Inventi Rapid: Pharm Analysis & Quality Assurance Vol.

2012, Issue 4
[ISSN 0976-3813]
2012 ppaqa 515, CCC: $10 Inventi Journals (P) Ltd
Published on Web 17/09/2012, www.inventi.in
REVIEW ARTICLE
INTRODUCTION
Chromatography is a non-destructive procedure for
separation and extraction of individual analyte from the
mixture. For many years, researchers have been finding
fast LC method. Smaller columns and faster flow rates
(amongst other parameters) have been used to check the
separation speed. Analysis at increased temperature is also
checked, it can increase diffusivity of analyte due to
reduction of viscosity. Using normal pressure and particle
size we can achieve high resolution
3
. So High performance
liquid chromatography (HPLC) is introduced 30 years ago.
We cannot get full advantage of small particle size in simple
HPLC. UPLC can be considered as new invention for liquid
chromatography. UPLC refers to Ultra Performance Liquid
Chromatography. Using UPLC technique we can get
increased sensitivity, resolution and speed. This system
operates at higher pressure than HPLC ,uses smaller particle
size(less than 2.5m) and mobile phase at higher linear
velocity. Simultaneously Super critical fluid chromatography
is also introduced since about 1982.To take the advantages
of both the technology Waters introduce AQUITY based new
product Ultra Performance Convergence Chromatograph (
UPC
2
), which expands the boundaries of reversed phase
liquid chromatography (LC) and of gas chromatography (GC)
separations and offers a replacement option for normal
phase chromatography applications
4, 5
.
UPC combines SFC with UPLC technology and expertise
in fluidics, kinetics, and thermodynamics to deliver fast,
reproducible separations. Supercritical CO
2
has very low
viscosity, so it increases mass transfer, and hence column
efficiency, even compared to hexane or water when used as
a mobile phase in this technique. Along with sub-2 m
particle column chemistries, UPC
2
System gives the ability
to precisely vary mobile phase strength, pressure, and
temperature. With this ability to fine-tune the resolving
power and selectivity of the system, one can exercise better
control over the retention of analytes for separating,

1
Post Graduation Department of Pharmaceutical Sciences, Sardar Patel
University, Vallabh Vidhyanagar- 388120, Gujarat, India.
E-mail: tanvi.divan@yahoo.in
*Corresponding author
detecting and quantifying Whether analyzing natural
products, traditional medicines, drugs, food additives or
contaminants, pesticides, surfactants, polymer additives, or
biofuels - all compounds that are often difficult to separate
by any other means. The UPC
2
System now takes its place
alongside LC and GC as a powerful complementary technique
for taking on the laboratory's toughest separations
challenges. UPC
2
can be coupled with a range of Waters
solutions previously considered incompatible with normal
phase chromatography and SFC. It is also best complement
to MS with low solvent load, high resolution, narrow peaks
and fast separation. Key benefit of system is use of CO
2
as a
mobile phase instead of toxic and expensive organic solvent
as a mobile phase. UPC
2
can analyze diverse range of analyte
with faster separation and superior resolution
1, 47
.
HISTORY AND NEED
UPLC
13

As demonstrated by Knox, Giddings and other authors,
small particle diameters induce an increase in efficiency,
optimal velocity and mass transfer. Therefore, fast
separations can be performed with relatively short
columns. However, only since 2004 columns packed with
sub-2 m porous particles are commercially available and
yield reliable performance. The use of columns packed with
sub-2 m particles while operating at high pressures was
developed ten years ago by Jorgenson and co-
workers and and Lee and co-workers. This strategy allows
a significant decrease (up to 20) in analysis time because
the latter is directly proportional to the maximal system
backpressure, as demonstrated elsewhere. Today,
numerous manufacturers commercialize analytical devices
that are able to handle pressure higher than 400 bar, such
as UPLC, for Ultra Performance Liquid Chromatography.
Columns compatible with extreme pressures are
commercially available. The packing material is a bridged
ethyl hybrid (BEH) technology, which improves mechanical
resistance.
SFC
Klesper, Corwin and Turner first demonstrated Supercritical
Fluid Chromatography (SFC) in Communications to the
Ultra Performance Convergence Chromatography (UPC
2
)
Paradigm Changed: SFC Based UPLC Technique
Divan Tanvi A
1*
, Parmar Shraddha J
1
, Patel Bhavana A
1
Abstracts: Ultra Performance Convergence Chromatography (UPC
2
) is a new category of separations science in chromatography
that makes the complex analysis routine. It combines the potential of SFC technique with available UPLC technique. Use of
compressed carbon dioxide CO2 (green solvent) and co-solvent as a mobile phase makes the technique inexpensive and non
toxic and it avoids use of toxic, expensive and volatile organic solvents. Along with sub-2 m particle column chemistries, system
gives the ability to precisely vary mobile phase strength, pressure, and temperature. With the ability to fine-tune the resolving
power and selectivity of the system, one can exercise better control over the retention of analytes like natural products,
traditional medicines, drugs, food additives or contaminants, pesticides, surfactants, polymer additives, or biofuels-all
compounds that are often difficult to separate by any other means. It is also best complement to MS with low solvent load, high
resolution, narrow peaks and fast separation. Waters Corporation has been awarded the prestigious 2012 Pittcon Editors Gold
Award for the Waters ACQUITY UPC
2
System as the best new product at the 2012 Pittsburgh Conference on Analytical Chemistry
and Applied Spectroscopy.
1

Inventi Rapid: Pharm Analysis & Quality Assurance Vol. 2012, Issue 4
[ISSN 0976-3813]
2012 ppaqa 515, CCC: $10 Inventi Journals (P) Ltd
Published on Web 17/09/2012, www.inventi.in
REVIEW ARTICLE
editor entitled High Pressure Gas Chromatography above
Critical Temperatures in 1962. Sie, Beersum and
Rijnders published a series of articles on High-Pressure
Gas Chromatography with Supercritical Fluids in
Separation Science (2-5) in 1966 and 1967. In these
articles, they extensively used carbon dioxide as mobile
phase. Since the 1980s, supercritical carbon dioxide
became the most preferred fluid in SFC and in
Supercritical Extraction (SFE) because it is inexpensive,
non-toxic, non-flammable and it has relatively low
critical temperature and pressure. In 2008, ultrafast SFC
also introduced
6
.
Super critical fluid extraction and ultra performance
liquid chromatography combination with MS detection is
also performed earlier
29, 41
.
In order to reduce the analysis time, maintain good
efficiency in liquid chromatography (LC) and to take
advantage of both above techniques, several solutions are
currently being investigated. For this purpose, the different
approaches were discussed.

Figure 1: Block diagram of ultra performance convergence chromatography

Figure 2: Normal phase HPLC separation of Anthralin and o-nitro
aniline.
17


Figure 3: UPC
2
separation of Anthralin and o-nitro aniline.
17

Figure 4: Normal phase HPLC analysis of Tolbutamide and
Tolazamide
12
.

Figure 5: Acquity UPC
2
analysis of Tolbutamide and
Tolazamide
12
.
2

Inventi Rapid: Pharm Analysis & Quality Assurance Vol. 2012, Issue 4
[ISSN 0976-3813]
2012 ppaqa 515, CCC: $10 Inventi Journals (P) Ltd
Published on Web 17/09/2012, www.inventi.in
REVIEW ARTICLE
For this previously super critical fluid chromatography
with different particle size (3 m, 1.5 m pellicular
particles and 1.8 m, 5 m) packed column is tried
2
.
Although many users have had success using this
approach, limitations in analytical SFC instrument design
and column availability have prevented the adoption of
small particle pSFC. Earlier attempts at sub-2 m pSFC
have resulted in many false starts due to the lack of
commercially available dedicated instrumentation. As for
analytical pSFC, many systems identified in publication
utilize instrument configurations limited by large system
volume and extra column volumes affecting the ability to
achieve optimal chromatographic performance. The scope
of this work explores the impact of a low dwell volume
and low extra column volume instrument configuration
with intent to determine any benefits in chromatographic
efficiency, resolution, sensitivity for sub 2 m particles
stationary phases. The advantages of overcoming these
instrumental performance limitations will allow a true
assessment of pSFC for compatible sub-2 m stationary
phases.
36

It appeared that columns packed with sub-2 m
particles under high-pressure conditions (UPLC) were well
adapted and this option represents an attractive alternative
to conventional LC.
To overcome the expanding need, challenge to get more
and better information faster, cost effectiveness new
technology based on existing technology is introduced by
Waters in 2011.
WATERS designed UPSFC system combining the
advantage of 2 m particle size and the efficiency of SFC
30,
46
.

This new system provides shorter run times, reduces
solvent usage &
Cuts the cost of analyses. Later, this system with MS
detection and other features is introduced as Ultra
Performance Convergence Chromatograph (UPC
2
).
PRINCIPLE
UPC
2
is based on principle of ultra performance
convergence chromatography
1
.
The UPC
2
is based on the principal of use of stationary
phase consisting of particles less than 2 m and super
critical solvent CO
2
as a mobile phase. By the van Deemter
equation, which is an empirical formula that describes the
relationship between linear velocity (flow rate) and plate
height (HETP or column efficiency), we can understand the
use of small particle size in column. The Van Deemter
curve, governed by an equation with three components
shows that the usable flow range for a good efficiency with
small diameter particles is much greater than for larger
diameters
8
.
H=A+B/v+Cv
Where A, B and C are constants and v is the linear
velocity, the carrier gas flow rate. The A term is
independent of velocity and represents "eddy" mixing. It is
smallest when the packed column particles are small and
uniform. The B term represents axial diffusion or the
natural diffusion tendency of molecules. This effect is
diminished at high flow rates and so this term is divided by
v. The C term is due to kinetic resistance to equilibrium in
the separation process. The kinetic resistance is the time
lag involved in moving from the gas phase to the packing
stationary phase and back again. The greater the flow of
gas, the more a molecule on the packing tends to lag behind
molecules in the mobile phase. Thus this term is
proportional to v. So if we decrease the particle size it
increases the column efficiency. Super critical fluids
(compressed CO
2
) can be used as a mobile phase because it
provides greater diffusivity, lower viscosity and lower
surface tension than other organic solvents. Solvating
power of super critical fluid is based on its density. As
pressure increases in system, solvating power of fluid
increases. So it decreases elution time
39
.
INSTRUMENTATION
UPC
2
system contain binary solvent manager, sample
manager, column manager and column heater with
temperature range of 20 to 90

C ,Acquity upc
2
manager,
Acquity upc
2
PDA detector and Acquity upc
2
instrument

Figure 6.Normal phase HPLC chromatogram (A) and UPC
2
chromatogram (B) of BINOL
18
.
3

Inventi Rapid: Pharm Analysis & Quality Assurance Vol. 2012, Issue 4
[ISSN 0976-3813]
2012 ppaqa 515, CCC: $10 Inventi Journals (P) Ltd
Published on Web 17/09/2012, www.inventi.in
REVIEW ARTICLE
control. System is provided with 200 prepacked, pre-
measured ready to use standards and reagents. System
uses compressed CO
2
and co-solvent as a mobile phase
which is stable, non flammable green solvent, inexpensive
and nontoxic solvent
10
.
Column Chemistry
15

The reduction and control of silanol activity on AQUITY
UPC
2
particles delivers, under SFC conditions, excellent
peak shapes, without use of mobile phase additives even
for well retained basic achiral compound. Four types of
columns with dimension of 2.1 to 4.6 mm I.D. upto 150 mm
in length are available which extends the selectivity range
of SFC separation.BEH 2-EP column provides good
retention ,peak shape and selectivity.BEH column provides
heightened interaction with polar group. CSH fluro-phenyl
provides good retention of weak bases and alternate
elution order for acidic and neutral compounds. Using HSS
C
18
SB column, glyceride analysis (Figure 1).
ADVANTAGES
1

Over Simple LC and HPLC
Instead of other organic liquid solvents, this system uses
compressed CO
2
gas as a mobile phase which is non
flammable green solvent, inexpensive and nontoxic solvent.
Low viscosity of mobile phase achieves higher diffusion
rate and enhanced mass transfer in UPC
2
.
Achiral analysis can be done with low solvent usage and
at lower cost than HPLC. Chiral analysis can be done at
much faster rate than normal phase HPLC.
With this system, analysis of diverse range of
compounds like hydrophobic and chiral compounds, lipids,
thermally-labile samples and polymers which cannot
analysed by other technique.
Over GC
Analysis can be done at much lower temperature with CO
2.
Over SFC
In UPC
2
SFC is combined with UPLC, so advantage of lower
particle size is also involved in it. Column efficiency
increases than SFC column.
APPLICATIONS
Enantiomeric Impurity Analysis
Most of the drugs are available in their enantiomeric form.
Only single enantiomeric form has high efficacy and better
pharmacological effect. So manufacturer of the enantiomer
have to remove other stereoisomer from the product. Using
UPC
2
, detection and quantification of low level
enantiomeric impurities in drug substance can be done
with high sensitive detection and repeatability. The high
detection sensitivity of the ACQUITY UPC
2
System enables
the identification and quantification of enantiomeric
impurities in drug substances.
9
Analysis of Tablet
Assay of tablet using normal phase HPLC takes more time
and consumes more solvents in separation. The UPC
2
System is an ideal solution for laboratories looking for
more efficient and cost effective methods for the analysis of
Dapsone tablets while enhancing health, safety, and
environmental concerns. ACQUITY UPC
2
System enables
seven time faster analysis of Dapsone tablet with
significant reduction in use of organic solvents
14
.
Assay of Drug Substance
UPC
2
technique also performs the assay of drug substances
with high quality results. A USP compendial HPLC method
for assay of drug substance Anthralin was successfully
converted to a high quality UPC
2
method at a cost of $0.05
per run (compared to $0.90) and was 1.6 times faster. In
this, each normal phase HPLC run used 16.4 ml of hexane
and 1.2 ml of dichloromethane. In contrast UPC
2
method
only uses 1.05 ml of methanol. This demonstrates the
reduction in use of organic solvents
17
.
Normal Phase HPLC separation of Anthralin and o-nitro
aniline is shown in Figure 2.
17
and UPC
2
separation of
Anthralin and o-nitro aniline shown in Figure 3.
17

Purity Estimation
The UPC
2
method used for the estimation of the
chromatographic purity of estradiol was three times faster
than the current normal phase HPLC method and reduced
cost per analysis by more than 100 times
11
.
Pesticide and Insecticide Analysis
Twenty five percent of pesticide currenty used are chiral
compounds. So there is increasing the demand of
stereoselective separation techniques and analytical assays
for evaluation of enantiomeric purity of pesticide.
Compared to chiral HPLC methods, UPC
2
offers a complete
baseline resolution of all permethrin isomers with
significantly shorter run time; ideal for pesticide
manufacturers who routinely perform diastereomer
analyses
45
.
Chromatographic Assay
This new Ultra Performance Convergence Chromatography
(UPC
2
) method produced data of equal or better quality
than the current HPLC method, was 10 times faster, and
consumed less solvent. eg. Tolbutamide assay with use
of tolazamide as an internal standard (Figure 4 and
Figure 5)
12
.
Organic Compound Analysis
Separation of organic compounds like binol can be done
by UPC
2
. Ultra Performance Convergence
Chromatography (UPC
2
) demonstrated a faster separation
of BINOL enantiomers (by 9 times) compared to normal
phase HPLC as well as appreciable cost savings per
analysis (Figure 6)
18
.
Enantiomeric Separation
The Waters ACQUITY UPC
2
System is ideal for laboratories
pursuing high throughput enantioselective analyses,
enantiomeric excess determination in a complex mixture or
matrix, and impurity profiling
7
.
4

Inventi Rapid: Pharm Analysis & Quality Assurance Vol. 2012, Issue 4
[ISSN 0976-3813]
2012 ppaqa 515, CCC: $10 Inventi Journals (P) Ltd
Published on Web 17/09/2012, www.inventi.in
REVIEW ARTICLE
Fat Soluble Vitamin Analysis
The use of Ultra Performance Convergence Chromatography
(UPC
2
) in fat-soluble vitamin analysis provides a single
viable technique that is cost-effective, sustainable, and a
green technology alternative that lowers the use of organic
solvents, provides fast analysis
19
.
Analysis of Formulated Lithographic Material
Lithographic material like photo resist and anti reflective
coatings is formulated for electronics industry. Many
products utilize various Dyes. Rapid analysis of dye
impurity, dye concentration can be done in less than two
minutes with UPC
2
system
48
.
CONCLUSION
From this review we can consider UPC
2
as a best analytical
separation technique for the separation of diverse range of
compounds. UPC
2
System expands the boundaries of
reverse phase liquid chromatography (LC) and of gas
chromatography (GC) separations and offers a replacement
option for normal phase chromatography applications.
Employing the principle of Ultra Performance Convergence
Chromatography, UPC
2
System is a novel analytical
system that gives laboratories an indispensable tool for
tackling tough-to-analyze compounds including
hydrophobic and chiral compounds, lipids, thermally-labile
samples and polymers. System uses CO
2
as a mobile phase
which is a green solvent and this technique provides better
selectivity, resolution and faster analysis than other
techniques. Waters Corporation has been awarded for the
Waters Acquity UPC
2
system in 2012.
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2
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Inventi Rapid: Pharm Analysis & Quality Assurance Vol. 2012, Issue 4
[ISSN 0976-3813]
2012 ppaqa 515, CCC: $10 Inventi Journals (P) Ltd
Published on Web 17/09/2012, www.inventi.in
REVIEW ARTICLE
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Cite this article as: Divan Tanvi A, Parmar Shraddha J,
Patel Bhavana A. Ultra Performance Convergence
Chromatography (UPC
2
) Paradigm Changed: SFC Based
UPLC Technique. Inventi Rapid: Pharm Analysis &
Quality Assurance, 2012(4): 1-6, 2012.
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