ACE®

Excel™ UHPLC Columns

info@ace-hplc.com

Advanced Chromatography Technologies Ltd

www.ace-hplc.com

Australia & New Zealand contact: infor@winlab.com.au or call +61 7 3205 5233

ACE®

HPLC / UHPLC Columns

Outline

Introduction

Key Product Points

Examples of Isocratic and Gradient HPLC UHPLC

Key Product Characteristics

Fixtures and Fittings

2

Australia & New Zealand contact: infor@winlab.com.au or call +61 7 3205 5233

ACE®

HPLC / UHPLC Columns

Key ACE®

Excel™ Features

Available as NEW high efficiency, ultra-inert 2mm silica particles

and suitable for UHPLC at 1000bar (15000psi)

Nine selectivities – including the unique C18-AR and C18-PFP

Highly reproducibility: column-to-column and batch-to-batch

Ultra-robust phases: NEW low dispersion column hardware and

NEW High Stability Column (HSC™) packing technology

Engineered with lower back pressures compared to other <2mm

phases due to 2mm particle size and frit technology

Fully scalable to ACE® 3mm, 5mm and 10mm

(Usable at lower pressures giving a performance boost to HPLC) Australia & New Zealand contact: infor@winlab.com.au or call +61 7 3205 5233

ACE®

HPLC / UHPLC Columns

Waters XBridge 5 C18150 x 4.6 mm1.00 ml/min

Waters ACQUITY 1.7 BEH C1850 x 2.1 mm0.21 ml/min

HPLC: 5µm C18tr4=13.60 mins

(L/dp = 3)

UHPLC: <2µm C18tr4=4.69 mins

(L/dp = 2.9)

UHPLC: ACE Excel 2µm C18-PFPtr4=0.66 mins

Rs = 1.9

ACE Excel 2 C18-PFP

30 x 2.1 mm1.30 ml/min

Aim: obtain Rs

≥ 1.7 in shortest possible time for mixture

Sample: 1) 1,2,dimethoxybenzene, 2) 1,3-dimethoxybenzene, 3) 1,3,5-trimethoxybenzene, 4) toluene (reference).Mobile phase 50:50 MeOH / H2O; Temperature 40°C

To maintain Rs and

reduce run time,

keep L / dp ratio

constant

Using UHPLC and selectivity, it

is possible to dramatically improve

resolution allowing shorter columns

& increased flow rates

Using ACE®

Excel™ C18-PFP Selectivity & Throughput (Isocratic)

~ x23

Quicker

All trademarks are recognised

4

Rs = 1.7

Rs = 1.1

Rs = 3.4

1 2

3

4

1 2

3

4

2

3

4

1

1

2

3

4

Australia & New Zealand contact: infor@winlab.com.au or call +61 7 3205 5233

ACE®

HPLC / UHPLC Columns

ACE Excel 2 C18-PFP

30 x 2.1 mm2.5 ml/min, tG = 0.7 min

max pressure: 914 bar1 min cycle time

ACE 5 C18100 x 4.6 mm

1 ml/min, tG = 29 minmax pressure: 92 bar

40 min cycle time

ACE Excel 2 C1850 x 2.1 mm

0.6 ml/min, tG = 5 minmax pressure: 367 bar

9 min cycle time

1, aspirin; 2, phenacetin; 3, 1,3-dinitrobenzene ; 4, ethylbenzoate; 5, nimesulide; 6, ibuprofen; 7, indomethacin.

Rs = 1.8

13

2

5

4

7

6, 22.49 min

Rs = 1.7

2.5 5

6, 4.17 min1

2

54

7

Rs = 1.5

3

min1.5 2 2.5 3 3.5 40.5 1

1

2

34

5

6

7, 0.61 min

Rs = 1.8 Rs = 1.9

Rs = 1.7

Rs

≥ 1.7

HPLC: 5µm C1822.49 mins

UHPLC: <2µm C184.17 mins

UHPLC: 2µm C18-PFP0.61 mins

min5 10 15 20 250

250

0

0

Using ACE®

Excel™ C18-PFP Selectivity & Throughput (Gradient)

Aim: obtain Rs

≥ 1.7 in shortest possible time for mixture

~ x25

Quicker

5

Australia & New Zealand contact: infor@winlab.com.au or call +61 7 3205 5233

ACE®

HPLC / UHPLC Columns

Specifically engineered for lower UHPLC backpressures

ACE®

Excel™ Has Typically Lower Back Pressure For UHPLC

min0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

3, 4

5

6

89

10

11

12

13

Agilent ZORBAX Eclipse 1.8 um XDB C18

Pmax: 540 bar

1 2

3

5

6

7

89

10

11

12

13 4

Waters ACQUITY 1.7um BEH C18

Pmax: 581 bar

1 2

1 2

3

4 5

6

7

8 9

10

11

12

13

ACE Excel 2 C18-AR

Pmax: 364 bar

1, 2

35

6

7

8 9 10

11

1213

4

Phenomenex Kinetex 1.7um C18

Pmax: 540 bar

50x2.1mm; conditions: A=5mM formic acid (aq); B=5mM formic acid in MeOH; tg= 3 to 100%B in 5 min; 0.6 ml/min; 40C; 254nm

1. Paracetamol 2. Hydrochlorothiazide 3. Methylphenylsulphoxide 4. Methylphenylsulphone 5. Aspirin 6. Phenacetin 7. 1,3-dinitrobenzene 8. 1,2,4-trimethoxybenzene 9. Ethylbenzoate 10. Nimesulide 11. Ibuprofen 12. Indomethacin 13. Mefenamic acid All trademarks are recognised

6

7

Australia & New Zealand contact: infor@winlab.com.au or call +61 7 3205 5233

ACE®

HPLC / UHPLC Columns

ACE®

Excel™ UHPLC Columns – Scalability & Reproducibility

ACE Excel 2 C18150 x 2.1mm0.21mL/min

ACE 3 C18150 x 3.0mm0.40mL/min

ACE 5 C18150 x 4.6mm1.00mL/min

ACE 10 C18150 x 21.2mm21.2mL/min

UHPLC

HPLC

Prep

LC2 310 6 754 8

MP: 35:65 v/v MeCN:0.1% TFA (aq); 22C; 254nm; 1. uracil; 2. 4-hydroxybenzoic acid; 3. acetylsalicyclic acid; 4. benzoic acid; 5. 2-hydroxybenzoic acid 6. ethyl paraben

3 µm

5 µm

10 µm

2 µm

1 2 34

5

6

Australia & New Zealand contact: infor@winlab.com.au or call +61 7 3205 5233

ACE®

HPLC / UHPLC Columns

NEW High Stability Column (HSC™) Packing Technology

Significantly Improves UHPLC Column Robustness

ACE®

Excel™ UHPLC Column Robustness

ACE® Excel™ 2 C18(with HSC™ Packing Technology)

Prototype 2 mm C18(without HSC™ Packing Technology)

1000 bar for ~2000 gradient runs Isocratic efficiency assessments every ~100 runs more demanding!

8

100x2.1mm; MPA 0.1% FA (aq); MPB: 0.1% FA in MeOH; 0.73mL/min; gradient: 20-90%B in 6 mins, 1 min hold; 3 min re-equilibration.

Australia & New Zealand contact: infor@winlab.com.au or call +61 7 3205 5233

ACE®

HPLC / UHPLC Columns

ACE®

Excel™ UHPLC Columns

pH and Temperature Stability:

For optimal column lifetime, as with ‘standard’ ACE columns,

the recommended pH operating range is 2-8

ACE Excel columns can be used outside of this pH range but

column lifetimes will vary with a number of factors (buffer,

temperature, solvents etc)*

ACE Excel columns can be operated at temperatures up to 100°C (but temperatures >60°C will reduce column lifetimes)

* For further information see FREE ACE booklet: ‘A Guide to HPLC and LC-MS

Buffer Selection’

Australia & New Zealand contact: infor@winlab.com.au or call +61 7 3205 5233

ACE®

HPLC / UHPLC Columns

ACE®

Excel™ C18-AR: Water Soluble Vitamins - Selectivity

1

2 3

4

5

6

7 ACE Excel 2 C18-AR Pmax: 209 bar

1 2, 3

4

5 6

7 PhenomenexKinetex 1.7 C18 Pmax: 315 bar

1

2 3

4

5 6

7 WatersACQUITY BEH C18 Pmax: 336 bar

min1 2 3 4 5

Agilent ZORBAXEclipse XDB-C18

1 2

3 5

6

7 Pmax: 299 bar

ACE Excel 2 C18

1

2 3

4 5, 6

7 Pmax: 216 bar

4

50x2.1mm; MP A: 20 mM KH2PO4, pH 2.7; MPB: 20 mM KH2PO4, pH 2.7 in MeOH/H2O (50:50 v/v); Gradient: 20 – 70 %B in 5 min; 0.31 ml/min; 40 °C; 205 nm1. Pyridoxine; 2. P-aminobenzoic acid; 3. Pantothenic acid; 4. Folic acid; 5. D-Biotin; 6. Cyanocobalamin; 7. Riboflavin

Australia & New Zealand contact: infor@winlab.com.au or call +61 7 3205 5233

ACE®

HPLC / UHPLC Columns

ACE®

Excel™ C18-PFP: Structural Isomers - Selectivity

ACE Excel 2 C18-PFP

16

4 7

5 2

3

P = 147 bar

Waters 1.7 ACQUITY BEH C18

1, 2, 3

6

4, 5

7

P = 237 bar

PhenomenexKinetex 2.6 C18

1, 2, 3

6 4, 5

7

P = 125 bar

min1 2 3 4 5 6 7 8 9

AgilentPoroshell 120 EC-C18

1, 2, 3

6 4, 5

7

P = 108 bar

50x2.1mm; MP: MeOH/H2O (50:50 v/v); 0.21 ml/min; 40 °C; 210/214 nm; 1. 1,2,3-trimethoxybenzene; 2. 1,2-dimethoxybenzene; 3. 1,2,4-trimethoxybenzene; 4. methoxybenzene; 5. 1,3-dimethoxybenzene; 6. 1,3,5-trimethoxybenzene; 7. toluene

1, 2, 3

6 4, 5

7

ACE Excel 2 C18

P = 150 bar

Australia & New Zealand contact: infor@winlab.com.au or call +61 7 3205 5233

ACE®

HPLC / UHPLC Columns

ACE®

Excel™ UHPLC Columns

A reusable ACE UHPLC column connector is available

Rated to 1720 bar (25,000 psi), these are suitable for at least 10

make / break cycles when correctly fitted

Suitable for use at temperature up to 100C

A torque wrench is recommended to ensure the correct force is

applied

EXL-CC = 1 / pk

EXL-CC10 = 10 / pk

EXL-TW =1 / pk

Australia & New Zealand contact: infor@winlab.com.au or call +61 7 3205 5233

ACE®

HPLC / UHPLC Columns

ACE®

Excel™ UHPLC Columns

A starter kit containing 1 x Torque Wrench and 4 x reusable

connectors is available

EXL-CCSK+ +++