Basic principles of HPLC

Introduction to the theory of HPLC

HPLC (High Performance Liquid Chromatography) depends The chromatogram below illustrates the most important pa-
on interaction of sample analytes with the stationary phase rameters which characterise a separation. These parameters
(packing) and the mobile phase to effect a separation. Fol- will be explained in the following paragraphs.
lowing are explanations of the separation mechanisms com-
monly used in HPLC.
In adsorption chromatography the stationary phase, prop-
erly speaking, is the liquid-solid interface Molecules are
reversibly bound to this surface by dipole-dipole interactions.
Since the strength of interaction with the surface is different w1/2 10% of

Injection
for different compounds, residence time at the stationary peak height
phase varies for different substances thus achieving separa-
tion. Liquid-solid adsorption chromatography is most often A B
used for polar, non-ionic organic compounds. w
Partition chromatography is the fundamental distribution
t’R1
mechanism in liquid-liquid chromatography, i. e. when both t0
mobile phase and stationary phase are liquids. Separation t’R2
tR1
by distribution is based on the relative solubility of the sam-
ple in the two phases. In normal phase partition chromatog- tR2
raphy the stationary phase is more polar than the mobile retention time
phase, in reversed phase (RP) chromatography the mobile
phase is more polar than the stationary phase. Stationary Explanation of the most important parameters to
phases may be either coated on to a support, or they may be characterise a separation:
chemically bonded to the surface. Normal phase partition
peak widths:
chromatography is used for very polar organic compounds,
w1/2 = peak width at half height
while reversed phase chromatography is commonly used for
w= band width of the peak (intersection point of the
nonpolar or weakly polar substances.
inflectional tangents with the zero line)
Ionic compounds are often better separated by ion peak symmetry is measured at 10% or peak height
exchange chromatography (IEC). In this case, the station- symmetry parameters:
ary phase consists of acidic or basic functional groups A= peak front at 10% of peak height to peak
bonded to the surface of a polymer matrix (resin or silica maximum
gel). Charged species in the mobile phase are attracted to B= peak maximum to peak end at 10% of peak
appropriate functional groups on the ion exchanger and height
thereby separated. retention times
Ion pairing chromatography is an alternative to ion t0 = dead time of a column = retention time of
exchange chromatography. Mixtures of acids, bases and an unretarded substance
neutral substances are often difficult to separate by ion t R1 , t R . . = retention times of components 1, 2 . .
exchange techniques. In these cases ion pairing chromatog- 2

raphy is applied. The stationary phases used are the same
reversed phases as developed for reversed phase chroma-
tography. An ionic organic compound, which forms an ion-
pair with a sample component of opposite charge, is added
to the mobile phase. This ion-pair is, chemically speaking, a
salt which behaves chromatographically like a non-ionic
organic molecule that can be separated by reversed phase
chromatography.
Size exclusion chromatography (SEC) or gel permeation
chromatography (GPC) uses as the stationary phase a
porous matrix which is permeated by mobile phase mole-
cules. Sample molecules small enough to enter the pore
structure are retarded, while larger molecules are excluded
and therefore rapidly carried through the column. Thus size
exclusion chromatography means separation of molecules
by size.
t′ R1, t′ R2 . . = net retention times of components 1, 2 . .
Retention: In an elution chromatographic separation sub-
stances differ from each other only in their residence time in
or at the stationary phase. From this the following time defini-
tions arise:
The total retention time ( t R1 or t R ) is the time, which is
2
needed by a sample component to migrate from column inlet
(sample injection) to the column end (detector).
The dead time t0 is the time required by an inert compound
to migrate from column inlet to column end without any retar-
dation by the stationary phase. Consequently, the dead time
is identical with the residence time of the sample compound
in the mobile phase.

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 Basic principles of HPLC

Introduction to the theory of HPLC

The net retention time ( t′ R1 or t′ R2) is the difference be-
tween total retention time and dead time. That is the time the t′ R = t R – t 0 t′ R = t R – t 0
1 1 2 2
sample component remains in the stationary phase.
The capacity factor k’ is a measure of the position of a sam-
ple peak in the chromatogram. It is specific for a given sub- tR – t0 tR – t0
1 2
k′ 1 = ----------------- k′ 2 = -----------------
stance. k’ depends on the stationary phase, the mobile t0 t0
phase, the temperature, quality of the packing etc.
The relative retention α, also known as separation factor, is
the ratio between two capacity factors, where the figure in k′
α = ------2-
the denominator is the reference compound. The relative k′ 1
retention describes the ability of a system of stationary and
mobile phase to discriminate between two compounds. It is
independent of column construction (length, quality of pack-
ing) and flow velocity. It depends on the temperature and the
properties of the mobile and stationary phases. Impurities in
the mobile phase (e.g. water content) strongly influence the
relative retention.
Instead of the mobile phase volumetric flow rate F (ml/min) it
is advantageous to use the linear velocity u (cm/sec). The L
u = ----
linear velocity is independent of the cross section of the col- t0
umn and proportional to the pressure drop in the column.
The linear velocity can be calculated by means of the dead
time, where L is the column length in cm and t0 the dead time
in sec.
The permeability K of a column describes its transmittance
for a mobile phase and characterises the hydraulic resist-
ance. The permeability of a column depends on mobile
phase, temperature, column length and pressure. A change
in permeability indicates a change in the packing (e.g. swell-
ing of ion exchangers, silica gel etc.).
The number of theoretical plates n characterises the qual- 2
tR 2  tR1 
ity of a column packing and mass transfer phenomena. The n = 16  -------1 or n = 5.54  ------------
larger n, the more complicated sample mixtures can be sep-  w  w 1 ⁄ 2
arated with the column.
The height equivalent of a theoretical plate h, HETP, is the
length, in which the chromatographic equilibrium between L
mobile and stationary phase is established. Since a large h = ---
n
number of theoretical plates is desired, h should be as small
as possible. There are, of course, no real plates in a chroma-
tographic column, since the packing is homogeneous. The
value of h is a criterion for the quality of a column. h values
depend on the particle size, the flow velocity, the mobile
phase (viscosity) and especially on the quality of a packing.
For practical reasons, the peak symmetry is measured at
10% of peak height, where A is the distance from peak front B
symmetry = ----
to peak maximum and B is the distance from peak maximum A
to peak end.
Ideally symmetry should be 1, i. e. A = B. With values below
1 one speaks of fronting, with values above 1 there is peak
tailing.

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 Basic principles of HPLC

HPLC troubleshooting
The following table lists frequent problems in HPLC and pos-
sible solutions for:
ideal peak shape
uncommon peak shapes
symmetry = 1 lack of sensitivity
poor sample recovery
pressure problems
baseline problems
leaks
changing retention times
As mentioned above, the ideal peak symmetry is 1.0.
Though broad peaks may have a symmetry value of 1, their
peak shape indicates a problem in the chromatographic sys-
tem. If the symmetry deviates largely from 1, it may provide
valuable information about problems in a separation system.

possible cause prevention / remedy

A uncommon peak shapes

1. broad peaks
1.1. early eluting analyte due to column dilute sample 1:10 and repeat the separation
overload
1.2. injection volume too large inject smaller volumes or reduce solvent strength for
injection to focus the sample components
1.3. viscosity of mobile phase is too high increase column temperature or use a solvent of low-
er viscosity
1.4. retention times too long use gradient elution or a stronger mobile phase for broad peak
isocratic elution
1.5. poor column efficiency use mobile phases of lower viscosity, elevated col- symmetry ~ 1
umn temperature, lower flow rate or a packing with
smaller particle size
1.6. peak broadening in the injection valve decrease size of the sample loop, or introduce an air
bubble in front and back of the sample in the loop
1.7. extra column volume of the LC system use zero dead volume fittings and connectors; use
too large smallest possible tubing diameter (<0,25 mm) and
matched size of fittings
1.8. volume of detector cell too large use smallest possible cell volume for the sensitivity required;
use a detector without heat exchanger in the system
1.9. detector time constant too slow adjust the time constant to the peak width
1.10. sampling rate of the data system is too low increase the sampling rate
1.11. only some peaks broad: late elution of flush the column with a strong eluent after each run,
analytes from a previous run or end gradient at a higher concentration

2. peak fronting
2.1. column overload decrease sample amount; increase column diameter; symmetry < 1
use a stationary phase with higher capacity
2.2. formation of channels in the column buy a new column or have the column repacked (we
will be glad to inform you about our refill service)

fronting

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 Basic principles of HPLC

HPLC troubleshooting

possible cause prevention / remedy

3. peak tailing
3.1. basic analytes: interactions with silanol use silica-based base deactivated RP phases (e.g.
groups NUCLEOSIL PROTECT I, NUCLEOSIL® HD or symmetry > 1
NUCLEOSIL® AB); use a competing base such as tri-
ethylamine; use a stronger mobile phase; switch to
polymer-based columns (e.g. NUCLEOGEL® RP)
3.2. sample components which can form only use high-purity silica packings (e.g.
chelates: metal traces in the packing NUCLEOSIL®, NUCLEOPREP®) with their very low
content of metal ions; add EDTA or another chelating
compound to the mobile phase; switch to polymer tailing
columns (e.g. NUCLEOGEL®)
3.3. silica-based column: silanol interac- decrease the pH value of the mobile phase to sup-
tions press ionisation of the silanol groups; increase the
buffer concentration; derivatise the sample to avoid
polar interactions
3.4. silica-based column: degradation at use RP columns with good surface shielding, polymer
high pH values columns or sterically protected phases; also see A. 3.1.
3.5. silica-based column: degradation at use temperatures below 50 °C
high temperatures
3.6. dead volume at the column head rotate injection valve quickly; use an injection valve
with pressure bypass; avoid pressure pulses / replace
the deteriorated column, or, if possible, open the up-
per end fitting and fill the void with the column pack-
ing or some silanised glass fibre wadding; have the
column repacked. With our VarioPrep® columns for
preparative HPLC you can compensate dead vol-
umes with the adjustable end fitting.
3.7. unswept dead volume minimise the number of connections; ensure that the
rotor seal is tight; check whether all fittings are tight
3.8. beginning of peak doubling see under double peaks

4. double peaks
4.1. simultaneous elution of an interfering use sample clean-up or fractionation prior to injection (e. g.
substance SPE with CHROMABOND® or CHROMAFIX®); improve
selectivity by choice of another mobile or stationary phase
4.2. simultaneous late elution of a sub- flush the column with a strong eluent after each run,
stance from a previous run or end gradient at a higher concentration
4.3. column overload see A. 2.1.
4.4. injection solvent too strong use a weaker solvent for the sample or a stronger
mobile phase double peak
4.5. sample volume too large if the sample is dissolved in the mobile phase, the in-
jection volume should be smaller than one-sixth of
the column volume
4.6. dead volume or formation of channels replace the column or, if possible, open the upper end
in the column fitting and fill the void with the same packing; have the
column repacked
4.7. plugged frit install an in-line filter with 0.5 µm pore size between
pump and injector to remove solids from the mobile
phase, or between injector and column, to filter parti-
culate matter from the sample / if possible, clean or
replace the plugged frit
4.8. unswept volume in the injector replace the rotor of the injection valve

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HPLC troubleshooting

possible cause prevention / remedy

5. negative peaks
5.1. RI detector: refractive index of the ana- reverse detector polarity to obtain positive peaks
lyte lower than that of the mobile phase
5.2. UV detector: absorption of the analyte use a mobile phase with lower UV absorption; if recy-
lower than absorption of the mobile cling solvent, use fresh HPLC grade eluent when the
phase recycled mobile phase starts to affect detection

negative peak

6. ghost peaks
6.1. contamination only use HPLC grade solvents / flush the column to remove impurities
6.2. late elution of an analyte from a previ- see A. 4.2.
ous run
6.3. unknown interfering substances in the use sample clean-up or fractionation prior to injection (e. g. SPE with
sample CHROMABOND® or CHROMAFIX®)
6.4. in ion pairing chromatography: prepare the sample in the mobile phase; reduce the injection volume
disturbed equilibrium
6.5. in peptide mapping: oxidation of prepare fresh trifluoroacetic acid solution daily; add an antioxidant
trifluoroacetic acid
6.6. in RP chromatography: contaminated check the suitability of the water by passing different amounts through the
water column and measure the peak height of the impurity as a function of en-
richment time; purify the water by running it through an old RP column or
use HPLC grade water
7. spikes
7.1. air bubbles in the mobile phase degas the mobile phase; install a back pressure re-
strictor at the detector outlet; ensure that all fittings
are tight
7.2. column was stored without endcaps always store columns tightly capped; flush reversed
phase columns with degassed methanol
Spikes

B lack of sensitivity
1. detector attenuation set too high reduce detector attenuation
2. not enough sample injected increase amount of sample for injection
3. sample loop of injector underfilled overfill loop with sample
4. sample loss during sample preparation use an internal standard for sample preparation and optimise your method
5. sample loss on column see paragraph C: poor sample recovery
6. autosampler line blocked check the flow and clear any blockages
7. peaks outside the linear range of the dilute or enrich the sample until the concentration is in the linear range of
detector the detector
8. only during first few injections:
sample absorption in sample loop of condition sample loop and column with concentrated sample
injector or column

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HPLC troubleshooting

possible cause prevention / remedy

C poor sample recovery

1. adsorption on stationary phase
2. chemisorption on stationary phase
3. acidic substances: <90% yield, irre-
versible adsorption on active groups
4. basic substances: <90% yield, irrevers-
ible adsorption on active groups
5. hydrophobic interactions between
stationary phase and biomolecules
6. adsorption of proteins
7. adsorption on tubing and other hard-
ware components
increase mobile phase strength to minimise adsorption; for basic com-
pounds add a competing base or use a base deactivated packing like
NUCLEOSIL® HD, NUCLEOSIL PROTECT I or NUCLEOSIL AB
ensure no reactive groups are present; use a column or separation mecha-
nism better suiting the problem; use polymer-based columns like e. g.
NUCLEOGEL®
use endcapped or polymeric stationary phases; acidify the mobile phase
use endcapped, base deactivated, sterically protected phases with dense
surface coverage or polymer-based reversed phase materials
(NUCLEOSIL® HD, NUCLEOGEL® RP); add a competing base to the
mobile phase
use short-chain reversed phase packings with 300 Å pore size; as an alter-
native you may use hydrophilic stationary phases or ion exchangers
use another HPLC mode to reduce nonspecific interactions; use a mobile
phase containing reagents which enhance solubility of the proteins, strong
acids or bases (only with polymer-based columns) or detergents like SDS
use inert tubing and fittings made from e.g. PEEK or titanium

D pressure problems
1. high back pressure
1.1. viscosity of mobile phase too high use a solvent of lower viscosity or increase the temperature
1.2. particle size of packing too small use a packing with larger particle size (e. g. 7 µm instead of 5 µm)
1.3. for polymer-based columns: swelling of use only solvents compatible with the column; check proper eluent compo-
the adsorbent caused by eluent chang- sition; consult instructions for use for solvent compatibility; use a column
es with a higher degree of cross-linking
1.4. salt precipitation especially in reversed phase chromatography with high proportions of or-
ganic solvents in the mobile phase; ensure that the solvent composition is
compatible with the buffer concentration; reduce the ionic strength and the
ratio organic : aqueous in the mobile phase; premix the mobile phase
1.5. contamination at the column inlet improve sample clean-up; use guard columns; backflush column with a
strong solvent in order to dissolve the impurity
1.6. microbial growth in the column use a mobile phase with at least 10% organic solvent; prepare fresh buffer
daily; add 0.02% sodium azide to aqueous mobile phases; for storage
equilibrate the column with at least 25% organic solvent and without buffer
1.7. plugged frit in in-line filter or guard replace frit or guard column
column
1.8. plugged frit at column inlet replace the end fitting or the frit
1.9. when the injector is disconnected from clean the injector or replace the rotor
the column: plugged injector

2. pressure fluctuations
2.1. air bubbles in the pump degas the solvent; flush the solvent with helium
2.2. leak in liquid lines between pump and tighten all fittings; replace defective fittings; tighten rotor in the injection
column valve

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 Basic principles of HPLC

HPLC troubleshooting

possible cause prevention / remedy

3. increasing pressure
3.1. accumulation of solids at the column filter sample and mobile phase; use an 0.5 µm in-line filter; disconnect the
head contaminated column and clean it by back-flushing; replace plugged inlet
frits; replace the guard column
3.2. in aqueous / organic solvent systems: ensure that the solvent composition is compatible with the buffer concen-
precipitation of buffer components tration; reduce the ionic strength and the ratio of organic : aqueous in the
mobile phase
3.3. plugged liquid lines systematically disconnect system components from the detector end to the
blockage; clean or replace the plugged component

4. decreasing pressure
4.1. insufficient flow from the pump loosen the cap on the mobile phase reservoir
4.2. leak in liquid lines between pump and tighten all fittings; replace defective fittings; tighten the rotor in the injection
column valve
4.3. leaking pump check valve or seals clean the check valve; replace defective check valves or seals
4.4. air bubbles in the pump degas all solvents; check for blockage between solvent reservoir and
pump; if necessary replace the frit in the inlet line

E baseline problems
1. baseline drifting to lower absorption
1.1. with gradient elution: UV absorption of use non-UV-absorbing HPLC grade solvents for your
mobile phase A mobile phases; if a UV-absorbing solvent is inevita-
ble, use a UV-absorbing additive in mobile phase B

2. baseline drifting to higher absorption

2.1. accumulation and elution of impurities use sample clean-up or fractionation prior to injection;
use only HPLC grade solvents; clean the contaminat-
ed column with a strong solvent
2.2. with gradient elution: UV absorption of use a higher wavelength of the UV detector; use non-
mobile phase B UV-absorbing HPLC grade solvents for your mobile
phases; if a UV-absorbing solvent is inevitable, use a
UV-absorbing additive in mobile phase A

3. undulating baseline
3.1. temperature changes in the room monitor or avoid changes in room temperature; iso-
late the column or use a column oven; cover the RI
detector to protect it from air currents

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 Basic principles of HPLC

HPLC troubleshooting

possible cause prevention / remedy

4. baseline noise
4.1. continuous: detector lamp problem or replace the UV lamp or clean the detector cell
dirty detector cell
4.2. periodic: pump pulses repair or replace the pulse damper; purge any air
from the pump; clean or replace the check valves
4.3. random: accumulation of impurities use sample clean-up or fractionation prior to injection;
use only HPLC grade solvents / backflush contami-
nated column with a strong solvent
4.4. spikes: air bubble in the detector see A. 7.1.
4.5. spikes: column temperature higher use lower working temperature
than the boiling point of the solvent
4.6. occasional sharp spikes: external elec- use a voltage stabiliser for your LC system or use an
trical interferences independent electrical circuit for your chromatogra-
phy equipment

5. baseline noise during gradient elution or isocratic proportioning

5.1. insufficient solvent mixing mix by hand, or – if possible – use solvents of lower viscosity; monitor pro-
portioning precision by spiking one solvent with a UV-absorbing substance
and measure the resulting detector output
5.2. malfunctioning proportioning valves clean or replace the proportioning valve; use partially premixed solvents

6. disturbance at dead time

6.1. air bubbles in the mobile phase degas the mobile phase or use premixed eluents
6.2. difference in refractive index between normal with many samples; if possible, use the mobile phase as solvent for
injection solvent and mobile phase the sample

F leaks
1. column loses stationary phase replace column!
2. serious leaks at column or fittings tighten loose fittings or use new fittings
3. serious leak at the detector replace defective detector seals or gaskets
4. serious leak at the injector replace worn or scratched valve rotors
5. serious leak at the pump replace defective pump seals; check the piston for scratches and replace
piston, if necessary

G changing retention times

1. decreasing retention times
1.1. column overloaded with sample reduce the amount of sample or use a column with larger diameter
1.2. increasing flow rate check and – if necessary – adjust the pump flow rate
1.3. active groups at the stationary phase use a mobile phase containing an organic solvent (modifier) or a competing
base, increase the buffer strength; use a packing with higher surface cover-
age
1.4. loss of bonded stationary phase replace column; for silica adsorbents use mobile phases between pH 2 and
pH 8

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 Basic principles of HPLC

HPLC troubleshooting

possible cause prevention / remedy

2. increasing retention times

2.1. changing mobile phase composition cover the solvent reservoirs; ensure that the gradient system supplies the
proper composition; if possible, mix the mobile phase by hand
2.2. decreasing flow rate check and – if necessary – adjust the pump flow rate; check for pump cavi-
tation; check for leaking pump seals and other leaks in the system
2.3. loss of bonded stationary phase for silica adsorbents use mobile phases between pH 2 and pH 8

3. fluctuating retention times

3.1. only during first few injections: active condition the column with concentrated sample
groups
3.2. insufficient buffer capacity use buffer concentrations above 20 mM
3.3. insufficient mixing of the mobile phase ensure that the gradient system supplies a mobile phase with constant
composition; compare with manually mixed eluents; use partially premixed
mobile phases
3.4. selective evaporation of one compo- cover the mobile phase reservoirs; avoid vigorous flushing with helium; pre-
nent from the mobile phase pare fresh mobile phase
3.5. accumulation of impurities flush the column occasionally with a strong solvent, replace the guard col-
umn more frequently
3.6. fluctuating column temperature ensure that the room temperature is constant; if necessary, thermostat or
isolate the column
3.7. Leaks see paragraph F

4. changing retention times resulting from insufficient equilibration

4.1. isocratic separation pass 10 to 15 column volumes of mobile phase through the column for
equilibration
4.2. gradient elution increase equilibration time with mobile phase A in order to obtain constant
retention times for early peaks, also: pass at least 10 column volumes of
eluent A through the column for gradient regeneration
4.3. reversed phase ion-pairing chromatog- increase the equilibration time; in ion-pairing chromatography sometimes
raphy 50 column volumes may be required for equilibration; long-chain ion-pair-
ing reagents require more time; if possible, use ion-pairing reagents with
shorter alkyl chains

182 MN