High Performance
Liquid Chromatography
HPLC
originally refered to:
High Pressure Liquid Chromatography
currently refers to:
High Precision Liquid Chromatography
– high pressure to be able to use small particle
size to allow proper separation at reasonable
flow rates
“Applications of
Liquid
Chromatography”
Partition chromatography
Adsorption, or liquid-solid
chromatography
Ion exchange chromatography
Size exclusion, or gel,
chromatography
Components
Solvent Reservoir and Degassing System
Pumps
Precolumns
Sample Injection System
Columns
Temperature Control
Detectors
“Schematic
of an
apparatus
for
HPLC”
HPLC - Resolution
• Resolution (RS) of a column provides a quantitative measure of its
ability to separate two analytes
Rs = Z /1/2(WA+WB)
Rs =
HPLC - Resolution
Capacity Factor (k’): Also called retention
factor. Is a measure for the position of a
sample peak in the chromatogram.
k’ = (tR1-to)/to
• specific for a given compound and constant
under constant conditions
• A function of column and mobile phase chemistry
• Primarily applicable under isocratic conditions
• In general, a change in the k’ of one peak will
move all peaks in the same direction.
Selectivity Factor (): Also called
separation or selectivity coefficient is
defined as
= k2’/k1’ = (tR2-to) / (tR1-to)
• A function of column and mobile phase chemistry
• Primarily applicable under isocratic conditions
• Changes in selectivity will affect different
Skoog and Leary:
compounds in different ways.
Principals of Instrumental Analysis, 4 th ed. Suanders, 1992
Phenomenex catalog, 1999
HPLC - Resolution
Theoretical Plates (N): The number of
theoretical plates characterizes the
quality or efficiency of a column.
N = 5.54 [(tR) / w1/2]2 (N = 16 (tR/W)2)
Skoog and Leary: Principals of Instrumental Analysis, 4 th ed. Suanders, 1992
RP Optimization
RP Optimization
Efisiensi Kolom: Pers. Van Deemter
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
(TLC vs Normal Phase and Reverse Phase HPLC)
Normal Phase (SiO2) TLC Normal Phase (SiO2)
a b c
a Time
0
b
Reverse Phase (C18)
x c b a
0 Time
RP-HPLC – Stationary Phase
Skoog and Leary: Principals of Instrumental Analysis, 5 th ed. Suanders, 1998
Effect of Solvent
Varying Selectivity
30% MeCN 45% MeOH
70% Water 55% Water
30x0.46 cm C-18, 1.5
Snyder and Kirkland, introduction
mL.min, to Modern Liquid
254 nm, 10 g each Chromatography, Wiley, 1979, p.
287.
pH
• Affects ionizable compounds
– organic acids
– organic bases
• In reversed phase we need to suppress
ionization as much as possible
• May need very precise pH control
Use of Buffers
• 0.1 pH unit ---> significant effect on
retention
• Buffer mobile phase for pH reproducibility
• pH of buffer should be within 1 pH unit of
pKa of acid (best at pH = pKa)
• Buffers weak (100 mM or less)
• Check solubility
Common buffers
Buffer pKa Values
Phosphate 2, 7
Acetate 4.75
Citrate 3.08, 4.77, 6.40
Useful buffering between pH 2-8.
Solvent Strength
Snyder and Kirkland, Introduction to Modern Liquid Chromatography, Wiley, 1979, p. 286.
Components
Solvent Reservoir and Degassing System
– isocratic elution - single solvent separation
technique
– gradiant elution - 2 or more solvents, varied
during separation
Isocratic mixture of
A (aqueous buffer)
and B (acetontrile)
Elusi isokratik:
"dilakukan dengan
pelarut tunggal (atau
campuran pelarut
konstan)"
Gradient Elution
Elusi gradien:
“perubahan
pelarut yang
terus menerus
komposisi untuk
meningkatkan
kekuatan eluen "
HPLC OF ANALGESICS - UV Detection
Gradient =
Standard Analgesics 0 min: 100% EtOAC (+ 0.2% HOAc)
3 min: 100% EtOAC (+ 0.2% HOAc)
2.82 min 5 min: 15% MeOH, 85% % EtOAc
Acetaminophen (+ 0.2%
HOAc)
8 min: 15% MeOH, 85% % EtOAc
(+ 0.2% HOAc)
10 min: 100% EtOAC (+ 0.2% HOAc)
1.48 min. SiO2
Aspirin Flow Rate = 1 mL/min
UV detector set at 240 nm
7.11 min.
Caffeine
Analgesic tR
Acetaminophen 2.82
Aspirin 1.48
1.35 min.
Ibuprofen Caffeine 7.11
Ibuprofen 1.35
Column: 5C18-PAQ
Standard Column size: 4.6mm I.D.-250mm
Mobile phase: Methanol/ 5mmol/l
Sodium 1-Hexanesulfonate,
20mmol/l Phosphoric Acid = 15/85
Flow rate: 1.0 ml/min
Temperature: 30°C
Detection: UV220nm
Injection Vol. 1.0 µl
Standard
Sample:
1; Citric Acid (10mg/ml)
Sample: minuman 2; L-Carnitine (20mg/ml)
3; Nicotinamide (0.2mg/ml)
4; Vitamin B6 [Pyridoxine] (0.2mg/ml)
5; Vitamin B1 [Thiamine] (0.2mg/ml)
6; Caffeine (0.2mg/ml)
7; Flavin Mononucleotide [FMN]
(0.2mg/ml)
Improvement in separation
efficiency by gradient
elution. Column: 1m x
2.1mm id, precision-bore
stainless; packing: 1%
PermaphaseR ODS.
Sample: 5L of chlorinated
benzenes in isopropanol.
Detector: UV photometer
(254 nm). Conditions:
temperature 60oC,
pressure, 1200 psi.
Components
Pumps
– 1000 - 6000 psi at rate of @ 3 mL/min
– reproducible to 2%
– screw driven syringe
– reciprocating pump
“A reciprocating
pump for HPLC.”
Components
Precolumns
– remove impurities from solvent
– saturates mobile phase with liquid of
stationary phase before the analytical column
Components
Sample Injection System
– sample valve
– syringe
“A sampling loop for liquid chromatography”
Components
http://www.youtube.com/watch?v=mPO7Tv2mIJU
Columns
– straight, 15 to 150 cm in length; 2 to 3 mm i.d.
– packing - silica gel, alumina, Celite
Normal phase – Polar Stationary Phase and Nonpolar Solvent
Reverse phase – Nonpolar Stationary Phase and Polar Solvent
Size exclusion (gel permeation) - Small molecules penetrate
into the pores within the packing while larger molecules only
partially penetrate the pores. The large molecules elute before
the smaller molecules.
Effect of Particle Size
Columns Cont.
Components
Temperature Control
Components
Detectors
– single wavelength UV
– tuneable UV
– diode-array
– refractive index
– MS
Performances of LC Detectors
Ultraviolet
detector cell for
HPLC
Diode-Array
Absorption
Spectra
“Absorption
spectra of the
eluent from a
mixture of
three steroids
taken at 5-second
intervals.”
