HPLC

Dr Naved Malek Assistant Professor Applied Chemistry Dept SVNIT

[email protected] +91-982 5746 660 +91-261 2201 794

Liquid-Column Chromatography
LC can be carried out using a glass tube hand-packed with a stationary phase (solid) through which a solvent is allowed to gravity-flow. So why do we need all the complicated high-tech equipment? One Word: SPEED A single analysis by "Classical" LC can take anywhere from 2 to 12 hours to carry out. HPLC allows an equivalent analysis to be done in 2 to 12 minutes. Reproducibility. A classical column must be freshly packed for each analysis, increasing the chance of errors. A single HPLC column can be used for hundreds or thousands of samples.

High Performance Liquid Chromatography (HPLC)

What is HPLC? Types of Separations Columns and Stationary Phases Mobile Phases and Their Role in Separations Injection in HPLC Detection in HPLC

Variations on Traditional HPLC Ion Chromatography Size Exclusion Chromatography

What is HPLC?
High Performance Liquid Chromatography High Pressure Liquid Chromatography (usually true] Hewlett Packard Liquid Chromatography High Priced Liquid Chromatography HPLC is really the automation of traditional liquid chromatography under conditions which provide for enhanced separations during shorter periods of time! Probably the most widely practiced form of quantitative, analytical chromatography practiced today due to the wide range of molecule types and sizes which can be separated using HPLC or variants of HPLC!!

What is HPLC?
High-Performance Liquid Chromatography Developed in 1960s as faster way to do column chromatography Advantages over traditional chromatography include:
Speed Adaptability resolution sensitivity columns reusable

HPLC
Popularity:
Widely applicable to numerous fields of study; both academic, industrial, and biomedical.

Great for separation of non-volatiles:

Amino acids, proteins, nucleic acids, hydrocarbons, carbohydrates, pharmaceuticals, pesticides, terpenids, pigments, antibiotics, steroids, vitamins, and various other organic and inorganic substances.

Generally, if a compound can be solublized in common solvents such as water, alcohol, acetonitrile, acetone then HPLC can probably be used.

HPLC
One of the most widely used analytical separation techniques. Uses a liquid mobile phase to separate components in a mixture Used high or low pressure to push solvent through a separation column Popular because:
Sensitive Accurate, quantitative methods can be used Great for separation of non-volatile components, heat labile compounds, and semi-volatile compounds. Non-destructive

Basic Hardware Components of HPLC

Solvent Delivery System (Pump) Injector (introduce samples) Column guard Column (containing stationary phase) Detectors (eyes ) Waste Collector Recorder (Data Collection)

Diagram of HPLC

Modern HPLC

Types of HPLC Separations

Normal Phase: Separation of polar analytes by partitioning onto a polar, bonded stationary phase. Reversed Phase: Separation of non-polar analytes by partitioning onto a non-polar, bonded stationary phase. Adsorption: In Between Normal and Reversed. Separation of moderately polar analytes using adsorption onto a pure stationary phase (e.g. alumina or silica) Ion Chromatography: Separation of organic and inorganic ions by their partitioning onto ionic stationary phases bonded to a solid support. Size Exclusion Chromatography: Separation of large molecules based in the paths they take through a maze of tunnels in the stationary phase.

What does the analyst do?

Select the correct type of separation for the analyte(s) of interest, based on the sample type (among other factors). Select an appropriate column (stationary phase) and mobile phase Select an appropriate detector based on whether universal or compoundspecific detection is required or available

Optimize the separation using standard mixtures

Analyze the standards and sample

Ideal pumps: 1) Ability to generate high pressure 2) Pulse-free output 3) Accurate control of flow 4) Corrosion resistant Role: Deliver the mobile phase Two groups of pumps: 1) Constant pressure 2) Constant volume Three types of pumps are available: 1) Reciprocating pumps (90% of Commercial HPLC; produce pulse flow) 2) Displacement pumps (produce flow that are independent of viscosity and back pressure) 3) Pneumatic pumps (cannot do gradient and pressure less than 2000psi)

Pumps
Reciprocating Pumps Displacement pumps Pneumatic Pumps

LC Pumping Systems
Reciprocating Pumps
Pulsed flow must be damped Small internal volume High output pressures Ready Adaptable for gradient elution Constant flow rates independent of column back-pressure or solvent viscosity Back and forth mechanism Flow independent of viscosity and back-pressure Limited solvent capacity Inconvenient to change solvents Screw-driven mechanism Inexpensive Pulse free Limited capacity and pressure Dependent on solvent viscosity and backpressure Not good for gradient elution

Displacement Pumps

Pneumatic Pumps

Typical HPLC Pump (runs to 4,000+ psi)

Most common injector is sample loop (5-500uL; 0.5-5uL)

Injection in HPLC
Usually 5 to 1000 L volumes, all directly onto the column not much worry about capacity since the columns have a large volume (packed). Injector is the last component before the column(s) A source of poor precision in HPLC errors of 2-3 %RSD are due just to injection other errors are added to this due to capillary action and the small dimensions/cavities inside the injector 6-PORT Rotary Valve is the standard manual injector Automatic injectors are available Two positions, load and inject in the typical injector Injection loop internal volume determines injection volume.

LOAD (the sample loop)

Inject (move the sample loop into the mobile phase flow)

Columns
Analytical column variables Length (10-30 cm) ID (4-10mm) Packing (many kinds) Particles sizes (3-10 m);pore sizes Most common columns 25cm x 4.6 id with 5m particles Preparative columns

Columns and Stationary Phases

HPLC is largely the domain of packed columns some research into microbore/capillary columns is going on. Molecules move too slowly to be able to reach and therefore spend time in the stationary phase of an open tubular column in HPLC. In solution, not the gas phase Larger molecules in HPLC vs. GC (generally) Stationary phases are particles which are usually about 1 to 20 m in average diameter (often irregularly shaped) In Adsorption chromatography, there is no additional phase on the stationary phase particles (silica, alumina, Fluorosil). In Partition chromatography, the stationary phase is coated on to (often bonded) a solid support (silica, alumina, divinylbenzene resin)

Stationary Phases
Polar (Normal Phase): Silica, alumina Cyano, amino or diol terminations on the bonded phase Non-Polar (Reversed Phase) C18 to about C8 terminations on the bonded phase Phenyl and cyano terminations on the bonded phase Mixtures of functional groups can be used!! Packed particles in a column require: Frits at the ends of the column to keep the particles in Filtering of samples to prevent clogging with debris High pressure pumps and check-valves Often a Guard Column to protect the analytical column

Optimization of Separations in HPLC

Correct choice of column so the above equilibrium has some meaningful (non-infinity, non-zero) equilibrium constants. Correct choice of mobile phase Decision on the type of mobile phase composition constant composition = isocratic varying composition = gradient elution Determination if flow rate should be constant usually it is Decision on heating the column heating HPLC columns can influence the above equilibrium.

The Mobile Phase in HPLC...

Must do the following: solvate the analyte molecules and the solvent they are in be suitable for the analyte to transfer back and forth between during the separation process Must be: compatible with the instrument (pumps, seals, fittings, detector, etc) compatible with the stationary phase readily available (often use liters/day) of adequate purity spectroscopic and trace-composition usually! Not too compressible (causes pump/flow problems)
Free of gases (which cause compressability problems)

Polarity Index for Mobile Phases..

The polarity index is a measure of the relative polarity of a solvent. It is used for identifying suitable mobile phase solvents. The more polar your solvent is, the higher the index. You want to try to choose a polarity index for your solvent (or solvent mixture) that optimizes the separation of analytes usually the index is a starting point the polarity of any mixture of solvents to make a mobile phase can be modeled to give a theoretical chromatogram Usually, optimization of solvent composition is experimental A similar number is the Eluent Strength (Eo] Increasing eluent strength or polarity index values mean increasing solvent polarity. Remember, the analyte(s) and samples must be mobile phase and stationary phase compatible!

Optimization of Mobile Phase Polarity Changing the mobile phase composition alters the separation.

Isocratic versus Gradient Elution

Isocratic elution has a constant mobile phase composition Can often use one pump! Mix solvents together ahead of time! Simpler, no mixing chamber required Limited flexibility, not used much in research mostly process chemistry or routine analysis. Gradient elution has a varying mobile phase composition Uses multiple pumps whose output is mixed together often 2-4 pumps (binary to quarternary systems) Changing mobile phase components changes the polarity index can be used to subsequently elute compounds that were previously (intentionally) stuck on the column Some additional wear on the stationary phase Column has to re-equiluibrate to original conditions after each run (takes additional time).

Detectors
Visualize separated compounds and translate the concentration changes into signals Using every conceivable physical and chemical properties Characteristics of an ideal detector
Adequate sensitivity Good stability and reproducibility Gives linear response to analysts that have several ranges magnitudes Short response time High reliability and ease of use Similarity in response toward all analyst Non-destructive

Detectors
Two basic types of detectors
Bulk property detectors response to a mobile-phase bulk property
Refractive index Dielectric constant Density

Solute property detectors response to solute property

Spectroscopy (IR, UV, MS, Fluorescence)

Detection in HPLC
Numerous Types (some obscure) Original HPLC Detectors were common laboratory instruments such as spectrophotometers, etc. you can even use a SPEC 20! Usually a narrow linear range (1E3, usually) Must be solvent -compatible, stable, etc. Universal respond to all analytes Analyte Specific respond to specific properties of analytes Non-destructive most Destructive MS and a few others.

Most Common HPLC Detectors

Absorbance (Absorption of UV-Vis based on Beers law) Fluorescence Electrochemical Refractive index Conductivity Mass spectrometry FI-IR Light scattering

Popular Types of Detectors

UV/Visible absorption detectors Fluorescence Refractive Index (RI) Electrochemical (ED) not in syllabus

Standard Absorbance Detector.

Single Beam UV-VIS instrument with a flow-through cell (cuvette) Can use any UV-VIS with a special flow cell
Extra connections lead to band-broadening if UV-VIS is far from HPLC column exit.

Usually utilize typical UV-VIS lamps and 254 nm default wavelenth

Can be set to other wavelengths (most) Simple filter detectors no longer widely used
adjustable wavelength units are cost-effective

Non-destructive, not-universal
not all compounds absorb light can pass sample through several cells at several different wavelenghts

Usually zeroed at the start of each run using an electronic software command. You can have real-time zeroing with a reference cell.

UV/Vis Absorbance Detectors

Compounds with strong UV/Vis chromophores Compounds with conjugated or nonconjugated double bonds; aromatic molecules Advantage: Simple, reliable, inexpensive, compatible with gradient elution and non-destructive Disadvantage: Not as sensitive as fluorescence detection, not-universal (only for molecules with chromophores) Samples of use: vitamins, carotenoids, phytonutrients UV/Vis - you can purchase fixed wavelength variable wavelength diode array

UV/Vis Absorbance Detectors

Light sources: Deuterium or tungsten filament sources

The Variable Wavelength UV Detector uses a monochromator (slits and a grating) to select one wavelength of light to pass through the sample cell.

The Photodiode Array Detector passes all wavelengths of light through the sample cell, then focuses each wavelength on a single sensor element.

Diode Array Detector (DAD)

The more common tool for research-grade HPLC instruments quite versatile... Advances in computer technology since ~1985 or so have lead to the development of Diode Array instruments Non-destructive, non-universal DAD scans a range of wavelengths every second or few seconds. At each point in the chromatogram one gets a complete UV-VIS spectrum! Huge volumes of data Detailed spectra for each peak and each region of each peak

Absorbance Detector Output

Fluorescence Detectors
Compounds with fluorophors By nature (carbamate pesticides, aflatoxins, vitamins, amino acids) By post-column derivatization Advantage: Highly sensitive, low background, highly selective (two distinct wave lengths instead of one in Ab detector), can solve co-elution problems, post-column derivatization can be used for this detection Disadvantage: Perceived difficulty of its use, more instrumental variables to account for during optimization, changes in fluorescence can occur with pH and viscosity Samples of use: vitamins E, drugs, aflatoxins

Fluorescence Detector Configuration

Refractive Index Detectors

Compounds that do not have strong UV/Vis chromophores, fluorophours, electrochemical activity or ionic conductivity Advantage: Universal in nature Disadvantage: Lack of sensitivity; impractical for gradient elution; instability of base line Samples of use: organic acids, sugars, fungal metabolites, oligosaccharides

Refractive Index Detector

One of a very few Universal HPLC detectors. Non-destructive Responds to analytes changing the RI of the mobile phase requires a separate reference flow of mobile phase Extremely temperature sensitive, usually heated sensitive to temp changes of +/- 0.001 C No longer really widely used Absorbance detectors are relatively cheap. Useful for process work, on-line monitoring, etc.

Recorder/Data Collections
Many recoding devices are available:
Strip-chart recorder (retention time/Peak areas or peak height) Integrator Computer controlled data collections

ELSD (Evaporative Light Scattering Detector)

Universal, destructive Useful for very large molecules, and a wide linear range Analytes are de-solvated in the detector Molecules pass through what is essentially a large cuvette for a UV-VIS instrument The reduction in light intensity detected (due to scattering by the analytes) is measured The larger and more concentrated a particular molecule is, the greater the scattering.

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