H.p.l.c. High performance liquid chromatography

Name :- Avdhesh Kumar
MSc. I Sem
Under the guidance of
Dr. ASHISH KUMAR
(Department of Biotechnology)
SANT GAHIRA GURU VISHWAVIDYALAYA,
SARGUJA, AMBIKAPUR, (CG.)

1. Introduction
2. Principle of HPLC
3. Instrument of HPLC
4. Types of HPLC
5. Separation method of HPLC
6. Application
7. Conclusion
8. Reference

 HPLC stands for ‘ High performance liquid
chromatography’ (sometimes referred to as high pressure
liquid chromatography)
 HPLC is a powerful tool in analysis it yields high
performance and high speed compared to traditional
columns chromatography because of the forcibly pumped
mobile phase
 HPLC is a chromatography technique that can separate
a mixture of compounds
 It is used in biochemistry and analytical to identify
quantify and purify the individual component of a
mixture

 Chromatography :- physical method in which separation of
components takes place between two phases a stationary
phase a mobile phase
 Stationary phase :- ( the substance to be separated
during chromatography) takes place it can be a solid, a
gel or a solid liquid combination
 Mobile phase:- solvent which carries the analyte ( a
liquid or gas )
Stationary phase Mobile phase

(2) Chromatography and its principle
 Chromatography is a separation technique which is
used to separate a mixture of compounds into its
individual components based on certain physical and
chemical properties

(3)Instrument of HPLC
Solvent reservoir
Pump
Sample injector
Column
Detectors
Recorder

Solvent reservoir
 Solvent can be polar or non-polar
 Solvent selection depends on type of sample
 Solvent as a carrier to the sample solution
 A sample solution is injected into the mobile phase of an
assay through the injector port

Pump
 Pumping system can be said to be the heart of HPLC , by
producing high pressures
 The role of the pump is to force a liquid (called the mobile
phase) through the liquid chromatograph at a specific flow rate,
exressed in milliliters per min. (mL/min)
 Normal flow rates in HPLC are in the 1-to 2-mL/mint range
 Typical pumps can reach pressures in the range of 6000-9000
psi(pound per square inch)

 Sample injector
 Typical sample volumes for manual injector are 5 to 20
microliters
 It is equipped with six port valves so that a sample can be
injected into the flow path at continuous pressure
 The knob is set to LOAD position for sample injection using
a syringe the sample is injected into the sample loop, which
is separated from the flow path
 The knob is turned to INJECT position and the eluent
travels through the loop from the pump and deliver the
sample to the column

Column
 The column stationary phase separates the sample
components of interest using various physical and chemical
parameters.
 It is usually made of stainless steel to withstand high pressure
caused by the pump to move the mobile phase through the
column packing other material include PEEK (Poly
Ethyl Ethyl Ketone) , stainless steel and glass
 The small particles inside the column are called the packing
what cause the high back pressure at normal flow rates
 Column packing is usually silica gel because of its particl
shape, surface properties , and pore structure give us a good
separation

 Detectors
 The detector can be detect the individual molecules that
elute from the column and convert the data into an electrical
signal
 A detector to measure the amount of those molecules
 The detector provides an output to a recorder or computer
that results in the liquid chromatogram
 Detector is selected based on the analyte or the sample
under detection
 Types of detector
1) Ultraviolet
2) Fluorescence
3) Refractive Index Detector
4) Mass Spectrometry

1) Ultraviolet detector
 This types of detector responds to substances that light
 The UV detector is mainly to separate and identify the
principal active components of a mixture
 UV detectors are the most versatile having the best
sensitivity
 UV detector cannot be used for testing substances that
are low in chromophores (colorless or virtually colorless)
as cannot absorb light at low range
 They are cost-effective and popular and are widely used
in industry

2) Fluorescence detector
 It is based on the fluorescent radiation emitted light by some
compounds
 This is a specific detector that senses only those substances
that emit light,
 The excitation source passes through the flow cell to a photo
detector while a monochromatic measure the emission wave
lenghth
 More sensitive and specific.

 Refractive Index Detector
The Refractive Index Detector uses a monochromator and is
one of the least sensitive detector
This detector is extremely useful for detecting those
compounds that are non-ionic do not absorb ultraviolet light
and do not fluoresce,
These chemical components included alcohols, sugars, fatty
acids, polymers and carbohydrates

1) Mass Spectrometry
(MS) ionizes atoms or molecules to facilitate their separation and detection in
accordance with their molecular masses and charges (mass to charge ratio).
MS is used in various applications, e.g., biochemicals and atomic physics.
The main processes that take place in a mass spectrometer are:
i. Sample Introduction: sample is converted into a gaseous phase (except
with gaseous samples or samples that are thermally unstable) and is
introduced through the inlet to the ionization chamber
ii. Ionization: gaseous sample is ionized to generate cations (in most cases
but a few types of MS work with anions)
iii. Separation: ions separate according to their mass/charge ratio by a mass
analyzer
iv. Detection: a detector is used to determine the species and quantity of each
ion

Recorder
 Frequently called the date system the computer not only
controls all the modules of the HPLC instrument but it takes
the signal from the detector and use it to determine the time
of elution (retention time) of the sample components
(qualitative analysis) and the amount of sample ( quantitative
analysis)
 The concentration of each detected component is calculated
from the area or height of the corresponding peak and
reported

(4)Types of HPLC
Normal phase
Reverse phase
Size exclusion
Ion exchange

 Normal phase
 In this column type the retention is governed by the
interaction of the polar parts of the stationary phase
 non polar than the mobile phase with respect to the sample
organic solvents such as hexane and ethyl acetate.

 Normal phase
Most polar Least polar

Reverse phase
 In this column the packing material is relative nonpolar and the
solvent is polar with respect to the sample, retention is the
result of the interaction of the nonpolar components of the
solutes and the nonpolar stationary phage
 Typical stationary phase are nonpolar hydrocarbons, waxy
liquids or bonded hydrocarbons (such as C18 , C8 etc.) and
the solvents are polar aqueous organic mixtures such
methanol-water or acetonitrile- water
the most common organic solvents used are acetonitrile,
methanol, and tetrahydrofuran (THF).

Reverse phase
Most non polar Least non polar

Reverse phase
Starting in the mid 1970s Reversed Phase Liquid Chromatography
(RPC) has become the standard technique to analyze small
molecular weight compounds. More recently, RPC has become an
accepted tool for the separation of peptides, proteins and other
biopolymers in the pharmaceutical, chemical and biochemical
industries.
TSKgel Reversed Phase Columns
1 Silica particles are most commonly used as the support in TSKgel
reversed phase columns, which is then derivatized with
octadecylisilane (ODS, C18) or smaller hydrocarbon chains such as
C1 (Methyl), C4 (Butyl), C5 (Phenyl), or C8 (Octyl).
2 Polymer-based methacrylate C4H5O2
-
supports have been introduced as an alternative to TSKgel silica-
based reversed phase columns, particularly for analyzing basic
compounds in their neutral state at high pH.

1Silica-based Columns
The silica-based TSKgel reversed phase column line offers a
variety of stationary phased designed for the analysis of either
large molecules, such as peptides, proteins and oligonucleotides,
or low molar mass analytes, such as:
pharmaceutical drugs
forensic compounds
derivatized amino acids
carbohydrates
steroids, lipids, fatty acids
TSKgel silica-based reversed phase columns are grouped into 2
product families:
b)Specialty Silica
Columns
a)Monomeric Bonded
Silica (8 nm) »

a) 8 nm Monomeric RP Silica Columns
For analysis of small molecules, the 8 nm pore size, 5 micron
particle size TSKgel 80Tm and 80Ts columns are offered in 4
different chemistries to suit a variety of needs. Combined with
monomeric bonding chemistry, these TSKgel reveresed phase
columns have an established reputation as general purpose
columns for pharmaceutical applications
i) TSKgel CN-80TS
TSKgel CN-80TS is based on a high-purity, metalfree silica with
10 nm pore size bonded to C3CN groups. It is completely
endcapped and the resulting effective pore size is 8 nm (80 Å).
TSKgel CN-80TS is an alternative to C18 (ODS) and C8 (Octyl)
phases. The cyano group is the least hydrophobic of the 80TS
phases available and in some cases is used under normal phase
conditions.
Column: TSKgel CN-80Ts, 5 μm, 4.6 mm ID × 15 cm L
Mobile phase: 50% MeOH(Methanol,)
Flow rate: 1.0 mL/min
Temperature: 25 ˚C
Samples: 1. uracil; 2. benzene; 3. toulene; 4. napthalene

ii) TSKgel Octyl-80TS
TSKgel Octyl-80TS is based on a high-purity, metal free silica with 10
nm pore size bonded to C8 groups. It is completely endcapped and
the resulting effective pore size is 8 nm (80 Å). TSKgel Octyl-80TS
columns reduce tailing when analyzing basic compounds. They have
a lower carbon load and hydrophobicity than the corresponding ODS
products.
Column: TSKgel Octyl-80Ts, 5 µm, 4.6 mm ID × 15 cm L
Mobile phase: 20 mmol/L KH2PO4 (Potassium dihydrogen phosphate) MeOH = 5/5/1(v/v/v)
Detection: UV/
Temperature: 25 °C
Injection vol.: 4 µL
Samples: 1. pranlukast hydrate, 0.2 mg/L; 2. isoamyl p-oxybenzoate;
(4-hydroxybenzoic acid isoamyl ester), 0.2 mg/L

b) Specialty RP Silica Columns
There are three specialty silica-based TSKgel reversed phase
columns: TSKgel ODS-120A, TSKgel ODS-120T, and TSKgel
OligoDNA-RP.
i) TSKgel ODS-120A
TSKgel ODS-120A columns are based on a 15 nm pore size silica base
support bonded with ODS groups. The bonding method results in a
polymeric coverage of C18 groups on the silica surface and an effective
pore size of 12 nm (120 Å). TSKgel ODS-120A is not endcapped. The silica
base support’s exclusion limit of 1.0 × 10^4 Da makes them a good choice
for the reversed phase chromatography of peptides and small proteins.
Column: TSKgel ODS-120A, 4.6 mm ID x 25 cm
Gradient: 40 min linear from 75% MeOH/25% H2O to 95% MeOH/5% H2O,
5min hold
Detection: UV
Temperature: 40°C
Sample: 5 mL mixture of:
1. Naphthalene, 2. Acenaphthylene
3. Acenaphthene, 4. Fluorene,
5. Phenanthrene, 6. Anthracene,
7. Fluoranthene, 8. Pyrene,
9. Benzo(a)anthracene, 10. Chrysene,
11. Benzo(b)fluoranthene, 12. Benzo(k)fluoranthene,
13. Benzo(a)pyrene, 14. Dibenzo(a,h)anthracene,
15. Benzo(g,h,i)perylene, 16. Indeno(1,2,3-cd)pyrene

2) Polymer-based Columns
The polymethacrylate-based TSKgel reversed phase columns are
available in a range of pore and particle sizes. Key advantages of
these polymer-based columns include:
Chemically stable from pH 2 to 12, allowing many basic
compounds to be analyzed in their uncharged form, thus reducing
secondary adsorption and improving peak shape
Improved recovery for peptides and proteins due to reduced
secondary interactions
The TSKgel polymer-based reversed phase columns are an
excellent choice for large molecular weight (MW) biomolecules
(>10,000 Da) and for analyzing small MW compounds at high pH
and are offered in 4 different chemistries

Column: TSKgel Octadecyl-2PW, 5 µm, 4.6 mm ID × 15 cm
Mobile phase: 30 min linear gradient from 0.1% TFA/CH3CN
from 90/10 to 30/70
Detection: UV @ 215 nm
Temperature: ambient
Samples: 1. met-enkephalin, 2. bradykinin
3. leu-enkephalin, 4. neurotensin
5. bombesin, 6. angiotensin I
7. somatostatin, 8. insulin (bovine)
a) TSKgel Octadecyl-2PW methacrylate C4H5O2
-
TSKgel Octadecyl-2PW is based on a highly cross-linked polymethacrylate base
matrix bonded with octadecyl groups. The polymeric matrix provides excellent
stability in high pH buffer systems and can withstand rigorous cleaning with
either acid or base.
The 12.5 nm (125 Å) pore size of TSKgel Octadecyl-2PW columns makes them
ideally suited for peptides and small proteins. They demonstrate faster analysis
than other competitive reversed phase polymeric columns

b) TSKgel Octadecyl-4PW
TSKgel Octadecyl-4PW is based on a large pore, highly cross-linked
polymethacrylate base matrix bonded with octadecyl groups. The polymeric
matrix provides excellent stability in high pH buffer systems and can
withstand rigorous cleaning with either acid or base.
The large pore size of TSKgel Octadecyl-4PW columns, 50 nm (500Å), allows
unhindered access to proteins and other large molar mass biopolymers. The
particle size offerings allow for analytical and semi-preparative scale
separations.
Column: TSKgel Octadecyl-4PW, 5 µm, 4.6 mm ID × 15 cm
Mobile phase: A. 0.2% TFA, pH 1.9
B. 0.05 mol/L phosphate buffer, pH 7.1
C. 0.2 mol/L NH3, pH 10.8
Gradient: 50 min. linear gradient from 0% to 80% CH3CN
Samples: 1. met-enkephalin, 2. bradykinin
3. leu-enkephalin, 4. neurotensin
5. bombesin, 6. angiotensin I
7. somatostatin, 8. insulin

Size exclusion
 In size exclusion the HPLC column is consisted of
substances which have controlled pore sizes and is able to
be filtered in an ordinarily phase according to its molecular
size . The columns are packed with 2 μm silica-based
beads shielded with a hydrophilic mobile phase methanol,
acetonitrile
 Small molecules penetrate into the pores within the
packing while larger molecules only partially penetrate the
pores. The large molecules elute before the smaller
molecules.

Size exclusion
Larger molecules Smaller molecules

Size exclusion
size Exclusion Chromatography (SEC) separates molecules
based on their size, or more precisely, their hydrodynamic
volume. It is based on the discrimination of individual sample
components by the pores of the packing material. Large sample
molecules cannot or can only partially penetrate the pores and
elute from the column first, whereas smaller molecules can
access all or a larger number of pores and elute later. SEC is
the only mode of chromatography that does not involve
interaction with a stationary phase by means of adsorption or
partitioning of the solutes.
The terms SEC, GFC (gel filtration chromatography) and GPC
(gel permeation chromatography) all refer to the same
chromatographic technique. In GFC an aqueous mobile phase
is used, while an organic mobile phase is employed in GPC.
The general term SEC covers both uses.

Size exclusion
Applications
a) SEC is the dominant mode of separation for natural and synthetic
polymers
b) GFC is the term used for the size-based separation of water-
soluble polymers, for example biopolymers or natural polymers
c) GPC is the term used for the size-based separation of polymers
soluble in organic solvents
a) TSKgel SEC Columns
Size exclusion chromatography columns are traditionally
packed with porous polystyrene divinylbenzene (PS-DVB) or
silica particles. PS-DVB columns are commonly used for the
analysis of synthetic polymers in organic solvents, while silica-
based columns are used for the separation of biopolymers.

Size exclusion
b) Aqueous SEC - Gel Filtration Chromatography
Gel Filtration Chromatography (GFC) is popular among
biochemists for the isolation of proteins, for the removal of
aggregates, to desalt a protein sample, to separate nucleic acid
fractions, or to characterize water-soluble polymers used in food
products, paints, pharmaceutical preparations, etc
c) Organic SEC - Gel Permeation Chromatography
Gel Permeation Chromatography (GPC) plays an important role in
the characterization of industrial organic-soluble and polar
organic-soluble polymers in the consumer, chemical and
petrochemical industries.

Size exclusion
(b) Aqueous Size Exclusion - GFC
(c) Organic Size Exclusion - GPC
(i) TSKgel Gel Filtration Chromatography Columns
Gel Filtration Chromatography (GFC) is popular among
biochemists for the separation of proteins or nucleic acid
fractions, to desalt a protein sample, or to characterize water-
soluble polymers used in food products, paints, and
pharmaceutical preparations.
(1)TSKgel SW-type silica-based columns, first introduced in
1977, are the biopharmaceutical industry's standard in gel
filtration chromatography of biomolecules.
(2)TSKgel PW-type polymer-based columns are the ideal choice
for analyses of water-soluble polymers.

Size exclusion
Proteins
The new TSKgel UP-SW columns, packed with 2 micron silica-based
particles, are optimized for the UHPLC analysis of proteins, such as
monoclonal antibodies. They are the result of over 40 years of experience in
gel filtration technology and provide the same outstanding quality and
reproducibility known from the renowned TSKgel SW series.
Column: TSKgel UP-SW2000, 2 µm,
4.6 mm ID × 30 cm
Mobile phase: 100 mmol/L phosphate buffer, pH 6.7,
100 mmol/L Na2SO4, 0.05% NaN3
Instruments: Agilent 1100 with Chemstation v. 2.18.18
Temperature: 25 °C
Samples: Peptide 1: Myoglobin 16.7 kDa
Peptide 2: RPDFC LEPPY TGPCK ARIIR
YFYNA KAGLC QTFVY GGCRA KRNNF
KSAED CMRTC GGA (58 aa) 6.5 kDa
Peptide 3: QRLGNQWAVGHLM (14 aa)
1.6 kDa

The TSKgel SW mAb products consist of three specialized columns
designed for the analysis of monoclonal antibodies (mAbs).
Compared to competitive columns, these stainless steel, silica-
based TSKgel columns offer reduced lot-to-lot variation, long
column life, reduction of unspecified adsorption, and superior
recovery of aggregates.ng
Column: TSKgel SuperSW mAb HR, 4 μm, 7.8 mm ID × 30 cm
Mobile phase: 0.1 mol/L phosphate/0.1 mol/L sulfate buffer + 0.05% NaN3
Temperature: 25 ºC
Injection vol.: 10 μL
Sample: human IgG (4.6 g/L) from Sigma Aldrich

TSKgel SW, SWXL and SuperSW columns are silica-based columns
optimized for the separation of proteins and nucleic acids using an
aqueous buffer as the mobile phase. A hydrophilic diol-type bonded
phase shields the silica surface from interacting with protein samples.
Compared to competitive HPLC columns, TSKgel SW-type columns
contain more pore volume per unit column volume, which results in
higher molar mass (MM) selectivity or better resolution. The columns
within the TSKgel SW-type group differ in pore size, offering the
ability to separate a wide range of compounds of varying molar mass
Columns: TSKgel SWXL columns, 7.8 mm ID × 30 cm
Mobile phase: 0.3 mol/L NaCl in 0.1 mol/L sodium phosphate buffer, pH 7.0
Samples: 1. thyroglobulin (6.6 × 10^5 Da)
2. IgG (1.56 × 10^5 Da)
3. bovine serum albumin (6.7 × 10^4 Da)
4. ovalbumin (4.3 × 10^4 Da)
5. peroxidase (4.02 × 10^4 Da)
6. β-lactoglobulin (3.5 × 10^4 Da)
7. myoglobin (1.69 × 10^4 Da)
8. ribonuclease A (1.37 × 10^4 Da)
9. cytochrome C (1.24 × 10^4 Da)
10. glycine tetramer (246 Da)

Water-soluble polymers
The TSKgel PW columns contain particles of 12 and 17 µm and deliver high
resolution separations. TSKgel PWXL columns are of smaller particle size
than the TSKgel PW columns, offering higher efficiency for optimal
resolution.
The main application areas for TSKgel PW and PWXL columns are the
analysis of celluloses, acrylamides, glycols, dextrans, polyvinylalcohol, and
oligosaccharides. Both the TSKgel PW and PWXL column lines include a
mixed bed column, prepared by mixing particles of different pore sizes,
which results in columns with an extended linear calibration curve. These
columns are useful as scouting columns, to quickly categorize the molar
mass (MM) profile of an unknown sample. Additionally, mixed bed TSKgel
PW and PWXL columns are highly useful in the characterization of samples
having a very broad MM distribution.

Type of Polymer Typical Sample Suitable Eluent
Nonionic hydrophilic polyyethylene glycol distilled water
Anionic Hydrophilic
sodium chondroitin sulfate,
sodium alginate, carboymethyl
cellulose, sodium polyacrylate,
sodium hyaluronate
Buffer or salt solution (e.g. 0.1
mol/L NaNO3)
Anionic Hydrophobic
sulfonated lignin sodium salt,
sodium polystyrenesulfonate
Buffer or salt solution with
organic solvent (e.g. 20%
CH3CN in 0.1 mol/L NaNO3)
Cationic Hydrophilic
glycol chitosan, DEAE-
dextran, poly(ethyleneimine),
poly(trimethylaminoethyl
methacrylate) iodide salt
0.5 mol/L acetic acid with 0.3
mol/L Na2SO4 or 0.8 mol/L
NaNO3
Cationic Hydrophobic
poly(4-
vinylbenzyltrimethylammoniu
m chloride), poly(N-methyl-2-
vinylpyridinium) iodide salt
0.5 mol/L acetic acid with 0.3
mol/L Na2SO4
Amphoteric Hydrophilic
peptides, proteins, poly- and
oligosaccharides, DNA, RNA
Buffer or salt solution (e.g. 0.1
mol/L NaNO3)
Amphoteric hydrophobic
blue dextran, collagen, gelatin,
hydrophobic proteins,
hydrophobic peptides
Buffer or salt solution with
organic solvent (e.g. 20%
CH3CN in 0.1 mol/L NaNO3 or
35-45% CH3CN in 0.1% TFA)

Three specialty columns are available within the TSKgel PWXL column
line. TSKgel G-DNA-PW is designed for the separation of large
polynucleotides, such as DNA and RNA fragments of 500-5,000 base
pairs. TSKgel G-Oligo-PW is the preferred column for the analysis of
oligosaccharides. TSKgel SuperOligoPW contains smaller particles and
semi-micro dimensions compared to the TSKgel G-Oligo-PW column,
enabling high speed separations with high resolution of aqueous
oligomers. Columns:
Mobile phase:
Flow rate:
Detection:
Samples:
TSKgel PWXL columns, all 7.8 mm ID × 30 cm
1. G3000PWXL, 2. G4000PWXL,
3. G5000PWXL, 4. G6000PWXL, 5. GMPWXL
0.2 mol/L phosphate buffer, pH 6.8
1.0 mL/min
UV @280 nm
25 °C
a. thyroglobulin (6.6 × 105 Da)
b. g-globulin (1.5 × 105 Da)
c. albumin (6.7 × 104 Da)
d. ovalbumin (4.3 × 104 Da)
e. b-lactoglobulin (3.6 × 104 Da)
f. myoglobin (1.69 × 104 Da)
g. cytochrome C (1.24 × 104 Da)

TSKgel PWXL-CP columns were specifically designed for the
analysis of water-soluble cationic polymers. Cationic groups are
introduced on the surface of the TSKgel PWXL-CP packing
material to prevent undesirable interaction of cationic polymers
and allow elution under low salt conditions.
Columns: TSKgel G3000PWXL-CP, 7 µm, 7.8 mm ID × 30 cm
TSKgel G5000PWXL-CP, 10 µm, 7.8 mm ID × 30 cm
TSKgel G6000PWXL-CP, 13 µm, 7.8 mm ID × 30 cm
Mobile phase: H2O with 0.1 mol/L NaNO3
Flow Rate: 1 mL/min
Detection: RI
Temperature: 25 °C
Samples: polyethylene oxides (PEO) standards
polyethylene glycols (PEG) standards

TSKgel SuperMultiporePW columns are packed with particles
containing a wide range of pore sizes. The unique multi-pore
technology creates a linear calibration curve within each particle,
resulting in better accuracy for molar mass determination
Columns: TSKgel SuperMultiporePW-N, 6.0 mm ID × 15cm
TSKgel SuperMultiporePW-M, 6.0 mm ID × 15 cm
TSKgel SuperMultiporePW-H, 6.0 mm ID × 15 cm
Mobile phase: H2O
Detection: RI
Temperature: 25 °C
Samples: polyethylene oxides (PEO) standards
polyethylene glycols (PEG) standards
ethylene glycol (EG) standards

(2) Organic Size Exclusion - GPC
Gel Permeation Chromatography (GPC) plays an important
role in the characterization of polar organic-soluble and
organic-soluble polymers in consumer, chemical and
petrochemical industries.
TSKgel Alpha and TSKgel SuperAW columns are designed
for the GPC analysis of polymers of intermediate polarity.
These columns contain highly crosslinked hydroxylated
polymethacrylate particles which minimize swelling in polar
organic solvents.
TSKgel H series GPC columns are optimized for the
analysis of organic-soluble polymers and are packed with
porous spherical polystyrene divinylbenzene (PS-DVB)
particles. This series includes TSKgel HXL, HHR, SuperH,
SuperHZ, and SuperMultiporeHZ columns. Each line of
columns within this series differs in degree of inertness,
solvent compatibility and operating temperature range.

Polar Organic-soluble Polymers
The TSKgel Alpha and SuperAW columns offer excellent stability in
100% aqueous to 100% organic solvents. Both column types are
highly applicable for polymers solubilized in methanol, acetonitrile,
DMSO, isopropanol, THF and HFIP. Examples of applications
include cleansing gel in methanol, degree of saponification of
polyvinylalcohol in HFIP, and sodium dodecylsulfate, polyimide, as
well as cellulose derivatives in DMF with 10 mmol/L LiBr.
A mixed bed column is available in each of the TSKgel Alpha and
SuperAW column line, which is prepared by mixing particles of
different pore sizes, resulting in columns with an extended linear
calibration curve. These columns are useful to quickly categorize
the molar mass (MM) profile of an unknown sample and are used
when a sample has a very broad MM distribution.
For high throughput applications, the smaller dimensions of the
TSKgel SuperAW columns provide equivalent resolution in half the
time and with minimal solvent waste compared to the TSKgel Alpha
columns

Column: TSKgel SuperAW columns, 6.0 mm ID × 15 cm
Mobile phase: A. H2O
B. MeOH containing 10 mmol/L LiBr
C. DMF containing 10 mmol/L LiBr
Detection: RI
Samples: polyethylene oxide (PEO), polyethylene glycol
(PEG),
and ethylene glycol (EG) standards
Column: TSKgel Alpha columns, 7.8 mm ID × 30 cm
Mobile phase: A. H2O
B. MeOH containing 10 mmol/L LiBr
C. DMF containing 10 mmol/L LiBr
Detection: RI
Temperature: A. 25 °C B. 25 °C C. 40 °C
Samples: A, B: polyethylene glycol (PEG) and polyethylene
oxide (PEO) standards
C. polystyrene (PS) standards

Organic-soluble Polymers
The TSKgel HXL columns are conventional GPC columns of 7.8 mm
ID x 30 cm. These columns show true size exclusion behavior for most
polymers, have limited solvent compatibility and maximum operating
temperatures of 60 and 80 °C. Included in the TSKgel HXL column line
are three mixed bed columns and a multi-pore column. Mixed bed
columns contain particles with different pore sizes, providing an
extended linear calibration curve. The multi-pore column provides a
linear calibration curve due to each particle containing a range of pore
sizes. One of the mixed bed columns is available for high temperature
analysis up to 140 °C.

Column size: 7.8 mm x 30 cm
Sample: polystyrene standards
Eluent: THF
Temperature: 25°C
Detection: UV@ 254 nm
Shipping Solvent Can Be Replaced With
Tetrahydrofuran 2,3
benzene, chloroform, toluene, xylene,
dichloromethane, dichloroethane
Acetone**
carbon tetrachloride, o-dichlorobenzene,
dimethylformamide, dodecane, dimethyl sulfoxide,
dioxane, ethylacetate, FC-113, hexane, pyridine,
hexafluoroisopropanol/chloroform, methyl ethyl
ketone, quinoline, cyclohexane
Chloroform**
m-cresol in chloroform, up to 10%
hexafluoroisopropanol in chloroform
Dimethylformamide
dimethyl sulfoxide, dioxane, tetrahydrofuran,
toluene
o-Dichlorobenzene 1-chloronaphthalene, trichlorobenzene

TSKgel SuperHZ columns are designed for high throughput/high
efficiency GPC applications. These columns are based on the TSKgel
HXL column line, differing only in dimensions (4.6 and 6.0 mm ID x 15
cm) and particle size
Column: TSKgel SuperHZ series, 4.6 mm ID x 15 cm
Eluent: polyethylene, polypropylene
Temp: 25°C
Sample: standard polystyrene
Inj. volume: 2 µL

Ion exchange
 In this column type the sample components are separated
based upon attractive ionic forces between molecules
carrying charges groups of opposite charge to those
charges on the stationary phase.
 Typical applications are
 Separation of peptides and proteins
 Analysis of charge isomers of proteins
 Quality control of recombinant proteins, such as monoclonal
antibodies (mAbs)
 Separation of oligonucleotides, siRNA, PCR fragments and
related nucleic acids
 Analysis of sugars, amino acids, nucleic acid bases, and
small drug candidates

Ion exchange
Ion Exchange Chromatography
Separation in Ion Exchange chromatography (IEC or IEX) is based
on reversible adsorption of charged solute molecules to
immobilized functional groups of opposite charge. Biomolecules
generally have charged groups on their surfaces, which change
with the buffer pH. Elution can be accomplished by changing the
ionic strength or the pH, of which changing the ionic strength by
increasing the salt concentration is most common.
IEC is further subdivided into cation exchange and anion exchange
chromatography. Anion and cation exchange phases are classified
as strong or weak, depending on how much the ionization state of
the functional groups vary with pH. A strong ion exchange phase
has the same charge density on its surface over a broad pH range,
whereas the charge density of a weak ion exchange phase
changes with pH, affecting its selectivity, which differs at different
pH values.

Ion exchange
Applications
Being a non-denaturing technique, IEC is one of the most frequently
used modes in the separation of biomolecules. Proteins have
numerous functional groups that can have both positive and negative
charges. IEC separates proteins according to their net surface charge,
which is dependent on the pH and ionic strength of the mobile phase
TSKgel Ion Exchange Columns
Bioscience offers a broad line of high efficiency columns for anion as
well as cation exchange chromatography. The product line
contains polymethacrylate- and silica-based columns for the analysis
of proteins, peptides, and nucleic acids. Polystyrene divinylbenzene-
based ion exchange columns are available for analyzing low molecular
weight sugars and drug candidates, as well as amino acids.
Hydrophilic polymer-based columns packed with nonporous resin
particles are available for the separation of protein, protein aggregates,
PEGylated proteins and charge isomers of mAbs.

Ion exchange
Anion Exchange Chromatography
Anion exchange chromatography is practiced with either a
strong or a weak anion exchange column, containing a
quarternary ammonium ion, or with a weak anion exchanger,
having either a tertiary or secondairy amine functional group,
such as DEAE (diethylaminoethyl). A counter ion, often Cl-,
maintains electroneutrality.
In ion exchange chromatography the pH of the mobile phase
buffer must be between the pI or pKa of the charged molecule
and the pKa of the charged groups on the solid support. For
instance, in anion exchange chromatography a molecule with a
pI of 6.8 is run in a mobile phase buffer at pH 8.0 with the solid
support pKa at 10.3

TSKgel Anion Exchange Chromatography Columns
Very efficient chromatography can be achieved with the nonporous TSKgel STAT columns
due to the novel bonding chemistry and the absence of micro-pores. Applications for the
TSKgel Q-STAT columns include the separation of: proteins, peptides, low molecular weight
nucleic acids, aggregates and charge isomers of monoclonal antibodies, PEGylated proteins,
oligo DNA, and siRNA. The TSKgel DNA-STAT column is ideal for the analysis of DNA
fragments, nucleic acids and nucleotides.
The pore structure and bonding chemistry of TSKgel BioAssist Q columns provides high
capacity for small to very large molar mass proteins and nucleic acids.
Columns: A: TSKgel BioAssist Q, 10 µm, 4.6 mm ID × 5 cm
B: Commercial Q-type product A, 5.0 mm ID × 5 cm
Mobile phase: 15 min linear gradient of NaCl from 0 to 1.0 mol/L
in 20 mmol/L Tris-HCl buffer, pH 8.0
Temperature: 25 ºC
Sample: mouse ascites fluid (3-fold dilution with initial eluent)

Proteins, peptides, DNA and RNA-derived oligonucleotides, and
other nucleic acid fragments are typical samples that are analyzed
or isolated on the TSKgel DEAE-5PW and TSKgel Super Q-
5PW columns.
Column: TSKgel DEAE-5PW, 10 µm, 6 mm ID × 15 cm (custom)
Mobile phase: 300 min linear gradient from 0.3 mol/L to
1.0 mol/L NaCl in 0.1 mol/L Tris-HCl, pH 7.6
Sample: total E. coli RNA
TSKgel DEAE-NPR nonporous columns are used for the high speed
separation and analysis of proteins and poly and oligonucleotides.
Applications for the TSKgel DNA-NPR nonporous columns include the
high efficiency separation of DNA fragments, PCR products, or
plasmids
Column: TSKgel DEAE-NPR, 2.5 µm, 4. 6 mm ID × 3.5 cm,
with guard column, 4.6 mm ID × 0.5 cm
Mobile phase: A: 0.02 mol/L Tris-HCI, pH 9.0
B: mobile phase A plus 1.0 mol/L NaCl
Gradient: 15 min linear gradient from 48% to 65% mobile phase B
Pressure: 14 MPa
Temperature: 40 ºC
Sample: Hae III digest of pBR322 DNA,
(base pair number for each peak is indicated

TSKgel DEAE-2SW and TSKgel QAE-2SW (both 5 µm, 12.5 nm) and
TSKgel DEAE-3SW (10 µm, 25 nm) are available for analyzing smaller
molar mass samples such as nucleotides, drug candidates,
catecholamines, and small peptides or proteins.
Column: TSKgel DEAE-2SW, 5 µm, 4.6 mm ID × 25 cm
Mobile phase: A: CH3CN in 0.1 mol/L phosphate, pH 3.0, 20/80
B: CH3CN in 0.5 mol/L phosphate, pH 3.0, 20/80
Gradient: 30 min linear gradient from buffer A to B
Samples: 1. AMP 2. IMP 3. GMP 4. ADP 5. ATP

TSKgel Sugar AXG columns are packed with 10 µm beads and are
used for the gradient separation and analysis of monosaccharides,
disaccharides, and sugar alcohols.
For isocratic separation of carbohydrates where lower and constant
back pressures may be generated, the 8 µm TSKgel Sugar
AXI column is optimal.
Column: TSKgel Sugar AXG, 10 µm, 4.6 mm ID × 15 cm
Mobile phase: 0.7 mol/L borate buffer, pH 8.6
Detection: RI
Temperature: 65 ºC
Samples: 1. alpha-D-glucopyranosyl-1,6-soribitol (GPS)
2. alpha-D-glucopyranosyl-1,6-mannitol (GPM)

Cation Exchange Chromatography
Cation exchange chromatography is practiced with either a strong or a
weak cation exchange column, containing a sulfonium ion, or with a
weak cation exchanger, having usually a carboxymethyl (CM)
functional group. A counter ion, often Na+, maintains electroneutrality.
In ion exchange chromatography the pH of the mobile phase buffer
must be between the pI or pKa of the charged molecule and the pKa of
the charged groups on the solid support. For example, a molecule with
a pI of 8.2 is run in a mobile phase buffer at pH 6.0 with the solid
support pKa at 1.2 in cation exchange chromatography.

TSKgel Cation Exchange Chromatography Columns
Very efficient chromatography can be achieved with the nonporous
TSKgel STAT columns due to the novel bonding chemistry and the
absence of micro-pores. Applications for the TSKgel CM-STAT and TSKgel
SP-STAT columns include the separation of proteins, protein
aggregates,charge variants of monoclonal antibodies, PEGylated proteins,
and peptide digests.
The pore structure and bonding chemistry of TSKgel BioAssist S columns
provides high capacity for medium to large molar mass proteins.
Columns: A: TSKgel BioAssist S, 7 µm, 4.6 mm ID × 5 cm
B: Conventional S type product C, 5.0 mm ID × 5 cm
C: Conventional S type product D, 4.6 mm ID × 5 cm
Mobile phase: A: 20 mmol/L sodium phosphate buffer, pH 6.5
B: 20 mmol/L sodium phosphate buffer containing 1.0 mol/L NaCl, pH 6.5
Gradient: 32 min (A-B)
Flow Rate: 0.8 mL/min
Temperature: 10 °C
Samples: 1. myoglobin, 1 g/L
2. α-chymotrypsinogen A, 2 g/L
3. ribonuclease A, 4 g/L
4. cytochrome C, 2 g/L
5. lysozyme, 2 g/L

TSKgel CM-5PW and TSKgel SP-5PW columns are an excellent
choice for analyzing biologically active molecules.
Columns: TSKgel SP-5PW and TSKgel CM-5PW, 10 µm, 7.5 mm ID × 7.5 cm
Mobile phase: 60 min linear gradient from 0 mol/L to 0.5 mol/L NaCl
in 0.02 mol/L phosphate, pH 7.0
Samples: 1. trypsinogen, 2. ribonuclease, 3. α-chymotrypsinogen
4. cytochrome C, 5. lysozyme

TSKgel SP-NPR nonporous columns are used for the separation and
analysis of proteins and peptides. They are particularly useful for
adeno-associated viruses and other large macromolecules.
Column: TSKgel SP-NPR, 2.5 µm, 4.6 mm ID × 3.5 cm
Mobile phase: A. 50 mmol/L HEPES, 1 mmol/L EDTA, 5 mmol/L MgCl, pH 7.5
B. 50 mmol/L HEPES, 1 mmol/L EDTA, 5 mmol/L MgCl,
pH 7.5 with 0.5 mol/L NaCl; linear gradient from 20% to 100%B
in 10 column volumes
Gradient: 0 min (0%B) 2 min (100%B)
Flow rate: 1 mL/min
Sample: purified adeno-associated virus

TSKgel CM-2SW and TSKgel SP-2SW (both 5 µm, 12.5 nm) and
TSKgel CM-3SW (10 µm, 25 nm) are available for analyzing smaller
molar mass samples such as nucleotides, drug candidates,
catecholamines, and small peptides or proteins.
Column: TSKgel SP-2SW, 5 µm, 4.6 mm ID × 25 cm
Mobile phase: 20%CH3CN in 0.2 mol/L phosphate, pH 3.0
Samples: 1. paraquat, 5 g/mL
2. diquat, 5 g/mL

TSKgel SCX columns are used for the separation and analysis of
organic acids, saccharides and alcohols.
Column: TSKgel SCX(H+), 5 µm, 7.8 mm ID × 30 cm × 2
Mobile phase: 0.05 mol/L HClO4
Detection: UV @ 210 nm, RI
Samples: 1. maltose, 2. glucose, 3. fructose
4. lactic acid, 5. acetic acid, 6. methanol
7. ethanol, 8. butyric acid

(6)Application
• Drug discovery
• Clinical analysis
• Forensic chemistry
• Drug metabolism study
• Envirornmental chemistry
• Diagnostic studies
• Cosmetic studies
• Biochemical genetics

ADVANTAGES
 Separation of volatile and non- volatile components
 Quick analysis
 High resolution
 More reproducibility
DISADVANTAGES
 High cost
 Complex to operate

(7)Conclusion of HPLC
 HPLC is a form of liquid chromatography used to
separate compounds that are dissolved in solution
 The different component in the mixture pass through the
column at differentiates due to differences in their
partition behavior between the mobile phase and the
stationary phase

o Introduction of HPLC – Agilent Technologies
o http://elchem.kaist.ac.kr/vt/chem-ed/sep/lc/hplc.htm
o Skoog, Holler, and neiman. Principles of Instrumental
analysis. 5th ed. Orland: Harcourt & Co, 1998
o https://www.separations.us.tosohbioscience.com/solutions
/hplc-columns/ion-exchange/anion-exchange
(1) Kampen WU, Voth M, Pinkert J, Krause A. Therapeutic status
of radiosynoviorthesis of the knee with yttrium [90Y] colloid inrheumatoid
arthritis and related indications. Rheumatology (Oxford)2007;46:16.
(2) Paganelli G, Bartolomei M, Grana C, Ferrari M, Rocca P, Chinol
M.Radioimmunotherapy of brain tumour. Neurol Res 2006;28:518
(3) Davies AJ. Radioimmunotherapy for B-cell lymphoma: Y-
90ibritumomab tiuxetan and I-131 tositumomab. Oncogene 2007;26:3614
(4) Marhol M. Ion exchangers in analytical chemistry. Prague: AcademiaPraga;
1982, pp. 25.
(5) Pillay KKS. A review of the radiation stability of ion exchangematerials. J
Radioanal Nucl Chem 1986;102:247.

H.p.l.c. High performance liquid chromatography

H.p.l.c. High performance liquid chromatography

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