Introduction to RP-HPLC
39
RP-HPLC – BASICS S.Karthikumar., M.Sc.,M.Phil.,M.Tech.,(Ph.D) Assistant Professor, Department of Biotechnology Kamaraj College of Engineering and Technology Virudhunagar-626001, Tamilnadu, INDIA [email protected] 1
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Transcript of Introduction to RP-HPLC
RP-HPLC – BASICSS.Karthikumar., M.Sc.,M.Phil.,M.Tech.,(Ph.D)
Assistant Professor, Department of Biotechnology
Kamaraj College of Engineering and Technology
Virudhunagar-626001, Tamilnadu, INDIA
3
Invention of Chromatography by M.Tswett
Ether
CaCO3
Chlorophyll
Chromatography
Colors
Advantages of High Performance Liquid Chromatography
• High separation capacity,
• Superior quantitative capability and reproducibility
• Moderate analytical conditions• Unlike GC, the sample does not need to be vaporized.
• Generally high sensitivity
• Low sample consumption
• Easy preparative separation and purification of samples
Fields in Which High Performance Liquid Chromatography Is Used
• Biogenic substances• Sugars, lipids, nucleic acids,
amino acids, proteins, peptides, steroids, amines, etc.
• Medical products• Drugs, antibiotics, etc.
• Food products• Vitamins, food additives, sugars, organic
acids, amino acids, etc.
• Environmental samples• Inorganic ions
• Hazardous organic substances, etc.
• Organic industrial products• Synthetic polymers, additives, surfactants,
etc.
Interaction Between Solutes, Stationary Phase, and Mobile Phase
• Differences in the interactions between the solutes and stationary and mobile phases enable separation.
Solute
Stationary
phaseMobile phase
Degree of adsorption,
solubility, ionicity, etc.
8
Stationary Phase and Mobile Phase Used in Normal Phase Mode
• Stationary Phase• Silica gel: -Si-OH
• Cyano type: -Si-CH2CH2CH2CN
• Amino type: -Si-CH2CH2CH2NH2
• Diol type: -Si-CH2CH2CH2OCH(OH)-CH2OH
• Mobile Phase• Basic solvents: Aliphatic hydrocarbons, aromatic hydrocarbons, etc.
• Additional solvents: Alcohols, ethers, etc.
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Relationship between Hydrogen Bonding and Retention Time in Normal Phase Mode
OH
HO
SiOH
SiOHStrong
Weak
Steric hindrance
Very weak
?
• Polar Functional Groups• -COOH
• Carboxyl groups
• -NH2
• Amino groups
• -OH• Hydroxyl groups
Reversed Phase Chromatography• Stationary phase: Low polarity
• Octadecyl group-bonded silicalgel (ODS)
• Mobile phase: High polarity• Water, methanol, acetonitrile
• Salt is sometimes added.
Stationary
phaseMobile phase
Normal
phase
High polarity
(hydrophilic)
Low polarity
(hydrophobic)
Reversed
phase
Low polarity
(hydrophobic)
High polarity
(hydrophilic)
• Nonpolar Functional Groups• -(CH2)nCH3
• Alkyl groups
• -C6H5
• Phenyl groups
Separation Column for Reversed Phase Chromatography
• C18 (ODS) type
• C8 (octyl) type
• C4 (butyl) type
• Phenyl type
• TMS type
• Cyano type
Si -O-Si
C18 (ODS)
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH3
12
Hydrophobic Interaction
H2O
H2OH2O
H2O
H2OH2O
H2O
Network of hydrogen bonds
H2O
H2OH2O
H2O
H2O H2O
H2O
Nonpolar solute
If a nonpolar
substance is added...
…the network is broken and...
H2OH2O H2O
H2OH2O H2O
H2O
Nonpolar solute
Nonpolar stationary phase
…the nonpolar substance
is pushed to a nonpolar
location.
Comparison of Normal Phase and Reversed Phase
• Normal Phase• Effective for separation of
structural isomers
• Offers separation selectivity not available with reversed phase
• Stabilizes slowly and is prone to fluctuations in retention time
• Eluents are expensive
• Reversed Phase• Wide range of applications
• Effective for separation of homologs
• Stationary phase has long service life
• Stabilizes quickly
• Eluents are inexpensive and easy to use
HPLC - Hardware
Solvent Delivery System, Degasser, Sample
Injection Unit, Column Oven
Pump
Sample injection unit
(injector)
Column
Column oven
(thermostatic
column chamber)
Detector
Eluent
(mobile phase)
Drain
Data processor
Degasser
Flow Channel Diagram for High Performance Liquid Chromatograph
18
Solvent Delivery Pump
• Performance Requirements• Capacity to withstand high load pressures.
• Flow rate does not fluctuate.
• Solvent replacement is easy.
• The flow rate setting range is wide and the flow rate is accurate.
19
Solvent Delivery Pump:Schematic Diagram of Plunger Pump
Motor and cam
Plunger
Plunger seal
Check
valves
Pump head
10 -100µL
Pump – Mixing & Flow Rate System
• Isocratic system• Constant eluent composition
• Gradient system• Varying eluent composition
• HPGE (High Pressure Gradient)
• LPGE (Low Pressure Gradient)
Difference in the Chromatogram
• In isocratic mode
Long analysis time!!
Poor separation!!
CH3OH / H2O = 6 / 4
CH3OH / H2O = 8 / 2
• If the eluent composition is changed gradually during analysis... Gradient system
• Problems caused by dissolved air in the eluent
• Unstable delivery by pump• More noise and large baseline drift
in detector cell
In order to avoid these problems, the eluent must be
degassed.
Degasser Heated stirring is used to reduce the dissolved airin the solution in the mobile phase bottle to thesaturated solubility level.
Mixing, Filtration, and Offline Degassing of the Eluent
Decompression
by aspirator
Ultrasonic
cleaning unit
Decompression
by aspirator
Membrane filter with pore
size of approx. 0.45 µm
Online Degasser
Gas-liquid separation membrane methodHelium purge method
Helium
cylinder
To draft
To pump
Eluent container
Regulator
Drain valve
To pump
Eluent container
Polymeric film tubeVacuum chamber
Preparing pH Buffer Solution
• Use a weak acid with a pKa value close to the desired pH value.
• Example: Preparing a buffer solution for a pH value of around 4.8. Use acetic acid, which has a pKa value of 4.8.
• Make the concentrations of HA and A- roughly equal. Mix an acid with its salt.
• Example: Mix acetic acid and sodium acetate so that they have the same molar concentration.
Buffer Solutions Used for HPLC Eluent
• Requirements• High buffering power at
prescribed pH.
• Does not adversely affect detection.
• Does not damage column or equipment.
• Inexpensive.
• Commonly Used Acids• Phosphoric acid
• pKa 2.1, 7.2, 12.3
• Acetic acid• pKa 4.8
• Citric acid• pKa 3.1, 4.8, 6.4
• Concentration• If only to adjust pH, 10
mmol/L is sufficient.
pH of Eluent and Retention of Ionic Solutes
COOH
COO
H
pH of eluent
Acidic
Alkaline
+
Increased
hydrophobicity
Increased
hydrophilicity
High RT
Low RT
Replacement of Eluent
• Mutually insoluble solvents must not be exchanged directly.
• Aqueous solutions containing salt and organic solvents must not be exchanged directly.
Water
Hexane
2-Propanol
Buffer solution
Water-soluble
organic solvent
Water
Sample Injection Unit (Injector)
INJECT position
LOAD position
From pump
To column
From pump
To column
Column Oven
• Air circulation heating type
• Block heating type• Aluminum block heater
• Insulated column jacket type• Water bath
HPLC Detectors
• UV-VIS absorbance detector
• Photodiode array-type UV-VIS absorbance detector
• Fluorescence detector
• Refractive index detector
• Evaporative light scattering detector
• Electrical conductivity detector
• Electrochemical detector
• Mass spectrometer
UV-VIS Absorbance Detector
A = e·C·l = –log (Eout / Ein)
l
C: Concentration
(A: absorbance, E: absorption coefficient)
Detection cell
Ein Eout
A
C
Comparison of Detectors
Note: The above table indicates general characteristics. There are exceptions.
Selectivity SensitivityPossibility of
Gradient System
AbsorbanceLight-absorbing
substancesng Possible
Fluorescence Fluorescent substances pg Possible
Differential
refractive indexNone µg Impossible
Evaporative light
scatteringNonvolatile substances µg Possible
Electrical
conductivityIonic substances ng Partially possible
ElectrochemicalOxidizing / reducing
substancespg Partially possible
Chromatogram
tR
t0
Inte
nsity o
f d
ete
cto
r sig
nal
Time
PeaktR : Retention time
h
A
t0 : Non-retention time
A : Peak area
h : Peak height
Retention Factor, k
tR
t0
Str
ength
of
dete
cto
r sig
nal
Time
tR: Retention time
t0: Non-retention time
0
0R
t
ttk
Theoretical Plate Number, N
W
W1/2
H1/2
H
2
.21
R
R
/
R
W
t
W
2
2
2
545
16
Area
Ht
tN
