LC Technical Note

LT152

GL Sciences Inc.

Metal-free Columns for HPLC

Part 1

Some amino acids, organic acids, tetracyclines and organophosphates are metal chelators. Chelation during
analysis of these compounds can distort the peak shapes and worsen the quantification accuracy. Residual
metals on the packing surface were assumed to cause these problems. However, in the case of microanalysis, it
has turned out that metals on the wetted parts such as tubing or frits also has a strong influence. Phosphatebuffers are widely used in metal chelator analysis to improve the peak shapes, however, phosphate buffers
cannot be used in LC/MS(/MS) because non-volatile salts precipitate from these buffers.
Metal-free columns have wetted parts made of PEEK instead of metal. In this technical note, some applications
using metal-free columns are shown and the advantages of metal-free columns are described.
(K.Kanno)

NNH

NH2
OH

Histidine

OO

Ethylenediaminetetraacetic acid (EDTA)POOHOHIn HPLC, analytes containing the functional groups above are easily affected by metals.

Metal Effects○ Peak tailing○ No elution○ Low sensitivityetc.

*Special inertization was carried out for the metal wetted parts of the HPLC systems used for this technical note.
Please contact for details.

GL Sciences LC Technical Note
RP Example 1. Tetracyclines
Tetracyclines are metal chelators, and their adsorption onto residual metals on the packing surface andmetal parts of the column results in poor performance.
By using a highly inert column with minimized residual metals, high sensitivity becomes possible withformate buffers, which are often used in LC/MS(/MS). Further sensitivity improvement is possible with ametal-free column.
1.
2.

<Stainless steel column>

Intensity, cps

ODS column A
Particle size：1.7 µm

Oxytetracycline
Tetracycline
2 mg/L each<Metal-free column><Stainless steel column>
InertSustainSwift C18
Particle size：1.9 µm

InertSustainSwift C18
Particle size：1.9 µm

200

200

200

180

180

180

160

160

160

140

140120

120

120

100

100

100

280
60

140
200

2.6

3.0
3.4
Time, min

3.8

60

60

40

40

20

200

2.6

3.0
3.4
Time, minConditions

180

1Different packing inertnessColumn

140280

2

3.80

2.6

3.03.4
Time, minDifferent column material
Stainless steel or PEEK

: InertSustainSwift C18 (1.9 μm, 50 x 2.1 mm I.D.)ODS column A (1.7 μm, 50 x 2.1 mm I.D.)

Mobile phase : A) 0.1 % HCOOH in CH3CN

2. TetracyclineB) 0.1 % HCOOH in H2O
A/B = 10/90 – 1.5 min – 10/90 – 2.5 min – 90/10 , v/v
Flow rate: 0.4 mL/min

Temperature : 40 ℃
Detection: LC/MS/MS (ESI, Positive, MRM)

Injection volume
Concentration : 2.0 mg/L: 10 mL

Q1
1. Oxytetracycline(OTC) 460.92. Tetracycline(TC)445.0

Q3426.2
154.0

Structures are created using Chemistry 4-D Draw which isprovided by ChemInnovation Software, Inc.

3.8

GL Sciences LC Technical Note
RP Example 2. Phosphate group-containing compounds
Phosphate group-containing compounds such as organophosphate pesticides and nucleotides easily form metalchelates. With a stainless steel column, analytes may not be able to elute from the column or distorted peaks
may be obtained. In this case, a metal-free column can be an effective tool.
50000 1:768.05>261.10(+) CE: -31.0

50000 1:768.05>261.10(+) CE: -31.0

45000

45000

40000

40000

35000

35000

30000

30000

25000

25000

20000

20000

15000

15000

10000

10000

5000

5000

Conditions
ColumnMobile phase: InertSustainSwift C18 (1.9 μm, 50 × 2.1 mm I.D)
: A) 5 mM HCOONH4 in H2OB) 5 mM HCOONH4 in CH3CN
A/B=98/2-2 min-80/20:
0.4 mL/min
Flow rate: 40 ℃
Temperature: LC/MS/MS (ESI, Positive, MRM)
Detection
Injection volume : 10 μL: Coenzyme A (Q1/Q3 = 768/261）
Analyte

1NH2OH
HS

NH

NH

OOO

O

O P O P O
CH
CH3 3 OH OH0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

min

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

min

Time(mm)Time(mm)<Stainless steel column><Metal-free column>

2Intensity, cps

6000

5000

5000

4000

4000

3000
2000
1000

1000

00

2

4

68Time, min

10<Stainless steel column>

12HO

2468Time, min<Metal-free column>

Fumonisins, kinds of mycotoxins, are strong metal chelators because of
their adjacent carboxyl and hydroxyl groups. Adsorption of fumonisins onto
the column inner wall and the filters of stainless steel columns distorts thepeak shapes.

Higher sensitivity
Better peak shapes

21500

OHOH

CH3Conditions
ColumnMobile phase
Flow rate
Temperature
Detection
Analyte

R

8000

300
0.0

4000
1 2 3 4 5 6 7 8 9 10 11Time (min)<Stainless steel column>

0.0

1 2 3 4 5 6 7 8 9 10 11Time (min)<Metal-free column>

NH

2. FMOC-Glufosinate: InertSustain C18 （3 μm , 100 ×2.1 mm I.D.）: A) 0.1 % HCOOH ,10 mM HCOONH4B) CH3CN
A/B = 60/40,v/v: 0.2 mL/min: 40 ℃: LC/MS/MS ( ESI , Positive. , MRM ): 1.Fumonisin B1 (Q1/Q3 = 722/334）
2.Fumonisin B2 (Q1/Q3 = 706/336）

1.2e4

600

OH

OH

R: FMOC
1. FMOC-GlyphosateOP

Fumonisin B1

1.6e4

900

O

O

2

2.0e4

1200

N

1

2.4e4

1R

P

3.2e4
2.8e4

1800

O

10RP Example 3. Fumonisins2100

1. Coenzyme A (500 mg/L)

2000

1

N

: InertSustain C18 (3 μm, 150 × 2.1 mm I.D)
: A) 5 mM HCOONH4 in H2OB) CH3CN
A/B=95/5 -9.3 min - 37/63
Flow rate: 0.25 mL/min
Temperature: 40 ℃
Detection: LC/MS/MS (ESI, Negative, MRM)
Injection volume : 10 μL
Analyte: 1. FMOC-Glyphosate (Q1/Q3 = 390/168）
2. FMOC-Glufosinate (Q1/Q3 = 402/190)
3. FMOC-AMPA(Q1/Q3 = 332/110) 2 µg/L each

3000

3NConditions
ColumnMobile phase

36000

ONO OHHO P O
OH00NFumonisin B2

GL Sciences LC Technical Note
HILIC Example 1. Aminoglycosides
Aminoglycoside antibiotics such as streptomycin possess amino sugar substructures and their adjacent hydroxyland amino groups contribute to metal chelation. The effects of stainless steel depends on the analyte; kanamycin
does not elute from the column, whereas spectinomycin elutes as a broad peak.
1.00e51.00e59.00e49.00e48.00e48.00e47.00e47.00e4

Intensity, cps

Intensity, cps

4. KM

6.00e46.00e45.00e45.00e44.00e44.00e43.00e4

1. SPCM

3.00e4

1. SPCM

2.00e42.00e41.00e41.00e4

2. SM
3.GEM

0.00

2. SM

3. GEM did not elute
4. KM did not elute
1234

56

7 8 9Time, min

0.00

10 11 12 13 14 15<Stainless steel column>

1234567 8 9Time, min

10 11 12 13 14 15<Metal-free column>
H2NNH

HNH

H2NO

HONH

OHRO

HOHN

NH2ROH

H2NOH

NH
O

1. Spectinomycin

OO

2. Streptomycin

HONH2HOO

OH O

HOHO

NH2

NH

3. Gentamycin

O
NH2

HO

4. Kanamycin

OH

O

HONH

OH
NH2

Conditions
Column: Inertsil Amide(5 μm, 150 x 2.1 mm I.D.)

Mobile phase : A) 0.1 % HCOOH in CH3CN
B) 0.1 % HCOOH in H2O
A/B = 70/30 – 10 min – 50/50 , v/v
Flow rate: 0.2 mL/min

Temperature : 40 ℃

Injection volume : 10 mL
Detection: LC/MS/MS (ESI, Positive, MRM)
Q1
1. Spectinomycin(SPCM) 351
2. Streptomycin(SM)3. Gentamycin(GEM)
4. Kanamycin(KM)
243

Concentration : 500 mg/L

OH

O

OHO

HOO

Q3
333
292
322
162

176
160

NH2

GL Sciences LC Technical Note
HILIC example 2. Amino acids
Amino acids containing multiple amino groups are easily affected by metals. There is no difference between
stainless steel columns and metal-free columns in the sensitivity of amino acids possessing only one amino group,
such as leucine and valine. On the other hand, the effects of column material are considerable on the sensitivity
and the peak shapes of amino acids include several amino groups, such as histidine and arginine.

Higher sensitivity
Better peak shapes
3.2e6
3.0e6
2.8e6
2.6e6
2.4e6

1. Leucine
2. Isoleucine
3. Valine
4. Histidine

3.0e6
2.8e6
2.6e6
2.4e6

4

2.2e6
Intensity, cps

2.0e6
1.8e6
1.6e6
1.4e6
1.2e6

1

1.0e6

52

8.0e5

3.0

4.0

5.0
6.0Time, min

7.0

1.4e6
1.2e6

12

8.0634.0e5

7
2.0

1.6e6

6.0e5

2.0e5
1.0

1.8e6

8.0e5

6

4.0e5

2.0e6

1.0e6

43

6.0e5

7

2.0e5
9.0

10.0

0.0

1.0

2.0

3.0

4.0

5.0
6.0
Time, min

7.0

8.0

Peak shape comparison for histidine

NNHOH

2.8e6

O

4. Histidine (His)

2.4e6
2.0e6

NH2

1.2e6

0.0

NH

H2NOH
O

NH

Stainless steel column Metal-free column

5. Arginine (Arg)

Conditions

NH2: Inertsil Amide(3 μm, 150 x 2.1 mm I.D.)OH

H2NMobile phase : A) 100 mM HCOONH4, 0.1 % HCOOH in 75 % CH3CN

6. Lysine (Lys)

B) 100 mM HCOONH4, 0.1 % HCOOH in H2O
A/B = 100/0 – 3 min – 100/0 – 5.5 min – 70/30 , v/v
Flow rate: 0.3 mL/min

Temperature : 40 ℃
Detection: LC/MS/MS (ESI, Positive, MRM)

Injection Volume
Concentration : 5 mM

10.0NH2

3.2e6Column

9.0<Metal-free column><Stainless steel column>

Intensity, cps

Intensity, cps

2.2e6

0.05

3.2e6

5. Arginine
6. Lysine
7. Cystine: 5.0 mL

1. Leucine
2. Isoleucine
3. Valine
4. Histidine5. Arginine
6. Lysine
7. Cystine

Q1 Q3132 86
132 86
118 72155 110
175 70147 84241 152

NH2OHO

3. Valine (Val)OGL Sciences LC Technical Note

Packing Material

Packing Material

PEEK

PEEK

Stainless SteelUHPLC-PEEK Columns

PEEK Columns

Analytical Columns List
InertSustain Series
・InertSustain C18・InertSustain C8・InertSustainSwift C18 ・InertSustain NH2・InertSustain AQ-C18

・InertSustain Phenylhexyl
・InertSustain Phenyl

Inertsil Series
・Inertsil ODS-4・Inertsil ODS-3・Inertsil ODS-SP・Inertsil ODS-P・Inertsil ODS-EP・Inertsil C8-4・Inertsil C8-3・Inertsil Ph-3・Inertsil WP300 C18・Inertsil WP300 C8・Inertsil WP300 C4・Inertsil Peptides C18・Inertsil HILIC・Inertsil Amide
・Inertsil Diol・Inertsil NH2・Inertsil WP300 Diol・Inertsil SIL-100A・Inertsil WP300 SIL・Inertsil CN-3

*Other packing materials are on request.
*Check https://www.glsciences.com/product/lc_columns/01853.html for details.

GL Sciences disclaims any and all responsibility for any injury or damage which may be caused by this data directly or indirectly.
We reserve the right to amend this information or data at any time and without any prior announcement.

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