Instrumentation of HPLC

Mobile Phase Considerations

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as there is interactive material that cannot be fully shown in this reference
i manual.
Aims and Objectives

Aims and Objectives

Aims

 To explain the purpose of the mobile phase in HPLC

 To outline the basic concepts of the Liquid Chromatographic process
 To describe the best way to prepare mobile phase components
 To highlight the potential problems caused by improper preparation and use of
mobile phases
 To describe the way that mobile phase composition effects retention and
selectivity in reverse phase HPLC
 To describe various physico-chemical properties of the typical solvents used for
HPLC
 To explain various methods for degassing HPLC mobile phases

Objectives

At the end of this Section you should be able to:

 Demonstrate an understanding of the purpose of the mobile phase in HPLC

 Describe the Liquid Chromatographic process and relate retention and selectivity
of analytes to different mobile phase compositions
 Describe suitable methods to prepare and degas mobile phases
 Demonstrate an understanding of how certain physico-chemical properties of
mobile phase constituents may affect an analysis
Content

The Mobile Phase –Introduction 3

The Liquid Chromatographic Process 4
Analyte Retention Processes 5
The Partition Coefficient 7
Retention and Selectivity 8
Solvent Type and Selectivity 10
Optimising Selectivity using Retention 11
Gradient Elution 13
Gradient steepness 14
Mobile Phase Delivery 15
Solvent miscibility 16
UV Cut Off 18
Other Solvent Considerations 19
Mobile Phase Preparation 21
Mixing 21
Filtration 22
Degassing 23
Outlet Degassing 24
Degassing / Column Damage 25
Methods of Degassing 25
Helium Sparging 27
Vacuum Degassing 27
Glossary 28

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The Mobile Phase –Introduction

In reverse phase HPLC, the stationary phase is an adsorbent packed into a column and
the mobile phase is composed of water and/or organic solvents plus other additives.

When an injection is made via the autosampler the sample is introduced into the mobile
phase flow path and swept into the column by the mobile phase.

When the sample enters the column it will partition (distribute), between the mobile and
stationary phase. The extent to which the sample partitions into either phase is governed
by its physico-chemical for that phase. By changing the chemical composition of the
mobile and/or stationary phase it is possible to effect the retention of various analytes –by
changing the partition ratio.

Important

Chromatography has been defined by the International Union of Pure and

Applied Chemistry (IUPAC) as:

‘a physical method of separation in which the components to be separated

are distributed between two phases, one of which is stationary whilst the
other moves in a definite direction’

A typical Liquid Chromatograph

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Where

Stage 1: Mobile phase reservoir. Solvent bottles. The mobile phase is stored here
prior to being pumped through the system.

Stage 2: Vacuum degassing. Modern HPLC Systems use on-line vacuum degassing to
remove dissolved gas from the mobile phase, this gives less noisy baselines and better
chromatographic performance.

Stage 3: The pump. The mobile phase then passes through the pump. Mixing of 2 or
more mobile phase components can be automated here using various different pump
configurations. Mobile phase components can be mixed in different ratios to alter the
mobile phase characteristics and therefore improve separation and resolution.

Stage 4: The injector. Here the sample is introduced into the mobile phase flow, without
interrupting the mobile phase flow or reducing the system pressure.

Stage 5: The column. The sample is partitioned between the mobile and stationary
phase. The degree to which the analyte partitions between the phases will govern its
retention.

Stage 6: The detector. The detector measures some physico-chemical property (eg UV
absorption) of the mobile phase as it elutes from the column. The response of the
detector will change as the sample components begin to elute.

The Liquid Chromatographic Process

The mobile phase is continuously pumped at a fixed flow rate through the system after
mixing (if required), by the pump. The injector is used to introduce a plug of sample into
the mobile phase without having to stop the mobile phase flow, and without introducing air
into the system.

The mixture of components is carried in a narrow band to the top of the column. Some
compounds in the sample mixture will have a greater preference for the stationary phase
than the mobile phase and will be retained in the column longer. Those components that
are not retained as strongly are transported more quickly through the column by the
mobile phase. The higher the surface area available to the sample (achieved using a
longer column or smaller particle size), the more opportunities for interaction with the
stationary phase and the greater the separation.

The detector is then used to respond to a physico-chemical property of the analyte

(perhaps a UV Chromophore consisting of a set of conjugated double bonds or an
aromatic group). This response is digitally amplified and sent to a data system where it is
recorded as the ‘chromatogram’.

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Representation of the chromatographic process

Analyte Retention Processes

The velocity at which the mobile phase moves through the analytical column is called the
‘flow rate’.

As the sample components pass though the column, driven by the mobile phase flow,
they will selectively partition themselves between the mobile phase and the stationary
phase.

If a sample component has similar chemical properties to that of the stationary phase it
will have a high affinity for this phase and will strongly interact with it - usually via
adsorption. The retention time (tR) (retention factor, k) of such a component will be
relatively high, as it will spend a longer time ‘immobilised’ onto the surface of the
stationary phase, rather than moving through the column whilst dissolved in the mobile
phase.

If a sample component has a high affinity for the mobile phase (i.e. is chemically similar to
the mobile phase) it will interact with the mobile phase more readily. The retention time of
such a component will be relatively low.

The chemical ‘nature’ of the mobile phase and the stationary (bonded) phase chemistry
can therefore both be altered to affect their affinity toward a particular analyte, in turn
affecting retention time.

In the next diagram the sample consists of two analytes represented by the blue and red
species. The sample enters the column as a homogenous mixture of these two
components. The red components have a higher affinity for the stationary phase than the
blue components and so they are ‘retained’ or ‘retarded’ more on the column leading to a
longer retention time than the blue species –the net result however, is a chromatographic
separation of the two components.
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The Partition Coefficient

The degree to which an analyte will partition between the two phases is governed by the
distribution co-efficient. This is defined by the following equation:

CSat
Kx 
CMob

Where CSat is the concentration of the compound in the stationary phase and CMob is the
concentration of the compound in the mobile phase, at equilibrium.

The value of Kx is constant for a particular compound under a given set of

chromatographic conditions.

Kx represents the equilibrium distribution (under a given set of analytical conditions) of the
analyte between the two phases. Because the mobile phase moves, a non-equilibrium
situation occurs at both the front and back of the analyte band in both the mobile and
stationary phases –this is illustrated in the example opposite. This results in a net
movement of analyte molecules at the front of the band into the mobile phase and those
at the back of the band into the stationary phase, to re-establish equilibrium conditions.

This causes the band of analyte to move through the column in the manner shown. The
time taken for adsorption and desorption to occur at both ends of the band will dictate the
speed of analyte transit through the column, and also the width of the chromatographic
peak for that analyte.

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In the presented chromatogram, compound B presents the highest partition
coefficient. Peaks that elute with a long retention time have a high affinity for the
stationary phase. The value of CSat in the distribution equation will be higher –
leading to a higher value for Kx.

Retention and Selectivity

The retention time (or capacity factor (k)) of an analyte is governed by its affinity for both
the mobile and stationary phases used for the separation.

By altering the composition of the mobile phase it is possible to affect the analyte solubility
in the mobile phase and hence alter its retention time.

Changes in mobile phase composition do not have an equal effect on the retention
behaviour of all compounds; hence selectivity (α) can also be altered. Chromatographic
selectivity describes the degree to which different analytes can be separated under a
given set of analytical conditions.

The most common and convenient method to alter the analyte solubility in the mobile
phase involves changing the amount and type of ‘organic modifier’ added. This is usually
an organic solvent used to alter the polarity of the mobile phase. In reverse phase HPLC
the modifier is usually a polar organic solvent, in normal phase chromatography a non-
polar organic solvent is used. In reverse phase chromatography the solvent system
polarity decreases as the percentage of organic modifier is increased. This usually has
the effect of increasing the affinity of analytes for the mobile phase and results in shorter
analyte retention times.

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Analytical conditions:
Column: C18 (15cm × 0.46cm × 5μm)
Flow: 2.00 mL/min
to: 1.28 min

 At low mobile phase organic solvent composition, the retention factor is high –the
analytes are interacting strongly with the stationary phase and are less soluble in
the mobile phase –the value of Kx is high
 Increasing the organic composition by 10% brings about 2-3 fold reduction in
retention factor
 When retention factors are very high or very low, the quality of the separation is
reduced. Retention factors below 1, for any of the components, generally indicate
the separation will be poor
 The selectivity between the chosen peaks changes constantly as the mobile phase
composition is altered
 Altering the mobile phase composition is both convenient and efficient for
changing the quality of the separation obtained

to: The volume of the mobile phase (or the corresponding time) required to
elute a component the concentration of which in the stationary phase is
negligible compared to that in the mobile phase. In other words, this
component is not retained at all by the stationary phase. Thus, the hold-up
volume (time) is equal to the Retention Volume (Time) of an unretained
compound. The hold-up volume (time) includes any volumes contributed by
the sample injector, the detector, and connectors.

To = Vo/Fc

Vo = Hold up volume (mL), Fc = Column flow rate (mL/min)

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Solvent Type and Selectivity

There are three fundamental properties of an organic solvent which will affect
chromatographic selectivity:

 Dipole Moment (Polarity)

 Acidity (Proton Donor)
 Basicity (Proton Acceptor)

Three solvents commonly used in reverse phase HPLC mobile phases whose properties
differ significantly are Methanol, Acetonitrile and Tetrahydrofuran (THF).

The properties of these three solvents can be mapped in a solvent selectivity diagram as
shown.

Relevant Colvent Properties

The diagram shows that THF presents the greatest dipole interaction followed by ACN
and then MeOH. THF is often considered the ‘strongest’ of the organic modifiers and
results in the largest changes in retention per %change in modifier concentration. MeOH
is the most ‘basic’ of the three solvents whilst acetonitrile is the most acidic. When
changing from one solvent to another it is more convenient to use the information from a
NOMOGRAM.

Solvent Nomogram for Reverse Phase HPLC Solvent

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A nomogram compares the relative elution strengths of each solvent. In this way an
equivalent mobile phase composition can be chosen that has same eluting power as the
original (i.e.the overall separation will occur in the same timeframe) but with differing
selectivity. In such cases the phases being compared are said to be ISOELUOTROPIC.

This means that the elution time of the last compound will be approximately the same,
whilst the selectivity between peaks within the chromatogram may be significantly
different.

In this way the separation conditions may be optimised, by altering the mobile phase
composition, in order to achieve the desired resolution (RS>1.7 for rugged baseline
separations) between all analytes.

Table 1. Physical Properties of Common Reverse Phase HPLC Solvents

Solvent εo (Al O ) εo (SiOH) εo (C18) P^
2 3
Tetrahydrofuran 0.45-0.62 0.53 3.7 4.0
Acetonitrile (ACN) 0.52-0.65 0.50-0.52 3.1 5.8
Methanol 0.95 0.70-0.73 1.0 5.1
Water - - - 10.2
o
ε - a measure of relative elution strength
P^- Snyder polarity index value

Optimising Selectivity using Retention

As well as altering the amount of organic modifier used to achieve a separation, it is also
possible to alter the type of organic modifier used. For example, in Reverse Phase HPLC
Acetonitrile and Methanol are often used interchangeably to affect a separation.

Nomograms can be used to alter the separation such that the overall retention time
remains similar, but the selectivity of the separation is altered. Such mobile phase
compositions are said to be ‘Isoeluotropic’.

In the next example a nomogram has been used to determine the composition of an
Acetonitrile based mobile phase that will give an equivalent run time, but may alter
selectivity, compared to a Methanol based equivalent. In this case, a satisfactory
separation could not be obtained using Methanol as the mobile phase modifier.

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 Optimum methanol based separation -50% MeOH / 50% H2O

In this classic experiment from the literature, –A nomogram is used to determine the
composition of a MeOH based mobile phase that will give an equivalent analysis time, but
may alter selectivity. 40% acetonitrile and 50% methanol are known to be isoelutropic so
the separation is attempted with methanol as the alternative organic modifier. The result
is a change in selectivity –however a different critical pair (a pair of unresolved peaks
within a chromatogram) has been created.

From this experiment it is possible to conclude:


Practical HPLC Method Development (2nd ed.), Snyder, L.R., Kirkland, J.J. and Glajch, J.L. 1997, Wiley & Sons, New York
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  Nomograms can be used to alter the selectivity of a separation without radically
altering the overall retention time
 If the desired selectivity cannot be achieved using one solvent, then changing to a
different organig modifier with isoelutropic composition can result in an altered
separation selectivity
 The isoelutropic mobile phase compositions result in separations of
APPROXIMATELY the same magnitude of retention
 The approximation becomes less accurate at extremes of organic modifier content
 For this separation would be IMPOSSIBLE to separate all components using
methanol as the organic modifier in the mobile phase
 The use of the nomogram (it is very important to access the on-line course!!!)
shows that modifier compositions of 40% acetonitrile or less ALL provide a suitable
separation
 40% acetonitrile will produce an isoelutropic separation with altered selectivity and
better overall chromatographic resolution
 33% acetonitrile produces the most suitable compromise between retention time
and resolution

Gradient Elution

HPLC is frequently used for the separation of samples containing analytes with a wide
range of sample chemistry and polarity. In such situations, a constant mobile phase
composition during the analysis, i.e. isocratic conditions, may not provide an acceptable
separation. Early eluting analytes may be poorly resolved whilst other analytes may have
unacceptably long retention times with poor peak shape and sensitivity. To solve these
problems, the composition of the mobile phase may be changed during the separation –
usually starting with low amounts of modifier, which is gradually increased during the
analysis. Two or sometimes three modifiers that differ in elution strength are sometimes
employed.

During gradient analysis the mobile phase composition is mixed on-line by the pumping
system. Two pumps heads can by employed to mix a two solvent (binary system), or one
pump head can be used in conjunction with an electronically controlled mixing device to
mix two (binary), three (ternary) or four (quaternary) solvent mixtures.

After sample introduction, the ratio of these solvents is programmed to vary either
continuously or in steps, resulting in enhanced separation efficiency. The next example
shows how it is possible to use a mobile phase gradient to overcome many of the
problems associated with isocratic analysis.

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Advantages of Gradient Elution:

 Improved resolution
 Increased detection
 Ability to separate complex samples
 Shorter analysis times
 Decrease in column deterioration due to strongly retained components

Other uses of Gradient Elution:

 Column cleaning
 Scouting runs for method development

Disadvantages of Gradient Elution:

 More expensive instrumentation

 Possible precipitation at interfaces, when using multiple proportioning valves
 Re-equilibration time adds to analysis time
 Instruments vary in their dwell volume (Vd), which can cause method transfer
problems

Gradient steepness

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Where tG is the gradient time (mins)

The gradient steepness has a profound effect on the resolution of the analytes in the
chromatogram. The same start and end compositions of the mobile phase are used in
these examples the gradient time (tG is the only parameter that is altered –which alters the
rate at which the mobile phase composition is changed during the analysis.

Mobile Phase Delivery

For both Isocratic and Gradient HPLC, the on-line mixing and delivery of the mobile phase
to the system is crucial. The composition of the mobile phase must be both accurate and
reproducible in order to achieve analytical objectives.

The ability to mix and change isocratic mobile phase composition in-line gives the user
flexibility to quickly optimise the separation and obtain the required resolution for analytes
of interest. The use of in-line mixing also enables the use of gradient elution methods –
giving rise the advantages already established.

The solvents that make up the mobile phase can be mixed in several ways, including:

Isocratic -the composition of the mobile phase remains constant throughout the analysis.
Mobile phase components can be pre-mixed and delivered via isocratic pump OR for
greater flexibility the two solvent mixture can be mixed in-line using a Binary or
Quaternary system. The composition of the mobile phase can then be quickly altered for
optimisation.

Isocratic Pump

Binary –this is an extremely accurate and reproducible way of mixing solvents under high
pressure as 2 pumps (or two pump heads on a single pump unit) are used. Binary HPLC
systems generally allow mixing of only 2 solvents.

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Binary Pump
Quaternary –mixing of any combination of up to 4 mobile phase solvents is possible. This
gives the user greater flexibility. Mixing is usually achieved with the use of an in-line (low
pressure) gradient proportioning valve. This method of mixing is less accurate and
precise than binary systems.

Quaternary Pump

Solvent miscibility

For analysts employing a variety of Modes of Chromatography (Normal Phase, Reverse

Phase, Ion-Pair Chromatography, Ion Exchange Chromatography etc.), there are various
factors that need to be considered when choosing a solvent for use in a particular
analysis.

Perhaps the most important consideration from a practical perspective is that of solvent
miscibility. That is –do the solvents mix well without precipitation or the formation of
immiscible emulsions. This applies to solvents that are being used for a particular
analysis as well as when changing instrument use from normal phase to reverse phase
and vice versa.

If immiscible solvents are used, mixing can become non-uniform and the formation of an
accurate and reproducible mobile phase composition is impossible. An unstable baseline
may result due to cavitation in the mixer and/or pump chamber and/or flow cell.
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Ultimately, de-wetting can occur in which traces of an immiscible solvent coat the internal
components of the HPLC instrument –leading to problems with mobile phase formation
and solubility issues with buffers and sample components.

Where:

1. Immiscible solvents may cause de-wetting in the vacuum degasser lines

(especially those constructed from polymer or plastic materials. This can case a
reduction in degassing efficiency and possible contamination of the solvent being
delivered
2. The pump is perhaps the most susceptible area for problems with immiscible
solvents. Cavitation may cause a lack of pumping capacity (low or unsteady
pressure reading) and the mobile phase composition may be inaccurate or
irreproducible. These problems might manifest themselves as unstable
backpressure readings or irreproducible retention times in gradient or isocratic
analysis
3. The autosampler may not function correctly if solvent de-wetting occurs. Reverse
phase mobile phases containing buffers or sample components may precipitate on
contact with certain solvents - causing blockages within the very narrow channels
in the valve or capillary tubing
4. Cavitation which leads to out gassing of air in column can be catastrophic. The air
is forced through the column destroying the silica packing material and causing
channels. This can lead to poor efficiency and/or fronting peaks
5. Cavitation that leads to out gassing in the detector flow cell can be the cause of
unstable baselines as the air bubbles produced may reside in the detector and
pulse with the natural (very small) pressure ripples from the pump

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Care should be taken when changing from one mobile phase system to another.

If the previous mobile phase is immiscible with the new one then the problems such as
cavitaiton and unstable baselines may result. In these cases, and situations where the
composition of the old mobile phase in unknown, it is a good practice to flush the system
though with water containing increasing amounts of isopropanol. Isopropanol is miscible
with all common solvents both organic and aqueous. The composition of isopropanol in
the flushing solvent is gradually increased to 100% (usually 0-100% isopropanol as a
gradient over 20 mins.).

You can use the Miscibility Calculator presented below to check the Miscibility of the
solvents you propose to use.

UV Cut Off

U.V. Detectors are commonly used in HPLC analysis. The UV absorbance of an analyte
containing a chromophore is plotted against time. The higher the UV absorbance the
higher the concentration of species present at that time.

When using UV detection it is necessary to select a solvent that has no significant UV

absorption at the wavelength at which measurements are to be taken.

Using a solvent with high UV cut-off at the selected wavelength can result in increased
noise level and a loss in sensitivity.

The UV cut-off must therefore be considered. A table of UV cut-off points for typical
HPLC solvents is shown next. Where possible, a solvent with a UV cut-off of 20nm below
the wave length selected for the analysis should be used.

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Table 2. The UV cut off points for some common HPLC solvents
Solvent UV cut off (nm)
Acetonitrile 190
Water 190
Cyclohexane 195
Hexane 200
Methanol 210
Ethanol 210
Diethyl Ether 220
Dichloromethane 220
Chloroform 240
Carbon tetrachloride 265
Tetrahydrofuran 210 (220)
toluene 285

In the next picture you can appreciate the effect of changing wavelength on sensitivity of
the analysis of some common beta-blocker molecules. Note that at lower wavelengths
the background (baseline) signal rises as the UV cut-off point of the solvent is
approached.

Effect of wave length on sensitivity

Other Solvent Considerations

Purity

The purity of the solvent is important as it can affect the sensitivity of the analysis. An
impure solvent can cause an increased level of baseline noise, as well as ghost peaks. It
is recommended that HPLC Grade or better solvents are used. Gradient grade solvents
are guaranteed not produce ghost peaks when using UV detection.

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 Table 3. Properties of selected HPLC solvents
o o
P^ Solvent ri UV η (cP) δ ε (SiO2) ε (Al2O3) BP
o
cutoff ( C)
0.0 cyclohexane 1.427 200 0.98 8.2 0.04 0.04 80.7
0.0 n-hexane 1.375 195 0.313 7.3 0.03 0.01 68.9
0.3 n-decane 1.412 210 0.92 7.8 0.04 174.1
0.4 i-octane 1.404 197-210 0.5 7.0 0.01 0.01 99.2
0.4 octane 1.395 0.5 99.2
1.7 butyl ether 0.7 147.2
1.7 carbon tetrachloride 1.466 265 0.97 8.6 0.12 0.18 76.5
1.8 triethyl amine 1.401 0.38 89.5
2.2 i-propyl ether 1.368 220 0.33-0.37 7.3 0.22 0.28 68.3
2.3 toluene 1.496 285 0.59 8.9 0.23 0.29 101.6
2.4 xylene, p 0.7 138.0
2.9 t-butyl methyl ether 1.370 210 0.27 0.35 55.2
3.0 benzene 1.501 280 0.65 9.2 0.25 0.32 80.1
3.3 benzyl ether 5.33 288.3
3.4 dichloromethane 1.424 232 0.44 9.6 0.32 0.4 40
3.4 methylene chloride 1.424 233 0.44 9.7 0.32 0.42 39.8
3.4-4.4 chloroform 1.443 245 0.57 9.2 0.26 0.36-0.4 61.2
3.7 dichloroethane 83.4
3.7 ethylene dichloride 1.445 230 0.79 9.7 0.38 0.49 83.5
3.9 butanol, 1- 117.2
3.9 i-butyl alcohol 3 117.7
4.2 tetrahydrofuran 1.408 212-230 0.55 9.1 0.35 0.45 66
4.3 ethyl acetate 1.370 256 0.46-0.47 9.1 0.38-0.48 0.58 77.1
4.3 propanol, 1- 2.3 97.2
4.3 propanol, 2- 1.380 210+ 2.35 0.82 82.4-
117.7
4.4 methyl acetate 1.362 260 0.37-0.45 9.2 0.46 0.6 56.3
4.5 cyclohexanone 2.24 155.7
4.5 methyl ethyl ketone 1.379 330 0.43 9.3 0.39 0.51 80
4.5 nitrobenzene 2.03 210.8
4.6 benzonitrile 1.22 191.1
4.8 dioxane, 1,4- 101
4.8 dioxane, p 1.54 101.3
5.2 ethanol 1.361 205-210 1.2 12 0.68 0.88 78.3
5.3 nitroethane 1.392 380 0.68 114
5.3 pyridine 1.510 305-330 0.94 10.7 0.71 115.3
5.4 acetone 1.359 330 0.32 9.6 0.47-0.53 0.56-0.58 56.3
5.5 benzyl alcohol 5.8 205.5
5.7 metoxyethanol, 2- 1.401 220 1.72 124.6
6.2 acetic acid 1.372 210 1.1-1.26 12.4 large 117.9
6.2 acetonitrile 1.344 190 0.37 11.7 0.5 0.55-0.65 81.6
6.4 dimethyl formamide, 1.431 268 0.90-0.92 11.5 153
n,n-
6.5 dimethyl sulfoxide 1.478 2.24 12.8 0.41 0.62 189
6.6 methanol 1.329 205 0.6 13.7 0.73 0.95 64.7
7.3 formamide 1.450 210 3.3-3.76 210.5
9 water 1.333 180 1 21 large 100

Where

P^: polarity index (Snyder)

η: (cP) at 20°C
δ: Hildebrand solubility parameter

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Viscosity

Lower viscosity solvents will in general give narrower peaks due to improved mass
transfer of analyte in the mobile phase. Viscosity is also important when considering
system backpressure. The more viscous the solvent the higher the system backpressure.

Refractive Index

A refractive index detector works by comparing the refractive index of a reference cell
filled with mobile phase to the refractive index of the mobile phase containing the analyte.
The greater the difference in refractive index the greater the concentration of species
present at that time and the larger the output signal.

Better detection limits will be achieved if the refractive index of the mobile phase varies
greatly from that of the sample.

Boiling Point

Important in preparative HPLC. A solvent with a low boiling point will be easier to remove
(by evaporation) from the sample.

Mobile Phase Preparation

Mobile Phase preparation can be divided into the following major Steps:

 Measure the appropriate volume of each solvent

 Adjust the pH of aqueous component using buffers
 Add any other required additives to the aqueous component of the mobile phase
 Mix solvents
 Filter mobile phaseξ
 Degas mobile phase
ξ
The filtering and degassing steps may be carried out in tandem.

Mixing

Most mobile phases are a mixture of at least two solvents. The volume of each solvent
should be measured independently and pH adjustment and additives added to the
aqueous component PRIOR to mixing the solvents.

This avoids problems associated with volume change on mixing certain solvents – a 60:40
mix of water:methanol may be incorrect by up to 10% due to latent heat of mixing affecting
the overall volume of the mixed solution.

Mixing is often carried out on-line by the HPC system – this tends to overcome the
problems when pre-mixing mobile phase components – which one has to do when using
isocratic pumping systems.

When using buffers in gradient analysis make sure they are entirely soluble in the full
range of expected mobile phase compositions. Less soluble buffers may precipitate as
the organic strength of the mobile phase is increased – mobile phases containing more
than 60% acetonitrile are particularly renowned for this phenomenon.

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Filtration

To protect the HPLC and your column from particulate matter, manufacturers recommend
filtering of mobile phases prior to use.

This task can be accomplished with simple vacuum filtration devices purchased through
HPLC supply catalogs. Add all buffers and modifiers prior to filtration. A vacuum is applied
to pull the solvent through a filter –which also acts to simultaneously degass the mobile
phase.

Handle filters with tweezers and make certain the filtration apparatus is clean at all times.
Nylon 66 is a good filter material for aqueous mobile phases, while PTFE is an excellent
filter for most organic solvents. Inorganic membranes are resistant to chemical
degradation from a wide range of HPLC solvents.

Be aware that Teflon filters cannot be used with water due to the material’s nonpolar
characteristics. HPLC mobile phase filters have a pore size of around 4.5 microns.

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Degassing

Mobile phase degassing is used to remove dissolved gases from the mobile phase. By
removing oxygen you also help prevent possible oxidative degradation of the sample and
mobile phase. Air in the mobile phase can cause a variety of problems:

Pumping reliability decreases. Air in the pumping system can cause irreproducible
system back-pressure, retention time drift and poor solvent mixing.

Air bubbles can get trapped in the check valves –causing a distinctive pressure ripple with
associated baseline appearance.

An air bubble in the piston chamber can cause a loss of pumping efficiency. A sinusoidal
base line is seen due the lower compressibility of air in the pump head. In extreme cases
it is possible that air will accumulate in the pump head which will stop the mobile phase
flow.

Back pressure may also rise.

Out gassing in the solvent mixing chamber of quaternary pumps may also cause
poor on-line solvent mixing characteristics –leading to irreproducible retention
times.

Important

Using inert materials such as stainless steel, sapphire, ruby and PEEK the
piston pumps can also give trouble free flow of solvent systems containing
buffers at pH extremes. Recent advances in column chemistry mean that the
full pH range can be used.

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Outlet Degassing

As well as air in the pump head another area where excessive air can be problematic is
the Flow cell.

An excessive amount of dissolved gas in the mobile phase can cause spurious peaks,
noisy and unstable chromatographic baselines.

As the mobile phase moves into the flow cell the relatively small volume of the capillary
tubing into the large volume of the flow cell it experiences a large change in pressure.
Any air dissolved in the solvent may degas into the flow cell causing baseline noise.

If excessive air accumulates in the flow cell it may agglomerate into a more significant air
bubble. In this case the bubble will move in time with the very small pressure variations
caused by the pump. As the air bubble passes in front of the flow cell windows – changes
in absorbance will occur – which manifest themselves as a undulating baseline or noise
spikes.

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Degassing / Column Damage

Air trapped in the column (as a result of poor storage or out gassing of dissolved gases
from the mobile phase), can cause severe mechanical destruction of silica based
stationary phase support materials.

Any air trapped in the column will move very rapidly once the pump is started and the
system pressure begins to rapidly rise. Air under very high pressure will effectively
destroy the packing material as it moves through the column –leaving a ‘channel’ shaped
space in its wake. Ultimately this channel will form a ‘path of least resistance’ for at least
part of the analyte slug and as there is relatively little silica surface within the channel it
will very quickly become saturated with analyte.

The remaining analyte will travel through the channel with very little retention –resulting in
very broad and obviously ‘fronting’ chromatographic peaks.

Methods of Degassing

There are four commonly used methods for degassing mobile phases:

 Helium Sparging
 Ultrasonic Degassing
 Vacuum Degassing
 Refluxing

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The relative efficiency of some of these methods is shown – the figure is based on the
percentage of oxygen removed from a set volume of methanol.

Broadly the degassing techniques are spilt into online and offline techniques –with online
techniques being preferred. Offline techniques might be highly effective – but once the
degassing stops and the eluent bottles are placed onto the HPLC system –ingress of air
will begin almost immediately unless the solvent is held under a blanket of inert gas.

Ultrasonic degassing is perhaps the least favoured of the off-line techniques. Not only is it
relatively inefficient, it also risks to loss of volatile mobile phase components (when the
phase has been pre-mixed) through solvent heating. This can lead to irreproducible
retention times.

Refluxing is perhaps the most effective degassing technique –however it is arduous and
not without risk when trying to degass large amounts of solvent at any one time. For
these reasons refluxing is rarely used.

Effective degassing with give flatter and more uniform baselines.

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Helium Sparging

Helium sparging removes approximately 80% of the dissolved gases by solubilising

dissolved gases from the mobile phase into itself. Helium however, is not particularly
liquid soluble and will agglomerate and out-gas, carrying the other dissolved gases with it.

Helium spargers can be simply constructed by connecting a length of PTFE tubing with a
frit at one end to a helium cylinder. Immerse the frit into the mobile phase and adjust the
regulator to release a gentle stream of bubbles. It is important that the helium is released
at the bottom of the eluent reservoir and permeates throughout the whole of the eluent.

Generally speaking 1 litre of mobile phase will be fully degassed with 1 litre of helium.

Excessive degassing with helium can cause the loss of more volatile mobile phase
components and may result in a poorly degassed phase – especially if the surface of the
eluent liquid can be seen to be ‘bubbling’.

Vacuum Degassing

The mobile phase enters the gas permeable tubing and passes in to the vacuum
chamber. Any dissolved gas will be drawn out from the mobile phase, thought the tubing
and into the lower pressure of the vacuum chamber.

An increased level of gas in the vacuum chamber will cause the pressure to increase and
level of vacuum to drop. The sensor monitors this change in pressure. Once a pre-
defined level is reached the control circuit opens the solenoid valve, turns the pump on
and the gas is removed from the vacuum chamber and the correct level of vacuum re-
established.

This method of degassing is perhaps the most popular for modern instruments and most
manufacturers will provide an online degassing unit such as that shown opposite. Whilst
online vacuum degassing is not the most efficient technique –it is both quick and
convenient –leading to it’s widespread adoption. Modern on-line vacuum degassers are

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capable of producing an eluent stream that satisfies all the stringent performance
characteristics of modern HPLC.

Glossary

Capacity Factor (k) - The capacity (or retention) factor is a means of measuring the
retention of an analyte on the chromatographic column.

The capacity factor is equal to the ratio of retention time of the analyte on the column to
the retention time of a non-retained compound. The non-retained compound has no
affinity for the stationary phase and elutes with the solvent front at a time to, which is also
known as the ‘hold up time’ or ‘dead time’.

Capacity factor is independent of some key variable factors including small flow rate
variations and column dimensions. Because of this, it is a useful parameter when
comparing retention of chromatographic peaks obtained using different HPLC systems.

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Critical Pair – a pair of unresolved peaks within a chromatogram.

Resolution (Rs) - The most important thing in HPLC is to obtain the ’optimum resolution in
the minimum time’. A resolution value of 1.5 or greater between two peaks will ensure
that the sample components are well separated - to a degree at which the area or height
of each peak may be accurately measured.

Resolution is calculated using the separation of two peaks in terms of their average peak
width at the base (tR2 > tR1).

In the case of two adjacent peaks it may be assumed that the peak width at the base w ≈
b1
w , and thus, the width of the second peak may be substituted for the average value.
b2

Selectivity (α) - The selectivity (or separation factor) (α) is a measure of the ability of the
chromatographic system to ‘chemically’ distinguish between sample components. It is
usually measured as a ratio of the retention factors of the two peaks in question and can
be visualised as the distance between the apices of the two peaks.

By definition, the selectivity is always greater than one – as when α is equal to one, the
two peaks are co-eluting (i.e. their capacity factor values would be identical). The greater
the selectivity value, the further apart the apex of the two peaks becomes.

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