In the Mightysil series lineup, a C8 and C4 with lower hydrophobicity is also offered mainly with silica gel based
reversed-phase fillers, particularly for ODS fillers that are mostly used for general purposes. Including the test items
concerning the elution behavior of each filler batch which were implemented in the GP series, additional items by analysis
subject according to its application were also implemented for the columns by application.

Mightysil RP-18GP

This is a difunctional ODS column which consists of an octadecylsilyl (ODS) agent bonded with 2
adjacent silanol groups.

Mightysil RP-18GP

This is high performance filler which is chemically bonded with monofunctional ODS group.

Mightysil RP-18GP Aqua

This is an ODS filler which demonstrates a stable retention time when using up to 100%water-based solutions as eluents.
This filler is effective particularly for analysis using a high polarity eluent, such as for the separationof samples with high hydrophilicity.

Mightysil RP-18(H)GP
Mightysil RP-18(L)GP

Compared with the conventional Mightysil RP-18 GP, this is an ODS filler introduced with octadecyl
ligands in a high density (H) or low density (L).

Mightysil RP-8GP
Mightysil RP-4GP

This filler consists of a highly purified spherical silica gel chemically bonded with the octyl groupand butyl group.
Since the hydrophobicity ligands are shorter than the RP-18 (ODS), the retentivity becomes
weaker for substances with a high level of hydrophobicity.

Filler Name

Particle Size
( m)

Pore Size(nm)

Specific Surface
2
Area (m /g)

Modification
Group

CarbonContent

Modification Type

5
3, 5
3, 5
5
5
5

14
12.5
13.5
12.5
13.5
12.5

270
330
270
390
270
330

C18
C18
C18
C18
C18
C8

15%
19%
15%
23%
15%
12%

Difunctional
Monofunctional
Monofunctional
Monofunctional
Monofunctional
Monofunctional

5

12.5

330

C4

4%

Monofunctional

Mightysil RP-18GP
Mightysil RP-18GP
Mightysil RP-18GP Aqua
Mightysil RP-18(H)GP
Mightysil RP-18(L)GP
Mightysil RP-8GP
Mightysil RP-4GP

Mightysil RP-18MS(Support LC/MS )This is an improved column of the conventional "Mightysil GP" series, and can be used reliably for
LC/MS. This column achieved lower back pressure, while maintaining high theoretical plates.
Moreover, the effluent which can be observed at the initial usage of the column has been reduced
to the utmost, by revising the cleaning process and filling method of the filler.

Mightysil RP-18PA(For analysis of
agricultural chemicals)This ODS filler is for analysis of agricultural chemicals where the nonspecific adsorption of oxine
copper has been suppressed to an extremely low level, by revising the deactivation method of theresidual silanol groups. This filler can be used as a common ODS column, and can also be reliably
used for other analyses of agricultural chemicals.

Mightysil DNPHThis column is for DNPH aldehydes measurement.

Mightysil Si60BDF

This column is for petroleum products test.

This filler which monomeric modified aminopropylsilyl based on a high purity silica-gel.
Mightysil NH 2

Aminopropyl type filler is often used in the analysis of the saccharide in particular, but is also
applied as the weak anion exchange filler and the reverse phase separation which used specificselectivity.

Mightysil Si60

This column is normal phase column using purity over 99.99% of high purity silica-gel.

Filler Name

Mightysil RP-18MS
Mightysil RP-18PA
Mightysil RP-18DNPH
Mightysil Si60BDH
Mightysil NH 2
Mightysil Si60

Particle Size
( m)

Pore Size(nm)

Specific Surface
Area (m2/g)

Modification
Group

CarbonContent

Modification Type

5

12.5
13.5
12.5
6
7
6

330
270
330
525
525
525

C18
C18
C18
NH 2
-

18
15
19
-

Monofunctional
Monofunctional
Monofunctional
Monofunctional
-

3

5
5
5
5
5

Supporting analysis validation Reversed-phase HPLC column

High purity silica gel Full end capping Reversed-phase fillers

High purity silica gel
Column;Mightysil RP-18 GP 150-4.6, 5 m

Eluent;Acetonitrile/ 20mM H 3 PO 4

Flow rate;

1.0mL/min

Temp;

40

Detection;

UV250nm5/95

Inj. Vol;

5 L

Sample;1. Oxine-

Column;Mightysil RP-18 GP 150-

Eluent;Methanol/ 20mM Buffer (pH7.6)

Flow rate;

0.5mL/min

Temp;

40

g/mL

Full end capping

Detection;

UV250nmInj. Vol;

5 L

ample;

m

1. Procainamide

40 g/mL

2.Acetyl Procainamide

45 g/mL

40/60*pH adjust; add 20mM K 2 HPO 4 3H 2 O to 20mM KH 2 PO 4

High durability

Variation of back pressure of column
Methanol-Water
Column;Mightysil RP-18 MS
150-2.0, 5 m

Flow rate;

1.0mL/min

Acetonitrile-Water

Durability test
Acidic eluent (pH1.5)

Basic eluent(pH10.0)

Gradient test

Column;Mightysil RP-18 GP 150-4.6, 5 m

Column;Mightysil RP-18 GP 150-4.6, 5 m

Eluent;Methanol/ 0.3M H 3 PO 4 (pH1.5)

Eluent;Methanol/ 20M K 2 HPO 4 (pH10.0)

Flow rate;

1.0mL/min

Flow rate;

1.0mL/min

B; 20

Temp;

Ambient

Temp;

Ambient

C; 80 Methanol

Sample;Anthracene

Sample;Anthracene

1/1

1/1

Column;Mightysil RP-18 GP 150-4.6, 5 m

Eluent;A; 10mM Phosphate buffer(pH4.7)
MethanolFlow rate;

1.0mL/min

Temp;

Ambient

Sample;AnthraceneRetention Time
RP-18(H) GP

RP-18GP

Retention Time(min.)

4
5

2

10

20

RP-18GP

6

3
1

0

RP-18GP
8

7

0

10

20

RP-18MS

RP-18(L) GP

0

10

20

RP-18GP Aqua

RP-8GP

0

2

3

4
NH2

O

N
N

5
NH2

6
HN

7
NH2

8
O

N
N

O

20

Column: 150-4.6 (5 m)Eluent:Acetonitrile/Water=30/70
Flow rate: 1.0mL/min
Temp.:
40
Detection: UV254nm
Inj. Vol.: 10 L
Samples: 1.Caffeine 2.Pyridine 3.Aniline 4.o-Toluidine
5.N-Methylaniline 6.o-Ethylaniline
7.p-Nitroanisole
8.N,N-Dimethylaniline

RP-4GP

1

10

N
NO2

N

Stereoselectivity
Column:Eluent;Flow rate;
Temp.;
Detection;
Inj. Vol.;
Samples:

546

7150-4.6 (5 m)Methanol/Water=80/20
1.0mL/min
40
UV254nm
5 L
1.Uracil,
2.Caffeine,
3.Phenol,
4.n-Butylbenzene,
5.o-Terphenyl,
6.n-Amylbenzene,
7.Tripheniylene

5
4

0

5

10

7

6

Retention Time (min.)k' Amylbenzene
k' Amylbenzene /k' Butylbenzene
k' Triphenylene /k' o-Terphenyl

RP-18GP
4.92
1.47
1.53

RP-18GP Aqua
4.99
1.47
1.47

RP-18GP
5.98
1.48
1.47

Figure of fillers
Si
O
Monofunctional filler(Monofictional)

Difunctional filler(Difunctional)

Mightysil RP-18GP

Mightysil RP-18GP

Surface of silica gel

Mightysil RP-18GP

C

Guarantee contents

Supporting in analysis validations in GLP GMP

Severe quality control

Three filler batches is prepared at all times

Provide Certificate of Analysis

Filling condition Theoretical plates

Test of physicochemical characteristics and the elution characteristics

Test contents and standard value of each filler batches
Test of the physicochemical
characteristics

RP-18

RP-8

RP-4

Measurement method

Test of the elution characteristics

RP-18

RP-8

RP-4

General description

Test contents and standard value of filling condition
Guaranteed item

RP-18

RP-8

RP-4

General descriptionDifunctional ODS silica gel
The "Mightysil RP-18 GPII" is a difunctional ODS column which consists of an octadecylsilyl (ODS) agent bonded with 2
adjacent silanol groups. Since the end capping is optimized to deactivate the residual silanol groups to the utmost, the peaktailing is suppressed to a minimum. Moreover, the high sensitivity of the hinokitiol which is known as a coordination compound
was realized.
This product is excellent in durability and can be used for high precision analysis therefore it can be used reliably for analysisof extensive samples, including samples separated by the conventional "Mightysil RP-18 GP."High sensitivity
Comparison with other products

ColumnEluentFlow rate
Temp.
Detection
Inj. Vol.
Sample

RP-18GP

RP-18GP

Competitor A
Column< coordination compound (Hinokitiol)>
15025mM Phosphoric acid/Acetonitrile=60/40
1.0mL/min
40
UV254nmCompetitor B
ColumnO
HO
Competitor C
ColumnCompetitor D
Column

Hinokitiol

Hinokitiol

RP-18GP

RP-18GP

A

B

C

D

Theoretical plate number

104160102257

87140

97560

11495790473

Peak symmetry

1.49

3.23

1.61

3.49

3.40

1.92

Excellent durability
Generally, the ODS silica gel filler is known to be hydrolyzed by a trifluoroacetic acid (TFA) solution, and deteriorates the column
performance irreversibly. Compared with the monofunctional ODS silica gel "Mightysil RP-18 GP," since the "Mightysil RP-18
GPII" consists of a structure where the ODS group is not separated easily, it demonstrates excellent durability even under
severe acidic conditions, and stable retention times can be obtained.
After a 0.5% TFA solution was supplied to the column forsufficient replacement, the column was sealed and left for apredetermined time at a room temperature of 15 to 20 °C.
After a predetermined time, the retention time of the anthracene
was measured, and compared with the initial retention time.
ColumnEluentFlow rate
Temp.
SampleMightysil RP-18GP 150-4.6 (5 m)
Mightysil RP-18GP 150-4.6 (5 m)
Acetonitrile/Water=75/25
1.0mL/min
40
AnthraceneDifference of stereoselectivity
Comparison of RP-18GP

Mightysil RP-18GP

Mightysil RP-18GP

Retention time (min.)Retention time (min.)Column:Eluent:Flow rate:
Temp:
Detection:

and RP-18GP

250-4.6(5 m)
Acetonitrile / Water = 75 / 25
1.0mL / min
40
UV254nmApplication DATA (Gradient analysis)
Analysis of PAHs

Retention time (min.)Column:Eluent:Flow rate:
Temp.:
Detection:
Inj. Vol.:

Mightysil RP-18GP
250-4.6 (Acetonitrile/Water=75/25 20min.
1.0mL/min
40
UV254nmSamples: 1.Naphthalene
3.Fluorene
5.Phenanthrene
7.Fluoranthene
9.Pyrene
11.Benzo[j]fluoranthene
13.Benzo[k]fluoranthene
15.Benzo[a]pyrene
17.Indeno[1,2,3-cd]pyrene

0.2 g/mLg/mL

0.2 g/mL
0.2 g/mL

10min.

2.Acenaphthylene
4.Acenaphthene
6.Anthracene
8.Benzo[a]anthracene
10.Chrysene
12.Benzo[b]fluoranthene
14.Benzo[e]pyrene
16.Dibenz[a,h]anthracene
18.Benzo[g,h,i]perylene

1.0 g/mL

0.2 g/mL
0.2 g/mLODS filler which is analyzed by a 100% water

Water repellent phenomenon of filler

Separation of hydrophilic compound
Mightysil RP-18 GP Aqua 250-4.6 5 m
Separation of amino acid

Separation of nucleic acidEluent;Flow rate;

Water 100%
1.0mL/minEluent;Flow rate;

3mmol/L KH 2 PO 4
1.0mL/min

Detection
Temp;

RI
35

Inj. Vol;
Sample;20 L
1.Alanine (0.1mg/mL)
3.Isoleucine (0.2mg/mL)

Detection;
Temp;
Inj. Vol;
Sample;

UV254nm
35
10 L
CMP, UMP, GMP, IMP, TMP

2.Valine (0.2mg/mL)
4.Leucine (0.2mg/mL)

AMP each 0.05mg/mL

Analysis of glucose oligomer
Flow rate;
Detection;
Temp.;
Inj. Vol.;
Sample;

1.0mL / min.
RI
Room Temp.(24)
20 L
Glucose Oligomer Mixtures(DP4 20)
Commercial Product 25mg/mL

Eluent Water / Acetonitrile

100 / 2.5

Eluent Water / Acetonitrile

100 / 2

Correlation between RP-18GP-Elution of steroids
RP-18GP

Eluent;Flow rate;
Detection;
Temp;
Inj. Vol;
Sample;Column;Acetonitrile/ 0.05mmol/L KH 2 PO 4
1.0mL/min
UV245nm
Ambient (22 )20 L
1. Cortisol
2. Cortisone
3. 6- -Methylprednisolone
250-4.6mm, 5 m

RP-18GP Aqua

30/70

Stability(1)Separation of dopamine

Columnsize;
Eluent;Flow rate;
Detection;
Temp;
Inj. Vol;
Sample;General ODS column
First time

Aqua
First time

72 hours later

72 hours later

250-4.6mm, 5 m
20mM KH 2 PO 4 / Methanol 99/1
including of 5mmol/L Sodium pentasulfonate
1.0mL/min
UV230nm
40
10 L
Dopamine HCl 0.1mg/mL(2)Separation of nucleic acid

<Analysis condition>
Column;Eluent;Flow rate;
Detection;
Temp;
Inj. Vol;
Sample;Mightysil RP-18 GP Aqua 150-4.6mm, 5 m
5mM KH 2 PO 4 buffer
0.5mL/min
UV254nm
35
5L
Nucleotide each 0.05mg/mL<Conditioning of column>
Acetonitrile/Water=50/50

Column ;Mightysil NH 2 150-4.6, 5 m

Eluent ;Acetonitrile/50mM NH 4 H 2 PO 4

Column ; Mightysil NH 2 150-4.6, 5 m
70/30

Eluent ;Acetonitirile/ Water= 80/ 20

Flow rate ; 1.0mL/min

Detec ion ;UV210nmFlow rat; 1.0mL/min

Temp ;

Inj. Vol ;10 L

Temp. ;

40

Sample;40
1.Riboflavine

20 g/mL

2.Pyridoxine HCl 20 g/mL

Inj. Vol ; 10 L

3.Nicotinic acid20 g/mL

4.Folic acid

Sample ;80 g/mL

Detection

1.Stevioside 0.5mg

Column ;Mightysil NH 2 150-4.6, 5 m

Column;Mightysil NH 2 250-4.6, 5 m

Eluent ;50mM Phosphate buffer (pH3 5)

Eluent;Acetonitrile/ Water
1.0ml/min

Flow rate ; 1.0mL/min

Detection ;UV254nmFlow rate;

Temp ;

Inj. Vol ;10 L

Detection;

RI

Inj. Vol;

10 L

Sample1.Fructose

40

Sample ; 1. 5’-CMP, 2. 5’-AMP, 3. 5’-UMP, 4. 5’-IMP, 5. 5’-GMP each 0.1mg/mL

2.Glucose
3.Sucrose
4.Maltose
5.Lactoseeach 10mg/mL

75/25

UV210nmColumn;

Mightysil Si60 150-4.6, 5 m

Column;Mightysil Si60 150-4.6, 5 m

Eluent;n-Heptane / 2-Propanol = 99 / 1

Eluent;n-Hexane /Diethylether /1.0mL/min

Flow rate;

1.0mL/min

Detection;

UV254nmFlow rate;

2-Propanol = 5 / 4 / 1

Range;

0.32AUFS

Detection;

UV240nm

Temp;

40

Range;

0.16AUFS

Inj. Vol;

5 L

Temp;

40

Sample;1.Dioctyl phthalate

Inj. Vol;

10 L

2.Di-n-butyl phthalate

Sample;1.Pr gesterone 0.02mg/L

3.Dirth l phthalate

2.Testosterone 0.02mg/L

4.Dimethyl phthalate

3.Hydrocortison

0.1 mg/L

each 0.1% in heptane

Column;Mightysil Si60 150-4.6, 5 m

Eluent;2,2,4-Trimethylpentane /Ethylacetate = 95 / 5

Flow

1.0mL/min

Column;Eluent;Flow rate;

Mightysil Si60 150-4.6, 5 m
n-Hexane / Ethylacetate=
37 / 63
1. mL/min

Detection;

UV280nm

UV 94nm

Range;

0.04AUFS

Range;

0.08AUFS

Temp;

40

Temp;

40

Inj. Vol;

20 L

Inj. Vol;

10 L

Sample;1.Catechin 0.05mg/ml

ate;
Detection;

Sample;1. -Tocopherol
2. -Tocopherol
3. -Tocopherol
4. -Tocopherol
each 0.1% in Trimethylpentane

T: + c ESI Full ms [ 100.00-2000.00]

RT: 10.28-20.97

NL: 2.00E6

100
Re
lati
ve
Ab
un
da
nc
e

R 80
el
ati
ve
60
Ab
un
da
nc 40
e
20

0
500

1000
m/z

1500

2000

NL: 2.13E5

2.782000003.58

100000nm=499.5-500.5
PDA
Sudan02_080407
144412

7.07

5.42

11.60

0
0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

Eluent:Acetonitrile/0.1%(v/v)Formic acid=90/10

19

Time (min)NL:
3.60E4

1.63
7.16

5.51

20000

2.88
0

1

2

3

4

5

6

TIC MS
Sudan02_0804
07144412

11.69

3.68

7

8

9

10

11

12

13

14

15

16

17

18

19

Time (min)NL: 8.52E2

2.87

m/z=
293.50-294.50 MS
Sudan02_08040714
4412

500
0
0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

Time (min)NL: 5.19E3

3.68

5000

m/z=
248.50-249.50 MS
Sudan02_08040714
4412

0
0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

HO
O2N

19

Time (min)

N N

m/z=
276.50-277.50 MS
Sudan02_08040714
4412

5000
0
1

2

3

4

5

N N

NL: 1.28E4

5.51
10000

0

HO

6

7

8

9

10

11

12

13

14

15

16

17

18

HO

HO

19

N N

N N

Time (min)NL: 1.62E4

7.16

m/z=
352.50-353.50 MS
Sudan02_08040714
4412

10000
0
0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

HO

19

Time (min)

N N

N N

NL: 9.87E3

11.69

m/z=
380.50-381.50 MS
Sudan02_08040714
4412

5000
0
0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

Time (min)RT:0.00 - 30.00 SM:15B

NL: 2.21E6
TIC F: ITMS + c ESI
Full ms [150.00-800.00] MS
Malachite_green01_
080403171158

2200000
2000000
1800000
1600000
1400000
1200000
1000000
800000600000400000200000
0

2

4

6

8

10

12

14
16
Time (min)

18

20

22

24

26

28

Malachite_green01_080403171158# 4068 RT:10.99 AV:1 NL:1.75E6
T: ITMS + c ESI Full ms [ 150.00-800.00]
329.351500000

1000000+

NN

330.295000000150240.18200

250

285.37313.42 331.28
300

391.64350

400

760.11
450

500

550

600

650

700

750

800

m/z
Malachite_green01_080403171158# 6548 RT:17.62 AV:1 NL:1.17E6
T: ITMS + c ESI Full ms [ 150.00-800.00]
331.37
1000000
800000N

N

600000400000332.372000000150210.39 239.34
200

250

284.39 329.33 333.42 363.29
300

350

637.48
400

450

500
m/z

550

600

650

729.15 761.16787.79
700

750

800

Eluent:A.0.01mol/L Ammonium formate
B. Acetonitrile

N N

Mightysil RP-18 PA

Column;Mightysil RP-18PA 150-4.6, 5 m

Eluent;

CH3CN / 50mM Phosphate buffer (pH3.5) 40/60

Flow rate;

1.0 L/min

Sample; Oxine-Copper 1 g/mL

Temp.;

40

Inj.Vol; 1 L

Detection;

UV230nmpH adjust;add KOH

Column;Mightysil RP-18 PA 150-4.6, 5 m

Eluent;CH 3 CN/25mM Phosphate buffer(pH3.30)=7/13

Flow rate;

1.0mL/min

Temp;

40

Detection;

UV

270nm(0UV

240nm(2.5 5.0min)

230nm(5.013.0min)

UV

240nm(13.016.0min)255nm(16.019.5min)

UV

245nm(19.525.0min)Inj. Vol;
Sample;5 L
1. Asuluam

2. Oxine-Cupper

3. Triclopyr

4. Thiram

5. Mecoprop

6. Flazasulfron,

7. Siduron

8. Halosulfuron-methyl

9. Azoxystrobineach 1 g/mL

Repeatability Guarantee

2.5min)1

Column;EluentMightysil RP-18 PA 150-4.6, 5 m
A; Acetonitrile/ Water/ Acetic acid = 10/ 90/ 0.3
B, Acetonitrile/ Water/ Acetic acid = 60/ 40/ 0.3

Gradient;

Min

A(%)

B(%)

0

100

0

25

7

25

30

75

25

Flow rate;

1.0mL/min

Temp;

40

Detection;

UV265nmInj. Vol;
Sample;L
1. Sulfadiazine, 2. Sulfamerazine, 3. Sulfadimidine,
4. Sulfamethoxypyridazine, 5. Sulfamonomethoxine, 6. Sulfamethoxazole,
7. Sulfisoxazole, 8. Sulfadimethoxine, 9. Sulfaquinoxaline sodium salteach 0.01mg/mL in Methanol

Column;Mightysil RP-18 PA 150-4.6, 5 m

Eluent;Acetonitrile/ Water = 70/ 30

Flow rate;

1.0mL/min

Temp;

40

Detection;

UV250nmInj. Vol;

5 L

Sample;1. Diflubenzuron, 2. Tebfenozide, 3. Hexaflumuron, 4. Teflubenzuron,
5. Lufenuron, 6. Flufenoxuron, 7, Chlorfluazuron
each 10mg/L in Acetonitrile

2
3 4
5

6
7

8
9

Column ;Mightysil DNPH 250-4.6

Eluent ;A : Water

Flow rate ;1mL/minDetection ;UV360nm

B : Methanol

Temp. ; 40
Standard;

Aldehydes-2,4-DNPH
standard stock solution 2
Cat.No.01106-96

Sample Conc. ;1ppm

Sample Vol. ;

10 L

1.

Formaldehyde-DNPH

2.

Acetaldehyde-DNPH(7.00min)(9.42min)3.

Acrolein-DNPH(13.25min)

4.

Acetone-DNPH(13.75min)

5.

Propionaldehyde-DNPH(14.81min)

6.

Crotonaldehyde-DNPH(19.81min)

7.

Butylaldehyde-DNPH(21.74min)

8.

Benzaldehyde-DNPH(24.59min)

9.

iso-Valeraldehyde-DNPH(25.40min)

10.

Valeraldehyde-DNPH(28.20min)

Column ;Mightysil DNPH 250-4.6

Eluent ;A : Water

Flow rate ;1mL/min

B : Acetonitrile
Detection ;UV360nmTemp. ;

40

Standard ;Aldehydes-2,4-DNPH
standard stock solution 2
Cat.No.01106-96

Sample Conc. ;1ppm

Sample Vol. ;

10 L

1.

Formaldehyde-DNPH(4.82min)2.

Acetaldehyde-DNPH(5.81min)3.

Acrolein-DNPH(7.09min)4.

Acetone-DNPH(7.33min)5.

Propionaldehyde-DNPH(7.85min)6.

Crotonaldehyde-DNPH(9.09min)7.

Butylaldehyde-DNPH(10.40min)

8.

Benzaldehyde-DNPH(11.13min)9.

iso- Valeraldehyde-DNPH(13.51min)

10.

Valeraldehyde-DNPH(14.25min)is confirmed

Expansion

Inspection of column efficiency

1-1 The column is shipped with the following solvent.
RP-18/RP-8
DNPH (H) (L)Analytical
I.D. 1mm

3mm

Analytical
I.D. 4.6mm 6mm
Preparative
I.D. 10mm

RP-4

Acetonitrile / Water
75/25

Si60/NH 2

Acetonitrile / Water
60/40
Acetonitrile / Water
75/25

n-Heptane

Methanol / Water
80/20

50mm

2 Starting Separation
2-1 Equilibrating

2-1-1 Reversed Phasea)
Organic solvent/water

Consider if the mobile phase you use is miscible with solvent in the column, then purge the column until the baseetc.), it is advised to place organic 100% step in equilibration and subsequent stepwise increasing of water isfollowed.

b)
Organic solvent/buffer

Basically same to a), in addition, the last step before the mobile phase should consist of organic/water, whichshould have the same ratio of eluent. Equilibration takes more time when the salt concentration is low (510mmol/L or less).

c)
Mobile phase of water orbuffer is 100%, or ofhigher water content

The surface of RP-18 is highly hydrophobic so water 100% mobile phase naturally has a lower affinity to stationary
phase. If the mobile phase you use has higher water content, we recommend gradually changing to the mobile
phase by stepwise increases of water through organic solvent100%, to gain stable separation. In case of separation
at water of 100%, it is advised to use "RP-18GP Aqua", which exhibits more stable retention in water at the 100%
mobile phase. e.g.)d)Equilibration of the mobile phase containing ion-pair reagent requires amore longer time. In general, it often takes

Mobile phase containing
ion-pair reagent

solvent over 100 ~ 200 times of column volume until completion. The procedure is as same as C).

2-1-2. Normal phase (Silica)
Organic solvent

After confirmation that the present solvent in the column is miscible with the preferred mobile phase, purge until you
gain a stable base line.
-Propanol

2-1-3. NH 2
Organic solvent
or
Organic solvent/
Aqueous solvent

In case of non-polar solvent use only, equilibration is the same as silica. If you use the mobile phase with water orpolar solvent like methanol, be aware that these solvents may not be miscible with the shipped one, you should
place an intermediate step to avoid failure of equilibration.

2-2. Caring for the column<Mobile phase>pH

Mightysil is a silica based material, so use between pH 2.0~ 7.5.

Quality of the solvent

HPLC grade solvents should be used and high purity reagents are recommended for buffer or ion-pair. Always filter
the mobile phase through membrane filters of 0.45µm or under .

Degas

Always degas the mobile phase. Often, It is not sufficient to only use the online-degasser equipped with HPLC
system.

Salt concentration

Salt concentration is preferable under 0.2mol/L. Confirm solubility of salts at changing mobile phases to prevent
precipitation of buffer.

<Handling>
Prevention of channeling

Physical shock or extremely rapid change in pressure when starting to pump or changing to different mobile
phases, will lead to channeling in the column and result in irreversible performance loss.

Pressure and flow
velocity

Use the column under pressure of 20MPa (About 200kgf/cm 2 or 2900psi). Higher flow rate leads to void the column
and result in performance loss.

Temperature

Do not exceed 80°C.

3. Washing or regenerating of the column

3-1. Reversed phase (DNPH)
GeneralRP-18 GP Aqua
Column used with
ion-pair reagent

Use acetonitrile. For heavy contamination, Tetrahydrofuran can be used.
If 100% water is used, the contaminant of mobile phase is well adsorbed to stationary phase, so changing ofretention or loss of performance tends to occur. In this case washing by acetonitrile will recover performance.
Generally washing takes a long time to eliminate ion-pair reagent from the surface of the stationary phase. Follow
these washing steps: to wash out the buffer purge the column 100 ~ 200times of column volume of solvent, desalt
from the mobile phase, then rinse the column with 100% acetonitrile until a stable base line is obtained.
e.g.) Acetonitrile/Buffer(Ion pair)=10/90
Acetonitrile/Water= 10/90
Acetonitrile 100

3-2. Silica gel
Wash with more polar solvent than actual used in the mobile phase. To avoid continuously deactivating silanol, thecolumn used for the non-polar solvent system should not be rinsed with methanol except in special cases.
e.g.)cleaning solvent (2-Propanol)
After washing, the column is re-equilibrated to the actually used mobile phase at procedure of 2-1-2 in reverse
order, 15~20 times of column volume are purged at each step.

3-3. NH 2
Normal phase solvent

Wash with polar solvent considering miscibility.
If you need to wash with water, the content should be below 30%. The stationary phase will be damaged at over
30%. Complete washing within as short a time as possible if water above 30% is needed.

Reversed phase solventng solvent (Acetonitrile/Water = 70/30)

4. StorageExample of Reducing Diameter of a Column for General-purpose Analysis Columns (150-4.6, 5 µm)

100-4.6mm I.D. (3 m)

150-4.6mm I.D. (5 m)

10.7MPa

5.9MPa150-3.0mm I.D. (5 m)Flow rate;
Cell path length;

1.0mL/min
10mm

Flow rate;
Cell path length;

0.5mL/min
10mm

Flow rate;
Cell path length;

0.2mL/min
5mm

100-3.0mm I.D. (3 m)

5.6MPa9.5MPa150-2.0mm I.D. (5 m)

100-2.0mm I.D. (3 m)

6.5MPa

11.6MPa50-2.0mm I.D. (3 m)(min)Column; Mightysil RP-18GP
Eluent; Acetonitrile/ Water
Temp.; 40
Detection UV254nm
Inj. Vol.; 2 L

30/ 709.1MPa(min)

Samples;
1. Caffeine
2. Pyridine
3. Aniline
4. o-Toluidine

35 g/mL
35 g/mL
85 g/mL
250 g/mL

5. N-Methylaniline
6. 2-Ethylaniline
7. 3-Nitroanisole
8. N,N-Dimethylaniline

50 g/mL
310 g/mL
280 g/mL
70 g/mLin Eluent

When you use the column of 1mm I.D. , 2mm I.D. , including the cell volume of the detector, it’s required optimization of the flow path system.

Column Size

150-4.6mm
I.D.

150-3mm
I.D.

150-2mmI.D.

100-4.6mm
I.D.

100-3mmI.D.

100-2mm
I.D.

50-2mmI.D.

Sensitivity

1

2

5

1

2

3 5*

3 5*

Time

1

1

1

2/3

2/3

2/3

1/3

1

1/2

1/5

2/3

1/3

2/15

1/15

1

1

1

1

1

1

1/2

Solvent
consumption
Efficiency

Scale up of Analytical to Preparative
Preparative (I.D. 10mm~50mm)
Length-I.D.
(mm)

Flow rate(mL/min)injection volume
( L)

Load
(mg)

250-4.6

1

1 15

0.05 1

250-6

1 2

10 25

0.1 2

250-104 6

20 1501 20

250-2015 20

50 1000

10 100250-50100 120500 2500

100 2000

Analytical column

Preparative column

Other information

The calculation of the flow rate
Because linear velocity (cm/min) is showed as (1), when under the conditions of the I.D. 4.6mm, flow rate 1.0mL/min is adapt to
I.D. 6mm, the flow rate at time of the combined linear velocity is 1.70 mL/min by (2)~(4).

Relations of the I.D. and cross-section and flow

I.D. (mm)Cross section2(cm )Flow rate3(cm /min)Using the upper expression, the change of flow rate is

2

0.03

0.19

shown by the change of I.D. to the right chart. The solvent

3

0.07

0.43

consumption can reduce by reduction of the flow rate due

4.6

0.17

1

to the smaller diameter.

6

0.28

1.70

10

0.79

4.73

20

3.14

18.90

Particlediameter ( m)The number oftheoretical

Columnpressure

5

1

1

3

1.7

2.8

Relations of the particle diameter and the numberof theoretical plates and column pressure
It shows the ratio of the respective values of the particle
diameter 3

when the number of theoretical plates andcolumn pressure of particle diameter 5 m is set to 1 to
the right table.

Guard column system

Only Analytical column

Equipped withcolumn system

Decrease in Theoretical Plate Height 0%

Decrease in Theoretical Plate Height 8%

Maker

Mightysil

Guard

Equipped withconventional precolumn

Decrease in Theoretical Plate Height 60%

Length of "h"type

No.10-32UNF
P=4.8mmAbt. 2.0mm
Abt. 3.5mm
Abt. 4.5mm
Abt. 2.3mm
Abt. 2.9mm
Abt. 2.5mm

A

No.10-32UNF
P=4.8mmAbt. 4.5mm

B

Old-Hitachi(655 or earlier)M6
P=6.0mmAbt. 4.7mm

C

Old-Shimadzuu(LC5A or earlier)No.10-32UNF
P=4.8mmAbt. 1.4mm

DType of connection

Connection TypeKANTO/Merck
WatersTOSOH
Shimadzu LC6A or later
Hitachi L6500 or later
Jasco 800 or later
Old-Jasco(BIP,TRI ROTAR or earlier)

Product name

Description

PackingCat. No.

Handy Connectors

One-piece PEEK finger tight fitting
For 1/16"capillary tube

5 pieces

18685-96of the length of "h"Metal ferrule and Nut
The length of "h" is fixed, it is caused a leak connection and a deadvolume.

Handy Connectors