High-Performance Liquid Chromatography

HPLC, when GC won’t cut it!!!

Components

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

HPLC-UV

Mobile Phases

A and B

HPLC Pump

syringe

6-port valveSample

loopHPLC column

Detector

MP waste

Jacket for controlling column temperature

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

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)

Columns

• Length (5-15 cm); much shorter than GC column• Diameter (4 mm down to 50m)• Particle size (3, 5, or 10 m)• 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 L/min

Reversed phase stationary phase

• Most common; n-octyldecyl, C18

Si-O-Si-(CH2)17-CH3

CH3

CH3

Si-O

-Si-(

CH

2)17

-CH

3

CH

3

CH

3

Si-O-Si-(CH2)17-CH3

CH3

CH3

Si-O

-Si-(C

H2 )

17 -CH

3

CH

3

CH

3

PP

C18 Phase designed to retain very polar compounds

Reverse-phase mobile phases

• Water

• Methanol

• Acetonitrile

• THF

• Additives, salts, acids, bases

• Ion pairing

Gradients in reverse-phase

• For complex mixtures

• Polar non-polar– Buffer A 100 % H2O

– Buffer B 100 % MeOH or acetonitrile

0 5 10 15 20 25 30 35 40 45

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

11.36 17.23

12.57

12.74

17.68

36.21

1.21 15.13 24.95

24.53

22.462.54

3.01 21.735.43 6.14 25.20

20.41 48.5527.31 37.1829.53 32.43 40.11 45.43

RP-HPLC Separation of a Tryptic Digest of BSA

HPLC Method Development• Isocratic, Fig 25-25 Harris• Find the best methanol separation• Use Table 25-25 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

Gradients

• First step – long, simple gradient– Adjust accordingly– Can become complex

• Do you need a gradient?Ift/tG > 0.25, then a gradient is appropriate

t = time between first and last peak

tG = time of gradient

RT: 0.00 - 35.03

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

8.59

10.02

12.56

3.152.99 13.933.6014.612.38

5.53 16.89 17.787.53 20.031.70 22.71 32.8429.14 31.2525.24 27.28

NL:2.27E8

Base Peak F: + c ESI Full ms [ 300.00-1300.00] MS chem361gbsaf

t = 22-8 = 14 min

tG = 22-4 min = 18 min

t/tG = 14/18 = 0.63 > 0.25

Normal Phase

• Bare silica – Mobile phases, (ethyl acetate/ hexane)

• HILIC columns– Attach polar groups to silica– Methanol to water

Ion Exchange

• Ion exchange resins– 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+

Ion Exchange Gradients

• Mobile Phase A – H2O

• Mobile Phase B – 500 mM NaAc

Ion chromatography

• Separation of small ionic species– PO4

3+, SO42-, BrO3-, NO2-, F-, Cl-, ect

– Mg2+, Na+, Ca2+, Li+, Ba2+, ect– -Detected by differences in conductivity

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

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

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

ICAT reagent

• Selectively capture cysteine-containing peptides

Wall of columnavidin

biotin

linker

iodoacetamide

CS A T WM

PA

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