Chromatography Techniques

Advances & Advantages in Analytical

Chemistry

Ms. Gnana Prasanna

Asst. Manager -Application Support
Spinco Biotech Pvt. Ltd.
Hyderabad
 Chromatography Techniques

o Concept of Chromatography

o Various Types of
Chromatography Techniques
o Principle & Instrumentation)
o HPLC, UFLC, LCMS, GC
& GCMS

o Applications
 Concept of Chromatography
• Chromatography is an analytical method that the
compounds are physically separated prior to measurement

• The main purpose of chromatography is to separate and

quantify the target sample in the matrix

Liquid
Chromatography

Gas
Chromatography
Chromatography

Supercritical-fluid
Chromatography
 History of chromatography
M. Tswett : first developer of chromatography

Petroleum ether

Chlorophylls

CaCO3
 Separation Mechanism

Compounds are separated because the molecules

move at different rates in the column.

1
2

column
 Separation Mechanism
Due to different interaction between stationary phase
and different sample, the molecules move at different
rate, therefore separation can be done.
Mobile Phase

1
2

Stronger Weaker
interaction interaction

Stationary Phase
 Chromato. . . ???
・ Chromatography
　　　 Method for Separation
・ Chromatograph
　　　 Instrument for Chromatography tR : Retention time
A : Area
・ Chromatogram h : Height

　　　 Data of Chromatography tR

Peak

Signal
. Chromatographer
h
Analyst A

Time
 Why use HPLC?
• Simultaneous Analysis
• High Resolution
• High Sensitivity (ppm-ppb)
• Good repeatability
• Small sample size
• Moderate analysis condition
- no need to vaporize the sample like GC
• Easy to fractionate the sample and purify
• No destructive for many detectors
 Scope of HPLC
Field Typical mixtures
Antibiotics, sedatives, steroids, analgesics, crude
Pharmaceuticals
drugs, cosmetics
Amino acids, proteins, peptides, carbohydrates,
Biochemical
lipids, enzymes, medicines, hormone
Mycotoxins, additives, saccharides, amino acids,
Food products
vitamins, fatty acid, coloring agents, antibacterials
Industrial Condensed aromatics, surfactants, propellants,
chemicals dyes, polymers, plasticizers
Forensic chemistry Drugs, poisons, blood alcohol, narcotics
Environmental Inorganic ions, organic acids, agricultural
field chemicals, pesticides, herbicides, phenols,
Bile acids, drug metabolites, urine extracts,
Clinical medicine
estrogens
 Separation Modes

• Normal phase chromatography

• Reversed phase chromatography

• Ion chromatography

• Size exclusion chromatography

• Affinity chromatography
 Scope of HPLC
106

Gel Size Gel

105
permeation exclusion filtration

Molecular weight
104

103
Normal Reversed Ion
phase phase exchange
102

Nonionic polar

Nonpolar Ionic
Water-insoluble Water-soluble

Increasing polarity
 Instrumentations

Modular HPLC
• Possible configurations
• Solvent delivery pumps
• Sample injectors
• Column ovens
• Detectors
Integrated HPLC
• LC-2010
 Isocratic System

Column
Detector
Injector
Pump Oven

Mobile Phase
Data
processor

Simple system with one pump and one solvent reservoir.

If more than one solvent is used, solvents should be premixed.
 Low-pressure Gradient System

Column Detector
Injector
Pump Oven
low pressure
gradient valve Data
processor

•One pump used to control 4 reservoirs;

•Mixing is done before pump.
•On-line degasser is necessary.
A B C D
 High-pressure Gradient System
A

pump

Mixer
B
Column Detector
pump Injector
Oven
Data
processor
C

pump • Excellent gradient accuracy.

• 2-3 pumps required - one pump per solvent used.
• On-line degassing may not be critical.
 Flexibility
 Modular type provides excellent flexibility.

Isocratic system Fully automated gradient system

 Outline of LC-2010
Reservior Tray

System Controller
Auto sampler

Column Oven
UV detector
Degassing Unit
Pump Unit

Low pressure
gradient device
 Detectors for HPLC

• UV-VIS Ultraviolet / Visible detector

• PDA Photodiode Array detector
• RF Fluorescence detector
• CDD Conductivity detector
• RID Refractive Index detector
• ECD Electrochemical detector
• ELSD Evaporative light scattering detector
• MS Mass spectrometer detector
Ultraviolet / Visible Detector
Advantage:
• Sensitivity is high
• Relative robust to temperature and flow rate change
• Compatible with gradient elution

Disadvantage:
• Only compounds with UV or visible absorption could
be detected.
Additional Functions
• Dual Wavelength mode
• Wavelength Time Program mode
• Wavelength Scan mode
 Photodiode Array Detector

Sample Cell Grating

One element detects

D2 / W lamp one absorbance at
one wavelength.

512 Elements Photodiode Array

 Photodiode Array Detector
3-D Data
Spectrum

Chromatogram
Absorbanc

gt
e

len
h ave
W
Time
 PDA Detector
Advantages:
• PDA Detector could analyze a sample simultaneously at many
different wavelengths.
• UV Visible spectra are useful for compound identification, checking
peak purity, as well as finding the optimum absorbance for the
compounds.
• UV Visible spectra of many compounds could be stored in the
spectrum libraries, which are useful for compound identification.
• Relatively robust to temperature and flow rate fluctuations
• Compatible with gradient elution.

Disadvantages:
• Slightly less sensitive than UV-Visible detector.
 Fluorescence of Compounds
Fluorescence is a type of luminescence in which the light
energy is released in the form of a photon in nanoseconds to
microseconds
Non-radiation transition
S1
Non-radiation transition
T1

Light absorption

Fluorescence
Phosphorescence

S0
Fluorescence Detector
 Fluorescence
Detector
Advantage
• Sensitivity is higher than UV-Vis detector
• Selectivity is high because relatively few compounds
fluorescence
• Compatible with gradient elution

Disadvanage
• Difficult to predict fluorescence
• Greatly affected by environment
– Solvent
– pH
– Temperature
– Viscosity
– Ionic strength
– Dissolved gas
 Refractive Index Detector
(1)

Photodiode
Reference Refraction

W Lamp
Sample
 Refractive Index Detector

Advantage
Responds to nearly all solutes
Unaffected by flow rate

Disadvantage
Not as sensitive as most other types of detectors
Could not be used with gradient elution
 Refractive Index Detector
Application Example
• Analytical Conditions
– Column : Shim-pack CLC-NH2
– Mobile phase : Acetonitrile / water
= 70/30
– Flow rate : 1.0 mL/min
– Temperature : Ambient
• Peaks
1. Glycerol
2. Xylose
3. Fructose
4. Glucose
5. Sucrose
6. Manose
7. Lactose
 Conductivity Detector
Principle

V K (conductivity) = I [A] / E [V]

=A [cm2] / L [cm] * k
I (k : specific conductivity)

k= (I/E)*(L/A)

A A

Electrodes L
 Conductivity Detector

Advantages:
• Respond to ionic compounds and suitable for ion
chromatography.
• High sensitivity for low concentration range

Disadvantages:
• Sensitive to the fluctuations in the solvent flow and mobile phase
composition
• Not compatible with gradient elution.
 Application Example (Anions)
• Analytical Conditions
– Column : Shim-pack IC-A3
– Mobile phase :
8.0 mM p-hydroxybenzoic acid
3.2 mM Bis-Tris *
– Flow rate : 1.5 mL/min
– Temperature : 40ºC
– Injection Volume : 100 µL
• Peaks
– 1. F- (1.4 ppm)
– 2. Cl- (10200 ppm)
– 3. NO2- (10 ppm)
– 4. Br- (43 ppm)
– 5. NO3- (44 ppm)
– 6. SO42- (431 ppm)

Bis-Tris : bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane

Application Example (Cations)

 Analytical Conditions
 Column : Shim-pack IC-C3
 Mobile phase : 2.0 mM Oxalic Acid
 Flow rate : 1.0 mL/min
 Temperature : 40ºC
 Injection volume : 100µL
 Peaks
 1. Na+ (8.25 ppm)
 2. NH4+ (0.01 ppm)
 3. K+ (1.66 ppm)
 4. Mg2+ (2.22 ppm)
 5. Ca2+ (11.85 ppm)
 Electrochemical Detector

Working
electrode
Reference
electrode

AUX electrode
 Principle of ECD Detection
Electrochemical detector responds to compounds that can
be oxidized or reduced, such as phenols, aromatic amines,
ketones, aldehydes.

R O + H+

e- Electrode
Glassy Carbon (GC)
Pt, Ag, Au
[ Applications ]
GC : phenol compounds
general use A
Pt : H2O2
Ag : halogen ion
Au : sugar analysis
 Electrochemical
Detector
Advantages:
• Selective as relatively few compounds are electro-active.
• Excellent sensitivity for low concentration range.

Disadvantages:
• Sensitive to temperature and flow rate fluctuations
• Not compatible with gradient elution.
• Aqueous or other polar solvents containing dissolved
electrolytes are required and they must be rigorously free from
oxygen.
Application Example
(catecolamines)
 Analytical Conditions
 Column : Shim-pack CLC-ODS
 Mobile phase :
80 mM phosphate buffer (pH=2.7)
100 mM NaNO3, 200 mg/l SOS
5 mg/l EDTA, 4 % acetonitrile
 Flow rate : 1.0 mL/min
 Applied Potential : + 0.8 V
 Temperature : 40 C
 Injection volume : 10 uL
 Peaks
 1. Noradrenalin ( 5 ppb)
 2. Adrenalin (5 ppb)
 3. Dopamine (5 ppb)
 Detector

Detection Pinciple

Three steps

• Nebulization

• Evaporation

• Detection
ELSD responds to compound that is less
volatile than that of the mobile phase

Shimadzu ELSD-LT
 Evaporative Light Scattering
Detector
Mobile
phase
PC Light

Gas
Detection

Nebulization
Evaporation
 Applications of ELSD

Food
（ Saccharides, fatty acids ）

Chemical Industry Pharmaceutical

（ Polymers, surfactants ） （ Impurities ）
 Evaporative Light Scattering
Detector
Advantages:
• Most compounds can be detected (universal detector)

• Compatible with gradient elution

Disadvantages:
• Mobile phase must be volatile.

• Nebulizing gas is required.

 　 Single Quadrupole LC/MS
System
HPLC Interface MS
Ionization probe Q-array Octopole Quadrupole Detector

Atmospheric
Pressure 10-3 ～ 10-4 Pa
80 ～ 150
Pa
TMP １ TMP ２

Rotary Pump
 Interface of LC-MS
Key Technology

HPLC Interface MS

 Aqueous/organic  To Remove solvent  High vacuum

solvent with buffers
 To Ionize analyte
 Non-volatile compounds molecules  Analyze ions, m/z

 Research on interfacing HPLC to MS began in the 1970s; API

(atmospheric pressure ionization) sources were commercialized in
1987.

 API interfaces: electrospray ionization (ESI) and atmospheric

pressure chemical ionization (APCI)
 Principles of ESI
Electro Spray Ionization

Vacuum
Drying
Nebulizing gas
gas

3-5 kV
Ions
HPLC
0.001-1 ml/min

ESI probe
+ ++ +
+ + ++++ ++
+ + + + +
++ + [M+H]+
++ +++ ++

 Ionization in liquid phase

 Ionization at room temperature
 Principles of APCI
Atmospheric Pressure Chemical Ionization
Heater (400oC)
Drying
Vacuum
Nebulisin gas
g gas
Corona
discharge
3-5 kV
HPLC
Ions
0.05 - 2 ml/min

ESI probe
Discharge to form primary ion:
N 2  N2 +
Gas phase ion – molecule reaction
with charge or proton transfer

 Evaporate LC elute into gas phase by a heater (400oC)

 Ionization in the gas phase by discharge, ion-molecule reaction
 Ionization diagram

Molecular
Weight

10,000 ESI

1,000 APCI

APPI
100 EI (GCMS)

Non-polar Very polar

polarity

- ESI has been most widely used in various LC-MS systems. More reference data are available
from open literature.
- APCI is chosen when its ionization effect is significantly better than ESI in certain analysis. “It is
difficult to generalize which class of compounds can be ionized by which probe, because there
are many exceptions.” (Britt E. Erickson, Today’s Chemist Feb 2001)
- APPI is chosen only when ESI and APCI could not ionize target compounds effectively.
 Ion Detector

Electron Multiplier

1. A series of dynodes maintained at

ever-increasing potentials
2. Ions strike the dynode surface,
resulting in the emission of
electrons.
3. These secondary electron are then
attracted to the next dynode where
more secondary electrons are
generated
4. ultimately resulting in a cascade of
electrons
 Ionization of Compounds in MS
Detector
• ESI
– drugs and their metabolites
– peptides
– proteins
– many kinds of natural product
(-OH, -NH2,-COOH, SO2, PO3 etc.)

• APCI
– pesticides
– steroids
– drugs
 What kind of benefits LC/MS users can
get ?
• Determination of MW
• Qualitative capability
• Selective quantitative
capability
m/z=100
• High sensitivity
A
B
TIC
A:100
B:100
D:150
C:150
m/z=150

C D
 Selection of Detectors

Detectors Type of compounds can be detected

UV-Vis & Compounds with chromophores, such as aromatic

PDA rings or multiple alternating double bonds.

Fluorescent compounds, usually with fused rings or

RF
highly conjugated planar system.

Charged compounds, such as inorganic ions and

CDD
organic acid.

For easily oxidized compounds like quinones or

ECD
amines.
For compounds that do not show characteristics
RID & ELSD usable by the other detectors, eg. polymers,
sccharides.
 Ultra Fast Liquid Chromatography
Advance in HPLC Technology Ultra Fast Liquid Chromatograph
Prominence UFLC Achieving higher speed without compromising quality

• Faster Faster Faster

• - More speed
•  　 Shorter run time while
• maintaining resolution
» - Higher linear velocity
» - Shorter column length

• ◆ Better Better Better

• - More resolution
–  　 Better resolution while
0.0 3.0 6.0 9.0 12.0 15.0
– maintaining run time
• - Higher theoretical plate
• numbers
• - Higher selectivity
• Goal: Enhancing Productivity

0.0 0.5 1.0 1.5 2.0 2.5

 Fast LC Application
◆ PTC-amino acids
→ 　 6 times faster than conventional column (5 m ， 4.6 mm i.d.  150 mm) 　　
→ 　 2 times faster than high speed column (3 m, 4.6 mm i.d.  75 mm)

mAU
250
1. Asp 2. Glu 3. Ser 1. Formaldehyde
4. Gly 5. His 6.Arg
18
2. Acetoaldehyde
7. Thr 8. Ala 9. Pro 3. Acetone
200 10. Ammonia 11. Tyr 12. Val 4. Acrolein
5. Propionaldehyde
13. Met 14.Cys-Cys 15. Ile
16. Leu 17. Phe 18. Lys 14 6. Crotonealdehyde
7. 2-Butanone
150 (each 400 pmol/1L) 11 12 15 16 8. Methacrolein
9
1 5 6 13 9. 17
n-Butylaldehyde

34 78 10. Benzaldehyde
11. Valeraldehyde
100 12. m-Tolualdehyde
2 13. Hexaldehyde
(each 120 nL/4L)

50 10

0
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 min

Chromatographic conditions; column: Shim-pack XR-ODS (2.2 m, 3.0 mm i.d.  75 mm), mobile phase: 10 mMol/L
(potassium)phosphate <pH 7.0> for A and acetonitrile for B (A/B: 95/5 for 0.3 min then 95/5 to 60/40 in 3.4 min),
flow rate: 1.2 mL/min, temperature: 40 C, detection: absorbance at 254 nm.
 Gas Chromatography (GC) & Gas
Chromatography Mass Spectrometry
(GCMS)
 What is Gas Chromatography?
•Gas Chromatography (GC) is an analytical Method where the
compounds in the sample are physically separated prior to

Measurement

What samples can be analyzed by GC?

•Organic Compounds, in general

•vaporable at 400deg C
•
 Typical GC System

A GC comprised of five main parts

Flow
Controller Injector Data
Detector Processor
Carrier gas
cylinder

Column / Oven
 Columns
• Column Materials

• Tubing
– Metal : SS, Cu, Al, Ni
– Glass : Pyrex, Fused silica
– Silco steel
– Teflon

• Packing Material
– Adsorbent (GSC)
– Dispersing (GLC)
 Type of Columns

Packed Capillary

I. D. 2~4 mm 0.05 ~ 1.00 mm

Length 0.5~10 m 10 ~ 105 m
Total plates ~4,000 ~100,000
Plate/meter ~1,500 ~1,500
Capacity ~10 g/peak ~50ng/peak
Film thickness 1~10 m 0.1~1 m
Phase ratio 5~50 50~500
Advantages of Capillary Column
 High resolution
 Short analysis time
 High detection limit
 Stable
 Thermally stable - Low bleeding
 Chemically stable - Solvent wash

 Inertness
 Much easier to connect MS
Short Analysis Time

Broad peaks
Incomplete separation

Sharp peaks
Complete separation

Shorten analysis time

 Injection System

• Direct injection system

– Packed Column injection system
– Wide bore capillary injection system

• Split injection system

• Splitless injection system

 Carrier Gas
 Gas used as a mobile phase
– Inert
– Minimize gaseous diffusion
– Pure
– Inexpensive
– Detector compatibility

 e.g N2, He, H2, Ar and etc.

 Purity and Flow Rate of
Carrier Gas
 The higher purity of the carrier gas will prolong the column
life and improve the detector sensitivity
 Purity
– Hydrogen > 99.9995%
– Helium > 99.9995%
– Nitrogen > 99.9995%
 Compress gas cylinder are usually certified only down to
10% of the original pressure
 Gas cylinders should be changed when they reach 10% of
their original pressure
 Recommended flow rate of carrier gas
– packed column 30 ~ 60 ml/min
– capillary column 0.5 ~ 20 ml/min
 Traps Installation
Location
Traps
Traps
GC GC

Traps

GCS
 Gas Chromatography :
Detectors
• Flame Ionization Detector (FID)
• Thermal Conductivity Detector (TCD)
• Flame Thermionic Detector(FTD)
• Flame Photometric Detector(FPD)
• Electron Capture Detector (ECD)
• Mass Spectrometer Detector (MSD)

TCD, FID, MSD are general detectors.

 FID (Flame Ionization
Detector)
• Respond to almost of organic compounds ( containing carbon)
<exception>
There is no response to C of carbonyl group or carboxyl group (C=O).
(CO, CO2, HCHO, HCOOH, etc.)

HCHO(Formaldehyde) CH3CHO(Acetaldehyde)

H-C-H CH3 - C - H
O No response O Response

• The main examples of application Analysis of organic compounds

 FID (Flame Ionization
Detector)

to Data processor
Organic compounds burn in hydrogen flame
and several ppm of those become the
Collector following ions.
Oxidation
Hydrogen CH CHO+ + e-
flame (O)
High Oxidation
voltage CN NO+ + CO + e-
2(O)

Air
Quartz
nozzle Hydrogen （ + Makeup
gas ）
Electric current flows when ions
Column are collected by collector
outlet
 Detector)
• Detection of all compounds except carrier gas is possible
• Helium is mainly used as carrier gas
　 (However, when analyzing helium and H2, N2 and Ar are used)
-->Thermal Conductivity Constant （ 10-6cal/sec ・ cm ・ degree C)

　　　 He:408 　　 H2 ： 547 　（ Very high ）

　　　 N2:73 　　 Ar:52 　　 O2:76 　　 H2O:60 Ethane:77
　　　 Methanol:52 　　 Acetone:40 　　 Chloroform:24 　 ...
• The main examples of application:
Gas analysis
Analysis of compounds undetectable by FID, such as water,
formaldehyde, and formic acid
FTD (Flame Thermionic Detector)
NPD (Nitrogen Phosphorus Detector)
• The detector which has high selectivity and high sensitivity
to organic nitrogen compounds, and to inorganic or
organic phosphorus compounds
(In the selectivity to the phosphorus compounds, FPD is
better.)

• No response to inorganic nitrogen compounds

• The main examples of application

Pharmaceuticals analysis, Organo-nitrogen pesticides /Organo-
phosphorus pesticides analysis
 FTD (Flame Thermionic Detector)
NPD (Nitrogen Phosphorus Detector)

to Data processor
When the platinum coil to which rubidium sulfate

controller
Power
adhered is heated by the electric current, plasma-
like atmosphere is generated into the surrounding
of rubidium sulfate. In this atmosphere, Rb*
(rubidium radical) is generated, and -CN and -
Collector PO2(generates by the oxidation of organic
phosphorus compounds) react as follows with
Rb2SO4 bead Rb*, and become ions.

High
CN + Rb * CN - + Rb +
voltage PO2 + Rb * PO2 - + Rb +
Quartz Air
nozzle Hydrogen （ + Makeup
gas ） Electric current flows when
ions are collected by collector
Column outlet
 ECD (Electron Capture Detector)

• The detector which has high selectivity and high sensitivity

to electrophilic compounds
(organic halogen compounds, organic metal compounds, and diketones)

• Since detector is equipped with radioisotope, the use

notification to Ministry of Science and Technology is
required in the case of installation.
• Main examples of application
Environmental analysis
Chlorine pesticides residue
•PCB residue Chlorine VOC in waste water
Organic mercury in environment
 ECD (Electron Capture Detector)

to Data processor
Collector
N2 used as carrier gas (makeup
gas) is ionized by the beta ray
Exhaust emitted from 63Ni.
 ray
N2 N2+ + e-
Electric current flows when electrons are
captured by collector （ Initial current ）

If electrophilic compounds enter then

63
Ni PCB + e- PCB-
Makeup gas Radiation Since PCB- is very greatly heavy compound
source
compared with e-, it requires time before
10mCi
reaching collector. --> 　 Ion current decreases.
Column
outlet
 FPD (Flame Photometric
Detector)
• The detector which has high selectivity and high sensitivity to
Phosphorus(P)compounds/Sulfur(S)compounds/Organic
Tin(Sn)compounds
• Selectivity of detection is the highest since it detects a
characteristic light of element which emits light in hydrogen
flame.

• Main examples of application

•Organo-phosphorus pesticides analysis
•Sulfur offensive odor analysis / Food flavor analysis
•Analysis of organic tin in marine products
 FPD (Flame Photometric Detector)
to Data processor

multiplier
Photo-
Combustion of S compounds, P
Air compounds, and Sn compounds
Filter emit the light of respectively
Quartz Only the light of specific inherent wavelength.
tube By letting it pass in a filter, only
wavelength can pass the filter
the light of inherent wavelength
For S(blue) ---394nm
reaches a photo-multiplier.
For P(yellow) ---526nm 　
For Sn(orange) ---610nm The intensity of light is amplified
Hydrogen and changed into electric signal by
(+Makeup gas) the photo-multiplier.

Column
outlet
 Similarity Search
using the Pesticide Library

In the Pesticide Library, 318

pesticides are registered for EI
spectrums of and 173
pesticides for NCI spectrums.
 Disease free GC/MS 　 Analytical Results
(x10,000,000)
TIC
1.25

IS margaric acid
IS(tropicacid)
1.00

hippunic acid
0.75

citnic acid

C24
0.50

succinic
0.25

10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0

Disease case
(x10,000,000)
TIC
methylmalonic acid

1.75

IS(tropicacid)

citnic acid
1.50

IS margaric acid
1.25
thiodiglycolic
succinic

hippunic acid
1.00

0.75 aconitic acid

C24
0.50

0.25

10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0

Which types of organic acids are increased?

How much are the increase and what are the correlation between them?
Specifying the disease is not so easy!