The document discusses the advancements and applications of nano liquid chromatography (NLC) and ultra performance liquid chromatography (UPLC) in pharmaceutical and biological analysis. NLC is notable for its high sensitivity and efficiency, reducing mobile phase consumption, particularly aiding proteomics research, while UPLC enhances resolution and speed of analysis at high pressures. Both techniques offer significant advantages over traditional HPLC, with various applications ranging from drug discovery to environmental sample analysis.

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