lecture 2 advanced separation science techniques
TRANSCRIPT
LECTURE 2
Advanced Separation Science Techniques Present and Future Separation Tools
Jack Henion, Ph.D. Emeritus Professor, Analytical Toxicology
Cornell University Ithaca, NY 14850
Lecture 2, Page 1
Contents
• HPLC
• HILIC
• Porous Graphitic Carbon
• UPLC
• Nano HPLC
• Capillary Electrophoresis (CE)
• Differential Mobility Spectrometry (DMS)
Lecture 2, Page 2
Schematic of HPLC System
Lecture 2, Page 3
The Resulting Chromatogram
tR
t0
minutes
wb
C
B A
Question: Is this an isocratic or gradient separation? What is the clue?
Lecture 2, Page 4
The Chromatographic Process
Least Retained Moderately Retained
Most Retained
Injected Sample Mixture
Lecture 2, Page 5
Retention and Differential Migration in LC
Mobile Phase
Equilibrium distribution of
compounds C and A
between stationary and
mobile phases
Cm Am
As Cs
S
Lecture 2, Page 6
Compound Polarity Differences Require Different Stationary Phases
‘Like Dissolves Like’ Choose a stationary phase that best
suits your sample mixture composition Lecture 2, Page 7
Comparison of Normal Phase vs.
Reversed-Phase Normal Phase
Reversed-Phase
Lecture 2, Page 8
The Five HPLC Modes Reversed-phase
ion exchange
ion pair
normal phase
size exclusion
Lecture 2, Page 9
HPLC
Pumping Systems
Injector
(Autosampler) Gradient Former
HPLC Pump(s)
HPLC Pump Characteristics: * Constructed of chemically inert materials. * Pulse-free and reproducible flow rate. * Flow rates from 0.05 mL/min to 2 mL/min. * Compatible with gradient elution. * Pressures up to 6000 psi. (400 bar).
Most common types of pumps: * Reciprocating pistons.
Key Issues: * Low pressure vs. high pressure mixing (helium sparging or no degassing). * Pulse dampner required which contributes delay to volume to gradient mixing. * Limitations regarding micro HPLC with gradients.
Lecture 2, Page 10
HPLC Column Dimensions
Lecture 2, Page 11
HPLC Column
HPLC Pump(s)
Injector
Detector
Column sizes: 4.6 mm i.d.: Flow = 1.0 mL/min 2.1 mm i.d.: Flow = 0.2 mL/min 1.0 mm i.d.: Flow = 0.050 mL/min 0.3 mm i.d.: Flow = 0.005 mL/min
0.075 mm i.d.: Flow = 0.0002 mL/min (200 nL/min)
Is the ‘condition’ of the bed packing important? Why Lecture 2, Page 12
Relationship Between Column Diameter and Particle Size
Lecture 2, Page 13
HPLC Resolution
Resolution is a function of three different factors:
1. Capacity factor, k’ 2. Column plate number, N. 3. Separation factor, alpha, or band spacing.
Changes in these three parameters are shown in figure.
Effect of different separation conditions on LC resolution (from J. W. Dolan and L.R. Snyder, “Troubleshooting LC/Systems”, Human Press, Clifton, NH 1989, p.97
Lecture 2, Page 14
Gradient HPLC System Two pumps and shorter run times
Lecture 2, Page 15
Key Parameters in HPLC Columns
• Column length – Long: (10-25 cm) gives more plates, more backpressure,
longer runs
– Short: (1-3 cm) gives fewer plates, lower backpressure, and short run times
• Column inside diameter (i.d.)
– Large: (4.6 mm and higher) employs higher mobile phase flow, reduced LC/MS sensitivity
– Small: (75 microns to 2.1 mm) employs lower mobile phase flow, higher sensitivity
Lecture 2, Page 16
Chromatographic Peak
Shape In Gradient Mode
HPLC
Contributed by: Tzipi Ben-Tzvi and Prof. Eli Grushka Institute of Chemistry The Hebrew University Jerusalem, Israel
Lecture 2, Page 17
Gradient
UA-velocity of mobile phase
U1=Ufront
U2=Urear
Lecture 2, Page 18
How Do We Characterize Peak Shape?
1.Tailing Factor = B/A
Lecture 2, Page 19
How Do We Characterize Peak Shape?
3
3/2
2( )
M
M2. Skew
Lecture 2, Page 20
Objectives
1. Comparing Peak shape in gradient and
isocratic separations
2. The dependence of peak shape on the time of gradient starts
3. The dependence of peak shape on the steepness of the gradient
Lecture 2, Page 21
Examples of peaks
-0.01
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
1900 1920 1940 1960 1980 2000 2020 2040 2060 2080 2100
Am
ou
nt
Transfer
Gradient starts after 570 transfers Skew=-0.34
-0.02
0
0.02
0.04
0.06
0.08
0.1
0.12
1000 1020 1040 1060 1080 1100 1120 1140 1160 1180 1200
Am
ou
nt
Transfer
Gradient starts after 30 transfers Skew=0.76
Lecture 2, Page 22
-0.01
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
1200 1700 2200
Am
ou
nt
Transfer
Grad start at 900
K=3 Skew2=0.259
K=1 Skew2=-1.16
K=0.5 Skew2=0.062
-0.01
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
1000 1500 2000
Am
ou
nt
Transfer
Grad start at 570
K=3 Skew2=-0.344
K=1 Skew2=-0.0262
K=0.5 Skew2=0.0432
-0.02
0
0.02
0.04
0.06
0.08
0.1
0.12
1050 1100 1150 1200
Am
ou
nt
Transfer
Grad start at 30
K=3 Skew2=0.706
K=1 Skew2=-0.134
K=0.5 Skew2=-0.032
Lecture 2, Page 23
Results – step gradient
-0.2
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1842 1852 1862 1872 1882 1892 1902 1912
Am
ou
nt
Transfer
Gradient start after 900 transfers, Skew2=0.59
The chromatogram is in the limited range Lecture 2, Page 24
Conclusions 1. The peak shape gradient elution can be different than
in isocratic separations.
2. The peak shape is a function of the gradient starting point.
3. Step gradient influences the peak shape more than continuous gradient.
4. When the slope of the gradient is higher the absolute values of the skew are greater.
5. A more accurate picture about the peak shape is obtained with skew measured in the limited range.
Lecture 2, Page 25