It is type of hplc ,its using sepration and identification method.

1. 1 NANO LIQUID CHROMATOGRAPHY
PRESENTED BY:
Mr. BORATE TUSHAR S.
Guided By:
Prof. Kashid Arun M.
Pharmaceutical chemistry
Presented By:
Mr. Borate Tushar S.
Mr. Wagh Viplav.p.
M. Pharm Sem III
Department Of Quality Assurance Techniques
STES’s
Sinhgad Institute of Pharmacy
Narhe, Pune 41

2. CONTENTS
 INTRODUCTION
 PRINCIPLE
 INSTRUMENTATION
 APPLICATIONS
 CONCLUSION
 REFERENCE
2

3. TYPES OF LIQUID CHROMATOGRAPHY
TECHNIQUES
 Rapid Resolution Liquid chromatography (RRLC)
 Ultra Performance Liquid chromatography (UPLC)
 Ultra Fast Liquid chromatography (UFLC)
 Nano Liquid chromatography (NANO LC)
3

4. RAPID RESOLUTION LIQUID CHROMATOGRAPHY
(RRLC)4

5. COLUMN5
Fig-Thermostatted Column Compartment Sl

6. ADVANTAGES OVER HPLC
 Increase in high-throughput.
 Reduction in the analysis cost.
 Short in analysis time due to the use of a shorter column size and
high resolution separation capability
 Highest analysis speed
 High sensitivity.
6

7. Ultra Performance Liquid Chromatography
(UPLC)
 UPLC refers to ultra performance liquid chromatography, which enhance
mainly in three areas
 1. High resolution peaks.
 2. Fast analysis
 3. High sensitivity
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Demo video..recent trade seminarShimadzu Nexera - UHPLC 3.0 - full version.mp4

8. ADVANTAGES OF UPLC
 Require less run time and enhance sensitivity.
 In chromatogram resolved peaks are obtained.
 Speedy analysis, quantify accurately analytes and related products.
 Time and cost both are reduced.
 Delivers real-time analysis in step with manufacturing processes.
 Assures end-product quality, including final release testing.
8

9. Ultra Fast Liquid chromatography (UFLC)9
Fig4- Ultra Fast Liquid chromatography (UFLC)
20AD PUMP
SPD M20A PDA
C18G
20 μL
LC SOL SOFTWARE

10. Nano Liquid Chromatography
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 Nano-liquid chromatography (nano-LC) is a recent developed micro fluidic
technique, mainly used for analytical purposes, offering some advantages
over conventional high-performance liquid chromatography (HPLC).
 Because of its features, this miniaturized technique has gained more and
more interest in various application fields resulting either alternative or
complementary to HPLC.
 Analysts separation takes place into capillary columns containing selected
stationary phases (SPs) under the effect of a mobile phase (MP) delivered at
low flow rates (10–700 nL/min)

11. 11
Fig-5 Scheme of a laboratory assembled nano-LC instrumentation.

12. HISTORY
 After a little modification in HPLC, in 1988 Karlsson and Novotny
were introduced the Nano Liquid chromatography (NanoLC)
technique.
 The huge increase in miniaturized LC systems has been driven by
biological applications and primarily proteomics research.
 Nano HPLC is also referred to as “Nano-bore HPLC” or “Nano-
scale HPLC”.12

13. ADVANTAGES
Significantly reduces solvent .
 Inner diameter reduction increases sensitivity or less sample
requirement.
 Does not increase system pressure.
Allows for simultaneous mass separation if LC chips are used.
13

14.  Very expensive,
limiting its wide spread
use.
Technical knowledge
about nano-LC is
required.
LIMITATIONS
14

15. COMPARISON BETWEEN HPLC & NANO LC
CHARACTERISTICS HPLC NANO LC
Particle size 3 to 10 μm 1.7 – 3 μm
Analytical column XTerraC18,
Alltima C18
Capillary HPLC,
Micro HPLC
Column dimensions
(length x I.D)
150 X 3.2 mm 125 mm X
0.05mm - 4.6mm
Column temperature 300C 25-350 C
Injection volume 5μL 10 nL-125 μL
Flow rate 0.01-5mL/min 20-200 nL/min15

16. PRINCIPLE
𝑯 = 𝑨 +
𝑩
µ
+ 𝑪µ
Where as,
A=Eddy’s diffusion
B=longitudinal diffusion
C=Concentration
µ=Linear Velocity
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The van Deemter equation,
Relationship between linear velocity (flow rate) and plate height.

17. PUMPS INJECTION
NANO
COLUMNS
DETECTION
SPLITLESSSPLIT
CAPILLARY
PACKED MONOLITHIC
17

18. 1)PUMP
 Reproducible nano flow rates and stability during the separation
Requires flow rates of 500 nL/min or less.
Two primary systems can be used in nano-LC:
1) Split System
2) Split less System
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19. Split systems can be divided into two groups:
a)The passive split system
 splitter divides the high flow of the pump between the
column and restrictor
 simple and relatively inexpensive, flow stability and
accuracy increase.
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20. b) The active split system
Improved flow stability and exhibit better
reproducibility than passive split systems
 Still the majority of the mobile phase is wasted.
The split less systems can be divided into two groups
a)solvent refill
b)continuous flow
20

21. SPLITLESS NANO LC PUMP
 Split less systems are
widely used in nano-LC.
 These systems prevent
solvent losses.
21

22. 2) INJECTION
22

23. INJECTION
 Direct injection setups can be used in nano LC setups.
 Sample is injected directly onto the column.
1)Nano litter injection
If low retained analytes dissolved in the mobile phase are injected
in to the column only few nano liters.
23

24. 2) On-line sample preconcentration
 Injection of samples in the nanoliter range is intended for analytes
their easy detection.
 Thus, it is not suitable for highly diluted samples..
 An accurate determination of proteomics, metabolomics or food
analysis.
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25. 3) COLUMNS
The commonly accepted standard internal diameter of nano LC
columns is 75 μm.
Column format provides a good compromise between sensitivity,
loadability and robustness.
 Two types of columns are used in nano LC
1) Packed columns
2) Monolithic columns
25

26. 1) Packed columns
 Made of Polyimide-coated
fused silica capillaries.
Frits strong pressures are used
to retain the stationary phase.
Columns: 5 μm, 3 μm, and now
down to sub 2 μm.
26

27. 2) Monolithic columns
A porous structure is formed throughout
the column eliminating the need for frits.
Monolithic stationary phases are single
rods of organic or inorganic material .
high porosity of these materials allows
higher flow rates of mobile phase,
reducing the separation time.
commercially available in lengths up to
50 cm.
27

28. 4) DETECTOR
Techniques for nano-LC are the same as
those employed for HPLC separations.
Commonly used nano-LC is Diode
array detection (DAD)
Its low cost and use of online detection.
The nano flow from the column
(frequently, 100–500 nL/min)28

29. DIODE ARRAY DETECTION (DAD)
ADVANTAGES
 Tungsten lamp offers
extended visible
wavelength range
 Optical unit of the
DAD is temp
controlled for optimum
signal quality
 Slit witdh can be
changed automatically
29

30. APPLICATIONS
 In food analysis
For determination of amino acids or polyamines in drinks.
 Separation of sulfonamides
Coupled with mass spectrometry was used for the simultaneous
determination
Discovery of Glycomics towards biomarker
Highly sensitive and quantitative method of separating and profiling
glycans.30

31.  The Mieux System is an
innovative system developed
for nano-LC for MS.
 This system is best suited for
proteome, metabolome, and
drug metabolism analyses
 high-separation and high-
sensitivity analysis.
 Pumping system can achieve a
gradient of 50 nL/min.
The Mieux System
31

32. FEATURES
 Pressure monitor enables full-time monitoring of pumping state.
 Various programs (auto flash, auto refill, auto drain) enable the System’s
high-speed equilibrium and full automatic pumping.
 Unique nano-spray stage and nano-spray column holder available
(optional).
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33.  Provide fast and reproducible
fluidic connections over the
complete flow path.
 Advanced pressue test quickly
diagnoses blocked capillaries
 Advanced Flow Control
 Software- Thermo Scientific™
Xcalibur™
THERMO SCIENTIFIC EASYnLC
1200 SYSTEM
33
Demo video..recent trade seminarThermo Fisher EASY-nLC-1000 Liquid
Chromatography System Predecessor to nLC-1200 - 9746.mp4

34. FEATURES
 Enables use of longer analytical columns for
higher peak capacity.
 Provides faster sample loading.
 Detailed event log for troubleshooting.
 Advanced pressure test quickly diagnoses
blocked capillaries
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35.  Optimize low flow separations.
 Flow delivery from 20 nL/min
up to50 μL/min at a maximum
pressure of 800 bar
 Nano, capillary, and micro LC
Zero sample-loss injection
 Software-DCMS Link™
software
ULTIMATE 3000 NANO LC SYSTEMS
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36.  The principal limitation at the present to wider use of nano-LC is
the high cost of the analytical instrumentation. However, the rapid
development of new equipment is overcoming this limitation,
expanding nano-LC to routine laboratories and industries.
 High-resolution columns
 Sample fractionation and automated Re-injection.
 Can perform multidimensional separations.
CONCLUSION
36

43. REFERENCES
 Hisashi Shimizu, Adelina Smirnova et. al; Extended-nano chromatography,
Journal of Chromatography 2016, 4(2), 1-10.
 Narayudu Yandamuri, Sathis Kumar Dinakaran et. al; Advanced study of
nano liquid chromatography and its application-a review, World Journal
of Pharmaceutical Research 2015, 4(4), 1355-1367.
 Haripriya, Sharadha Srikanthet et. al; a Review on rapid resolution liquid
chromatography of agilent1200 rrlc series, world journal of pharmacy
and pharmaceutical sciences 2015, 4(7), 1581-1595.
 Mariana Roberto Gama, Carol H et. al; Nano-liquid chromatography in
pharmaceutical and biomedical research, Journal of Chromatographic
Science 2013, 3(4), 694–703.
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44.  Salvatore Fanali; Nano-liquid chromatography applied to enantiomers
separation, Journal of Chromatography, 2016,4(2), 1-15.
 Nikalje Pratima, Baheti Shraddha, et. al; Review of ultra performance
liquid chromatography and its applications, International Journal of
Research in Pharmacy and Science, 2013 (1), 19-40.
 http://www.jcscientific.com/english/Product/T15/36.html/
(Accessed on: 15/11/17).
 Yamato Scientific Nano LC System Mieux, http://www.yamato-
scientific.com/product/other/mieux.html (Accessed on: 16/11/17).
 http://www.shimadzu.com/an/hplc/prominence/nano2.html
(Accessed on : 17/11/17).
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