1. Institute of Chemical Sciences
Ultra Performance Liquid Chromatography
(UPLC)
Presented by Mazhar Abbas
Roll No MPH-12-20
Semester st
Institute of Chemical Sciences
Bahauddin Zakariya University, Multan, Pakistan.

2. Types of Liquid Chromatography
(TLC) Paper
Chrom.
Gravity Chrom.
Tsvett, 1903
Flash Chrom.
1978
HPLC 1952
UPLC 2004

3. About UPLC
By using smaller particles, speed and peak
capacity can be extended to new limits. It
also provides good resolution.
According to Van Deemter, decrease in
particle size less than 2.5 mm, a significant
gain in efficiency is achieved.

4. Van Deemter plot illustrating the evolution of particle sizes
We see that H decreases drastically as dp becomes smaller, and there is an added benefit because the right side of the curve becomes more flat, thus enabling higher speeds without loss of column efficiency.
Van Deemter plot illustrating the
evolution of particle sizes

5. I:It is an overlay of both conventional (3 µm) HPLCand 1
I:It is an overlay of both conventional (3 µm) HPLCand 1.7 µm UPLC for a five component sample mixture.
II:It is an expansion of the first 0.6 minutes of the overlay to show the increased speed of UPLC, while resolution is still maintained.

6. Principle of UPLC Reducing the particle diameter of a
packing material, column length.
The back pressure requred for the use
of the small particle column.
3. Monolithic columns provide lower flow
resistance than conventional columns.
4. Columns can be operated at high flow rates.

7. Theory of separations using small particles
The fundamental resolution equation
Efficiency is proportional
to column length and
inversely proportional to
the particle size:
Resulation (Rs) is directly propotional to the square root of N. But N is inversely propotional to the particle size (dp)
As the particle size is lowered by a factor of three, from, for example, 5 micro m (HPLC-scale) to 1.7 micro m (UPLC-scale), N is
increased by three and resolution by the square root of three or 1.7. N is also inversely proportional to the square of the peak width
So as the particle size decreases to increase N and subsequently Rs, an increase in sensitivity is obtained, since narrower peaks are taller peaks. Narrower peaks also mean more peak capacity per unit time in gradient sepration

8. Method optimization on UPLC
Applying the scaling factor to the mobile
phase.
2. The mobile phase flow rate was increased
until limited by column back-pressure.
3. Reducing the total run time by increasing
organic solvent content was more
economical.

9. Comparison between HPLC and UPLC
HPLC Assay
UPLC Assay
Column
C18, 50 x 4.6 mm, 4 µm particles
BEH C18, 50 x 2.1 mm, 1.7 µm particles
Flow rate
3.0 ml / min
0.6 ml / min
Injection volume
20 µl
3 µl partial loop fill OR 5 µl full loop fill.
Total run time
10min
1.5min
Total solvent consumption (including 0.5 min of delay time in between injections)
Acetinitril:10.5ml Water: 21.0 ml
Acetinitril:0.53ml Water: 0.66 ml
Plate count
2000
7500
USP resolution
3.2
3.4
Lower limit of quantitation (LOQ)
~ 0.2 µg/ml
~ µg/ml

10. Ethylene Bridged Hybrid [BEH]
The 1.7 µm Ethylene Bridged Hybrid [BEH] particle is one of the key enablers behind UPLC® technology. It is available in three different pore sizes [130Å, 200Å and 300Å]

11. Temperature Control in UPLC
Temperature control is needed to get
Reproducibility
Solubility
Stability

12. Temperature control system
1. Oven
2. Heater Block
3. Water bath
Temperature control system

13. Instrumentation
I. Pump
II. Solvent system
III. Sample injector
IV. Column
V. Detector

14. Schematic diagram of instrumentation of UPLC

15. I. Pumps
The most important advantages of pumps are: higher resolution, faster analyses, and increased sample load capacity.
Pump Module – types
• Isocratic pump - delivers constant mobile phase composition; solvent must be pre-mixed
• Gradient pump - delivers variable mobile
phase composition;
a. Binary gradient pump
b. Quaternary gradient pump

16. Gradient vs. Isocratic Conditions
• Best for simple separations
• Often used in quality control applications that support
and are in close proximity to a manufacturing process.
Gradient
• Best for the analysis of complex samples
• Often used in method development for unknown
mixtures.
• Linear gradients are most popular

18. II. Solvent system
A. Mobile phases
several common properties:
a. Purity of the solvent
b. Detector compatibility
c. Chemical inertness
d. Low viscosity
B. Mobile phase reservoir
The most common type of solvent reservoir is a glass bottle. Most of the manufacturers supply these bottles with special caps, Teflon tubing and filters to connect to the pump inlet and to the purge gas (helium) used to remove dissolved air.

19. III. Sample Injection
Manual Injector:
User manually loads sample into the injector using a
syringe.
2. And then turns the handle to
inject sample into the
flowing mobile phase which
transports the sample into the
beginning (head) of the column,
which is at high pressure.

20. Auto sampler:
User loads vials filled with sample solution into the
auto sampler tray (100 samples)
2. And the auto sampler automatically
a. Measures the appropriate sample volume,
b. Injects the sample,
c. Then flushes the injector to be ready for the next sample, etc., until all sample vials are processed for unattended automatic operation

21. Types of columns in UPLC
IV. The columns
Types of columns in UPLC
1. Analytical column [internal diameter (i.d.) mm;
lengths 15 – 250 mm]
2. Preparative column (i.d. > 4.6 mm; lengths 50 – 250 mm)
3. Capillary column (i.d mm; various lengths)
4. Nano column (i.d. < 0.1 mm, or sometimes stated as < 100 µm)

22. a. Porous, polymeric beds:
Column packing
a. Porous, polymeric beds:
based on styrene-divinyl benzene co-polymer.
b. Porous layer beds:
silica or modified silica on an spherical inert core (e.g. glass beds).
c. Totally porous silica particles:
(diameter < 10 µm) with narrow size range.
Particles of diameter > 20 µm are usually dry packed,
while particles of diameter < 20 µm are slurry packed,
in which particles are suspended in a suitable solvent
and the slurry so obtained is driven into column under
pressure.

23. V. Detector
1. Spectroscopic Detection
2. Refractive Index Detection
3. Fluorescence Detection

24. 1. Ultraviolet (UV) Absorption
An ultraviolet light beam is directed through a flow cell and a sensor measures the light passing through the cell. If a compound elutes from the column that absorbs this light energy, it will change the amount of light energy falling on the sensor. The resulting change in this electrical signal is amplified and directed to a recorder or data system.

25. 2. Refractive Index (RI) Detection
The ability of a compound or solvent to deflect light provides a way to detect it.
Analytes change the refractive index of the light in a proportional amount to the concentration.
Heat can change the RI of the mobile phase so thermo control important
RI is ideal for analyzing complex sugars and carbohydrates which have no chromophores, fluorescence or electrochemical activities

26. Refractive Index detector

27. 3. Fluorescence Detection
Compared to UV-Vis detectors fluorescence detectors offer a higher sensitivity and selectivity that allows quantifying and identifying compounds and impurities in complex matrices at extremely low concentration levels (trace level analysis).

28. VI. Data system
Since the detector signal is electronic, using modern data collection techniques can aid the signal analysis. In addition, some systems can store data in a retrievable form for highly sophisticated computer analysis at a later time.

29. 9. Advantages of UPLC
High resolution
High speed
It is up to 9 times faster, has up to twice the
resolution and three times the sensitivity than
that of HPLC.
Quick and ease of instrument handling
High sensitivity.
Even neon particles can be separated easily.
UPLC (BEH C18 columns) can be used at
broad range of pH ranging from 2 to 12.

30. 10. Applications One of the most common compromises in HPLC is
sacrificing resolution for speed. The UPLC increase the resolution in shorter run times and it generates more information faster without sacrificing resolution. These newer technique fulfils the market needs.
B. Together with an UPLC System merged with
a mass spectrometer, it enables scientists to
attain more information about the identity of
pharmaceutical, biological, industrial and
environmental compounds than conventional
UV/Vis-based detector systems

31. 11. Other applications
From injection of a poly-drug reference standard and
whole blood extract, separation and identification of
amphetamine, methamphetamine, ephedrine,
pseudoephedrine and ketamine in less than
3 min using the UPLC method.
Determination of Coumarone in UPLC-Electro spray-Tandem Mass Spectrometry.
UPLC used in bioanalysis, metabolite identification and
method development.

32. 12. Conclusion
Now a day’s various pharmaceutical companies are going to use HPLC and UPLC as separation techniques to increase the marketing needs. The new technology in chemistry and instrumentation provides more information per unit of work as UPLC begins to fulfill the promise of increased speed, resolution and sensitivity predicted for liquid chromatography

33. THANK YOU