compound between 2 phases:
solid stationary phase & liquid mobile phase
(i.e. Liquid Chromatography) done under high
pressure.
Modes of separation:
1- partition.
2- adsorption.
3- ion exchange.
Separation, Qualification, Quantitation: Analytical.
Chromatography
| Gas Chromatography | LiquidChromatography |
Mobile phase : Gas | Mobile phase : Liquid |
Volatility of sample | Solubility of sample |
M.W. < 50 | M.W. > 500 |
Thermal Stability | Ambient temperature |
Difference of boiling point | Difference of boiling |
Suitable for substances that can't be separate by GC [non-volatile substances]That need volatilization or derivatization to be volatile.
short time for analysis.
Elution
Elution is described by minutes Or by Capacity Factor [K]
The higher the affinity between substance [analyte] & mobile phase leads to shorter time for analysis [& vice versa].
Only compounds with different capacity factors can be separated by HPLC.
1.Physico-chemical properties of the analyte.
2.Composition & flow rate of the mobile phase.
3.Surface area , particle size & column length of
stationary phase.
The chromatographic system
10ml/min.
Usually one or more pump used under control of a
computer.
Pumps should be capable of delivering mobile phase at a constant reproducible flow rate. - Stability of flow rate & pressure. - Easy to use, use of variable solvents. - Pulse elimination system.
Notice for Pump operation
Degassing of solvent.
2. Do not use when solvent is absent.
3. Rinse with water after use of buffer.
4. Check Miscibility if multiple solvents.
5. Pump priming before use.
6. Pulse control.
7. Check pump pressure.
Types of elution
There are 2 types of elution :
1- Isocratic elution : fixed ratio of mobile phase
components during the analysis.
2- Gradient elution (mobile phase programming) :the components of mobile phase ratio is changed
through out the analysis for separation of components very close in polarity.
Elution
Isocratic elution for simple mixtures.
Gradient elution for complex mixture.
DO NOT used detectors that are sensitive to
change in solvent composition [Refractometer].
Isocratic & gradient elution
Mobile phase
The elution solvent (mobile phase):
HPLC grade [highly pure].
Particle free & degassed.
Water used should be of specific quality with low conductivity & low UV absorption.
Low Viscosity.
Miscibility of solvents (miscibility difference : below 15).
Solubility of the sample in the mobile phase.
Injectors
Sample dissolved in suitable solvent [with the same composition of mobile phase] giving.
particle-free solution injected into system.
Injector composed of:
Injection pore: in which needle introduced.
Fixed volume loop: which hold certain volume of the sample transferred into chromatograph.
Injector
-Manual injector (Valve injector): low accuracy.
-Automatic injector: high accuracy & speed.
Change loop volume for injection volume change
Column (stationary phase)
According to the mode of separation:
1- Normal phase:
Stationary phase (Polar)
obile phase (Non-Polar)
2-reversed phase
Stationary phase (Non-Polar)
M0bile phase (Polar)
Normal phase chromatography
Stationary phase (Polar)
Mobile phase (Non-Polar)
Non polar material elutes first.
Reversed phase chromatography
Stationary phase (Non polar)
Mobile phase (Polar)
P0lar material elutes first.
Bonded type stationary phase
Most polar column silica bonded with very polar nitrile group (CN).
Least polar column Octadecyl silane [C18]composed of a core of silica chemically bound to
an organic phase [Long chain HC].
Types of columns
Aalytical columns: of least diameter 2-5 mm & particle size = 3-10µm.
Preparative & semi preparative columns: larger diameter.
columns shouldn’t be heated more than 60C to avoid decomposition of stationary phase.
Choice of the column
Column separates mixed compounds to individual compound.
Column Selection
- Nature of the backing Material.
- Particle Size.
- Shape of the particle.
- Pore Size.
Particle Size & shape
If particle size is small → ↑↑ resolution & back pressure
Characteristics of stationary phase
• Pore size & distribution :
- Small Pore = higher efficiency = less band broadening
• Spherical or Irregular :
- Irregular = higher Surface area = higher carbon load
- Spherical = lower pressures & better structural stability = longer life
• Particle size
- Smaller particle = higher operating pressure = higher efficiency
Detectors
UV spectrophotometer is used consist of a flow cell placed at end of column to receive portion of eluted solute.
Light of specific wavelength pass through the cell, some parts are absorbed, the others are transmitted
Specific sample has high absorbance to specific wavelength
Amount of absorbed light (A) is proportional to the
concentration of sample
A = constant X b X c
UV detector
Types of UV detectors
There are several types of UV detectors:
1- detectors operating at fixed wavelength [= 254 nm] emitted by low-pressure Hg lump].
2- detectors operating at variable l depending on lmax of the components leading to increased sensitivity [from deuterium or xenon lamp with monochromator or filter to generate monochromatic radiation at selected l].
3- Multi-wavelength detectors : measure at 2 or more selected l at the same time.
Other detectors
Refractometer ,
Fluorimetric.
Potentiometric.
Voltametric.
Polarographic or electrochemical detectors
Data collection
Data collection:
when a solute in eluted will give an absorption peak,its height or area is corresponding to the concentration of solute.
Old manual detectors: give printout of peaks or peak areas.
New detectors: provide chromatograms with accompanied peak areas & heights.
Retention time
Retention time “ t ” : time spent from injection till appearance of tip of the peak
Retention time is characteristic for each compound so a standard of the analyte must be injected to assure retention time of the substance.
Data collection
Peak area = h X W/2 = h X W h/2
Where:
h = height of the peak.
W = width at peak base line.
W h/2 = width at half height of the peak.
Capacity Factor
VR = Retention volume
V0 = Retention volume of solvent
t R = Retention time
t 0 = Retention time of solvent
t’R = Actual retention time
Selectivity
Column Efficiency
Resolution 
Resolution
Quantitative HPLC
Automatic injectors [Auto samples] | Manual injectors [a syringe] |
increases degree of accuracy in volume injected | decreases degree of accuracy in volume injected |
Use external standardto construct calibration curve to determine concentration. | Use internal standard to cancel error which may occur during manual |
External standard: ·Injectedseparately in each run. ·Of the same substance of analyte. ·comparepeak response [height or area]. | Internal standard: ·Injected with fixed concentration together with test or standard solution. ·Of different substance than analyte. ·Mustnot interfere with peaks measured. ·We compare peak response ratio. |
Ideal chromatogram
1.Peaks should be sharp & symmetric.
2. Peaks should be well separated from one another (each peak should start at the baseline and comes back down to the baseline).
3.The base line should be straight (with no drifting up or down & no underlying impurities).
4.The base line should be free from background or electric noise.
5.The chromatographic run time should be reasonable (15 minutes is ideal).
6.The developed procedure should have good sensitivity. When the analysis is repeated under the same conditions, the chromatogram should be reproducible
Esam Othman
Hanaa Hashem
