an adsorbent (the stationary phase) and a solvent (the mobile phase). The stationary phase is made up of very small particles contained in a steel column. Due to the small particle size (3-5 um), pressure is required to force the mobile phase through the stationary phase. There are a wide variety of stationary phases available for HPLC. We will discuss normal phase (Silica gel), although reverse phase (silica gel in which a 18 carbon hydrocarbon is covalently bound to the surface of the silica) columns are currently one of the most commonly used HPLC stationary phases.

HPLC Advantages of HPLC Limitations Types of HPLC separation modes
Can analyze both volatile and non volatile compounds High sensitivity (detector dependent) Wide spread applications Biological samples, hydrocarbons, carbohydrates, organometallic, pharmaceuticals, polymers, pesticides … Limitations Can be expensive (instrument and supplies) Need more experienced operator (Separation more sophisticated than GC or TLC) Lower separation efficiencies than GC Types of HPLC separation modes Partition (absorption) Adsorption Ion-Exchange Size Exclusion (Gel Permeation)

Separation Performance
Mechanics behind a separation and how to measure Rate Theory (General) Van Deemter Equation (Specific) First principals Kinetic Performance How temperature, pressure, volume and heat flow effects chemical and physical processes Retention mechanisms Thermodynamics

Steps to develop method
1 Information on Sample, define separation goals 2 Define separation mode, procedure, pretreatment 3 Choose Detector and settings 4 Choose LC Method, preliminary run 5 Optimize separation conditions 6 Check for problems, challenge method 7 Validate method for release

Applications of Liquid Chromatography
Information on Sample, define separation goals

What application is appropriate?
What are you trying to separate? Polarity/Molecule weight of expected constituents in sample Large and small molecules A complex mixture or clean sample What is the precision required? Drug substance assay (spec = 98.0 – 102.0%) Who will run the assay routinely?

Some things don’t change
Partition/Absorption: Reversed-phase HPLC; solute dissolves in the bulk of stationary phase Adsorption: Normal phase HPLC; solute stays on surface Ionic charge density: ion exchange retention Molecular size: size exclusion (gel filtration, gel permeation)

Adsorption Equilibrium
Adsorption vs. Absorption Adsorption is accumulation of molecules on a surface (a surface layer of molecules) in contact with an air or water phase Absorption is dissolution of molecules within a phase, e.g., within an organic phase in contact with an air or water phase Adsorption Absorption PHASE I PHASE 2

Adsorption Equilibrium
Adsorption vs. Absorption Adsorption is accumulation of molecules on a surface (a surface layer of molecules) in contact with an air or water phase Absorption is dissolution of molecules within a phase, e.g., within an organic phase in contact with an air or water phase Adsorption Absorption PHASE I Silicon Dioxide C18 PHASE 2

Normal Phase and Reverse Phase HPLC
What can you say about a, b, c?

Normal Phase vs. Reverse Phase HPLC
Skoog and Leary: Principals of Instrumental Analysis, 5th ed. Suanders, 1998

Instrumentation Mobile Phase (A) Pump Pulse Damper Vent Valve Injector
Mobile Phase (B) Column Waste Detector

Detectors and Selectivity
Evaporative Light Scattering Refractive index UV fixed wavelength UV variable wavelength – absorption Fluorescence - excitation and emission Nitrogen chemiluminescence - nitrogen Electrochemical - redox potential Mass spectrometer - mass / charge Increasing selectivity

UV Detector Optics (Waters Model 486)
Lamp Monochromator (grating) Beam Splitter Mirror Flow cell (sample, reference) Dual diodes (sample, ref.) Reference Sample

Diode Array Detectors Currently most common detector
Flexibility in wavelength(s) Capable of performing UV/VIS spectra match What is the difference between the two detectors? Hint, how many wavelengths are actually being read by the sensor?

Diode Array 3-dimensional Data: Chromatogram at 280 nm UV spectrum of each peak 3D plots

Diode Array Detectors Collection of full spectrum enables faster methods development Helps on troubleshooting Faster to collect full spectra across sample than to guess at what wavelength to collect Can always extract out single wavelength Ensures you don’t miss anything due to your wavelength selection Disadvantage: Large amounts of data storage required

Fluorescence Detector Optics
Advantages: Higher sensitivity than UV/VIS and subsequently lower limits of detection Disadvantages: Not many analytes fluoresce Therefore have to chemically label analytes which can effect the separation Light source more expensive

Refractive Index Detector Schematic
Measurement based on Snells law When a beam of light passes from one medium into another, the wave velocity and direction changes. The change in direction is called refraction. The relationship between the angle of incidence and the angle of refraction is expressed in Snell's Law of refraction. Initial mobile phase is used as the reference Sensitive to temperature pressure

Evaporative Light-Scattering Detector
Selectivity is Low: Response is proportional to the mass of the analyte; unlike optical absorption detectors, the detection sensitivity in ELSD is independent of the compound’s spectral properties. Operating Principle Evaporative light-scattering detectors for LC measure, in an absolute sense, the amount of light scattered by particles of mobile phase that have been dried through evaporation. In general, evaporative light-scattering detectors deliver a signal for all compounds that do not evaporate or decompose during the mobile-phase evaporation stage Three stages of the detector: 1. Nebulization: A nebulizer combines a gas flow of air or nitrogen with the column effluent to produce an aerosol of minute droplets. 2. Mobile-phase evaporation: The aerosol is introduced into a heated drift tube in which the mobile phase evaporates and leaves behind a particulate form of the target compound. Evaporation is in a heated zone, with a temperature set by users, and the useful temperature range is a matter of distinction between the various instruments on the market. 3. Detection: Light striking the dried particles that exit the drift tube is scattered, and the photons are detected by a photodiode or photomultiplier tube at a fixed angle from the incident light.Theoretically, except for highly volatile analytes (for example, ethanol in wine), most compounds can be detected.

Electrochemical Detection
Great for sugars and vitamins Many do not have inherent UV/VIS signal Disadvantage Passivation of the system by passing Nitric acid through to remove contaminants that will interfere with the detection mode

Dynamic Range What is the target concentration? How much sample should be injected? Example: For active and impurity determinations, a wide dynamic range is needed. For UV detectors, target the UV absorbance at the analysis wavelength to be about 1.5 to If it is too low, then the impurity detection limits will not be adequate. If too high, linearity will be poor. When a sample concentration is not limited, it is the detector’s linear dynamic range that determines sensitivity.

Mobile Phases Liquid or solvent mixture Degased to get rid of bubbles
Interferes with detection Sparging with inert gas In-line Vacuum degassing most popular Mobile phase selection based on sample constituents Must be soluble Samples should be stable in the mobile phase Compatible with instrumentation (columns, injectors) plus detection (wavelength interference) Vacuum Porous Tubing (Teflon)

When Should a Mobile Phase be Buffered?
In reversed phase HPLC, the retention of analytes is related to their hydrophobicity. The more hydrophobic the analyte, the longer it is retained. When an analyte is ionized, it becomes less hydrophobic and, therefore, its retention decreases. Acids lose a proton and become ionized when pH increases and Bases gain a proton and become ionized when pH decreases. Therefore, when separating mixtures containing acids and/or bases by reversed phase HPLC, it is necessary to control the pH of the mobile phase using an appropriate buffer in order to achieve reproducible results.

Separations sensitive to pH
Not very robust separation (small changes in pH, large changes in resolution and retention) Try shifting pH

Common Buffers A buffer concentration in the range of 25 to 50 mM is adequate for most reversed phase applications. This concentration is also low enough to avoid problems with precipitation when significant amounts of organic modifiers are used in the mobile phase and, in the case of phosphate buffers, low enough to minimize the abrasive effect on pump seals. It is seldom advisable to use a buffer concentration less than 10 mM. Buffer pKa Buffer Range UV Cutoff (nm) Phosphate 2.1 200 7.2 12.3 Formic acid* 3.8 210 Acetic acid* 4.8 Citrate 3.1 230 4.7 5.4 Tris 8.3 205 Triethylamine* 11.0 Pyrrolidine 11.3 * Volatile buffers

Considerations in the Selection of Mobile Phase Buffers for Reversed Phase
Phosphate is more soluble in CH3OH/water than in CH3CN/ water or THF/water. Ammonia salts are more soluble in organic/water mobile phases than potassium salts, and potassium salts are more soluble than sodium salts. TFA and TEA degrade with time and increase their UV absorbance. Mobile phases containing these buffers should be made fresh often. Citrate buffers attack stainless steel. When using these buffers, be sure to flush them out of the system as soon as you complete your assay. Microbial growth can quickly occur in buffered mobile phases that contain little or no organic modifier. This growth will accumulate on column inlets and damage chromatographic performance. These mobile phases should be made fresh daily and pre-column filters should be used to protect columns Using boiled water to prepare buffered mobile phase and storing it under refrigeration will help reduce the problem of microbial growth or add some organic modifier At pH greater than 7, phosphate buffers accelerate the dissolution of silica and severely shorten the lifetime of silica-based HPLC columns. Organic buffers should be used at pH higher than 8

Preparing a buffered mobile phase
Prepare an aqueous buffer solution of the desired concentration and pH. Measure the pH of the solution and adjust, if necessary, to the desired pH. When adjusting the pH of a buffer solution, make sure to wait until the solution reaches equilibrium after adding acid or base before measuring the pH. Combine the aqueous buffer solution with the appropriate organic modifier, e.g., methanol or acetonitrile, to produce the desired mobile phase. NOTE: You may choose to measure and adjust the pH of the final mobile phase rather than the aqueous buffer solution. However, measuring the pH of an aqueous/organic solution is not as accurate as measuring the pH of a purely aqueous solution, so it is highly recommended that you measure and adjust pH before adding the organic modifier. But if you do decide to measure and adjust the pH of the mobile phase, you should always do it the same way each time you prepare mobile phase including using the same glass and reference electrodes.

Pumps Ideally: Reciprocating Pump: Generate up to 6000 psi of pressure
Pulse free Flow rate from 0.1  10 mL/min +/- 0.5% or better flow rate Easily operated Made of inert, corrosive resistant materials (Teflon + Stainless Steel) Reciprocating Pump: Motor driven piston moves in and out of solvent chamber Check valves ensure flow direction Advantages: High pressures can be generated rather easily Small internal volumes aid in gradient capabilities Constant flow rate capabilities (positive displacement pump) Disadvantages: Pulsed flow – hence typical to use a secondary pump out of phase to compensate

Example Pump Unit Agilent 1200 Quaternary Pump

Quaternary Pump Design
Agilent 1200 Design (Typical low pressure mixing)

Pump Care Flush with water after running a buffer, (note there are special procedures when using reverse phase columns.) Replace seals in a timely manner. Maintain check valves. Do not allow solids in the mobile phase

Injector Inject a well defined plug into the solvent stream Enable injection to occur at low pressure (atmospheric from vial) Use 6 port valve Load depends on size of sample loop – typically between uL Modern injectors can also “partial” fill loop to provide programmable injection volumes independent of hardware installation LOAD INJECT

Injectors Traditionally incorporated into an auto sampler
Enables samples to be aligned with injectors Traditionally >100 samples can be loaded and ran without interruption

Routine Care of Injectors
Never use a pointed or bevel tip needle. Rinse after the use of buffer solutions. Avoid abrasive particles by filtering samples before injection. Use burr-free tubing to avoid metals shavings from getting into the injector.

General Plumbing

If Your Fittings Leak Good Bad
Check to make sure your tubing is seated properly The fitting may not be tightened enough You may be using incompatible fittings Valco fittings versus Rheodyne – like Ford versus Chevy parts Check the condition of the nut and ferrule Ferrule will get “Squashed” over time inhibiting the ability for it to seal Sometimes a leaking connection has nothing at all to do with the nut and ferrule, but with the receiving port NOTE: Using fittings made of material that is incompatible with your mobile phase is a sure way of creating leaks Good Bad Because of the nature of polymer-based fittings, the same degree of care does not have to be taken when choosing the proper fitting to mate with a specific manufacturer's receiving port. Primarily, the only two characteristics of the fitting which must be known are the geometry (coned or flat-bottom) of the receiving port and the thread dimensions. Also, and again unlike stainless steel fittings, polymer-based fittings do not permanently attach to a piece of tubing and usually do not require the use of any tool (besides your fingers!) to properly tighten and use. Additionally, these fittings often come in a variety of polymers, including PEEK™, Teflon®, Tefzel®, Delrin®, PPS, polypropylene, and others, for maximum cost and solvent resistance flexibility. Check to make sure your tubing is seated properly When using universal Fingertight fittings, the tubing must bottom out in the fitting before the nut and ferrule are tightened. If, after tightening the fitting a gentle tug disengages your tubing, remove fitting and try again. The fitting may not be tightened enough Stainless steel nuts and ferrules require a wrench to tighten them, even after repeated use. Fingertights also require a good turn with your fingers, but not with a wrench as this may damage the fitting. You may be using incompatible fittings Make sure you are using a nut and ferrule that are compatible with the components of your system. To avoid this problem and ensure compatibility, use Upchurch Scientific®'s universal Fingertight fittings. Because the ferrule does not permanently swage onto your tubing, a Fingertight can be used over and over again with most types of systems. Check the condition of the nut and ferrule After repeated use, nuts (and especially ferrules) will gradually become deformed to the point of being incapable of creating the seal they were designed to make. Always keep an extra supply of all the nuts and ferrules you are using so that you can replace them quickly and avoid unnecessary down time. Sometimes a leaking connection has nothing at all to do with the nut and ferrule, but with the receiving port Female fittings that have had stainless steel fittings swaged into them are especially susceptible to damage. Check the receiving port for visible burrs or scratches and replace if necessary. NOTE: Using fittings made of material that is incompatible with your mobile phase is a sure way of creating leaks--and possibly causing permanent destruction of the fittings.

HPLC Columns Column Properties: Diameters: 1mm – 4.6 mm Column Packing
Volumetric flow rate related to the cross sectional area Column Packing Silica particles 1 um – 10 um Porous and non porous Derivatized to achieve different desired surface chemistries

Silica gel electron micrographs
Irregular to spherical gels Irregular was common due to milling which was initially used to reduce particle size Direct synthetic methods to produce and control size of silica gel has progressed significantly Produce non-porous Low surface area Produce porous (Pelliculer) Higher surface area Completely porous Highest surface area Loading Capacity

Reaction of Silica and Functional Silanes
General Reaction: SiOH + RSiCl SiOSiR HCl

Treat your column well Minimize pressure surges
Maintain pumps to minimize pressure swings resulting from inconsistent flow rates. Use a guard column and/or an in-line 0.5 µm filter. Place each of these before the column and after the injector. An in-line filter will catch large particulates and a guard column will prevent strongly adsorbed materials from reaching your analytical column. Frequently flush columns with a strong solvent. Flushing with 100% acetonitrile is often adequate, but if stronger solvents are needed, consider methylene chloride (CH2Cl2). Less-polar solvents, like CH2Cl2, are strong solvents in reversed-phase chromatography. Many strong solvents are immiscible with aqueous containing mobile phases. Remember to flush the column and HPLC system with isopropanol prior to and after the use of CH2Cl2. Pretreat "dirty" samples to minimize strongly retained components and particulates Solid phase extraction, filtering sample through 0.45 µm filters, or high- speed centrifugation are useful pretreatment techniques. Use column temperatures of less than 60°C. Check column manufacturer specifications. Keep mobile phase pH between 3 and 7 for silica based columns. If operating outside the pH range, choose a column designed for low pH (StableBond) or high pH (Eclipse XDB). When storing columns, purge out salts and buffers. Leave column in pure acetonitrile. This prevents precipitation of buffer salts in the column. Acetonitrile is a good storage solvent because aqueous and alcohol mobile phases can increase the rate of stationary phase hydrolysis.

Selectivity Changes Column to column changes in selectivity needs to be minimized through quality column selection Many vendors include the column to column reproducibility tests as a means to lower your risk in this area for methods development

Classifying Selectivity Changes
Run samples on both columns to determine what the root cause of the selectivity change may be: Bonded phase test to determine if stationary phase surface coverage has changed Silica test will help determine if there is some differences in the polar nature of the reverse phase column

Automated Screening Column 1 Column 2 Column 3 Column 4 Column 5
Method Development Workstation: an Agilent 1100 HPLC that is configured with a column switcher and customized to handle multiple mobile phases. Column 1 Column 2 Column 3 Column 4 Column 5 Column 6 1 2 3 4 5 6

Optimization Traditional DOE Experiments Input parameters:
Mobile Phase, pH, Ionic Strength Injection Volume Column Identity Detection parameters Sample Preparation considerations Sample diversity – lot to lot variability Analyst to analyst Lab to lab

Methods Validation Accuracy Detection Limit and Quantitation Limit
Linearity Precision Repeatability Injection Repeatability Analysis Repeatability Intermediate Precision Reproducibility Range Recovery Robustness Sample Solution Stability Specificity/selectivity

System Suitability Capacity factor Precision/injection repeatability
Relative retention Resolution Tailing factor Theoretical plate number Ensuring equipment being used to analyze samples is performing at a level that can produce trustable results

Direct spectroscopic measurements Complicated via sample matrix hv hv Complex samples cause interference problems with direct spectroscopic measurements Chromatography can help improve quantitative and qualitative measurements by separating these components prior to making the spectroscopic measurements Increased time for analysis and subsequent costs

Role of HPLC in Pharma Premier tool to address following highlighted guidelines Q1 Stability Q2 Analytical Validation Q3 Impurities Q6 Specifications Q7 Good Manufacturing Practices Q8 Pharmaceutical Development Q9 Quality Risk Management Q10 Pharmaceutical Quality Systems

Capillary Electrophoresis Techniques

Capillary Electrokinetic Separations
Outline Brief review of theory Capillary zone electrophoresis (CZE) Capillary gel electrophoresis (CGE) Capillary electrochromatography (CEC) Capillary isoelectric focusing (CIEF) Micellar electrokinetic capillary chromatography (MEKC) Extremely High Plate Counts! 100,000 Plate Count

Capillary Electrophoresis
Electrophoresis: The differential movement or migration of ions by attraction or repulsion in an electric field Anode Cathode Basic Design of Instrumentation: E=V/d Buffer Anode Cathode Detector The cathode is defined as the electrode of an electrochemical cell at which reduction occurs (i.e. electrons are added to cations to create neutral atoms). The cathode supplies electrons to the positively charged cations in the cell. The simplest electrophoretic separations are based on ion charge / size

Types of Molecules that can be Separated by Capillary Electrophoresis
Proteins Peptides Amino acids Nucleic acids (RNA and DNA) - also analyzed by slab gel electrophoresis Inorganic ions Organic bases Organic acids Whole cells

The Basis of Electrophoretic Separations
Migration Velocity: Where: v = migration velocity of charged particle in the potential field (cm sec -1) ep = electrophoretic mobility (cm2 V-1 sec-1) E = field strength (V cm -1) V = applied voltage (V) L = length of capillary (cm) Electrophoretic mobility: q = charge on ion  = viscosity r = ion radius Frictional retarding forces

Inside the Capillary: The Zeta Potential
Inside wall of the capillary is covered by silanol groups (SiOH) that are deprotonated (SiO-) at pH > 2 SiO- attracts cations to the inside wall of the capillary The distribution of charge at the surface is described by the Stern double-layer model and results in the zeta potential Top figure: R. N. Zare (Stanford University), bottom figure: Royal Society of Chemistry The Zeta Potential () is the potential at any given point in double layer (decreases with increasing distance from capillary wall). The Stern model (see any good Surface Chemistry textbook for details, or look at pg. 566 in Skoog et al.) is also referred to as the electrical double-layer model. Note: diffuse layer rich in + charges but still mobile

Silanols fully ionized above pH = 9
Electroosmosis Electroosmotic flow is due to a “slip” layer at the surface being uniformily pulled towards cathode Excess cations in the diffuse Stern double- layer flow towards the cathode, exceeding the opposite flow towards the anode Net flow occurs as solvated cations drag along the solution Silanols fully ionized above pH = 9 Top figure: R. N. Zare (Stanford University), bottom figure: Royal Society of Chemistry Note – electroosmosis is the mobile phase “pump” of capillary zone electrophoresis (CZE) It is the uneven distribution of cations in the diffuse layer that force the net flow towards the cathode. The net flow becomes quite large for high pH situations – a 50 mM pH 8 buffer flows through a 50-cm capillary at 5 cm/min with 25 kV applied potential (see page781 of Skoog et al.)

Electroosmotic Flow (EOF)
Net flow is large at higher pH: A 50 mM pH 8 buffer flows through a 50-cm capillary at 5 cm/min with 25 kV applied potential Key factors that affect electroosmotic mobility: dielectric constant and viscosity of buffer (controls double-layer compression) EOF can be quenched by protection of silanols or low pH Electroosmotic mobility: Where: v = electroosomotic mobility o = dielectric constant of a vacuum  = dielectric constant of the buffer  = Zeta potential  = viscosity E = electric field

Electroosmotic Flow Profile
- driving force (charge along capillary wall) - no pressure drop is encountered - flow velocity is uniform across the capillary Cathode Anode Electroosmotic flow profile Frictional forces at the column walls - cause a pressure drop across the column High Pressure Low Hydrodynamic flow profile Result: electroosmotic flow does not contribute significantly to band broadening like pressure-driven flow in LC and related techniques

Controlling Electroosmotic Flow (EOF)
Want to control EOF velocity: Variable Result Notes Electric Field Proportional change in EOF Joule heating may result Buffer pH EOF decreased at low pH, increased at high pH Best method to control EOF, but may change charge of analytes Ionic Strength Decreases  and EOF with increasing buffer concentration High ionic strength means high current and Joule heating Organic Modifiers Decreases  and EOF with increasing modifier Complex effects Surfactant Adsorbs to capillary wall through hydrophobic or ionic interactions Anionic surfactants increase EOF Cationic surfactants decrease EOF Neutral hydrophilic poymer Adsorbs to capillary wall via hydrophobic interactions Decreases EOF by shielding surface charge, also increases viscosity Covalent coating Chemically bonded to capillary wall Many possibilities Temperature Changes viscosity Easy to control

Electrophoresis and Electroosmosis
Combining the two effects for migration velocity of an ion (also applies to neutrals, but with ep = 0): At pH > 2, cations flow to cathode because of positive contributions from both ep and eo At pH > 2, anions flow to anode because of a negative contribution from ep, but can be pulled the other way by a positive contribution from eo (if EOF is strong enough) At pH > 2, neutrals flow to the cathode because of eo only Note: neutrals all come out together in basic CE-only separations

Electrophoresis and Electroosmosis
A pictorial representation of the combined effect in a capillary, when EO is faster than EP (the common case):

The Electropherogram Detectors are placed at the cathode since under common conditions, all species are driven in this direction by EOF Detectors similar to those used in LC, typically UV absorption, fluorescence, and MS Sensitive detectors are needed for small concentrations in CE The general layout of an electropherogram:

CE Theory The unprecedented resolution of CE is a consequence of the its extremely high efficiency Van Deemter Equation: relates the plate height H to the velocity of the carrier gas or liquid Where A, B, C are constants, and a lower value of H corresponds to a higher separation efficiency

CE Theory In CE, a very narrow open-tubular capillary is used
No A term (multipath) because tube is open No C term (mass transfer) because there is no stationary phase Only the B term (longitudinal diffusion) remains: Cross-section of a capillary:

Sample Injection in CE Hydrodynamic injection Electrokinetic injection
uses a pressure difference between the two ends of the capillary Vc = Pd4 t 128Lt Vc, calculated volume of injection P, pressure difference d, diameter of the column t, injection time , viscosity Electrokinetic injection uses a voltage difference between the two ends of the capillary Qi = Vapp( kb/ka)tr2Ci Q, moles of analyte vapp, velocity t, injection time kb/ka ratio of conductivities (separation buffer and sample) r , capillary radius Ci molar concentration of analyte

Joule Heating Joule heating is a consequence of the resistance of the solution to the flow of current if heat is not sufficiently dissipated from the system the resulting temperature and density gradients can reduce separation efficiency Heat dissipation is key to CE operation: Power per unit capillary P/L  r2 For smaller capillaries heat is dissipated due to the large surface area to volume ratio capillary internal surface area = 2 r L capillary internal volume =  r2 L End result: high potentials can be applied for extremely fast separations (30kV)

Advantages and Disadvantages of CE
Offers new selectivity, an alternative to HPLC Easy and predictable selectivity High separation efficiency (105 to 106 theoretical plates) Small sample sizes (1-10 ul) Fast separations (1 to 45 min) Can be automated Quantitation (linear) Easily coupled to MS Different “modes” (to be discussed) Disadvantages Cannot do preparative scale separations Low concentrations and large volumes difficult “Sticky” compounds Species that are difficult to dissolve Reproducibility problems

Common Modes of CE in Analytical Chemistry
Capillary Zone electrophoresis (CZE) Capillary gel electrophoresis (CGE) Capillary electrochromatography (CEC) Capillary isoelectric focusing (CIEF) Capillary isotachophoresis (CITP) Micellar electrokinetic capillary chromatography (MEKC)

Capillary Zone Electrophoresis (CZE)
Capillary Zone Electrophoresis (CZE), also known as free-solution CE (FSCE), is the simplest form of CE (what we’ve been talking about). The separation mechanism is based on differences in the charge and ionic radius of the analytes. Fundamental to CZE are homogeneity of the buffer solution and constant field strength throughout the length of the capillary. The separation relies principally on the pH controlled dissociation of acidic groups on the solute or the protonation of basic functions on the solute.

Capillary Gel Electrophoresis (CGE)
Capillary Gel Electrophoresis (CGE) is the adaptation of traditional gel electrophoresis into the capillary using polymers in solution to create a molecular sieve also known as replaceable physical gel. This allows analytes having similar charge-to-mass ratios to also be resolved by size. This technique is commonly employed in SDS-Gel molecular weight analysis of proteins and in applications of DNA sequencing and genotyping.

Capillary Isoelectric Focusing (CIEF)
Capillary Isoelectric Focusing (CIEF) allows amphoteric molecules, such as proteins, to be separated by electrophoresis in a pH gradient generated between the cathode and anode. A solute will migrate to a point where its net charge is zero. At the solute’s isoelectric point (pI), migration stops and the sample is focused into a tight zone. In CIEF, once a solute has focused at its pI, the zone is mobilized past the detector by either pressure or chemical means. This technique is commonly employed in protein characterization as a mechanism to determine a protein's isoelectric point.

Capillary Electrochromatography (CEC)
Capillary Electrochromatography (CEC) is a hybrid separation method CEC couples the high separation efficiency of CZE with the selectivity of HPLC Uses an electric field rather than hydraulic pressure to propel the mobile phase through a packed bed Because there is minimal backpressure, it is possible to use small-diameter packings and achieve very high efficiencies Its most useful application appears to be in the form of on-line analyte concentration that can be used to concentrate a given sample prior to separation by CZE

An Example of CEC

Micellar Electrokinetic Capillary Chromatography
Micellar Electrokinetic Capillary Chromatography (MECC OR MEKC) is a mode of electrokinetic chromatography in which surfactants are added to the buffer solution at concentrations that form micelles. The separation principle of MEKC is based on a differential partition between the micelle and the solvent (a pseudo-stationary phase). This principle can be employed with charged or neutral solutes and may involve stationary or mobile micelles. MEKC has great utility in separating mixtures that contain both ionic and neutral species, and has become valuable in the separation of very hydrophobic pharmaceuticals from their very polar metabolites. Analytes travel in here Sodium dodecyl sulfate: polar headgroup, non-polar tails

Micellar Electrokinetic Capillary Chromatography
The MEKC surfactants are surface active agents such as soap or synthetic detergents with polar and non-polar regions. At low concentration, the surfactants are evenly distributed At high concentration the surfactants form micelles. The most hydrophobic molecules will stay in the hydrophobic region on the surfactant micelle. Less hydrophobic molecules will partition less strongly into the micelle. Small polar molecules in the electrolyte move faster than molecules associated with the surfatant micelles. The voltage causes the negatively charged micelles to flow slower than the bulk flow (endoosmotic flow).

Capillary Electrophoresis: Summary
CE is based on the principles of electrophoresis The speed of movement or migration of solutes in CE is determined by their charge and size. Small highly charged solutes will migrate more quickly then large less charged solutes. Bulk movement of solutes is caused by EOF The speed of EOF can be adjusted by changing the buffer pH The flow profile of EOF is flat, yielding high separation efficiencies

Sample Preparation

Objectives of Sample Preparation
Range: The analyte concentration is in the measurable range of the instrument Selectivity: The purity (interference) complements the chromatographic system Recovery: The recovery is quantitative or reproducible from sample to sample Stability: The analyte is stable until the sample is analyzed Compatibility: The sample extract’s solvent is compatible with the chromatographic system

Sample Preparation Discussion
Types of Samples Drug Substances Drug Products (Tablets, Capsules, Transdermal Patches, Oral Solutions) Plasma samples General Principles Solubility Physiochemical interactions Phase equilibrium Specific Techniques Liquid-Liquid extraction Solid Phase extractions Solid phase microextraction Protein precipitation Derivitization Standards Internal/External Standards Standard addition

Drug Substance (API) Sample
Relatively pure Choice of solvent is relatively simple Should result in clear solution Need high precision

Tablets and Capsules Excipients: inactive ingredients Sample prep
Chosen for their solid state properties Fillers Cellulose, lactose, calcium phosphate Glidants or lubricants (<5%) Magnesium stearate Disintegrants Sodium starch glycolate, polyvinylpyrolidone (<1%) Others Gelatin, colors, flavors, polymers Sample prep Disintegration, dissolution of active Overcome adsorption to fillers

Transdermal Patch Excipients
Insoluble adhesive: silicone, polybutylene Inert laminates for backing and release liner Manufacturing processes: may involve manipulation of solvent strength resulting in a solid solution Dissolve adhesive, then extract drug or Dissolve drug directly Inject extract

Oral Solution Water, cosolvent (glycerin, propylene glycol)
Direct injection Dilute, then inject

Plasma Samples Proteins Trace levels of drug
Bound to protein Drug, metabolites, drug conjugates with amino acids Bound, free drug

General Principles Solubility Physicochemical interactions
Ionic, hydrophobic interactions, hydrogen bonds Sample prep also involves covalent bonds Conjugate metabolites, derivatives Phase equilibrium

Solubility Understand Saturation Solubility Super saturation
pH dependence Ionic Strength Co-solvents in small amounts can have a dramatic effect Acetaminophen in water: 11 mg/mL Acetaminophen in 2% ethanol: >20 mg/mL

Solvent Extraction Methods
Extraction of organic acids and bases utilizing their un-ionized and ionized forms. Salts of organic bases (sulfates and hydrochlorides) are often water soluble and their free bases are soluble in polar organic solvents like chloroform and ethanol or mixtures of these. Sodium and potassium salts of organic acids are water soluble and their un-ionized acids are usually soluble in organic liquids. These properties are used as an advantage in designing extraction procedures.

liquid-liquid extraction isolation from liquid sample partitioning
commonly available equipment and reagents solid phase extraction step-gradient liquid chromatography mechanisms supplies are commonly available, including automated devices column-switching in-line biological prep combines various liquid chromatography selectivities complex computer controlled valve system solid phase microextraction isolation from liquid samples direct immersion or headspace partition or adsorption from solution or gas inexpensive segments of coated fused silica protein precipitation protein removal for plasma sample analysis decreases protein solubility using special reagents ultrafiltration molecular weight selective membranes uses special filtration and low speed centrifuge dialysis molecular weight selective membranes combined with osmotic pressure dialysis membranes drug conjugates hydrolysis chemical hydrolysis of drug metabolites hydrolysis direct HPLC injection analysis of plasma samples with no sample preparation achieves molecular weight selectivity using specially designed bonded silica or maintains protein solubility with surfactants some methods require expensive columns Technique Application Principles Requirements derivatization chemical reaction to improve separation or detection treatment of active hydrogen functional group with electrophillic reagent small volumes of reactive derivatization reagents

Application of liquid/liquid extraction: sample preparation of drugs from a transdermal patch
Dissolve adhesive in hexane Extract drug from hexane into water:methanol Inject water:methanol layer onto HPLC

Liquid extractions Poor efficiency can be overcome by pooling multiple extracts Sensitivity can be enhanced by evaporating large volume of extract and re-dissolving in a smaller volume

Solid Phase Extraction (SPE)
Based on the use of prepared cartridges of a small amount of stationary phase. Similar to HPLC bonded silica, larger particle size (about 40 um) The column is washed with 5-10 volumes of solvent to condition the column (for C18, it’s methanol, then water). Analyte is loaded onto column in a solvent which is too weak to elute it from the column. The sample solvent passes through the column leaving the analyte and impurities stuck to the adsorbent bed. The column is washed with a solvent that will elute as many impurities as possible while leaving the analyte on the column. The analyte is eluted with an appropriate solvent. Analyte may be injected, evaporated to concentrate, derivatized, etc.

Solid Phase Extraction (SPE)

Considerations with SPE
Flow rate : not too fast. Capacity of sorbent gel: 1-5% of their mass (100 mg cartridge 1-5 mg). pH Careful choice of washing solvents. Elution solvent must overcome the interaction of the analyte with the bonded phase as well as with the silanols.

Specialty SPE Borate gels: immobilized alkyl boronic acids have selective affinity for 1,2 or 1,3 diols. Immunoaffinity gels: based on immobilized ligands which have a high affinity for a particular analyte.

Solid Phase Micro-extraction
Based on use of capillary GC column technology Developed in 1989 Eliminate problems associated with Liquid Liquid extraction and SPE Integration of steps Sampling Extraction Concentration Sample introduction Single solvent free step

Applications for SPME Include the following:
Volatile Organic Compounds Organic Volatile Impurities Fat soluble vitamins in tablets Drugs, alcohol, and organic solvents in liquid biological fluids Determination of fatty acids and esters Quality of fermentation products Flavor profiles Biologically active leachables Fast analysis for protein identification Microextraction of drugs Toxic impurities Stimulants and narcotics Surfactants Pesticides and herbicides Explosives Air- and water-born organic volatiles

Step 1: Extraction Expose sample as solution or gas. Sample and standard matrix should be same. Until equilibrium is reached Absorption, diffusion to inner layer Step 2: Transfer of SPME to chromatograph Beware of desorption of analyte or adsorption of contaminants Step 3: Desorption 100%: must be quantitative Thermal desorption inside the GC splitless injector for fiber configuration Flow through mobile phase HPLC or GC for tube configuration Can’t use split flow due to sensitivity

Solid Phase Micro-Extraction
SPME is an equilibrium sampling method: The amount of analyte absorbed by the fiber coating at equilibrium is proportional to analyte concentration in a sample. This relation is described by the following equation: where n = analyte mass absorbed by the fiber coating, Kfs is the fiber coating/sample matrix distribution constant, Vf is the fiber coating volume, Vs is the sample volume, Co is the initial concentration of a given analyte in a sample. Fused Silica Fiber KfsVfVsCo KfsVf + Vs n = Sample matrix Polymeric coating

Protein Precipitation
For blood or plasma samples Addition of protein denaturing agent and subsequent centrifugation Acetonitrile, methanol, perchloric acid, trichloroacetic acid Direct injection of supernatant Typically releases drug from non-covalent protein binding

Ultrafiltration Membrane filters with MW cutoff below albumin
Used to measure free drug levels

Analytical Derivatization
Enhance detectability: sensitivity, selectivity For HPLC analysis For Mass spec analysis GC Required prior to GC if compound is highly polar or non- volatile. In the underivatised GC the strongest base gives poor peak shape. due mainly to polar hydroxyl groups. The polar groups can be masked with a reaction with trifluoroacetic anhydride (TFA). This reagent is useful because it is very reactive, boils at 40oC and leaves no residue. It will not react with tertiary amines.

Derivatives for HPLC Add a chromophore Add a fluorescent group
Add a bulky group to facilitate chiral recognition on a chiral column Add a second chiral center to form a diastereomer to facilitate separation on an achiral column

External Standards HPLC instrumentation is capable of high precision and thus amenable to the use of external standards for quantitative analysis If complete recovery can be guaranteed then the area of a HPLC peak obtained from a known weight of a formulation can be compared directly with a calibration curve constructed using a series of solutions containing varying concentrations of a pure standard. The use of a single point calibration can also be justified since in QC applications the content of the formulation is unlikely to vary by  10% from the stated content.

Internal Standards Poor recovery or erratic recovery during sample preparation Poor control of solvent volumes Very useful for formulation with complicated sample preparation. Some liquid extractions may be less than quantitative (<100%), e.g. creams, ointments, controlled release patches and other advanced dosage forms. Biological samples. Reponse factor is based on ratio of the analyte relative to the IS i.e. a ratio of the areas of the peaks obtained for equal amounts of IS and analyte. (Ideally this would be close to one for equal amounts of both.) The response factor may be based on a single point calibration or more typically a mulit-point calibration curve. Once the response factor is determined the sample is extracted with a solution containing the same concentration of IS as was used in determining the response factor (or a solution which after dilution will yield an extract in which the IS is at the same concentration as in the calibration solution). As long as the solution containing the fixed concentration of internal standard is added to the sample in a precisely measured volume, any loss of sample is compensated for since the losses of the analyte will be the same. Also a cure for poor injection precision. Structure similarity is not as critical if injection precision is the only goal, not extraction.

Properties of an internal standard
Stable Resolved from analyte and excipients. Should elute near the analyte. For a given weight should produce a detector response similar to that of the analyte. Ideally should be closely related in structure to the analyte

Method of standard addition
Even using an internal standard, a standard solution must be made. If the sample is unique and a standard matrix cannot be obtained that suitably mimics the sample….standard addition. Example: you work for a forensic lab that needs a brain alcohol level. The sample is used as its own standard. Known amounts of standard are added to at least two aliquots of the sample. Extract and inject, including unspiked sample. Plot the response v. added concentration The (-)x-intercept is the sample concentration

4 measurements Sample concentration is -x intercept

HPLC Sample Solvent Injection Effects
Avoid distortion of the chromatographic peak Injection solvent should be mobile phase or weaker solvent strength Can cause tailing, fronting, splitting or RT shift. Other factors: injection volume, retention time/volume, pH, ionic strength, buffering capacity of MP. Solutions: Use weaker solvent for sample, std Inject less volume Dilute with weak solvent (water for RP HPLC) Change solvents: evaporate and reconstitute

Detector selectivity can save lots of sample prep method development and analysis time.
Nitrogen detectors GC, HPLC Mass detectors Allow monitoring only selective ions that may be unique to the analyte. May be M+1,M-1 or fragment.

Current Trends Minimize sample preparation
Dilute and shoot Filter? The best sample preparation is no sample preparation: precise, accurate Direct injection Direct Measurement NIR Raman Selective detectors (mass spec, tandem mass spec)

End of Talk

Theory, Application of TLC, HPLC, CE Separation Methods: Part 2

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Theory, Application of TLC, HPLC, CE Separation Methods: Part 2

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