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HPLC. The best application fields of various chromatographic modes. GC Volatile, thermostable compounds LC Polar, non volatile. thermolabile EKC Ionic compounds. The role of interaction types in various chromatographic modes. Advantages of various chromatographic modes.

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The best application fields of various chromatographic modes
The best application fields of various chromatographic modes

GC

Volatile, thermostable compounds

LC

Polar, non volatile. thermolabile

EKC

Ionic compounds





Resolution efficiency selectivity
Resolution-efficiency- selectivity modes

HPLC can produce high selectivity,

but moderate efficieny (< 100 000 tp).

At least, α = 1.3 is required for baseline separation.


Band broadening in hplc
Band broadening in HPLC modes

The HPLC uses packed columns.

The diffusion processes are much slower in HPLC than GC.


Van deemter curve in hplc
Van Deemter curve in HPLC modes

The slow diffusion causes increasing HETP values

as function of linear flow of mobile phase.


Schematic view of high performance liquid chromatography hplc instrument
Schematic view of high performance liquid chromatography (HPLC) instrument

Degassing is important to gain smooth baseline.


An pp to date hplc instrument
An pp to date (HPLC) instrumentHPLC instrument

  • Pumps upto 300 bar

  • The degassing is important


Pump (HPLC) instrument

Pump head

Motor & Cam

Check valves

Plunger

Plunger seal


Pump of hplc instrument
Pump of HPLC instrument (HPLC) instrument

Pulsation of system is decreased with two pumps,

working in opposite periods.


Gradient system
Gradient system (HPLC) instrument

  • Isocratic system

    • Fixed (un-changeable) mixing ratio during analysis

  • Gradient system

    • Changeable mixing ratio during analysis

      • HPGE (High Pressure Gradient, mixing after pumps)

      • LPGE (Low Pressure Gradient, mixing before pumps)


Mobile phase pump with 4 eluents
Mobile phase pump with 4 eluents (HPLC) instrument

Low Pressure Gradient


Aim of gradient problems in isocratic mode
Aim of gradient (HPLC) instrument - problems in isocratic mode -

  • in isocratic mode

Methanol / water = 6 / 4

Long analysis time, low signal to noise ratio

Methanol / water = 8 / 2

Poor separations

(Column : ODS type)


Aim of gradient solution

95% (HPLC) instrument

30%

Aim of gradient - solution -

  • Gradual change of the mixing ratio during analysis

Methanol concentration

in mobile phase

Short analysis time

&

Excellent separation, good signal to noise ratio


Polarity of eluents
Polarity of eluents (HPLC) instrument


Rotary valve injection in hplcben
Rotary valve injection in HPLCben (HPLC) instrument

The loop injector introduces exact volume of sample.


On line spe hplc arrangement
On-line SPE-HPLC arrangement (HPLC) instrument

Precolumn is in the loop. Precolumn is good for sample concentration.



Integrated precoumn hplc
Integrated precoumn HPLC concentration

The precolumn protect the main column, against thedepositionof matrixcomponents, and dissolution of stationary phase.

Main columns have 15-25 cm length and 2- 4,6mm I.D.


Dead volume
Dead volume concentration

  • Dead volume may cause problems such as poor peak separations and poor reproducibility.

Male nut

Dead volume

Tube

Poor connection

Excellent connection


Sample vs hplc mode
Sample vs. HPLC mode concentration


The diameters and porosity of sample influence of efficieny
The diameters and porosity of sample influence of efficieny concentration

The efficiency increase with the decrease of packing diameter. However the mobile phase pressure has limits (~ 250 att), wichallows3-5 µmsize of packing material.

The increased porosity increased the loadability. However the deep holes are badly washed.

Spherical particles are the best.


Various hplc packings
Various HPLC packings concentration

Goodnes: monolith > spherical > irregular


New type of packings
New type of packings concentration

The limited depths of holes improves the efficiency.


New trend the use of 1 8 m diameter packings
New trend the use of 1.8 concentrationµm diameter packings

Very high pressure, short columns and fast analyses


Different molecular weight molecules requires different poremsizes
Different molecular weight molecules requires different poremsizes

Bigger molecules need bigger pore size..




Retention order on reverse vs normal phase packings
Retention order on poremsizesreverse vs. normal phase packings


Polarity of solvent
Polarity of solvent poremsizes

The strongest mobile phase is hexane in reversed phase mode.

The strongest mobile phase is acetic acid in normal phase mode.


Bonded silica reversed phase hplc packing
Bonded silica poremsizes(Reversed phase HPLC packing)

Revers phase s are used in 80 % of HPLC analyses.


Stationary phase
Stationary phase poremsizes

Reversed phase packings:

  • C18

  • C8

  • C4

  • Cinao

  • Diol

    Normal

    Specials: chiral, ion exchange, gel

Increasing polarity→


Most frequently used hplc stationary phase c 18
Most frequently used HPLC stationary phase C poremsizes18

Apolar compounds have big retention

Mobile phases are mixture of water, methanol acetonitrile.


Condition process of c 18 stationary phase
Condition process of C poremsizes18 stationary phase

A methanol wash reqires for the activation of

C18 stationary phase.


Column polarity retention time
Column polarity - Retention time poremsizes

OH

C18 (ODS)

weak

strong

CH3


Mobile phase polarity retention time
Mobile phase polarity - Retention time poremsizes

Mobile phase: Methanol /Water

Methanol / Water

60 / 40

Methanol / Water

70 / 30

Methanol / Water

80 / 20


Influence of strength of mobile phes on c 18 stationary phase
Influence of strength of mobile phes on C poremsizes18 stationary phase

A decrease of mobile phase strength results in

increases of resolution values and retention times.


Hplc analysis of basic herbicides
HPLC analysis of basic herbicides poremsizes

Amines need specially deactivated packings





Normal phase adsorption chromatography
Normal phase, poremsizesAdsorption chromatography

The molecules of sample is solved in mobile phase, but they touch only in the surface of stationary phase.


Ion excange chromatography
Ion excange chromatography poremsizes

The ions of stationary phase interact with the oppositely

charged molecules of sample.


Ion chromatogram of anaions
Ion chromatogram of anaions poremsizes

The stationary phase is anionic ionexchange resin.



Size excusion gel chromatography
Size excusion (gel) chromatography poremsizes

The voluminous molecules elute fast because they are

excluded from the small diameter pores, therefore they

interact in less extent.



Specially designes stationary phase for carbamate pesticides
Specially designes stationary phase for carbamate pesticides poremsizes

Carbamate can not be analysed with GC,

because they are thermolabiles.


Molecular imprintesd mip stationary phases
Molecular imprintesd (MIP) stationary phases poremsizes

They are very selective,

but low efficiency

packings


Various hplc detectors
Various HPLC detectors poremsizes

Electrochemical S

Mass spectrometric U

Fluorescent S

Ultraviolett S

Refractive U

Light scaterring U

S, selective; U, univeral


Uv uv vis detector

E poremsizesin

A

C

UV/UV-VIS detector

C : Concentration

Cell

Eout

D2 / W Lamps

l

A= e·C·l= –log (Eout / Ein)

(A : Absorbance)


External standard
External standard poremsizes

Area

Concentration

A1

Calibration curve

C1

A4

A2

A3

C2

Peak area

A2

A3

C3

A1

A4

C1

C2

C3

C4

C4

Concentration


Internal standard
Internal standard poremsizes

Concentration

Area

Internal

Target

standard

A1

AIS

Calibration curve

C1

CIS

A4 /AIS

A2

AIS

A3 /AIS

C2

CIS

Area: Target / Internal standard

A2 /AIS

A3

AIS

C3

CIS

A1/AIS

A4

AIS

C1/CIS

C2 /CIS

C3 /CIS

C4 /CIS

C4

CIS

Concentration: Target / Internal standard





Light scattering hplc detector
Light scattering HPLC detector groups

Universal, sensitive


Refractive index detector rid 10a
Refractive index detector groups(RID-10A)

Photodiode

Reference

W Lamp

Sample



Lc ms ms is appropriate for compound identification
LC/MS-MS is appropriate for compound identification groups

First MS→Ionic adduct with soft ionization

Second MS→fragmentation with EI ionization



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