Nano LC Miniature LC Liquid chromatography Capillary LC Chromatography Advanced chromatography Instrumentation

1. NANO LIQUID CHROMATOGRAPHY
BY
Dr.A.S.CHARAN

2. Nano-liquid chromatography (nano-LC)
• Nano-liquid chromatography (nano-LC), first introduced by Karlsson
and Novotny in 1988.
• It is a miniaturized liquid chromatographic technique where the
separation of analytes takes place on a capillary column containing
selected stationary phases (SPs).
• Assigned to those techniques employing capillaries of internal
diameter (I.D.) ≤ 100 µm and flow rates in the range 25-800 nL/min.
• In contrast, capillary liquid chromatography (CLC) makes use of 150-
500 mm I.D. columns and flow rates in the range 1-20 µL/min.

3. Advantages of Nano-LC over HPLC
• In nano-LC, the relatively low flow rate represents an interesting
advantage over conventional techniques, allowing the perfect coupling
with mass spectrometry (MS).
• In addition, costs are reduced because of the limited use of mobile
phases, and consequently, low waste makes this technique ecofriendly.
• In addition, peak dilution during the chromatographic run is reduced
with an increase of mass sensitivity.
• Finally high efficiency, good resolution, and short analysis times are
currently obtained.
• Used for the analysis of proteomics, pharmaceutical, chiral, food and
beverages, environmental, forensic and toxicology, etc

4. Principles & theory of Nano-LC
• A miniaturized chromatographic system is based on the same
principles applied to conventional chromatography, e.g., van Deemter
equation, injection volume, backpressure, partition or other
mechanisms, analyte-SP interactions; on the same experimental
procedures, i.e., isocratic/gradient elution mode, choice of MP,
temperature, and so on.
• However, there are important differences concerning the
instrumentation employed, such as pumps, injectors, columns, and
detectors.

5. IMPROVING SENSITIVITY & REDUCING THE
CHROMATOGRAPHIC DILUTION
• The use of lower MP volumes is one of the advantages in reducing
the column i.d.
• This is advantageous not only in reducing costs but also in
improving the chromatographic performances.
• In chromatography, after injecting the sample into the column as a
small plug, the MP moves analytes toward the detector producing
a band enlargement that depends on several experimental
parameters.
• Samples are diluted during the chromatographic separation
process.

6. • The following equation calculates the sample dilution D:
where C0 and Cmax are the concentrations of the sample when injected and at
the peak maximum, respectively, r the column radius, L the column length, H
the plate height, ϵ the column porosity, and Vinj is the injected volume.
• From this equation, it is clear that the most important parameter is the column
radius.
• The reduction of the radius produces a consistent chromatographic dilution.

7. EFFICIENCY
AND EXTRA COLUMN BAND BROADENING
• The efficiency of a capillary column can be described considering the
van Deemter equation where the height equivalent to a theoretical
plate (H) vs the linear velocity is plotted.
• When making comparison studies regarding packed columns in
different MP conditions and particle diameter, the reduced equation
proposed by Kennedy and Knox is often used.

8. where h is the reduced height equivalent to a theoretical plate, ν the reduced linear
velocity, dp the particle diameter, and Dm the diffusion coefficient of the sample in the
MP.
• In order to achieve the desired good efficiency, it is necessary to evaluate all
parameters influencing the extra column band broadening.
• To solve this problem, attention must be paid in selecting carefully the following
parts of the system: (i) detector and detector cell, (ii) valve injection, and (iii) tubes
connections.

9. INSTRUMENTATION

10. INSTRUMENTATION IN NANO-LC
• MICROFLUIDIC PUMP SYSTEMS
• NANO-VOLUMES' INJECTION
• COLUMNS
• DETECTORS

11. MICROFLUIDIC PUMP SYSTEMS
• Delivering MPs at μL and nL/min is a challenging issue in microfluidic
techniques.
• Pumps must be capable of delivering MPs in both isocratic and
gradient modes in a precise and reproducible way.
• In addition, they have to be compatible with a large number of
solvents (organic, acidic, basic) and operate at high pressures (400
and 1000 bar, for conventional HPLC and UHPLC, respectively).
• Two primary systems can be used in nano-LC: split and splitless
pumps, the latter being commercially available.

12. Split systems
• Split systems divide high flows (mL/min)
from conventional HPLC pumps by using a
flow restrictor between the pump and the
miniaturized column.
• These systems allow for the use of the
usual HPLC pumps with an easily
constructed nano flow restrictor.
• However, split systems may lead to variable
split ratios and low reproducibility of the
nano flow, decreasing the repeatability of
the separation.
• Reproducible gradient elution is very
difficult to achieve, especially with
homemade splitting devices: the different
viscosities of the mixed solvents can cause
back-pressure fluctuations, limiting this
elution mode
• These systems prevent solvent losses and
have more reproducible nano flow rates.
• Syringe pumps using a single reservoir with
a limited volume are better than split
systems.
• But continuous flow pumps, similar to
conventional reciprocating pumps with two
pistons per channel, are currently the most
widely used pump model.
• Continuous flow pumps can be used in both
isocratic and gradient elution at nano flows
and adjustments of the desired nano flow
rates are easily achieved.
Splitless systems

13. Active flow splitting systems; (a) and (b) represent arrangements with fixed splitter (c) shows a configuration with
an adjustable splitter where the electronic controller systematically drives the electro magnetic proportional valve
(EMPV) according to the flow detected by the calibrated flow meter.

14. Splitless nanoflow gradient pump arrangement; (a) solvent refill
system; (b) continuous flow system

15. The continuous flow nano LC×µLC for both modulation valve positions. The nanoflow from
the D1 nanocolumn fills one loop while the other loop is arranged to the microflow path and
fast separation on the D2 microcolumn is performed.

16. Injection Systems
• The volume of the injected sample is another important parameter
that needs great attention in chromatography.
• since a non-appropriate injection volume can cause serious problems,
deteriorating the analytes' separation due to extra column band
broadening.
• Therefore, in any chromatographic system the sample must be
introduced as a zone as narrow as possible.
• The maximum sample volume that can be injected into a column
depends on several parameters such as the length of the column (L),
the particle diameter (dp), the column diameter (dc), and the
retention factor (k).

17. • Therefore, dedicated valves, commercially available, with internal
loop with volumes of 40–60 nL are used.
• Alternatively, loops of higher volumes are employed; however, in this
case only part of the sample solution is injected controlling the time.
• Modern nano-LC instrumentation makes use of automatic sample
injection systems where the volumes, usually μL, are reduced to nL
utilizing a split.

18. Nano LC sample injection approaches; Direct injection with an internal (a), and with an
external loop (b); On-line pre-concentration on vented column (c), and by column
switching

19. Nano columns

21. • Although columns of 10 mm i.d. can be employed, nano-LC columns
of 75 mm i.d. are the most frequently used.
• This i.d. column provides a good compromise between detectability,
loadability and robustness in nano-LC separations.
• In general, nano-LC columns are made of polyimide-coated fused
silica capillaries that present flexibility, high mechanical resistance
and a variety of internal dimensions, but stainless steel and titanium
tubes are also used for nano columns.
• They can be packed with silica-based particles, filled by a monolithic
bed or, less commonly, wall-coated with appropriate organic or
inorganic materials.

22. Types of Columns
• Packed Capillary Columns
• These SPs allow the separation of analytes by partition, size exclusion,
adsorption, affinity, and/or ion-exchange mechanisms.
• Monolithic Capillary Columns
• Monolithic stationary phases are single rods of organic or inorganic material
that are produced inside the capillary column.
• Open Tubular Capillary Columns
• Open tubular (OT) columns, where the SP is directly bonded or adsorbed to
the inner wall of the capillary, usually have an I.D. between 10 and 60 µm.

24. DETECTORS
• The types of detection for nano-LC are the same as those employed for
HPLC separations.
• They include UV-visible, fluorescence, conductivity, and more frequently,
MS.
• Diode array detection (DAD) is commonly used in nano-LC, because of its
low cost, wide range of applicability and use of online detection.
• However due to the short path length of the nano column, detectability is
limited when on-column detection is applied. This is overcome by the use
of specially configured detection cells with low sample volume that
provide longer light paths.
• Laser induced fluorescence (20) and inductively coupled plasma MS (21)
are also used in nano-LC detection.

26. Hyphenation of Nano LC
• Nano-LC-NMR

27. • Nano-LC-Mass Spectrometry

28. APPLICATIONS OF NANO LC

38. THANK YOU