HIGH PERFORMANCE LIQUID CHROMATOGRAPHY(HPLC)
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
• 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. In this lab we will use a 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.
http://www.chemistry.nmsu.edu/Instrumentation/Waters_HPLC_MS_TitlePg.html
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
http://www.labhut.com/education/flash/introduction07.php
TLC vs High Performance Liquid Chromatography (HPLC)HPLC Optimization
HPLC – Optimizing Separation
Skoog and Leary: Principals of Instrumental Analysis, 5 th ed. Suanders, 1998
Schematic Presentation of a Chromatogram
HPLC - ResolutionHPLC - Resolution
• Resolution Resolution (R(RSS)) of a column provides a quantitative measure of its of a column provides a quantitative measure of its
ability to separate two analytesability to separate two analytes
Rs = Z /1/2(WA+WB)
Rs =
HPLC - ResolutionHPLC - Resolution
Rs
Skoog and Leary: Principals of Instrumental Analysis, 4 th ed. Suanders, 1992
HPLC - ResolutionHPLC - 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 compounds in different ways.
Skoog and Leary: Principals of Instrumental Analysis, 4 th ed. Suanders, 1992
HPLC – Capacity Factor
HPLC – Selectivity Factor
HPLC - ResolutionHPLC - 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 (t(N = 16 (tRR/W)/W)22))
Skoog and Leary: Principals of Instrumental Analysis, 4 th ed. Suanders, 1992
Phenomenex catalog, 1999
HPLC - ResolutionHPLC - 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 (t(N = 16 (tRR/W)/W)22))
Plate Height (H): The height equivalent to a theoretical plate (HEPT = H)
H = L / N
Resolution (Rs) depends on the number of theoretical plates:
Rs =
Skoog and Leary: Principals of Instrumental Analysis, 4th ed. Suanders, 1992
Skoog and Leary: Principals of Instrumental Analysis, 4 th ed. Suanders, 1992
HPLC - General Elution ProblemHPLC - General Elution Problem
Skoog and Leary: Principals of Instrumental Analysis, 5 th ed. Suanders, 1998
x
a
b
c
a b c
c b a
0
0
Time
Time
Normal Phase (SiO2)
Reverse Phase (C18)
Normal Phase (SiO2) TLC
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY(TLC vs Normal Phase and Reverse Phase HPLC)
Skoog and Leary: Principals of Instrumental Analysis, 5th ed. Suanders, 1998
Reverse Phase HPLC
Normal Phase vs. Reverse Phase HPLC
Skoog and Leary: Principals of Instrumental Analysis, 5 th ed. Suanders, 1998
RP-HPLC – Stationary Phase
Skoog and Leary: Principals of Instrumental Analysis, 5 th ed. Suanders, 1998
RP-HPLC – Mobile Phase vs k’
Skoog and Leary: Principals of Instrumental Analysis, 5 th ed. Suanders, 1998
Skoog and Leary: Principals of Instrumental Analysis, 5th ed. Suanders, 1998
RP-HPLC – Mobile Phase (k’, )
RP-HPLC – Mobile Phase ()
Skoog and Leary: Principals of Instrumental Analysis, 5 th ed. Suanders, 1998
RP-HPLC - Example
Alltech Chromatography Sourcebook, 2004-04 catalog
RP-HPLC - Optimization
Alltech Chromatography Sourcebook, 2004-04 catalog
RP-HPLC – Gradient Elution
Alltech Chromatography Sourcebook, 2004-04 catalog
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Alltech Chromatography Sourcebook, 2004-04 catalog
HPLC – Resolution vs Column Efficiency (N, H)HPLC – Resolution vs Column Efficiency (N, H)
van Deemter Equation H = A + B/u +(Cs + Cm)u
H = L / N
Skoog and Leary: Principals of Instrumental Analysis, 5 th ed. Suanders, 1998
HPLC - Column EfficiencyHPLC - Column Efficiency
Skoog and Leary: Principals of Instrumental Analysis, 5 th ed. Suanders, 1998
van Deemter Equation H = A + B/u +Cu
HPLC - Column EfficiencyHPLC - Column Efficiency
Skoog and Leary: Principals of Instrumental Analysis, 5 th ed. Suanders, 1998
HPLC - Column EfficiencyHPLC - Column Efficiency
H = H = AA + B/ + B/uu + C + Cuu
A = 2A = 2 d dpp
1.1. depends on particle size distribution, the depends on particle size distribution, the narrower the distribution the smaller the narrower the distribution the smaller the
2.2. ddpp = particle size = particle size
3.3. Independent of mobile phase flow rateIndependent of mobile phase flow rate
4.4. Also known as eddy diffusionAlso known as eddy diffusion
Skoog and Leary: Principals of Instrumental Analysis, 5th ed. Suanders, 1998
HPLC - Column EfficiencyHPLC - Column Efficiencyparticle sizeparticle size
Skoog and Leary: Principals of Instrumental Analysis, 5 th ed. Suanders, 1998
HPLC Column EfficiencyHPLC Column Efficiency
Longitudinal Diffusion (B)Longitudinal Diffusion (B)
H = A + H = A + B/B/uu + C + Cuu
B/u = 2B/u = 2DDMM/u/u
1. = constant depending on
quality of packing
2. DM is the mobile phase diffusion coefficient
3. Inversely related to mobile phase flow rate
HPLC Column EfficiencyHPLC Column Efficiency
Mass Transfer Mass Transfer (Cs + Cm)
H = A + B/u + (Cs + Cm)u
CS = fS(k’)df2 / DS
CM = fM(k’)dp2 / DM
• DDMM is the mobile phase is the mobile phase
diffusion coefficientdiffusion coefficient
• DDSS is the stationary phase is the stationary phase
diffusion coefficientdiffusion coefficient
• ddff is film thickness is film thickness
• ddpp is particle size is particle size
• Directly related to mobile Directly related to mobile phase flow ratephase flow rate
Skoog and Leary: Principals of Instrumental Analysis, 5th ed. Suanders, 1998
RP-HPLC – Variables
Alltech Chromatography Sourcebook, 2004-04 catalog
1.35 min.Ibuprofen
7.11 min.Caffeine
1.48 min.Aspirin
2.82 minAcetaminophen
Analgesic Retention
Time
Acetaminophen 2.82
Aspirin 1.48
Caffeine 7.11
Ibuprofen 1.35
Gradient = 0 min: 100% EtOAC (+ 0.2% HOAc) 3 min: 100% EtOAC (+ 0.2% HOAc) 5 min: 15% MeOH, 85% % EtOAc (+ 0.2% HOAc) 8 min: 15% MeOH, 85% % EtOAc (+ 0.2% HOAc) 10 min: 100% EtOAC (+ 0.2% HOAc)SiO2
Flow Rate = 1 mL/minUV detector set at 240 nm
HPLC OF ANALGESICS - UV Detection
Standard Analgesics
Question
The peak areas of aspirin and acetaminophen are very different, even though they are present in equal amounts (250mg/tablet) in Excedrin ES.
Caffeine is present at ~ ¼ the concentration of aspirin (65 mg/tablet vs. 250 mg/tablet), but it’s peak area is greater than the peak area of aspirin.
WHY? UV Absorbance of analgesics vs UV setting of detector
Area %Aspirin 19.5%Acetaminophen 50.0%Caffeine 20.5%
Excedrin ES250 mg aspirin250 mg acetaminophen65 mg caffeine
HPLC OF ANALGESICS - UV Detection
Detector set at 240 nm
Detector set at 254 nm
Detector set at 280 nm
UV MaxAspirin 225, 296 nmAcetaminophen 248 nmCaffeine 272 nm
Area %Aspirin 19.5%Acetaminophen 50.0%Caffeine 20.5%
Area %Aspirin 7.3%Acetaminophen 81.9%Caffeine 10.8%
Area %Aspirin 24.8%Acetaminophen 39.3%Caffeine 35.9%
HPLC: Peak Area vs Detector setting
Figure 2. HPLC (SiO2) of crude tumeric extract.
Gradient 0-2 min, 4% EtOAc/Hexane; 2-9 min 4 to 80% EtOAc;
9-11 min , 80% EtOAc/hexane; 11-13 min, 80 to 4% EtOAc/hex, 13-15 min, 4% EtOAc /hexane.
(A) Detector set at 420 nm. (B) Detector set at 254 nm. (C) Detector set at 254 nm (0-3.5 min), 420
nm 3.5-15 min.
(A)
(B)
(C)
HPLC – UV Detection