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.
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
http://www.chemistry.nmsu.edu/Instrumentation/Waters_HPLC_MS_TitlePg.html
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
TLC vs High Performance Liquid Chromatography (HPLC)
HPLC Optimization
http://www.labhut.com/education/flash/introduction07.php
HPLC – Optimizing Separation
Skoog and Leary: Principals of Instrumental Analysis, 5 th ed. Suanders, 1998
Schematic Presentation of a Chromatogram
HPLC - Resolution
• Resolution (RS) of a column provides a quantitative measure of its
ability to separate two analytes
Rs = DZ /1/2(WA+WB)
Rs =
HPLC - Resolution
Rs
Skoog and Leary: Principals of Instrumental Analysis, 4 th ed. Suanders, 1992
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 (a): Also called
separation or selectivity coefficient is
defined as
a = 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 - Resolution
Theoretical Plates (N): The number of
theoretical plates characterizes the
quality or efficiency of a column.
N = 5.54 [(tR) / w1/2]2
Skoog and Leary: Principals of Instrumental Analysis, 4 th ed. Suanders, 1992
(N = 16 (tR/W)2)
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)
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 Problem
Skoog and Leary: Principals of Instrumental Analysis, 5 th ed. Suanders, 1998
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
(TLC vs Normal Phase and Reverse Phase HPLC)
Normal Phase (SiO2) TLC
Normal Phase (SiO2)
a
b
a
c
Time
0
b
Reverse Phase (C18)
c
c
x
0
b
Time
a
Reverse Phase HPLC
Skoog and Leary: Principals of Instrumental Analysis,
5th ed. Suanders, 1998
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
RP-HPLC – Mobile Phase (k’, a)
Skoog and Leary: Principals of Instrumental Analysis,
5th ed. Suanders, 1998
RP-HPLC – Mobile Phase (a)
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
Alltech Chromatography Sourcebook, 2004-04 catalog
Alltech Chromatography Sourcebook, 2004-04 catalog
HPLC – Resolution vs Column Efficiency (N, H)
H=L/N
van Deemter Equation
H = A + B/u +(Cs + Cm)u
Skoog and Leary: Principals of Instrumental Analysis, 5 th ed. Suanders, 1998
HPLC - Column Efficiency
Skoog and Leary: Principals of Instrumental Analysis, 5 th ed. Suanders, 1998
HPLC - Column Efficiency
van Deemter Equation
H = A + B/u +Cu
Skoog and Leary: Principals of Instrumental Analysis, 5 th ed. Suanders, 1998
HPLC - Column Efficiency
H = A + B/u + Cu
A = 2l d p
1.
2.
3.
4.
l depends on particle size distribution, the
narrower the distribution the smaller the l
dp = particle size
Independent of mobile phase flow rate
Also known as eddy diffusion
Skoog and Leary: Principals of Instrumental Analysis, 5th ed. Suanders, 1998
HPLC - Column Efficiency
particle size
Skoog and Leary: Principals of Instrumental Analysis, 5 th ed. Suanders, 1998
HPLC Column Efficiency
Longitudinal Diffusion (B)
H = A + B/u + Cu
B/u = 2gDM/u
1.
g = constant depending on
quality of packing
2.
DM is the mobile phase
diffusion coefficient
3.
Inversely related to mobile
phase flow rate
HPLC Column Efficiency
Mass Transfer (Cs + Cm)
H = A + B/u + (Cs + Cm)u
CS = fS(k’)df2 / DS
CM = fM(k’)dp2 / DM
•
•
•
•
•
DM is the mobile phase
diffusion coefficient
DS is the stationary phase
diffusion coefficient
df is film thickness
dp is particle size
Directly related to mobile
phase flow rate
Skoog and Leary: Principals of Instrumental Analysis,
5th ed. Suanders, 1998
RP-HPLC – Variables
Alltech Chromatography Sourcebook, 2004-04 catalog
HPLC OF ANALGESICS - UV Detection
Standard Analgesics
2.82 min
Acetaminophen
1.48 min.
Aspirin
7.11 min.
Caffeine
1.35 min.
Ibuprofen
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/min
UV detector set at 240 nm
Analgesic
Retention
Time
Acetaminophen
2.82
Aspirin
1.48
Caffeine
7.11
Ibuprofen
1.35
HPLC OF ANALGESICS - UV Detection
Area %
Excedrin ES
250 mg aspirin
250 mg acetaminophen
65 mg caffeine
Aspirin
19.5%
Acetaminophen 50.0%
Caffeine
20.5%
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
HPLC: Peak Area vs
Detector setting
Detector set at 240 nm
UV Max
Aspirin
225, 296 nm
Acetaminophen 248 nm
Caffeine
272 nm
Area %
Aspirin
19.5%
Acetaminophen 50.0%
Caffeine
20.5%
Detector set at 254 nm
Area %
Aspirin
7.3%
Acetaminophen 81.9%
Caffeine
10.8%
Detector set
at 280 nm
Area %
Aspirin
24.8%
Acetaminophen 39.3%
Caffeine
35.9%
HPLC – UV Detection
(A)
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.
(B)
(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.
(C)