Introduction, Classification of HPLC Systems, Advantages over HPLC, Instrumentation, Applications, Ultra Performance Liquid Chromatography, Advantages, Disadvantages, Instrumentation of UPLC, Comparision between UPLC, HPLC & NanoLC, Applications of UPLC, References.

1. NANO LIQUID
CHROMATOGRAPHY & ULTRA
PERFORMANCE LIQUID
CHROMATOGRAPHY
Srinivasa Rao college of Pharmacy, P.M.palem, Visakhapatnam
2016-2018
By
M.Trilochana M.Pharm
Pharmaceutical analysis and quality assurance

2. Contents:
 Introduction
 Classification of HPLC systems
 Advantages over HPLC
 Instrumentation
 Applications

3.  Nano High performance liquid chromatography
(Nano HPLC) is an important qualitative and
quantitative technique, generally used for the
estimation of pharmaceutical and biological
samples.
 After a little modification in HPLC, in 1988
Karlsson and Novotny were introduced the Nano
Liquid chromatography (NanoLC) technique.
 NanoLC systems has been driven by biological
applications and primarily proteomics research.

4.  Classification of HPLC systems :
Description Internal diameter of
Column
Flow Rate
Nano LC 10-100 μm 24-4000 nL/min
Capillary HPLC 150-500 μm 0.4-200 μL/min
Micro HPLC 1.0-2.1 mm 50-1000 μL/min
Normal HPLC 4.0-5.0 mm 1.0 -10.0 mL/min
Preparative HPLC >10 mm > 20 mL/min

5. ADVANTAGES OVER HPLC :
 Significantly reduces the mobile phase consumption
and subsequent waste production.
 Internal diameter reduction increases sensitivity
and/or less sample requirement.
 Significantly cheaper, quicker than its conventional
counterpart.
 Increased detection sensitivity in MS because of
lower flow rates in smaller columns.
 High separation efficiency and possibility to analyse
very small amount of solute.
 Significantly increased the resolution power for
complex sample analysis.

6.  Nano HPLC is of two types :
1. On-chip microfluidic columns
2. LC that has the flow rate on the scale of
nano liters per minute(nl/min)
Lab-on-chip
The Micro fabricated
chips includes a column,
frits/filters, an injector,
and a detector.

7.  A relatively recent development in nanoLC is
the introduction of chip-based structures.
 Chip based systems aim at integrating the
connections , columns, and spray needle in
one device to make installation and operation
of nanoLC system easier.
 This is also called as “Lab-on-a-chip.”
 LOC devices make use of microfluidic
techniques, which use small amounts of fluids
(10-9 to 10-18 litres), and channels with
dimensions of tens to hundreds of micrometres.

8. INSTRUMENTATION :
 Pumps : Split pumps and Splitless pumps.
 Columns: Internal diameter of nanoLC is
75μm.
 Injectors: Direct injection setups can be used
in nano-LC systems.
 Detectors: Diode array detector (DAD)

9. Applications :
 The study of proteins and nucleic acids in
proteomics and genomics.
 Separation of sulfonamides : Nano-liquid
chromatography coupled with mass
spectrometry was used for the simultaneous
determination of 18 sulfonamides.

10.  Water analysis : Quantitation of
perfluorooctanoic acid (PFOA) and
perfluorooctane sulphonate (PFOS) in surface
water by using a combination of on-line solid-
phase extraction, nano-liquid chromatography,
and nano spray mass spectrometry.
 Nano-LC for glyco bioanalysis : Structural
heterogeneity of glyco conjugates and glycans
in biological matrices.
 Determination of abused drugs and
metabolites in human hair

11.  Other applications :
 In the analysis of Biological and
environmental samples: When small amounts
of samples are available such as blood of
infants, cerebrospinal fluid , hormones,
enzymes and xenobiotics at nano levels.
 High throughput screening (HTS) and drug
discovery where the limits of detection are
very low.
 Proteomic and genomic research.
 Detection of accumulated drug samples in the
body.

12. Ultra performance liquid
chromatography
 Introduction
 Advantages
 Disadvantages
 Instrumentation
 Comparison between UPLC, HPLC &
NanoLC
 Applications

13.  Ultra Performance Liquid Chromatography
(UPLC) is a relatively new technique giving
new possibilities in liquid chromatography,
especially concerning decrease of time and
solvent consumption.
 The separation on UPLC is performed under
very high pressures (up to 100 MPa) .
 It improves in three areas: chromatographic
resolution, speed and sensitivity analysis.

14.  The UPLC is based on the principle of use of
stationary phase consisting of particles less
than 2 μm .

15. Advantages :
 Decreases run time and increases sensitivity
 Provides the selectivity, sensitivity, and dynamic
range of LC analysis
 Maintaining resolution performance.
 Expands scope of Multi residue Methods
 UPLC’s fast resolving power quickly quantifies
related and unrelated compounds
 Faster analysis through the use of a novel
separation material of very fine particle size

16. Cont..
 Operation cost is reduced
 Less solvent consumption
 Reduces process cycle times, so that more
product can be produced with existing
resources
 Increases sample throughput and enables
manufacturers to produce more material.
 Delivers real-time analysis in step with
manufacturing processes
 Assures end-product quality, including final
release testing

17. Disadvantages :
 Due to increased pressure requires more
maintenance and reduces the life of the
columns of this type.
 So far performance similar or even higher has
been demonstrated by using stationary phases
of size around 2 μm without the adverse effects
of high pressure.
 In addition, the phases of less than 2 μm are
generally non-regenerable.

18. Instrumentation :
 The Acquity UPLC system consists of :
 Binary solvent manager
 Sample manager including the column heater
 Optional Sample manager
 Pumps
 Detector

19.  Comparison between UPLC, HPLC, and
NanoLC
Characteristics HPLC UPLC Nano LC
Particle size 3 to 10 μ Less than 2 μ 1.7 – 3 μ
Analytical column XTerraC18,
Alltima C18
Acquity UPLCbeh
C18,C8,rp
Capillary HPLC,
Micro HPLC
Column
dimensions
(length x I.D)
150 X 3.2 mm 150 X 2.1 mm 125 mm X
0.05mm - 4.6mm
Column
temperature
300 C 650 C 25-350 C
Injection volume 5μL 2μL 10 nL-125 μL
Flow rate 0.01-5mL/min 0.6 mL/min 20-200 nL/min

20.  Applications of UPLC :
 Drug Discovery : UPLC improves the drug
discovery process by means of high
throughput screening, combinational
chemistry.
 Analysis of Dosage form : It provides high
speed, accuracy and reproducible results for
isocratic and gradient analysis of drugs and
their related substance. Thus method
development time decrease.

21.  Analysis of amino acids : UPLC used from
accurate, reliable and reproducible analysis of
amino acids.
 Determination of Pesticides : UPLC couples
with triple Quadra-pole tandem mass
spectroscopy will help in identification of trace
level of pesticides from water.
 High throughput quantitative analysis :
UPLC coupled with time of flight mass
spectroscopy give the metabolic stability assay.

22.  Bioanalysis / Bioequivalence Studies : UPLC
delivers excellent chromatographic resolution
and sensitivity.
 Dissolution Testing : UPLC provides precise
and reliable automated online sample
acquisition.

23. References:
 http://www.justchromatography.com/hplc/nano-
liquid-chromatography
 http://www.chromatographyonline.com/nano-
lc-principles-evolution-and-state-art-
technique?id=&sk=&date=&%0A%09%09%0
9&pageID=4
 https://www.researchgate.net/publication/27473
4689
 https://www.news-medical.net/life-
sciences/What-is-Lab-on-a-Chip.aspx