Transcript of Ultra Performance Liquid Chromatography (UPLC) Institute of Chemical Sciences Bahauddin Zakariya...
- Slide 1
- Ultra Performance Liquid Chromatography (UPLC) Institute of
Chemical Sciences Bahauddin Zakariya University, Multan, Pakistan.
Presented by Mazhar Abbas Roll No. MPH-12-20 Semester 1 st
- Slide 2
- Types of Liquid Chromatography Gravity Chrom. Tsvett, 1903
Flash Chrom. 1978 HPLC 1952UPLC 2004(TLC) Paper Chrom. 2
- Slide 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. 3
- Slide 4
- Van Deemter plot illustrating the evolution of particle sizes
4
- Slide 5
- I:It is an overlay of both conventional (3 m) HPLCand 1.7 m
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. 5
- Slide 6
- Principle of UPLC 1.Reducing the particle diameter of a packing
material, column length. 2.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. 6
- Slide 7
- Theory of separations using small particles The fundamental
resolution equation Efficiency is proportional to column length and
inversely proportional to the particle size: 7
- Slide 8
- Method optimization on UPLC 1.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. 8
- Slide 9
- HPLC AssayUPLC Assay Column C18, 50 x 4.6 mm, 4 m particles BEH
C18, 50 x 2.1 mm, 1.7 m particles Flow rate3.0 ml / min0.6 ml / min
Injection volume20 l 3 l partial loop fill OR 5 l full loop fill.
Total run time10min1.5min 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 count20007500
USPUSP resolution3.23.4 Lower limit of quantitation (LOQ)~ 0.2
g/ml~ 0.054 g/ml 9 Comparison between HPLC and UPLC
- Slide 10
- 10 Ethylene Bridged Hybrid [BEH] The 1.7 m Ethylene Bridged
Hybrid [BEH] particle is one of the key enablers behind UPLC
technology. It is available in three different pore sizes [130, 200
and 300]
- Slide 11
- Temperature Control in UPLC Temperature control is needed to
get Reproducibility Solubility Stability 11
- Slide 12
- Temperature control system 1. Oven 2. Heater Block 3. Water
bath Temperature control system 12
- Slide 13
- Instrumentation I. Pump II. Solvent system III. Sample injector
IV. Column V. Detector 13
- Slide 14
- Schematic diagram of instrumentation of UPLC 14
- Slide 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 15
- Slide 16
- Gradient vs. Isocratic Conditions Isocratic 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
16
- Slide 17
- 17
- Slide 18
- II. Solvent system A. Mobile phasesseveral 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. 18
- Slide 19
- III. Sample Injection Manual Injector: 1.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. 19
- Slide 20
- Auto sampler: 1.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 20
- Slide 21
- IV. The columns Types of columns in UPLC 1. Analytical column
[internal diameter (i.d.) 1.0 - 4.6-mm; lengths 15 250 mm] 2.
Preparative column (i.d. > 4.6 mm; lengths 50 250 mm) 3.
Capillary column (i.d. 0.1 - 1.0 mm; various lengths) 4. Nano
column (i.d. < 0.1 mm, or sometimes stated as < 100 m)
21
- Slide 22
- Column packing a. Porous, polymeric beds: b. Porous layer beds:
based on styrene-divinyl benzene co-polymer. silica or modified
silica on an spherical inert core (e.g. glass beds). c. Totally
porous silica particles: (diameter < 10 m) with narrow size
range. Particles of diameter > 20 m are usually dry packed,
while particles of diameter < 20 m are slurry packed, in which
particles are suspended in a suitable solvent and the slurry so
obtained is driven into column under pressure. 22
- Slide 23
- V. Detector 1. Spectroscopic Detection 2. Refractive Index
Detection 3. Fluorescence Detection 23
- Slide 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. 24
- Slide 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 25
- Slide 26
- Refractive Index detector
- Slide 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). 27
- Slide 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. 28
- Slide 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. 29
- Slide 30
- 10. Applications A.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 30
- Slide 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. 31
- Slide 32
- 12. Conclusion Now a days 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 32
- Slide 33
- 33
- Slide 34
- 34