LC Technical Note LT152 Metal free Columns for HPLC Part 1 · Metal‐free columns have wetted parts made of PEEK instead of metal. In this technical note, some applications using

LC Technical Note

GL Sciences Inc.

LT152 Metal‐free Columns for HPLC Part 1

Some amino acids, organic acids, tetracyclines and organophosphates are metal chelators. Chelation duringanalysis of these compounds can distort the peak shapes and worsen the quantification accuracy. Residualmetals on the packing surface were assumed to cause these problems. However, in the case of microanalysis, ithas 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 bufferscannot 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 applicationsusing metal‐free columns are shown and the advantages of metal‐free columns are described.

(K.Kanno)

NHN

OH

NH2

O

Metal Effects○ Peak tailing○ No elution○ Low sensitivity

etc.

In HPLC, analytes containing the functional groups above are easily affected by metals.

Histidine

Ethylenediaminetetra‐acetic acid (EDTA) P

OOH

O

OH

*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

Tetracyclines are metal chelators, and their adsorption onto residual metals on the packing surface and

metal parts of the column results in poor performance.

By using a highly inert column with minimized residual metals, high sensitivity becomes possible with

formate buffers, which are often used in LC/MS(/MS). Further sensitivity improvement is possible with a

metal‐free column.

<Stainless steel column>ODS column A

Particle size：1.7 µm

1

2

Conditions

Column : 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

B) 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 : 10 mL

Concentration : 2.0 mg/L

Q1 Q31. Oxytetracycline(OTC) 460.9 426.22. Tetracycline(TC) 445.0 154.0

Structures are created using Chemistry 4‐D Draw which is provided by ChemInnovation Software, Inc.

2. Tetracycline

1. Oxytetracycline2. Tetracycline

2 mg/L each

RP Example 1. Tetracyclines

2.6 3.0 3.4 3.8Time, min

0

20

40

60

80

100

120

140

160

180

200

Inte

nsity

, cp

s

2.6 3.0 3.4 3.8Time, min

0

20

40

60

80

100

120

140

160

180

200

2.6 3.0 3.4 3.8Time, min

0

20

40

60

80

100

120

140

160

180

200

1

2

1

2

Different column materialDifferent packing inertness

Stainless steel or PEEK

<Stainless steel column>InertSustainSwift C18Particle size：1.9 µm

<Metal‐free column>InertSustainSwift C18Particle size：1.9 µm


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 peaksmay be obtained. In this case, a metal‐free column can be an effective tool.

1. Coenzyme A (500 mg/L)

RP Example 2. Phosphate group‐containing compounds

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 min

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

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

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 min

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

50000 1:768.05>261.10(+) CE: -31.0: InertSustainSwift C18 (1.9 μm, 50 × 2.1 mm I.D): A) 5 mM HCOONH4 in H2OB) 5 mM HCOONH4 in CH3CNA/B=98/2‐2 min‐80/20: 0.4 mL/min: 40 ℃: LC/MS/MS (ESI, Positive, MRM) : 10 μL: Coenzyme A (Q1/Q3 = 768/261）

ConditionsColumnMobile phase

Flow rateTemperatureDetectionInjection volumeAnalyte

SHNH NH

O O

OH

CH3CH3

OO

OHO

P

O

OH

O

OH

O

O

P

POH

OH O

N

N

N

N

NH2

Time(mm)Time(mm)

1

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

RP Example 3. Fumonisins: InertSustain C18 （3 μm , 100 ×2.1 mm I.D.）: A) 0.1 % HCOOH ,10 mM HCOONH4

B) CH3CNA/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）

Fumonisin B1

Fumonisin B2Time (min)

1

22100

1800

1500

900

300

1200

600

1 2 3 4 5 6 7 8 9 10 110.0

1 2 3 4 5 6 7 8 9 10 11

Time (min)

2.8e4

2.4e4

8000

4000

0.0

2.0e4

1.6e4

1.2e4

3.2e4

2

1

2 4 6 8 10Time, min

0

1000

2000

3000

4000

5000

6000

Inte

nsity

, cps

: InertSustain C18 (3 μm, 150 × 2.1 mm I.D): A) 5 mM HCOONH4 in H2OB) CH3CNA/B=95/5 -9.3 min - 37/63

: 0.25 mL/min: 40 ℃: LC/MS/MS (ESI, Negative, MRM) : 10 μL: 1. FMOC-Glyphosate (Q1/Q3 = 390/168）2. FMOC-Glufosinate (Q1/Q3 = 402/190)3. FMOC-AMPA (Q1/Q3 = 332/110) 2 g/L each

PHO

O

OH

NOH

OR

R: FMOC

1. FMOC‐Glyphosate

2 4 6 8 10Time, min

0

1000

2000

3000

4000

5000

6000

1

2

3

1 2

3

PCH3 OH

OO

OHNH

R

2. FMOC‐Glufosinate

<Stainless steel column> <Metal‐free column>



Flow rateTemperatureDetectionInjection volumeAnalyte



Flow rateTemperatureDetectionAnalyte

Higher sensitivityBetter peak shapes

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; kanamycindoes not elute from the column, whereas spectinomycin elutes as a broad peak.

HILIC Example 1. Aminoglycosides

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15Time, min

0.00

1.00e4

2.00e4

3.00e4

4.00e4

5.00e4

6.00e4

7.00e4

8.00e4

9.00e4

1.00e5

Intensity, cps

4. KM

1. SPCM

2. SM

1. SPCM

3. GEM did not elute4. KM did not elute


Conditions

Column : Inertsil Amide

(5 μm, 150 x 2.1 mm I.D.)



A/B = 70/30 – 10 min – 50/50 , v/v




Detection : LC/MS/MS (ESI, Positive, MRM)Q1 Q3

1. Spectinomycin(SPCM) 351 3332. Streptomycin(SM) 292 1763. Gentamycin(GEM) 322 1604. Kanamycin(KM) 243 162

Concentration : 500 mg/L

1. Spectinomycin

4. Kanamycin

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15Time, min

0.00

1.00e4

2.00e4

3.00e4

4.00e4

5.00e4

6.00e4

7.00e4

8.00e4

9.00e4

1.00e5

Intensity, cps

O

HO OH

O

HO

NH2

NH2

O

HO OH

OH

HO

NH2

NH2

O

NH

HO

OH

O

O O

NH2

HO NH2 HNR

R

H2N

H

3. Gentamycin

OO

O

O

O

NH

NH

OH

OHHO

NH

H2N

H2N

NH

HO NH

OH

HO

OH

2. Streptomycin

3.GEM

2. SM



Amino acids containing multiple amino groups are easily affected by metals. There is no difference betweenstainless 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 sensitivityand the peak shapes of amino acids include several amino groups, such as histidine and arginine.

HILIC example 2. Amino acids

Conditions

Column : Inertsil Amide

(3 μm, 150 x 2.1 mm I.D.)

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

B) 100 mM HCOONH4, 0.1 % HCOOH in H2O

A/B = 100/0 – 3 min – 100/0 – 5.5 min – 70/30 , v/v




Injection Volume : 5.0 mL

Concentration : 5 mM

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0Time, min

0.0

2.0e5

4.0e5

6.0e5

8.0e5

1.0e6

1.2e6

1.4e6

1.6e6

1.8e6

2.0e6

2.2e6

2.4e6

2.6e6

2.8e6

3.0e6

3.2e6

Inte

nsi

ty, c

ps

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0Time, min

0.0

2.0e5

4.0e5

6.0e5

8.0e5

1.0e6

1.2e6

1.4e6

1.6e6

1.8e6

2.0e6

2.2e6

2.4e6

2.6e6

2.8e6

3.0e6

3.2e6

Inte

nsi

ty, c

ps

1


2

34

5

6

7

12

3

4

5

6

7

1. Leucine2. Isoleucine3. Valine4. Histidine

5. Arginine6. Lysine7. Cystine

Q1 Q3

1. Leucine 132 86

2. Isoleucine 132 86

3. Valine 118 72

4. Histidine 155 110

5. Arginine 175 70

6. Lysine 147 84

7. Cystine 241 152

4. Histidine (His)

H2N NH OH

O

NH2

NH

NHN

OH

NH2

O

OH

O

NH2

H2N

6. Lysine (Lys)

5. Arginine (Arg)

0.0

1.2e6

2.0e6

2.4e6

2.8e6

3.2e6

Inte

nsity

, cps

Peak shape comparison for histidine

Stainless steel column Metal‐free column

OH

O

NH2

3. Valine (Val)

Higher sensitivityBetter peak shapes


GL Sciences, Inc. USA4733 Torrance Blvd. Suite 255Torrance, CA 90503Phone: 310‐265‐4424Fax: 310‐265‐4425Email: [email protected]: www.glsciencesinc.com

GL Sciences, Inc. Japan22‐1 Nishishinjuku 6‐ChomeShinjuku‐ku, Tokyo, 163‐1130, JapanPhone: +81‐3‐5323‐6620Fax: +81‐3‐5323‐6621Email: [email protected]: www.glsciences.com

International DistributorsVisit our Website at www.glsciences.com/distributors

GL Sciences B.V.De Sleutel 95652 AS EindhovenThe NetherlandsPhone: +31 (0)40 254 95 31Email: [email protected]: www.glsciences.eu

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.

Packing Material

PEEK

Packing Material

PEEK

Stainless Steel

UHPLC‐PEEK Columns PEEK Columns

InertSustain Series

・InertSustain C18・InertSustainSwift C18・InertSustain AQ‐C18

・Inertsil ODS‐4・Inertsil ODS‐3・Inertsil ODS‐SP・Inertsil ODS‐P・Inertsil ODS‐EP

・InertSustain Phenylhexyl・InertSustain Phenyl

Inertsil Series

・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

・InertSustain C8・InertSustain NH2

Analytical Columns List

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

LC Technical Note

GL Sciences Inc.

LT155 Metal‐free Columns for HPLC Part2

The advantages of metal‐free columns are described in LT152 “Metal‐free columns for HPLC Part1”. Thistechnical note focuses on the packing inertness (e.g. residual metals) as a sequel to LT152.

(K.Kanno)

Oxine copper, a kind of pesticide, is a metal chelator and a better peak is expected to be obtained with a metal‐free column. In the chromatograms below, two different packings are compared between stainless steel andmetal‐free columns. The highly inert packing increases the peak height more significantly, and the effects ofmetal‐free columns are more pronounced.

RP Example 1. Oxine copper

Stainless steel Metal‐free Stainless steel Metal‐free

Conditions

Column : ODS columns (2 μm, 50 x 2.1 mm I.D.)



A/B = 5/95 – 5 min – 100/0 , v/v





Analyte : Q1 Q3

1. Oxine copper 146 128Concentration : 2.0 mg/L


1. Oxine cupper

N

O

Cu

O

N

Inertness★★★★★

InertSustain C18 Particle size：2 mm

Inertness★★★

Brand‐A Particle size：2 mm

1 2

Time, min

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

Intensity, cps

1 2

Time, min

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

Intensity, cps

1 2

Time, min

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

Intensity, cps

1 2

Time, min

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

Intensity, cps

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


Inertness of the packing and the column material is compared in analysis of 17 pesticides. The differences in thepeak heights are smaller than the case of RP Example 1. Oxine copper, however, the more highly inert columnsgenerally yield higher peak heights. Even for analysis of compounds not categorized as metal chelators, a morehighly inert packing can highlight the effects of metal‐free columns.

RP Example 2. Pesticides

Conditions




A/B = 5/95 – 5 min – 100/0 – 3 min , v/v




Injection volume : 5.0 mL

Concentration : 20 mg/L each

1 2 3 4 5 6 7 8 9

Time, min

0

1 e5

2 e5

3 e5

4 e5

5 e5

6 e5

7 e5

8 e5

9 e5

Intensity, cps

1 2 3 4 5 6 7 8 9

Time, min

0

1 e5

2 e5

3 e5

4 e5

5 e5

6 e5

7 e5

8 e5

9 e5

Intensity, cps

1 2 3 4 5 6 7 8 9

Time, min

0

1 e5

2 e5

3 e5

4 e5

5 e5

6 e5

7 e5

8 e5

9 e5

Intensity, cps

1 2 3 4 5 6 7 8 9

Time, min

0

1 e5

2 e5

3 e5

4 e5

5 e5

6 e5

7 e5

8 e5

9 e5

Intensity, cps



Inertness★★★


Q1 Q3 DP CE

1. Oxine Copper 146 128 80 34

2. Asulam 231 156 61 17

3. Methomyl 163 88 41 13

4. Tricyclazole 190 163 76 33

5. MPP oxon sulfone 295 217 60 20

6. Thiodicarb 355 88 56 33

7. Carbofuran 222 165 50 19

8. Thiuram 241 88 60 20

9. MPP oxon 263 231 60 20

10. Bensulfuron methyl 411 149 71 29

11. Flazasulfuron 408 182 60 20

12. Siduron 233 137 81 23

13. Azoxystrobin 404 372 65 19

14. Dymron 269 151 64 20

15. Iprodione 330 245 61 21

16. Bensulide 398 314 71 17

17. Carpropamid 334 139 66 27

1

CUR CAD IS TEM GS1 GS210 7 5500 500 80 80


As shown in LT152, tetracyclines easily adsorb onto residual metals on the packing surface or metal parts of the

column by chelation, and this adsorption has a bad influence on the analysis. Like RP Example 1. Oxine copper,

the less residual metals are on the packing, the more significantly the peak heights increase. In the analysis of

metal chelators, not only the column material but also the inertness of the packing are important.

Stainless steel

Conditions




A/B = 10/90 – 1 min – 10/90 – 3 min – 90/10 , v/v





Analyte : Q1 Q3

1. Oxytetracycline(OTC)460 4262. Tetracycline(TC) 445 4103. Chlortetracycline(CTC)479 444

*Conditions are partly different from those of tetracycline analysis in LT152.


2. Tetracycline

1. Oxytetracycline2. Tetracycline3. Chlortetracycline

1.0 2.0 3.0 4.0 5.0Time, min

0

400

800

1200

1600

2000

2400

2800

Inte

nsity

, cp

s

1.0 2.0 3.0 4.0 5.0Time, min

0

400

800

1200

1600

2000

2400

2800

Inte

nsity

, cp

s

1.0 2.0 3.0 4.0 5.0Time, min

0

400

800

1200

1600

2000

2400

2800

Inte

nsity

, cp

s

1.0 2.0 3.0 4.0 5.0Time, min

0

400

800

1200

1600

2000

2400

2800

Inte

nsity

, cp

s

Metal‐free Stainless steel Metal‐free

1 23

12

3

1

2

3

1

2

3

Maximum height increase: 12% (1.OTC)

Maximum height increase: 55% (2. TC)



Inertness★★★


CUR CAD IS TEM GS1 GS210 7 5500 700 80 80


GL Sciences, Inc. USA4733 Torrance Blvd. Suite 255Torrance, CA 90503Phone: 310‐265‐4424Fax: 310‐265‐4425Email: [email protected]: www.glsciencesinc.com

GL Sciences, Inc. Japan22‐1 Nishishinjuku 6‐ChomeShinjuku‐ku, Tokyo, 163‐1130, JapanPhone: +81‐3‐5323‐6620Fax: +81‐3‐5323‐6621Email: [email protected]: www.glsciences.com

International DistributorsVisit our Website at www.glsciences.com/distributors

GL Sciences B.V.De Sleutel 95652 AS EindhovenThe NetherlandsPhone: +31 (0)40 254 95 31Email: [email protected]: www.glsciences.eu

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.

Packing Material

PEEK

Packing Material

PEEK

Stainless Steel

UHPLC‐PEEK ColumnsPEEK Columns

InertSustain Series

・InertSustain C18・InertSustainSwift C18・InertSustain AQ‐C18

・Inertsil ODS‐4・Inertsil ODS‐3・Inertsil ODS‐SP・Inertsil ODS‐P・Inertsil ODS‐EP

・InertSustain Phenylhexyl・InertSustain Phenyl

Inertsil Series

・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

・InertSustain C8・InertSustain NH2

Analytical Columns List

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

Documents

LC Technical Note LT152 Metal free Columns for HPLC Part 1 · Metal‐free columns have wetted parts made of PEEK instead of metal. In this technical note, some applications using