2. I. Introduction
• RP: separates molecules on the basis of
their reversible interaction with the
hydrofobic surface of a chromatographic
medium
• The proteins are unfolded and thus
denatured during the chromatography.
• Since mid ‘70s
3. II. Hydrofobic interactions
• RP-HPLC is mainly based on the reversible hydrofobic
interactions between amino acid side chains on the protein
and the hydrophobic surface on the chromatographic medium
(stationary phase).
• Hydrofobic binding is the result of an entropic effect that has
preference:
Hydrophobic parts of
proteins bind to long
carbonyl chains
More hydrophilic proteins
bind less well
Reverse phase chromatography
• Conditions at start are usually hydrophilic
solvent (e.g. buffered water) such that a high
degree of organised water surrounds the
protein surface and stationary phase
• Binding of the protein to the stationary
phase minimizes the amount of exposed
hydrophobic surface on the protein and
stationary phase:
surface = 4 πr2 , volume = 3/4 πr3
Ratio surf./vol. decreases with increasing r
• This causes reduction of water organisation
and thus corresponding increase in entropy,
creating an energetic more favourable
situation for the proteins to associate with the
stationary phase.
4. • The composition of the mobile phase is gradually
changed as to bring the proteins differentially into the
mobile phase (desorption).
• A RP-HPLC column is therefore run with an increasing
concentration of hydrophobic solvent.
• Proteins are concentrated and purified.
20% ACN
50% ACN
Start gradient
Gradient elution:
5. III. Standard conditions for RP-
HPLC
III.1. Stationary phase
• Silica-based: mechanically strong, high binding capacity.
• Beads have pores that increase the active surface
Silica glass Silica gel
Silicagel: chemical structure
Silicagel beads
6. • Macroporous packing (pores > 300Å) with beads of 5-10
µm diameter most frequently used.
• Attachment of functional groups on the beads:
– C4, C8 (octyl) alkyl chains for more hydrophobic analytes
– C18 (octadecyl) alkyl chains for more hydrophilic analytes
• Smaller beads give better performance (Van Deemter
curve!), however, backpressure will increase.
Preparative (> 10 mg)
Analytical (< 10 mg)
7. • Recent advancement: on-chip liquid chromatography
e.g. etched pillar array chip can be directly coupled to mass
spectrometer
105 - 106 theoretical plates (50 cm)!
8. III.2. Mobile phase
All reagents must be of the highest purity (‘HPLC-purity’) in order to avoid
concentration of contaminants.
III.2.1. Organic solvent
• Concentration changes cause changes in the capacity factor k = ms/mm of the
proteins. Very important because resolution (capacity to separate two
compounds 1 and 2) is dependent on solvent efficiency: α = k2/k1
• Separation of a complex mixture of proteins with slightly different
hydrophobicy requires gradient elution whereby k gradually changes and the
proteins elute from the column one by one (in principle).
Protein 1 Protein 2
L/H=N=16.(TR/w)2
9. • Frequently used organic solventia are (in order of elutropic
strenght):1-propanol >2-propanol > tetrahydrofuraan > ethanol >
acetonitrile > methanol, with acetonitrile most used. ACN and
methanol are less viscous and are UV transparent.
• Elution is followed by measurement of UV absorption. In case of
proteins: absorption at 280 nm, in case of peptides (or proteins
without Trp, Tyr and Phe): 214 nm (absorption at peptide bond).
Methanol and
acetonitrile
10. • Sample evaporation:
Eluted samples can be concentrated via evaporation (centrifugal
lyophilization) with evaporation of acid (see below) and organic
solvent. However, usually part of the sample is lost since
proteins/peptides tend to ‘stick’ to glass or polypropylene of the tube.
Addition of a non-ionic detergent (e.g. Tween-20; 0.015%)) may
improve sample recovery. Also, the sample can be completely dried
down and resolubilized in new buffer (see also SPE).
Vacuum centrifuge
Cooling trap
Vacuum pump
11. • Solid phase extraction (SPE):
Reverse phase chromatography is frequently used to change the buffer
of a protein sample or remove the salt (no retention of salt by the
column) (solid phase extraction or SPE). Here, no hydrophobic
gradient is used (e.g. 100% ACN) for elution.
Sample
containing
salts
Salts do not bind or
are easily washed
away
Hydrophobic
solvent in step 4
(e.g. ACN,
methanol etc.)
All proteins
and/or peptides
Evaporate all solvent
and re-solubilize in
new buffer
12. III.2.2. pH of mobile phase
• Most silica packings have a restricted pH range: between 2 and 8
– < 2: hydrolysis of bound active phase
– > 8: hydroxide ions solubilize silica
– However, under these circumstances residual silanol groups on the matrix
still react with basic analytes and result in peak ‘tailing’:
• Best pH region between 2 en 4.
– Good solubility of sample
– Ion suppression of the silanol groups and AA side chains is possible by ‘ion
pairing’
• Most frequently used acids for ion pairing (conc.: 50-100mM):
– Trifluor acetic acid (TFA)
– Heptafluoro-butyrinic acid (HFBA)
– Ortho-phosphoric acid (H3PO4)
13. III.2.3. Agents for ion-pairing
• Most frequently used: TFA -> dissociates in CF3COO- and H+
– Dual use: proton as well as anion have an effect (resp. pH en ion pairing
effect)
– Evaporates easily
– Low UV absorption.
• pH 2-8:
– Silanol groups protonated
– Under these conditions Arg and Lys are protonated-> reaction with ion pairing
agent (negative ions)
– ‘Bulky’ alkyl chain is linked to these AAs:
• Causing the protein or peptide to unfold and to expose more hydrophobic regions -> stronger
binding to stationary phase
• Alkyl moiety of TFA interacts with stationary phase -> stronger binding of protein/peptide to
stationary phase
• Results in sharper peaks (no tailing).
14.
15. • Figure shows a standard mixture of
peptides in solventia containing 0.01 M
TFA, PFPA or HFBA.
• Retention times of peptides can vary
considerably according to the used acid:
average elution time decreases with
decreasing hydrophobicity of ion pairing
agent
• Going from HFBA to TFA, some peptides
decrease more in retention times as
compared to others (e.g. ACTH1-24 and
ACTH1-39). This corresponds to a relatively
high number of basic AAs present in
ACTH1-24 and ACTH1-39.
1: Methionine enkephaline (1)
2: ACTH1-24 (9)
3: a-MSH (3)
4: HuACTH1-39 (12)
5: Somatostatin (3)
6: Bovine Insulin (6)
7: Hu Calcitonine (3)
(Number of basic residues at pH2, inclusive
His, is shown between brackets)
HFBA = Heptafluorobutyrinic acid
PFPA = pentafluoropropionic acid
TFA = Trifluoracetic acid
• Retention of the protein or peptide is influenced by type and concentration of
ion-pairing reagent (see figure).
16. IV. Microcolumn Reverse Phase
chromatography
IV.1. General
• Advantages:
– Concentration of samples: analyte is eluted in small volumes (peak volumes): 20-60µl as
compared to 500-1500 µl with 4.6 mm id. column.
– Higher sensitivity of protein detection by UV detector
– Low backpressure causing columns to be washed and loaded (not run!) quickly.
• According to the internal diameter (i.d.), microcolumns can be divided into:
microcolumn
17. IV.2. Principle
• Efficiency = N=16.(TR/w)2. Not only at the time of elution, but also within the column: N=16.(y/x)2.
• The peak width w is dependent on the volume of the sample loaded: large volumes -> large x -> result
in broader peaks (= large w) and vice versa.
• So do not overload column!
18. IV.2. Principle
• Consider two colums with different i.d. but same lenght: eluent peak volume is proportional with the
square of the ratios of the column diameters: Peak volume A = (rA/rB)2 x peak volume B.
• With other words: at the same position the decrease in peak volume in the column with the smallest
i.d. will be larger as compared to the decrease in column i.d.. And decreasing peak volume =
decreasing w = increasing N = increasing efficiency!
→ in a column with smaller internal diameter less sample is loaded but elutes in a smaller volume so
the concentration of the eluens is similar compared to a column with larger i.d.. (see figure)
Vice versa: if the same (low) amount of analyte is loaded on a column with smaller diameter, the
concentration (and thus the peak) will be higher -> higher sensitivity.
i.d.: 1 mm
Flow: 50µl/min
Load: 1 µg
Peak vol.: 22 µl
Conc.: 1µg/22µl
2.1 mm i.d.
200µl/min
4 µg
100 µl
1µg/25µl
4.6 mm i.d.
1000µl/min
20 µg
450 µl
1µg/22.5µl
Legend: a b c
450µl = (4.6 mm/1 mm)2 x peak volume B = 21,16 x peak volume B => peak volume B = 22µl
Although the column diameter is only 4.6x reduced, 20x less sample can be loaded on the 1mm i.d.
column in order to obtain the same concentration (peak hight and width) at elution.
1 mm 2,1 mm 4,6 mm
19. IV.3. Adaptation of optimal flow rate
• Same ratio: Flow A = (rA/rB)2 x flow B.
• Optimal flow rate of 1 ml/min for a 4.6 mm column (Flow a) ->
1 ml/min. = (4.6mm/1.5mm)2 . x ml /min or x= (1ml/min)/9.4 = ±
100µl/min for a 1.5 mm column
• Nanocolumns-> nanoliters/min.!
20. IV.4. Capacity and resolution of microcolumns
• Dependent on:
– Matrix: diameter and porosity of beads
– Column dimensions
• ‘Optimal’ loading quantities exist (to prevent overloading):
– 2.5-5 µg (1 mm i.d.)
– 10-20 µg (2.1 mm i.d.)
– 30-100 µg (4.6 mm i.d.)
If exceeded -> peak broadening -> increase in peak volume -> decrease in
resolution and column efficiency N:
* Flow rates were adapted to column diameters.
21. IV.5. Adaptations of the HPLC
system
• Pump:
– Past: flows in ml/min-range
– Now: µLC or nano-LC requires
stabile flows in µl or nl/min-range
-> nanopumps
• Detector
– Sensitive (e.g. UV detector):
measure small volumes
– Flowcell must be small enough to
keep extracolumnal peak
broadening (‘dead volume’) to a
minimum.
• Connections:
– Reduce diameter of capillaries
and fittings in order to reduce
extracolumnal peak broadening
since effect of ‘dead volume’ is
greater on columns with a small
diameter.
Plate Count
Extra-
Column
Volume
(µL)
Column A
4.6 x 150 mm,
5 µm
Column B
3.0 x 150 mm,
5 µm
10 12,600 11,200
50 11,300 9,295
100 9,400 5,750
22. V. 2D-LC
1st dimension (HPLC1)
2nd dimension
(HPLC2)
Sample Separation only in
1st dimension
Separation only in
2nd dimension
• Higher complexities: ”peak saturation”
• Proteins need to be separated by two chromatographies (2D-LC): where every system is
independent of the other (different separation principles; orthogonal).
• Usually: strong cation exchange chromatography (SCX) combined with RP.
• Peak capacity (Pc):
The number of peaks that can be separated theoretically with good resolution (Rs>1) to fill the
complete chromatogram (separation space).
→ hence: determined by resolution RS and separation space (time).
• The Pc of an orthogonal multidimensional setup can be given as follows:
n
n
tot Pc
Pc
Pc
Pc
Pc
= −1
2
1 ...
23. General: eluent of the SCX is
loaded on a RP column. The RP
run removes the salt and at the
same time performs a second
separation.
Offline:
Elution by means of salt
gradient: fractions are collected
and injected into the RP circuit.
Online:
1. Stepwise elution by means of
‘salt plugs’. No gradient but
several isocratic*
chromatographies with
stepwise increase of salt.
Each plug results in the
elution of a ‘packet’ of
analytes -> loaded on a short
trapcolumn by means of a
valve.
2. Valve switches -> trap
column is now part of the RP
circuit, allowing the analytes
to be separated on the
analytical RP column by
gradient chromatography.
*Isocratic = use of same mobile phase throughout the elution process
24. • Valve switching:
Isocratic
pump
Injector
Valve
2 1
3 6
4 5
C18 trapcolumn
C18 RP-column
Fraction
collector
Multiple
wavelength
detector
Capillary pump
Valve
C18 trapcolumn
Isocratic
pump
Injector
Fraction
collector
Multiple
wavelength
detector
Capillary pump
Valve
WASTE WASTE
2 1
3 6
4 5
Position 1: loading of trapcolumn Position 2: RP-analysis
C18 RP-column
SCX-column SCX-column
min
0 5 10 15 20 25 30 35
mAU
0
50
100
150
200
250
300
350
400
MWD1 A, Sig=214,8 Ref=off (08042108042101.D)
28.748
30.338
23.350
18.709
21.250
14.692
9.865
26.827
15.309
19.873
16.835
18.325
33.906
10.261
31.033
33.185
25.705
22.049
12.843
35.030
37.023
27.799
29.883
24.214
22.667
26.297
36.156
17.908
8.055
19.295
16.584
32.637
38.267
11.945
20.353
11.268
13.998
MWD1 B, Sig=280,8 Ref=off (08042108042101.D)
28.752
21.235
10.285
12.838
14.490
11.965
PMP1, Solvent B
6-port valve switching:
https://www.youtube.com/watch?v=LJ5UJoN2s0s (takes time to upload)
https://www.youtube.com/watch?v=nTVcOk0fi7s (en Français!)
Mind you: In these examples the trap column is exchanged by a capillary
loop!
25. • Online (vs offline):
• Automation possible
• Less sample loss because of less sample manipulation
(sometimes losses on trapcolumn however)
• Saltplugs: not optimal separation in the first dimension.
• First and second dimension have to be optimized in function of
each other, hence less efficient separation of both dimensions.
26. • Example – columns:
– 1ste dimension:
• Strong cation exchange (SCX)
• Sulfonic acid (SO3
-) as stationary phase
• 0,8 x 50mm (ID x L)
– 2de dimension:
• RP column
• C18-chains as stationary phase
• 0,3 x 150mm
– Trapcolumn:
• RP column
• C18-chains as stationary phase
• 0,5 x 10mm
27. • Example - pumps:
– Isocratic pump:
• 1ste dimension
• No gradient possible
• Elution by means of salt plugs (increasing concentration NaCl)
• Higher flow volumes (± 100 µl/min.), faster injection
• Mobile phase: 0,01% FA / 3% ACN in H2O
– Binairy pump:
• 2de dimension
• Gradient pump
• Elution by means of ACN-gradient
• Lower flow volumes (capillary pump) ± 5 µl/min.
• Mobile phase:
– solvent A: 0,1% FA in H2O
– solvent B: 0,1% FA / 5% H2O in ACN
