Chromapedia
Complete Guide
2
Chapter 1
Basic Principles of Flash and Prep HPLC
Types of chromatography techniques
5
Purification workflow
5
Adsorption chromatography: flash versus prep HPLC
6
The chromatography system
8
The chromatogram
10
Normal-phase versus reversed-phase
14
Stationary phases
15
Mobile phases
20
Summary of common stationary and mobile phases in adsorption chromatography
26
Chapter 2
Consumables and Sample Loading
Consumables in flash and prep HPLC
29
Cartridge & column handling
31
Self-Packing of empty flash cartridges and glass columns
32
Sample loading in flash and prep HPLC
35
Chapter 3
Detection Methods
Detection methods in flash and prep HPLC
39
UV detector
39
Evaporative light scattering (ELS) detector
42
Mass spectrometry (MS) detector
44
Refractive index (RI) detector
44
Fluorescence detector
45
Combination of UV and ELS detectors
46
Chapter 4
Method development
Method development in flash and prep HPLC
49
Definition of the purification method (flash or prep HPLC)
49
Analysis of sample solubility
49
Screening of suitable conditions for separation (mobile & stationary phase)
50
Upscaling to flash or prep HPLC
54
Improving resolution
58
References
69
Notes
70
3
Basic Principles
of Flash and
Prep HPLC
Basic
Basic Principles
Principles of
of Flash
Flash and
and Prep
Prep HPLC
HPLC
Chapter 1
4
Basic Principles of Flash and Prep HPLC
Types of chromatography techniques
Chromatography is one of the most powerful methods for separating a sample, such as a
synthesized mixture or a biological crude extract, into its single components. The separation
process is based on the components partitioning between two phases: a stationary phase
with a large surface and a mobile phase which moves through the stationary phase.
Chromatography is classified into two types: liquid or gas chromatography. The difference is
related to the physical state of the mobile phase. In gas chromatography, the mobile phase is
a gas which transports the sample through a solid stationary phase, whereas in liquid chromatography the mobile phase is a solvent. The interaction of the compounds with the stationary phase (= mode of separation) is governed by differences in polarity, size, or specific
binding affinities. The mode of separation determines the type of liquid chromatography:
Type of liquid chromatography
Mode of separation based on
Adsorption chromatography
(Normal- and reversed-phase)
Polarity
Affinity chromatography
Specific binding interaction
Size exclusion chromatography
Molecular sizes
Ion exchange chromatography
Charge
Table 1: Types of liquid chromatography
Purification workflow
Adsorption chromatography is the main part of a typical isolation and purification workflow
for drugs, chemicals, flavors and others. Initially, the compounds are chemically synthesized
or extracted from plants, bacteria or other living organisms. Evaporation is used to concentrate the material to simplify downstream processing. If the sample is new and unfamiliar, a
screen for optimal separation conditions is performed, usually by thin-layer chromatography
(TLC) or analytical high-pressure liquid chromatography (HPLC) methods. Once suitable
conditions have been found, the procedure is upscaled to preparative chromatography. In
the preparative step, the target compound is purified in high quantities either by flash chromatography, prep HPLC or a combination of both. If the techniques are used together, flash
chromatography is applied for the pre-purification step and prep HPLC to achieve the final
high purity. After successful separation of the compounds, a second concentration step is
performed either by evaporation or freeze drying. At this point, the compound is ready for
analysis of its purity and function by other techniques, including melting point analysis, analytical HPLC, enzymatic assays and others. The complete workflow is shown in the following
figure:
5
Basic
Basic Principles
Principles of
of Flash
Flash and
and Prep
Prep HPLC
HPLC
Extraction /
Synthesis
Evaporation
Decreasing the
sample volume
Analytical
Chromatography
TLC (NP)
Screening for the optimal condition for the
following preparative
chromatography step
Analytical
HPLC (RP)
Preparative
Chromatography
Flash Pre
purification
Separation of the
target compound
Prep HPLC
High resolution
purification
Evaporation /
Freeze Drying
Decreasing the
sample volume
Analysis purity /
function
Figure 1: Purification workflow
Adsorption chromatography: flash versus prep HPLC
Adsorption chromatography was the very first form of chromatography, discovered more
than 100 years ago by Mikhail Tsvet, a Russian-Italian botanist who used adsorption chromatography to separate the pigments in plants. Since then it has developed very rapidly into
a major technique used in any synthesis or extraction lab.
In adsorption chromatography, the separation is governed by the interactions of the sample components with the stationary and mobile phase. Compounds with different chemical
properties (polarities) display varying affinities, or strengths of adhesion, towards the stationary and mobile phase. Affinity is influenced by two molecular properties, adsorption and
desorption. Adsorption refers to the ability of a certain component to stick to the stationary
phase. Desorption, or solubility, describes how well a component of the mixture dissolves
in the mobile phase. The speed with which individual sample components migrate through
the stationary phase depends on their adsorption/desorption properties as shown in the
following figure:
Adsorption
Figure 2: Adsorption versus desorption
6
Desorption
Basic Principles of Flash and Prep HPLC
Adsorption chromatography can be either preparative or analytical. Analytical chromatography is normally performed with smaller amounts of material to establish the presence or
quantify the relative proportions of components in a mixture. It is either done via TLC or
column chromatography (= analytical HPLC). The difference is the type of stationary phase:
in TLC the stationary phase is coated on a glass or aluminum plate whereas in column chromatography the stationary phase is packed in a column.
The primary objective of preparative chromatography is to separate or purify the components of a mixture for downstream applications. Here, the goal is to produce a large quantity
of pure compound. Preparative chromatography could be either gravity fed chromatography (low pressure), flash chromatography (medium pressure), or prep HPLC (high pressure).
These different chromatography types differ by the pressure generated by the flow of the
mobile phase through the stationary phase. Gravity feed chromatography, also known as
open column chromatography, uses only gravity to push the substances through the column
which is rather time intensive. Modern chromatography systems use a pump (flash and prep
HPLC) which allows for faster speed and makes the separation process more efficient. The
pros and cons of the traditional and modern ways are summarized in the following table:
Open column chromatography
Flash / prep HPLC
Pros
Pros
Fast process
• High resolution
• High flow rates
• High automation
Low expenses for instrumentation, maintenance and consumables
Low pressure
Low solvent consumption
• High resolution
Cons
Very time consuming
• Low resolution
• Low flow rates
High flexibility
• Compatibility with large variety of consumables and detectors
High solvent consumption
• Low resolution
High reproducability
• Stabile conditions
Low flexibility
• Not compatible with high performance
consumables or detectors
No fume hood needed (if fraction collector
closed)
Cons
Low reproducability
• No regulated flow or pressure
Instrument & maintenance costs
Consumable costs
Fume hood needed
Table 2: Open column chromatography versus flash & prep HPLC
7
Basic
Basic Principles
Principles of
of Flash
Flash and
and Prep
Prep HPLC
HPLC
The first publication using elevated pressure was at the end of the seventies with a method called flash chromatography. Besides this, attempts were made to increase the size of
the analytical HPLC systems and thus make them available also for preparative work (prep
HPLC). Nowadays, both techniques are frequently used but for different objectives: flash
chromatography is mainly used as a pre-purification step to purify large sample quantities at
a decent resolution, whereas in prep HPLC the goal is to achieve highest resolution (purity)
under the condition of lower loading capacities. Therefore, the two techniques differ in the
material used for the stationary phase (different particle size), the dimension of the cartridge
or column (internal diameter (ID) and lengths) and flow rate of the mobile phase as shown in
the following table:
Flash
Prep HPLC
Particle size
15 - 63 μm
5 - 15 μm
Column ID
12 - 115 mm
10 -70 mm
Flow rate
15 - 250 mL/min
5 - 100 mL/min
Loading capacity
< 300 g
< 10 g
Max pressure
50 bar
300 bar
Table 3: Differences between flash and prep HPLC
The chromatography system
A complete chromatography system is comprised of a mobile phase reservoir, a pump which
moves the mobile phase through the system, a sample injection unit for sample loading, the
column/cartridge packed with the stationary phase where the actual separation occurs, a
detector linked to a computer to measure changes in the composition of the eluate and a
fraction collector to gather all fractions with different compositions. All components are described in more detail below.
Mobile phase reservoir
The reservoir needs to be large enough to hold enough mobile phase to ensure the stationary phase never dries out. For gradient elution, solvents are stored separately from each
other and several mobile phase reservoirs are attached to the system.
Pump
The pump must maintain a flow rate that is high enough to counter the back pressure generated by the stationary phase packed in the column or cartridge.
Injection unit
The injection unit is essential for reliable and homogeneous sample delivery to the packing
material. Sample loading should occur without introduction of any air into the column and
should be performed in the most direct manner possible.
8
Basic Principles of Flash and Prep HPLC
Column/cartridge
The column or cartridge is the actual site where the separation takes place. It`s filled with the
stationary phase and its hardware should be robust enough to resist to pressures generated
by the flow of the mobile phase passing through it.
Detector
The detector is needed to visualize the separated component bands as they elute from the
column. The detector indicates when to switch sample collection vessels based on a signal
matching the composition of the sample. Since the characteristics of the sample components can vary greatly, several types of detectors have been developed (see Chromapedia
volume 1).
Fraction collector and waste
When the mobile phase exits the detector, it can be either collected or sent to waste. Collection is needed in cases where the mobile phase contains a separated band with a desired
component that is needed for further processing and evaluation.
Computer
The detector is wired to a computer, the system component that records the electrical signal
needed to generate a chromatogram. The chromatogram is used to identify and quantify the
concentration of sample constituents and to enable proper collection.
System connections
All system components are connected with high-pressure tubing, valves and fittings to form
a continuous structure through which the mobile phase, sample and the resulting separated
component bands can flow.
A depiction of a typical liquid chromatography set-up is illustrated below:
Pump
Injection valve
Waste diverter valve
Column
Mobile
phase
reservoir
Detector
Fraction collector
Waste
Computer data station
Figure 3: Setup of a liquid chromatography system
9
Basic
Basic Principles
Principles of
of Flash
Flash and
and Prep
Prep HPLC
HPLC
The chromatogram
Results of a chromatographic separation are visualized with a chromatogram. Detected signals are referred to as peaks. Each peak represents the response of the detector to a different compound. The entire record of all detector signals is called a chromatogram.
Several measurable parameters of a chromatogram are important for data analysis and
quality control of the separations:
•
•
•
•
•
•
t0: the dead time of the column, represents the time required by a solvent to flow through a
packed column at a given flow rate
tR: retention time, refers to the time from injection of the substance to the peak maximum
w: the base width of the peak, measured between the inflectional tangents
a: the value of the front half-width of the peak
b: the value of the back half-width of the peak
b0.5: the peak width at the half-height
The x-axis measures time in minutes, whereas concentration values are plotted on the y-axis.
tR
h/2
b 0.5
h/2
t0
a
b
h10%
w
0
1
2
3
4
5
6
Time (min)
Figure 4: Chromatogram
An ideal chromatography peak has a sharp symmetrical shape as a Gaussian peak with a
narrow width on a flat baseline. Obtaining good peak shapes is essential to efficiently separating the target compounds from other components in a mixture. However, narrow widths
are typical for analytical chromatograms. Narrow widths do not apply to preparative chromatography, as higher sample loading generally leads to peak broadening.
10
Basic Principles of Flash and Prep HPLC
As long as the peaks are symmetrical, the sample flows homogenously through the stationary phase. Only in the case of abnormal peak shapes, the components flow gets disturbed,
which can be caused by the sample, the solvent, or the equipment including the cartridge
or column.
There are two ways to determine the shape of a peak:
•
•
asymmetry factor
tailing factor
The asymmetry factor (As) is measured at 10 % (of peak height) above the baseline and is
defined as:
a is the front half-width of the peak at 10 % of the peak height
b is the back half-width of the peak at 10 % of the peak height
The tailing factor measures the peak width at 5 % (of peak height) above the baseline and
is defined as:
a is the front half-width at 5 % peak height
b is the back half-width at 5 % peak height
A Tf or As of 1 is indicative of a perfectly symmetrical peak. Below or above 1, the peak
shows either fronting or tailing:
Fronting
Gauss Curve
Tailing
Tf or A s < 1
Tf or A s = 1
Tf or A s > 1
Common causes and suggestions on how to overcome these issues are discussed below.
It is worth mentioning that the causes of peak distortion can vary greatly and might not be
addressed here.
11
Basic
Basic Principles
Principles of
of Flash
Flash and
and Prep
Prep HPLC
HPLC
Peak tailing
• Secondary interactions
Ionized silanol groups can interact with compounds possessing amine and other basic functional
groups. As a result, not all molecules travel through the column at the same speed which causes
tailing at the peak. To counter this problem, the pH of the mobile phase could be reduced to
suppress ionization of silanol groups.
• Dead volume
Excessive column dead volume in a chromatography system leads to diffusion of the solute molecules and can therefore tail the peak. This will mostly influence the peak shape of the early eluting
peaks. Later eluting compounds will be more influenced by the packing material itself, as these
slower compounds reside longer in the material.
• Sample solvent
In case the sample is not completely soluble in the starting condition, the peak might tail because
not all molecules travel homogenously through the stationary phase. Therefore, it is important to
make sure the sample is always fully solubilized. Peak tailing could also be due to a sample solvent that has a much higher elution strength than the mobile phase. During the run through the
stationary phase the elution strength decreases by diffusion effects with the mobile phase, which
leads to different retention times of the components. To avoid this problem, it is recommended to
use the mobile phase as an injection solvent or to ensure the elution strength of the mobile phase
matches the starting conditions of the purification.
• Column overload
Peak tailing could arise when the column gets overloaded so that the concentration of compounds in the mobile phase is very high, whereas the concentration on the stationary phase is
rather low. The high concentration peak will move through the column more rapidly than the low
concentration peak. Lastly, the peak will have a sharp front and a sloping tail. The problem could
be eliminated by reducing the sample loads, diluting the sample, solid loading or switching to a
bigger column or cartridge.
Peak fronting
• Column overload
Column overload can also lead to fronting. The reason for that is vice versa to peak tailing: it
happens if the concentration of compounds in the stationary phase is very high, whereas the
concentration in the mobile phase is rather low. In this case, the high concentrations peak will
move through the column more slowly than the lower concentration peak and the peak will be
distorted with a sloping front and a sharp tail. The problem could be eliminated as described
above by reducing the sample loads, diluting the sample, solid loading or switching to a bigger
column or cartridge.
12
Basic Principles of Flash and Prep HPLC
• Packing bed deformation
The creation of a void at the inlet of the column or the presence of channels in the packing bed
can lead to peak fronting. This can happen if the column has been used for a long time and is at
its shelf life limit. However, if the issue occurs suddenly, it might be related to running the cartridge
at excessively high pressure. This can happen in cases, such as the outlet frit or tubing becoming
clogged by particles from the solvent. Hence it is recommended to always use clean solvents
and filter the sample.
Peak splitting
Besides peak fronting and tailing, another abnormal peak shape can occur which is peak
splitting. Peak splitting describes a Gaussian peak with a shoulder or a twin:
If peak splitting occurs, it is important to check if the splitting happens on one or two peaks
or all of the peaks are affected. If only a single peak is affected, it is most likely due to a
problem with the chemistry (separation problem). In this case the problem can be solved
eventually by optimizing the mobile phase conditions. Another solution could selecting another stationary phase to improve the separation.
But often, all peaks are affected by peak splitting and in this case, the issue is related to the
column:
•
The presence of a void at the inlet of the column or channeling in the packing bed will
lead to peak splitting. When using pre-packed cartridges, it is not possible to correct
these issues so using a new cartridge is the only option.
•
Partially clogged inlet frits can also lead to peak splitting. Filtration of samples before
injection can prevent the splitting from happening. The use of solid loading injection
techniques can also help eliminate the problem.
13
Basic
Basic Principles
Principles of
of Flash
Flash and
and Prep
Prep HPLC
HPLC
Normal-phase versus reversed-phase
In general, flash and prep HPLC can be performed under two conditions: normal-phase and
reversed-phase. These conditions differ in the polarity of the stationary and mobile phase
and therefore also in their applications. In normal-phase chromatography, the stationary
phase is polar or hydrophilic and the mobile phase is rather non-polar or hydrophobic in
nature. As individual components travel through the column, their polar groups interact with
the polar groups of the stationary phase. Low polarity substances have a lower affinity for
the stationary phase and are eluted first. To elute the hydrophilic components, the use of
more polar solvents in the mobile phase is required. Generally, the polarity of the solvents
increases during a run to achieve best separation results of the components.
UNPOLAR
POLAR
Carboxylic acids
Amids
Sulfones/Sulfoxides
Alcohols/Amines
Esters/Aldehydes/Ketones
Nitro compounds
Ethers
Sulfides
Aromatics Organic halgen compounds
Olefins
Alkanes
Figure 5: Normal-phase conditions
In reversed-phase chromatography, the phases are reversed so that the stationary phase is
non-polar, and the mobile phase is rather polar in nature. Here, hydrophobic molecules adsorb to the hydrophobic stationary phase, whereas hydrophilic molecules pass through the
column faster and are eluted first. Hydrophobic molecules can later be eluted by decreasing
the polarity of the mobile phase by using an organic, non-polar solvent. Generally, the polarity of the solvents during a run decreases.
POLAR
UNPOLAR
Figure 6: Reversed-phase conditions
14
Aliphatics
Organic halogen compounds
Ketones
Ethers/aldehydes
Amines
Alcohol/phenols
Carboxylic acids
Basic Principles of Flash and Prep HPLC
Stationary phases
The stationary phase needs to retain the target compound, but neither too strongly nor to weakly.
In the case of no adsorption, the compound runs with the mobile phase through the column and
likely fails to separate from the impurities or other compounds in the mixture. If the compound is
too sticky to the stationary phase, it will take too much time and solvent to elute it.
Adsorption is mainly influenced by the polarity of the stationary phase, but other parameters
have an impact as well. These parameters are discussed in more detail below.
Normal-phase stationary phases
Bare (= unbonded) silica is the most widely used stationary phase in chromatography. It’s a porous
form of silicon dioxide, consisting of an irregular tridimensional framework of alternating silicon
and oxygen atoms.
HO
Si
O
Si
HO
O
O
O Si
Si
O
O
O
O
O
Si
O
Si
HO
O
Si
HO
Si
O
Si
O
HO
Figure 7: Silica molecular structure
The silica material is a porous material where the silanol groups are spread over the entire
surface of the packing material, including the pores.
HO
HO
HO
SiO2
HO
HO
HO
HO
Figure 8: Polar surface of silica
Silica is slightly acidic, and stable in the pH range of 2 to 8. Its bulk density is about 0.4 to
0.8 g/cm3.
Unbonded silica is considered a cost-effective stationary phase with high sample capacity
and strong mechanical resistance. This type of packing material is a good choice for separating high and medium polar compounds and is mainly used for flash applications.
Other silica phases, called bonded phases, can be used for normal-phase applications as
well. They feature lower polarities and are therefore good alternatives in cases where the
retention of polar compounds on silica is too strong.
15
Basic
Basic Principles
Principles of
of Flash
Flash and
and Prep
Prep HPLC
HPLC
HO
H2N
H2N
H2N
Si
O
Si
O
Si
O
HO
HO
HO HO HO
O
O
O
Si
O
Amino
Si
O
Si
O
Diol
Figure 9: Surface of amino- and diol- bonded silica
Alumina is another rather polar phase and can be used in place of unbonded silica. It features a network of aluminum and oxygen atoms. The active points of this packing material
are the Al3+ centers and the connecting O2- atoms. Alumina is typically basic but can also be
obtained in neutral or acidic form. Alumina gels are available in particle sizes similar to those
of silica gels. The density is about 0.9 g/cm3. For neutral alumina, the sequence of elution is
very similar to that of silica gels.
OO
OH
OH
OH
OH
AI
AI
AI
AI
AI
O
O
O
O
O
O
Figure 10: Alumina structure
Alumina provides superior pH stability (pH 2-12) over silica and its neutral form is frequently
used for acid sensitive compounds, which cannot be purified with silica. Even though alumina has a lower loading capacity than silica, alumina offers a unique separation performance
for some applications.
Reversed-phase stationary phases
Reversed-phase silica consists of non-polar organic groups, such as octadecyl (C18), octyl (C8)
or butyl (C4) alkyl chains bonded to the silanol groups of the silica gel via the stable Si-O-Si-Cbond.
Si
O
Si
O
Si
O
C4
Figure 11: C4, C8 and C18-bonded silica
16
Si
O
Si
O
C8
Si
O
Si
O
Si
O
C18
Si
O
Basic Principles of Flash and Prep HPLC
Typically, C18 with its long alkyl chain is used for compounds smaller than 2000g/mol,
whereas C8 and C4 give better results for molecules with higher molar mass (up to
10000g/mol).
Besides normal-phase, amino and diol phases can be used for reversed-phase applications.
Since their polarity is somewhere between unpolar and polar, this makes amino and diol
phases the allrounders in chromatography. They are ideal phases in cases where the retention of rather unpolar compounds on C18 is too strong.
Important parameters to keep in mind in reversed-phase chromatography are carbon loading and endcapping. These parameters play an important role for retention as described
below.
Endcapping
After functionalization (e.g. with C18 chains), the silica still has unreacted silanol groups (free
-OH groups on the silica surface).
Unpolar endcapping is used to limit the interaction of the free acidic silanols with basic compounds. A smaller hydrophobe such as trimethylsilyl fits into the tight space between the
C18 groups and reacts with the active silanol and therefore renders the silica non-acidic and
non-polar. This minimizes non-specific binding of the basic compounds with the surface and
improves the chromatographic peak shape.
On the other hand, polar endcapping is used to stabilize the C18 alkyl chains in a 100 % water environment. Many polar compounds prefer highly aqueous mobile phases for solubility
reasons and can be retained only with a minimal concentration of organic modifier. Standard
C18 shows phase collapse and poor resolution resulting from these conditions. The polar
endcapping of C18 allows phase stabilization and better retention of polar compounds in a
highly aqueous environment.
O Si
O Si
O Si
O Si
O Si
O Si
O Si
O Si
O Si
O Si
No endcapping
Unpolar
O Si
O Si
Polar
Unpolar
O Si
O Si
Polar
Unpolar endcapping
O Si
Polar endcapping
Figure 12: Different types of endcapping
17
Basic
Basic Principles
Principles of
of Flash
Flash and
and Prep
Prep HPLC
HPLC
Carbon content
Carbon loading refers to the covalent attachment of hydrocarbons to a silica gel. It depends
on the relative surface coverage and chain length of the bonded functional groups. Higher
carbon content generally results in better resolution, but also in longer run times.
O Si
O Si
O Si
O Si
O Si
O Si
O Si
O Si
Figure 13: High and low-carbon content silica gel
Particle size
Particle size is one of the most important properties of any stationary phase. Smaller silica
particle sizes in a cartridge or column correspond to higher total surface area. Higher surface
area increases the number of possible adsorption and desorption steps of the separation
and therefore improves column efficiency.
The smaller the particle size, the better the resolution and purity of the final product. Although switching to smaller silica gel particles is relatively easy, a limitation to consider is
adverse backpressure effects.
Decreasing the particle size by half increases the pressure value by a factor of 3-4 as shown
in the following figure:
10 µm
Pressure: 23 bar
20 µm
Pressure: 6 bar
40 µm
Pressure: 2 bar
80 µm
Pressure: 0.5 bar
Time (mins)
Time (mins)
Time (mins)
Time (mins)
Figure 14: Impact of particle size on back pressure
For flash chromatography, standard particle sizes range from 40-60 µm, but for more complex samples 20-30 µm are commonly used. For prep HPLC, the particle sizes range from
5-15 µm, which allows highest purity separations.
18
Basic Principles of Flash and Prep HPLC
Particle shape
The shape of silica gel particles could be either spherical or irregular. Spherical particles help
to achieve a more homogenous flow through the column than irregular particles, which ultimately leads to sharper peaks. Higher resolution is achieved thanks to the fact that spherical
particles can be more densely and uniformly packed in a column or cartridge. The production
of spherical particles is very complex, so the costs of such material is much higher than for
irregular particles.
Generally, spherical particles are useful for complex applications, irregular particles are sufficient for basic applications.
Spherical particles
Irregular particles
Homogenous flow
Irregular flow
Figure 15: Sample flow through spherical and irregular silica particles
Pore size
Silica particles can have a range of pore sizes. The standard pore size used in flash chromatography and prep HPLC is around 60 to 100 Å. These pore sizes are well suited for small molecules
with sizes of up to 5 kDa, because the pores are well accessible, the surface for interaction is
large and the resolution is high. As a rule of thumb, for easy access to the pores, the pore size
should be at least three times the diameter of the molecule. For bigger molecules, especially
peptides or proteins bigger than 5 kDa, larger pore size silica of 200 to 300 Å should be used for
optimal separations. In general, adsorption chromatography is useful for peptides with a maximal
size of 100 kDa. Size exclusion chromatography is needed to separate any molecules larger than
100 kDa.
Small pore diameter
Large pore diameter
Figure 16: Pore size differences
19
Basic
Basic Principles
Principles of
of Flash
Flash and
and Prep
Prep HPLC
HPLC
Surface area
Typically, silica particles have a very high surface area, a property which is required for good resolution. Their specific surface area is equal to the sum of its internal and external surface area and
therefore affected by the particle size and the pore size. But since more than 99 % of the surface
area is contained in the internal pore structure, the pore size plays the most important role. Generally, the smaller the pore size, the higher the surface area and the better the separation. However,
this is only true for molecules that can easily diffuse into the pores, so the ratio of compound size
and pore size is crucial to fulfil this rule.
The typical surface area of silica for purification of small molecules (> 5 kDa) is at around
500-600 m2 /g, whereas for bonded silica it is at 200-300 m2 /g. For alumina, the surface area
is much lower at around 150 m2 /g.
Mobile phases
The most important characteristics of the mobile phase are its ability to solubilize the sample
and to transport the target compound through the stationary phase neither too quickly nor
too slowly. The speed is dependent on the strength of the solvent and its ability to displace
or elute a substrate from the active sites of the stationary phase. In this sense, solvents are
described as weak or strong and usually a combination of both is used during the separation
process.
Apart from solvent strength, there are other factors which affect the suitability of a solvent
for a particular separation problem. These properties include the dipole parameter, the proton donor parameter and the proton acceptor parameter, which all have an impact on the
separation.
Solvents in normal-phase chromatography
In normal-phase chromatography, the solvents used are rather non-polar or hydrophobic. Typical
solvent combinations of a weak and strong solvent are hexane / ethyl acetate and dichloromethane/methanol. Dichloromethane/methanol has a higher polarity than hexane/ethyl acetate, so it
is better suited to the separation of more polar compounds. The table below offers an overview
of other common solvents in normal-phase chromatography indicating their polarity and other
characteristics. More polar solvents have higher solvent strengths.
20
Basic Principles of Flash and Prep HPLC
Solvent
Polarity E°
Dipole
parameter
Proton
acceptor
parameter
Proton donor
parameter
n-Pentane
0.00
0
0
0
Hexane
0.00
0
0
0
Petroleum ether
0.01
0
0
0
Cyclohexane
0.04
0
0
0
Xylene
0.26
0
0.5
0
Isopropyl ether
0.28
0.5
0.5
0
Toluene
0.29
0
0.5
0
Diethyl ether
0.38
2
2
0
Chloroform
0.40
3
0.5
3
Dichloromethane
0.42
5.5
0.5
0
Tetrahydrofuran
0.45
4
3
0
Acetone
0.56
5
2.5
0
Dioxane
0.56
4
3
0
Ethyl acetate
0.58
3
2
0
Table 4: Solvents in normal-phase chromatography
Solvents in reversed-phase chromatography
In reversed-phase chromatography the mobile phase is rather polar in nature and therefore water
becomes the weakest solvent. The value of the solvent strength increases with decreasing polarity. Frequently used mobile phases in reversed-phase chromatography are mixtures of water and
a second water-miscible solvent, such as methanol, acetonitrile or ethanol.
21
Basic
Basic Principles
Principles of
of Flash
Flash and
and Prep
Prep HPLC
HPLC
Solvent
Polarity E°
Dipole
parameter
Proton
acceptor
parameter
Proton donor
parameter
Acetonitrile
0.65
8
2.5
0
Propanol
0.71
2.5
4
4
Ethanol
0.88
4
5
5
Methanol
0.95
5
7.5
7.5
Water
Large
Large
Large
Large
Table 5: Solvents in reversed-phase
Miscibility
Miscibility is a property that describes how well two substances mix together, or how well they
dissolve each other at any concentration to form a homogeneous solution. The mobile phase is
often made up of a mixture of solvents that need to be miscible. A miscibility table between various solvents can be found below.
n-Pentane
Hexane
Petroleum ether
Cyclohexane
Xylene
Diisopropyl ether
Toluene
Diethyl ether
Chloroform
Dichloromethane
Tetrahydrofuran
Acetone
Dioxane
Ethyl Acetate
Acetonitrile
n-Propyl alcohol
Ethanol
Methanol
Water
Figure 17: Solvent miscibility
22
Basic Principles of Flash and Prep HPLC
UV absorption and boiling point
When using a UV detector, the solvents should not absorb UV light at the wavelength used by the
detector in order to avoid interference and to allow a proper collection. Solvents with low boiling
points are advantageous for evaporative light scattering detection (ELSD), as they allow work at
lower temperature, which is beneficial for compound stability. Also, a low boiling point makes any
post-run concentration step via evaporation much easier and faster.
Solvent
UV limit (= UV cutoff)
Boiling point °C
Hexane
210
69
Cyclohexane
210
81
Ethanol
210
78
Methanol
210
64
Dichloromethane
245
40
Ethyl acetate
260
78
Acetone
330
56
Acetonitrile
200
81
Toluene
285
111
Dipropylether
220
90
Chloroform
245
62
Water
190
100
Propanol
210
82
Tetrahydrofurane
220
66
Dichloroethane
230
84
N,N-Dimethylformamide
270
153
Dimethyl sulfoxide
265
189
Benzene
280
80
Table 6: UV limits and boiling point of solvents
23
Basic
Basic Principles
Principles of
of Flash
Flash and
and Prep
Prep HPLC
HPLC
Toxicity
The most toxic part of preparative chromatography involves the organic solvents in the mobile
phase. In general, normal phase solvents, especially hexane and dichloromethane (DCM), are
more toxic than reversed phase solvents. From frequently used reversed-phase solvents, acetonitrile is the most toxic, followed by methanol and then ethanol as least toxic. Users could try to
reduce the toxicity levels of their purifications by minimizing the amount of organic solvent in the
mobile phase. This could be achieved by using columns with reduced length, internal diameter
and particle size. Alternatively, toxic solvents could be substituted for less toxic mobile phases,
such as isopropanol and DMSO or water. For isocratic analysis, a plausible option is to use a
mobile phase recycler to additionally minimize hazardous waste.
Viscosity
Viscosity describes a fluid's resistance to flow. Solvents with low viscosity are favored as they
are less “thick” and they minimize column or cartridge back pressure. Viscosity is especially important when using mixtures of specific ratios of water and organic solvents with high viscosity.
When comparing common normal-phase solvents, ethyl acetate and dichloromethane are more
viscous than hexane. From the most frequently used solvents in reversed-phase chromatography, ethanol is the most viscous, followed by methanol and then acetonitrile (see graph below).
viscosity
[cP] at 20 °C
3
3
n-propanol
2
2
ethanol
1
1
water
methanol
acetonitrile
0
20
Figure 18: Viscosities of different water mixtures
24
40
60
80
100 % [w/w] organic
Basic Principles of Flash and Prep HPLC
Solvent modifiers
pH control of the mobile phase can have a high impact on the retention of an ionic compound
and its peak shape. Neutral compounds are not affected by the pH. Since ionic compounds are
soluble in water and at risk of non-reversible interactions with the ionizable silanols, pH control
is mainly happening in reversed-phase applications. Before adjusting the pH of the solvent, it is
important to know that the silica backbone Si – 0 – Si hydrolyzes at pH 8.0, whereas alumina is
stable throughout a pH range of 2-13. Solvent modifiers are typically used at concentrations of
one percent or less and buffer effectively at up to 1 pH unit above or below their pK.
Acidic and basic compounds may form their conjugates at a neutral pH and lead to poor
peak shape or even double peaks. In case of acidic analytes, the pH is reduced, so the
compound gets non-ionized. In general, ion-suppressed analytes show better retention than
ionized analytes under reversed-phase applications. With basic compounds, the ion-suppressed form may be at a high pH that is not suitable for the stationary phase. However,
most bases are traditionally purified at low pH and show good separation results under
these conditions.
An additional issue impacting retention of acidic and basic compounds is the potential ionization of silanols on the silica surface at mid pH. Typically, these silanols become deprotonated and negatively charged. This can result in increased retention of positively-charged,
basic ions, which can result in ion-exchange interactions, a type of secondary interactions.
The result is often peak tailing due to an interaction other than the partitioning that is expected with a reversed-phase column. This effect can be avoided by working at a low pH where
the silanols are non-ionized or by adding a competing amine such as ammonium hydroxide
to the mobile phase. Through this, the silanols attract the amine additive in the mobile phase,
which neutralizes them.
There are several different pH modifiers that are suitable for pH control of the solvent. However, it’s important to consider the type of detector used for the application. UV detectors
require use of additives with a low UV cutoff and for ELSD detectors, a certain volatility is
needed to get sufficient results. The following table shows the most common mobile phase
modifiers.
Modifier
UV cutoff
pKa
Triethylamine (TEA)
267 nm
10.8
Trifluroacetic acid (TFA)
210 nm
0.3
Acetic acid
210 nm
4.8
Ammonium acetate/ formate
200 nm
9.2
Formic acid
210 nm
3.8
Table 7: Modifiers in adsorption chromatography
25
Basic
Basic Principles
Principles of
of Flash
Flash and
and Prep
Prep HPLC
HPLC
Summary of common stationary and mobile phases
in adsorption chromatography
Stationary
phase
Types of adsorption
chromatography
Surface polarity
Silica
Normal phase
Polar
C18
Reversed phase
Unpolar
C18 WP
Reversed phase
Unpolar
Amino
Normal & reversed phase
Medium polar
Diol
Normal & reversed phase
Low polar
Alumina
Normal phase
Polar
26
Basic Principles of Flash and Prep HPLC
Suitable solvent system
Retention
Remarks
High retention of high to
medium polar compounds
•
•
•
Hexane / ethyl acetate
Dichloromethane /
methanol
No water
•
•
•
Water / methanol
Water / acetonitrile
Water / ethanol
•
•
•
Water / methanol
Water / acetonitrile
Water / ethanol
No retention of highly
unpolar componds
Most popluar polar phase
Too strong retention of
extremely high polar
compounds
High retention of mid to
unpolar compounds
Most popular unpolar phase
No retention of high polar
compounds
High retention of mid to
unpolar compounds
No retention of high polar
compounds
See C18 and Silica
High retention of medium
polar compounds
See C18 and Silica
High retention of low polar
compounds
Wide pores (200-250 A: ideal
for proteins / peptides >2000
Da)
Ideal for carbohydrates &
amines
High retention of high to
medium polar compounds
•
•
Hexane / ethyl acetate
Dichloromethane /
methanol
No retention of high unpolar
compounds
Available as neutral, acidic
and basic types
Too strong retention of
extremely high polar
compounds
27
Consumables
and Sample
Loading
Basic
Principles
of Sample
Flash and
Prep HPLC
Consumables
and
Loading
Chapter 2
28
BasicConsumables
Principles of Flash
and Sample
and Prep
Loading
HPLC
Consumables in flash and prep HPLC
Users of preparative liquid chromatography have a wide range of choices when it comes to
consumables. The selection of consumables is of high importance: the separation of compounds in a synthetic mixture or a natural extract happens in the cartridge or column. The
chromatographic system only makes the separation process efficient and convenient.
The user can select from:
Flash cartridges (prepacked or
empty)
Glass columns (empty)
Prep HPLC columns (prepacked)
There is a larger variety of consumables for flash chromatography compared to prep HPLC. This
is because for flash applications, the user can either choose cartridges (prepacked or empty) or
glass columns (empty). In prep HPLC, the columns are always prefilled.
There are several factors that need to be considered before choosing a consumable:
•
•
•
•
•
Difficulty of the separation
Required purity
Amount of sample
Purification throughput
Available instrumentation
Flash and prep HPLC consumables have distinctive benefits. Prep HPLC columns are usually
used for high efficiency separations performed at high back pressures. They are limited in their
size and therefore not suitable for applications where large amounts of compound are required.
Flash consumables are used for purifications that do not require high efficiency, but sample
amounts are high. Often, flash is used as a pre-purification step followed by prep HPLC to reach
highest purity. The differences between flash and prep HPLC are shown in the table below.
Flash
Prep HPLC
Particle size
15-63 μm
5-15 μm
Column ID
12-115 mm
10-70 mm
Flow rate
15-200 mL/min
5-100 mL/min
Loading capacity
< 300 g
< 10g
Max pressure
50 bar
300 bar
Table 1: Flash versus Prep HPLC
29
Basic
Principles
of Sample
Flash and
Prep HPLC
Consumables
and
Loading
There are usually three options for consumables for flash applications: prepacked flash cartridges,
empty flash cartridges or empty glass columns. The choice of the consumable depends on the
requirements of the application. Pros and cons are discussed in the following table:
Advantage
•
Prepacked flash cartridge
(Polypropylene tube filled
with media, typically silica)
•
•
high flexibility (wide
range of media and
sizes available)
high quality due to professional packing
no investment costs
Disadvantage
•
•
•
Empty flash cartridge
(Polypropylene tube for
self-packing)
•
•
low consumable costs
(only bulk silica)
no investment costs
•
•
•
•
•
Empty glass column
(glass column for selfpacking)
•
•
low consumable costs
(only bulk silica)
pressure rating rather
high (up to 50 bar)
high flexibility in terms of
size (g to kg)
long lifetime (columns
can be refilled)
•
•
•
•
pressure limits for most
cartridges rather low
(typically 10-15 bar)
high consumable costs
time consuming filling
and cleaning procedure
low to medium level
separation results
filling creates fine dust
particles (influence on
user safety)
no refilling possible
high investment costs
time consuming filling
and cleaning procedure
low to medium level
separation results
filling creates fine dust
particles (influence on
user safety)
Table 2: Consumables for flash applications
Prep HPLC columns are typically not self-packed. The main goal of prep HPLC is high resolution, which is only achievable if the material is perfectly packed. This is done via a slurry filling
so that prep HPLC columns are delivered with silica and solvent (usually isopropanol / ethanol).
The hardware material of prep HPLC columns is stainless steel to handle high back pressures.
30
BasicConsumables
Principles of Flash
and Sample
and Prep
Loading
HPLC
Cartridge & column handling
Proper handling of a cartridge or column is of high importance. It can have a major impact on
the column’s lifetime and on the quality of results obtained.
Equilibration
The equilibration of flash and prep HPLC consumables facilitates a uniform and steady flow of
sample and solvent by removing any trapped gasses during the separation process. This helps
to avoid undesired separation issues, such as unresolved compounds and low purity fractions.
The equilibration time of a cartridge or column is governed by its dimensions. Typically, a column
or cartridge is equilibrated by flushing it with 5-10 column volumes (CV) of the starting solvent.
For flash cartridges, the process of equilibration generates heat due to solvent adsorption on the
dry silica media. The amount of generated heat mainly depends on the following parameters:
•
•
•
Solvent type
Flow rate
Amount of silica
Since silica is a polar material, the adsorption of polar solvents is stronger and leads to more
heat generation than the adsorption of less polar solvents. Higher flow rates result in higher
amounts of generated heat. In smaller cartridges, heat can easily disappear due to a relatively
short residence time and easy transfer to the cartridge wall. In larger-sized cartridges, which
contain higher amounts of silica, heat transfer via the wall is slower, which leads to build-up of
more heat in the cartridge. High amounts of trapped heat can reach such high levels that the
cartridge might be damaged due to associated pressure increases. The heat can also influence
the chromatographic process, via the adsorption and desorption steps, which can reduce the
separation efficiency. The generated heat can also cause partial or complete evaporation of
semi-volatile compounds.
To counteract these issues, it is recommended to reduce the amount of polar solvents in the
equilibrium mixture. It might also be helpful to reduce the flow rate during equilibration. Once
the packed silica bed is properly wetted, the heat generation is controlled, and the best possible
separation yield can be achieved. Since prep HPLC columns are packed as slurries, they get
delivered wetted and therefore heating during equilibration won't be observed.
Storage and Cleaning
Generally, it’s not recommended to store normal phase media (unbonded silica). This polar material gets deactivated when it comes in contact with polar solvents or water traces. And once
the silica is deactivated, it is not possible reactivate it. Deactivated silica does not provide the
same quality separation as previously achieved, since selectivity changes.
Reversed phase media (bonded silica such as C18, C8, C4, Amino & Diol) can be used for
several separations, because this hydrophobic stationary phase is washable with organic solvents. Highly retained contaminants can be removed, and the cartridge or column can be stored
for longer time periods. Though reversed phase media cost more than silica media, they can be
31
Basic
Principles
of Sample
Flash and
Prep HPLC
Consumables
and
Loading
reused, which might balance out the cost difference. Once wetted, it is recommended to never
dry out a reversed phase cartridge or column. Drying out induces channeling due to expansion
and contraction of the stationary phase. The consumable should be stored wet and well-capped
in 20 % water and 80 % organic solvent such as acetonitrile, methanol, or ethanol. The organic
content is necessary to avoid bacterial/algae growth. Since the hardware of the flash cartridges
is polypropylene, it is not possible to store consumables in 100 % hexane as it will soften the
hardware.
Further recommendations
•
•
•
•
Do not exceed the maximum pressure limit and avoid pressure shocks which can lead to
channeling or gaps in the silica bed.
Never use 100 % water with standard C18 material, since it will lead to phase collapse and
negatively affect the separation. Special water-resistant C18 phases are available for applications requiring 100 % water.
Keep the pH range between 2-8 if not stated differently in the supplier documentation.
Otherwise, the silica might be damaged.
Use guard columns for prep HPLC columns to avoid irreversible adsorption of impurities at
the silica material and therefore extend the lifetime of the column.
Self-Packing of empty flash cartridges and glass columns
If the user decides to pack a cartridge or column himself, it is important for him to achieve a homogeneous particle distribution and uniform packing density. However, the quality of a manually
packed consumable is usually not comparable to those columns that are professionally packed:
separation efficiency and reproducibility are on a lower level. Self-packing is a suitable solution for cases when the user wants to purify simple mixtures in the most economical way.
Since a large variety of prepacked flash cartridges are available, they are rarely used for
self-packing. Also, reuse of the polypropylene hardware is limited, as it is not very robust when
it comes to reassembly. Therefore, glass columns are generally users’ first choice for self- and
re-packing.
There are two ways to fill a glass column: dry and slurry packing. Generally, dry packing is a fast
and easy process that uses compressed gas, but the packing density is not as dense. Therefore, it is only ideal for medium to big sized silica particles (25-200 μm). For smaller particles its
recommended to use a slurry of silica and solvent, filled via a pump into the glass column.
Generally, any packing process can be divided into three phases:
•
•
•
Preparation
Filling
Compacting
Below is a short description of a dry packing and liquid packing method.
32
BasicConsumables
Principles of Flash
and Sample
and Prep
Loading
HPLC
Dry packing of glass columns
The materials needed for the dry packing process include a separation column, filling vessel,
silica, nitrogen cylinder and a funnel. During the preparation step, the column must be washed
until clean and left to dry completely. The column gets clamped vertically and the filling vessel
screwed on. The silica is then loosely introduced with the help of a funnel. The filling vessel is
packed with the silica to such an extent that at least 10 % of the column volume is in excess.
Importantly, the column should never be tapped or caused to vibrate, which would lead to the
inevitable separation of the particles and render the column useless. In the final stage, nitrogen
is blown from the nitrogen cylinder through the column until a hissing is no longer audible. The
main valve of the nitrogen cylinder must be closed, and the pressure must be completely let
down. This procedure is essential since otherwise the column bed will come apart when the
nitrogen hose is detached. The column is now ready for equilibration.
Silica
Funnel
Filling
vessel
Nitrogen cylinder
Separation
column
Filling
Compacting
Figure 1: Dry filling of glass columns
The equilibration of a dry-filled column is carried out directly with the solvent required for the
separation. It is recommended to discard the first 100 to 200 ml of liquid from a freshly filled
column and only then to circulate the solvent. This precautionary step prevents any fine silica
gel particles that have been washed out from entering the pump. As soon as air stops emerging
from the column and the base line of the detector is stable, the separation can be carried out.
33
Basic
Principles
of Sample
Flash and
Prep HPLC
Consumables
and
Loading
Slurry packing of glass columns
The material needed for the process includes a separation column, slurry filling vessel, silica,
solvent, funnel and a beaker. First, the column is washed, cleaned and then dried. The silica
gel is suspended in the solvent. A typical ratio is 1 g silica to 2-3 ml solvent as shown with the
examples in the table below:
Column
ID x L (mm)
Silica gel (g)
Slurrying process (ml of
solvent)
26 x 460
150
300 – 600
36 x 460
270
500 – 900
49 x 460
470
800 – 1200
70 x 460
1000
2000 – 2500
Table 3: Ratio Silica to solvents in glass columns
The column is clamped vertically, the slurry filling vessel is screwed on and the slurry is introduced into the column from the filling vessel. The filling vessel is filled up completely with solvent
and the column inlet is connected to the pump. Via the pump, 1-2 column volumes of solvent
are pumped till the silica is nicely compacted.
Columns that are packed with the slurry method do not require an additional equilibration step
and can be used immediately after the flow of 1-2 column volumes through the adsorbing material.
Silica /
Solvent slurry
Funnel
Pump
Filling
vessel
Solvent
Separation
column
Filling
Figure 2: Slurry filling of glass columns
34
Compacting
BasicConsumables
Principles of Flash
and Sample
and Prep
Loading
HPLC
Sample loading in flash and prep HPLC
In preparative chromatography, loading of the sample is of primary importance but could prove
to be challenging. The mixture to be purified should be applied in the correct concentration to
the cartridge or column bed as compactly as possible to achieve a narrow horizontal band. If
the sample volume is too high, the band is considerably wider, and the separation becomes less
efficient.
Loading capacities
Loading capacities are quite different between flash and prep HPLC. Since flash chromatography
is usually used as a pre-purification step, high resolution is not a priority, so loading capacities
tend to be much higher than those used with prep HPLC. In prep HPLC, the goal is more focused
on getting highest purities, so baseline separation is a must.
There is also a considerable difference in loading capacities between normal phase and reversed
phase silica, such as C18, amino or diol. Since unbonded silica has a larger surface area than is
chromatographically available, this type of stationary phase has a higher loading capacity. Quantitatively, the loading capacity of normal phase silica is usually around 10 times higher than that of
bonded silica media. The loading capacity (%) is represented as follows:
grams of sample/grams of media in the cartridge or column x 100
Example: On a 120 g silica cartridge with a loading capacity of 10 %, the maximal sample
amount is 12 g
Standard silica (particle size 40-60 μm) typically provides 10 % loading capacity and can reach up
to 30 % with use of smaller particles (15-30 μm). However, it is important to know that loading capacity, regardless of whether the chromatography is reversed or normal phase, is always governed
by sample complexity. For a complex sample with multiple compounds, the determining factor
for complexity is how well separated the target compound is from its nearest eluting neighbors.
Usually, complex samples can be loaded only in small quantities.
Sample loading techniques
In liquid chromatography, a sample can be loaded via two different ways: solid or liquid. In liquid
loading, the sample is dissolved in a solvent and injected directly on the cartridge or column. With
solid loading, the sample, which consists of a solid mixture of crude sample and support material
such as silica, is placed in front of the separation cartridge.
Solvent
Crude sample
Crude sample
Support material
(e.g. silica)
Figure 3: Liquid and solid sample
35
Basic
Principles
of Sample
Flash and
Prep HPLC
Consumables
and
Loading
Each technique has its own special aspects to consider, as discussed in more detail below.
Liquid loading is an approach used for samples that are well dissolved in the starting solvent. It’s a technique used both in flash as well as prep HPLC applications. A weak solvent
is recommended with this technique, as use of strong solvents reduces the resolution. Liquid
loading is considered the easiest and fastest way, but the potential of sample loss should be
considered. The following factors need to be taken into account:
•
•
•
•
Solubility of compounds in the starting solvent – the sample needs to be fully soluble because
precipitation in the injection system or on top of the column might create excessive pressure
in the system and lead to eventual sample loss.
Polarity of the dissolution solvent – if polar solvents are used to dissolve the sample, they
might adsorb onto the polar silica cartridge matrix and negatively affect the separation of
more polar compounds (later eluting compounds on a silica material).
Volume of the sample solvent – the bigger the sample volume, the higher the risk the sample
starts migrating into the packing bed right from the beginning, causing band broadening and
reduced resolution. The ideal sample volume should not be more than 10 % of the purification column volume. The more retentive the sample is, the more volume can be loaded.
Sample quantity – each cartridge or column has a defined loading capacity. The ideal sample quantity should not exceed the maximal loading capacity of the purification cartridge or
column.
In flash chromatography, the liquid samples are usually introduced with the help of a syringe
manually and directly on top of the cartridge (see picture below). Manual loading on a prep
HPLC column is not possible due to high back pressure. Therefore, the loading is done via a
dedicated sample injection valve as shown in the following picture:
Syringe
(liquid sample)
Syringe
(liquid sample)
Injection
valve
Flash
cartridge
Prep
HPLC
column
Figure 4: Liquid loading in flash and prep HPLC
Solid loading is a technique used only for flash applications and for samples that are only soluble
in strong solvents or for samples with sticky or strong impurities that cannot be easily removed.
This approach is also beneficial in improving resolution by reducing band broadening and subsequent tailing effects. Generally, solid loading is slower, but it allows for higher resolution than liquid
loading. The recommended sample quantity should not exceed the maximal loading capacity of
the flash cartridge.
36
BasicConsumables
Principles of Flash
and Sample
and Prep
Loading
HPLC
Solid loading is usually accomplished with the following steps:
•
•
•
•
•
•
•
The crude sample is dissolved in a suitable polar solvent.
This mixture is then incubated for a few minutes in an ultrasonic bath to enhance the solubility.
The mixture is filtered to get rid of material that has not fully dissolved.
Silica is added to the mixture at 5 times the weight of the crude sample.
The solvent is removed completely by evaporation.
Finally, the solid sample, which now consists of a mixture of crude sample and silica, is
packed into a precolumn (solid loader), which is then fitted in front of the separation cartridge
into the solvent flow.
The components to be separated are constantly eluted from the precolumn into the actual
separation cartridge.
Solid loader
(solid sample)
Flash
cartridge
Figure 5: Solid loading in flash chromatography
The support material plays a vital role in the solid loading technique: the crude sample gets absorbed which enables a better transfer and distribution of the eluted compounds. It also keeps
the sample in place by making it stationary. This is advantageous for challenging samples such
as oily extracts.
Unbonded silica is the most commonly used sorbent but may be not the best choice. Generally,
it is recommended to use the same type of silica in the separation cartridge for the support material. This avoids unwanted chemical interaction or irreversible adsorption of the sample’s compounds. A useful alternative material is celithe, which shows no interaction with any substance
due to its chemically neutral properties.
Technique
Advantage
Disadvantage
Liquid loading
Fast and easy
Reduced resolution
Solid loading
High resolution
Time consuming
Table 4: Differences solid and liquid loading
37
Detection
Methods
Detection Methods
Chapter 3
38
Basic Principles of Flash
Detection
and Prep
Methods
HPLC
Detection methods in flash and prep HPLC
When performing chromatography separation, it is essential to determine the precise point
when a substance is eluted from the column or cartridge to achieve optimal fractionation. A
chromatography detector is a device used to detect the presence of individual substances as
they elute from the column or cartridge. The detector converts a change in effluent levels into
an electric signal which is recorded by a data system.
In preparative liquid chromatography, the detectors used must accommodate high flow rates
and high sample concentrations associated with the process. Various detector types exist
for this purpose and each type of detector has its specific advantages and limitations. In the
following, five commonly used detector types in liquid chromatography are discussed in more
detail.
UV detector
UV detectors are the most frequently used detectors in preparative chromatography. A UV detector is a selective detector, meaning it only measures substances which absorb light of a selected wavelength in the ultraviolet range (200-400nm) or visible range (400-800nm). Substances which are suitable for UV detection include compounds with a chromophoric group, such as
•
•
•
•
•
aromatic ring
two conjugated double bonds
double bond adjacent to an atom with one electron pair
carbonyl group
bromine, iodine or sulfur
The UV detector measures the change in intensity of a UV light beam passing through a solution.
The absorption of the light is related to the concentration of the solution which the light beam is
traveling through. This relationship is described by the Lambert-Beer Law:
E = Extinction [dimensionless]
ε = Extinction coefficient [M–1· cm–1]
c = Solution concentration [mol/l]
d = Path length of the light beam through the solution [cm]
Every solvent has a UV absorbance cutoff wavelength. At wavelengths below this value the solvent
itself absorbs all the light. When using a UV detector, it is necessary to select a solvent which has
no significant UV absorption at the wavelength at which measurements are to be taken. Otherwise, the signal of the substance and the solvent will overlap, resulting in incorrect fractionation.
39
Detection Methods
Typical critical solvents used are acetone, toluene and benzene, as indicated in the following
table:
Solvent
UV limit/cutoff
(nm)
Solvent
UV limit/cutoff
(nm)
Acetone
330
Dipropylether
220
Toluene
285
Hexane
210
Benzene
285
Cyclohexane
210
N,NDimethylformamide
275
Ethanol
210
Methanol
210
Dimethyl sulfoxide
275
Propanol
210
Ethyl acetate
260
Heptane
200
Dichloromethane
245
Acetonitrile
200
Chloroform
245
Water
190
Dichloroethane
230
Tetrahydrofurane
220
Table 1: Critical solvents used in flash and prep HPLC
If the absorption spectrum of a compound is not known, it is useful to use multiple wavelengths
simultaneously or even a diode array detector (DAD), which can record the whole UV spectrum.
The DAD can also confirm purity and compound identity by showing the absorption spectrum of
each peak, eliminating post fraction thin layer chromatography (TLC).
For example, the DAD analysis of caffeine, vanillin and piperine is shown below:
Figure 1: Typical chromatogram with average UV signal
(200- 400nm) of the three compounds caffeine, vanillin
and piperine
40
Figure 2: 3D dataset of UV signals (200-400nm) of
caffeine, vanille and piperine indicating the absorption
maxima
Basic Principles of Flash
Detection
and Prep
Methods
HPLC
Scan at 6.95 min
Figure 3: Detailed UV absorption spectrum of the vanillin peak
Advantages of UV detection
Limitations of UV detection
•
•
•
•
•
•
•
•
Easy to use
Reliable
Relatively inexpensive
Solvent gradient compatible
Non-destructive to sample
Relatively sensitive (depending on
compound activity)
Specific
•
Poor response if compound doesn’t
have a good chromophoric group
Solvents limited by UV cutoff especially
at low UV wavelengths
Table 2: Advantages and limitations of UV detection
41
Detection Methods
Evaporative light scattering (ELS) detector
The general mechanism of ELSD involves measuring the amount of light scattered by particles of
solvent which have been dried through evaporation. The process consists of three steps: nebulization, solvent evaporation and detection (see picture below). During nebulization, a nebulizer
combines a gas flow of air or nitrogen with the column or cartridge effluent to produce an aerosol
of tiny droplets. In the next step, the droplets are introduced into a drift tube where the mobile
phase evaporates and leaves behind a particulate form of the target compound. In the last step,
light strikes the dried particles which exit the drift tube, the light is scattered, and the resulting
photons are detected by a photodiode.
Step 1. Nebulization
Step 2. Mobile Phase Evaporation
Step 3. Detection
Figure 4: Process of ELSD
ELSD is described mathematically by equations which are governed by particle size. The peak
area (A) is related to the quantity of analyte in the column (m):
A = amb
Where m is the solute mass, and a and b are constants which depend on variety of factors,
such as the size of the particles, the concentration and type of the target substances, the gas
flowrate, the mobile phase flow rate, and the temperature of the drift tube.
ELS detectors are a preferred choice for purification of compounds without a chromophoric group. Such compounds include carbohydrates, lipids, fats and polymers. The function of
ELS detectors remains undisturbed by mobile phase variations and gradient baseline shifting.
Sensitivity in ELSD is independent of the compound’s physical and chemical properties and is
therefore governed only by the absolute quantity of the compound. ELSD is a mass-dependent
detector, meaning a high signal indicates that a large amount of compound is eluting. As a
semi-quantitative technique, it provides valuable information on the ratio of the compounds in
the sample.
ELSD can detect nearly all compounds, except for highly volatile analytes, such as ethanol in
wine. In general, the compound of interest must be less volatile than the mobile phase. Any
modifier in the mobile phase needs to be also volatile. Since ELSD is a destructive detector,
sample amount transferred to the ELSD should be as minimal as possible.
The lower the boiling point of the mobile phase, the easier it is for the solvent to evaporate. Mobile
phases with high boiling points (e.g. DMS, DMF, toluene or water), either need to be evaporated
at high temperatures, with the risk of destroying the target substances, or nebulized into extremely tiny droplets, which allow evaporation even at room temperature.
42
Basic Principles of Flash
Detection
and Prep
Methods
HPLC
Solvent
Boiling point °C
Dimethyl sulfoxide
189
N,N-Dimethylformamide
153
Toluene
111
Water
100
Propanol
97
Dipropylether
90
Dichloroethane
84
Acetonitrile
82
Cyclohexane
81
Benzene
80
Ethyl acetate
77
Ethanol
78
Hexane
69
Tetrahydrofuran
66
Methanol
64
Chloroform
62
Acetone
56
Dichloromethane
40
Table 3: Boiling points of typical solvents used in flash and prep HPLC
Advantages of ELSD
Limitations of ELSD
•
•
•
•
•
•
Detects any compound less volatile
than the mobile phase
Good sensitivity
Straightforward operation
Gradient compatible – maintains stable
baselines during gradients
•
Only volatile mobile phases can be used
The sample must be less volatile than
the mobile phase
ELSD is a destructive technique
Table 4: Advantages and limitations of ELSD
43
Detection Methods
Mass spectrometry (MS) detector
Liquid chromatography separates compounds according to their physio-chemical properties,
whereas mass spectrometry (MS) differentiates compounds by mass. The mass spectrometer,
as a chromatography detector, enables the identification of species corresponding to each chromatographic peak based on its unique mass spectrum.
The basic components of a liquid chromatography system coupled with a MS detector are shown
below:
Figure 5: Components of a liquid chromatography system with MS detection
The workflow involves converting molecules from the chromatography eluent to a charged or
ionized state. The mass analyzer is the component of the mass spectrometer which takes ionized
masses and separates them based on charge-to-mass ratios. The analyzer then outputs them to
the detector where they are recognized and converted to digital output.
Advantages of MS detection
Limitations of MS detection
•
•
•
•
•
Good sensitivity
Very selective
Provides structural information
Purchase price is very high
Frequent maintenance is needed
Table 5: Advantages and limitations of MS detection
Refractive index (RI) detector
The refractive index detector measures changes in the refraction of light caused by a medium
as it flows through a measuring cell. The detector is non-selective as it registers all substances
which flow through the cell. Refractive index detectors measure according to the following principle:
= Difference between the refractive indices
nG = Refractive index of the dissolved sample
nL = Refractive index of the pure solvent
ni = Refractive index of the sample
c = Concentration of the sample
Since the refractive index detector is not specific, it is universally applicable. This type of detector
is however not well suited to applications with gradient elution, since the eluate is always compared with the pure solvent in a reference cell. The system is therefore sensitive to changes in
the composition of the mobile phase. In addition, the detector is highly sensitive to temperature
changes.
44
Basic Principles of Flash
Detection
and Prep
Methods
HPLC
Advantages of RI detection
Limitations of RI detection
•
•
•
•
Universal nature of the detector
response
Good linear dynamic range - ~4 orders
of magnitude
Easy to operate
•
•
Cannot be used with solvent
gradients
Low sensitivity
Very sensitive to temperature
and pressure fluctuations
Table 6: Advantages and limitations of RI detection
Fluorescence detector
When compounds with specific functional groups are excited by shorter wavelength energy, they
emit higher wavelength radiation or fluorescence. Fluorescence intensity is dictated by both the
excitation and emission wavelength, which enables the selective detection of some components
over others. Approximately 15% of all compounds have natural fluorescence. Some of these
compounds include aliphatic and alicyclic compounds with carbonyl groups and compounds
with highly conjugated double bonds. Aromatic components with conjugated pi-electrons give
off the strongest fluorescence activity.
Fluorescence detectors offer one of the highest sensitivity levels among existing detectors. The
sensitivity of fluorescence detectors is 10 to 1000 times higher compared to UV detectors.
Advantages of Fluoresence detection
Limitations of Fluoresence detection
•
•
•
•
•
Very sensitive
Very selective
Generally insensitive to flow and
temperature changes
•
•
•
Limited linearity
Not many compounds naturally
fluorescent
Derivatization complicated method
Complicated to use – must
have firm grasp on both chemical and
instrument variables
Some chemicals, such as oxygen, may
quench fluorescence - must degas well
Table 7: Advantages and limitations of fluorescence detection
45
Detection Methods
Combination of UV and ELS detectors
In general, users in preparative chromatography require detectors which are universal and easy
to use, with high sensitivity, with non-destructive technology and without mobile phase effects.
However, none of the currently available detectors fulfill all these criteria as shown in the following
table.
Detector
name
Compounds
detected
Gradient
compatible
Destructive
Easy to
use
UV
Chromophoric
compounds
Dependent
on compound
activity
Yes
No
Yes
ELSD
Non volatile
compounds
Medium
Yes
Yes
Yes
MS
Ionizable
compounds
Medium
Yes
Yes
No
RI
Universal
Low
No
No
Yes
Fluorescence
Fluorescent
compounds
High
Yes
No
No
Sensitivity
Table 8: Summary of detection methods in flash and prep HPLC
UV detection is the default method in preparative chromatography, since it is an established
technique and a lot of molecules absorb UV light. However, for some applications, UV detection alone may not provide the needed purity levels. Especially in synthesis, the detection of all
by-products in the reaction mixture is crucial for an efficient separation and sometimes the user
is not certain if all compounds are UV active. Therefore, to be on the safe side, a combination
with another detector, preferably ELSD, is often used to meet the needs of the user.
Both UV and ELS detectors are easy to operate, gradient compatible and feature good levels
of sensitivity. The fact ELSD is destructive can be overcome by choosing a detector with lowest
sample loss in the µl range. Besides this, the combination of UV and ELSD allows the detection of
almost any compound: chromophoric and non-chromophoric, as well as volatile and non–volatile
(see figures below).
46
Basic Principles of Flash
Detection
and Prep
Methods
HPLC
Solvent %
ELSD
UV1
UV2
Time (min)
H 3C
O
H3C
H
H
O
O
CH3
CH3
H3C H
CH3
OCH3
OH
H
H2N
O
Cholesteryl acetate
(non-chromophoric)
HO
Methyl Paraben
(chromophoric)
4- Aminobenzoic
acid (chromophoric)
Figure 6: Separation of chromophoric and non-chromophoric compounds in a mixture
Time (min)
O
N
O
O
N
N
N
Caffeine (non-volatile)
O
H
HO
O
HO
OCH3
Vanillin (high volatile)
O
Piperine (non-volatile)
Figure 7: Separation of volatile and non-volatile compounds in a mixture
47
Method
development
Method development
Chapter 4
48
Basic Principles of Flash
Method
anddevelopment
Prep HPLC
Method development in flash and prep HPLC
Prior to the purification of an unknown target compound by preparative liquid chromatography, a detailed analysis of the samples solubility and separation behavior is needed. This
analysis allows users to achieve reproducible and efficient results. Once the suitable conditions have been found, the procedure can be upscaled. Depending on the needs, the target
compound is purified in high quantities either by flash chromatography, prep HPLC or a
combination of both.
The general approach for method development is as follows:
• Definition of purification method (flash or prep HPLC)
• Analysis of sample solubility
• Screening of suitable conditions for separation (mobile & stationary phase)
• Upscaling to flash or prep HPLC
• Improving resolution
Definition of the purification method (flash or prep HPLC)
In preparative liquid chromatography, the user can choose between two purification methods:
flash or prep HPLC. Both techniques are frequently used but for different objectives: flash
chromatography is mainly used as a pre-purification step to purify large sample quantities
at a decent resolution, whereas in prep HPLC the goal is to achieve the highest resolution
(purity) possible under the condition of lower loading capacities. Depending on the choice,
the subsequent steps explained below partially differ.
Analysis of sample solubility
The first step a user has to perform with an unknown sample is to test its solubility. Generally, similar likes similar, which means polar compounds are soluble in rather polar solvents
(water, acetonitrile = ACN, ethanol or methanol) and unpolar compounds are soluble in unpolar solvents (organic solvents, such as hexane or ethyl acetate).
The easiest way to purify a sample is in its liquid form, as liquid samples can be injected
directly on a cartridge or column. To achieve this, the samples’ solvent needs to be fully
compatible with the starting conditions of the flash or prep HPLC method: unpolar for normal phase and polar for reversed phase (see figure 1). Consequently, mixtures containing
unpolar target compounds are best purified with normal phase whereas mixtures containing
polar and water-soluble compounds are best purified via reversed phase.
On the other hand, retention is a crucial factor for getting a target compound separated from
all other compounds of a mixture. To obtain efficient separation, the compound must have
a similar polarity to the stationary phase, so polar compounds are best purified via normal
phase whereas unpolar compounds are best purified via reversed phase. Consequently,
easy injection and high retention are at odds with each other and the users have to decide
which of these factors is more important for their application.
49
Method development
When it comes to prep HPLC, the user has no choice: the sample needs to be fully soluble
in the starting condition. This is because liquid loading is the only way to inject a sample into
a prep HPLC column. In flash chromatography, the situation is different. In case the user’s
preference is to get highest retention, the sample can be loaded via dry loading because
it is likely not compatible with the starting condition of the type of method (normal or reversed phase). However, even in flash chromatography, liquid loading is usually the preferred
method to inject the sample on the flash cartridge.
UNPOLAR
POLAR
POLAR
UNPOLAR
Figure 1: Difference between normal phase and reversed phase
Screening of suitable conditions for separation
(mobile & stationary phase)
In order to save time, costs and avoid complications during large scale purifications, it is
recommended to perform a detailed screening of stationary and mobile phases prior to using
a cartridge or a column. There are two common approaches for performing such screens, thin
layer chromatography (TLC) or analytical HPLC. Traditionally, TLC is used to screen for a flash
method and analytical HPLC for a prep HPLC method.
TLC
A TLC screening is the fastest, easiest and most economical way to determine the appropriate stationary and mobile phase for a specific sample. The equipment is cheap and available
in nearly every lab. On the other hand, the plate length is limited and hence separation takes
place only up to a certain length and in an open system which can be affected by humidity and
temperature.
50
Basic Principles of Flash
Method
anddevelopment
Prep HPLC
TLC and R f value
In the first step of the TLC screening, one selects a silica coated plate made of glass or aluminum. Ideally the silica on the TLC plate and in the cartridge should be identical (type and pore
size) so that the TLC conditions can be successfully applied to the cartridge. The liquid sample
(some µl of a 1% sample mixture) is spotted at the start position of the plate and the chosen
mobile phase runs through the plate via capillary action. The solvent front is run nearly the whole
way through the plate and the different spots on the plate are then examined (see Figure 2).
Solvent front
a
b
Starting line
Figure 2: TLC plate with distance of the solvent (a) and distance of the analyte (b)
To analyze how well the target compound has been separated, the Rf (retention factor) is calculated using the following formula:
retention factor (Rf)= distance analyte (b)/distance solvent front (a)
Rf = 1 means no retention on TLC (moving up with solvent front) and Rf = 0 means no affinity for
the solvent (staying at starting line). Ideally, Rf values should range between 0.2 and 0.5.
If the Rf value is too low, the compound is too sticky to the stationary phase. If these TLC
conditions are transferred to column chromatography, the sample elution would take too long,
wasting time and solvent. A Rf value that is too high indicates that the interaction between the
compound and the stationary phase is too weak. These conditions could lead to lower resolution when upscaling to a flash cartridge.
Importantly, the spots around the target compounds should be as far away as possible from
each other to ensure high purity in the upscaled experiment.
Using TLC to determine the most appropriate stationary and mobile phase
If the TLC run results in Rf values that are between 0.2 and 0.5, then the selected mobile and
stationary phase are well-suited for flash chromatography. If the Rf values are lower or higher,
then a stationary or mobile phase with different polarities should be chosen.
In case of low Rf values when using silica TLC plates (normal phase), it`s either recommended
to change the solvent system or to use less polar phases, such as amino or diol, for the flash
51
Method development
application. In case of high Rf values, the interaction of the compound with the polar surface
of the silica TLC plate is too weak and besides screening for the proper solvent, C18 (reversed
phase) material could be used for upscaling. As reversed phase silica is more expensive than
regular silica it is recommended to perform an additional TLC screen on a C18 TLC plate (reversed phase) to confirm run quality, prior to upscaling to column chromatography.
Mobile phase screening via the Snyder selectivity triangle
TLC screening helps to find the most suitable solvent system for a specific application. It is
recommended to start with the most commonly used solvents, such as:
Normal phase (silica): hexane, ethyl acetate, dichloromethane (DCM) and methanol
Reversed phase (bonded silica, commonly C18): water, ethanol, methanol and acetonitrile (ACN)
If these mobile phases fail to provide satisfactory results, it would be worthwhile to examine the
Snyder selectivity triangle for other options. The diagram below offers various solvent choices
grouped together in so called selectivity groups based on their properties as proton acceptors,
donors or dipoles. Choosing a solvent from groups as far away from each other as possible on
the triangle guarantees the highest difference in separation power (= selectivity).
I
Isopropyl ether
Diethly ether
Triethyl emine
II
Propanol
Ethanol
Methanol
III
Tetraydrofuran
Pydrine
Dimethylforamide
IV
Proton
acceptor
II
IV
VIII
Acetic acid
Formamide
I
Dichloromethane
1,2-Dichloromethane
VI
Dioxane
Ethyl acetate
Acetone
Acetonitril
VII
Toluene
Benzene
VIII
Chloroform
III
VI
VII
Proton
donor
V
V
Dipole
Figure 3: Snyder triangle with selectivity groups I-VIII
How to perform a TLC screening using different mobile phases is illustrated with the following
example (see figure below). Here, a mixture of three compounds is run with solvents from
different selectivity groups. The most effective separation of all three compounds is achieved
using selectivity group VI. If there is only interest in component 1 (top mark), the choice would
be selectivity group V.
52
Basic Principles of Flash
Method
anddevelopment
Prep HPLC
Figure 4: Sample running on TLC plate at four different solvent conditions (selectivity groups I, V, VI and VIII)
In case the compounds are running too fast with the solvent and interacting too weakly with the
surface of the silica, the strength of the solvent needs to be reduced. Solvent strength is the
property of displacing a substance from the active sites of the stationary phase. In this sense,
solvents are described as weak or strong. If the solvents are arranged in order of increasing
strength, a so called eluotropic series is obtained. The following table shows the different solvent
strengths used in normal and reversed phase.
Normal phase
solvent
Solvent strength
Reversed phase
solvent
Solvent strength
Hexane
0.1
Water
0
Cyclohexane
0.2
Methanol
3
Isopropyl ether
2.4
Acetonitrile
3.1
Toluene
2.4
Ethanol
3.6
Xylene
2.5
Isopropanol
4.2
Diethyl ether
2.8
Dioxane
3.5
Tetrahydrofuran
4.0
Chloroform
4.3
Dichloromethane
4.3
Ethyl acetate
4.4
Acetone
5.1
Table 1: Solvent strengths of normal and reversed phase solvents
53
Method development
An example is given below. On the left-hand, a Dichloromethane (DCM) TLC plate was used
where the compounds end up running too fast. By adding hexane (3/1 ratio hexane/ DCM) the
retention could be reduced.
Figure 5: Sample separation on a TLC plate at two different solvent conditions (left: 100% DCM; right: 3/1 hexane/ DCM)
Analytical HPLC
In contrast to TLC, analytical HPLC can be performed with gradients, which brings major advantages. It is the preferred screening method for upscaling to a prep HPLC column. But using analytical HPLC requires fully automated equipment that can be rather expensive. Commonly, a C18
phase (reversed phase) is used due to economic reasons. The C18 phase can be reused, as it
can be washed with organic solvents to remove strongly retained contaminants. This is different
to unbonded silica which cannot be reused after washing (resulting in decreasing performance).
Sample concentrations and flow rates are chosen based on the proposal of the manufacturer.
Usually sample amounts are between 1-10 mg and flow rates between 0.1-10 mL/min. The
aim is to get the target compound baseline separated from the impurities in a minimal amount
of time and at maximal loading. For screening with an analytical column, the same approach to
select mobile and stationary phases can be used as for TLC. One difference is that for reversed
phase, common solvents are water mixed with methanol, ethanol or acetonitrile (ACN).
Upscaling to flash or prep HPLC
Upscaling to a flash cartridge or a prep HPLC column can be achieved based on the analytical
data from TLC or analytical HPLC. As a first step, it is advisable to use a small-sized flash cartridge or prep HPLC column and to upscale to bigger sizes at a later step if needed.
Upscaling from TLC
Using the R f to calculate column volume
It is also possible to use the Rf value to calculate the column volume (CV). The CV is the volume
inside of a packed cartridge or column that is not occupied by the media. This volume includes
both the interstitial volume (volume outside of the particles) and the media’s own internal po-
54
Basic Principles of Flash
Method
anddevelopment
Prep HPLC
rosity (pore volume). Combined, the two volumes usually constitute 70% to 80% of the packed
column’s volume.
From the CV, it is possible to calculate the amount of volume of mobile phase that is required to
elute a compound from a cartridge. This number can be easily determined using the retention
factor (Rf) with the following formula:
CV = 1/Rf
The correlation between the CV and Rf values is shown below:
B
A
A Rf 0.50
B Rf 0.40
CV
2.0 2.5
Figure 6: Sample run on a TLC plate with two separated spots or compounds (left) and on a flash cartridge with two
separated peaks (right)
The smaller the Rf, the more solvent (CV) is needed to elute the compound from the cartridge:
R f value
Column
volume
CV
R f value
Column
volume
CV
R f value
Column
volume
CV
0.9
1.11
0.6
1.67
0.3
3.33
0.8
1.25
0.5
2.00
0.2
5.00
0.7
1.43
0.4
2.50
0.1
10.0
Table 2: Correlation between Rf and CV
Ideal conditions for upscaling to flash chromatography are given at Rf values of 0.2 to 0.5, which
corresponds to 5-2 CV. The ΔC should be >1 to ensure a good separation and purity of the
target compound. The CV of a flash cartridge can be found in its technical data.
55
Method development
Using R f for calculating a gradient
A gradient elution (stepwise change of the solvent ratio) is usually needed to separate the target
compound sufficiently from the impurities. Even though an isocratic elution (constant ratio of
the solvents) is easy to run, separation results are mostly poor. With so-called gradient elution,
the composition of the mobile phase varies by increasing the amount of solvent with higher
eluting strength. Consequently, at the beginning of the run when the mobile phase strength is
low, the analyte will be concentrated into the stationary phase at the head of the cartridge. As
the mobile phase strength increases, the analyte will begin to partition into the mobile phase
and move along the cartridge. At the some point, the analyte may be wholly partitioned into the
mobile phase and will be moving with the same velocity as the mobile phase. Gradients usually
generate less peak broadening than isocratic elution.
Gradients can be classified into linear and step gradients. Using a linear gradient means that
the solvent composition gets gradually changed whereas in a step gradient, a series of linked
isocratic steps is used to separate and elute the sample components. A linear gradient is simpler
to handle compared to a step gradient, but liner gradients also feature lower separation power
as shown in the following figure:
80
80
60
60
%
100
%
100
40
40
20
20
0
0
0
2
4
6
8 10 12 14 16 18 20 22 24
Time (min)
Absorption
Step
0 2
4
6
8 10 12 14 16 18 20 22 24
Time (min)
%B
Absorption
%B
Linear
Figure 7: Sample separation on a flash cartridge using different gradient types (step and linear). More compounds
(peaks) of the mixture are visible using the step gradient.
A gradient screening can be performed via TLC as well. The following example shows how the
separation on a TLC plate can be affected by the solvent strengths and how linear and step
gradients influence the separation on a flash cartridge.
Figure 8: Sample separation on TLC plates at different solvent conditions (% of solvent where B= strong solvent)
56
Basic Principles of Flash
Method
anddevelopment
Prep HPLC
Upscaling from analytical HPLC
Calculation of the loading, flow rate and column dimensions
Upscaling from analytical to prep HPLC is possible with the help of a few formulas. The easiest
approach is to keep the particle size of the silica as well as the lengths of the analytical column
the same as that of the prep HPLC column. In this case, the following formulas for the loading
(volume or concentration), flow rate and diameter are applicable:
LoadingA= LoadingB x (DiameterA/DiameterB)2 x (LengthA/LengthB)
Flow rateA= Flow rateB x (DiameterA/DiameterB)2
A= prep HPLC column
B= analytical HPLC column
To finalize the method, the same gradient profile (ratio of solvents and time) is used for prep HPLC.
Example:
Analytical run
Prep HPLC run
Column ID: 4.6 mm
Column ID: 30 mm
Column Lengths: 150 mm
Column Lengths: 150 mm
Particle Size: 5 microm
Particle Size: 5 microm
Loading: 0.1 g
Loading: 4.25 g
Flow rate: 1 mL/min
Flow rate: 42.5 mL/min
Gradient 10-90% B: 15 min
Gradient 10-90% B: 15 min
Typical sequence and CV of a flash or prep HPLC run
A typical flash or prep HPLC method consists of four steps: equilibration (preparation of the
cartridge or column), injection (loading of the sample), separation (gradient step) and rinsing
(cleaning of the cartridge or column). The equilibration step of a cartridge usually takes longer
than for a prep HPLC column, as the cartridges are delivered dry and therefore need time to get
wet. The following rule of thumb exists related to CV:
Equilibration: 4-6 CV with starting condition of the gradient (weak solvent)
Injection volume: max. 0.1 CV
Separation: 4-6 CV of gradient from weak to strong solvent
Rinsing: 2-3 CV of strong solvent
Generally, the CV of a method strongly depends on the sample’s retention and the user’s needs.
For example, purifying high retentive samples at high resolution will need more CV.
57
Method development
Improving resolution
In chromatography, resolution (R) describes the relative positions (retention times) of two adjacent peaks in a chromatogram. It’s a measure of how well two elution peaks can be differentiated
and it is calculated as follows:
tR1/2: retention time of compound 1/2, refers to the time from injection of the substance to the peak maximum
w1/2: the base width of the peak 1/2, measured between the inflectional tangents
b0.5(1/2): width of peak ½ at the half-height
tR2
tR1
h
/2
h
/2
h
/2
b 0.5(1)
b 0.5(2)
h
/2
w1
w2
Time
Figure 9: Chromatogram with two peaks (two compounds) with different retention times (tR). The x-axis measures the
time in minutes, whereas concentration values are plotted on the y-axis.
The resolution is a specific characteristic of a column or cartridge and is suitable for comparison
purposes using the same test mixture. The smaller the R, the shorter is the distance between
the two peaks. Between R = 2 and 1.5, the peaks start to overlap, which means some portion
of the compounds are eluting at the same time and hence are not separated. For example, if two
peaks are assumed to have a Gaussian distribution and have the same peak height and peak
width, at R = 1.5 the overlap is 0.15%, while at R = 1 the overlap is already at 0.23%. Generally,
the user has to decide how much overlap is tolerable. For most flash applications R = 1-1.5 is
enough and gives an optimal time/ benefit ratio, whereas in prep HPLC, baseline separation is
a must.
58
Basic Principles of Flash
Method
anddevelopment
Prep HPLC
R = 1.5
R = 2.0
R = 4.0
Time (min)
Time (min)
Time (min)
Figure 10: Different resolutions with an effect on peak overlapping
To achieve optimal resolution, three conditions need to be fulfilled:
•
•
•
The compounds must be retained on the stationary phase. This process is quantified by a
parameter named retention
The compounds must be retained differently. This is quantified by a parameter called selectivity
Obtained peaks must be sufficiently narrow in order to avoid or reduce overlapping. This is
quantified by a parameter termed efficiency
Signal Intensity
tR3
tR2
tR1
tR0
Retention
Selectivity
Efficiency
Time, t
Figure 11: Retention, selectivity and efficiency of a chromatogram
These three major factors, column efficiency, selectivity factor and retention factor, can be expressed mathematically with the familiar resolution equation:
Rs: resolution
N: efficiency
α: selectivity
k: retention
In the following sections, the different parameters and their influence on resolution are discussed
in more detail.
59
Method development
Selectivity
This factor, also known as the separation factor, relates primarily to the chemistry of the system, which can be either the stationary phase or mobile phase chemistry. By altering these two
characteristics, a change in the distance between two neighboring peaks can occur. The selectivity factor α is expressed as follows:
tR1/2: retention time of compound 1/2, refers to the time from injection of the substance to the peak maximum
tR0: dead time which refers to the elution time of an unretained compound or the time needed for the solvent to travel
through the cartridge
If the two compounds elute at the same time and therefore cannot be separated, the selectivity
factor is 1. The larger this value, the simpler is the separation as shown in the following figure:
α = 1.1
Poor separation,
thus low selectivity
α = 1.3
Partial separation,
thus best selectivity
α=2
Baseline separation,
thus best selectivity
Figure 12: Separations with different selectivity factors α
The selectivity factor is the parameter that affects resolution most. Therefore, screening of the
mobile and stationary phase should be the first step during method development as previously
discussed.
Retention
The retention factor is the ratio of the amount of time a compound spends in the stationary
phase to the time the compound spends in the mobile phase. The stronger the interactions of
the compound with the surface, the longer the retention and the higher the retention factor. This
factor is influenced primarily by:
•
•
•
60
mobile phase composition
the nature of the stationary phase
the length of the column or cartridge
Basic Principles of Flash
Method
anddevelopment
Prep HPLC
Manipulating mobile phase composition is the easiest way to increase the retention factor. This
is frequently done by using a mobile phase that is a weaker solvent. When the mobile phase has
lower solvent strength, the solutes spend more time interacting with the stationary phase and
need a longer time to elute.
The retention factor k (also known as capacity factor) is described by the following formula:
tR: retention time of the compound, refers to the time from injection of the substance to the peak maximum
tR0: dead time which refers to the elution time of an unretained compound or the time needed for the solvent to travel
through the cartridge.
Large k values will give a long separation time, whereas small k values give short separation
times. K values ideal for practical work are between about 1 and 5. This corresponds to a Rf
value of 0.5-0.2 in TLC. K values above 5 only provide a minimal increase in resolution as shown
in the following figure:
4.5
Resolution
4
3.5
3
Area where k has little effect
2.5
2
1.5
1
0.5
1<k<5-k has greatest effect
0
0
5
10
15
20
25
k
Figure 13: Correlation between the retention factor k and resolution
The retention factor is easy to influence but is rather limited in affecting resolution.
61
Method development
Column efficiency
The efficiency is expressed by the number of theoretical plates, N, which indicates the
performance of a cartridge or column. A higher number of results in the less dispersion, and
narrower, more efficient peaks. The number of theoretical plates is calculated as follows:
tR: retention time of the compound, refers to the time from injection of the substance to the peak maximum
w: the base width of the peak, measured between the inflectional tangents
b0.5: width of peak ½ at the half-height
Each plate represents the theoretical distance required for one adsorption-desorption step of
sample components between the stationary and mobile phase (see figure below).
Desorption
One theoretical plate
Adsorption
Analyte
Figure 14: Description of a theoretical plate
The more theoretical plates available within a column or cartridge, the more equilibrations between the stationary and mobile phases are possible and the better the quality of the separation.
For a column with a given length L, a high number of plates corresponds to a lower distance
between each plate, a parameter also known as plate height (H). The plate height is often called
“height equivalent to a theoretical plate” (HETP) and can be expressed with the following formula:
The plate height term measures how efficiently the column has been packed. For high efficiency
separations, N is high and the H is low.
62
Basic Principles of Flash
Method
anddevelopment
Prep HPLC
The number of theoretical plates is affected by many parameters as shown in the following
figure:
Mobile phase factors
•
•
•
•
Viscosity
Chemical nature
Rate of diffusion into packing
Linear velocity (flow rate/area of column cross section)
Solute factors
•
•
•
•
•
•
•
Chemical nature
Sample load on column
Solvent used for injection
Concentration of injected sample
Rate of diffusion in each phase
Rate of mass transfer between both phases
Capacity factor k
Stationary phase factors
•
•
•
•
•
•
•
Particle size
Particle size distribution
Particle shape
Pore size
Pore shape
Surface area
Surface chemistry
Numerical factors
•
•
•
•
Peak shape
Peak asymmetry
Method of calculation
Accuracy and precision of measurements
Mechanical factors
•
•
•
•
•
Homogeneity of packed bed structure
Extent of voids and channels within column bed
Excessive dead volume in LC system
Improper sample distribution at column inlet
Excessive sample remixing after column outlet
Figure 15: Different ways to influence the efficiency factor
63
Method development
The efficiency factor is predominantly dependent on the particle size of the stationary phase, the
linear flow rate of the solvent and the sample size, which are discussed in more detail below.
Increasing the column length only helps in theory, as it has a significant effect on the run time and
the pressure and can lead to diffusive effect that causes the peak width to broaden.
The efficiency factor can be easily altered and gives satisfactory results in terms of improving
resolution.
Particle size
The smaller the particle size of the silica, the higher is the total surface. This increases the possible adsorption and desorption steps, or the number of theoretical plates. The following figure
shows the relationship between the number of plates and the particle size:
Number of theoretical plates N
12000
10000
8000
6000
4000
2000
0
0
20
40
60
80
100
120
140
160
Particle size (microns)
Figure 16: Correlation between particle size and number of theoretical plates N
The effect on a separation is shown in the following two chromatograms using silica in different
particle sizes:
ELSD (mV)
Solvent %
ELSD
UV1
UV3
663
0.50
359
0.50
530
0.40
287
0.40
0.30
215
0.20
143
398
AB
265
AB
133
0
0
2
4
6
8
10
12
0.10
71
0.00
-1
0.30
AB
0.20
AB
0.10
0.00
0
2
4
6
8
Time (min)
Time (min)
Silica, 50 μm particle size
Silica, 15 μm particle size
Figure 17: Separations on a flash cartridge using different silica particle sizes
64
UV2
10
12
Basic Principles of Flash
Method
anddevelopment
Prep HPLC
Decreasing the particle size is an easy and fast approach for increasing resolution. But this approach should be applied with care, as small particles come together with an obvious drawback.
The smaller the particles, the more pressure is needed to push the mobile phase through the
column or cartridge. The pressure generated during a run is influenced by several parameters:
ΔP: change in pressure (bar)
F: flow rate (mL/min)
L: column length (mm)
K: specific column permeability
dp: particle size (μm)
r: column radius (mm)
n: solvent viscosity (cP) (1 for water, 2 for water/
MeOH 50/50)
The particle size along with the column radius are the two main factors that influence pressure.
Pressure values are inversely proportional to the square of both. Decreasing the particle size
by half increases the pressure by a factor of 4. Therefore, it is very important take this into
consideration when changing the particle size.
Flow rate
The flow rate (velocity) of the mobile phase also plays a crucial role for the separation. The plate
height H reaches a minimal height at a certain flow rate (optimal velocity) and increases again
with increasing flow rate, as seen in the figure below.
Plate height
Optimum velocity
Minimum plate
height
Mobile phase velocity
Figure 18: Correlation between mobile phase flow rate (velocity) and plate height H
The relationship between flow rate and plate height H can be also explained by the constants in
the van Deemter equation.
H: Plate height
dp: Particle size
u: Flow rate
65
Method development
Below the optimal flow rate, the analysis time, as well as the longitudinal diffusion, will increase
significantly, ultimately influencing the efficiency. Above the optimal flow rate, the adsorption of
the analyte to the stationary phase results in some of the sample lagging behind leading to low
efficiency as well. Another drawback is the higher pressure generated by higher flow rates.
Low efficiency
Long analysis time
High pressure
Low efficiency
Optimal flow rate
Figure 19: Effects below and above the optimal flow rate
The practical flow rates are usually higher than the optimum as slow flow rates result in very long
separation times. Hence, the user needs to find a good balance between time and resolution
quality.
The effect of the flow rate on the efficiency differs with the particle size. The effect is more pronounced for big particles and becomes less significant for small particles. This means small
particles allow the process to speed up without losing much on resolution as shown in the
following figure:
1000
50 μm
900
30 μm
H*= plate height
800
700
25 μm
600
500
400
300
200
15 μm
100
10 μm
0
0
20
40
60
Flow rate (mL/min)
Figure 20: Correlation between the flow rate and the plate height H.
Loading
The loading refers to the amount of substance introduced into a column or cartridge, expressed
in grams of substance per gram of adsorbent. The relationship of plate height and efficiency is
as follows:
66
Plate height (H)
Basic Principles of Flash
Method
anddevelopment
Prep HPLC
•
•
•
Loading B0 = 200 μg/g SiO2
Analyt. loadings: B<B0
Prep. loadings: B>B0
Loading (B) B0
Figure 21: Dependence of the plate height (H) on the loading (B).
The point of inflection is referred to as the linear capacity B0. This point is defined as the load at
which a 10% increase of the plate height (H) occurs.
The loading capacity per cartridge or column differs for each run and depends on the particular
selectivity factor α and the retention factor k. But common maximal loadings for normal phase
separations are between 100-300 mg/g silica, whereas for reversed phase the loading is 3-4
times lower than that. Overloading of the cartridge or columns (B>B0) decreases the efficiency
but is nearly always done in preparative chromatography. It is impossible to work with very low
loading capacities as the process run times would be substantially increased. An example of
chromatograms with a different sample loading is given below:
0
5
2,5 mg/g Silica gel
10
0
5
25 mg/g Silica gel
10
0
5
10
50 mg/g Silica gel
Column 26 x 460 mm, Silica 40 - 63 μm
Eluent Petroleum elher/Ethyl acetate 4:1. 85 mL/min
Figure 22: Effect of the sample amount on efficiency
As an increasing amount of sample is loaded, neighboring peaks begin to overlap as the examples above show. Peak overlap results in material loss, but it enables much more sample
to be loaded per run, so that the process is faster, and resources can be saved. In analytical
chromatography, the actual loading is lower than the capacity of the packing material (B>B0) as
the objective of analytical chromatography is to achieve high resolution rather than to load high
sample amounts.
67
68
References
Aced G. and Möckel HJ. (1991). Liquid-chromatographie Apparative, theoretische und methodische Grundlagen der HPLC. Weinheim, Germany: VCH.
Hostettmann K. et al. (2010). Handbook of Strategies for the Isolation of Bioactive Natural
Products. Beijing, China: Science Press.
Pitt JJ. (2009). Principles and Applications of Liquid Chromatography-Mass Spectrometry in
Clinical Biochemistry. 30(1):19-34.
Schmidt-Traub H. et al. (2012). Preparative Chromatography. (2nd ed.). Weinheim, Germany:
Wiley-VCH Verlag.
Talamona A. (2005). Laboratory Chromatography Guide. Flawil, Switzerland: BÜCHI
Labortechnik AG.
Young C.S. and Dolan J.W. (2003). Success with Evaporative Light-Scattering Detection.
LCGC Europe. https://pdfs.semanticscholar.org/86fe/6906062968f3e659798669428c1a119f063a.pdf
69
Notes
70
71
BÜCHI Labortechnik AG Meierseggstrasse 40 Switzerland – 9230 Flawil
T+41 71 394 63 63 marketing@buchi.com www.buchi.com