This document compares HPLC and UPLC and provides an overview of UPLC. UPLC uses smaller particle sizes of 1.75-1.8 μm compared to 3-10 μm for HPLC, which allows for higher pressures over 15,000 psi, improved precision, faster analysis times, and higher resolution. The key factors that enable improved performance in UPLC are increased efficiency through smaller particle sizes and improved selectivity, retentivity, and efficiency. UPLC also provides increased sensitivity, faster run times, and the ability to analyze more samples due to its enhanced resolving power. The document discusses UPLC stationary phases, columns, instrumentation including pumps, columns, and detectors, as well as applications of UPLC in areas like method

1. (Ultra Pressure Liquid
Chromatography)
Presented by: Guided by:
Ms. Sonali R.Phadke Dr. Asha B. Thomas
M.Pharm :sem-I(QAT)
UPLC-An overview 1

2. Comparison between HPLC and
UPLC :
HPLC
 dp(3-10)µm
 Inlet pressure 5000 psi
 Lower precision comparative
in sample introduction.
 Detector that uses large flow
rates and larger detection
cells.
 Some are equipped with
automated sampling devices.
 Slower with lower resolution.
UPLC
 dp.(1.75-1.8)µm
 Inlet pressure more than
15000psi
 Higher precision in sample
introduction.
 Detector that uses small flow
rates and low detection
limits.
 Most of are equipped with
automated sampling devices.
 Rapid with higher resolution.
UPLC-An overview 2

3. The fundamental resolution
equation :
Rs=
𝑁
4
(
𝛼−1
𝛼
) (
𝑘
𝑘+1
)
efficiency selectivity retensivity
In UPLC, increasing N (efficiency) is the primary
focus.
As Rs is proportional to square root of N
Rs∝ √𝑁
If N ↑ 3𝑋 , 𝑅𝑠 ↑ 1.7𝑋
UPLC-An overview 3

4. Improving particle size:
Efficiency is inversely proportional to particle size so
N∝
1
𝑑𝑃
Rs ↑ 1.7X
(i.e. Rs∝ √𝑁)
UPLC-An overview 4

5. Resolution (constant column length):
Optimal flow rate is inversely proportional to dp
Fopt ∝ 1/𝑑𝑝
Isocratic analysis time is Inversely proportional to F
Therefore
T ↓3X
UPLC-An overview 5

6. Peak width and sensitivity
Efficiency E, is inversely proportional to square of peak
width.
N∝ 1/W2
Peak height is inversely proportional to peak width
Height ∝ 1/w
sensitivity ↑ 1.7 x
UPLC-An overview 6

7. Sensitivity increases:
 Assuming same efficiency, peak height is inversely
proportional to column length,
Height=1/L
For same length column length L decreased
proportional to particle size dp
(N=1x, Rs=1x)
Sensitivity ↑ 3𝑥
UPLC-An overview 7

8. Backpressure increases:
Backpressure (p) is directly proportional to column
length
P∝ 𝐿
For constant L/dp, Backpressure is inversely
proportional to the square to particle size,dp
P∝ 1/dp2
P↑ 𝟗𝑿
UPLC-An overview 8

9. HPLC vs UPLC
UPLC-An overview 9

10. Why to migrate from HPLC to UPLC?
 Acquire results in less time and
with more resolution.
 More information-faster
 More robust method-more
confidence
 Better situational response
 More samples analyzed per
system, per scientist
UPLC-An overview 10

11. UPLC stationary phases :
 Mechanical strength and stability of columns were
critical issues due to high pressure.
 Classical gel-sol method used for generation of
durable columns which are superior than silica
particles
 Made by incorporating carbon in form of methyl
groups
 Mechanically strong, better efficiency and can be used
over extended range of pH.
UPLC-An overview 11

12. Ethylene Hybrid Bridge(EHB) particles :
 This technology increases the stability of the 1.7 µm
particles by bridging the methyl group in the silica
matrix.
 Can withstand high pressure and pH.
 Efficiency of column is directly proportional to column
length and inversely proportional to particle size.
 Pore diameters are 130 Å and 300Å.
UPLC-An overview 12

13. High Strength Silica (HSS) particles
 Only UPLC certified
100% silica particles
having 1.8 𝜇
 These are tested for
15000 psi pressure
 Include tribonded C18
ligand
 Long column life and
UPLC efficiencies
UPLC-An overview 13

14. Column chemistries:
UPLC-An overview 14

15. Stationary phases:
UPLC-An overview 15

16. Charged surface Hybrid(CHB) technology
These particles
incorporate a low level
surface hybrid charge
improves sample
loadability and peak
asymmetry in low ionic
strength mobile phases.
UPLC-An overview 16

17. Instrumentation :
UPLC-An overview 17

18. • Acquity UPLC system consists :
 Binary solvent manager
Two individual serial flow pumps to deliver a parallel
binary gradient. provides steady pulse free solvent flow
at analytical flow rate(1-2 ml/min).
 Sample manager including sample heater
Injects sample draws from vial in to chromatographic
flow stream. Can perform injection approx. at 15 sec.
Also controls column temperature.
 Optional sample manager
Stores micro meter or vial plates and to and from the
sample manages, automating their processing and
increasing through put.
UPLC-An overview 18

19. Injecting the sample:
UPLC-An overview 19

20. Pumps: pumps
Constant
flow pumps
Reciprocating
piston pumps
Dual piston
pumps
Constant
pressure
pumps
UPLC-An overview 20

21. UPLC columns:
UPLC-An overview 21

22. Detectors:
detectors
Optical
detector
Tunable UV
detector
Evaporating
light scattering
detector
Florescence
detector
UPLC-An overview 22

23. Tunable UV detector:
 These are used for UV/visible
range
 Consists light guided flow
cell like optical fiber
 contains volume of 500
nanolitre and pathlength of
10 mm, and high sensitivity
flow cell with volume 2.4
microliters and 25 mm
pathlength.
 Operates at wavelength
ranging 190-700 nm
 Empower and MassLynx
software is used for
operations
UPLC-An overview 23

24. Applications:
 Identification of metabolites in in vitro discovery
studies.
 Bioequivalence study
 Detection of impurities
 Dissolution testing in relatively high potent drug
sample
 Method development/validation
 Forced degradation studies
 Impurity profiling
UPLC-An overview 24

25. References :
 Waters AAPS 2007 seminars, November 12-13,2007
 UPLC: a preeminent technique in pharmaceutical
analysis by Ashok Kumar et al, Acta Poloniae
Pharmaceutica-Drug research, vol.69 No.3pp.371-
380,2012
 Ultra Performance Liquid Chromatography: An
Indroduction And Review by T. Sunil Kumar Reddy et
al,IJPRA,vol 2,issue 1, 2012,24-31
 Ultra Performance Liquid Chromatography : A Review
by Patil V.P. et al.IRJP2(6) 2011 39-44
 Ultra Performance Liquid Chromatography: A
chromatography technique by Uttam Singh Baghel et
al.IJPQA.2010;2(1):19-25
UPLC-An overview 25

26. Thank you
UPLC-An overview 26