Two dimensional

liquid
chromatography
Jero Victor Wilson
1st m pharm
Pharmaceutical analysis

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 CONTENTS
• INTRODUCTION
• HISTORY
• MODES
• PRINCIPLE
• TYPES
• INSTRUMENTATION
• METHODS FOR TRANSFER
• APPLICATIONS
• ADVANTAGES AND DISADVANTAGES
• CONCLUSION

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 INTRODUCTION
• Two-dimensional chromatography is a type of chromatographic
technique in which the injected sample is separated by passing
through two different separation stages.
• Two different chromatographic columns are connected in sequence,
and the effluent from the first system is transferred onto the second
column.
• Typically the second column has a different separation mechanism, so
that bands that are poorly resolved from the first column may be
completely separated in the second column.

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• Two dimensional liquid chromatography (2D-LC) combines two
separate analyses of liquid chromatography into one data analysis.
• Two-dimensional liquid chromatography is a significant addition to the
family of LC techniques, which includes one-dimensional isocratic and
gradient elution LC.

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• Isocratic: A separation in which the mobile phase composition remains
constant throughout the procedure is
termed isocratic (meaning constant composition).
• The example of these the percentage of methanol throughout the
procedure will remain constant i.e 10%
• Gradient : A separation in which the mobile phase composition is
changed during the separation process is described as a gradient
elution.
• One example is a gradient starting at 10% methanol and ending at
90% methanol after 20 minutes.

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 HISTORY
• The origins of modern two-dimensional liquid chromatography (2D-LC)
can be traced to the late 1970’s and early 1980’s.
• The proof-of-principle experiments along with conceptual and
theoretical work clearly made the case that 2D-LC offered more
potential resolving power compared to one-dimensional liquid
chromatography (1D-LC)
• In the 1990’s 2D-LC played a key role in the separation of complex and
difficult-to-separate materials encountered in the fields of proteomics
and polymer chemistry.
• In 1990, Bushey and Jorgenson worked on the comprehensive 2D-LC
separation of a 14 component mixture of proteins using a combination
of size exclusion and ion-exchange chromatography.
• Now high resolution 2D-LC separations can be carried out in less than an
hour.

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 MODES
• 2D-LC separations can be carried out either in off-line or in on-line
mode.
• Off-line 2D-LC- It can be performed with a conventional HPLC system.
• Fractions from the first column are collected and reinjected into a
second dimension at a later time.
• The same instrument can be used for both dimensions by only changing
stationary phase and/or mobile phase.
• The off-line mode is convenient with no time constraint in the second
dimension. However, this mode is time consuming, not suitable for
automation and not reproducible.

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• Online mode-In contrast, on-line 2D-LC is faster, easy to automate
and, in any case reproducible.
• Fractions from the first dimension are continuously transferred via a
specific interface to the second dimension in order to be further
separated.
• The peak capacity in on-line 2D-LC is usually lower than in off-line
2D-LC. However it is far much higher than in 1D-LC.

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 PRINCIPLE
• LC techniques offer a variety of separation mechanisms such as:
o Normal phase
o reversed phase,
o size exclusion, and
o affinity chromatography
• They are characterized by different selectivities.

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 TYPES
• There are two major classifications of 2D liquid
chromatography. These include:

• Comprehensive 2D liquid chromatography (LCxLC)

• Heart-cutting 2D liquid chromatography (LC-LC).

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 COMPREHENSIVE 2D-LC
(LCXLC)
• In comprehensive 2D-LC, all the peaks from a column elution are fully
sampled, but it has been deemed unnecessary to transfer the entire
sample from the first to the second column.
• A portion of the sample is sent to waste while the rest is sent to the
sampling valve.

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HEART-CUTTING 2D-LC (LC-LC)
• In heart-cutting 2D-LC specific peaks are targeted with only a small
portion of the peak being injected onto a second column.
• Heart-cutting 2D-LC has proven to be quite useful for sample analysis
of substances that are not very complex provided they have similar
retention behavior.
• Compared to comprehensive 2D-LC, heart-cutting 2D-LC provides an
effective technique with much less system setup and a much lower
operating cost.

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INSTRUMENTATION
1D-LC

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2D-LC

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 AUTOSAMPLER
• An autosampler normally consist of a robotic device
• It can either bring the sample to a sampling station, or bring a
sampling device to the sample that stays on a tray (or carousel) along
with other samples.

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 DETECTORS
• UV detectors (PDA detectors)
• Fluorescence detector
• Mass spectrometer
• Refractive Index Detector
• Conductivity detector

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 METHODS FOR TRANSFER
Three different methods can be used to transfer the fractions from the
first to the second dimension.
(i) Direct transfer,
(ii) Temporary storage in sample loops
(iii) Temporary storage in trapping columns.

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 DIRECT TRANSFER
Direct transfer is exclusively used in LC-LC, generally with a 2-position 6-
port switching valve. By switching the valve from position 1 to position 2,
a fraction of 1D-eluent is transferred and focused onto 2D-column. After
switching the valve back to its initial position (position 1), the
compounds are eluted by 2D eluent.

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 LOOP STORAGE
• Unlike direct transfer, loop transfer can be used both in isocratic and
gradient elution mode.
• In this setup, the valve is equipped with one (in LC-LC) or several (in
LC × LC) loop(s), thereby allowing to collect the fractions from the first
dimension and to store them before injection in the second dimension.
• Interfaces based on a 6-port switching valve equipped with a single
loop are well adapted to LC-LC analyses where only one fraction of
the first dimension has to be transferred to the second dimension.

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 LOOP STORAGE

• (a) conventional 8-port valve and (b) dual 4-port valve.

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 TRAP COLUMNS
• Sample loops can be replaced by trap columns with a 10-port
valve.
• Compared to sample loops, trap columns are expected to
enhance sensitivity (by sample enrichment) and to reduce
injection issues.

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 TRAP COLUMNS
• The compounds eluted from 1D-column are focused on the trap
column before being backflushed to 2D column.
• Such a system was able to perform multi heart-cutting achiral-chiral
analysis in a single step. Each compound separated on 1D column was
individually stored on a chiral cartridge and then analyzed on 2D chiral
column.

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 ADDITIONAL SET-UPS
MAKE-UP FLOW TECHNIQUE
Make-up flow (also called Assistant flow) technique consists in
adding a post-column flow after the first separation in order to
reduce the eluent strength of 1D mobile phase before sending the
fractions in 2D, thereby limiting injection issues by better focusing the
sample onto the column.

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CHROMATOGRAM

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 APPLICATIONS OF 2D-LC IN
PHARMACEUTICAL ANALYSIS

THE APPLICATIONS OF 2D-LC IN PHARMACEUTICAL ANALYSIS IS AS

FOLLOWS:
 TRACE ANALYSIS
 CHIRAL ANALYSIS
 IMPURITY PROFILING
 HERBAL MEDICINE
 BIOANALYSIS
 FOOD TESTING

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 TRACE ANALYSIS
• In pharmaceutical analysis, peak co-elution is a major concern
since impurities can co-elute with API or with other components.
• A major application of 2D-LC is directed towards the separation
of peaks which co-elute in conventional 1D-LC methods.
• This is of prime importance, for example, for peak purity
assessment.
• LC-LC with a heart cut of the fraction containing API and its co-
eluted impurities can address this problem.

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 TRACE ANALYSIS
• Two different approaches were done:
• This first approach allowed the development of sensitive methods
in the context, for example, of pharmacokinetics or therapeutic
drug monitoring (TDM) studies.
• Others applied 2D-LC techniques for separation, detection and
sometimes identification of low level during the early stages of
drug development.

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 CHIRAL ANALYSIS
• For separating enantiomers, heart-cutting can be useful.
• In this case, although the compounds were well separated on the chiral
column, a first C18-dimension with only a small sample fraction sent to 2D,
allowed to significantly extend the lifetime of the chiral column.
• Targeted chiral compounds are either collected and stored into loops or
cartridge before transfer or directly transferred to an enantioselective
second dimension column that can separate their enantiomers
• The main benefit of this method is that the chiral separation can be
enhanced by excluding interfering substances from the second column.

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 CHIRAL ANALYSIS
Simultaneous analysis of Paracetamol and Ketorolac enantiomers
based on achiral-chiral 2D-LC approach.

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 IMPURITY PROFILING
• 2D-LC separations must be taken into consideration for impurity
profiling during pharmaceutical degradation studies as well as for
the analysis of drug mixtures.
• On-line LCxLC separation of an API and its related impurities.

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COMPREHENSIVE 2D-LC ANALYSIS OF CHINESE
HERBAL MEDICINE
• Chinese herbal medicine (CHM) is one aspect of traditional Chinese
medicine (TCM) and uses single plants or preparations of several
plants.
• The effectiveness of CHM depends on the synergistic effects of
multiple components in the plants.
• The plants used in CHM present extremely complex samples.
• Therefore, comprehensive two dimensional liquid chromatography
(comprehensive 2D-LC) is the method of choice for their analysis.

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• Comprehensive 2D-LC is ideally suited for the analysis of herbal
formulations, especially when combining two RP phases in the two
separation dimensions.

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BIOANALYSIS

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 FOOD TESTING-POLYPHENOLS
IN BEVERAGES
• Polyphenols are well known natural anti-oxidants that are present in
many fruits that are used for juices, wines or other beverages like
beers, lemonades etc.
• It is a free radical scavenger with positive effects against cancer.
• The matrix is highly complex therefore they are ideal for the 2D-LC
analysis.

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• The qualitative and quantitative analysis is performed by
comprehensive 2D-LC coupling two reverse phase seperations.

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 Advantages of 2D-LC
• High resolving power
• Overlapping of peaks in the first dimensions are resolved.
• Large increase in peak capacity.
• Separate mixtures that one dimensional liquid chromatography
otherwise cannot separate effectively.
• It can separate closely related compounds.

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 Disadvantages
• The very long timescale (typically several hours to tens of hours) of
comprehensive 2DLC is clearly its chief drawback.

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 CONCLUSION
• 2D-LC is a great tool that can help to solve analytical problems faster
than ever before.
• It’s only one small step from 1D-LC to 2D-LC.
• It is a reliable and reproducible method and especiall y useful in the
pharmaceutical industry.

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 REFERENCES
• https://www.sciencedirect.com/topics/chemistry/2d-liquid-
chromatography
• https://www.agilent.com/cs/library/primers/public/5991-2359EN.pdf
• http://www.chromatographyonline.com/recent-advances-two-
dimensional-liquid-chromatography-pharmaceutical-and-
biopharmaceutical-analysis
• https://en.wikipedia.org/wiki/Two-dimensional_chromatography
• https://pubs.acs.org/doi/pdf/10.1021/acs.analchem.6b03506
• https://theanalyticalscientist.com/fileadmin/tas/issues/14-
11/images/1114-Agilent_Supplement_new.pdf

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