L-column2 ODS

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Chemicals Evaluation and Research Institute, Japan

1 2 3 4 5 6 7 8 9

L-column2 ODS exceeds even the high performance of L-column ODS by virtue of its advanced new end-capping method. It accommodates the analysis of acidic, basic and chelating compounds.

■ Characteristics of L-column2 ODS・　Sharper peaks for acidic, basic and chelating compounds due to extremely low silanol adsorption.・　Superior peak shapes in both acetonitrile/water and methanol/water mobile phases makes L-column2 ODS convenient to use.・　Economical due to high durability in a wide range of pH and temperature.・　Uniform lot to lot reproducibility of analyses due to extensive quality control measures.

Next generation high performance silica-based ODS column　　

L-column2 ODS 2 μm, 3 μm, 5 μm120 ÅpH 2–9L1

Average particle sizeAverage pore sizeRange of pHUSP category

L-column2 ODS

L-column ODS

Brand D-1

［Analytical conditions］Column: 4.6×150 mm (C18, 5 μm)Mobile phase: CH3CN/25 mM Phosphate buffer pH 7.0 (40/60)Flow rate: 1 mL/minTemp.: 40 ºCDetection: UV 220 nmInj.vol.: 1 μL

1 2 3 4 5 6 7 8 9

1 2 3 4 5 6 7 8 9

1 2 3 4 5 6 7 8 9

1 2 3 4 5 7 6 8 9

1 2 3 4 5 6 7 8 9

Brand C-3

Brand F-2

Brand E-1

■ Comparison between L-column2 ODS and other columnsBasic compounds show peak tailing due to their adsorption by residual silanol groups. The shape of the peaks depends notably on the amount of silanol groups (Fig. 3).

0　 5 10 15 20 25 30 35 40 45　　　　　 　 Time (min)

■ Residual silanol groupsThe level of residual silanol groups is measured by FT-IR spectrum. The spectra of C18 without end-capping and the fully end-capped L-column2 ODS are shown (Fig. 2). The spectrum region for C-H and O-H provides quantitative information as well as qualitative identification. FT-IR spectra show virtually no presence of silanol groups on L-column2 ODS. In addition, the spectrum region for O-H (the right spectra) shows that L-column2 ODS has the least residual silanol groups of any column tested.

Not end-capped C18 L-column2 ODSC-H

Fig. 2 FT-IR spectra for L-column2 ODS.

-OH of silanols

4000 3500 3000 2500　　　 (cm-1)

4000 3500 3000 2500　　　 (cm-1)

End-capping

C-H

Fig. 3 Chromatograms of basic compounds using L-column2 ODS and other columns.Application No. 2011

1. Fexofenadine (50 mg/L); 2. Chlorpheniramine (50 mg/L)3. Triprolidine (50 mg/L); 4. Diphenhydramine (50 mg/L)5. Diphenylpyraline (100 mg/L); 6. Homochlorcyclizine (200 mg/L)7. Hydroxyzine (200 mg/L); 8. Astemizole (50 mg/L)9. Promethazine (200 mg/L)(in CH3CN)

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L-column2 ODSChemicals Evaluation and Research Institute, Japan

L-column2 O

DS

L-column O

DS

Brand C

-3

Brand D

-1

Brand E

-1

Brand F

-2

1.16

41.36

4

1.20

5

1.24

9

1.15

8

←N

.D.

1.09

1

1.23

9

1.09

1

1.09

8

1.13

4

1.06

1

0

1.0

2.0

3.0

4.0

7.0

US

P ta

iling

fact

or

1

［Analytical conditions］Column: 4.6×150 mm (C18, 5 μm)Moble phase: CH3CN/20 mM H3PO4 (2/98)Flow rate: 1 mL/min; Temp.: 40 ºC; Detection: UV 210 nm; Inj.vol.: 1 μL

1. Formic acid (0.5 %)2. Acetic acid (1 %)(in H2O)

1

1111

1 2 3 4　　　　 Time (min)

2 2 2222

1

［Analytical conditions］Column: 4.6×150 mm (C18, 5 μm)Moble phase: CH3CN/20 mM H3PO4 (40/60)Flow rate: 1 mL/min; Temp.: 40 ºC; Detection: UV 240 nm; Inj.vol.: 10 μL

1. Hinokitiol (0.5 mg/L)(in CH3CN)

1↓

1

11

■ HinokitiolO

OH

H3C CH3

■ Formic acid ■ Acetic acid

O

H H

O

H3C H

3.29

9

6.63

8

2.03

4

1.27

7

1.15

8

3.66

1

Acidic compounds also show peak tailing on poorly end-capped columns. Superior-performance columns provide sharp peaks of basic and acidic compounds.

Brand C-3L-column ODSL-column2 ODS Brand D-1 Brand E-1 Brand F-2

N.D.

Chelating compounds are adsorbed by metal impurities present on the surface of base silica gels. Fewer metal impurities and higher end-capping surface coverage provide sharper peaks of chelating compounds.

Brand C-3L-column ODSL-column2 ODS Brand D-1 Brand E-1 Brand F-2

5 6 7 8 9 10　　　　 Time (min)

6 7 8 9 10 11　　　　 Time (min)

0 3 6 9 12 15　　　　 Time (min)

7 8 9 10 11 12　　　　 Time (min)

1 2 3 4　　　　 Time (min)

1 2 3 4　　　　 Time (min)

1 2 3 4　　　　 Time (min)

1 2 3 4　　　　 Time (min)

1 2 3 4　　　　 Time (min)

Application No. 2008

Application No. 2009

1.08

0

2.10

62.57

9

2.31

9

1.63

12.03

7

1.26

7

1.95

8

1.82

8

1.22

7

1.99

5

1.11

9

■ 5. Diphenylpyraline ■ 9. Promethazine

NO CH3CH

S

N

N CH3H3C

CH3H

Fig. 4　Chromatograms of acidic compounds, formic acid and acetic acid, using L-column2 ODS and other columns.

Fig. 5 Chromatograms of chelating compound, hinokitiol, using L-column2 ODS and other columns.

Fig. 6 Comparison between tailing factors of the adsorptive compounds for L-column2 ODS and those for other columns.

Acidic compounds

Basic compounds

Chelating compound

6 7 8 9 10 11　　　　 Time (min)

5 6 7 8 9 10　　　　 Time (min)

L-column2 ODS

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Chemicals Evaluation and Research Institute, Japan

■ Low adsorptionBasic compounds show peak tailing due to their adsorption by residual silanol groups. Therefore, adsorption is inhibited using an acidic mobile phase or a mobile phase including methanol to control peak tailing. Because the residual silanol groups show intrinsic activity using a neutral mobile phase or a mobile phase including acetonitrile as an organic solvent, peak tailing of basic compounds due to adsorption occurs when using poorly end-capped columns.

L-column2 ODS is perfectly end-capped, so it does not adsorb analytes using any composition of mobile phases and provides a superior peak shape. While peak tailing of basic compounds using a neutral mobile phase or a mobile phase including acetonitrile occurs with almost all C18 columns, peak tailing of basic compounds using these mobile phases does not occur with L-column2 ODS (Fig. 7). Therefore, it can be used in a wide range of compositions of mobile phases. This is an important point when selecting a column.

■ Improved durabilityA durability test was carried out under high temperature conditions which accelerate deterioration of columns. L-column2 ODS was stable for the longest time. Although it is silica-based, it shows superior durability even under alkaline conditions due to the extremely dense end-capping.

[Accelerated acidic mobile phase lifetime test]Under acidic conditions, below pH 1, both the end-capping group and the ODS group are hydrolyzed. Retention time decreases with the decrease of ODS groups. Resolution decreases with the progression of the hydrolysis. L-column2 ODS resists hydrolysis even under these harsh conditions to maintain retention and resolution for an extended lifetime (Fig. 8).

[Accelerated alkaline mobile phase lifetime test]Dissolution of the base silica is accelerated in alkaline mobile phase. Efficiency (theoretical plate number) drops suddenly in these conditions. L-column2 ODS has superior durability under high pH conditions (pH 10) due to the protection of the silica surface afforded by the advanced end-capping process (Fig. 9).

Moble phase： CH3OH/10 mM Ammonium acetate (65/35)CH3OH/25 mM Phosphate buffer pH 7.0 (80/20)CH3CN/10 mM Ammonium acetate (40/60)CH3CN/25 mM Phosphate buffer pH 7.0 (55/45)

［Analytical conditions］Column: 4.6×150 mm (C18, 5 μm)Flow rate: 1 mL/min; Temp.: 40 ºC; Detection: UV 220 nm; Inj.vol.: 5 μLSample: Amitriptyline

L-column2 O

DS

L-column O

DS

Brand C

-3

Brand D

-1

Brand E

-1

Brand F

-2

0.96

6

1.15

5

1.22

2

1.05

4

1.16

9

1.05

6

1.32

1

1.35

91.67

7

1.05

3

1.13

1.962.

412.56

1.34

1.14

0

1.0

2.0

3.0

US

P ta

iling

fact

or

1.27

7

2.03 2.

188

1.32

91.6

2.76

4

2.80

2

1.08

5

Fig. 7　Difference of tailing factor by kind of solvent (amytriptyline).

[Durability test contidions]Column: 4.6×150 mm (C18, 5 μm)Mobile phase: 50 mM TEA in CH3OH/50 mM TEA in H2O (10/90)Flow rate: 1 mL/min; Temp.: 50 ºC; Sample: Naphthalene

0

20

40

60

80

100

0 　 20 40 60 80 100 Time (Hour)

Sta

bilit

y of

Num

ber o

f the

oret

ical

pla

tes

(%)

[Durability test contidions]Column: 4.6×150 mm (C18, 5 μm)Mobile phase: TFA/CH3OH/H2O (2/10/90)Flow rate: 1 mL/min; Temp.: 90 ºC; Sample: Naphthalene

Fig.8　Accelerated durability test with acidic mobile phase (under pH 1).

0

20

40

60

80

100

0 50 100 150 200 250 300 Time (Hour)

Sta

bilit

y of

Num

ber o

f the

oret

ical

pla

tes

(%)

Fig.9　Accelerated durability test with alkaline mobile phase (pH 10).

L-column2 ODSL-column ODSBrand D-1Brand F-2Brand H-1

L-column2 ODSL-column ODSBrand D-1Brand F-2Brand H-1

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L-column2 ODSChemicals Evaluation and Research Institute, Japan

■ Superior reproducibilityVariation between product lots due to residual silanol groups is prevented by superior end-capping. Although reproducibility of retention times of basic compounds is poor between product lots, the coefficient of variation of the retention times between the product lots is under 1 % for L-column2 ODS (Fig. 10). Regardless of the product lot, L-column2 ODS provides the same results in HPLC analysis because of thorough quality control.

■ CartificationSpecifications and test results of each product lot as well as test results for each column are supplied with the column (Fig. 11). In addtion we support method validation by supplying columns from three different media lots.

［Analytical conditions］Column: L-column2 ODS (C18, 5μm, 120 Å) 4.6×150 mmMobile phase: CH3CN/25 mM Phosphate buffer pH 7.0 (60/40)Flow rate: 1 mL/min; Temp.: 40 ºC; Detection: UV 220 nm; Inj.vol.: 1 μL

Lot.C

Lot.B

Lot.A

2 4 6 8 10 12 14 16　　　　　 Time (min)

12 4

3 56

Lot.ALot.BLot.CCV(%)

tR (1)5.1155.1805.1340.647

tR (2)5.7965.8685.8230.626

tR (3)7.0087.1037.0220.725

tR(4)7.6477.7557.6870.707

tR (5)9.5469.6699.6090.640

tR (6)12.5012.6412.600.575

Fig. 10　Reproducibility between product lots(basic drugs: antidepressants).

Results of Chromatogram Lot No. E0000

10.00 20.00 min Test_C

5.00 10.00 min Test_A

1.00 9.00 min Test_B

5.00 10.00 min Test_E

2.00 4.00 min Test_F

5.00 10.00 min Test_D

CCeerrtt ii ff iiccaattee oo ff aannaallyyssiissL-column2 ODS Lot No. E0000

Analytical Results for Packing Materials Specification Result

Analysis of unbonded Silica gel Median Particle Size(d50)* [mm] 4.3-4.5 4.44 Surface Area** [m2/g] 330-370 332 Median Pore Diameter** [nm] 10.5-13.5 11.9 Pore Volume** [mL/g] 0.95-1.15 0.99 Metal contents***

Al [ppm] < 5.0 0.3 Fe [ppm] < 10.0 1.5 Na [ppm] < 10.0 0.5 Ti [ppm] < 0.5 0.1

* Coulter counter method, ** BET method, *** ICP -AES

Analytical Results for L-column2

Carbon content [%] 16.8-18.8 17.5

Chromatographic Results Test A k(Naphthalene) 3.400-3.800 3.667Test B k(Pyridine)/k(Phenol) 0.340-0.420 0.385Test C k(Caffeine)/k(Phenol) 0.410-0.490 0.450Test C k(Amylbenzene)/k(Butylbenzene) 1.440-1.480 1.462Test C k(Triphenylene)/k(o-Terphenyl) 1.600-1.780 1.629Test D k(Acenaphthene)/k(Amitriptyline) 0.890-0.950 0.890Test E k(Oxine-copper)/k(Caffeine) 0.170-0.200 0.180Test F N(Formic acid) >8000 10699

Approved: Date: - -

L-column2

1. Doxepin (20 mg/L)2. Imipramine hydrochloride (50 mg/L)3. Amitriptyline (50 mg/L)4. Mianserin (100 mg/L)5. Clomipramine (100 mg/L)6. Trimipramine (100 mg/L)(in CH3CN)

Table 1　Silica gel test specifications (extract)■ Base silica gelHigh purity silica gel, in which metal impurities are reduced to the absolute limit, is used as the starting material, facilitating analysis of chelating compounds (Table 1).

Metal impurities

Al

Fe

Ti

Mg

Content (ppm)

5.0

10.0

0.5

5.0

≤≤≤≤

Specifications silica

Fig. 11　Certificate at each product lot of packing materials.

[Items of quality assurance]Physical properties of the base silica gel:Median particle size (d50)Surface areaMedian pore diameterPore volumeMetal content

Properties of the media:Capacity factor of a standardAdsorptive property for basic compoundsAdsorptive property for acidic compoundsAdsorptive property for chelating compoundsSurface hydrophobicityPlanar and non-planar compounds separation performance,etc.

Quality of packing as measured by theoretical plate number

tR: Retention time (min)