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Chromatography Techniques
Advances & Advantages in Analytical Chemistry
Mr.Rambabu
Executive -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
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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
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History of chromatography
M. Tswett : first developer of chromatography
Petroleum ether
Chlorophylls
CaCO3
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Separation Mechanism
Compounds are separated because the molecules
move at different rates in the column.
1
2
column
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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
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Chromato. . . ???
・Chromatography
Method for Separation
・Chromatograph
tR : Retention time
Instrument for Chromatography
A : Area
・Chromatogram h : Height
Data of Chromatography tR
Peak
Signal
. Chromatographer
h
Analyst A
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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
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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 chemicals,
field pesticides, herbicides, phenols,
Bile acids, drug metabolites, urine extracts,
Clinical medicine
estrogens
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Separation Modes
• Normal phase chromatography
• Reversed phase chromatography
• Ion chromatography
• Size exclusion chromatography
• Affinity chromatography
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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
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Increasing polarity
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Instrumentations
Modular HPLC
• Possible configurations
• Solvent delivery pumps
• Sample injectors
• Column ovens
• Detectors
Integrated HPLC
• LC-2010
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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.
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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
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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.
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Flexibility
Modular type provides excellent flexibility.
Isocratic system Fully automated gradient system
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Outline of LC-2010
Reservior Tray
System Controller
Auto sampler
Column Oven
UV detector
Degassing Unit
Pump Unit
Low pressure
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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
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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
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Photodiode Array Detector
Sample Cell Grating
One element detects
D2 / W lamp one absorbance at
one wavelength.
512 Elements Photodiode Array
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Photodiode Array Detector
3-D Data
Spectrum
Chromatogram
Absorbance
Time
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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.
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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
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Fluorescence Detector
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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
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Refractive Index Detector (1)
Photodiode
Reference Refraction
W Lamp
Sample
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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
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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 http://pharmacy2011foru.blo
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SHIMADZU GROUP
130th
….the future of
HPLC
1.World’s first web control
◆ World’s first Web Control
Internet Explorer to access to the Prominence (no special software required)
Status summary
・To show working
condition of the
systems in a
working group,
system check result,
HPLC Explorer and maintenance
information.
* Displays all
HPLC systems
on the network
* One workgroup
includes max 10
HPLC systems. Instrument monitor
・To monitor
parameter setting
conditions and
baseline of chosen
system.
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1.World’s first web control
◆ Access to the instruments via Ethernet
--> CBM-20A/20Alite
- No need to dedicate a client PC
- Any PC on network can access to Prominence system.
PC-Bからprominence-Aにログイン
PC-Aからprominence-Aにログイン
Prominenceでの使用環境
prominence-Aからログアウト
PC-Aからprominence-A/Bをモニタ
PC-Bからprominence-A/Bをモニタ
従来の使用環境
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CBM-20A/20Alite Features --Network example--
Reserve
room AP Office
to backbone network
Control/data acquisition of Control/data acquisition of Monitor/setting of prominence
prominence B, C, D, E, and prominence B, C, D, E, and B, C, D, E, and
communication with backbone communication with backbone communication with backbone
network network network
LCsolution LCsolution Client PC
prominence E LCsolution AP
Control/Data acquisition of Prominence E
Workgroup 1 Workgroup 2
Lab
prominence A Empower prominence B CLASS-VP prominence C LCsolution prominence D LCsolution
SPD-M20A
Monitor/setting of prominence A
Data acquisition by Empower
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Control/data acquisition of prominence B Control/data acquisition of prominence C, D Control/data acquisition of prominence C, D
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2.World’s only system with data buffering
◆ LC workstation connection via network
HPLC system control and data processing by
LCsolution/CLASS-VP on the network..
◆ World's only data buffering capability
Even in case network problem occurs, precious
analytical result is secured. (Max. one analysis data
less than 24 hours at 500ms sampling.)
◆ Automation capability CBM-20A
Auto-purge, auto-startup, auto-shutdown are
possible for modular type HPLC systems for the
first time.
CBM-20Alite
to be installed inside solvent delivery unit or
autosampler
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3.World’s quietest HPLC pump
Low pressure GE
valve
World's best solvent delivery performance
→ LC-20AD/20AB
・ Flow rate resolution 3nL/step is
achieved with micro stroke volume plunger
・ Ideal front-end for LC-MS for ultra trace
high sensitivity analysis
* Wide flow rate range from 0.0001 to
10 ml/min
Inside view of LC-20AT
LC-20AD LC-20AT LC-20AB
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4.World’s Lowest Degassing volume
World's lowest degassing volume
DGU-20A3/20A5
Low Capacity Degasser uses Teflon AF Film and Degassed Flow capacity
is as low as 170 micro liter.
Degassing volume allows a dramatic decrease in time required for
replacing mobile phases and stabilization.
Specifications :
• Number of Flow lines : DGU-20A3 – 3 lines, DGU-20A5 – 5 lines
• Maximum operating Flow rate : 20 ml / min per flow line
• Internal capacity : 380 ul / flow line
• Tubing 3 mm OD x 1.5 mm id PTFE tubing
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5. World’s fastest Auto Sampler
◆ World's fastest sample injection
Fast needle drive technology developed for the LC-
2010HT and SIL-HT is further improved, and achieved
the world's fastest - 10 seconds for 10uL injection.
◆ ◆ In MTP/DWP setting, vials can be used.
Control rack to accommodate 10 x 1.5mL vials is
available. SIL-20AC (right) and Rack Changer
(left)
Sample Rack Control Rack
◆ Rack Changer for large number of sample
processing
Switching max 12x MTP/DWP plates, continuous
analysis is possible. * Standard model and Cooling
model (Rack Changer C)
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6. World’s Cleanest Auto-Sampler
◆Near zero sample carry over
Needle tip and seal shapes are modified. World's best low sample carry over
performance is achieved. Using optional rinsing kit, multi-liquid rinse is possible.
SIL-HT SIL-20A
Remained sample
reduced to 1/4
Minimized touch area
SIL-20A provides good result even With rinsing pump, sample carryover is
with adsorptive compound. not detected.
with HPLC from
vendor A:
0.08 %
0.0007 % Not
detected
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Sample carryover test using Chlor-hexidine
7. World’s most sensitive PDA detector
◆ World's highest sensitivity
Noise level is reduced by reducing noise derived from
electrics.
<Noise>
SPD-M20A: less than 0.6×10-5AU (high sensitivity mode)
◆ Improved stability
Baseline is more stabilized with light source correction
mechanism. SPD-M20A
Temperature controlled cell as standard to minimize baseline
change by room temperature change.
◆ D2 lamp/W lamp simultaneous lit mode
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8. World’s most sensitive UV-VIS detector
◆ World's highest sensitivity
Noise level is reduced by reducing noise derived from
electrics.
<Noise>
SPD-20A/20AV: less than 0.25×10-5AU
◆ ◆ Improved stability
Baseline is more stabilized with light source correction SPD-20A/20AV
mechanism.
Temperature controlled cell as standard to minimize baseline
change by room temperature change.
◆ Low Pressure Mercury Lamp is incorporated
Easy wavelength calibration in UV region.
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SPD-20A/20AV/M20A Performance
--Temperature controlled cell--
◆ Temperature controlled cell is equipped as standard so that
influence of ambient temperature change to analysis is
reduced.
・Stable baseline improves analysis reliability for trace component analysis.
without cell temperature control
セル温調なし セル温調40℃
cell temperature controlled at 40deg.C
mAU ℃ mAU ℃
40 40
Cell temperature セル温度
セル温度 35 Cell temperature 35
Room Room
室温
Temperature 室温
Temperature
15 30 15 30
25 25
10 10
ベースライン
Baseline 20 20
5 5
Baseline
ベースライン
0 0
0 30 60
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90 120 min 0 30 60 90 120 min
Effect of temperature controlled cell --baseline change when ambient
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temperature is changed from 20 deg.C to 30 deg.C.
9. World’s best front-end HPLC for
LC-MS/MS
◆ Higher throughput
• World's fastest sample injection makes analysis cycle less than 1 minute possible.
• Ideal front-end for LC-MS for ultra trace high sensitivity analysis
• Rack changer processes upto 12 MTP/DWP for high throughput use.
• Data reliability improvement for ultra trace sample analysis
• Powerful support for LC-MS/LC-MSMS analyses
0 0.5 1 1.5 2 2.5 3 min
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10. World’s best LC Virtual advisor
• Shimadzu Lc Virtual
advisor
• 24/7/365
• www.shimadzu.com/Lcva
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Summary
• World’s first web-based instrument control
• World’s only system with Data Buffering
capability
• World’s quietest HPLC pump
• World’s lowest degassing volume
• World’s fastest Auto-sampler
• World’s cleanest Auto-sampler
• World’s highest sensitive PDA detector
• World’s most sensitive UV-VIS detector
• World’s best Front End HPLC for LC-MS/MS
• World’s best LC Virtual advisor multimedia tool
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eXtreme Resolution LC
Prominence UFLCXR series
Analytical & Instruments Division
LC Business Unit
New drivers for customer to
buy New HPLC
Driver Productivity Improvement Better quality of
analysis
Why they •Shorten development time Can assure better
need •Improve Cost/Analysis quality of their Products.
this?
Who need Pharma Lead Discovery, High •API provider etc.
this? throughput screening •Who needs impurity
analysis
What is •Fast injection cycle •Hi-Resolution
needed •High sample capacity •Wide dynamic range
for HPLC •MS compatibility( Low carry •Low carry over
over)
•Longevity
Our UFLC+XR-ODS UFLC XR+XR-ODSⅡ
Solution
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High speed & High
resolution
High throughput
UFLC
High throughput
screening etc.
UFLC XR
Impurity analysis
for quality control etc.
Conventional
HPLC
Resolution
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Design concept of UFLC-XR
• No compromise in basic performance!
– Waters gave up direct injection in UPLC! that result in poor
reproducibility, less linearity and much carry over.
– Agilent could not improve their fatal design that will not
satisfy both High Speed and Low Carry over at the same
time.
– Thanks to our DIIMS technology (Direct Injection with
Isolated Metering System), Only Shimadzu can achieve
High Speed, Low Carry over, High precision and good
linearity at the same time.
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To realize extreme resolution
Prominence UFLC-XR series have
• Optimized flow line for less dispersion.
– tubing,needle seal, Valve
• Increased pressure tolerance
– up to 66MPa
• Higher resolution column
– 2.2um core with 8nm pore
• Enhanced DIIMS for high performance in eXtream
pressure.
– Same repeatability and low carry over even in
60MPa.
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Outline of Prominence UFLC-XR series
• Maximum allowable pressure: 66MPa
• New Products for Prominence UFLCXR series
– LC-20ADXR ：New
– SIL-20AXR / SIL-20ACXR ：New
– CBM-20A ：F/W Ver.1.2
– LCsolution ：Ver.1.23
– Shim-pack XR-ODSII Column ：New
• Upgrade KIT for current 20A products
are available
＊for LC-20AD
＊for SIL-20A/C series
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Solvent delivery pump LC-20ADXR
• What’s new in LC-20ADXR
– Pressure Sensor ：70MPa scale pressure sensor
– Drain Valve ：Modified Sealing performance
– Inlet Check Valve ：Modified ball/seat design
– Plunger Seal ：GFPUHMWPE
– Seal wash KIT ：Standard
– F/W version ：Ver. 1.21
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Auto sampler SIL-20A/CXR
• What’s new in SIL-20A/CXR
– Enhanced DIIMS
– High pressure valve ASSY ： Reduced volume,
higher pressure
– Needle Seal ： Reduced volume, higher
pressure
– Sample loop ： Reduced volume
– Outlet tube ： Improved handling
– F/W ： Ver. 1.20
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Shim-pack XR-ODSⅡ
•
Column
For higher resolution analysis
– Raised Maximum pressure from 30MPa to 60MPa
– Decreased pore size from 12nm to 8nm to realize high
separation.
– Added 150mm length for high separation use.
• Line up for Shim-pack XR-ODSⅡ
ID Length Max. Press Pore size
2mm 75，100， 60MPa 8nm
150mm
3mm 75，100， 60MPa 8nm
150mm
Ref: XR-ODS column
ID Length Max. Press Pore size
2mm 30, 50, 75，100mm 30MPa 12nm
3mm 30, 50, 75，100mm 30MPa 12nm
4.6mm 30, 50, 75，100mm 30MPa
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Performance of UFLC-XR series
• Fast and sharp.
– Double the resolution with 1/4 of
separation time.
mAU
150
1 Shim-pack XR-ODSⅡ (3 mm i.d. 150 mm , 2.2 m)
Flow rate : 1.3 mL/min
2
125
3 N for Peak 7 : 28,100
100
4
75 5
6
50
7
25
0
0.0 0.5 1.0 1.5 2.0 2.5 min
mAU
3.0
125 1 Shim-pack VP-ODS (4.6 mm i.d. 150 mm , 4.6 m) Peaks; 1: Acetophenone
100
2 Flow rate : 1.0 mL/min 2: Propiophenone
3 N for Peak 7 : 14,800 3: Butyrophenone
75 4 4: Valerophenone
5
50
6
5: Hexanophenone
7
25 6: Heptanophenone
0
7: Octanophenone
0.0 2.5 5.0 7.5 10.0 min
12.5
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Performance of UFLC-XR series
• extreme resolution
mAU
100
1
75
Shim-pack XR-ODSⅡ (3 mm i.d. 100 mm , 2.2 m)
50 2 4
3
5 6 8 Chromatographic conditions;
9
25 7
10 11
12 13 mobile phase
0
A: water/THF (8/2,v/v)
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 min B: acetonitrile
100
mAU (Gradient Elution)
1
75
1. Formaldehyde
2. Acetoaldehyde
Shim-pack XR-ODS (3 mm i.d. 75 mm , 2.2 m)
3. Acetone
50 2 3 4 4. Acrolein
5 8 5. Propionaldehyde
6
25
7 9 6. Crotonealdehyde
10 11 12 13 7. 2-Butanone
8. Methacrolein
0 9. n-Butylaldehyde
0.0 1.0 2.0 3.0 4.0 5.0 min 10. Benzaldehyde
11. Valeraldehyde
12. m-Tolualdehyde
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UFLC Applications
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Fast LC Application (1)
◆ PTC-amino acids
→ 6 times faster than conventional column (5m，4.6 mm i.d.
150 mm)
mAU
2. than high speed column (3m, 4.6 mm i.d. 75
→ 2 times faster Glu
250
1. Asp 3. Ser 1. Formaldehyde
18
2. Acetoaldehyde
mm)
4. Gly 5. His 6.Arg
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
14 6. Crotonealdehyde
16. Leu 17. Phe 18. Lys 7. 2-Butanone
150 (each 400 pmol/1L) 11 12 16 8. Methacrolein
9 15
1 5 6 13 9.17
n-Butylaldehyde
34 78 10. Benzaldehyde
11. Valeraldehyde
100 12. m-Tolualdehyde
2 13. Hexaldehyde
(each 120 nL/4L)
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 (2.2m, 3.0 mm i.d. 75
mm), mobile phase: 10 mMol/L (potassium)phosphate <pH 7.0> for A and acetonitrile
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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,
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temperature: 40 C, detection: absorbance at 254 nm.
Fast LC Application (2)
◆ 2,4-DNP-aldehydes/ketones
→ 6 times faster than conventional column (5m，4.6 mm
i.d. 150 mm)
mAU
→ 3 times faster than high speed column (3m, 4.6 mm
100
1 1. Formaldehyde
i.d. 75 mm)
2. Acetoaldehyde
3. Acetone
4. Acrolein
75 5. Propionaldehyde
6. Crotonealdehyde
7. 2-Butanone
8. Methacrolein
9. n-Butylaldehyde
50 2 4 10. Benzaldehyde
3
11. Valeraldehyde
5 8 12. m-Tolualdehyde
6 13. Hexaldehyde
7 9 (each 120 nL/4L)
25 10 11 12
13
0
0.0 1.0 2.0 3.0 4.0 5.0 min
Chromatographic conditions; column: Shim-pack XR-ODS (2.2m, 3.0 mm i.d. 75
mm), mobile phase: water/THF (8/2,v/v) for A and acetonitrile for B (A/B: 8/2 to 5/5 in
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5 min), flow rate: 1.2 mL/min, temperature: 50 C, detection: absorbance at 360 nm.
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2,4-DNP-Aldehydes/ Ketones ＵＦＬＣ-LCMS
7.5 (x10,000) (x10,000)
Shim-pack XR-ODS + APPI-MS 5.0 Bland-A ODS (5 µm) + APPI-MS
7.0
6.5 2 3 8 10 11 13
1 4.0
12
6.0 4 5 9
7 3.0
5.5
6
5.0 2.0
4.5
1.0
4.0
3.5 0.0
0.0 5.0 10.0 15.0 20.0 25.0 min
3.0
(x10,000)
2.5 5.0 Bland-A ODS (5 µm) + APCI-MS
2.0 4.0
1.5
3.0
1.0
2.0
0.5
1.0
0.0
0.0 1.0 2.0 3.0 4.0 5.0 min 0.0
Detector: LCMS-2010EV 0.0 5.0 10.0 15.0 20.0 25.0 min
Column: Shim-pack XR-ODS (50 mmL x 2.0 mmI.D. Particle size: 2.2 µm) or
Bland-A ODS (150mmL x 2.0 mmI.D. Particle size: 5 µm)
Mobile phase: A; water/tetrahydrofuran = 80/20
B; acetonitrile
Flow rate: 0.5 mL/min (Shim-pack XR-ODS), 0.2 mL/min (Bland-A ODS)
Injection volume: 2 µL (27pg each)
Time program: 20%B (0 min) - 50%B (4.0 min) - 50%B (4.2 min)
APPI negative, SIM, m/z 209 (1: formaldehyde), m/z 223 (2: acetaldehyde), m/z 237 (3: acetone, 5: propionaldehyde),
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m/z 235 (4: acrolein), m/z 249 (6: crotonaldehyde, 8: methacrolein), m/z 251 (7: 2-butanone, 9: n-butyraldehyde),
m/z 285 (10: benzaldehyde), m/z 265 (11: valeraldehyde), m/z 299 (12: m-tolualdehyde), m/z 279 (13: hexaldehyde)
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LCMS spectrum of 2,4-DNP-Aldehydes and Ketones
Inten. (x1,000,000) Inten. (x1,000,000) Inten. (x1,000,000)
467 537 1.25 421
1.5 1 1.00 2 3
1.00
0.75
1.0 0.75
0.50
0.50
0.5
701 0.25 0.25 210 632
268 807 843
233
935
0.0 0.00 0.00
250 500 750 1000 m/z 250 500 750 1000 m/z 250 500 750 1000 m/z
Inten. (x1,000,000) Inten. (x100,000) Inten. (x100,000)
1.00 537 407 451
7.5
4 5 6
7.5
0.75
5.0
0.50 5.0
815
268 2.5 611 677 903
0.25 203 2.5
225
207319
806 1075
0.00 0.0 0.0
250 500 750 1000 m/z 250 500 750 1000 m/z 250 500 750 1000 m/z
Inten. (x100,000) Inten. (x1,000,000) Inten. (x100,000)
763 1.00 747 7.5 557
1.00 7 8 9
381
0.75 373
0.75 5.0
0.50
0.50
2.5
0.25 0.25
215 251 327
281 347
441
0.00 0.00 0.0
250 500 750 1000 m/z 250 500 750 1000 m/z 250 500 750 1000 m/z
Inten. (x100,000) Inten. (x1,000,000) Inten. (x100,000)
784.4 1.25 834.6 972.5
5.0 10 11 12
1.00 5.0
0.75
2.5
0.50 2.5
740.6 0.25 928.3
0.0
702.5
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0.00 0.0
890.5
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250 500 750 1000 m/z 250 500 750 1000 m/z 250 500 750 1000 m/z
Fast LC Application (3)
◆ Tryptic digest of ovalbumin
→ High-speed separation by using the column with 19,500 in
theoretical plate number
→ Peak width at half height is 1.2 sec
The
（ 40mAU average of the three peaks between 2.5 and 3.5
1. Formaldehyde
2. Acetoaldehyde
minutes）
3. Acetone
4. Acrolein
5. Propionaldehyde
6. Crotonealdehyde
30 7. 2-Butanone
8. Methacrolein
9. n-Butylaldehyde
10. Benzaldehyde
20 11. Valeraldehyde
12. m-Tolualdehyde
13. Hexaldehyde
(each 120 nL/4L)
10
0
0.0 2.5 5.0 7.5 min
Chromatographic conditions; column: Shim-pack XR-ODS (2.2m, 3.0 mm i.d. 100
mm), mobile phase: 0.1 % TFA aqueous solution for A and 0.1 % TFA acetonitrile solution
min), flow rate: 1.0 mL/min, temperature: 50 C, detection:
for B (A/B: 9/1 to 5/5 in 10 http://pharmacy2011foru.blo
absorbance at 214 nm, sample: 800 pmol/4L.
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Pigments analysis Column Shim-pack XR-ODS
mA U
(3.0 mm i.d.×75 mm)
500 Mobile A) 50mmol/L
Peaks Phase (Anmonium) Acetate
1: Yello w 4 7: Green 3 Buffer (pH 4.7)
2: Red 2 8: Blue 1 B) 50mmol/L
400 8 (Anmonium) Acetate
3: Blue 2 9: Red 106 Buffer (pH 4.7) /
4: Red 102 10: Red 104 Acetonitrile (1/1)
5: Yello w 5 11: Red 3 A/B) 90/10 to 0/100 in
9 5.5 min
300 6: Red 40 12: Red 105
0/100 for 1.5 min
90/10 for 2 min
and end
Flow Rate 1.2 mL/min
200
10 Temperatu 40 C
7 re
3 Detection Maxplot at 300 - 600 nm
11 12
5 6
100 1 2 Sample 4 L
4 Volume
0
0 1 2 3 4 5 6 min
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Synthesis Color Pigments ＵＦＬＣ-LCMS
8.0 (x1,000,000)
1
2 3 4 8 10
7.0 6 12
5
11
6.0
9
7
5.0
TIC
4.0 m/z 467
m/z 537
m/z 421
3.0
m/z 407
m/z 451
2.0 m/z 763
m/z 747
1.0
m/z 557
m/z 784.5
m/z 834.5
0.0 m/z 972.5
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 min
Detector: LCMS-2010EV
Column: Shim-pack XR-ODS (50 mmL x 2.0 mmI.D. Particle size: 2.2 µm)
Mobile phase: A; 20mM ammonium acetate buffer (pH 4.7)
B; 20mM ammonium acetate buffer (pH 4.7)/acetonitrile = 1/1
Flow rate: 0.5 mL/min
Injection volume: 5 µL
Time program: 10%B (0 min) - 100%B (5.0 min)
DUIS, negative, Scan m/z 200-1200, 1: tartrazine, 2: amaranth, 3: indigo carmine, 4: new coccine, 5: sunset yellow FCF,
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6: allura red AC, 7: fast green FCF, 8: brilliant blue FCF, 9: acid red, 10: phloxine B, 11: erythrosine, 12: rose bengal
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