How to use HPLC Simulator Tool

General infomation

HPLC simulator, as the name already implies, simulates a virtual HPLC experiment. If you don´t know what HPLC is, please check
the in level 1 provided slides about chromatographic separation methods (“normal phase and reversed phase chromatography”).

This tool was adapted from a free, open-source HPLC simulator version in order to help with the understanding of chromatography in
general and with adjustable parameters in particular. Doing real HPLC experiments, you will have to consider some further aspects,
but the tool nevertheless gives you a good idea about the general process.

The E-learning platform Analytics+ is divided into three different levels, with level 1 being the provided learn material. Level 2
deepens the acquired knowledge by providing the possibility to test different HPLC settings and their effects on chromatographic
separation („learning by doing“ & „trial and error“). You will be able to directly assess the impact on the chromatogram after changing
certain parameters. Level 3 represents a „test situation“ to apply your acquired knowledge by planning your own HPLC experiment.
Therefore HPLC simulator tool is divided into two levels, i.e. level 2 and level 3.

We hope you enjoy the application. Please give us feedback by answering the questionnaire!

If you have questions please contact c.kaufmann@tum.de or t.letzel@tum.de

Content
Level 2 Mobile phase composition Level 3 Column selection
• Solvent A/Solvent B Substance selection
• Elution mode Elution selection
• Gradient Chromatographic properties
• Isocratic & solvent B fraction in % Column properties
• Pre-column volume Validation results
• Gradient values Chromatogram
Chromatographic properties
• Temperature
• Injection volume
• Flow rate
Stationary phase composition
• Column selection
• Column length, inner diameter, particle size
• Reduced van Deemter terms
Compounds
• Compounds selection
• Retention time
• k´ value
• Sigma value
• W (pmol)
Results
• Dwell volume & Dwell time
• HETP
• Theoretical plates
• Backpressure
• Eluent viscosity
• Reduced plate height
Level 2
The mobile phase composition can be altered by selecting solvent A and B.
A (hydrophilic/aqeuous solvent): current possible option is only water
B (hydrophobic/organic solvent): select either methanol or acetonitrile

Elution mode is either isocratic (= constant solvent composition during the entire
experimental time) or gradient elution (= mobile phase composition changes over
time)

Selection of solvent B fraction in % only applies for „isocratic elution“, in which the
% of B remains constant throughout the experiment. Low % of B (= low organic
solvent content of methanol or acetonitrile) consequently results in a higher
proportion of A (= hydrophilic solvent water), which makes the mobile phase more
polar. Thus non-polar compounds (i.e. low solubility in the polar mobile phase) will
be retained longer on the non-polar stationary phase. And vice versa.

Please find more information about „mobile phase“, „isocratic“ and „gradient“ elution
on slides „Elution“ and „Reversed Phase Chromatography“ (Level 1 –
Chromatography)
Elution mode is either isocratic (constant solvent composition during the entire
experimental time) or gradient elution (mobile phase composition changes over
time).

Selection „pre-column volume“ is only available with „gradient elution mode“ and is
divided into mixing and non-mixing volume. Mixing volume mainly consists of the
volume of the pump and the solvent mixer. A large mixing volume causes a delayed
and dispersed gradient reaching the column. Non-mixing volume consists of the
volume of e.g. tubings and connectors. The non-mixing volume has no effect on the
gradient like the mixing volume, but results in a longer time for the gradient to reach
the column inlet.

Table „Gradient values“ belongs to the gradient elution mode. It contains

information about changes in the gradient composition over time, e.g. proportion of
solvent B increases from 5% to 55% between minute 0 and 5 etc. Consequently
solvent A decreases from 95% to 45% in the same time span.

Please find more information about „mobile phase“, „isocratic“ and „gradient“ elution
on slides „Elution“ and „Reversed Phase Chromatography“ (Level 1 –
Chromatography)
Mixing and non-mixing volume
Section „Chromatographic properties“ enables the adjustment of column
temperature as well as the injection volume of the sample to be analyzed and the
flow rate of the mobile phase.

Increasing the temperature of the column decreases the retention times.

This effect is due to the increase of the average diffusion coefficient of compounds
and the decrease of eluent viscosity and backpressure. Furthermore due to the
heating, the solubility of non-polar compounds is improved, which results in earlier
retention times.

The injection volume (10 to 100 µL) determines the amount of the sample, which is
carried to the column by the mobile phase flow, i.e flow rate (10 to 2000 µL). An
optimal injection volume/flow rate proportion is 1/10.

Please find more information about „mobile phase“, „isocratic“ and „gradient“ elution
on slides „Elution“, „Chromatographic parameters and van Deemter equation“ and
„Reversed Phase Chromatography“ (Level 1 – Chromatography)
In section „Stationary phase properties“ you may choose between two
different C18 columns in „Column selection“.
By altering the „Column length“, its „Inner diameter“ as well as the
„Particle size“, you may learn about the effect of those parameters on
the chromatographic separation.

„Reduced van Deemter parameters“ are used to calculate the „reduced

plate height“, which is calculated by means of the following equation:

𝐵
ℎ=𝐴+ +𝑐∗𝑣
𝑣
• h: Reduced plate height
• A: Eddy diffusion
• B: Random molecular diffusion
• C: Mass transfer effects
• V: Flow velocity

Please find more information about

„van Deemter Terms“, „Stationary Phases“ and
„Columns“ on slides „Chromatographic
paramaters and van Deemter equation“ and
„Reversed Phase Chromatography“
(Level 1 – Chromatography)
Select the „Compound(s)“, which you want to chromatographically
separate. You may also change the concentration(s) by douple clicking
of the respective field. After typing the desired value, click „save“.

A chromatogramm is generated after you complete the selection of

settings and compounds.
The compounds, which were selected, are displayed as chromatogram.
You may now change the setting to test their effects on the e.g. retention times of the compounds,
For instance you can switch between gradient and isocratic elution, increase or decrease the
temperture etc.
k´or capacity factor is used as a means to describe the amount of time, which an analyte needs to
elute from the column. Usually k´ ought to be between 1 and 5. A value below 1 corresponds with a
very fast elution, which results in difficulties to determine an accurate retention time. Value above
20 belong to compounds, which show a very late elution. E.g. by using an isocratic elution
compared to a gradient elution, k´ values will distinctly increase.
Sigma
W (pmol) is the amount of the compound injected onto the virtual chromatographic column, i.e.
𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑎 𝑐𝑜𝑚𝑝𝑜𝑢𝑛𝑑
.
𝑖𝑛𝑗𝑒𝑐𝑡𝑖𝑜𝑛 𝑣𝑜𝑙𝑢𝑚𝑒

For instance the injection of a sample with the concentration of 30 µM in 10 µL injection volume
results in a W (pmol) of 300.
 30 µM = 30 µmol/L = 300 pmol/10 µL
Dwell volume is available with gradient elution and is determined by the fixed values of mixing volume (200 µL)
and non-mixing volume (200 µL) added together (= 400 µL). It is a measure of the total volume before the column
inlet. The dwell volume causes the gradient to be delayed in reaching the column. Dwell time is dependent on the
dwell volume (here 400 µL) and the flow rate. The highter the flow rate, the lower the dwell time, i.e. dwell volume
flow through time. Is is calculated according to the following equation:
𝑛𝑜𝑛 − 𝑚𝑖𝑥𝑖𝑛𝑔 𝑣𝑜𝑙𝑢𝑚𝑒 + 𝑚𝑖𝑥𝑖𝑛𝑔 𝑣𝑜𝑙𝑢𝑚𝑒
𝑓𝑙𝑜𝑤 𝑟𝑎𝑡𝑒
HETP means „height equivalent to a theoretical plate“ in cm. It is calculated as follows:
HETP = h*dp with h being the reduced plate height (cm) and dp being the particle diameter (cm).

Theoretical plates indicates the number of theoretical plates in the column. It is calculated by means of the
𝐿
followng equation: Nt = with L being the column length and HETP being the height equivalent to a
𝐻𝐸𝑇𝑃
theoretical plate. The higher the number, the more efficient the separation.
Backpressure is dependent on the flow rate, eluent viscosity and column, i.e. length, diameter, particle
size. Most HPLC instruments have a maximum backpressure of 400 bar, which is therefore set as limit
with the HPLC simulator. There are however UHPLC instruments, which allow pressure up to 1000bar and
higher.
Eluent viscosity is dependent on the mobile phase composition as well as the temperature. The eluent
viscosity is useful for the calculation of the backpressure and the average diffusion coefficient (= molecule
dispersion and mixture along the column).
Reduced plate height indicates the performance of a HPLC column under a particular set of conditions. It is most
commonly calculated using the following equation:
𝐻𝐸𝑇𝑃
ℎ=
𝑑𝑝
• h: Reduced plate height
• HETP: Height equivalent of a theoretical plate
• dp: particle size
h is dimensionless and allows the estimation of the performance of a “good” column independent of particle size,
due to the normalization of HETP with dp.
The more theoretical plates the better your resolution.
Level 3
Whereas Level 2 was designed to provide a „trial and
error“ version of the HPLC simulator, level 3 requires a
basic understanding of HPLC parameters.
In contrast to level 2, here you´ll get immediate feedback
about whether or not your selection of particular
parameters is reasonable or not. The feedback is provided
in the form of color coding in section „validation results“,
with red being complete wrong, yellow partly wrong/right
and green being the right choice.
However, more than one selection can be right or wrong.
You can only proceed to the next step after getting a
„green“ or „yellow“ feedback.

Comparable to level 2 in section „column selection“ two

C18 columns are provided.
After selecting a column, you may choose the compounds
with which you want to proceed to eventually generate a
chromatogram.
„Substance selection“ provides the compound names and
their respective logD. Before you can proceed to the next
step you have to find a solvent“ to properly solve the
compounds you selected (find more information about
logD in Level 1 slides „Polarity and Solubility“).
After successfully solving the compounds, you may now
select the mobile phase solvents A and B as well as
whether you want to perform an isocratic or gradient
elution.
With isocratic elution, you have to adjust the slide bar in
order to increase or decrease the % B solvent fraction of
the mobile phase.
With gradient elution a timetable and the respective
increase of the organic solvent proportion (%B) as well as
the pre-column volume (compare manual for HPLC
simulator Level 2) are given.
The injection volume (10 to 100 µL) determines the
amount of the sample, which is carried to the column
by the mobile phase flow, i.e flow rate (10 to 2000
µL).
Select the desired column length, the column inner
diameter and the particle size.

After successfully completing (i.e. green or yellow

feedback in „validation results“) the selection of
„column properties“ a chromatogram is generated,
which contains the compounds you selected in step
2 of the process.