HPLC Application Systems

Lin Yuan
Customer Support Center

Shimadzu (Asia Pacific)

1

HPLC Application Systems

Amino acid analysis
Organic acid analysis
Reducing sugar analysis
Carbamate pesticide analysis
GPC analysis
Bromate analysis

Amino Acid Analysis System

Amino Acid Analysis

1958 Spackman, Stein and Moore

First automatic separation and quantitation of amino acids

NH2
R

UV-210nm

COOH

H
4

Derivatization of Amino Acids

Postcolumn Derivatization
Amino Acid itself

Reagent
Detector

Column

Precolumn Derivatization
Derivatized Amino Acid
Detector

Column

Postcolumn Derivatization
Amino Acid itself

Reagent

Column

Detector

Column : Cation Exchange Column

Reagent : Ninhydrin method --- UV/VIS detector
OPA method --- Fluorescence detector

The separation column for postcolumn derivatization

Cation Exchange Column is used.

Resolution of cation exchange column is not as
good as reversed phase column.
It takes long time to analyze all the amino acids
completely.
protein hydrolysates (45 min)
physiological fluids (160 min)

Ninhydrin Method

Primary and Secondary amino (imino acid) acid

Detection : 440nm (proline) or 570nm (primary)
Reaction Temperature : 130C
Reaction speed : Fast
Reagent stability : unstable (must keep it cool)
Derivatized product stability : stable
8

Ninhydrin Method
Column Oven
Amino Acid itself

130C

Cation Exchange Column

Reaction
Heater

UV/VIS
detector

Ninhydrin Reagent
9

OPA Method
(o-phthalaldehyde)

Primary amino acid

Detection : Fluorescence (Ex=350nm, Em=450nm)
Reaction Temperature : Ambient
Reaction speed : Fast
Reagent stability : Less stable
Derivatized product stability : Less stable
10

Proline or Hydroxyproline analysis

Proline and Hydroxyproline can not

react with OPA reagent.
Must be oxidized before reaction of
OPA using
Hypochlorite
Chloramine-T

11

Auxiliary Reagent for OPA reaction

The reaction between OPA and primary amino

acids takes place in the presence of a thiol.

ethanethiol
2-mercaptoethanol
3-mercaptopropionic acid
(non-volatile)

12

OPA Method
Column Oven
Amino Acid itself

Cation Exchange Column

Hypochrolite

Fluorescence
detector

OPA reagent
13

Comparison
Ninhydrin vs OPA

Ninhydrin

OPA

Reaction

primary, secondary amine

primary amine

Reaction Temp.

130 C

Ambient

Reaction Speed

Fast

Fast

Detection

Visible detector

Fluoresces detector

Reagent Stability

unstable

less stable

14

Precolumn Derivatization
Derivatized Amino Acid
Column

Detector

Column :

Reversed Phase Column (ODS)

Reagent :

OPA (Fluorescence)
FMOC (Fluorescence)
Fluorescamine (Fluorescence)
DANS-Cl (Fluorescence)
PITC (UV)
15

The separation column for

precolumn derivatization
Reversed Phase Column (ODS) is used.
Due to reversed phase column, peak resolution is
excellent.
It takes a short time to analyze several types of
amino acids.
protein hydrolysates
physiological fluids

16

OPA Method
(o-phthalaldehyde)

Primary amino acid

Detection : Fluorescence (Ex=350nm, Em=450nm)
Reaction Temperature : Ambient
Reaction speed : Fast
17

Chromatogram of OPA precolumn derivatization

physiological amino acid standard

Shim-pack CLC-ODS

18

FMOC Method
(9-fluorenylmethyoxycarbonyl chloride)

popular reagent for N

terminal protection of
peptide

19

Fluorescamine Method

O
O
O

Rapid reaction with primary

R-NH2

amino acid at pH=7

O
R
N
O
OH

Easy to hydrolyze

OH

20

10

DANS-Cl Method
(1-dimethylaminonaphthalene-5-sufonyl chloride)

Low reactivity with

primary and secondary
amino acid

21

PITC Method
(phenylisothiocyanate)

PITC

Primary and Secondary amino

acid (alkaline condition)
Long reaction time

PTC

Poor sensitivity due to use of

UV detector

22

11

Chromatogram of PTC - amino acids

a standard solution of amino acids

STR ODS-II
23

Automatic Amino Acid Analyzer

Which one is better to adopt for automatic amino
acid analyzer

"Postcolumn" or "Precolumn"?
To control reaction time, postcolumn method is
easier. That is why repeatability obtained by
postcolumn is much better.
Postcolumn repeatability - RSD is around 1%
Precolumn repeatability - RSD is more than 2%
24

12

Amino Acid Analysis System

Post-Column OPA Fluorescence
Reaction Regent A
MP-A

MP-C

Reaction Regent B

MP-B

DGU-20A5

PRR-2A

Reaction Coil Kit

FCV-11ALS
RF-10AXL
LC-20AB
SIL-20AC
Shim-pack
Amino-Na/Li

Mixer
ISC-30/S0504 Na

CTO-20AC

Cation Exchange Chromatography

25

Separation Column
Strong acidic cation exchanger resin
(styrene-divinylbenzene copolymer with sulfonic groups)

Na type and Li type

Shim-pack AMINO-Na

for hydrolysis amino acid

Shim-pack AMINO-Li

for physiological amino acid

26

13

Chromatogram of
hydrolysis amino acids

27

Chromatogram of
physiological amino acids

28

14

Mobile Phase
[ Na type ]

A solution : 0.2N sodium citrate

(adjusted to pH 3.2 with perchlorite acid)
B solution : 0.6N sodium citrate / 0.2M boric acid
(adjusted to pH 10.0 with 4M sodium hydroxide)
C solution : 0.2N sodium hydroxide(for column washing)

[ Li type ]

A solution : 0.15N lithium citrate / 7% methyl cellosolve

(adjusted to pH 2.6 with boric acid)
B solution : 0.3N lithium citrate / 0.2M boric acid
(adjusted to pH 10.0 with 4M lithium hydroxide)
C solution : 0.2N lithium hydroxide(for column washing)
29

pH gradient for separation

H

H
R

C
NH3+

COOH

H
COOH

NH2

COO

NH2

Separation is carried out by

ionic force

30

15

N-acetylcystine
as a auxiliary agent

31

Ammonia Trap Column

without
trap column

Ammonia
in Mobile Phase

with
trap column

32

16

Repeatability (250 pmol each)

Asparatic acid

Rretention time

Area value

Retention time

retention time

Area value

0.2

1.2

Methionine

0.4

1.1

Threonine

0.2

1.0

Isoleucine

0.3

0.9

Serine

0.2

1.3

Leucine

0.4

1.3

Glutamic acid

0.2

1.3

Tyrosine

0.3

1.2

Proline

0.2

0.9

Phynylalanine

0.3

1.3

Glycine

0.2

0.9

Histidine

0.1

1.1

Alanine

0.3

1.0

Lysine

0.1

1.5

Cystine

0.4

1.6

Arginine

0.1

0.9

Valine

0.4

1.0

(n=10)
33

Linearity

34

17

Chromatogram of high sensitivity

25 pmol amino acids standard

35

Amino Acid Analysis

Amino Acids in Vinegar

SHIMADZU APPLICATION NEWS LAAN-A-LC-E025 No. L323

36

18

Pretreatment of Sample
Defatting
Removal of Protein
Protein Hydrolysis
Sample Dilution
Filtration

37

Defatting
Fat components are affected to analytical column.
Add low polarity solvent
ethyl acetate
chloroform
hexane etc.

After separation of two phases, the aqueous phase must

be filtered by 0.45 m membrane filter.

38

19

Removal of Protein
Proteins will interfere with separation of amino acid.
Removal methods
Acidic Solvent
Organic Solvent
Ultra Filtration

Protein precipitate must be removed by centrifuge (3000 rpm, 5

min) or filtration.

39

Removal of Protein
Acidic Solvent Method

Trichloroacetic acid
(5-10% aqueous solution)

Perchloric acid
(0.1 - 1% aqueous solution)

After removal of the protein, neutralization is

required using NaOH or LiOH solution.

(Acidic sample will affect retention time.)

40

20

Removal of Protein
Organic Solvent Method

Acetonitrile
Ethanol
Polystyrene column is week to organic solvent.
In case of organic solvent method, better to inject the sample
as small as possible.

41

Removal of Protein
Ultra Filtration Method

Effective
Costly

42

21

Analysis of free amino acids

in rat serum

43

Analysis of free amino acids

in bovine urine

44

22

Analysis of free amino acids

in porcine blood powder

45

Analysis of free amino acids

in tea leaf

46

23

Protein Hydrolysis (1)

Hydrochloric acid Hydrolysis Method
6N - HCl 110C (N2 atmosphere)
22 - 72 hours

Tryptophan, Cysteine, Methionine are affected

(another method).
Asparagine and Glutamine will be converted to
acidic form.

47

Protein Hydrolysis (2)

Alkaline Hydrolysis Method
4N NaOH 110C (N2 atmosphere)
16 - 24 hours

This method is for Tryptophan.

48

24

Protein Hydrolysis (3)

Performic Acid Oxidation Method
A/B=5/95 110C (N2 atmosphere)
A:30% hydrogen peroxide
B:90% formic acid
22-72 hours

This method is for Cysteine, Cystine and Methionine.

49

Example of

Hydrochloric acid Hydrolysis Method

Analysis of hydrolysis
amino acids of insulin
obtained from pancreas

50

25

Sample Dilution
0.2N sodium citrate buffer (pH 2.2)
0.2N lithium citrate buffer (pH 2.2)
will be used for dilution solvent.
Optimum concentration after dilution is about
5 nmol/ml - 10 mmol/ml.

51

Filtration
Aqueous membrane filter of 0.45 mm
Sample should be filtered before injection.

52

26

Organic Acid Analysis System

53

Organic Acid Analysis System

Unique post-column pH-buffered electroconductivity method (Japanese
Patent No. 2017498) is ideal for the selective and highly sensitive
detection of organic acids.
Compared to conventional methods, such as the UV shortwavelength
method or a simple conductivity method, this system improves
quantitation reliability.
Compared to the post-column VIS absorption detection methods using
pH indicators, this system has higher sensitivity, better linearity, and is
easier to use.
Complex samples, which usually require troublesome pretreatment, can
be analyzed after simple pretreatment techniques like dilution and
filtration.

54

27

Organic Acid Analysis

Flow diagram

55

Analytical Condition
Column:

Shim-pack SCR-102H

Mobile phase:

5mM p-toluene sulfonic acid

Flow rate:

0.8ml/min

Reaction reagent:

5mM p-toluene sulfonic acid

20mM Bis-tris
0.1mM EDTA

Reaction reagent flow rate: 0.8ml/min

Temperature:

45C

Detector:

conductivity detector
56

28

Principle
Ion exclusion chromatography is suitable to separation of organic
acids, but it is difficult to combine with the electroconductivity
detection, because the acidic mobile phase increases the
background conductivity, and decreases the response to target
organic acids.
Post-column pH buffered method permits the combination of ion
exclusion chromatography and conductivity detector.

R-COOH

RCOO- + H+

57

Conductivity Detector Principle

V
I

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

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

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

Electrodes

58

29

59

60

30

Organic Acid Analysis

References
Food Analysis Guidebook (C180-E060)
Analysis of Organic Acid in Beer

HPLC Food Analysis Application Databook (C190-E078)

Analyses of organic acids in soy sauce, beer and lemon juice.
Analyses of organic acids in tomato, umeboshi (pickled Japanese
plum), roasted meat gravy, and seaweed.

Application News
L213 Application of HPLC Organic Acids Analysis System Comparison with UV Detection -

61

Reducing Sugar Analysis System

62

31

Reducing Sugar Analysis System

Unique post-column fluorescence detection method (Japanese
Patent No. 1471088) with arginine as the reagent, is a highly
sensitive and selective method for sugars.
This system is suitable for the analysis of complex samples,
like fermentation products, and for ultra trace analysis, such
as determination of sugar components in glycoprotein (except
saccharose).

63

System Flow Diagram

64

32

Analysis Example

65

Reducing Sugar Analysis System

with single pump

Flow diagram of reducing sugar analysis system with single pump

66

33

Analysis Example
with single pump

67

Carbamate Pesticide Analysis System

68

34

Carbamate Pesticide Analysis System

The carbamate pesticide analysis system, with
post-column reaction detection, provides highsensitivity, high accuracy analysis. This system
can detect 100 pg of methiocarb.

69

Principle of measurement
H3CHN
O
RO

a chemical formula of N-methylcarbamate

70

35

System Flow Diagram

1.9. Degassing units

2. Low pressure gradient unit
3.10. Pumps
4. Sample injector
6. Column oven
7. Column
8. Chemical reaction box
11. Mixer
12. Fluorescence detector
M1,M2. Mobile phase
R1,R2. Reaction reagent

71

Analytical Conditions

72

36

Retention Index

73

Analysis of lemon juice

Spike analysis of Carbaryl

74

37

Pretreatment of lemon juice

Sample
Cut finely
20g: 500mL Stainless tube for Centrifuge
Water 10ml
Acetone 40ml
Homogenize
Centrifuge 6000G, 15min

Acetone layer

Residues
Acetone 40mL
Mixture
Centrifuge 6000G, 15min

Acetone layer

Residues
75

Replace in 300ml flask

Take upper layer (7-9mL)

Evaporate until about 1/2 volume

Replace in 300ml separatory funnel

5mL of sample applied

to GPC clean up system

Saturated NaCl(aq) 150mL

Dichloromethane 40mL, 5min x 2times
Take each fraction
Replace in 200mL flask
Sodium Sulfate anhydrous for dehydration
Filter with glass filter
Evaporation to dryness
Dichloromethane : Cyclohexane (1:1)

Evaporation to 10mL
(1mL=1g)
1 mL of sample applied
to C18 cartridge column
Methyl alcohol 5mL

Up to 10mL (1mL=2g)
Evaporation to 1ml (1mL=1g)
Centrifuge (3000rpm, 5min)
HPLC system
76

38

Reference
Food Analysis Guidebook (C180-E060)
Analysis of N-Methylcarbamate Pesticides in Lemons
Analysis of Carbofuran in Water

Shimadzu Review
Analysis of N-Methylcarbamate Pesticides in Food by High
Performance Liquid Chromatography (available on GCS)

77

GPC System

78

39

Size Exclusion Chromatography

Aqueous Solvent
Gel Filtration Chromatography (GFC)
mainly biological science field

Mobile Phase

Organic Solvent
Gel Permeation Chromatography (GPC)
mainly polymer science field

79

SEC Column
Sample solution

to Detector
Crosslinked particle with
macro~micropores

SEC column is packed

with porous polymer
particles
80

40

Pictures of porous polymer particles

6m

10m

Exclusion Limit Molecular

Weight : 4,000,000

Exclusion Limit Molecular

Weight100,000

81

How to make porous polymer particles

~Suspension Polymerization~

Oil-phase

Stirring

Water-phase

82

41

Porous polymer particle = Packing material

Heating

83

Model Figure of Pore Distribution

Macropore

Micropore

84

42

SEC : Separation According to Size of Molecule

Elution in Decreasing Order of Size

85

Elution in Decreasing Order of Size

minimum pore

maximum pore

Larger than
maximum pore size
Exclusion Limit
Molecular Weight

Exclusion Limit Volume

Elution Volume
86

43

Three Steps for SEC Analysis

Preparing calibration curve

using standard samples

MW

Measuring
unknown sample

Elution volume

Elution volume

MW

Calculating
molecular weight based on
the calibration curve

Elution volume
87

Calibration Curve
= Standard Line for Calculation of MW

Vo
Vi

1.E+07
1.E+07

Exclusion Limit Molecular Weight

Vg

MMooleleccuulrlraar r wweeigighht t

1.E+06
1.E+06

within Vi

1.E+05
1.E+05

Vt =
15ml

1.E+04
1.E+04
1.E+03
1.E+03
1.E+02
1.E+02

Vi

Vo

Vg

Interstitial Volume Pore Volume

00 Limit
Exclusion
Volume

55

Solid Volume

10
10

15
15

Volume(mL)
Volume(mL)

Vt = Vo + Vi + Vg

88

44

Gentle slope gives better separation

GPC KF-800 series

107

106

Large pore
KF-805

105
104

103

Small pore
KF-801

102

89

Standard Samples and Columns for GPC

~ Organic Eluent ~

PS: Polystyrene

MW

PMMA: Poly(methyl methacrylate)

Elution volume

PEG: Poly(ethylene glycol)

Shodex GPC
KF-801, 802, 802.5, 803, ..;
K-801, 802, ..;
KD-801, 802, ..;
LF-804
HFIP-803, 804, ..

90

45

Example of GPC Analysis

~ Organic Eluent ~

Phenoxy resin

Columns : Shodex GPC LF-804

Eluent

: THF

Flow rate : 1.0mL/min

Detector : RI
Column temp. : 40

91

Average Molecular Weight Distribution

Mn : number average molecular weight

Mw : weight average molecular weight

tensile strength, impact resistance

brittleness

Mz : Z-average molecular weight

flexibility, stiffness

Mn < Mw < Mz
92

46

Average Molecular Weight Distribution

Mn =

W
=
Ni

MiNi = Hi
Ni (Hi/Mi)

WiMi
Mw =
=
W

(Mi Ni) = (MiHi)

(MiNi) Hi
2

Mi Hi
Mz =
(MiHi)
2

d = Mw/Mn

d: polydispersity
93

Calibration Curve

Inject the standard polymer sample one by one to

know the relationship between molecular weight and
retention time.
Chromatogram

No.
1
2
3
4
5
6
7
8
9
10
11

time(min)
22.0
22.6
23.4
25.0
27.4
31.0
33.8
38.4
39.8
42.8
46.8

mol. wet.
5500000
1800000
860000
400000
160000
50000
20000
4000
2000
600
80

Calibration curve

Retention time

94

47

Actual Sample Injection

GPC software is required to calculate the average molecular
weight distribution.

95

Bromate Analysis System

96

48

Bromate Analysis System

Background
Different Analysis Methods from EPA
Shimadzu Methods
- Analytical conditions
- Sample pretreatment

97

What is DBPs
Disinfection is an essential part of drinking water treatment in
the 20th century, which was a major factor in reducing typhoid
and cholera.
However, the disinfectants themselves can react with naturallyoccurring materials in the water to form unintended organic
and inorganic byproducts which may pose health risks.
NOM + disinfectants DBP
NOM naturally-occurring materials

DBP disinfection by-products

98

49

Common DBPs
Disinfectants
Chlorine dioxide

Disinfection by-products
Inorganic oxyhalide,
eg. chlorite and chlorate

Hypochlorite

Chlorate

Ozone

Bromate

99

Ozonating Process

Copy from , <>,

100

50

Maximum Contaminant Level in Water

(MCL)
Both the World Health Organization (WHO) and the U.S.
Enviromental Protection Agency (EPA) have listed bromate as
a potential carcinogen at the low g/L level.
1993, WHO specifies a MCL of 25 g/L for bromate.
2003, WHO suggests a MCL of 10 g/L for bromate.
2003, EPAs Stage 1 D/DBP rule specifies a MCL of 10 g/L for
bromate.

101

Potassium Bromate in Food

Flour manufacturers and bakers use
potassium bromate as a flour improver
to strengthen the bread dough. It makes
the bread softer and whiter.
In addition, potassium bromate is used
in treating barley in beer making.
It has been used for the improvement of
the quality of fish paste products in
Japan (Ministry of Health and Welfare,
Japan, 1979).
102

51

History of Potassium Bromate in Bread

1914, potassium bromate was first used
for baking products in US.
The previous acceptable level of used for
baking products was made temporary
with a maximum treatment level of 75 mg
KBrO3 per kg flour.
1990s, potassium bromate treated-flour
is not allowed in EU.
August 1999, it is banned in Australia.
1st July 2005, all potassium bromate
treated-flour is banned in China.
103

EPA Methods for Bromate

EPA Method 300.0 (IC), August, 1993.

EPA Method 321.8 (IC/ICP-MS), December, 1997.
EPA Method 317.0 (IC-PCR), July, 2001.
EPA Method 326.0 (IC-PCR), June, 2002.

104

52

EPA Method 300.0

Separation: Anion exchange

Detection: Conductivity detector with suppressor

105

EPA Method 321.8

Separation: Anion exchange
Nebulization
Desolvation
Atomization
Ionization

Detection: ICP- MS
Inductively Coupled Plasma Mass Spectrometer.
Both mass 79 and mass 81 are monitored.
The signal at Mass 79 is used for quantitation.
The signal at Mass 81 provides isotope ratio information which
can be used to screen for potential polyatomic interferences.

106

53

EPA Method 317.0

Ion chromatography with post-column reagent method
Seperation: Anion exchange
Eluent: 9.0 mM Na2CO3.
Detector 1: Suppressed conductivity detector.
Detector 2: Absorption (450nm).
Reaction reagent: o-dianisidine (ODA).
Temperature: 80C.

107

EPA Method 326.0

Ion chromatography with post-column reagent method
Seperation: Anion exchange
Eluent: 9.0 mM Na2CO3.
Detector 1: Suppressed Conductivity Detector.
Detector 2: Absorption (352nm).
Reaction reagent: ammonium molybdate + potassium iodide.
Temperature: 80C.

108

54

Method (1)

Analytical Conditions

109

Method (1)

System Configuration
Injector

Chemical reaction box

Pump

Detector

CRB-6A

Column

Mobile phase

Pump

Reaction reagent
110

55

Method (1)

Sample Pretreatment - Bread

111

Method (2)

Post-column Ion Chromatography

Analytical Conditions
<Seperation>
Column:
Shim-pack IC-SA2 (250mm L.4.0mm I.D.)
Mobile phase:
12mM NaHCO3+0.6mM Na2CO3
Flow rate:
1.0mL/min.
Temperature:
40C
<Post-column Reaction>
First Reaction
Reagent:
1.5M KBr + 1.0M H2SO4
Flow Rate:
0.4mL/min.
Temperature:
40C
Second Reaction
Reagent:
1.2mM NaNO2
Flow rate:
0.2mL/min.
Temperature:
40C
Detection:
268nm
112

56

Method (2)

System Configuration

113

Method (2)

Post Column Reaction

114

57

Method (3)
Analytical Conditions
<Seperation>
Column:
Shim-pack IC-Bromate (150mm L.4.0mm I.D.)
Mobile phase:
1.7mM NaHCO3+ 1.8mM Na2CO3
Flow rate:
1.0mL/min.
Temperature:
40C
<Post-column Reaction>
First Reaction
Reagent:
1.5M KBr+1.0M H2SO4
Flow Rate:
0.4mL/min.
Temperature:
40C
Second Reaction
Reagent:
1.2mM NaNO2
Flow rate:
0.2mL/min.
Temperature:
40C
Detection:
268nm
115

Method (3)

Bromate & Chlorite

Shim-pack IC-Bromate is a high-performance anion exchange
column designed to provide good separation of bromate and
chlorite, which elute at similar timings.

116

58

Shimadzu HPLC Application Systems

References
IMD GCS website
https://svc2.shimadzu.co.jp/imd1mk/products/lc/system/system_index.htm

C180-E060
Food Product Analyses
Analysis Guidebook

C190-E078
Food Analysis Applications
HPLC Data book

117

118

59