09 11 2006 Chromatography
Short Description
物理生化學-09-11-2006 Chromatography-黃文鑫...
Description
WenHsin Huang
Principles of Chromatography Principles of Chromatography
•Introduction to Analytical Separations –Solvent Extraction –What is Chromatography? –A Plumber’ s View of Chromatography –Efficiency of Separation –Why Bands Spread
Introduction to Analytical Separations
Wen-Hsin Huang Ph. D. NDMC 2006/9/7
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Solvent Extraction
Simple Separations
Solute particles only have one choice of solvent
•Extraction –transfer of solute from one phase to another –phase can be gas, solid, liquid
Solute particles Solute particles now have two choose preferred choices of solvent solvent
•Liquid/liquid extraction –2 immiscible solvents used –typically aqueous solvent and organic solvent
add second immiscible solvent
•you know water and oil don’ t mix
–organic solvents less dense than water –organic solvents more dense than water •chloroform, carbon tetrachloride, dichloromethane 2006/9/7
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shake
Legend: aqueous solvent organic solvent solute particles
•diethyl ether, toluene, hexane
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Solvent Extraction
Phase Partitioning If we have a solute (S) that is partitioned between two phases (1 & 2) then we can write an equilibrium expression for this equilibrium.
•Like dissolves like –solute chooses solvent most like itself –polar compounds (and ionic compounds) choose water (which is very polar) –nonpolar compounds choose nonpolar organic solvents
S1 S 2
AS [S ] K 2 2 AS1 [ S1 ] Partition Coefficient (K)
• Convention: Organic phase = phase 2 2006/9/7
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Distribution Coefficient
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Example of Extraction with n-Hexane The distribution coefficient for compound Z between n-hexane and water is 6.25. Calculate the percent Z remaining in 25.0 mL of water that was originally 0.0600 M in Z after the extraction with the following volumes of n-hexane. A) One 25.0-mL portion 13.8%
The distribution coefficient (D) is used in place of the partition coefficient when dealing with a species that has more than one chemical form.
total _ concentration _ in _ phase _ 2 D total _ concentration _ in _ phase _ 1
B) Two 12.5-mL portions
1.90%
C) Five 5.00-mL portions
4.99 x 10-3%
D) Ten 2.50-mL portions
2.49 x 10-7%
It is more efficient to do several small extractions rather than one large extraction. 2006/9/7
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Solvent Extraction (pH effects)
Solvent Extraction (pH effects)
•Charges of acidic and basic species change with pH
•Distribution of HA between two solvents –two equilibria to consider •Ka equilibrium •D equilibrium
–neutral species generally more soluble in organic solvent –charged species generally more soluble in aqueous solution
•Distribution coefficient (D) describes distribution of species between two phases –Takes into account all forms of a compound (i.e. H2A, HA-, A2-) –Different from partition coefficient (K) 2006/9/7
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organic
HA
aqueous
HA
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Solvent Extraction (pH effects)
D=
[HA]1 + [A-]1
[HA ]2 K [HA ]1 •Substitute and rearrange to get K[H] D [H ] K a
[B]2 [B]1 + [BH+]1
•Ionic species only found in aqueous layer 2006/9/7
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[H][ A ] K [HA ] Ka [ A ] a [HA ] [H ] •Also have K expression for [HA]
total conc. in phase 1 (aqueous) D=
H+ + A-
•Distribution of HA between two phases
total conc. in phase 2 (organic)
[HA]2
Ka
Solvent Extraction (pH effects)
•Distribution of acids and bases between two phases D=
H+ + A-
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Eq’ n 12
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Solvent Extraction (pH effects) D for a base (B)
K[H] D [H ] K a
KK a D K a [H]
•Adjust pH to effectively extract an acid or base (and its conjugates) into aqueous layer •Consider base B with a pKa of 9.0
Eq’ ns
–pH < 9.0, B hydrolyzes so majority of B (in the form BH+) in water layer (low D value)
•Distribution of acids and bases between two phases is pH-dependent •To find D, need to know
0
[ B] K org [ B ]aq [ H ][ B]aq Ka [ BH ]aq
–pH of solution –Ka of acid or conjugate acid –K of acid or base 2006/9/7
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History of Chromatography
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pKa
-4
mainly BH+ 2
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pH
mainly B 8
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Fig.14
Tswett’ s Experiment
Mikhail Tswett is credited with the invention of chromatography.
The separated species appeared as colored bands on the column.
-2
-6
[ B ]org K Ka D KB [ B ]aq [ BH ]aq K a [ H ]
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Developed a technique that separated various plant pigments (e.g., chlorophylls and xanthophylls) by passing solutions through glass columns filled with finely ground CaCO3.
log D
D for an acid (HA)
Solvent Extraction (pH effects)
Ground up plant leaves and added petroleum ether.
Filled column with chalk. M. S. Tswett Russian Scientist (1872-1919)
Method forgotten for many years.
Hence Greek chroma = color and graphein = writing 2006/9/7
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What is Chromatography? Tswett’ s Experiment
•Method to separate components in a mixture based on different distribution coefficients between the two phases •Same principle as solvent extraction (like dissolves like), but one phase is “ stationary” and one phase is “ mobile” –no longer working with organic and aqueous layers in a separatory funnel –working in a column (which is just a tube)
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What is Chromatography? Types of Chromatography
•Two phases: mobile and stationary •Mobile phase is solvent moving through column
•Adsorption Chromatography •Partition Chromatography •Ion-exchange Chromatography •Molecular Exclusion Chromatography •Affinity Chromatography
–liquid (Methanol, water, buffer) –gas (He, H2, N2)
•Stationary phase fixed inside column –viscous liquid coated on inside of column –solid particles packed inside column
•Solutes have different affinities for mobile phase and stationary phase 2006/9/7
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Adsorption Chromatography
Partition Chromatography
Stationary phase Mobile phase
Stationary phase
Mobile phase
Solid
Liquid on solid support
Liquid or Gas
Liquid or Gas
How it separates
How it separates
–solute adsorbs on to stationary phase surface
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–solute dissolves into liquid coating
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Molecular Exclusion Chromatography
Ion-Exchange Chromatography Stationary phase Anions or cations covalently bound to solid stationary phase
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Mobile phase Liquid
Stationary phase
Mobile phase
Porous gel
Liquid
How it separates –small molecules trapped in pores of stationary phase
How it separates solute ions of opposite charge attracted to stationary phase
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Also called gel filtration, gel permeation, or size-exclusion chromatography 23
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Classification Liquid chromatography (LC) (Mobile phase a liquid)
Affinity Chromatography Stationary phase
Mobile phase
Immobilized molecules on Liquid liquid or solid stationary phase
How it separates –Molecules with specific shape dock with ligands Gas chromatography (GC) (Mobile phase a gas)
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Basic Principles
Specific Method Stationary Phase Liquid-liquid or Liquid adsorbed onto partition solid
Type of equilibrium Partition between immiscible liquids
Liquid-bonded phase
Organic species bonded to solid surface
Partition between liquid and bonded surface
Liquid-solid, or adsorption
Solid
Adsorption
Ion exchange
Ion-exchange resin
Ion exchange
Size exclusion or Interstices of polymeric molecularsolid exclusion
Sieving
Affinity Covalently bonded Chromatography molecule (eg antibody)
Se l e c t i veAf f i ni t y–( e gs pe c i f i c Protein/antibody bind together)
Gas-liquid
Liquid adsorbed onto solid surface
Partition between gas and liquid
Gas-bonded phase
Organic species bonded to solid surface
Partition between gas and bonded liquid
Gas-solid
Solid
Adsorption
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Simple Column
Two phases considered: Fresh eluent Initial band of A and B
1) Mobile Phase: solvent moving through the column. 2) Stationary Phase: stays in place inside of the column.
Column Packing
“ Eluent”
COLUMN
“Eluate”
Porous Disk
Process is called “ elution”
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Chromatogram
Chromatographic Terms
A chromatogram is a graph showing the detector response (proportional to concentration) as solutes emerge from a chromato-graphic column as a function of time or volume. Detector
tm = “dead time”which is the minimum time for mobile phase to pass through the column tr = “retention time” which is the time required for the solute (analyte) to pass through the column.
• All solutes spend equal time in mobile phase
t’ adjusted retention r= “ time”describes time solutes spend in stationary phase
t rt r t m
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•kcapacity factor describes how long solute retained on column
= “relative retention” which is the ratio of adjusted retention times.
t t k' r m tm
t r 2 t r 1 >1 always
k’= “capacity factor”
time _ in _ stationary t r k time _ in _ mobile tm
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=
time spent in stationary phase time spent in mobile phase
•Higher kindicates solute retained longer •If k= 0, solute unretained •If k= 1, solute spent same amount of time in stationary phase as in mobile phase
then separation between two components
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A Plumber’ s View…
Chromatographic Terms
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A Plumber’ s View…
Partition Coefficient Relationship
•kdescribes time spent in two phases •kalso describes number of moles of solute partitioned between two phases
CV k' s s Cm Vm
time _ in _ stationary moles _ in _ stationary tr k time _ in _ mobile moles _ in _ mobile tm
[S ] V V t k s s K s r [ S ]m Vm Vm t m
Eq’ n
•K Partition coefficient compares concentration of solute in one phase to concentration of solute in the other
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[ S] C V K 2 s k' K s [S]1 Cm Vm WenHsin Huang, NDMC
t k K r 2 2 2 t r k1 K1 1 What this says is that analytes with different partition coefficients will have different retention times.
Eq’ n 33
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Partition coefficient describes conc. of solute in SP and MP
•q fraction of solute in mobile phase molesm Cm Vm q moless molesm Cm Vm Cs Vs 1 1 q CV V 1 s s 1 K s Cm Vm Vm
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k' (1 q) 1 k '
fraction in MP
fraction in SP
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Review
A Plumber’ s View…
1 q 1 k'
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C K s Cm
Capacity factor describes time solute spends (or moles of solute) in SP and MP
V t t k' r m K s tm Vm
Relative retention compares retention times (or partition coefficients or capacity factors) of solute 1 and 2
t ' k' K r 2 2 2 t r1' k '1 K 1
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Chromatographic Peaks
Resolution of peaks
•Solutes elute from a column in a Gaussian peak shape –w –w1/2
Resolution is a measure of how well two peaks are separated.
width of peak at base width of peak at half-height
As tr grows, resolution between two peaks improves.
average retention time w1/2=2.35
h
1/2h
t ( t t ) resolution r r1 r 2 w av ( w 1 w 2 ) / 2
R=0.50
R=0.75
R=1.00
R=1.50
w=4
t0
tr
time
Peak width (w) is defined as the baseline width (4). 2006/9/7
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Plate Height (H)
Resolution Example
Bands broaden as they travel through the column.
Two solutes have retention times and widths of tr1 = 235 s, w1 = 8 sec; tr2 = 250 s, w2 = 10 s. Resolution = 1.7 Plate height (H) relates the amount of broadening to the linear distance traveled. H = plate height x = distance traveled 2 = variance of peak 2006/9/7
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2 H x
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Evaluating Separation Efficiency
Evaluating Separation Efficiency
•“ N”in chromatography is analogous to “ n” in liquid-liquid extractions
•Plate theory –Breaks separation up into many discrete stages –Stages represent individual equilibria
–each extraction represents a theoretical plate
[S]2
•N (Theoretical plate) represents each equilibrium between MP and SP
[S]1 •Each time a solute molecule enters the SP from the mobile phase is a theoretical plate 2006/9/7
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•Higher N corresponds to better separation –indicates solute molecules enter SP a higher number of times 41
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Evaluating Separation Efficiency
•Distribution of retention times for a solute •Peak shape represents differences in behavior of solute molecules during separation
–column –analyte –retention time of analyte –width (at base or at half-height) of peak
t 16t N r 2 2r w 5.55t N 2 r w 1/ 2 2006/9/7
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2
average retention time
2
N is dimensionless so measure tr and w (or w1/2) in same units (i.e. time) WenHsin Huang, NDMC
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Chromatographic Peaks
•N is dependent on
2
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w1/2=2.35
h
1/2h w=4
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t0
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tr
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time 44
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Evaluating Separation Efficiency
Evaluating Separation Efficiency
•Do not want solute band to spread out as it travels through column •H (Plate height) represents the relationship between the width of a solute band to the distance traveled through column •Lower H corresponds to better separation (higher efficiency) •H also called HETP
•H related to N and length of column (L) Report H in units L H of length (i.e. cm) N •Resolution related to N
R
–Height Equivalent to a Theoretical Plate –One solute equilibrium between SP and MP is one theoretical plate (N) –Equilibrium occurs in one HETP 2006/9/7
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k '2 N 1 4 1 k ' av •Increase L to increase R L L H N N H •Increase to increase R
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The number of theoretical plates (N) increases as the efficiency of the separation increases.
Eq’ n
16t 2 t 2 N 2r r 2 w
Derived
or
5.55t 2 t 2 N 2 r r2 w1
–to change , must change nature of MP and SP to change relative affinity analytes have for SP WenHsin Huang, NDMC
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Number of Theoretical Plates (N)
t ' k' K r 2 2 2 t r1' k'1 K 1
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Eq’ n
–R is dimensionless –k 2 = capacity factor for solute retained longer on column (higher tr)
Evaluating Separation Efficiency R
k '2 N 1 4 1 k'av
2
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Example
Factors Affecting Resolution The greater the resolution, the better the separation. The more theoretical plates, the better the separation.
A solute with a retention time of 302 s has a width of 11 s on a column that is 15 m long. Find both N and H for this separation.
R
R N 1 k 2 0
N = 1.2 x 10-4 H = 1.2 mm
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N 4
k 2 1 1 k ave
R L R0 R0 WenHsin Huang, NDMC
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Why Do Bands Broaden?
Principles of Chromatography
Solute invariably spreads apart as it travels (diffusions) through a chromatographic column.
•Introduction to Analytical Separations –Solvent Extraction –What is Chromatography? –A Plumber’ s View of Chromatography –Efficiency of Separation Why Do Bands Spread
The observed variance is a sum of variances from all the broadening mechanisms. 2 obs i2
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Where Does Broadening Occur?
Where Does Broadening Occur? Inside the column:
Outside the column: Width of injection plug Mixing in detector dead volume
Pump
Multiple paths Longitudinal diffusion Equilibration time
Pump
Injector I
I Column
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Injector
Detector
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Column
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Multiple Paths (A)
Detector
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Multiple Paths (A)
•Solute molecules can choose many different paths through packed columns
Band of 3 solute molecules traveling through packed column
Band spreads
packed column
open tubular column layer of SP coated on inside of column
SP particles packed inside column
time Occurs only in packed columns.
Compare to solute traveling through a straw 2006/9/7
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Compare to solute traveling through a bean bag WenHsin Huang, NDMC
Smaller particles reduce the plate height.
HA
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• Open tubular columns do not provide multiple paths for solute during tm (no A term)
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Longitudinal Diffusion (B /ux)
Equilibration Time (Cux)
•S molecules travel through column in a band •[S] varies throughout band
•Finite time required to allow solute molecules to equilibrate between MP and SP •If flow rate too high, MP will “ leave behind” molecules in SP (i.e., Solute in stationary remains
–some solute molecules get ahead of band –some solute molecules lag behind band
“stuck”while solute in mobile phase moves forward.)
•Keep SP thin to decrease equilibration time
Caused by diffusion of solute in the mobile phase. Faster flow rate means less time, thus less diffusion.
H B/ux so, increase ux (flow rate) to reduce problem
start as band travels 2006/9/7
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Direction of travel
MP SP bandwidth
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•Three terms in van Deemter equation correspond to three sources of band spreading or broadening
H A Cu x ux
•A term Equilibration Time
•B term •C term
• Break equation up into A, B, and C terms
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–A
H independent of flow rate
–B/ux
H inversely proportional to flow rate
–Cux
H proportional to flow rate WenHsin Huang, NDMC
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Band Spreading
Putting the three factors together yields the Van Deemter equation that helps predict how the column flow rate will affect the theoretical plate height. B
Longitudinal Diffusion
bandwidth
H Cux so decrease ux to reduce problem WenHsin Huang, NDMC
Van Deemter Equation
Multiple Paths
MP SP
slow eq.
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Multiple Paths term (also called Eddy Diffusion) Longitudinal Diffusion term Equilibration Time term
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van Deemter plot
Column Type Affects H
H
B ux
–high resolution (low H) –high sensitivity –low analysis time
A
Flow rate
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–not useful for large-scale analyses WenHsin Huang, NDMC
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•Overloaded column (high [S]) –so much S enters SP that the SP starts to look more like S than it looks like original SP –most S molecules retained longer on column Observed peak in chromatogram
Band shape
•In reality, K changes as [S]total changes •If [S] is too high or too low, peak shape deviates from Gaussian shape
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•Disadvantages
Asymmetric Band Shapes
•Expect Gaussian peak shape from solute S eluting from column, independent of [S]total •Separation based on K C K s Cm
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–useful for large-scale analyses –A term in van Deemter equation increases H
•Disadvantages
Asymmetric Band Shapes
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•Advantages
•Advantages
Hmin
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SP particles packed inside column
layer of SP coated on inside of column
Cux
uopt
packed column
open tubular column
•Plot dependence of H on individual terms •Sum and find optimum ux to use to minimize band spreading
Majority of S retained on column longer 63
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Faster-moving (less retained) S reaches detector first
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Asymmetric Band Shapes
Asymmetric Band Shapes
•Underloading (low [S])
•Load less sample to reduce overloading problem •Protect groups on SP to reduce number of hot sites to reduce tailing problem
–“ hot sites”on SP more available when less S molecules trying to enter SP –some (minority) S get “ stuck”on hot sites –some S retained longer on column Observed peak in chromatogram
Band shape
overloaded
Constant K (slope) results in ideal peak shape
Cs tailed Slower-moving (more retained) S reaches WenHsin Huang, NDMCdetector last
Minority of S retained on column longer 2006/9/7
Cm
Fig. 23-19 65
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Quantitative Analysis
Chromatographer’ s Triangle Relationship
•In general, detectors can tell us –“ Yes, something is eluting from the column.”
•Use calibration methods to determine how much of a compound is eluting from column Peak Area
Resolution
Speed
Capacity
-b x= m concentration (M)
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Finding Peak Area •A = ½ wbaseh •A w½ h •cut-and-weigh •computer integration
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Quantitative Analysis
Example
•Generally use an internal standard
When 1.06 mmol of 1-pentanol and 1.53 mmol of 1-hexanol were separated by GC, they gave relative peak areas of 922 and 1570 units, respectively. When 0.57 mmol of pentanol was added to an unknown containing hexanol, the relative chromatographic peak areas were 843:816 (pentanol:hexanol). How much hexanol did the unknown contain?
–internal standard is a known amount of a compound different from analyte –compare analytical signal from analyte to analytical signal from internal standard –not the same as method of standard additions –internal standards discussed in Ch. 4
Ax A F s [ X] [ S] 2006/9/7
Ax area of analyte peak As area of standard peak [X] concentration of analyte [S] concentration of standard F response factor WenHsin Huang, NDMC
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GUARD
•Analyte
•Constructed of steel or plastic •5 –30 cm length •1 –5 mm i.d. •Easily contaminated and degraded •Guard column
–Soluble in MP (more likely than being volatile)
•Mobile phase –Liquid •Methanol, Water, Acetonitrile, Hexane
–Plays more active role in separation
•Stationary phase
–Contains same SP –Dust, other particles, strongly adsorbed solutes retained on guard column –Expendable –Extends life of analytical column
–Liquid (partition) or solid (adsorption)
•High Performance Liquid Chromatography(HPLC) –High pressure used to force solvent through column (as opposed to gravity)
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LC Columns
Liquid Chromatography
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A N A L Y T I C A L 72
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LC Columns
LC Columns
•Packing particle diameter (dp) crucial
•Almost exclusively use packed columns •Solutes move much slower through liquids than through gases
–Typical particle diameters are 3 –10 m –Decrease particle diameter to decrease H
Fig. 21-14 S
S
H (m)
•Provide more uniform flow (low A) •Less time needed for solute to get to particle (low C) 10m 60
–Time needed to diffuse to SP in open tubular column is too long
40 20
5m
10
3m 0
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LC –Adsorption Chromatography
HO
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Si O
Si O Si
O Si O
Si
Solvent Solute
O
O
Si O
O Si
Fig. 21-10
Si
•Solute is displaced by solvent molecule
OH OH
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SP
OH
OH
Deprotonated silanols (Si –O-) are “ hot sites” that can lead to tailing 2006/9/7
MP Flow
OH
O
8
•Solvent molecules compete with solute molecules for sites on SP
–Pure, spherical, microporous particles
Silica gel with silanol groups on surface
6
Sponge-like Structure
Aggregate of Particles
•Permeable to solvent •Very high surface area •Use when pH < 8
4
LC –Adsorption Chromatography
Microporous Silica Particles
•Solid SP –Silica gel
2
Flow WenHsin rate (mL/min) Huang, NDMC
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OH
OH 75
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LC –Adsorption Chromatography
Isocratic vs. Gradient Elution •Isocratic elution
•Forcible desolvation of solute by solvent is nearly independent of solute identity
–One solvent or one constant solvent mixture used as MP throughout entire separation
–Dependent on solvent identity
•Gradient elution
o) •Eluent strength (
–Adjust solvent mixture through separation –Adjusting eluent strength –May speed up separation process
–Ability of solvent to displace solute
•Elutropic series –Relative eluent strengths of common solvents –Must know about relative polarity of SP/MP
o values of solvents for •Table 21-2 includes adsorption chromatography on silica o –polarity,
•Eluent strength increases as MP becomes more like SP 2006/9/7
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Isocratic vs. Gradient Elution Hexane °=0.01
Acetonitrile °=0.52
Start w/ 100% Benzene then add Acetonitrile
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Fig. 21-18 & Fig. 21-19
isocratic elutions
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Isocratic vs. Gradient Elution •Previous example illustrated balance of good resolution and reasonable retention times •Gradient elution may help –Improve resolution –Shorten retention times (and total analysis time) –Improve peak shapes
gradient elution 79
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LC –Partition Chromatography
LC Phases
•Liquid SP coated on solid support
•Normal-phase Chromatography
–Often, liquid SP covalently attached to surface of silica gel particle
–SP –MP
CH3 Si
O Si
o –More polar solvent has higher
R
•More attracted to SP and able to displace solute
•Reversed-phase Chromatography
CH3 support
–SP –MP
liquid SP
Common polar R groups
Common nonpolar R groups
(CH2)3NH2
(CH2)17CH3 (or C18)
(CH2)3C N (CH2)2OCH2CH(OH)CH2OH
(CH2)7CH3 (or C8) (CH2)3C6H5
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polar weakly polar or non polar
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nonpolar or weakly polar polar
o –Less polar solvent has higher
•More attracted to SP and able to displace solute
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LC Phases
WenHsin Huang, NDMC
HPLC Instrumental Design •Already covered columns •Focus now on
•Normal-phase developed first •Reversed-phase is more common
–Injector –Detectors
–Many choices of polar solvents in which solutes are commonly soluble •Water, methanol
Pump
–Nonpolar SP less likely to have hot sites
Injector I
•Less peak tailing
Column
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Detector
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Detectors
HPLC 6-way Injection Valve
•Questions to ask when evaluating an LC detector:
Fig. 21-17 waste
waste Sample loop
–Is the detection universal? –Is the detector response linear? –What is the limit of detection (LOD)? –Is the detector useful with gradient elution?
Sample loop
column
•In other words, is the detector insensitive to solvent composition?
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UV Detector (w/ flow cell)
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UV Detector
•Common HPLC detector –Many solutes absorb UV light
•Will cover during spectrophotometry
Eluate out
–Any UV spectrophotometers discussed previously are good LC detectors
Flow cell
Light source
•UV cutoff wavelengths –Below given , MP absorbs UV radiation
Detector
–MP absorbance overwhelms analyte absorbance
Eluate in Fig. 21-20 2006/9/7
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UV Detector
Fluorescence Detector
•Applicable for UV chromophores Essentially linear
•Similar idea to UV detector •Few molecules fluoresce •Derivatize solutes with fluorescent tag
–Will discuss deviations from linearity in CH 19
LOD ~ 0.1 ng Good detection method with gradient elution
–Derivatize mixture of solutes before separation –Derivatize solutes as they elute from column before detection •Post-column Derivatization
–Use solvents that do not absorb UV radiation
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Refractive Index Detector
Fluorescence Detector
•Compare refractive indices of eluate & reference
Applicable to fluorophores Essentially linear –Will discuss deviations from linearity (Reference)
LOD ~ 0.001 ng Fine with gradient elution
•Cannot use with gradient elution –reference composition cannot exactly match solvent composition during experiment
–Use solvents that do not fluoresce
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(MP solvent & analyte = eluate)
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Comparison of LC Detectors
Refractive Index Detector Type
Essentially universal LOD ~ 100 ng Essentially non-linear
UV-VIS
–Linear range very small
Disaster with gradient elution Finicky –Need constant T 2006/9/7
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Approx LOD Approx Comments Linear range 10 -10 g 10 4 For light absorbing compounds
Fluorescence
10-14 g
10 5
For Fluorescent compounds
MS
10-7-10-9 g
10 5
Universal detector
-8
Electrochemical conductometric
10 g/mL
Electrochemical amperometric
10- 10-10 –11 g 10 5
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10
5
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Specific detector; for all ions Specfic detector; electroactve compounds 94
Liquid Chromatography (LC) Especially high-performance liquid chromatography (HPLC). Thet e r m“ hi g h-pe r f o r ma nc e ”r e f e r st ot he use of packed columns with very small packing particles (diam. 5-10 m) giving greatly enhance resolution.
Note: several types of LC. In addition to partition (as described so far) - there are also ionexchange and size-exclusion chromatography using liquid mobile phases. We will concentrate only on partition. 2006/9/7
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