HPLC Seminar

HPLC seminar
1. Introduction
High Performance Liquid Chromatography (HPLC) is one mode of chromatography, the most widely used analytical technique. Chromatographic processes can be defined as separation techniques involving mass-transfer between stationary and mobile phases. HPLC utilizes a liquid mobile phase to separate the components of a mixture. These components (or analytes) are first dissolved in a solvent, and then forced to flow through a chromatographic column under high pressure. In the column, the mixture is resolved into its components. The amount of resolution is important, and is dependent upon the extent of interaction between the solute components and the stationary phase. The stationary phase is defined as the immobile packing material in the column. The interaction of the solute with mobile and stationary phases can be manipulated through different choices of both solvents and stationary phases. As a result, HPLC acquires a high degree of versatility not found in other chromatographic systems and has the ability to easily separate a wide variety of chemical mixtures.
History of HPLC
Prior to the 1970's, few reliable chromatographic methods were commercially available to the laboratory scientist. During the 1970's, most chemical separations were carried out using a variety of techniques including open-column chromatography, paper chromatography, and thin-layer chromatography. However, these chromatographic techniques were inadequate for quantification of compounds and did not achive sufficiently high resolution to distinguish between similar compounds. During this time, pressure liquid chromatography began to be used to decrease flowthrough time, thus reducing purification times of compounds being isolated by column chromatogaphy. However, flow rates were inconsistent, and the question of whether it was better to have constant flow rate or constant pressure was debated. (Analytical Chem. vol 62, no. 19, Oct 1, 1990). High pressure liquid chromatography was developed in the mid-1970's and quickly improved with the development of column packing materials and the additional convenience of on-line detectors. In the late 1970's, new methods including reverse phase liquid chromatography allowed for improved separation between very similar compounds. By the 1980's HPLC was commonly used for the separation of chemical compounds. New techniques improved separation, identification, purification and quantification far above those obtained using previous techniques. Computers and automation added to the convenience of HPLC. Additional column types giving better reproducibility were introduced and such terms as micro-column, affinity columns, and Fast HPLC began to immerge. The past decade has seen a vast undertaking in the development of micro-columns, and other specialized columns. The dimensions of the typical HPLC column are: XXX mm in length with an internal diameter between 3-5 mm. The usual diameter of micro-columns, or capillary columns, ranges from 3 m to 200 m. Fast HPLC utilizes a column that is shorter than the typical column. A Fast HPLC column is about 3 mm long and is packed with smaller particles. Currently, one has the option of selecting from a lot of columns for the separation of compounds, as well as a variety of detectors to interface with the HPLC in order to obtain optimal analysis of the compound. Although HPLC is widely considered to be a technique mainly for biotechnological, biomedical, and biochemical research as well as for the pharmaceutical industry,in actual fact these fields currently comprise only about 50% of HPLC users(Analytical Chem. vol 62, no.19, Oct 1, 1990). Currently HPLC is used in a variety of fields and industries including the cosmetics, energy, food, and environmental industries.
What is HPLC ?
1. Introduction
H P L C
: : : :
High Performance (Pressure) Liquid Chromatography
GC : Gas chromatography TLC : Thin layer chromatography IC : Ion chromatography
What is HPLC used for ?

1. Introduction
1. Separation of mixed components 2. Qualitative analysis / Quantitative analysis 3. Preparation of interest components Separation analysis and/or preparation of interest components
Separation and Analysis

1. Introduction
A A C B C A B C A C
A B C
Separation
B C
Qualitative analysis What are components A, B and C ? Quantitative analysis What is the concentration of components A, B and C ?
Results obtained by HPLC

1. Introduction
C A B
Chromatogram containing three peaks

Qualitative analysis (identification) and Quantitative analysis (determination) Can be performed using the information contained in the chromatogram
Chromatography : Method Chromatogram : Results Chromatograph : Instrument
Chromatogram
1. Introduction
Sample IN
Mobile phase IN column baseline
Sample IN F Mobile phase IN A C B A BC D E E D
Chromatogram
Identification
What is component A?
1. Introduction
C A B
Sample
Caffeine
Component A elutes the same time as a caffeine peak. Component A is identified as caffeine.
Determination
What is the concentration of component A?
1. Introduction
C A B
Caffeine (1mg/ml) 5ul injection (5ug)
Peak area (or height) is proportional to the concentration (or amount) of the component. The concentration of component A(caffeine) is determined by comparing the peak area with that of the standard caffeine peak.
Separation Mechanism
1. Introduction
Separation is determined by column (packing material) and mobile phase (solvent).
Mobile phase elutes components. Packing materials retain components in the column.
Mobile phase (solvent)

A B C C A B
Column time Packing material
C>B>A
Five modes in HPLC

1. Introduction
LC mode Normal phase chromatography Packing materials Silica gel Mobile phase n-Hexane/IPE MeOH/Water THF Buffer sol. Buffer sol. Interaction Adsorption Hydrophobic Gel permeation Ion exchange Affinity
Reversed phase chromatography Silica-C18(ODS) Size exclusion chromatography Ion exchange chromatography Affinity chromatography Porous polymer Ion exchange gel Packings with ligand
HPLC Basic Instrumentation

1. Introduction
Solvent Delivery Injector Column Separation Mobile phase Pump Sample Injection
Detector
Data Processor
HPLC Instrumentation
1. Introduction
Column oven
Data processor
Pump
Injector
Column
Detector
Drain
Gradient Elution Unit
Auto sampler
Reagent pump
Fraction collector
System Controller
The JASCO advanced technology team has again met the challenge and designed a new line of HPLC instruments, The LC-1500series more than satisfies in response to the growing demand for greatly expanded HPLC analyses in the fields of not only biochemistry, pharmaceutical and medical science, but also in the areas of among other organic and inorganic compounds, foods, agricultural sciences, polymeric and natural substances and pollution. The LC-1500 series comprises pumps, detectors, autosamplers, its own column oven and other units each having built-in intelligence and incorporating many features with much higher levels of operability and reliability in addition to multiple functions, higher performance and higher accuracy than before, making them the most advanced instruments available.
2. Parameters used in HPLC
Parameters used in HPLC
Retention parameters Column efficiency parameters Peak symmetry parameters Condition for Separation Retention : When a component in a sample interacts with the stationary phase in the column and a delay in elution occurs. Column efficiency : Goodness of a column
Retention parameters
tR : retention time (the time between the injection point and the maximum detector response for
correspondent compound)
vR : retention volume (tR x eluent flow rate) k : capacity factor t0 : the time required for the component not retained by the column to pass through the column
tR tR - t0 t0 k =
tR - t0 t0
Column efficiency
The number of theoretical plates N is given by: 4 method FWHM method 5 method h x 0.044 h x 0.5 h
tR
W4 N = 16 ( tR / W4 )2
W1/2 2 N = 25 ( tR / W5 )2 N = 5.545 ( tR / W0.5) W5
The height of the theoretical plate H is given by: H=L/N L : Column length
Peak symmetry
S : Symmetry factor ( T : Tailing factor )
h x 0.05 f W0.05 W0.05 S= 2f
S = 1 : The peak is completely symmetric. S > 1 : Tailing S < 1 : Leading
Degree of separation
tR2 tR1 k1 k2 Separation factor : W1 W2 k2 = k1 Resolution : Rs = 2 x tR2 - tR1 W1 + W2
Condition for good separation

A larger Rs value means a better separation. 1 4 - 1 k2 1 + k2 N
Rs = k2
1 + k2 - 1
: Capacity term increases the retention time : Selectivity term increases the time interval between peaks : Column efficiency term produce narrow peaks
Parameters and selectivity

Longer retention time
Larger
Improved column efficiency
Review of Sections 1 and 2
What is HPLC ? What is HPLC used for ? What is Separation and Analysis ? Qualitative and Quantitative analysis from chromatogram HPLC Parameters
What is HPLC ? What is HPLC used for ? What is Separation and Analysis ? H : High P : Performance (Pressure) Qualitative and Quantitative analysis from L chromatogram : Liquid C : Chromatography HPLC Parameters
What is HPLC ? What is HPLC used for ? What is Separation and Analysis ? Qualitative and Quantitative analysis from 1. chromatogramSeparation of mixed components
2. Qualitative analysis / Quantitative analysis 3. Preparation of HPLC Parametersinterest components
What is HPLC ? What is HPLC used for ? What is Separation and Analysis ? Qualitative and Quantitative analysis from chromatogram Qualitative analysis
What are components A, B and C ? HPLC Parameters Quantitative analysis What is the concentration of components A, B and C ?
Review of Section 1 and 2

Qualitative analysis (identification) and quantitative analysis (determination) can ? be performed using the information Contained in the chromatogram.
What is HPLC
What is HPLC used for ?
What is Separation and Analysis ? Qualitative and Quantitative analysis from chromatogram HPLC Parameters
What is HPLC ? What is

Retention parameters HPLC used for ?efficiency parameters Column Peak symmetry parameters Condition for and Analysis What is Separation Separation
Qualitative and Quantitative analysis from chromatogram HPLC Parameters
3. Separation mode
Column and mobile phase solvent
3. Separation mode
Sample and Analytical method

What is the sample ? Concentration of the interested component Contaminant
In which materials ? In what concentration ? Which sample ? With which technique ?
Characteristics of the sample - Structure - Molecular weight - pKa - Solubility Analytical technique - Column - Mobile phase - Detector - Sample preparation
3. Separation mode
Sample information
Merck Index Great Chemical Dictionary Great BioChemical Dictionary Reports based on other measurement techniques
3. Separation mode
Method information
Society magazines Journal of Chromatography. Analytical Chemist Manufacturer JASCO Application data
3. Separation mode
HPLC separation mode
HPLC separation mode Normal phase chromatography (NP) Reversed phase chromatography (RP) Size exclusion chromatography (SEC) Ion exchange chromatography (IEX) Affinity chromatography
3. Separation mode
Separation modes and features

Mode
Normal phase chromatography Reversed phase chromatography
Stationary phase
Silica gel
Mobile phase
Organic solvent (n-Hexane/IPE) MeOH/Water
Interaction
Adsorption
Feature
Fat-soluble
Silica-ODS (Silica-C18)
Hydrophobic
Most widely used
Size exclusion Chromatography Non-aqueous (GPC) Porous Polymer Aqueous (GFC) Aqueous porous Polymer Ion exchange Chromatography Affinity Chromatography Ion exchange gel
Organic solvent (THF) Gel permeation Molecular weight distribution Buffer solution Gel permeation Protein Separation Buffer solution Ion exchange Separation of ionic substances Purification of enzymes and proteins
Packing with ligand
Buffer solution
Affinity
GPC : Gel Permeation Chromatography GFC : Gel Filtration Chromatography
3. Separation mode
Solvent used in HPLC and range of Application

Solvent Polarity 0.1 0.1 -0.2 1.9 2.4 2.4 2.8 2.7 3.1 3.9 4.0 4.0 4.4 3.9 4.1 4.7 4.8 5.1 4.3 6.0 5.8 6.4 7.2 5.1 10.2 E0 0.01 0.01 0.04 0.54 0.28 0.29 0.38 0.32 0.42 0.7 0.82 0.57 0.58 0.82 0.40 0.51 0.56 0.56 0.88 0.65 0.75 0.95 R.I. 1.389 1.372 1.423 1.398 1.365 1.494 1.350 1.498 1.421 1.397 1.385 1.405 1.370 1.384 1.443 1.376 1.420 1.356 1.359 1.370 1.341 1.428 1.477 1.326 1.333 b.p. 99 69 81 89 68 110 35 80 40 118 97 66 77 82 61 80 101 56 78 118 82 153 189 65 100 Viscosity UV cut off 0.47 0.30 0.90 0.36 0.38 0.55 0.24 0.60 0.41 2.6 1.9 0.46 0.43 1.9 0.53 0.38 1.2 0.30 1.08 1.1 0.34 0.80 2.00 0.54 0.89 197 190 200 220* 285 218 280 233 210 240 212* 256 205 245 329 215 330 210 190 268 268 205
200
UV transmittance
250 300 350
Isoctane LCn-Hexane Cyclohexane Triethylamine i-Proryl ether Toluene Ethyl ether Benzene Methylene chloride n-Butanol n-Propanol Tetrahydrofuran Ethyl acetate i-Propanol Chloroform Methylethyl ketone Dioxane Acetone Ethanol Acetic acid Acetonitrile Dimethylformamide Dinethylsulfoxide Methanol Water
3. Separation mode
Polar compounds
Polar compound
Non-polar compound
H H O H H C H H
Bonding electrons are not shared evenly. The end of the bond with electrons becomes partially negative. The end of the bondwithout electrons becomes partially positive.
Polar compounds are soluble in polar solvents. Non-polar compounds are soluble in non-polar solvents.
3. Separation mode
Normal Phase Chromatography

Interaction : Packing materials : Adsorption Polar ex. Silica gel Silica-NH2 Silica-CN Silica-OH ex. n-Hex/CH2CL2 iso-Oct/IPA iso-Oct/AcOEt
Mobile phase :
Non-polar
Sample :
Fat-soluble Different polarity
3. Separation mode

Packing material
The most popular packing material is silica gel. It is believed that silanol radicals ( -Si-OH ) on the surface of silica gel act as the active site and the sample is separated.
O H
O H
O H
O H
O H
Si
Si Si
the surface of silica gel
O H
3. Separation mode

Interaction
OH OH H2N O 2N
OH
H2N
NO2
OH
H2N
OH OH OH
O 2N
3. Separation mode

Mobile phase solvents n-Hexane iso-Octane Chloroform Dichloromethane Ethylacetate Isopropylalchol Tetrahydrofran Dioxane Acetonitrile Ethanol Methanol Amines Acids n-Hex iso-Oct CHCl3 CH2Cl2 AcOEt IPA THF CH3CN EtOH MeOH
High Low
Polarity
3. Separation mode

Retention behavior
Low A B C
n-Hex/AcOEt(60/40)
A Polarity of Mobile phase B C D
n-Hex/AcOEt(50/50)
A BC D
Polarity of sample components A
<
<
<
High
n-Hex/AcOEt(30/70)
3. Separation mode
Reversed Phase Chromatography

Interaction : Packing materials : Hydrophobic Non-polar ex. Silica-C18 Silica-C8 Polymer ex. MeOH/H2O CH3CN/H2O MeOH/Buffer sol.
Mobile phase :
Polar
Sample :
Having different length of carbon chain
3. Separation mode

CH3 OSiCH2(CH2)16CH3
Silica-C18 Packing materials

Commonly used packing materials are hydrocarbons having 18 carbon atoms (called the Octadecyl radical) which are chemically bonded to silica gel (SilicaODS).Since the surface of the Silica-ODS is covered with hydrocarbon, the polarity of the packing material itself is very low.
Si
CH3 CH3 OSiCH2(CH2)16CH3 CH3 CH3 OSiCH3
Si
CH3 CH3 OSiCH2(CH2)16CH3 CH3
3. Separation mode

Hydrophobic Interaction
CH3
CH2COOCH3
CH3
CH2COOCH3
Silica-C18 (ODS)
3. Separation mode

Mobile phase solvents
Main solvent : MeOH - H2O CH3CN - H2O EtOH IPA THF DMF Acid Salt Ion-pairing agent
Sub solvent :
Additive :
3.Separation mode

Retention behavior in reversed phase HPLC
CH3CN/H2O
(70/30) (60/40) (50/50)
A B C A B C A B C Carbon chain length of sample A < B < C A : p-Hydroxy ethyl benzoate B : p-Hydroxy propyl benzoate C : p-Hydroxy butyl benzoate
0 5 0 5 0 5 10 (min)
Low
Polarity of Mobile phase Column : Finepak SIL C18
High
3.Separation mode

Length of packing materials carbon chains and retention time A
Finepak SIL C18 A B Finepak SIL C8 C B C
A
Mobile phaseCH3CN/H2O(40/60)
Finepak SIL C1 A : p-Hydroxy ethyl benzoate B : p-Hydroxy propyl benzoate C : p-Hydroxy butyl benzoate
0 5
B C
10
15
20
25
30 (min)
3.Separation mode
Reversed Phase and Normal Phase Chromatography

Comparison of Reversed Phase and Normal Phase
Normal phase Stationary phase Mobile phase Interaction Elution order

High polarity Low polarity Adsorption Low to High (Polarity)
Reversed phase
Low polarity High polarity Hydrophobic Short to Long (Length of Carbon chain)
3.Separation mode
Reversed Phase and Normal Phase Chromatography

Comparison of Reversed Phase and Normal Phase
Reversed Phase Reversed Phase Chromatography Chromatography
Finepak SIL C18 Finepak SIL C18 MeOH MeOH
VA VD
Normal Phase Normal Phase Chromatography Chromatography

n-Hexane/IPA(96/4) n-Hexane/IPA(96/4)
VE VD 10 (min) 0 10 20 (min)
Finepak SIL Finepak SIL
VA
3.Separation mode
Ion-exchange Chromatography
Interaction : Ion-exchange
Stationary phase: Anion exchange gel Cation exchange gel Mobile phase : Sample : Buffer solution Ionic substances (Cations or Anions)
3.Separation mode
Ion-exchange Gel
SO3- Na +
NR3
+ Cl -
SO3- Na + SO3- Na +
NR3+ Cl NR3+ Cl -
SO3- Na +
NR3+ Cl -
Cation exchange gel
Anion exchange gel
3.Separation mode
Mobile phase solvents used for Ion-exchange
Buffer solution Salt concentration pH (Hydrogen ion concentration) Type of salt Additive (Organic solvent)
SO3 - Na + S+ SO3 Na
+
Na SO3 S
+
3.Separation mode
Application data of Ion-exchange chromatography
Separation of polyphosphoric acid
1. 2E+ 05 uV
1 5.89
Column
2 9.49
Mobile phase
1. 0E+ 05
Reactor
4 17.79 3 13.58 5 21.65 6 24.65
8. 0E+ 04
7 26.95
Detection
8 28.83
: Finepak GEL SA-121 (6.0mmI.D. POLY_003.CH x 100mmL) : A= 0.1M KCl + 1% EDTA-4Na (pH 10.0 adjusted HCl) B= 1.0M KCl + 1% EDTA-4Na (pH 10.0 adjusted HCl) gradient : 1.8MH2 SO4(1L), (NH4o7)6MO24-4H2O (5g), Sand of zinc metal(0.6g) : 830nm
10 31.71
11 32.86 12 33.84 13 34.72 14 35.50 15 36.21 16 36.85 17 37.43 18 37.95 19 38.38 20 38.72
6. 0E+ 04
4. 0E+ 04
2. 0E+ 04
0. 0E+ 00
9 30.40
10. 0
20. 0
30. 00
40. 00
[m i n]
3.Separation mode
Ion Chromatography
Summary of Ion Chromatography
Purpose : Separation of inorganic ions, organic acids
Stationary phase: Anion exchange gel Cation exchange gel Mobile phase : Detection : Buffer solution Conductivity detector
3.Separation mode
Ion Chromatography
Suppressor and Non-suppressor
P
Mobile phase
pump
P
Mobile phase
pump
injector
injector
column
column
suppressor
Conductivity detector
Conductivity detector
3.Separation mode
Ion Chromatography
Cation measurement data
Sample Column Mobile phase Detector : Tap water : Shodex IC YK-421 : 5mM tartaric acid+2mM Gibicolin acid : Conductivity detector (CD-5)
Na+ 5.276ppm
Ca2+ 12.386ppm
K+ 0.785ppm
Mg2+ 1.829ppm
10
15
20(min)
3.Separation mode
Ion Chromatography
Anion measurement data
Sample : Tap water Column : Shodex IC I-524A Mobile phase : 2mM phthalic acid+1.84mM tris +300mM boric acid(pH4.0) Detector : Conductivity detector (CD-5)
Cl- 6.029ppm
F- 0.111ppm
SO42- 10.426ppm
NO3- 6.694ppm
10
15
20
25(min)
3.Separation mode
Size Exclusion Chromatography (SEC)

GPC and GFC
Non-aqueous SEC Interaction Packing Mobile phase Sample : : : : :
GPC (Gel Permeation Chromatography)

Gel permeation Cross-Linked porous Polystyrene Organic solvent (THF, CHCl3, DMF) Molecular weight distribution of polymer Synthetic Oligomer separation
Aqueous SEC Interaction Packing Mobile phase Sample
: : : : :
GFC (Gel Filtration Chromatography)

Gel permeation Hydrophilic silica gel / Hydrophilic porous polymer Buffer solution Separation of Water-soluble polymers (proteins, nucleic acid, sugar) oligomers
3.Separation mode

SEC Separation mechanism
B
Mobile phase
A D A D C D C
Small pore
Packing material
C B D
A+B
3.Separation mode

Gel permeation chromatography and calibration curve
1. 0E+ 05
uV
RI
Column : Shodex GPC KF-806Lx 2 Column Mobile phase : THF

PS-8420K PS-900K PS-110K
5. 00 10. 00 15. 00
6. 0E+ 04
4. 0E+ 04
2. 0E+ 04
0. 0E+ 00
PS-18.1K PS-2.98K PS-Oligomer

20. 00 25. 00
8. 0E+ 04
30. 00
35. 00 m n] [ i
3.Separation mode

Peak analysis of polymer to calculate molecular weight distribution
no 1 2 3 Vi 10.0 10.5 11.0 Hi 74 156 318
H1 V1
H2 H3
25.0 (min)
10.0
15.0
20.0
V2 V3
Retention time
3.Separation mode

Molecular weight calculation
N 1 2 3 Vi 11.12 11.37 11.62 Hi 74 387 1539 hi Mn = Mw = Mz = D Hi/ HiMi/ HiMi2/ Hi/Mi = 15.5104 Hi = 28.6104 HiMi = 46.5104 Mi 2094050 1734413 1432619 mi Hi/Mi Hi/Mi HiMi HiMi HiMi2 HiMi2
= Mw/Mn = 1.84
3.Separation mode

Column selection
Molecular weight of the sample : Exclusion limit molecular weight Ability to dissolve the sample Molecular weight distribution : Applicable to packing materials : Range of calibration curve
3.Separation mode

Column suited to the sample in terms of molecular weight
Shodex A-8012
EPIKOTE 3-8 1001 2 1 n=0
Shodex A-8032
2 1 3 4 5 6 7 8 n=0
EPIKOTE 828
15
20
25 min
30
40 min
Eluent : THF
Flow rate : 1.0ml/min
3.Separation mode

Solvent and column
Solvent
THF
Column
Finepak GEL 101F Shodex KF series Finepak GEL 101C Shodex K series Shodex KD series Shodex SB series Shodex KS series
CHCl3
DMF H2O, Buffer solution
3.Separation mode

Calibration curves for columns
Eluent : THF
3.Separation mode
Columns for exclusive use

Columns for Exclusive use
Amino acids : Aapak (Cation exchange) Organic acid : Shodex Ionpak KC-811 (ion exclusion and partition & adsorption) Sugar : Shodex Ionpak KS series (aqueous SEC) Shodex Sugar series (ligand exchange) Finepak SIL NH2-5 (Normal phase) Finepak GEL SA-121 (Strong anion exchange)
N-methyl carbamate : Carbamatepak (Reversed phase)
3.Separation mode

Amino Acid Analysis
Column : AApak Na II-H Mobile phase : Sodium citrate buffer Stepwise gradient Detection : OPA post label Ex 345nm Em 445nm Sample : Sake NH3 Leu Ala Gly ThrSer Pro Glu Val
Met
Tyr
Phe
Lys His
Asp
Ile
Arg
3.Separation mode

Organic Acid Analysis
Column : Shodex Ionpak KC811x2 Mobile phase : Detection : BTB post label UV 445nm Sample : Sake
citric pyrvic malic
succinic
lactic
pyroglutamic
acetic
3.Separation mode
Ion suppression method & Ion-pair chromatography

Separation method to analyze ionic compounds by reversed-phase chromatography Ion suppression method Ion pair chromatography : Acidic ion components : Basic ion components / Acidic ion components
3.Separation mode

Diagram of Ion suppression method
Add phosphoric acid
H+ H
+
HA
H+ H+ HA H+ H+ H+
A-
Silica-C18
H+ H+ A
-
Silica-C18
HA H+ H+
AA: Sample
+ H+
HA
H+ : Hydrogen ion
HA
: Sample
3.Separation mode

Chromatogram when Ion suppression method is used
Benzoic acid p-Hydroxy ethyl benzoate
Finepak SIL C1 CH3CN/H2O (40/60)
Finepak SIL C1 CH3CN/0.2% H3PO4 (40/60)
propyl butyl
10 (min)
10 (min)
3.Separation mode

Diagram of Ion-pair chromatography
SO3SO 3
+ R4 - N
+NR
Silica-C18 SO3-
SO3-
Silica-C18
+NR
Add Ion-pair reagent
SO - + 3 N R
SO3 +NR
- + NR 4
SO3SO3- + NR4
SO 3
-
Silica-C18
SO 3
SO - +
3
NR
SO3 -
3.Separation mode

Chromatogram when Ion-pair chromatography is used
A A B B
Without Ion-pair reagent
With Ion-pair reagent
Typical ion reagents

Acidic ions : Tetra alkyl ammonium halide Basic ions : l-Alkyl sulfonate
3.Separation mode

Ion-pair chromatography
Effects of basic additives
- Stable pH - Longer retention time - Ion pair reagent effect
3.Separation mode

Acid and basic sample for Reversed phase LC
Method Ion suppression Sample
Weak acidic sample
Reversed phase
phosphoric acid acetic acid perchloric acid trifluoroacetic acid Tetra alkyl ammonium halide l-Alkykl surfonate (acetic acid) (trifluoroacetic acid) phosphate citrate
Ion pair
Acidic sample Basic sample
Addition of salt
Review of Section 3
4 separation modes Polarity of packing material and solvent Change of mobile phase and elution Ion suppression method and Ion pair method Salt effect
4. Gradient elution method

For separation of a sample containing many components
MeOH(100)
Mobile phase
Gradient MeOH/Water (50/50) 0.1M KH2PO4 Step wise 0.01M 0 5 10 Time(min) 15 20 25 0.5M

Advantage of gradient elution method
Isocratic elution method Gradient elution method
A
Finepak SIL C18 MeOH/1% AcOH(40/60)
B * * A
MeOH/1% AcOH(30/70) MeOH/1% AcOH 30/7045/55 Linear Gradient16min
A : Chlorogenic acid B : Rutin * : Impurity

Precautions in gradient elution method
- Can the gradient save time ? - Reproducibility - Baseline - Ghost peak - Salt

Effect of temperature on retention time
60*C 1 2 3 4 Finepak SIL C18 CH3CN/H2O(90/10) Sample 1. Benzene 2. Anthracene 3. Pyrene 4. Benz(a)pyrene 4
40 *C
20 *C
2 3 4
10
12 (min)
Review of Section 4
Gradient elution method Temperature effect
5. Detector
5. HPLC detectors
HPLC detectors
UV-VIS(Absorption) PDA (Absorption) Differential refractometer(Refractive index) Fluorometric (Florescence) Electrochemical (ECD) (Oxidation -reduction) Conductivity Mass Chiral (OR) Circular dichroism (CD)
5. HPLC detectors
UV/Vis detector
- Selective detection minimizing effects from other components - High sensitivity detection at maximum absorption wavelength
5. HPLC detectors
Improved selectivity
Traditional medicine berberine impurity berberine
impurity
7.385
Wavelength=260nm
Wavelength=340nm
7.395
5. HPLC detectors
Improved sensitivity
Saccharin (SAC) and sorbin acid (SOR)
230nm
265nm
SOR
0nm
SAC
12.250
SAC
SOR
10
20
10
Wavelength programming
Fixed wavelength at 265nm
12.467
3.575
3.608
20
5. HPLC detectors
UV spectrum measurement to find wavelength effective for wavelength programming
1.0 Diazepam (DZP) Absorbance
Nitrazepam (NZP) 0.5
Chronazepam (CZP) 210 250 300 Wavelength(nm) 350 0
5. HPLC detectors
Wavelength programming and fixed wavelength
Blood serum NZP : 420ng/ml CZP : 130ng/ml DZP : 440ng/ml
NZP
DZP
NZP 0 5 CZP DZP 10 Fixed wavelength
310nm 250nm
CZP
10
Wavelength programming
5. HPLC detectors
Optics of Multi-wavelength detector
I2 lamp D2 lamp
Grating UV/Vis detector Photo diode
lamp
Grating
Photo diode
Cell
Cell Photodiode array
5. HPLC detectors
Multi-wavelength detector 3D chromatogram
5. HPLC detectors
Multi-wavelength detector Cont. data
5. HPLC detectors
Features of Multi-wavelength detector
1. 2. 3. 4. Spectrum collection at any time Library search Purity check Quantitative analysis at 6 wavelengths
5. HPLC detectors
Principle of Fluorescence detector
(S1) Excited state(S2) (S3)
excitation
emission
H (fluorescence)
S Ground state 0 Mobile phase
5. HPLC detectors
Features of Fluorescence detector
1. Selective detection 2. Detection at any excitation or emission wavelength 3. High sensitivity
5. HPLC detectors
Wavelength programming by Fluorescence detector
Wavelength programming 0 6.6 10.0 min 450 525 nm VB2 Fixed wavelength Ex=275nm Em=400nm Fixed wavelength Ex=450nm Em=525nm
Ex 275 240 Em 400 350
VB2 Phosphate
VB2 Phosphate 10 20 min 0 10
VB6
VB6
VB1
10
20 min
20 min
Column : Finepak SIL C18S Mobil phase : MeOH/Phosphate Buffer Gradient
VB2
5. HPLC detectors
Selectivity of UV detector and Fluorescence detector
UV detector
Fluorescence detector
5. HPLC detectors
Principle of RI detection
light i i
light
r0
Solvent
Sample and solvent
r0>r
5. HPLC detectors
UV detector and RI detector
RI detector
UV detector
5. HPLC detectors
Considerations for IR detection
1. 2. 3. 4. Temperature change Replacement of solvent (reference cell and sample cell) Unstable when solvent mixed Replacement of solvent inside column
5. HPLC detectors
Detectors
UV Sensitivity Detection selectivity Temperature Influence ng selective Fluorescence pg highly selective RI g universal
small
small
large
Gradient elution possible
possible
impossible
5. HPLC detectors
Label method
Samples absorb less UV/Vis light . Samples do not fluoresce.
Improved sensitivity and selectivity required
Label method
5. HPLC detectors
Label method
Pre-label method
reagent sample (reaction) pump injector column
Post-label method
injector
column reactor
reagent
detector detector
5. HPLC detectors
Label method
Post-label method
Aminoacid
0PA ninhydrine guanidine brom thymol blue ethylenediamine THI NAD HSD
Fluorescence Absorption in Visible range Fluorescence Absorption in Visible range Fluorescence Fluorescence Fluorescence Fluorescence
Sugar Organic acid Catecholamine
Bile acid
5. HPLC detectors
Pre and Post column derivatization method
Pre-column LC system required Standard system
Post-column Reaction system is required
Reproducibility Operation Reagents Applicability
less than post-column good for all samples wide range spot only reagents limited routine
5. HPLC detectors
Pre-column derivatization method
Dabcyl-Cl
CH3 N CH3 N=N SO2 Cl
Amino acid
R
+
H2 N O
O H
pH8.170 12min R CH3 N CH3 N=N SO2 N H O O H
Dabcyl - Amino acid
5. HPLC detectors
Separation of Dabcyl - Amino acid
4.0E+04 uV
Wavelength : 465nm
3.5E+04
DABS-OH
NH3
40pmol each
15 16 8 14 3 56 17 9 7 13 12 11 10
1 2 3.0E+04
1.Asp 2.Glu 3.Ser 4.Thr 5.Gly 6.Ala 7.Arg 8.Pro 9.Val
10.Met 11.Ile 12.Leu 13.Phe 14.Cystine 15.Lys 16.His 17.Tyr
2.5E+04
2.0E+04
5.00
10.00
15.00
20.00
25.00 [min]
5. HPLC detectors
Post-column derivatization method
Amino acid
CHO
2-mercapto ethanol
+ HS CH2 CH2 OH + NH2 C
COOH R H
CHO
orthophthalaldehyde (OPA)
S CH2 H N C R CH2 OH
COOH
Derivative compound
5. HPLC detectors
Post column derivatization method
Ex : 345nm Em : 455nm
CysSO3H
Val
Asp Thr Ser Glu Pro
Gly Ala Cys
Met
Ile Leu
His
Phe
Tyr
Lys
20
40
60 (min)
Trp
Arg
Review of Section 5
Detectors Selectivity and sensitivity Pre-/Post- column derivatization methods
6. Data processing
6. Data processing
Data processing in HPLC

1. Qualitative analysis 2. Quantitative analysis 3. Molecular weight distribution
6. Data processing
Qualitative analysis
1. 2. 3. Retention time Retention volume of the standard sample Sample components are collected after separation, and subjected to spectrometric analysis such as IR, NMR and MS.
6. Data processing
Identification from retention time

tR A B
Standard sample
Unknown sample
6. Data processing
Standard addition method
Standard addition
Target peak
6. Data processing Standard addition method

Retention time of standard sample is different from unknown sample
Standard sample
Unknown sample
Unknown sample and Standard sample
6. Data processing Identification using a different instruments after preparative analysis Identification from retention time
Limitation:
On flow UV spectrum On flow emission spectrum Multi-channel detector
Preparative analysis
Spectrum measurement using a different instrument
6. Data processing
Quantitative analysis
How much component A ?
The amount of a component can be calculated from the peak height and peak area of the chromatogram. Standard sample (1mg/ml)
A
Injection of 10g
Unknown sample
A
Injection of 10g
6. Data processing
Calibration method
External standard sample Internal standard sample
6. Data processing
External standard sample

Thiamiral in serum
Concentration(g/ml)
Finepak SIL C18T-5 CH3CN/10mM KH2PO4 aq. (50:50) UV 288nm
Peak area
Thiamiral
Thiamiral
6. Data processing
Internal standard sample

Anticonvulsants in serum
Standard sample Calibration curve
1PB 2DPH 3CBZ ISPhenacetin
Unknown sample
Peak area
concentration(g/ml) Concentration ratio

Finepak SIL C18T CH3CN/5mM KH2PO4 aq.
6. Data processing
Guide for selecting the internal standard sample

No overlapping peaks No Components included in unknown sample Chemical and physical stability High purity
6. Data processing
External standard and Internal standard samples

External standard injection volume impossible Internal standard volume to be added possible
Error Correction of Pre-treatment loss
6. Data processing
Caution when using an Integrator

One point calibration Integrator
True curve
error
large small
large
6. Data processing
Caution when using an Integrator

Two point calibration Integrator
True curve
error
large
small
large
6. Data processing
Baseline
6. Data processing Considerations when performing quantitative analysis

Standard sample Integrator Micro syringe Sample preparation Concentration change of standard sample Contamination
Review of Section 6
Identification 1. Retention time 2. Standard sample 3. After preparative analysis, measure spectrum using a different method Quantitative analysis 1. External standard sample 2. Internal standard sample 3. Items to consider when performing quantitative analysis
7. Sample preparation
Sample preparation
Cause Sample is not liquid. Concentration is too high. Concentration is too low. Contains foreign particles Problem not possible to inject over load for column / out of detection range cannot detect clogged up Countermeasures extraction / dissolving dilution concentration / derivative centrifugation / filtration solvent extraction /derivative solvent extraction /derivative pH adjustment
Includes components which damage column Includes interference for separation Solvent unsuitable quantitation error deterioration of column
Sample preparation Method

Filtration Extraction Concentration 0.45um, 0.2um membrance filter Solvent extraction Solid phase extraction Evaporation Solid phase exraction (Bond Elut) Fused drying Organic acid
Deprotaination Homonization
Solid phase extraction

1. Activation Wash with MeOH Activate With H2O 2. Load sample
Sample Contaminant
3. Wash
Wash with H2O orH2O/MeOH
4. Elute target compound

MeOH or Eluting solvent
Contaminant Vacuum
Target compound
Removing contaminants which have strong retention

1. Activate
Wash with MeOH Activate with proper solvent
3. Elute a target compound
2. Load sample
Target sample Compound which has strong retention
Using vacuum or pressure
Compound which has strong retention
Target sample Vacuum
Concentration
1. Activate 2. Load and concentrate target sample
target sample
3. Elute target sample
Wash with MeOH
Activate with H2O
Elute with MeOH
pump
Small amount of Target sample
Target compound is concentrated. Vacuum
Considerations when preparing sample

Recovery rate Contamination
Review of Section 7
1. The most appropriate preparation method depends on various factors including the sample(target compound), the amount of target compound in the sample, and the kinds of contaminant. 2. Consider such factors as the sample state, amount, running cost, running time, and handling.
8. Procedure for developing analytical conditions
Procedure for developing analytical conditions

Step one : clear analytical purpose, and research the target compound. (1) Molecular weight Molecular structure Functional group (2) Solubility, stability UV, FP absorption (3) Amount of concentration, contaminant (4) Application data reference literature, magazines Development analytical conditions (trial and error) (1) When attempting to develop analytical conditions, use an appropriate concentration of standard solution (2) Check the detection limit and detection method (3) Prepare sample (4) Check contaminat and target compound peak separation
Step two :
Procedure for developing analytical conditions

Step three : Establish analytical condition for routine analysis (1) Linearity of calibration curve (2) Reproducibility of analysis (3) Check for contaminants that retain strongly in the column (4) Check for Correlation with other methods. Routine quantitative analysis (1) Lifetime of column (2) Running cost (3) Develop analytical procedure (SOP) (4) Check HPLC and column performance.
Step Four

HPLC Seminar

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HPLC seminar

High Performance (Pressure) Liquid Chromatography

GC : Gas chromatography TLC : Thin layer chromatography IC : Ion chromatography

What is HPLC used for ?

Separation and Analysis

Results obtained by HPLC

Chromatogram containing three peaks

Chromatography : Method Chromatogram : Results Chromatograph : Instrument

Mobile phase IN column baseline

Sample IN F Mobile phase IN A C B A BC D E E D

Caffeine (1mg/ml) 5ul injection (5ug)

Column time Packing material

Five modes in HPLC

HPLC Basic Instrumentation

Gradient Elution Unit

2. Parameters used in HPLC

2. Parameters used in HPLC

Parameters used in HPLC

2. Parameters used in HPLC

2. Parameters used in HPLC

W1/2 2 N = 25 ( tR / W5 )2 N = 5.545 ( tR / W0.5) W5

2. Parameters used in HPLC

h x 0.05 f W0.05 W0.05 S= 2f

S = 1 : The peak is completely symmetric. S > 1 : Tailing S < 1 : Leading

2. Parameters used in HPLC

tR2 tR1 k1 k2 Separation factor : W1 W2 k2 = k1 Resolution : Rs = 2 x tR2 - tR1 W1 + W2

2. Parameters used in HPLC

Condition for good separation

2. Parameters used in HPLC

Parameters and selectivity

Improved column efficiency

Review of Sections 1 and 2

Review of Sections 1 and 2

Review of Sections 1 and 2

2. Qualitative analysis / Quantitative analysis 3. Preparation of HPLC Parametersinterest components

Review of Sections 1 and 2

Review of Section 1 and 2

What is HPLC used for ?

Review of Sections 1 and 2

What is HPLC ? What is

Qualitative and Quantitative analysis from chromatogram HPLC Parameters

Column and mobile phase solvent

Sample and Analytical method

In which materials ? In what concentration ? Which sample ? With which technique ?

HPLC separation mode

Separation modes and features

Most widely used

Packing with ligand

GPC : Gel Permeation Chromatography GFC : Gel Filtration Chromatography

Solvent used in HPLC and range of Application

Normal Phase Chromatography

Fat-soluble Different polarity

Normal Phase Chromatography

the surface of silica gel

Normal Phase Chromatography

Normal Phase Chromatography

Normal Phase Chromatography

Polarity of sample components A

Reversed Phase Chromatography

Having different length of carbon chain

Reversed Phase Chromatography

Silica-C18 Packing materials

CH3 CH3 OSiCH2(CH2)16CH3 CH3 CH3 OSiCH3