1. High-Performance Liquid Chromatography HPLC, when GC won’t cut it!!!

2. Components Mobile phase reservoirs HPLC Pump(s) Mixing valves
Sample injector (manual or auto)
Column
Detector
Plumming
Mobile phase waste container

3. HPLC-UV HPLC Pump Jacket for controlling column temperature
6-port valve
Mobile Phases
A and B
Sample loop
HPLC column
syringe
MP waste
Detector

4. HPLC Separations
Different analytes have different equilibria between the mobile phase and stationary phase
Equilibrium is dynamic; thus we can view it as a given analyte molecule spending a fraction of time dissolved in the mobile phase
Since different solutes gave different fractions, a separation of the analytes occur as they are pushed through the column by the mobile phase

5. Types of HPLC
Reverse-phase (polar mobile phase/non-polar stationary phase/somewhat polar analytes)
Normal Phase (non-polar mobile phase/polar stationary phase/non-polar analytes)
Adsorption (non-polar mobile phase/polar stationary phase/non-polar analytes); isomer separation
Ion-Exchange (salts/ionic stationary phase)
Size-exclusion (aqueous/gel for large MW solutes, >104)

6. Columns Length (5-15 cm); much shorter than GC column
Diameter (4 mm down to 50mm)
Particle size (3, 5, or 10 mm)
Different phases bonded to silica
Typically detection limit is decreased by decreasing the column diameter
Optimal linear flow rate conserved; so optimal volumetric flow rate decreases with the square of the radius
4 mm/ 1.0 mL/min; 1 mm/60 mL/min

7. Reversed phase stationary phase
Most common; n-octyldecyl, C18
Si-O-Si-(CH2)17-CH3
CH3
Si-O-Si-(CH2)17-CH3
CH3
Si-O-Si-(CH2)17-CH3
CH3
Si-O-Si-(CH2)17-CH3
CH3

8. C18 Phase designed to retain very polar compounds

9. Reverse-phase mobile phases
Water
Methanol
Acetonitrile
THF
Additives, salts, acids, bases
Ion pairing

10. Gradients in reverse-phase
For complex mixtures
Polar non-polar
Buffer A 100 % H2O
Buffer B 100 % MeOH or acetonitrile

11. RP-HPLC Separation of a Tryptic Digest of BSA
11.36
100
17.23 95 90 85 80
12.57 75
12.74 70 65
Relative Abundance 60 55 50 45
17.68 40
36.21 35
1.21
15.13
24.95 30 25
24.53 20
2.54
22.46 15
3.01
5.43
6.14
21.73 10
25.20
20.41 5
27.31
29.53
32.43
37.18
48.55
40.11
45.43 5 10 15 20 25 30 35 40 45
Time (min)

12. HPLC Method Development
Isocratic, Fig Harris
Find the best methanol separation
Use Table to guide you in finding the best acetonitrile and THF separations
Based on separations try binary mixtures
Methanol, 38 %
Acetonitrile, 30 %
THF, 22 %
19 % MeOH/15% acetonitrile, 15 % acetonitrile/11% THF, 19 % MeOH/11% THF
Trinary mixture, 13:10:7
Temperature/computer simulations

13. Gradients First step Do you need a gradient? long, simple gradient
Adjust accordingly
Can become complex
Do you need a gradient?
If Dt/tG > 0.25, then a gradient is appropriate
Dt = time between first and last peak
tG = time of gradient

14. Dt = 22-8 = 14 min
tG = 22-4 min = 18 min
Dt/tG = 14/18 = 0.63 > 0.25

15. Normal Phase Bare silica HILIC columns
Mobile phases, (ethyl acetate/ hexane)
HILIC columns
Attach polar groups to silica
Methanol to water

16. Ion Exchange Ion exchange resins Bound to polystyrene support
Strong cation, -SO3-H+
Weak cation, - COO-H+
Strong anion, - N(CH3)3+OH-
Weak anion, - NH3+OH-
Bound to polystyrene support
Mechanism
RSO3-H P RSO3-P H+

17. Ion Exchange Gradients
Mobile Phase A – H2O
Mobile Phase B – 500 mM NaAc

18. Ion chromatography Separation of small ionic species
PO43+, SO42-, BrO3-, NO2-, F-, Cl-, ect
Mg2+, Na+, Ca2+, Li+, Ba2+, ect
-Detected by differences in conductivity

19. Size Exclusion Chromatography
Stationary phase is a gel
Fractionates sample on the basis of size
Elution volume vs. molecular weight
Pore size of the gel defines the MW range
Exclusion limit – (10 6), permeation limit (103)
Ve = V0 + KVi
Large molecules can not diffuse into the pore, Ve = V0

20. Stationary and Mobile phases
Gel filtration – hydrophilic packing (styrene and divinylbenzene) and aqueous mobile phase
Gel permeation –hydrophobic packing (sulfanated divinylbenzenes and polyacrylamides) and non-polar organic mobile phases

21. Affinity Chromatography
A “handle” is attached to a solid support, which is packed into a column
This handle selectively binds to a certain analyte or group of analytes
Examples
Antibodies to capture specific proteins
avidin binds to biotin

22. ICAT reagent Selectively capture cysteine-containing peptides
Wall of column
avidin
biotin
linker
iodoacetamide C S A T W M P

23. TLC Glass plates coated with thin layer of coated particles
Apply sample with capillary tube or syringe or fancy applicators
Develop plate
Rf = dr /dm, retardation factor