: 1Principles of Instrumental Analysis D'A'Skoog 2 Instrumental Analysis C'D'Chiristian and J'G'Oei

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Instrumental Analysis D.A.Skoog2-
Instrumental Analysis C.D.Chiristian and
J.G.Oeilly 3- Physical and chemical methods of
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Introduction

• Plant Pigments
• Chlorophylls
• Xanthophylls
• Martin synge(1952 Nobel Prize)

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A general description chromatography

• Stationary phase
• Mobile phase

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Classification of chromatography

• Column chromatography
• Planer chromatography

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Linear chromatography

• Partition coefficient
• K Cs/Cm
• Cs , is molar analytical
• Cm , solute concentration in the mobile phase

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Elution chromatography on columns

• Elution is defined as a process whereby a solute
  is washed through a column by additions of fresh
  solvent.
• Retention time (tR)
• Qualitative Quantitative Chromatography

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Chromatograms

• If a detector that responds to solute
  concentration is placed at the end of the column
  its signal is plotted as function of time (or
  of volume of the added mobile phase), a series of
  peaks is obtained, such a plot , called a
  chromatogram.

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Theories of elution chromatography

• The effects of migration rates zone broadening
  on resolution
• Plate theory
• Kinetic theory
• LNH
• NL/H

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Retention time

• VL/tR
• UL/tM
• Where , V , u tM are average Linear rate of
  solute migration , average Linear rate of
  movement of the molecules of the mobile phase
  dead time
  
  
  

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The definition of plate height

• H?2/L
• NL2/?2
• Where H, is the Plate height , L , is the column
  length ?2 is the variance of measurements

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Experimental Evaluation of N H
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Definition of N

• W4C
• ? ? / (L/to)
• ? LW/4tR
• H LW2/16(tR)2
• N 16 (tR/W)2
• N 5.54 (tR/W1/2)

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Band broadening

• The parameters that effect to the band broadening
  are as follow
• 1. Eddy diffusion
• 2. Longitudinal diffusion
• 3. Mass transfer
• H A ( B/U) CU

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Eddy diffusion

• Zone broadening in the mobile phase arises in
  part from the multitude of path ways by which a
  molecule ( or ion) can find its ways through a
  packed column.

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Longitudinal diffusion (B/U)

• Longitudinal diffusions results from the
  tendency of molecule to migrate from the
  concentrated center of a band toward more dilute
  regions on either side.

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Mass transfer

• Mass transfer to and from the stationary phase
  (Cusp)
• Effect of column
• Mass transfer in the mobile phase ( CMU)
• Effect of fluent

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Relationship between retention time and partition
coefficient

• A solute migrates only when it is in the mobile
  phase
• V U ( fraction of time the solute spends in the
  mobile phase)
• V U x no. moles solute in mobile
• total no. moles solute

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V U

• V U( CMVM)/(CMVM CSVS) U(1/(1
  (CSVS/CMVM))
• V U ( 1/(1 (Kvs/VM)))

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The capacity factor , K

• K, is related to the migration rate of solute
• K KVS/VM
• V U ( 1/(1K))
• L/tR L/tm (1/(1K))
• K (tR-tM)/tM tR/tM

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The selectivity factor, a

• The selectivity factor is the ability of column
  to resolve two or more solutes in a column
• a KB/KA
• Where KB is the partition coefficient for the
  more strongly retained solute, B
• Or a KB/KA
• Or a ((tR)B tM)/((tR)A tm)

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Column Resolution

• RS Z/(WA/2) (WB/2) 2?Z/(wA wB) 2(tR)B
  (tR)A /(wA wB)
• Resolution more than 1.0 is good.

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Relationship between resolution and column
properties

• If wA ? wB ? w then
• Rs ((tR)B (tR)A )/w
• Rs ((tR)B (tR)A )/(tR)B x (N)1/2/4
• Rs (KB KA )/(1 KB) x (N)1/2/4
• Rs (N)1/2/4 ((a -1 )/a ) (KB/(1 KB) )

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Relationship between , N, Rs, K and a

• N 16 R2s(a /(a -1))2 ((1 KB)/KB)
• Rs (N)1/2/4(a -1)(KB/(1KB))
• Or N 16 R2s(1/(a -1))2 x ((1KB)/KB)

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Relationship between , Rs and elution time

• VB L/(tR)B
• (tR)B (NH(1KB))/U
• (tR)B (16R2sH)/U x (a /(a -1)2 x ((1KB)3) /
  (KB)2

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Optimization of column performance

• Variation in a
• Variation in U
• Variation in K
• Variation in Rs
• Variation in N
• Variation in H

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Variation in K

• We had Rs and (tR)B so
• Rs (QKB)/(1KB)
• And
• (tR)B Q ((1KB)3)/(KB)2

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Optimization of the a

• 1) Changing the composition of the mobile phase
• 2) Changing the pH of the mobile phase
• 3) Changing the column temperature
• 4) changing the composition of the stationary
  phase
• 5) Using special chemical effects

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The different K in practice
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Quantitative Analysis

• Analysis based on
• 1) Peak height
• 2) Peak areas Normalization
• 3) Calibration standards
• 4) Peak Integration
• 5) the Internal standard method

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Qualitative analysis

• X- Retention time or Retention volume
• XX- Relative Retention time
• XXX- Retention Index or Kovats Retention Index

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Define each of the following terms

• A- stationary phase
• B- mobile phase
• C- eluent
• D- solid support
• E- retention factor

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• Calculate the resolution of two peaks, t1 131sec
  t2 137sec, if the average peak width is
  8.0sec. Would the resolution of these two peaks
  be considered good ?

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Gas Chromatography
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Instrumentation of GC

• Carrier gas supply
• Sample injection system
• Separation column
• Oven
• Detector
• Recorder

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Sample injection system

• Overload
• Dilute samples
• Tenax-GC

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Types of Columns in GC

• Packed columns
• Capillary columns

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Packed columns

• Column dimensions
• -Stainless steal , copper or aluminum
• -Length 2 to 3 meters or 15cm (Coils)
• -Inside diameters 2 to 4mm.

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Packed columns

• -types of supports
• -specific surface area at least 1m2/g
• -made from diatomaceous earth (chromosorb P, wand
  G)

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Packed columns

• Particle size supports
• 1) Usual size 60-80 mesh (250- 170µm)
• 80 to 100 mesh ( 170-149 µm)

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Packed columns

• Adsorption solid supports
• Problem present of polar sites
• Hydrolyzed silicate surface

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Some craters of liquid phase in GC

• 1- low volatility
• Thermal stability
• Chemical inertness
• Solvent characteristics
• These are the reasons why we use inert gas in GC
  as carrier gas.

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Open Tubular Capillary Columns

• 1- Wall Coated Open Tubular (WCOT)
• 2- Support-Coated Open Tubular (SCOT)
• 3- Fused Silica Open Tubular (FSOT)

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Characteristics of SCOT and WCOT
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Column coating

• 1- High specific area
• 2- chemical inert
• 3-diatoma
• 4-deatomized soil

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Rohrshneider and polarity of columns

• 1- squalene, polarity0
• 2- A selected compound polarity100
• 3- The others are between 0-100

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X- Some papers about polarity of compound in
column
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Oven and column thermostating

• A- simple ( Isothermal) thermostating
• B- programmed thempreature GC

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Detectors in GC

• Dozens of detectors (more than 100) have been
  investigated and used during the development of
  gas chromatography. Only four , however , have
  found widespread use
• 1- Thermal conductivity
• 2- Flame ionization
• X- Thermionic
• 4- Electron capture

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Characteristics of the ideal detector for GC

• 1- adequate sensitivity
• 2- good stability and reproducibility
• 3- A linear response
• 4- A temperature range
• 5- A short response time
• 6- high reliability
• 7- similarity in response towards all solutes
• 8- nondestructive of sample

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Thermal Conductivity detectors (TCD(
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Flame Ionization Detectors (FID)

• 1- for most organic compounds
• 2- H2 and air or O2 is needed
• 3-insensitive towards noncobustible gases
• H2O, CO2, SO2, and NOx

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Advantages and disadvantages of FID and TCD

• 1- FID is not sensitive to H2O
• 2-TCD is nondistructive
• 3- TCD is simple
• 4- sensitivity of TCD ( 10-8g/ml) is less than
  FID ( 10-13g/ml)
• 5-FID is not general detector
• 6- FID is not sensitive to rear gases and N2 and
  O2
• 7- the noises in FID is less than TCD

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ECD

• 1- effluent from the column
• 2- ? emitter ( 63Ni or 3T)
• 3- electron of emitter ionized the carrier gas
• 4- some compounds adsorb the electron
• 5- change in the normal current of the detector

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Applications of ECD

• 1- chlorinated insecticides
• 2- peroxides
• 3- halogens
• 4- quinones
• 5- nitrogroups
• I- AMINES
• II- ALCOHOLS
• III- HYDROCARBONS

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Reaserch Projects

• X- name some common stationary phases in GC
• XX- what are Mcreynold constants
• XXX- how can we choice stationary phase
• IV- what is GSC ( some papers)
• V- what is GLC ( some papers )

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GC/MS

• 1- Advantages
• 2- Disadvantages

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GC/FT-IR

• 1- separating
• 2- identifying

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? Explain why a liquid sample should be injected
rapidly onto a GC column
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? Why is a solid support soetimes silanized
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? How temperature programmed GC is able to give a
faster separation than an isothermal separation?
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X- Introduce a method for the determination of
ethanol in blood
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XX- Introduce a method for the determination of
tolune in a sample
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XXX- Introduce a method for the determination of
gasoline in oil
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High- Performance Liquid Chromatography (HPLC)
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Comparison of GC and LC

• 1- GC is better for the samples with high
  volatilities
• 2- In the GC the mobile phase is a gas , but in
  the LC is liquid
• 3- the permability of liquids is 105 times lower
  than gases
• 4-the viscosity of liquids is 102 times grater
  than gases

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Limiting number of theoretical plates

• (Nlim)LC/(Nlim)GC (?GDG)/(?LDL)103
• (? G)/(? L) 102
• (DG)/(DL) 105

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HPLC

• 1- Introduction
• 2- column chromatography
• 3- planer chromatography

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Column liquid chromatography

• 1- partion chromatography
• 2- adsorption or liquid solid chromatography
• 3- Ion exchange chromatography
• 4- Exclusion or gel chromatography

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Column efficiency in LC

• 1- effect of particle size of packings
• 2- extra column band broadening in LC
• 3- effect of sample size

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Istruments for HPLC

• 1- solvent reservoirs
• 2- pump
• 3- injection valve
• 4- column
• 5- detector
• 6- recorder

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Mobile phase and solvent reservoirs

• 1- Isocratic elution
• 2- gradient elution

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Pumping systems

• The requierments for pums
• 1- the generation of pressure of up to 6000psi
• 2- pulse free output
• 3- flow rates from 0.1 10ml/min
• 4- flow control and flow reproducibility of 0.5
  relative or better
• 5- corrosion resistant

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Some types of pumps

• 1- Reciprocating
• 2- Syringe or displacement
• 3- Pneumatic or constant pressure

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Reciprocating pumps

• 1- It is not pulse free
• 2- low sample internal volume ( 35-400ul)
• 3- gradient elution
• 4- high pressure ( up to 10000psi)

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Displasment pumps

• 1- sample internal volume ( 250ml)
• 2- pulse free
• 3- syringe like chamber
• 4- It dos not depend to viscosity of solvent
• 5- gradient elution

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Pneumatic pumps

• 1- pulse free
• 2- maximum pressure 2000psi ( disadvantage )
• 3- isocratic elution

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Sample injection systems

• 1- syringe injections
• 2- stop flow injection
• 3- sampling valves

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Analytical columns

• 1- cost 200 1000
• 2- inside diameter, 4 -10 mm
• 3- length, 25cm
• 4- N, 40000 60000 plates/meter
• 5- particle size of packing, 5 10um

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Guard column

• 1- removing particulate matter and contaminates
• 2- increasing the life of the analytical column
• 3- saturation the mobile phase with the
  stationary phase
• 4- large particle size ( than s. phase )

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X- Post column
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Detectors

• Some characteristics
• 1- sensitivity
• 2- linear response
• 3- free from flow rate sensitivity
• 4- nondistructive of the sample

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The types of detectors

• 1- general detectors
• MS
• Refractive index
• 2- specific detectors
• IR
• UV-Visible
• Polarography
• Florometry

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Electrochemical detectors

• 1- high sensitivity
• 2- simplicity
• 3- convenience
• 4- applicability

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X- HPLC - MS
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Mobile phases

• 1- high purity
• 2- ready availability
• 3- A boiling point that is 20 to 50Co above the
  column temperature
• 4- low viscosity
• 5- low reactivity
• 6- immiscibility with the s. phase
• 7- compatibility with the detector
• 8- limited flammability and toxicity

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Types of mobile phase

• Normal phase ( polar s. phase )
• Reversed phase ( polar M. phase )

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Partition Chromatography

• Some parameters that effect on selectivity factor
  and K
• 1- solvent solute interaction in mobile phase
• 2- dispersion interactions
• 3- dipole interactions
• 4- dielectic interaction
• 5- molecular complex formation

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Solvent strength and K

• P log (Kg)e log(kg)d log (kg)n
• PAB ?ApA ?BpB
• K2/K1 10(p1- p2 )/2
• K2/K1 10(p2 P1)/2

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Classification of solvents

• X- Snyder classification of solvents

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Packing for partition chromatography

• Siloxane packing
• Ester formation
• Silicon/carbon
• Silicon/nitrogen bond

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X- Ion pair chromatography
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LSC

• Silica - alumina s. phase
• E0 of the solvent

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Ion- Exchange chromatography
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Size exchange chromatography

• Vt vg vi v0
• Ve v0 kvi

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Application of size -E- chromatography

• Gel filtration C. ( polar solvent )
• Gel permeation C. ( non-polar solvent)

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Comparison, advantages, disadvantages

• 1- short time for separation
• 2- non-distructive
• 3- sharp peaks
• 4- high sensitivity

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Supercritical Fluid Chromatography ( SFC)
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Operating variables and instrumentation

• 1- effect of pressure
• 2- stationary phases ( packed and capillary)
• 3- mobile phase ( in most case is CO2)
• 4- detectors ( MS, UV, R.F., FID and)

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Planer Chromatography
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Planer chromatography

• 1- paper chromatography (PC)
• 2- Thin Layer Chromatography ( TLC)

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TLC

• 1- glass plate
• 2- s. phase
• 3- m. phase
• 4. origin line
• 5. solvent front

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Performance characteristics of TLC

• 1- the retardation factor
• 2- H and N
• 3- the capacity factor, K and R

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Chromatogram development

• 1- ascending flow
• 2-horizontal flow
• 3- one dimensional TLC
• 4- two dimensional TLC

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Qualitative and Quantitative analysis in TLC

• RF
• travel distance of analyte
• Rx
• travel distance of standard substancea
• X- quantitative ?

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Electrophoresis and Electrochromatography

• 1- electrophoresis is defined as the migration of
  particles through a solution under the influence
  of an electric filed.
• X- electrochromatography

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